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The rat ovarian surface epithelium in vitro Adams, Anne Theresa 1982

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THE RAT OVARIAN SURFACE EPITHELIUM IN VITRO by ANNE THERESA ADAMS B.Sc • r The University of Toronto, 1962 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1982 (c^Anne Theresa Adams, 1982 DOCTOR OF PHILOSOPHY i n In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Zoology  The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 March 3, 1982 nF-fi (7/1V) ABSTRACT The ovarian surface epithelium, a very small portion of the to t a l mass of the ovary, i s generally thought to be the s i t e of origin of over 85% of ovarian cancers. Such cancers are c l a s s i f i e d with the "Common E p i t h e l i a l Tumours" of the ovary. In most i n d u s t r i a l i z e d countries, malignancies of the ovary rank fourth in cancer deaths in women; over 70% of these neoplasms have spread beyond the ovary when f i r s t diagnosed. Experimental approaches to the study of carcinogenesis in this tissue have been limited by the lack of pure populations of ovarian surface e p i t h e l i a l c e l l s . Studies done on rodents in  vivo suggest that both chemicals and C-type RNA viruses can induce ovarian cancers similar to those which are said to arise from the surface epithelium. However, the c e l l of o r i g i n cannot be proven in such studies. The purpose of thi s project was to develop a model for ovarian cancers of surface* ep-itheliad- o r i g i n based on-carcinogenesis i_n v i t r o . To thi s end a method was devised to culture the rat ovarian surface epithelium in pure form. These cultured c e l l s , whose identity has been confirmed by morphological, histochemical and u l t r a s t r u c t u r a l means, are polygonal with clear cytoplasm, have well-defined borders, and grow in confluent monolayers. Their morphology i s quite d i s t i n c t from those of other ovarian c e l l s in v i t r o . Cultured rat ovarian surface e p i t h e l i a l c e l l s are histochemically positive for 17^-hydroxysteroid dehydrogenase, and negative for A5-30-hydroxysteroid dehydrogenase, the same as in frozen sections of whole rat ovary. U l t r a s t r u c t u r a l l y , cultured surface e p i t h e l i a l c e l l s have basal laminae, m i c r o v i l l i , a p i c a l i n t e r c e l l u l a r junctions, large nuclei, abundant rough endoplasmic reticulum, Golgi complexes, perinuclear bundles of microfilaments, and numerous v e s i c l e s . Although the ovarian suface epithelium is suspected of being an estrogen target tissue, there is no previous report of estrogen receptors in these c e l l s . In this study cultured rat ovarian surface e p i t h e l i a l c e l l s have been shown by autoradiographic means to exhibit estrogen receptor-like a c t i v i t y . Translocation of t r i t i a t e d e s t r a d i o l from cytoplasm to nucleus, and estrogen-specific binding have been demonstrated. E s t r a d i o l was shown to be mitogenic for cultured ovarian surface e p i t h e l i a l c e l l s . From these re s u l t s , the surface e p i t h e l i a l .cells of the ovary should be considered an estrogen target tissue. Kirsten murine sarcoma virus was used to produce three transformed c e l l lines from pure, f i r s t passage cultures of these c e l l s . These three lines retained 170-hydroxysteroid dehydrogenase a c t i v i t y and showed s l i g h t A5-3p-hydroxysteroid dehydrogenase a c t i v i t y . Tumours resu l t i n g when these c e l l s were injected into immunosuppressed female rats were highly malignant and resembled h i s t o l o g i c a l l y human endometrioid stromal sarcomas of the ovary. This neoplasm i s classed with the "Common E p i t h e l i a l Tumours" of the ovary, but is generally not iv considered a derivative of the surface epithelium. In l i g h t of t h i s study, perhaps this tumour should: be considered, to be of surface e p i t h e l i a l o r i g i n . A continuous c e l l l i n e a r i s i n g from pure cultures of rat ovarian surface e p i t h e l i a l c e l l s produced structures ir\ v i t r o resembling those found in ovarian serous p a p i l l a r y cystadenomas of borderline malignancy. This tumour is classed as a common e p i t h e l i a l ovarian tumour. Hence, in t h i s study the rat ovarian surface epithelium has been cultured in pure form, has been characterized for a number of properties by several investigative techniques, and has been shown to be susceptible to transformation by an oncogenic v i r u s ; This work supports the theory that the "Common E p i t h e l i a l Tumours" of the ovary are, in fact, derived from the surface epithelium. The a v a i l a b i l i t y of cultured ovarian surface e p i t h e l i a l c e l l s should allow investigation into factors which make thi s tissue so susceptible to malignant transformation. From such cultures could come markers suitable for use in tests to detect ovarian cancers at an early stage. The culture of pure rat ovarian surface epithelium, as described herein, could readily be used to study chemical, physical and v i r a l carcinogenesis in this tissue to produce experimental models of cancers a r i s i n g in the ovarian surface epithelium. V • ACKNOWLEDGMENTS I wish to thank Dr.. Nelly Auersperg for her advice, guidance and support throughout th i s investigation. To the other members of my research committee, Drs. A. B. Acton, A. F. Burton, P. B Clement and C. V. Finnegan, I wish to express my appreciation for expert advice on this research project and for c r i t i c a l examination of the thesis manuscript. Drs. A. Adler, J. B. Hudson and K. M. McErlane gave valuable help with certain specialized procedures. I would l i k e to thank Drs. S.M.Friedman and C. E. Slonecker of the Department of Anatomy for the f a c i l i t i e s to carry out t h i s research. To Mr. K. S. Wong and Ms. D. Mauldin I extend thanks for expert technical advice and assitance. A special thanks goes to my husband, Robert, whose encouragement and support were esse n t i a l for the completion of this project. To him goes cred i t for the preparation of thi s computer-typescript. Thanks go also to our children, Marion, Vale r i e , Alex and Andrew who helped in many ways. During this investigation the author was recipient of scholarships from the. Natural Sciences and Engineering Research Council of Canada, of an H. R. MacMillan Family Fellowship from the University of B r i t i s h Columbia, and of studentships from the National Cancer Institute of Canada. This research was supported by grants from the National Cancer Ins t i t u t e of Canada to Dr. N. Auersperg. v i TABLE OF CONTENTS ABSTRACT . i i ACKNOWLEDGMENTS v TABLE OF CONTENTS vi LIST OF FIGURES x LIST OF TABLES x i CHAPTER I. INTRODUCTION . .... 1 . THE OVARIAN SURFACE EPITHELIUM IN VIVO 2 a) History 2 b) Development 5 c) Structure 6 d) Function 8 2 . OVARIAN CANCER ...... 9 a) Origin 9 b) Natural Course of Ovarian Cancer 11 c) Epidemiology and Etiology 12 3. ANIMAL MODELS FOR OVARIAN NEOPLASTIC TRANSFORMATION 14 a) Spontaneous Ovarian Tumours in Animals 14 b) Experimental Models for Ovarian Tumourigenesis .. 14 4. THE PROBLEM 15 5. THIS RESEARCH PROJECT: THE RAT OVARIAN SURFACE EPITHELIUM IN VITRO 17 a) Culture Of The Rat Ovarian Surface Epithelium ... 17 b) I d e n t i f i c a t i o n of the Cultured Rat Ovarian Surface Epithelium 19 c) Autoradiographic Investigation of Cultured Rat Ovarian Surface E p i t h e l i a l C e l l s for Evidence of Estrogen Receptors 24 d) Transformation of the Cultured Rat Ovarian Surface Epithelium by Kirsten Murine Sarcoma Virus 26 6. THE QUESTIONS 30 v i i CHAPTER I I . MATERIALS AND METHODS . . 32 1. CELL CULTURE ... .... 33. a) Ovarian Cultures-...' 33 b) Peritoneal C e l l and Muscle Fascia Fibroblast Cultures 43 2. HISTOCHEMISTRY ........ 44 a) Dehydrogenase Testing of Ovarian Cryostat Sections 45 b) L i p i d as Determined by O i l Red 0 Staining 46 c) Dehydrogenase Testing of Cultured C e l l s 46 d) O i l Red 0 Staining for L i p i d in Cultured C e l l s . . . 47 3. BIOCHEMICAL DETERMINATION OF HYDROXYSTEROID DEHYDROGENASE ACTIVITY IN CULTURED CELLS 48 a) Calibration Chromatograms Using Radioinert Steroids . . 49 b) P u r i f i c a t i o n of Radioactive Steroids 50 c) Culture of Ce l l s for Incubation with 1*C-' Steroids 50 d) Incubation of C e l l s with Radioactive Steroid .... 51 e) Extraction of Steroids from Medium 52 f) Chromatography of Extracts 53 g) Autoradiographic Detection of Radioactive Products 54 h) R e c r y s t a l l i z a t i o n of Putative 1"C-Estrone from ROSE Cultures with Radioinert Estrone 54 i) Determination of Spec i f i c A c t i v i t y of Estrone and E s t r a d i o l Crystals and Mother Liquors 56 j) R e c r y s t a l l i z a t i o n of Putative 1"C-progesterone'f-rom ROSE*: C e l l Incubations 5,7 4 ELECTRON MICROSCOPY OF CULTURED ROSE CELLS 57 . 5 TRANSFORMATION OF CULTURED ROSE CELLS BY THE KIRSTEN MURINE SARCOMA VIRUS 58 a) Infection of Cultures with KiMSV 59 b) Histochemistry of Transformed C e l l s 60 c) Tumourigenesis Testing of Transformed C e l l s 60 d) Testing of C e l l s for Evidence of V i r a l Transformation 62 6 AUTORADIOGRAPHIC INVESTIGATION OF TISSUES AND CULTURED CELLS FOR THE PRESENCE OF ESTROGEN RECEPTOR ACTIVITY 63 a) Preparation of Tissue Specimens for v i i i Autoradiography .. 63 b) Fixation and Labelling of Cultured C e l l s for Autoradiography 65 c) Coating of Sections and Cultured C e l l s with Autoradiographic Emulsion 67 d) Development and Staining of Autoradiograms 68 e) Evaluation of Autoradiograms .................... 68 CHAPTER I I I . RESULTS ...70 1". CELL CULTURE ....... . "70 a) Ovarian Cultures 70 b) Peritoneal C e l l and Muscle Fascia Fibroblast Culture '..-.• 88 2. HISTOCHEMISTRY 91 a) Dehydrogenase Testing of Ovarian Cryostat Sections 91 b) L i p i d as Determined by O i l Red 0 in Frozen Sections 92 c) Dehydrogenase Testing of Cultured C e l l s 92 d) O i l Red 0 Staining for L i p i d in Cultured C e l l s - . . 98 3. BIOCHEMICAL DETERMINATION OF HYDROXYSTEROID DEHYDROGENASE ENZYME ACTIVITY IN CULTURED CELLS 98 a) 1 4C-Pregnenolone Incubations 98 b) 1 "C-Estradiol Incubations ...100 c) R e c r y s t a l l i z a t i o n Of Radioactive Estrone To Constant Specific A c t i v i t y 102 4. ELECTRON MICROSCOPY OF CULTURED ROSE CELLS 103 5. TRANSFORMATION OF CULTURED CELLS BY THE KIRSTEN MURINE SARCOMA VIRUS 106 a) Transformation of C e l l s by KiMSV ...106 b) Histochemical Testing of KiMSV-Transformed C e l l s 110 c) Tumourigenesis Testing of Transformed C e l l s 114 d) Evidence for V i r a l Transformation of Cultured C e l l s 127 6. AUTORADIOGRAPHIC INVESTIGATION OF TISSUES AND CULTURED CELLS FOR THE PRESENCE OF ESTROGEN RECEPTOR ACTIVITY 128 a) Autoradiography Of Sections 128 b) Autoradiography of Cultured ROSE Ce l l s 131' 7. CONTINUOUS CELL LINES ROSE-199 AND ROSE-239 138 ix a) ROSE-199 ... .. 138 b) ROSE-239 . .. . ... ..148 CHAPTER IV. DISCUSSION ........... ..150 1. IDENTIFICATION OF RAT OVARIAN SURFACE EPITHELIAL CELLS IN CULTURE .......150 .2 A COMPARISON OF GROWTH CURVES FOR DIFFERENT OVARIAN CULTURES .. . 154 3. FURTHER CHARACTERIZATION OF CULTURED ROSE CELLS ....156 4. AN EVALUATION OF AUTORADIOGRAPHIC TECHNIQUES 162 5. DEVELOPMENT OF MODELS FOR OVARIAN CANCER 164 a) Transformation Of ROSE C e l l s With Kirsten Murine Sarcoma Virus 165 b.) Continuous Line ROSE-199, A Tumour In V i t r o ....169 6. CONCLUSION 171 CHAPTER V. SUMMARY 174 BIBLIOGRAPHY 176 APPENDIX I. SOURCES OF MATERIALS 188 APPENDIX II . PREPARATION OF SOLUTIONS 194 APPENDIX I I I . PROCEDURES FOR HISTOLOGY, HISTOCHEMISTRY AND ELECTRON MICROSCOPY ..201 APPENDIX IV. TECHNIQUES FOR STEROID BIOCHEMISTRY 206 APPENDIX V. TECHNIQUES INVOLVED WITH KIRSTEN VIRUS 212 APPENDIX VI. AUTORADIOGRAPHIC DETECTION OF ESTROGEN RECEPTORS 215 X LI ST OF FIGURES FIGURE 1. Culture method for ROSE c e l l s .... 40 FIGURE 2. C e l l types in.cultures from whole ovaries 72 FIGURE 3. Growth curves for mixed c e l l cultures from whole ovary . . . 74 FIGURE 4. Removal of ovarian surface c e l l s by dis s o c i a t i v e enzymes 79 FIGURE 5. Cultured rat ovarian surface e p i t h e l i a l c e l l s . 82 FIGURE 6. Growth curves for f i r s t passage ROSE c e l l s .... 84 FIGURE 7. Culture morphology of peritoneal c e l l s 90 FIGURE 8. Culture morphology of muscle facia f i b r o b l a s t s 90 FIGURE 9. Hydroxysteroid dehydrogenase histochemistry of the rat ovarian surface epithelium 94 FIGURE 10. Lactate dehydrogenase and l i p i d histochemistry of the rat ovarian surface epithelium 95 FIGURE 11. Ultrastructure of ROSE cells, in culture 105 FIGURE 12. V i r a l transformation of cultured ROSE' c e l l s ...109 FIGURE 13. Hydroxysteroid dehydrogenase histochemistry of KiMSV-transformed ROSE c e l l s 113 FIGURE 14. Histopathology of tumours from KiMSV-transf ormed ROSE l i n e s 120 FIGURE 15. Histopathology of tumours from KiMSV-transformed l i n e s of ovarian and peritoneal o r i g i n 121 FIGURE 16. Ultrastructure of tumours derived from KiMSV-transf ormed ROSE c e l l s 123 FIGURE 17. Histopathology of a tumour a r i s i n g in a rat injected with ROSE-1 99 c e l l s .. 124 FIGURE 18. Evidence for v i r a l transformation of cultured c e l l s 124 FIGURE 19. Autoradiography of sections of ovary and uterus 130 FIGURE 20. Autoradiograms of cultured ROSE c e l l s 130 FIGURE 21. Grains per" c e l l and" N/C ve-rsus/ c e i L area* v ...... 1 34 -FIGURE 22. Culture morphology of ROSE-199 c e l l s ..140 FIGURE 23. C e l l densities of ROSE-199 cultures 141 FIGURE 24. Growth curves for ROSE-199 c e l l s at 25th passage 143 FIGURE 25. Multilayered structures formed by ROSE-199 c e l l s in v i t r o 146 xi LIST OF TABLES TABLE I. Enzyme di s s o c i a t i o n treatments used on whole ovaries 38 TABLE II. Incubations performed 52 TABLE I I I . Chromatography done 53 TABLE IV. Cultures infected with KiMSV 60 TABLE V. Evaluation of cultures of c e l l s removed from the ovarian surface by enzymes 77 TABLE VI. Effect of e s t r a d i o l on the mitotic index and binucleate index of f i r s t passage ROSE c e l l s . . 87 TABLE VII. Histochemical data for ovarian cryostat sect ions . . . 92 TABLE VIII. Histochemical data for cultured c e l l s 96 TABLE IX. A comparison of a c t i v i t i e s of hydroxysteroid dehydrogenases in ovarian cryostat sections and cultured c e l l s 1 97 TABLE X. Summary of chromatographic results 99 TABLE XI. Radioactivity in progesterone and estrone regions of paper chromatograms .101 TABLE XII. Specific a c t i v i t i e s for cr y s t a l s and mother liquors from r e c r y s t a l l i z a t i o n s 102 TABLE XIII. Summary of KiMSV transformed c e l l l i n e s ....107 TABLE XIV. Summary of histochemical data for KiMSV-transformed c e l l s 111 TABLE XV. Gross observations on tumours produced by KiMSV-transformed c e l l s at subcutaneous injection s i t e s 1 115 TABLE XVI. Gross observations on tumours produced by KiMSV-transformed c e l l s injected i n t r a p e r i t o n e a l l y 1 116 TABLE XVII. Histopathology of tumours derived from KiMSV-transformed c e l l s 118 TABLE XVIII. Comparison of autoradiograms^ of' ROSE'"cells*-la b e l l e d after f i x a t i o n with those l a b e l l e d l i v e ..133 TABLE XIX. Pulse-chase analysis of t r i t i a t e d e s t r a d i o l movement in l i v e - l a b e l l e d ROSE c e l l s 1 135 TABLE XX. Steroid competition in ROSE c e l l s 136 TABLE XXI. C e l l densities of ROSE-239 cultures 148 1 CHAPTER I. INTRODUCTION The surface epithelium of the mammalian ovary represents a small fracti o n of the t o t a l mass of the ovary. This simple epithelium, ranging from squamous to columnar in many species, covers the ovary and is continuous with the mesothelium of the peritoneal cavity at the hilum of the ovary. In spite of i t s apparent insignificance the ovarian surface epithelium has had a long, co l o u r f u l and controversial history as the "germinal" epithelium, that i s , the source of oocytes. It has been suggested as the developmental source of ovarian c e l l types other than oocytes. The surface c e l l s of the ovary have a considerable c l i n i c a l significance as th i s tissue i s considered to be the s i t e of or i g i n of over 85% of ovarian cancers. It i s this l a t t e r aspect of the ovarian surface epithelium which prompted the undertaking of the research presented in this thesis. Before launching into the c l i n i c a l relevance of the surface epithelium of the ovary, I would l i k e to dwell f i r s t on the h i s t o r i c a l , developmental, s t r u c t u r a l and functional facets of t h i s interesting tissue. 2 1. THE OVARIAN SURFACE EPITHELIUM IN VIVO a) Hi story It was Waldeyer in 1870 who originated the idea of the ovarian surface epithelium as the "germinal" epithelium, or source of oocytes in mammals. He claimed that the entire surface of the peritoneal cavity in certain lower vertebrates (e.g. amphibians) had.the potential to produce oocytes, but that in mammals t h i s oogenetic potential had shrunk to the surface c e l l s of the ovary only. In the ensuing 80 years there was much observation and manipulation of thi s tissue in e f f o r t s to prove or disprove i t s germinality. Three schools of thought vied with one another in the f i r s t half of thi s century. The f i r s t claimed that oocytes were derived from the'germinal epithelium both prenatally and c y c l i c a l l y with each estrous cycle. A second stated that oocytes were derived from primordial germ c e l l s migrating from the yolk sac to the gonadal ridge in the early embroyo. The t h i r d , a group of fence-s i t t e r s , accepted that oocytes were derived from both sources. Most reports supporting the idea of an ovarian germinal epithelium were based on subjective interpretations of hi s t o l o g i c data. Edgar Allen, the chief proponent of the germinal epithelium concept in the 1920's to 1940's, based his view on the two observations in ovaries of mice that mitosis in the germinal epithelium varied in phase with the estrous cycle, and that numerous oocytes were clo s e l y associated with the epithelium (1923). He claimed that fresh oocytes were produced 3 each cycle, and that ones from the previous cycle deteriorated. In a later study (Stein and A l l e n , 1942) estrone injected into the rat o v a r i a l bursa stimulated a great increase in mitoses and, according to the interpretation of the researchers, a new wave of oocytes. Latta and Pederson (1942) found carbon p a r t i c l e s in both the germinal epithelium and underlying oocytes of rats injected i n t r a p e r i t o n e a l l y with India ink. They concluded from such observations that carbon-labelled oocytes must have been derived from the overlying germinal e p i t h e l i a l c e l l s . In 1940 Long claimed to have cultured" the mouse germinal epithelium. He supported his claim by pointing out the presence of oocytes in the v i c i n i t y of the cultured e p i t h e l i a l c e l l s . Although this controversy generated much rather subjective research on the "germinal" epithelium, there was the occasional good piece of work. Cunningham, in 1922, set out to map the l i n i n g c e l l s of the peritoneal cavity for regional differences as detected by variations in the pattern of uptake of v i t a l dyes injected into rabbits either i-fiti?aivew0us-iL'y'-o«-' fcntrape^Horceaii-y'. He found that the c e l l s covering the ovary d i f f e r e d in their uptake of v i t a l dyes from other peritoneal mesothelial c e l l s . The question was e f f e c t i v e l y answered by Witschi (1948) who s e r i a l l y sectioned early human embryos and traced the migration of the primordial germ c e l l s from the yolk sac to the primitive gonads. These germ c e l l s undergo mitosis during migration and before f o l l i c l e formation in the" ovary. In an extensive revieV, considering the evidence from a large number of often crude 4 experiments on the germinal epithelium, Zuckerman (1951) concludes that "the view that oogenesis continues, throughout, reproductive l i f e i s very insecurely based." However, he did leave open the question of prenatal derivation of oocytes from the surface epithelium of the. ovary. Evidence that oocytes persist in a state of suspended meiosis from prenatal or early natal l i f e u n t i l ovulation had to wait for the dawn of the t r i t i a t e d thymidine era. Rudkin reports that pregnant mice injected at mid-gestation, with t r i t i a t e d thymidine produced daughters whose oocytes retained radioactive nuclei from mid-fetal age to maturity at six weeks (Rudkin and Greich, 1962). Other ovarian tissues, with the exception of some f o l l i c l e c e l l s in primordial f o l l i c l e s , showed no l a b e l . Loss of label implies repeated mitosis after the l a b e l l i n g period. In a similar experiment using neonate rabbits Kennelly has showed that la b e l l e d does when superovulated at ages 20 to 50 weeks produced mostly radioactive ova (Kennelly and Foote, 1966). These observations' support- the view- that oocytes arise from primordial germ c e l l s and not the ovarian surface epithelium, and that a portion of these oocytes persist u n t i l ovulation at sexual maturity. The view held today is that mammalian oocytes are derived only from primordial germ c e l l s , and that the ovarian surface epithelium is not a germinal one. A few diehards remain; Ludwig (1965) claims that the ovary in adolescent g i r l s undergoes neo-oogenesis from the germinal epithelium. Anatomy texts published 5 as late as the the 1970's (Grouch, 1973; Jacob and Francone, 1970) continue to promulgate this, error (that the ovarian surface epithelium is the source of oocytes) to unwitting students. The term "germinal epithelium" i s f a l l i n g into disuse in the current l i t e r a t u r e , b) Development The ovarian surface epithelium is continuous with the mesothelium l i n i n g the peritoneal cavity. The peritoneal cavity f i r s t arises as coelomic spaces in the l a t e r a l mesoderm of early embryos (Moore, 1977). These coelomic spaces coalesce to become the intraembryonic coelom which at a later phase i s divided into pleu r a l , p e r i c a r d i a l and peritoneal spaces. The coelomic spaces are lined by a simple epithelium, the mesothelium, which i s morphologically e p i t h e l i a l , but i s mesenchymal (that i s , primitive connective tissue) in o r i g i n . The peritoneal mesothelium i s generally squamous, but the epithelium covering the ovary varies from squamous to columnar in many species. In histology texts i t i s often given as- an example of a- simple' cuboidal epithelium. The s i t e of development of the ovary l i e s very close to the s i t e of invagination of the Mullerian (paramesonephric) duct, which i s the primordium for oviduct, uterus and upper vagina. Hence the e p i t h e l i a l l i n i n g of Mullerian duct derivatives and the ovarian surface epithelium are embryologically closely related, both being derived from the coelomic epithelium. This fact w i l l be important later in the discussion of neoplastic 6 transformation of the ovarian surface epithelium. According to one school, the surface epithelium of the developing ovary becomes s t r a t i f i e d during early development and gives r i s e to cords of c e l l s , primary and secondary sex cords, the l a t t e r of which envelop primordial germ c e l l s and give r i s e to granulosa c e l l s (Moore, 1977; Teilum, 1976). Others dispute this role and claim that primary sex cords (in the mouse) are of stromal o r i g i n and become the rete o v a r i i which gives r i s e to granulosa c e l l s (Byskov and Linte.rn-Moore, 1973; Stein and Anderson, 1979). Both these theories are based on the interpretation of complex h i s t o l o g i c a l specimens. It i s d i f f i c u l t to say which is closer to r e a l i t y , although the second view seems to have more influence in recent years, c) Structure After the intense investigation prompted by the germinal epithelium before 1950 there was a l u l l in research on the ovarian surface epithelium for more than a decade. Most of the work done in recent years has been u l t r a s t r u c t u r a l and histochemical in nature, and has been done on the ovarian surface epithelium in s i t u . The surface c e l l s of the ovary, as studied u l t r a s t r u c t u r a l l y i_n s i t u in several mammalian species, are seen to be e p i t h e l i a l c e l l s , squamous to columnar in shape, resting on well-defined basal laminae and joined by focal tight junctions, gap junctions and desmosomes. The apical surfaces are covered with numerous m i c r o v i l l i and the occasional ci l i u m . •7 These c e l l s usually have large, irregular. nuclei, many mitochondria with lamellar cristae,. abundant rough, endoplasmic reticulum (RER), well-developed Golgi complexes, perinuclear filaments, some l i p i d , and numerous vesicles both coated and uncoated (Anderson et a l . , 1976; Donaldson, 1976; Papadaki and Beilby, 1971; Weakley, 1969; Wischnitzer, 1965). Although some claim that the surface c e l l s of the ovary, are just mesothelial in nature (Parmley and Woodruff, 1974), Blaustein and Lee (1979) have shown that there are many u l t r a s t r u c t u r a l and histochemical differences between the covering c e l l s of the ovary and those covering the rest of the peritoneal cavity. Peritoneal mesothelial c e l l s are a l l squamous, have fewer m i c r o v i l l i , no c i l i a , and few desmosomes. In a three dimensional analysis involving both scanning and transmission electron microscopy Motta (1980) and co-workers have developed a picture of the surface epithelium of the adult ovary as marked by evaginations (papillae) and invaginations (crypts and cords) which are due* to the- p r o l i f e r a t i v e - activity-of these c e l l s . The crypts and cords often become detached from the surface to become cysts and nests of c e l l s . Noting s i m i l a r i t i e s between cord c e l l s , granulosa c e l l s and i n t e r s t i t i a l c e l l s , Motta speculates that the c e l l s in the surface epithelium might be a continuous source of d i f f e r e n t somatic c e l l s in the mature ovary. He then proceeds to dub these c e l l s a germinative (sic) epithelium. 8 d) Function The function of the ovarian surface epithelium i s not self evident, and is s t i l l a topic of speculation and research. These c e l l s p r o l i f e r a t e after ovulation and aid in healing of the ruptured ovarian surface. Bjersing and Cajander (1974) have suggested that the ovarian surface epithelium plays an active role in the process of ovulation. In a series of three studies (Bjersing and Cajander, 1974a,b,c) on induced ovulation in rabbits, they describe, via l i g h t microscopy and scanning electron microscopy, hypertrophy of ovarian surface c e l l s overlying preovulatory f o l l i c l e s , and the development of numerous large intracytoplasmic bodies. In a later study (Cajander and Bjersing, 1975) they demonstrate that these cytoplasmic bodies contain the lysosomal marker enzyme, acid phosphatase. They speculate that these bodies are lysosomes releasing degradative enzymes which weaken the f o l l i c u l a r wall, thus f a c i l i t a t i n g ovulation. Rawson and Espey (1977) claim, however, that development of these lysosomal bodies- r e f l e c t a physiological response of the ovarian surface to the trauma of stretching due to the rapidly enlarging f o l l i c l e . They speculate that these lysosomes may be preparatory for healing and clean-up processes after rupture of the f o l l i c l e . In u l t r a s t r u c t u r a l studies employing tracers such as horseradish peroxidase (Anderson et a l . , 1976; Donaldson, 1976), and in early studies using India ink- and v i t a l dyes"' (Dat'ta and Pederson, 1942; Cunningham, 1922), the ovarian surface c e l l s 9 were shown to be phagocytic with a b i l i t y to transport material from the ovary to the peritoneal lumen and in the reverse d i r e c t i o n . What bearing this a c t i v i t y has. on the function of the ovarian surface epithelium is not known. 2. OVARIAN CANCER a) Origin According to the currently held view of the histogenesis of ovarian tumours, two thirds of a l l these tumours, and 85% of malignant tumours, arise from the surface epithelium (Scully, 1977a; Woodruff and Jul i a n , 1970). The World Health Organization (WHO) c l a s s i f i c a t i o n of ovarian tumours l i s t s over 90 di f f e r e n t tumour types, with half being malignant v a r i e t i e s (Scully, 1977a). It i s also, s t r i k i n g that the ovary i s a very common s i t e for development of metastases from malignancies of other organs. Those tumours- a:r-i;si"ng from the surface- epithelium are. termed the "Common E p i t h e l i a l Tumours" of the ovary in the WHO l i s t i n g . Surface e p i t h e l i a l tumours are c l a s s i f i e d according to the c e l l type predominating. The major s u b - c l a s s i f i c a t i o n s of this group are the serous, mucinous, endometrioid, clear c e l l , Brenner, mixed e p i t h e l i a l , and u n c l a s s i f i e d e p i t h e l i a l tumours, and the undifferentiated carcinomas. These are l i s t e d in order of frequency. A l l but the last two groups are further divided into benign, borderline malignant, and malignant forms. These terms indicate the degree of atypism of the e p i t h e l i a l component 10 ( s t r a t i f i c a t i o n , nuclear atypism, increased mitoses) and the presence or absence of invasion of the stromal compartment by the epithelium. Benign forms show no e p i t h e l i a l atypism or stromal invasion. Tumours of borderline malignancy exhibit e p i t h e l i a l atypism and no stromal invasion, but can metastasize by seeding into the peritoneal cavity. Such borderline malignancies are r e l a t i v e l y slow growing and have a more favourable prognosis than frankly malignant forms. Malignant tumours demonstrate e p i t h e l i a l atypism, invasion of stroma, and metastasis by peritoneal seeding. Tumours c l a s s i f i e d as serous, mucinous and endometrioid are the so-called Mullerian-type which, although not of Mullerian duct o r i g i n , do exhibit h i s t o l o g i c a l and secretory c h a r a c t e r i s t i c s of Fallopian tube, endocervix and endometrium respectively. Recall that the ovarian surface epithelium and the Mullerian duct e p i t h e l i a are derived from coelomic epithelium, and that their s i t e s of o r i g i n l i e close together in the embryo. The claim that the common e p i t h e l i a l tumours of the ovary are derived from the surface epithelium is based on s t r i c t l y h i s t o l o g i c a l evidence. Frequently the surface epithelium forms inclusion cysts during the late reproductive and menopausal periods. These cysts are often found in conjunction with a spectrum of neoplastic growth from benign to borderline malignant to malignant. Although several levels o-f neoplast-rc change may be found in the same ovary, there i s no real proof 11 that each malignancy has evolved through these d i f f e r e n t l e v e l s . H i s t o l o g i c a l l y , there is. much evidence,, circumstantial, though i t may be, that the common e p i t h e l i a l tumours of the ovary do arise from the surface epithelium (Czernobilsky, 1977; Scully, 1977a; Woodruff and Jul i a n , 1970). b) Natural Course of Ovarian Cancer Although the di f f e r e n t types of ovarian cancer vary greatly in virulence, the overall five-year survival rate for ovarian cancer i s 30 to 35%. This, poor situation has not improved, much in the la s t few decades. The main reason for the generally poor prognosis for ovarian cancer i s that some 70% of cases are detected after spread beyond the ovary, rendering these cases incurable by surgery. There are usually no early symptoms of ovarian cancer, and the advanced disease exhibits vague symptoms caused by crowding of abdominal structures. The ovaries are not readily accessible for examination. Although careinoembryonic antigen (CEA, an antigen associated with a wide variety of neoplasms) has- been detected' by- - immunoperoxidase^ techniques*-in' some c e l l s of sections of mucinous cystadenocarcinomas of the ovary (Crum and Fenoglio, 1980), there are currently no cy t o l o g i c a l markers s p e c i f i c for malignant surface e p i t h e l i a l c e l l s e x foliated into the peritoneal f l u i d . The a v a i l a b i l i t y of such markers could lead to detection of ovarian cancer at an e a r l i e r stage. Complications in the v course of this disease', and' result in'g* death, are usually caused by intraperitoneal spread leading to 12 ascites, i n t e s t i n a l obstruction and cachexia. Distant metastases via lymphatic or hematogenous spread are, not common., and tend to happen only in advanced cases. Most ovarian cancer patients who die of their disease die from complications of the peritoneal tumour burden, not from distant metastases, c) Epidemiology and Etioloqy In most developed countries cancer of the Ovary ranks fourth in cancer deaths in women, being exceeded only by cancers of the breast, large intestine and lung. Epidemiologically, ovarian cancer is similar to cancers of the breast and colon with respect to geographic and r a c i a l d i s t r i b u t i o n (Lingeman, 1974). The incidence of ovarian cancer is generally higher in in d u s t r i a l i z e d countries. Japan i s a notable exception to t h i s rule as i t has one of the lowest incidences of ovarian cancer (2 per 100,000 compared with Sweden's 15 per 100,000). However, Japanese people who s e t t l e in western countries (USA-Hawaii) and take up western l i v i n g styles (especially diet) soon develop ovarian cancer at rates- t y p i c a l of western, developed- countries. Cases of ovarian cancer have been reported in age groups from infancy to the tenth decade. Tumours of the common e p i t h e l i a l c l a s s i f i c a t i o n appear almost e n t i r e l y after puberty, and peak in the f i f t i e s . Host factors contributing to increased risk for ovarian cancer include a f a m i l i a l history of gynecologic cancer, a personal history of breast cancer, n u l l i p a r i t y or low parity, and peripubertal infection with rubella, measles and mumps (McGowan et a l . >- —"1 979). Ovarian 13 cancers, mainly serous cystadenocarcinomas, have shown up in several families with a pattern suggestive of genetic transmission of predisposition to this cancer as a Mendelian autosomal dominant t r a i t (Fraumeni et a l . , 1975). Knowledge of the etiology of ovarian cancer i s at best sketchy. It has been said that "incessant" ovulation, resulting in repeated rupture of the ovarian surface, i s a factor in the development of ovarian neoplasia (Fathalla, 1971). Use of oral contraceptives which i n h i b i t ovulation has. been associated with a somewhat reduced risk of ovarian cancer (McGowan et a l . , 1979). The connection of ovarian cancer with i n d u s t r i a l i z a t i o n suggests environmental or l i f e - s t y l e carcinogenesis (Mattison and Thorgeirsson, 1978; Lingeman, 1974) but.asbestos i s the only s p e c i f i c substance to be implicated. There i s some evidence that viruses may be involved in the development of ovarian cancer. Peripubertal infection with the rubella virus leads to an almost fourfold increase in risk for developing ovarian cancer. Similar infection with measles and mumps leads to lesser increases in risk (McGowan et a l . , 1979; Menczer et a l . , 1979). Reverse transcriptase, t y p i c a l of that from C-type retroviruses, has been found in ovarian cancers of surface e p i t h e l i a l o r i g i n (Gerard et a l . , 1978). The ovary is exposed in the peritoneal cavity to environmental agents, chemical and b i o l o g i c a l , which enter via the genital t r a c t . However, the peritoneal mesothelium is s i m i l a r l y exposed and yet malignancies ari s e rarely from th i s 1 4 tissue. Perhaps rupture caused by "incessant" ovulation and close proximity . to ovarian. hormones are key factors distinguishing the ovarian surface epithelium from the l i n i n g of the rest of the peritoneal cavity. 3. ANIMAL MODELS FOR OVARIAN NEOPLASTIC TRANSFORMATION a) Spontaneous Ovarian Tumours in AnimaIs . Spontaneous ovarian cancers in animals are generally rare and tend to be of types not commonly found in humans. These tumours are often granulosa and thecal c e l l tumours of low malignancy (Lingeman 1974; Cotchin, 1977). Two exceptions to this statement are the domestic fowl which develops ovarian adenocarcinomas i f kept, in a state of constant egg laying (Wilson, 1958), and certain purebred canine breeds which have a high incidence of adenocarcinomas resembling the common human type (Hayes and Young, 1978). b) Experimental Models for Ovarian Tumourigenesis Work done on experimental induction of ovarian tumours has mainly involved studies on rodents in vivo. When X-radiation or chemical carcinogens (e.g. 7,12-dimethyl benzanthracene) are applied to the whole animal or to the ovaries alone, ovarian tumours are frequently produced. The commonest of these were granulosa c e l l tumours of benign or low-grade malignant form, tumours of a type rare in humans ( J u l l , 1973). This mode of tumourigenesis i s thought to be caused by excess production of 15 gonadotroph!ns resulting from f a i l u r e of f o l l i c u l o g e n e s i s due to oocyte death caused by X-rays and, carcinogens. Similar, tumour production can be achieved i f ovaries are transplanted to the spleen, thus preventing estrogen feedback to the p i t u i t a r y and leading to excess release of gonadotropins ( J u l l , 1973). If carcinogen-impregnated sutures are stitched into rat ovaries a high incidence of adenocarcinomas results after periods of three or more months (Sekiya et a l . , 1979). Rapp and Todaro (1980) have reported development of a careinoma-indueing variant of a mouse C-type retrovirus capable of inducing a low but s i g n i f i c a n t rate of ovarian adenocarcinomas when injected intraperitoneally into neonate Swiss mice. In a l l these in_ vivo experiments i t is not possible to prove the c e l l u l a r origins of resulting ovarian tumours. There is some evidence from these models that both carcinogens and C-type retroviruses can produce ovarian adenocarcinomas which resemble those found in humans. 4. THE PROBLEM Ovarian cancer i s a disease causing considerable suffering and loss of l i f e . A major stumbling block to improving i t s poor prognosis and low survival rate is a lack of means for detection of the disease at an early stage when i t i s s t i l l curable by surgery. Early c y t o l o g i c a l detection has been prevented by lack 16 of suitable markers which could ident i f y exfoliated malignant ovarian c e l l s (especially of the surface, epithelium.) in peritoneal f l u i d s . In spite of the obvious c l i n i c a l importance of the ovarian surface epithelium as putative source of most ovarian cancers, experimental approaches to the study of this tissue have been limited by the u n a v a i l a b i l i t y of pure populations of these c e l l s . The goal of obtaining such pure populations is made d i f f i c u l t because the tissue represents such a minor fraction of the mass of the whole ovary. In vivo animal models.have not provided d e f i n i t i v e evidence for carcinogenesis in the ovarian surface epithelium. There i s no di r e c t proof that the ovarian surface epithelium i s , in fact, the tissue of or i g i n of the "common e p i t h e l i a l tumours" of the ovary. A l l of our knowledge concerning these c e l l s comes from specimens fixed for li g h t or electron microscopy. It therefore seemed desirable to culture the ovarian surface epithelium in pure form for characterization of i t s dynamic properties and for carcinogenesis t e s t i n g . 17 5. THIS RESEARCH PROJECT: THE RAT OVARIAN SURFACE  EPITHELIUM IN VITRO In-order to.provide a basis for testing the theory that ovarian cancers of the common e p i t h e l i a l type are derived from the surface epithelium t h i s project has been concerned, with culturing the surface epithelium of the rat ovary. The plan involved characterizing such cultures by morphologic, histochemical and u l t r a s t r u c t u r a l means, and attempting transformation of these cells, by an oncogenic RNA virus, a) Culture Of The Rat Ovarian Surface Epithelium There were a number of approaches taken to culture the ovarian surface epithelium. One way was to explant pieces of rat ovary and examine any outgrowing c e l l s . Both Scott (1952) and Long (1940) have described e p i t h e l i a l outgrowths from pieces of rodent ovaries. Scott attempted no i d e n t i f i c a t i o n of these c e l l s . Long claimed the c e l l u l a r sheets to be mouse "germinal" e p i t h e l i a l in o r i g i n as "proved" by the presence of oocytes in the c e l l sheets. Dissociative enzymes have been used with considerable success in preparing many tissues for c e l l culture. Collagenase has been reported as an e f f i c i e n t and gentle agent for mammary gland and embryonic tissues (Lasfargues, 1970) and ovarian tumours (Blackwood, 1970). A mixture of collagenase and hyaluronidase followed by trypsin and DNa'se was used to dissociate bovine corpora lutea to single c e l l suspension (Gospodarowicz and Gospodarowicz, 1972a). Pronase has been used 18 to dissociate rodent ovaries into f o l l i c l e s and corpora lutea (Grob, 1964). Such vigorous d i s s o c i a t i o n of whole ovaries, did not suit our purposes. Collagenase acts to hydrolyse collagen in the i n t e r c e l l u l a r spaces without damaging the c e l l s . It can be used with complete culture medium, thus maintaining c e l l s in ideal conditions during incubation. Trypsin, a p r o t e o l y t i c enzyme for many protein types, but not collagen, hydrolyses proteins on the c e l l membrane and in the e x t r a c e l l u l a r matrix, causing c e l l dispersion along with membrane damage. Trypsin i s often used in the presence of ethylene diamine tetraacetic acid (EDTA) in calcium-free, magnesium-free solutions. The EDTA i s a chelating agent which binds calcium ions necessary for c e l l to c e l l adhesion. Hyaluronidase of t e s t i c u l a r o r i g i n can hydrolyse hyaluronic acid, chondroitin and chondroitin sulphate A and C, a l l components of the* ground substance, between connective tissue c e l l s (Cameron, 1966). In t h i s project various combinations of these agents were t r i e d under gentle conditions in an e f f o r t to remove only the surface epithelium of whole rat ovaries. 19 b) I d e n t i f i c a t i o n of the Cultured Rat Ovarian Surface  Epithelium i) Morphology There are many, well-substantiated reports of the culture of several d i f f e r e n t ovarian c e l l types. Many of these culture techniques involve the use of large ovaries from which the desired tissue can be readily dissected, for example granulosa, thecal and stromal tissue of equine and human ovaries (Channing, 1969a,b) and bovine l u t e a l tissue (Gospodarowicz and' Gospodarowicz, 1972,b). Granulosa c e l l s from small rat ovaries have been cultured by a method e f f e c t i v e l y guaranteeing the tissue of o r i g i n of the cultured c e l l s (Redmond et a l . , 1970). Published micrographs of granulosa, thecal, stromal and l u t e a l c e l l s provide a morphological basis against which any cultured ovarian c e l l s suspected of being of surface e p i t h e l i a l o r i g i n can be compared, i i) Histochemistry There i s a large bank of published data on the histochemical detection of enzymes involved in the biosynthesis and metabolism of steroids. Two of these enzymes figure prominently in the l i t e r a t u r e , namely A5-3p-hydroxysteroid dehydrogenase (A5-30-HSDH) and 17p-HSDH. Histochemical tests for these two enzymes have potential for distinguishing the ovarian surface epithelium from other ovarian c e l l types. The enzyme complex A5-3p-HSDH occupies a key position in 20 ovarian steroid biosynthesis as i t i s necessary for the production of progesterone, and, ultimately estrogens,.,. In vivo, th i s enzyme complex catalyses the conversion of pregnenolone to progesterone. In the A5-3p-HSDH complex a dehydrogenase converts the 30-hydroxyl group to a 3-keto group with NAD as co-factor. An isomerase, 3-ketosteroid A4,A5-isomerase, a l t e r s the double bond position from A5 to A4. These two enzyme a c t i v i t i e s are always associated. Location of the complex has been variously reported in the smooth endoplasmic reticulum, in mitochondria, or in both these organelles (Robertson, 1979; Gower, 1975). In histochemical tests based on the method of Levy (Levy et a l . , 1959) using dehydroepiandrosterone as substrate, A5-30-HSDH a c t i v i t y has been reported in corpora lutea, theca interna, and i n t e r s t i t i a l c e l l s in frozen sections of several species (Levy et a l . , 1959; B a i l l i e et a l . , 1966). These tissues are considered major s i t e s of steroid biosynthesis. The surface epithelium was negative*- for this? enzyme- in- a l l reports- seen.. The enzyme 17p-HSDH is act u a l l y an oxido-reductase which catalyses the interconversion of e s t r a d i o l and estrone in vivo. The i n t r a c e l l u l a r location of thi s enzyme, as determined biochemically, i s microsomal although i t has also been found in the nuclear fracti o n and free in the cytosol (Bitar et a l . , 1979). In biochemical studies t h i s enzyme uses either NAD or NADP as co-factor. With e s t r a d i o l or testosterone as substrate, marked 17 21 HSDH a c t i v i t y has been histochemically demonstrated in the surface epithelium of ovaries, of mouse,, rat, rabbit and human ( B a i l l i e et a l . , 1966; Hart et a l . , 1966; Blaustein and Lee, 1979). The.rest of the ovary generally tests negative for thi s enzyme, although corpora lutea, theca interna and a t r e t i c granulosa c e l l s have been reported as staining weakly. Levy also noted intense histochemical staining for lactate dehydrogenase in the rat ovarian surface epithelium. Cultured granulosa and thecal c e l l s from several species are histochemically positive for both A5-3p-HSDH and 170-HSDH (Stadnicka, 1976, 1977; Stadnicka and Stoklosowa, 1976; Fischer and Kahn, 1972). Cultured bovine l u t e a l c e l l s are positive for A5-30-HSDH (Gospodarowicz et a l . , 1972b). The histochemical detection of dehydrogenase a c t i v i t y depends on dehydrogenation of the substrate with production of reduced co-factor (e.g. NAD to NADH). The reduced co-factor is dehydrogenated by enzymes present in the c e l l , diaphorases, which transfer hydrogen to the tetrazolium s a l t in the testing solution. Reduced tetrazolium precipitates as dark blue formazan, the end marker for a positive test (Troyer, 1980). The presence of c e l l u l a r diaphorases i s essential for these t e s t s . The enzyme NAD diaphorase is intensely active in most tissues of the ovary, namely theca interna, i n t e r s t i t i a l tissue, corpora lutea, granulosa c e l l s of a t r e t i c f o l l i c l e s , the surface epithelium and walls of blood vessels. A much weaker reaction' is seen in normal, healthy granulosa and in connective tissue 22 stroma ( B a i l l i e et a l . , 1966). The d i s t r i b u t i o n of NADP diaphorase i s very, similar in the rat except that the surface epithelium, which is r i c h in NAD diaphorase, displays very weak NADP diaphorase. Hence a positive histochemical reaction for the hydroxysteroid dehydrogenases also implies presence of the diaphorase corresponding to the enzyme used. For purposes of this study NAD was the co-factor of choice. Histochemical testing for dehydrogenase a c t i v i t y i s technically simple but not without drawbacks (Troyer, 1980). Soluble dehydrogenase can diffuse to other s i t e s resulting in false p o s i t i v e s . Use of the wrong co-factor ( i . e . NADP instead of NAD) can lead to false negatives i f the tissue tested lacks the corresponding diaphorase. Use of a pH much in excess of 7 can lead to a "nothing dehydrogenase" reaction l i k e l y caused by reduction of co-factor by sulfhydryl groups. Any c e l l s containing high endogenous levels of reduced co-factor w i l l test positive with or without added substrate. Endogenous substrates in high concentrations- could- lead'to false p o s i t i v e s . Thus, i t is essential in these tests to use control cultures or sections treated with dehydrogenase testing solution lacking substrate. C e l l s of the peritoneal mesothelium are histochemically negative for the enzyme 170-HSDH (Blaustein and Lee, 1979) and can be used as further negative controls for histochemical tests involving the cultured ovarian surface, epithelium. 23 i i i ) Biochemical Detection of Hydroxysteroid  Dehydrogenase A c t i v i t y The presence of hydroxysteroid dehydrogenase a c t i v i t y in cultured c e l l s or tissues can be d e f i n i t i v e l y ' shown by incubation of c e l l s with radioactive substrate. Products are subsequently isolated and i d e n t i f i e d . Although such procedures are more certain than the histochemical ones just described, they are technically much more complex and require c e l l populations of a single c e l l type. A c t i v i t y of enzymes A5-3p-HSDH and 170-HSDH were investigated in cultured c e l l s by incubation with 1 UC-pregnenolone and 1"C-estradiol respectively.. Any . resulting radioactive products were detected and separated chromatographically, i d e n t i f i e d tentatively by chromatography and d e f i n i t i v e l y by r e c r y s t a l l i z a t i o n with authentic radioinert steroid to constant s p e c i f i c a c t i v i t y . iv) Ultrastructure of the Rat Ovarian Surface  Epithelium in V i t r o There i s a wealth of u l t r a s t r u c t u r a l information on the ovarian surface epithelium ij\ s i t u from several species as discussed e a r l i e r in this chapter. These data along with reports of the ultrastructure of other c e l l types ij\ s i t u ( A l b e r t i n i and Anderson, 1974; McKerns, 1969) served as a basis against which the ultrastructure of cultured c e l l s thought to be of surface e p i t h e l i a l o r i g i n was compared. 24 c) Autoradiographic Investigation of Cultured Rat Ovarian  Surface Epithel.ial C,e 1 Is, for, Byidenee, of Estro.qen Receptors There is some evidence that the ovarian surface.epithelium might be an estrogen target tissue. Surface epithelium treated in vivo by direct application of estrone showed a marked increase in mitoses (Stein and Alle n , 1942). This tissue in several species is positive for 17p-HSDH, 'an enzyme often found in known estrogen target tissues such as uterus and mammary gland (Gurpide and Marks, 1981; Patinawin et a l . , 1980; Pollow et a l . , 1977). Estrogen receptor a c t i v i t y has been demonstrated biochemically in homogenates of whole ovary, and of ovarian cancers thought to be derived from the surface epithelium ( G a l l i et a l . , 1981). There has been no report of estrogen receptors in the normal surface epithelium. By autoradiographic techniques Stumpf noted estrogen receptors in granulosa, thecal and l u t e a l c e l l s in rat ovaries with l i t t l e l a b e l l i n g of ova and stroma (Stumpf, 1969). He doesn-' t mention. the- surface epithelium. Biochemical determinations of estrogen receptors require a gram of tissue, or the order of one b i l l i o n c e l l s (Puca, 1970; G a l l i et a l . , 1981). Such biochemical determinations for estrogen receptors were not feasible for this study. A number of autoradiographic techniques have been developed for the detection of estrogen receptors in tissues. Stumpf (Stumpf et a l . , 1969) uses a laborious and t r i c k y technique 25 involving i_n vivo l a b e l l i n g and dry mounting of freeze-dried sections on emulsion coated s l i d e s (done in a darkroom) followed by exposure at -20°C for over six months. This technique was deemed not worth trying. Exposure times of such great length are fraught with ar t i f a c t s . s u c h as image fading (Rogers, 1979). A variation of Stumpf ' s. method (Tuohimaa, 1970), using vapour-fixed sections obviates the section mounting in a darkroom, but s t i l l requires large amounts of t r i t i a t e d steroid , for in_ vivo l a b e l l i n g , and also exposure times of three to six months. A t h i r d method (Uriel et a l . , 1973) involves l a b e l l i n g of tissues as sections following f i x a t i o n . U r i e l claims that after f i x a t i o n with certain f i x a t i v e s Such as absolute ethanol and glutaraldehyde, estrogen receptors retain their estrogen-s p e c i f i c a f f i n i t y . This method is technically simple, economic of t r i t i a t e d steroid, and requires exposures of the order of only three weeks. Results presented by U r i e l of rat uterine sections prepared by this method resemble results achieved by the more laborious techniques. U r i e l ' s method leads' to l a b e l l i n g predominantly in the cytoplasm, whereas i_n vivo l a b e l l i n g gives predominantly nuclear l a b e l l i n g . U r i e l ' s method method was t r i e d on ovarian sections and fixed cultures. Labelling of l i v e c e l l s was also t r i e d using a method much simpler than that reported by Weiller (1974) who based his method on that of Stumpf (1969). In l i v e l a b e l l e d c e l l s evidence for translocation of l a b e l l e d steroids from cytoplasm to nucleus was sought. Live 26 lab e l l e d c e l l s , and also c e l l s l a b e l l e d after ethanol f i x a t i o n , were tested for estrogen s p e c i f i c i t y of their estrogen, binding, by competing excess radioinert e s t r a d i o l with the t r i t i a t e d s teroid. d) Transformation of the Cultured Rat Ovarian Surface  Epithelium by Kirsten Murine Sarcoma Virus Animal models of ovarian carcinogenesis i_n vivo have suggested that both chemicals (S.e.kiya et a l . , 1979) and a. C-type mouse careinoma-indueing virus (Rapp and Todaro, 1980) can induce adenocarcinomas of the ovary resembling types of human ovarian cancer. A major d i f f i c u l t y with i_n vivo carcinogenesis is that the c e l l type of or i g i n of tumours cannot be proven. The use of in v i t r o transformation of c e l l s of proven o r i g i n in pure culture could avoid t h i s p i t f a l l . In a review a r t i c l e on the r e l a t i o n s h i p of in v i t r o transformation to tumour formation in vivo, Ponten (1976) concludes that there is "remarkably good correspondence- between events- in v i t r o and in vivo-." Retroviruses of the C-type w i l l often transform the majority of c e l l s in a culture in a r e l a t i v e l y short time. Chemical carcinogens on the other hand tend to transform small proportions of treated c e l l s over longer periods. Rodent c e l l s tend to undergo spontaneous transformation at a r e l a t i v e l y high rate (Ponten, 1976). Transformation of c e l l s by an experimental agent must be proved to be other than spontaneous'. Chemical transformation i s d i f f i c u l t to prove, although antigenic changes 27 have been demonstrated in chemically transformed c e l l s (Baldwin et a l . , 1978). Spontaneous transformants generally, show no such antigenic a l t e r a t i o n (Pitot, 1978). On the other hand, transformation by RNA oncogenic viruses can be readily shown by a number of techniques. There i s l i t t l e evidence that retroviruses are involved in human tumourigenesis. Retroviruses have been isolated from human leukemias and retrovirus-1ike p a r t i c l e s observed in milk and mammary carcinomas (zurHausen, 1980). P a r t i c l e s with biochemical c h a r a c t e r i s t i c s of reverse transcriptase t y p i c a l of C-type retroviruses have been detected in human ovarian adenocarcinomas of the common e p i t h e l i a l type (Gerard et a l . , 1978). These "sightings" contribute to circumstantial evidence for involvement of retroviruses in human cancers, but w i l l never prove i t . The dire c t experimental approach of transformation of a given c e l l type by retroviruses might shed more l i g h t on the problem. From such experiments one develops animal models which s t i l l do not- prove the involvement' of'* retrov-rr;uses' iin- huma-n-cancers. The virus chosen for th i s work is the Kirsten murine sarcoma v i r u s . This virus was o r i g i n a l l y isolated as a mouse erythroblastosis virus (MEV) (Kirsten et a l . , 1967a) which induced p r o l i f e r a t i o n of basophilic erythroblasts in_ vivo but did not cause transformation i_n v i t r o . After jjn vivo passage in rats the virus acquired the a b i l i t y to- induce sarcomas in vivo' in rats and mice (Kirsten et a l . , 1967b), and also 28 transformation in v i t r o . The virus was then renamed the Kirsten murine sarcoma v i r u s . Roy-Burman and Klement (1975) have shown that in passage through rats the o r i g i n a l MEV l o s t approximately 30% of i t s genomic sequence, which was replaced with a roughly equal amount of r a t - s p e c i f i c sequences. A detailed account of the genome of th i s virus i s given by Andersson (1980). Wyke (1981) speculates that these animal sequences, "oncogenes," when reintroduced into the c e l l under the control of v i r a l t r a n s c r i p t i o n a l promotors lead to expression of the oncogenes and can result in neoplastic transformation of the c e l l . The Kirsten sarcoma vi r u s , although capable of transformation on i t s own, cannot replicate without a "helper" v i r u s . The helper virus used in KiMSV stocks is the so-called Kirsten murine leukemia virus, KiMuLV. The KiMuLV i s non-focus-forming in infected c e l l s . C e l l s transformed by Kirsten sarcoma virus stocks can be "producer" c e l l s , that i s , they can be capable Of producing i n f e c t i v e virus, or they can be "non-producer" c e l l s . A number of tests are available for examining c e l l s for evidence of transformation by KiMSV-like viruses. Producer c e l l s w i l l produce virus detectable by electron microscopy, and by focus-forming a b i l i t y of m i l l i p o r e - f i l t e r e d growth medium from transformed c e l l s . Production of RNA retroviruses can be shown by incorporation of t r i t i a t e d uridine by transformed c e l l s to produce l a b e l l e d p a r t i c l e s i d e n t i f i a b l e as retroviruses by 29 sucrose density gradient centrifugation. The above tests do not prove that the c e l l s were transformed s p e c i f i c a l l y by KiMSV. Ce l l s can be i d e n t i f i e d as KiMSV transformants by the presence of antigens s p e c i f i c for the Kirsten sarcoma virus (Auersperg et al.-, 1977). This l a s t test can be used on both producer and non-producer c e l l s . Transformation of cultured c e l l s i s marked by many changes in morphology and behavior. Some of the c r i t e r i a for transformation include "immortality" of transformed c e l l s in  v i t r o (that i s , they can be passaged i n d e f i n i t e l y ) , loss of contact i n h i b i t i o n of r e p l i c a t i o n (leading to saturation densities much higher than those of normal c e l l s ) , loss of contact i n h i b i t i o n of movement (resulting in p i l i n g up of c e l l s ) , and acquisition of a rounded-up or spindle-shaped morphology (Pitot, 1978). The a b i l i t y of c e l l s to grow in soft agar, that i s , evidence of loss of anchorage dependence, i s often stated as indicating malignant transformation. However, the most conclusive-evidence for malignant transformation is- the production of malignant tumours i_n vivo by injection of transformed c e l l s into syngeneic or immunosuppressed animals. In the case of c e l l s transformed by virus i t is advisable to use syngeneic, immunosuppressed animals because of the antigenicity of virus-transformed c e l l s . The Kirsten sarcoma virus has been used to transform cultured, steroid-producing c e l l s of adrenocortical o r i g i n after short-term culture. These c e l l s retained d i f f e r e n t i a t e d c e l l 30 markers (steroidogenic enzyme a c t i v i t y ) . Lines of KiMSV-transformed adrenocortical c e l l s produced carcinomas,, both well, d i f f e r e n t i a t e d and anaplastic, and sarcomas (Auersperg et a l . , 1977, 1981). Techniques used for attempted transformation of cultured rat ovarian surface e p i t h e l i a l c e l l s were those employed in the adrenal c e l l study. The ovarian c e l l s were infected after short-term culture in order to improve the chance of retaining d i f f e r e n t i a t e d c e l l markers on transformation. Transformed rat ovarian surface e p i t h e l i a l c e l l s were tested for evidence of v i r a l transformation. They were characterized for tumourigenic a b i l i t y . Any resulting tumours were c l a s s i f i e d h istopathologically and compared to human ovarian cancers. 6. THE QUESTIONS Are the common e p i t h e l i a l ovarian cancers in humans derived from the ovarian surface epithelium? If so, what i s i t about the ovarian surface epithelium that makes i t so susceptible to malignant transformation? To answer the f i r s t question, ovarian surface epithelium of the rat was cultured in pure form, and tested for s u s c e p t i b i l i t y to transformation by an oncogenic virus, the Kirsten murine sarcoma virus, and tested for tumourigenicity. This type of experiment would produce direct evidence on carcinogenesis in the ovarian surface- epithelium-, rather than the circumstantial evidence provided by h i s t o l o g i c a l 31 investigations of tumours i n s i t u . To shed some l i g h t on the second question, cultured ovarian surface e p i t h e l i a l c e l l s were characterized according to their morphology, behaviour and u l t r a s t r u c t u r e . These c e l l s were investigated histochemically, biochemically and autoradiographically for evidence indicating that the surface epithelium of the ovary might be an estrogen target tissue. 32 CHAPTER II . MATERIALS AND METHODS The main thrust of this.research was to culture rat ovarian surface e p i t h e l i a l (ROSE) c e l l s as a model for human e p i t h e l i a l ovarian cancers which comprise some 85% of a l l ovarian cancers. Hence i t was necessary f i r s t to establish c r i t e r i a for identi f y i n g .ROSE c e l l s in culture. This i d e n t i f i c a t i o n was based on a morphological comparison of suspected ROSE c e l l s with other cultured ovarian c e l l types, an u l t r a s t r u c t u r a l comparison with the ovarian surface epithelium in s i t u in several mammalian species, and histochemical tests done to determine a c t i v i t y of the enzymes A5-3p-hydroxysteroid dehydrogenase (A5-30-HSDH), 17p-hydroxysteroid . dehydrogenase (170-HSDH) and lactate dehydrogenase in the surface epithelium and other ovarian c e l l types both _in vivo and i_n v i t r o . Once i d e n t i f i e d , cultured ROSE c e l l s were characterized by morphology, growth c h a r a c t e r i s t i c s , ultrastructure, biochemical determination of the metabolism of the steroids e s t r a d i o l and; pregnenolone, and by an autoradiographic investigation of these c e l l s for estrogen receptor a c t i v i t y . Cultured ROSE c e l l s were subjected to transformation by the C type retrovirus, Kirsten murine sarcoma virus (KiMSV). It was necessary to refine culture methods to obtain morphologically pure ROSE c e l l cultures for such transformation tests to be certain that resulting : transformed' c e l l s ' and- tumours' resulting from such c e l l s were, in fact, of ROSE c e l l o r i g i n . 33 As control c e l l s for histochemistry and tumourigenesis, peritoneal c e l l s from the mesentery of the small intestine were cultured. As control cells, for steroidogenesis, muscle fascia f i b r o b l a s t s were grown. 1. CELL CULTURE Sources of materials used are in Appendix I. Preparation of solutions used in c e l l culture, histology, histochemistry and electron microscopy are found in Appendix I I . a) Ovarian Cultures The c e l l culture experiments discussed in thi s section are given in the order done. They represent a progression from mixed c e l l cultures from whole ovaries in which ovarian surface e p i t h e l i a l (ROSE) c e l l s were f i r s t t entatively i d e n t i f i e d , through several attempts* at; producing- cultures* 'enriched' in ROSE c e l l s , and ultimately to a reproducible technique whereby cultures of f i r s t passage pure ROSE c e l l s could be produced. i) Mixed C e l l Cultures from Whole Ovaries These ovarian cultures were grown to see which c e l l types could be cultured from mechanically dissociated ovaries, and were used for histochemical tests and morphological comparisons to e s t a b l i s h the identity of ROSE c e l l s _in v i t r o . 34 Animals used were three month old Fischer 344 rats which were k i l l e d by ce r v i c a l d i s l o c a t i o n a f t e r brief ether, or chloroform anaesthesia. Ovaries were a s e p t i c a l l y removed and ca r e f u l l y cleaned of extraneous tissue (bursa, fat) under a dissecting microscope. Ovaries were cut into small explants with sciss o r s , spread on dry 35 mm p l a s t i c culture dishes (one ovary per dish) and allowed to dry for a few minutes to allow adhesion of tissue to the dish. To each dish was added 1.5 ml of 25% FBS/WM medium (Waymouth medium MB752/1 with 100 IU of p e n i c i l l i n G per ml and 100 *ig streptomycin per ml, enriched' with 25% f e t a l bovine serum). Cultures were maintained in a humidified incubator with an atmosphere of 5% C0 2 and 95% a i r at 37°C. Medium was changed on the t h i r d day due to acid pH and was changed every t h i r d or fourth day afterwards. After one week cultures were either fixed in ethanol and stained with toluidine blue (2% aqueous) for l i g h t microscopy (see appendix I I I ) , subcultured for growth curves and l i f e span tests, or used- for histochemistry (see:' Section:- 2; for-histochemistry). Cultures for growth curves were subcultured using trypsin (0.125% in Ca + +Mg + +-free Hanks Balanced Salt Solution), and seeded in 35 mm culture dishes (about 20 dishes) at 20-30,000 c e l l s per dish in 10% FBS/WM. Two cultures were trypsinized and c e l l s counted by hemacytometer every second day. Li f e spans were determined for these cultures by subculturing after a week to 25 cm2 p l a s t i c culture flasks at 150-400,000 c e l l s per flask in 10% FBS/WM. Each flask was subcultured to two flasks at approximately weekly i n t e r v a l s . The l i f e spans 35 were performed in th i s way as controls for KiMSV transformed c e l l s derived from mixed cell . o v a r i a n cultures. i i) Mixed C e l l , Whole Ovary Cultures from Ovaries  Kept in Organ Culture Before Mineing The purpose of th i s technique was to injure or k i l l inner c e l l s by i n s u f f i c i e n t nutrients and oxygen while maintaining the surface c e l l s in good health in the hope that cultures resulting from these ovaries would be 'enriched' in surface e p i t h e l i a l c e l l s . Ovaries from 3 month old rats were c a r e f u l l y cleaned and set on s t e r i l e stainless steel mesh grids in 35 mm culture dishes containing 2 ml of 25% FBS/WM medium at a density of 2 ovaries per dish. Ovaries were kept in organ culture for periods ranging from 1 to 7 days and then minced as before for culture. Specimens from each time period were also fixed, whole, in 10% phosphate buffered formalin and prepared for examination by l i g h t microscopy* to detect signs- of c e l l - death. A series of ovaries was kept for 10, 14, 17, 21, 24 or 28 days in organ culture and then fixed in formalin for l i g h t microscopy. Cultures were kept for up to 3 weeks with medium changes approximately every 4-5 days, fixed in ethanol, stained with toluidine blue and examined by l i g h t microscopy for types of c e l l s and amount of growth. 36 i i i ) Mixed C e l l , Whole Ovary Cultures from Ovaries  Subject to Drying. Before Mincing, The intent of th i s brief experiment, a converse of the previous one, was to k i l l surface e p i t h e l i a l c e l l s on ovaries subjected to drying times of 1/2 and 1 hours, culture the treated ovary by mincing, and examine the resulting culture for c e l l types and r e l a t i v e proportions of each. One would anticipate a reduction in the proportion of ROSE c e l l s . This would serve as additional evidence for i d e n t i f i c a t i o n of cultured ROSE c e l l s . Ovaries from 3 month old animals were cleaned of extraneous tissue, rinsed by a brief dip in s t e r i l e d i s t i l l e d water, touched to a s t e r i l e dish to remove excess water, and set on a dry stain l e s s steel mesh gr i d in covered culture dishes in a C0 2 incubator at 37°C for drying periods of 1/2 hour and 1 hour. Ovaries were minced for culture as previously described, with medium changed on days 3 and 6, and were fixed in ethanol and stained with toluidine blue on day 7. Cultures were examined for c e l l types and r e l a t i v e proportions of each. 37 iv) Attempt at Removing Ovarian Surface Epithelium  Using Hypertonic Buffer Solutions Ovaries from 3 month old animals were c a r e f u l l y cleaned and then placed either whole or halved in one of the following solutions (provided by L. Jasch - Dept. of Anatomy, UBC) at 37°C with a g i t a t i o n , according to the method of Jasch (Jasch 1979): i) 111mM NaCl, 5.6mM KC1, lOOmM Na2HP04 at pH 7.5 (osmolality 400 m osmoles) i i ) 111mM. NaCl, 5.6mM KCl, Hepes buffer 1OmM EDTA + sucrose to get 400 m osmole solutions. Tissue specimens were examined frequently under a dissecting microscope over periods of up to 2 hours to detect any l i f t i n g of the surface epithelium. Any c e l l s removed by this process, either loose in the buffer or in sheets, were placed in culture in 25% FBS/WM medium. Residual ovarian tissue was fixed in 10% phosphate buffered formalin for h i s t o l o g i c a l examination. v) Removal of the Surface Epithelium from Whole  Ovaries By Enzyme Dissociation In an attempt to produce a large y i e l d of ovarian surface e p i t h e l i a l c e l l s uncontaminated by other c e l l types whole ovaries were incubated in solutions of di s s o c i a t i v e enzymes with and without vortexing. Ovaries from 3 month old animals were c a r e f u l l y cleaned of contaminating tissue- with great care* taken not to injure the ovarian surface. For each enzyme treatment 38 TABLE I. ENZYME DISSOCIATION TREATMENTS USED ON WHOLE OVARIES SOLUTION INCUBATION TIME (MIN) VORTEX MIXING NOTATION FOR CULTURES PRODUCED C 45 SC, RC C ' 90 •. - SC, RC c 135 ' • - ' • SC, RC c 45 • + SCV, RCV c 60 + SCV, RCV H 60 - SH, RH ' H 60 + SHV, RHV (TE) 30 • - S(TE) ; (TE) 30 + S(TE)V, R(TE)V H then C 3 0 , 45 + • SHCV, RHCV H then C then (TE) 3 0 , 4 5 , 15 + SHC(TE)V, RHC(TE)V C then (TE) 4 5 , 15 + SC(TE)V, RC(TE)V C + H 60 + S(CH)V, R(CH)V H then (TE) 6 0 , 30 - SH(TE) H then (TE) 6 0 , 30 + SH(TE)V, RH(TE)V (TE) then H 3 0 , 60 + S(TE)HV, R(TE)HV A l l enzyme solutions were f i l t e r s t e r i l i z e d . C H (CH) 0 . 1 % collagenase in 5% FBS/WM 0 . 1 % hyaluronidase in buffered Hanks BSS 0 . 1 % collagena-se- +' 0 .1 %:-hy.a-luron-idase in buffered Hanks BSS (TE) - 0 .125% trypsin + 0 .02% ethylene diamine tetraacetic acid (EDTA) in Hanks BSS vortex mixing - generally for 3 0 - 4 5 seconds cultures derived from removed surface c e l l s cultures derived from minced residual ovaries V S R (see table I for combinations used) 3 ovaries were incubated in 5 ml of solution in 25 cm2 culture flasks at 37°C. After treatment, c e l l s were spun down at 150 g for 4 minutes and seeded in two 25 cm2 flasks with 5'ml' 25% FBS/WM'me'dium per' flask ( l a b e l l e d surface cultures 'S'). The residual ovaries 39 were processed as- follows: one was fixed in phosphate 10% buffered formalin for h i s t o l o g i c a l examination,,,, and,, the other two were minced and cultured with one ovary per 35 mm culture dish (labelled residual cultures 'R'). A l l cultures (S and . R) were kept for one week with medium changed as demanded by acid pH, and then were fixed in ethanol and stained with toluidine blue for l i g h t microscopic examination. vi) Production of Pure F i r s t Passage ROSE C e l l s by  Explant inq and Subculture Whole ovaries from 3 month animals were cleaned of extraneous tissue, cut straight across with sharp scissors avoiding the hilum region (producing 3 pieces, 2 polar and 1 central containing the hilum), and the polar pieces were explanted (cut side down) on dry culture dishes with one explant per dish (see Figure 1). Explanted pieces were allowed to dry for 5 minutes to permit adhesion before 1.5 ml of 25% FBS/WM medium was added. These primary cultures were incubated at 37°C in an atmosphere of 5% C0 2, 95% a i r in a humidified incubator. Medium was changed on day 4. The region of the hilum contains the t r a n s i t i o n of surface c e l l types from ovarian surface e p i t h e l i a l to peritoneal mesothelial. Hence i t i s undesirable to cut the ovary through the hilum to produce explants for culture. For the purpose of obtaining primary cultures for techniques not requiring pure ROSE c e l l s , the central piece, containing the hilum, can also be used. Such cultures tend to be contaminated with non-ROSE c e l l s 40 e FIGURE K_ CULTURE METHOD FOR ROSE CELLS Diagram to demonstrate the culturing method for ROSE c e l l s . In a, a rat ovary, 6 mm in diameter, i s sectioned with sharp scissors to produce two explants, avoiding the central region containing the hilum (H). In b, the cut pieces are placed cut-side down on dry 35 mm p l a s t i c culture dishes and allowed to dry for 5 min before 1.5 ml of medium are added. In c and d, l a t e r a l and surface views of the outgrowths of ROSE c e l l s ringing the explant 5 days after explanting are shown. In e, after removal of explants and any contaminating c e l l types, several primary cultures are trypsinized, and first-passage cultures are set up in 16 mm wells at 5X10 3 c e l l s / w e l l . loosened from the upper cut surface. After 6-7 days the explant was removed and the primary 41 cultures were scraped clean of contaminating c e l l s . Cultures were trypsinized (0.125% trypsin, in Ca.++ ,,Mg++-f ree Hanks Balanced. Salt Solution . BSS) for 15-20 minutes at room temperature, centrifuged at 150 g for 4 minutes, resuspended in 25% FBS/WM medium, counted by hemacytometer and seeded in 16 mm Linbro wells at 3,000-7,000 c e l l s per well. After two days growth these f i r s t passage ROSE cultures were checked morphologically for purity (that i s , only ROSE c e l l colonies present) by scanning with an inverted microscope. Such pure ROSE cultures were deemed' suitable for use in v i r a l transformation experiments. v i i ) Growth Curves for ROSE C e l l s Three series of f i r s t passage cultures derived from week-old primary cultures and grown in 25% FBS/WM were set up to determine growth curves for these c e l l s . In one series c e l l s were seeded in 35 mm dishes at 2,600 cells/cm 2 (25,000 c e l l s per dish). In the other two series c e l l s were seeded in 16 mm Linbro wells at either 3,500 cells/cm 2 (7,000 c e l l s per well) or 17,500 cells/cm 2 (35,000 c e l l s per well). For each series two cultures were counted every second day u n t i l growth reached a plateau. The counting procedure involved t r y p s i n i z i n g cultures in measured volumes to produce a suspension of single c e l l s , and counting c e l l s with a hemacytometer. 42 v i i i ) L i f e Span Determinations for ROSE Ce l l s Four sets of cultures were set up in an attempt to determine the l i f e span of ROSE c e l l s in culture. In the f i r s t two sets, week-old primary cultures grown in 25% FBS/WM were passaged into 16 mm wells at 2,500 cells/cm 2 (5,000 c e l l s per well). Half of these cultures were grown in 10% FBS/WM (series 215) and the. other half in 25% FBS/WM (series 218). At stationary growth cultures reached c e l l densities of 12,500 cells/cm 2 (series 215) and 18,000 cells/cm 2 (series.218 ) . These cultures were then passaged to 35 mm dishes at 5,000 cells/cm 2, that i s , at d i l u t i o n s of 1:2 and 1:3 respectively. Cultures were thereafter passaged at a d i l u t i o n of 1:2 at confluence or stationary growth. The second two sets of cultures were derived from week-old primary cultures grown in 10% (series 239) and 25% (series 241) FBS/WM. F i r s t passage cultures in 16 mm wells were seeded at 10,000 cells/cm 2 (series 239) and 12,000 cells/cm 2 (series 241) and reached densities of 34,000 cells/cm 2 and 36,000 cells/cm 2 respectively at stationary growth. These cultures were passaged to 35 mm dishes at densities of 14,000 cells/cm 2 and 10,000 cells/ c m 2 , that i s , at d i l u t i o n s of approximately 1:3 and 1:4. At each subsequent passage c e l l s were subcultured at a d i l u t i o n of 1:2 at confluence or stationary growth. 43 ix) Effect of Added Es t r a d i o l on the Growth of  Cultured ROSE Ce l l s A brief experiment was performed to determine i f es t r a d i o l added to the culture medium had any effect on the growth of ROSE c e l l s . Twenty four primary cultures set up in 25% FBS/WM were subcultured on day seven to eighteen 35 mm dishes at 30,000 c e l l s / d i s h in 2 ml of 25% FBS/WM. To 6 dishes, 5j#g/ml es t r a d i o l was added; to 6 others, lO^g/ml e s t r a d i o l was added; to the remaining 6 (control dishes) no es t r a d i o l was added. On days 3, 6 and 9 two dishes from each set were fixed in ethanol and stained with toluidine blue. Cultures were evaluated for percent mitotic figures and percent multinucleated c e l l s . b) Peritoneal C e l l and Muscle Fascia Fibroblast Cultures C e l l s of peritoneal o r i g i n were cultured as control c e l l s for histochemistry and v i r a l transformation experiments. Pieces of transparent mesentery cut from the mesentery of the small intestine of 3 month old female rats were spread out on dry 35 mm culture dishes (usually one piece per dish) and allowed to dry for 2-3 minutes to permit adhesion. To each dish 1.5 ml of 25% FBS/WM medium was added, and cultures were incubated at 37°C in 5% C0 2, 95% a i r in a humidified incubator. Medium was changed on the fourth day and whenever required by the a c i d i t y of the medium (but always at least once weekly). In an alternate culture method for peritoneal c e l l s pieces of mesentery from the small intestine were incubated in various 44 enzyme solutions with or without vortex mixing. Treatments used were 0.1% collagenase in 5% FBS/WM. for 90. minutes and 135 minutes, .0.1 % hyaluronidase in Waymouth medium for 90 minutes, and 0.125% trypsin plus 0.02% EDTA for 60 minutes, this l a t t e r treatment both with and without 45 seconds of vortex mixing at the end of the incubation period. Cultures of muscle fasci a f i b r o b l a s t s (MFF) were used as controls for ROSE c e l l s in experiments involving incubation of c e l l s with radioactive steroids ( 1 "C-pregn.enolone and 1 4C-e s t r a d i o l ) . Fibrous tissue was removed from between layers of abdominal muscle in 3 month old male and female rats, was spread on dry 35 mm culture dishes, allowed to dry for a few minutes, and then incubated in 15% FBS/WM at 37°C in an atmosphere of 5% C0 2, 95% a i r in a humidified incubator. 2. HISTOCHEMISTRY Histochemical tests, in pa r t i c u l a r those for a c t i v i t y of the enzymes A5-3p-hydroxysteroid dehydrogenase (A5-3p-HSDH) and 170-HSDH, were used as part of the i d e n t i f i c a t i o n procedure for the rat ovarian surface epithelium i_n v i t r o . These tests were also used to characterize KiMSV transformed ovarian c e l l s . In  vivo, the enzyme A5-30-HSDH catalyses the conversion of pregnenolone to progesterone, but in these tests, dehydroepiandrosterone (DHEA) is used because i t i s more soluble than pregnenolone in the testing solutions. The enzyme 17p-HSDH 45 catalyses the interconversion in vivo of es t r a d i o l and estrone, but the histochemical, tests. employed, detect only the dehydrogenation reaction, v i z . e s t r a d i o l to estrone.. Tests for lactate dehydrogenase (LDH) were t r i e d because i t was noted (Levy et a l . , 1959) that the ovarian, surface epithelium reacted strongly for thi s enzyme. Lactate dehydrogenase catalyses the conversion in vivo of lactate to pyruvate. It is an ubiquitous enzyme and i t s presence i s not a d e f i n i t i v e test. a) Dehydrogenase Testing of Ovarian Cryostat Sections, Ovaries from 3 month old rats were mounted on cryostat chucks with an aqueous mounting medium and frozen in l i q u i d nitrogen as quickly as possible after removal from the animal. The frozen tissue was sectioned at 8>< in a cryostat at -20°C. Sections were picked up on room temperature glass coverslips (0.17 mm thick) and allowed to dry for approximately 30 minutes inside the refrigerated cabinet of the cryostat. After a 5 minute rinse in 0.1 m phosphate buffer (pH 7.2-7.4) sections were incubated in the dehydrogenase testing solution at 37°C for 30-45 minutes. The testing solution used was that found in Levy (Levy et a l . , 1959) except that NN-dimethyl formamide was used as the steroid solvent instead of propylene glycol (see also Appendix I I ) . In this fashion, ovarian sections were tested for the a c t i v i t y of the enzymes A5-30-HSDH, 170-HSDH and LDH. DHEA was used as substrate for A5-30-HSDH; es t r a d i o l or testosterone was used for 17p-HSDH; sodium lactate was used' for LDH. For controls, sections were incubated in testing solution lacking 46 the substrate. After s u f f i c i e n t incubation (staining checked by microscope) sections were fixed in 10%. formal in in 5,0% ethanol for 15 minutes, counterstained in 1% aqueous safranin '0' for 1 minute, rinsed b r i e f l y in d i s t i l l e d . water, and mounted on a microscope s l i d e with Farrant's aqueous, mounting medium. These preparations were assessed soon after mounting by microscopy and photography since the staining i s not stable for long periods. Storage was at 4°C. b) L i p i d as Determined by Oil. Red 0, Staining Sections were stained for the presence of l i p i d by O i l Red 0 (Culling, ; 1974; see also Appendix III) immediately after sectioning, and counterstained with haematoxylin (Appendix I I I ) . O i l Red 0 was also used as a counterstain for A5-30-HSDH and 170-HSDH staining after incubation with the dehydrogenase testing solution and before f i x a t i o n . c) Dehydrogenase Testing of Cultured C e l l s Primary mixed c e l l cultures from minced whole ovaries, ROSE c e l l s , peritoneal c e l l s , and f i r s t passage c e l l s of muscle fascia f i b r o b l a s t s were tested for A5-3p-HSDH, I7p-HSDH and LDH a c t i v i t y using DHEA, es t r a d i o l and sodium lactate, respectively, as substrates. The testing solution was the same as that used for cryostat sections except that for the steroid dehydrogenase tests the concentration of steroid was doubled. This change was necessary to reduce incubation times since the' cultured' c e l l layer was thinner than the cryostat sections. C e l l s were tested 47 fresh or after freezing at -20°C. Monolayers in 35 mm culture dishes were rinsed in phosphate buffer after- removal of explants to remove traces of serum in the culture medium. Steroids in the f e t a l bovine serum used in the growth medium and in the ovarian explants could lead to false positive staining, even in the control cultures. Cultures were incubated in 1.5 ml of testing solution for 2 to 2.5 hours at 37°C. Some cultures were counterstained for l i p i d with O i l Red 0 (Appendix I I I ) . Cultures were fixed for 5 minutes in phosphate buffered 10% formalin, rinsed b r i e f l y in water and mounted with a cover s l i p using Farrant's medium. Preparations were assessed soon after mounting and stored at 4°C. The steroid dehydrogenase tests were also performed according to Fischer's method (Fischer and Kahn, 1972). Testing solutions used in th i s method were much more concentrated (Appendix II) than those used in Levy's method. d) O i l Red 0 Staining for L i p i d in Cultured C e l l s Fresh, formalin-fixed or frozen cultures of ovarian, peritoneal, and muscle fascia f i b r o b l a s t c e l l s were stained for l i p i d using O i l Red 0 and counterstained with haematoxylin (Appendix I I I ) . 48 3. BIOCHEMICAL DETERMINATION OF HYDROXYSTEROID  DEHYDROGENASE. ACTIVITY, IN CULTURED CELLS These experiments were done to confirm the histochemical results, 'for ,A5-3*-HSDH and 1 7p-HSDH; a c t i v i t y in ROSE c e l l s , to quantify t h i s a c t i v i t y , and to detect other products resulting from the incubation of ROSE c e l l s with 1"C-pregnenolone and 1"C-es t r a d i o l . I_n vivo the enzyme A5-3p-HSDH catalyses the conversion of pregnenolone to progesterone, and the enzyme 17p-HSDH the interoonversion of e s t r a d i o l and estrone.. As negative controls for thi s test, peritoneal c e l l s were used, but when these showed similar a c t i v i t y to ROSE c e l l s ( i . e . similar radioactive spots on chromatograms) muscle fascia f i b r o b l a s t s were used. The procedure used involved incubation of l i v e c e l l s with 1"C-steroid, extraction of the medium with dichloromethane, separation and p u r i f i c a t i o n of products by either thin layer chromatography (TLC) or p a r t i t i o n paper chromatography (PPC), detection of radioactive spots by X-ray f i l m autoradiography, elution of radioactive products, and i d e n t i f i c a t i o n of such products by r e c r y s t a l l i z a t i o n with radioinert steroid to constant s p e c i f i c a c t i v i t y . Appendix IV should be consulted for the detailed techniques involved in these procedures. 49 a) Calibration Chromatograms Using Radioinert Steroids The following steroids, in 100 »qm aliquots, were chromatographed on system I (ligroine) and system II (benzene:hexane = 1:1) on paper: pregnenolone, 17a-hydroxypregnenolone, 20c-dihydropregnenolone, progesterone, 17a-hydroxyprogesterone, 20a-dihydroprogesterone, testosterone, e s t r a d i o l , estrone and e s t r i o l . A l l these steroids as well as A 4-androstenedione and dehydroepiandrosterone (DHEA) were run on system III (benzene:ethyl acetate = 3:1) on TLC plates. Steroids other than pregnenolone, progesterone, e s t r a d i o l and estrone were tested since i t is possible that other c e l l u l a r enzyme a c t i v i t y could obscure products of the p a r t i c u l a r enzyme studied. For example, 17a-hydroxylases and 20a-reductases acting on pregnenolone and any progesterone resulting from A5-30-HSDH a c t i v i t y could diminish the y i e l d of progesterone and obscure A5-30-HSDH a c t i v i t y . Location of the d i f f e r e n t steroids was found by exposing dry chromatograms to u l t r a v i o l e t radiation (UV), thus detecting UV absorbing steroids which contain the structure A4,3-keto in the A ring, such as progesterone, 17a-hydroxyprogesterone, 20a-dihydroprogesterone, A4-androstenedione and testosterone, or a saturated A ring as found in estrone, e s t r a d i o l and e s t r i o l . Most steroids containing a hydroxyl group are positive for the phosphomolybdic acid (PMA) test (Appendix IV). 50 b) P u r i f i c a t i o n of Radioactive Steroids By paper chromatography on System I , the 1 uC-pregnenolone as purchased was found to be contaminated with 1 4C-progesterone. Stock 1"C-pregnenolone was routinely p u r i f i e d by paper chromatography on System I. The p u r i f i e d pregnenolone was eluted, dried down under nitrogen, redissolved in a measured amount of ethanol, and a 1 »il aliquot counted in a s c i n t i l l a t i o n counter. Stock 1 "C-estrad'iol as received was tested by paper chromatography Systems I and II and then by thin layer chromatography on System I I I . There was no sign of ra d i o a c t i v i t y except in the es t r a d i o l region. Hence the stock 1 " C - e s t r a d i o l required no p u r i f i c a t i o n before use, but was dried down, redissolved in 1 ml of ethanol and a 1 jil aliquot counted in a s c i n t i l l a t i o n counter. c) Culture of C e l l s for Incubation with 1"C-Steroids i) ROSE C e l l s Primary ROSE c e l l s were set up as previously outlined. On the 6th to 8th day cultures were scraped free of contaminating c e l l s , trypsinized, and seeded in 25 cm2 f l a s k s . Several seeding densities were used, ranging from 2 explant cultures per flask (approximately 50,000 c e l l s ) up to 12 explant cultures per flask (approximately 300,000 c e l l s ) . Cultures of high purity ( i . e . estimated greater than 90% ROSE c e l l s ) and varying in c e l l density from half confluent to near confluent were chosen for 51 incubation with radioactive s t e r o i d . Those seeded at 300,000 c e l l s per flask were ready for use within a wee.k of subculturing. . i i) Peritoneal C e l l s and Muscle Fascia Fibroblasts Primary cultures of peritoneal c e l l s were set up as previously outlined and at 6 to 8 days seeded in 25 cm2 flasks at 100,000 c e l l s per flask. Muscle fascia f i b r o b l a s t (MFF) cultures were set up as before using tissues from both male and female rats, and subcultured after 3" to 4 weeks to 25 cm2 flasks at 100,000 c e l l s per flask. At densities from one half to near confluence these flasks were incubated with radioactive steroids. d) Incubation of C e l l s with Radioactive Steroid Cultures in 25 cm2 flasks were incubated with 3 ml of 10% FBS/WM medium containing 400,000 cpm of radioactive steroid, for 7 hours at 37°C with gentle rocking. Flasks without c e l l s but with 3 ml of 10% FBS/WM containing 400,000 cpm of radioactive steroid were also incubated. These medium blanks were used to detect any substrate reactions caused by components of the serum or medium. Incubation medium was saved; c e l l s were trypsinized and counted in 3 ml of trypsin, and 24 ml of acetone was added to the combined medium, trypsin and c e l l s . The acetone was used to disrupt c e l l u l a r membranes. The blank medium was also combined with trypsin and acetone5. Incubations performed' are l i s t e d in Table I I . 52 TABLE I I . INCUBATIONS PERFOI WED CELL TYPE1 1"C-PREGNENOLONE 1 "C-ESTRADIOL ROSE peritoneal MFF (female) MFF (male) medium blanks 8 6 ND2 NA3 3 ' 11 4 4 4 6 1 - a l l . cultures incubated were f i r s t passage 2 - ND - not done 3 - NA - done by Auersperg et a l . , 1977 e) Extraction of Steroids from Medium Incubation mixtures were centrifuged to remove c e l l u l a r debris, and the acetone was blown off at 40°C under nitrogen. When a sample was to be used for thin layer chromatography, 50 »ig of each radioinert c a r r i e r steroid was added before extraction. To samples for paper chromatography 100-150 of each steroid was added. Steroids added to the i n i t i a l i aC-pregeneolone medium were pregnenolone, progesterone, 17a-hydroxypregnenolone*, 20a-dihydropreg;nenolone> 1-7a-hydroxyprogesterone and 20a~dihydroprogesterone. For the i n i t i a l 1 " C - e s t r a d i o l extraction, estrone, e s t r a d i o l , e s t r i o l , testosterone, A*-androstenedione and DHEA were used. In later incubations only pregnenolone and progesterone were required when 1"C-pregnenolone was substrate; only estrone and e s t r a d i o l were needed when 1 u C - e s t r a d i o l was substrate. Medium samples and blanks were extracted twice with 6 volumes of methylene dichloride with vigorous shaking. Aqueous and methylene dichloride phases were separated by f i l t r a t i o n through Whatman 53 phase separation f i l t e r paper. After evaporation to dryness, whole extracts were dissolved in 1 ml of ethano.l and. 20 MI aliquots counted in a s c i n t i l l a t i o n counter. f) Chromatography of Extracts Dried extracts were dissolved in a few drops of chloroform and spotted onto chromatography paper or thin layer plates (see Appendix IV for d e t a i l s ) . For each chromatograph a side s t r i p TABLE III CHROMATOGRAPHY DONE CELL TYPE SUBSTRATE PAPER SYSTEM I 2 PAPER SYSTEM I I 3 TLC SYSTEM 111" ROSE ROSE peritoneal peritoneal MFF (female) MFF (male) 7 blank blank 1*C-preg 5  1 flC-E76 C-preg 1 « C _ 1 "C~-E2 1«C-E 2  1 f tC-E 2  1"C-preg 1 4C-E 2 8 2 1 1 4 2 2 2 2 1 1 2 2 1 3 1 _ 2 _ 3 _ « _ 5 _ 6 _ 7 _ see Appendix IV for d e t a i l s of chromatographic techniques l i g r o i n e as mobile phase benzene/hexane 1:1 as mobile phase benzene/ethyl acetate 3:1 as mobile phase 1"C-pregnenolone 1 "C-estradiol male MFF c e l l s were incubated with 1"C-pregnenolone by Auersperg et a l in 1977 containing radioinert steroids was included. System I paper chromatograms were usually run for • 2-2-23 hours* and"' Syst'e'iri"' IT paper chromatograms for 23-25 hours. System III TLC plates were 54 allowed to run only u n t i l the solvent front reached.the top of the plate, usually 1.5 to 2 hours. The chromatograms done are l i s t e d in Table I I I . g) Autoradiographic Detection of Radioactive Products Chromatograms were dried for 24 hours, stapled to X-ray film, and exposed in l i g h t - t i g h t cassettes for 4 to 5 days. Chromatograms were removed from the f i l m and the fil m developed (Appendix IV).... Radioactive regions were located and outlined in pencil on the chromatogram. For tentative i d e n t i f i c a t i o n , the positions of the radioactive spots were compared with - those of radioinert steroids by UV absorbance in the radioactive s t r i p s , and with-UV absorbance and PMA spots on the radioinert s t r i p s . Radioactive spots were cut out and eluted in chloroform/methanol 1:1, twice for 2 hours at room temperature with gentle a g i t a t i o n . Elutants were transferred to v i a l s , dried down, redissolved in 1 ml ethanol and 20 aliquots were counted. h) R e c r y s t a l l i z a t i o n of Putative 1"C-Estrone from ROSE Cultures with Radioinert Estrone Radioactive specimens eluted from the estrone region of paper chromatograms were subjected to r e c r y s t a l l i z a t i o n with radioinert estrone to confirm or deny the radioactive product as 1 *C-estrone. Identity as estrone was confirmed i f the radioactive material could be r e c r y s t a l l i z e d with radioinert estrone to constant specific- a c t i v i t y . Constant s p e c i f i c a c t i v i t y was defined to be reached i f three sequential c r y s t a l s 55 had s p e c i f i c a c t i v i t i e s (counts per min per mg or cpm/mg) d i f f e r i n g by no more than 5% from the, mean of, their .specific, a c t i v i t i e s . Two 1"C-estrone specimens were pooled, ( 54,200 cpm with 26.4 mg of estrone - to assure a beginning s p e c i f i c a c t i v i t y of about 2,000 cpm/mg), and r e c r y s t a l l i z e d (in 50 ml centrifuge tubes with conical ends) from the following sequence of solvents: methanol, ethanol, acetone, benzene, methanol and acetone as described by Schwers (Schwers et a l . , 1965). At each r e c r y s t a l l i z a t i o n , estrone c r y s t a l s were dissolved in the b o i l i n g solvent and the solution concentrated u n t i l c r y s t a l s began to form. The tube was removed from heat and, after room temperature was reached, was refrigerated overnight at 4°C. Following centrifugation at 4°C and 2,000 rpm for 10 minutes, the mother liquor (ML) was c a r e f u l l y removed by decantation or Pasteur pipette and saved. The c r y s t a l (XL) was washed with cold solvent (the next one in the series except that methanol was used instead of acetone' since' estrone is very soluble in acetone), recentrifuged i f necessary, and the washings transferred to the ML. After drying and redissolving in 1 ml of a suitable solvent (eg. acetone), a 20 »/l aliquot was counted in a s c i n t i l l a t i o n counter. The f i r s t two c r y s t a l s , XL1 and XL2, were used e n t i r e l y for the next r e c r y s t a l l i z a t i o n , but approximately 1 mg samples of c r y s t a l s XL3 through XL6 were saved for determination of s p e c i f i c a c t i v i t y . Samples of 1 " O e s t r a d i o l eluted from paper chromatograms 56 were s i m i l a r l y r e c r y s t a l l i z e d with radioinert e s t r a d i o l except that the solvent sequence used was methanol,., benzene,. hexane/acetone (1:1), benzene, methanol, hexane/acetone (Schwers et a l 1965) i) Determination of Specific A c t i v i t y of Estrone and  Est r a d i o l Crystals and Mother Liquors Each sample of c r y s t a l and mother liquor to be analysed was, dissolved in 1 ml of a suitable solvent and divided and saved as follows: 0.5 ml as backup, two 0.1 ml aliquots for gas liquid" chromatography (GLC) to determine the mass of either estrone or es t r a d i o l in the specimen, and 0.3 ml for s c i n t i l l a t i o n . The GLC was a Hewlett Packard model #5830A f i t t e d with an OV225 column (in the laboratory of Dr. K. McErlane, Faculty of Pharmaceutical Sciences, UBC). Conditions for the GLC and preparation of the column are given in Appendix IV. A l l estrone (E,) and es t r a d i o l (E 2) specimens analysed were f i r s t derivatized 1 to their: corresponding? t r i m e t h y l s i l y l (TMS) ether derivatives as outlined in Appendix IV. If these r e l a t i v e l y polar steroids are analysed by GLC in their underivatized states they are retained for excessively long periods in the GLC column. Derivatization to TMS ethers produces much more v o l a t i l e compounds which pass f a i r l y quickly through appropriately prepared GLC columns. A response curve for the GLC was prepared using known quantities of E, and E 2 in proportions E,/E 2 equal to 1/4, 1/2, 1, 2, 4 and 6. The ratios of areas of E, and E 2 peaks on the GLC printouts were plotted 57 against the rati o s of weights (Appendix IV). The mass of either E, or :-E2 in a specimen was determined by GLC analysis, using a known weight of an internal standard added to each specimen. For E, specimens E 2 was used as the internal standard; for E 2, Ey was the internal standard. By ca l c u l a t i n g the r a t i o of areas of E, and E 2 peaks in the unknown specimen, and consulting the response curves, masses were readily determined for the unknown estrogen. At least three i to 2 nl aliquots from each specimen were tested by GLC analysis. From s c i n t i l l a t i o n counting of the 0.3 ml samples and the mass as determined by GLC, the s p e c i f i c a c t i v i t i e s were calculated. j) R e c r y s t a l l i z a t i o n of Putative 1"C-progesterone from  ROSE C e l l Incubations Due to breakdown of the GLC in t h i s lab and the lack of alternate equipment elsewhere, the confirmation of 1"C-progesterone production by ROSE c e l l s could not be confirmed by r e c r y s t a l l i z a t i o n to constant s p e c i f i c a c t i v i t y . 4 ELECTRON MICROSCOPY OF CULTURED ROSE CELLS Descriptions of the ultrastructure of the ovarian surface epithelium _in s i t u in several species have been reported (Anderson et a l 1976, Donaldson 1976, Papadaki and Beilby 1971, Weakley 1969, Wischnitzer 1965). Cultured 1 ROSE c e l l s were examined electron microscopically to compare c h a r a c t e r i s t i c s of 58 the c e l l s in v i t r o with those in vivo. Cultures were fixed with glutaraldehyde in Millonig's buffer, post fixed with osmium tetroxide, dehydrated in an alcohol series, and embedded in Epon. A complete outline of this procedure i s given in Appendix III with preparation of solutions in Appendix I I . Epon blocks were removed from dishes, trimmed.and sectioned with glass or diamond knives on a Reichert OM-U2 ultramicrotome. Sections were picked' up on carbon coated copper grids and stained with uranyl acetate followed by lead c i t r a t e (Appendix I I I ) . Sections were examined and photographed on a Zeiss EM-10 electron microscope. 5 TRANSFORMATION OF CULTURED ROSE CELLS BY THE KIRSTEN  MURINE SARCOMA VIRUS Cultures of ROSE c e l l s were infected by Kirsten Murine Scarcoma Virus (KiMSV) to test their s u s c e p t i b i l i t y to transformation by RNA oncogenic viruses. A l l resulting transformed c e l l l i n e s were tested for tumourigenicity by injecti o n into immunosupressed animals. Tumours were c l a s s i f i e d according to their histopathology. The virus used, KiMSV, was derived as reported by Roy-Burman (Roy-Burman and Klement, 1975) and assayed as given in Auersperg (Auersperg et a l . , 1977). (See Appendix V.) virus 59 suspensions were stored in Waymouth medium enriched with 5% heat-treated FBS and frozen under liquid, nitrogen. Non heat-treated FBS may deactivate the vi r u s . a) Infection of Cultures with KiMSV Ce l l s to be infected with KiMSV were cultured in 35 mm. pl a s t i c culture dishes or 16 mm wells. Since the virus is incorporated into the c e l l u l a r genome only, i f the c e l l s are synthesizing DNA, i t is important that cultures s t i l l have many dividing c e l l s when the virus is added. C e l l s were incubated in virus suspension (Table IV) for one hour at 37°C before an equal volume of 25% FBS/WM (non heat-treated FBS) was added. Cultures were subcultured as warranted, f i r s t to 35 mm dishes and, as c e l l numbers allowed, to 25 cm2 f l a s k s . Fully morphologically transformed c e l l s were maintained on 10% FBS/WM. For each transformed l i n e , c e l l s at early passages (4 to 8) were frozen in l i q u i d nitrogen (Appendix I I ) . As a p i l o t project to see1 i f any- ovarian c e l l s ; were transformable by KiMSV, mixed c e l l cultures from minced whole ovaries were infected. Primary ROSE cultures were infected but as these proved to be impure, due to fibr o b l a s t s underlying ROSE c e l l s near the explant, pure f i r s t passage ROSE cultures were ultimately infected. As controls for histochemistry of transformed ovarian c e l l s , peritoneal cultures consisting of both e p i t h e l i o i d and f i b r o b l a s t - l i k e c e l l s were infected with virus. As negative controls for transformation and tumourigenesis, a continuous c e l l l i n e from non-virus-infected, 60 TABLE IV. CULH PURES INFECT! ID WITH KiMSV CULTURE " • , TYPE - . NUMBER OF CULTURES 1FPU 2 OF VIRUS PER CULTURE primary ovarian (mixed c e l l ) primary ROSE . , pure f i r s t passage ROSE primary mixed c e l l peritoneal 6 .. 4 16 wells 4 2 1 .4 x 107 8 x 105 2 x 105 1 .4 x 107 8 x 10s 1 - unless otherwise, noted, cultures are. in 3 5 mm dishes 2 - FFU - focus forming units. pure, ROSE cultures was used. Table IV outlines the numbers and types of cultures infected with KiMSV. b) Histochemistry of Transformed C e l l s A l l r e s u l t i n g l i n e s of transformed c e l l s were subjected to histochemical testing for A5-30-HSDH and 170-HSDH a c t i v i t y and for l i p i d by Oil- Red 0 staining-. c) Tumourigenesis Testing of Transformed C e l l s A l l transformed c e l l l i n e s were tested for tumourigenicity by subcutaneous (s.c.) or intraperitoneal (i.p.) injection of 3 to 5 m i l l i o n early passage c e l l s (usually 5th or 6th) into immunosuppressed, 4 week old, female Fischer rats. Subcutaneous injection was just anterior to the l e f t haunch and the intraperitoneal in j e c t i o n was given in the midline, midway between diaphragm and pubis. Immunosuppression was achieved by / 61 subjecting animals to a sublethal dose (400 rads) of whole body X-radiation (280 KV X-ray) and waiting 24 to 36 hours before injection to allow c i r c u l a t i n g lymphocytes to disappear. At least 3 animals were injected for each s i t e for each l i n e . Rats injected with virus-transformed c e l l s were housed in C l e v e l containment (Appendix V), and fed lab chow and water ad 1ibiturn. The animals were clos e l y watched for the development of tumours. When tumours reached a suitable size (approximately 1 cm in diameter in s.c. sites) or i f the animals appeared sick or exhibited pain (especially the i.p. injected r a t s ) , animals were s a c r i f i c e d by c e r v i c a l d i s l o c a t i o n and autopsied. Autopsies were thorough only in the region of s.c. i n j e c t i o n and abdominal cavity, as relevant. Tumour specimens were fixed in 10% phosphate buffered formalin for. histopathological examination, and small pieces were fixed in 2.5% glutaraldehyde (in Millonig's buffer) for examination by electron microscopy. The presence or absence of ascites f l u i d was noted, and, where possible, the volume was measured by syringe. As negative controls for tumourigenesis, c e l l s of a continuous l i n e of ROSE o r i g i n were used; 3 to 5 m i l l i o n c e l l s from the 18th, 19th and 25th passages were injected i.p. or s.c. into twelve, 4 week old, immunosuppressed, female Fischer rats. These control animals were kept one year, or u n t i l development of i l l n e s s or a tumour. 62 d) Testing of C e l l s for Evidence of V i r a l Transformation Morphologically transformed lines derived from virus-infected, pure, f i r s t passage ROSE cultures were subjected, to three tests to show whether they were, in fact, v i r a l l y transformed. Cultures were prepared for electron microscopic analysis (Appendix III) and examined for u l t r a s t r u c t u r a l evidence of C-type retroviruses either budding from c e l l u l a r surfaces or lying between c e l l s . Mi 1 1 i p o r e - f i l t e r e d , day-old medium from transformed cultures was added to subconfluent cultures of a rat kidney c e l l l i n e (NRK - normal rat kidney) and the cultures monitored for development of foci of transformed c e l l s . Subconfluent cultures of transformed c e l l s in 75 cm2 flasks were incubated with 5-[ 3H]-uridine and the medium subjected to d i f f e r e n t i a l centrifugation on sucrose density gradients to detect l a b e l l e d p a r t i c l e s of a density c h a r a c t e r i s t i c of the Kirsten virus (Auersperg et a l . , 1977). The l a b e l l i n g and sucrose density gradient analysis was performed by Dr. J . B. Hudson, Division of Medical Microbiology, Department of Pathology, UBC. 63 6 AUTORADIOGRAPHIC INVESTIGATION OF TISSUES AND CULTURED  CELLS FOR THE: PRESENCE OF ESTROGEN, RECEPTOR ACTIVITY., Although the presence of estrogen receptors has been demonstrated in ovarian cancers . of surface e p i t h e l i a l o r i g i n there i s no report of such receptors in normal ovarian surface epithelium. In t h i s study the rat ovarian surface epithelium, both i_n s i t u and i_n v i t r o , was examined by autoradiographic techniques for evidence of estrogen receptor a c t i v i t y , that i s , evidence of estrogen s p e c i f i c i t y and of translocation of l a b e l l e d estrogen from cytoplasm to nucleus. a) Preparation of Tissue Specimens for Autoradiography i) . Fixation Tissue specimens were fixed overnight in absolute ethanol at 4°C, cleared in xylene and imbedded in p a r a f f i n , or else they were fixed in neutral, phosphate buffered 2.5% glutaraldehyde, dehydrated, cleared, and wax imbedded as outlined by U r i e l ( U r i e l et a l . , 1973). According to U r i e l , such f i x a t i o n techniques preserve the estrogen a f f i n i t y of the estrogen receptors. Whole ovaries, with bursa s t i l l in place, were removed from 3 month old rats and immediately fixed. As positive controls, pieces of uterus, and as negative controls, pieces of skeletal muscle (thigh) were s i m i l a r l y prepared. 64 i i) Sectioning Wax imbedded tissue was sectioned at 5 M. and sections were mounted on glass microscope s l i d e s (Appendix VI). Several sections of ovary, uterus and muscle were mounted on each s l i d e . A s u f f i c i e n t number of slides was prepared to allow s l i d e s to be developed at weekly intervals from 1 to 4 weeks. Sections were prepared for autoradiography within 48 hours of sectioning to minimize oxidation and hence inactivation of the estrogen receptors. i i i ) Labelling SI ides with T r i t i a t e d E stradiol Paraffin sections were rehydrated to phosphate buffered saline (PBS) via xylene and a graded alcohol series. Rehydrated sections were incubated for 30 minutes at room temperature in PBS containing approximately 1 »*Ci per ml of 2,4,6,7-3H-es t r a d i o l ( 3H-E 2). Slides were then washed in two 20 minute rinses of PBS, rinsed very b r i e f l y in d i s t i l l e d water, and allowed to a i r dry a t room temperature-. Some' srl*i-de>s; - were l e f t . unlabelled as controls for positive chemography ( i . e . chemical reduction of Ag + ions to Ag atoms in the emulsion). iv) Steroid Competition Experiment Using Ethanol-fixed  Sect ions Sections were processed as above by l a b e l l i n g with 3H-E 2 except that for some sli d e s the PBS wash contained one of the following radioinert steroids at 4 ^gm/ml: estrone, e s t r a d i o l , progesterone or testosterone (Uriel et a l . , 1973). 65 b) Fixation and Labelling of Cultured C e l l s for  Autoradiography Primary cultures of ROSE c e l l s were grown on p l a s t i c coverslips (Thermanox). Week old cultures were generally used. C e l l s were either l a b e l l e d after ethanol f i x a t i o n or la b e l l e d l i v e and then freeze-dried. i) Labelling of Ethanol Fixed Cultures Cultures were prerinsed for 10 minutes in PBS to remove traces of FBS, fixed in absolute ethanol for 20 minutes at 4°C, rehydrated for one minute in PBS, and incubated for 30 minutes at room temperature in PBS containing 3H-E 2 at 1 >#Ci/ml. Following incubation, coverslips were rinsed in two 20 minute rinses of PBS with gentle a g i t a t i o n , rinsed very b r i e f l y in d i s t i l l e d water, and allowed to a i r dry at room temperature. Some cultures were l e f t unlabelled as controls for positive chemography and unintentional exposure to radiation. (See Appendix VI.) i i) Steroid Competition Experiment Using C e l l s  Labelled After Ethanol Fixat ion Cultured c e l l s on p l a s t i c coverslips were la b e l l e d with 3H-E 2 as above, but some were washed with PBS containing either radioinert e s t r a d i o l or progesterone at 2 ygm/ml of buffer. This concentration of- radioinert steroid is approximately' 600 times that of the t r i t i a t e d e s t r a d i o l used in l a b e l l i n g . 66 i i i) Labellinq of Live C e l l s Week old primary cultures, with explants removed, were thoroughly rinsed in two 10 minute changes of serum-free Waymouth medium to remove traces of serum. Fetal bovine serum contains many steroids, including estrogens, and.thus would compete with the 3H-E 2 used in l a b e l l i n g . Cultures were then incubated in Waymouth medium containing 1 »#Ci/ml of 3H-E 2 for one hour at 37°C in a humidified incubator with an atmosphere of 5% C0 2, 95% a i r , or at room temperature and atmosphere. After incubation, cultures were rinsed in two 20 minute changes of Waymouth medium,, rinsed very b r i e f l y in d i s t i l l e d water, and then immediately l e f t to freeze-dry in a dessicator at -20°C. Some cultures were l e f t unlabelled as controls for positive chemography and unintentional exposure to radiation. iv) Steroid Competition Experiments with L i v e - l a b e l l e d  C e l l s This experiment was performed to test for estrogen s p e c i f i c i t y of estrogen binding by l i v i n g c e l l s . Cultures were processed as above except that the following l a b e l l i n g solutions were used: - Waymouth medium with 3H-E 2 at 1 vCi/ml, Waymouth medium with 3H-E 2 at 1 MCi/ml plus 2 j/gm/ml of radioinert e s t r a d i o l , - Waymouth medium with 3H-E 2 at 1 »»Ci/ml plus 2 ygm/ml of radioinert progesterone. 67 The concentrations of -ra.di ©inert steroids., used were approximately 600 times that of the 3H-E 2 used for l a b e l l i n g . v) Pulse-Chase Experiment with L i v e - l a b e l l e d C e l l s This experiment was done to test for translocation of label from cytoplasm to nucleus with time. Week-old primary cultures were labelled (pulse) as in section b ( i i i ) but during the post l a b e l l i n g phase cultures were l e f t for periods of 0.75, 2.5, 4.5 or 6.5 hours (chase) in Waymouth medium containing 1-2% FBS. The 0.75 hour minimum chase was required as the usual washing time. A small amount of FBS was added to the 'chase' medium to maintain the c e l l s in reasonably healthy condition and to reduce l i f t i n g of c e l l s off the growth surface. After the chase, coverslips were dipped very b r i e f l y in d i s t i l l e d water and freeze-dried in a dessicator at -20°C. c) Coating of Sections and Cultured Cells- with' Autoradiographic Emulsion Labelled and control sections and cultures were coated in autoradiographic emulsion (Kodak NTB-3 dil u t e d 1:1 with d i s t i l l e d water) and allowed to dry at room temperature. Coated preparations were stored in l i g h t - t i g h t boxes and exposed at 4°C for 1 to 5 weeks. (See Appendix VI.) 68 d) Development.and Staining of Autoradiograms Slides and coverslips coated in emulsion and exposed for periods of 1 to 5 weeks were developed according to Leighton's method (Leighton et a l . , 1980) (see also Appendix VI), washed well in tap water and stained. Sections were stained b r i e f l y in haematoxylin (a minute or less) and eosin. Since s i l v e r grains in the autoradiogram are d e s t a b i l i z e d and lost by extremes of pH (especially acid) i t was necessary not to overstain with haematoxylin. The gelatin of the emulsion take.s up eosin very strongly. Coverslips were stained in toluidine blue for 1-2 minutes, then very b r i e f l y in eosin. e) Evaluation of Autoradiograms Sectioned material was not evaluated quantitatively. Cultures on p l a s t i c coverslips were covered by a glass cove r s l i p using d i s t i l l e d water as a temporary mounting medium only while counting s i l v e r grains. S i l v e r grains were counted with the aid of a grid in the microscope eyepiece and a hand t a l l y . It was found best to count a whole (or half) f i e l d (at x25 objective) at one time, e.g. by t o t a l grains per f i e l d , t o t a l grains over nuclei, and t o t a l grains over blank areas between c e l l s , and then c a l c u l a t i n g t o t a l cytoplasmic grains by subtraction. In some tests, however, involving the number of grains per c e l l versus c e l l area, individual c e l l s were counted. Background grains due to steroid' a f f i n i t y of serum-proteins 4 was1 determined by counting grains in c e l l - f r e e f i e l d s . Unlabelled 69 control cultures were evaluated for s i l v e r grains to determine background due to posit i v e chemo.gr a phy and radiation from sources other than the t r i t i a t e d e s t r a d i o l used for l a b e l l i n g . 70 CHAPTER III..- RESULTS The results of experiments performed are presented here in the same order as in Materials and Methods. An additional section has been added after autoradiography to deal with the description of two continuous c e l l l i n e s a r i s i n g from non-virus-, infected ROSE cultures. 1. CELL CULTURE a) Ovarian Cultures i) Mixed C e l l Cultures from Whole Ovaries It was in mixed c e l l ovarian cultures, produced by mincing whole ovaries, that rat ovarian surface e p i t h e l i a l (ROSE) c e l l s were f i r s t tentatively i d e n t i f i e d . In these cultures four c e l l types were generally found. The predominant c e l l type had a d i s t i n c t e p i t h e l i a l morphology (Figure 2a) which did not resemble published pictures of: other- ovarian cells-,- namely granulosa (Channing, 1969a, 1969b; Fischer et a l . , 1972), thecal (Stadnicka et a l . , 1976; Stadnicka, 1976), l u t e a l (Gospodarowicz et a l . , 1972b), or stromal (Channing, 1969a,b). These c e l l s were polygonal in shape with clear cytoplasm, had well-defined borders, and grew in confluent monolayers. These e p i t h e l i a l c e l l s were morphologically, histochemically and u l t r a s t r u c t u r a l l y shown to be surface e p i t h e l i a l in o r i g i n in the course of t h i s work. A report published by Long in 1940 claiming to have cultured the mouse 'germinal' epithelium 71 FIGURE 2: CELL TYPES IN CULTURES FROM WHOLE OVARIES 2a. E p i t h e l i o i d c e l l s with clear cytoplasm and well-defined borders i d e n t i f i e d as ovarian surface e p i t h e l i a l c e l l s , from mixed c e l l cultures derived from minced whole ovaries of 3-month-old rats. 2b. Granulosa c e l l s from f o l l i c l e s of ovaries from prepubertal rats stimulated with pregnant mare serum gonadotrophin. 2c. Spindle-shaped c e l l s resembling fi b r o b l a s t s from mixed c e l l ovarian cultures, tentatively i d e n t i f i e d as stromal c e l l s . 2d. Large, polygonal, l i p i d - f i l l e d c e l l s t e n t a t i v e l y i d e n t i f i e d as l u t e a l c e l l s in mixed c e l l ovarian cultures. 2a,b,c,d. Ethanol-fixed, toluidine blue, x145 2d % 73 presented micrographs of cultured c e l l s strongly resembling ROSE c e l l s . The other c e l l types in mixed cultures were tentatively i d e n t i f i e d by histochemistry and by comparison of culture morphology with published reports as granulosa, stromal (Figures 2c) and l u t e a l (Figure 2d) in that order of predominance. In Figure 2b i s seen the culture morphology of granulosa c e l l s cultured from the f o l l i c l e s of prepubertal rats treated with pregnant mare serum gonadotropins (Harrison and Auersperg, 1 981 ). Growth curves for mixed c e l l ovarian cultures are shown in Figure 3. In both series c e l l s deteriorated rapidly after day 16. These two curves d i f f e r e d considerably, with that for series (a) reaching almost double the c e l l number (approximately three population doublings) at stationary growth as that in curve (b) which underwent two population doublings. Neither series exhibited an exponential growth phase, but i f the time taken to progress from 50,000 to 100,000 c e l l s was compared for both curves, series (a) had; a doubling time-of 2.5-daiys', and-series (b) 4.5 days. The difference between these two curves was due perhaps to the heterogeneous nature of these cultures. In the two l i f e span determinations attempted for mixed c e l l ovarian cultures one ceased to grow after the t h i r d passage and the other resulted in a continuous c e l l l i n e . In both determinations only fibroblast-1ike c e l l s remained after the second passage. The continuous l i n e (125) was- carried 26 passages before being discontinued. Growth curves for L125 at FIGURE 3: GROWTH CURVES FOR MIXED CELL CULTURES FROM WHOLE OVARY 75 the 16th passage were very similar, possibly because of the uniformity of c e l l , composition of this line.. Cultures seeded at 5,000 cells/cm 2 in 10% FBS/WM reached densities of 26,000 cells/cm 2 (2 population doublings) at. stationary growth. i i) Mixed C e l l Cultures from Ovaries Kept in Organ  Culture Before Mincing This was a f i r s t attempt at producing cultures enriched in surface e p i t h e l i a l c e l l s . When whole ovaries were kept in organ culture for 1 to 7 days in order to k i l l the inner cells', and then minced as before, the proportion of ROSE c e l l s was somewhat increased but the y i e l d was greatly reduced and the cultures far from pure. This method was judged not to be a good one for growing ROSE c e l l s , and was pursued no further. The v i a b i l i t y of ROSE c e l l s was reduced by prolonged organ culture but the v i a b i l i t y of underlying c e l l s was not reduced enough. i i i ) Mixed C e l l Cultures from Ovaries Subject to Drying  Before Mineinq This experiment, the converse of that in the preceeding section, was performed to help confirm the identity of cultured ROSE c e l l s . Cultures produced from ovaries dried for periods of 30 and 60 minutes showed a reduced number and size of ROSE c e l l colonies when compared with cultures from fresh ovaries. The reduction of ROSE c e l l growth was greater after the longer drying period. The actual amount of growth for other c e l l types (especially luteal) increased over that in cultures from freshly minced ovaries. This result showed the importance of reducing 76 exposure of the ovaries to a i r in order to minimize damage to the surface epithelium. This brief 'drying' experiment,, along, with i t s converse in which ovaries were kept in organ culture before mincing, tended to confirm the i d e n t i f i c a t i o n of the ovarian surface epithelium in culture, but i t merited no further analysis. iv) . Attempts at Removing the Ovarian Surface Epithelium by Treatment of Ovaries by Hypertonic  Buffer Solutions This method was o r i g i n a l l y developed to separate the e p i t h e l i a l layers from underlying mesenchyme in regenerating newt limbs (Jasch, 1979). Although very successful when used on newt limb regenerates, i t separated very l i t t l e surface tissue from rat ovaries and the few single c e l l s and small sheets produced were not viable in culture. v) Removal of the Surface Epithelium from Whole  Ovaries by Enzyme Pi ssoc iat ion A summary of results for these enzyme dis s o c i a t i o n t r i a l s i s given in Table V. See Table I for an explanation of the terms used. The results can be placed in three categories, namely good y i e l d , poor y i e l d , and impure. The good enzyme treatments, SCV, SH(TE) AND SH(TE)V, produced high y i e l d s of c e l l s which resulted in cultures consisting mainly of ROSE c e l l s as judged by morphology. H i s t o l o g i c a l examination of residual ovaries showed stretches of surface denuded" of surface 1 epithelium with l i t t l e erosion into subsurface structures such 77 TABLE V. EVALUATION OF CULTURES OF CELLS REMOVED. FROM. THE, OVARIAN SURFACE BY ENZYMES c e l l culture 1 y i e l d composition SC low mostly ROSE SH very low no growth SHV low mixed SHCV low mixed S(TE) low mostly ROSE S(TE)V moderate mostly ROSE S(TE)H low mixed S(TE)HV moderate mixed SCV high mostly ROSE SH(TE) high. mostly ROSE SH(TE)V high mostly ROSE" S(CH)V high mixed SC(TE)V high mixed SHC(TE)V high mixed 1 - see Table I in Materials and Methods for d e f i n i t i o n s of abbreviations used. as corpora lutea and f o l l i c l e s (Figure 4a). In cultures produced from minced residual ovaries ( i . e . RCV, RH(TE), RH(TE)V) there was reduced ROSE c e l l growth with many of the ROSE colonies not associated with an explant as in cultures from untreated ovaries. Such colonies l i k e l y resulted from sheets of surface epithelium loosened by the enzyme treatment and l i f t e d off during mincing. Enzyme treatments producing poor yie l d s of c e l l s (as shown in cultures SC, SH, SHV, S(TE), S(TE), S(TE)V, S(TE)H AND S(TE)HV) l e f t residual ovaries showing l i t t l e loss of surface epithelium (Figure 4b). Residual ovary cultures (RC, RH etc.) were much l i k e those from untreated ovaries. In general, vortex 78 FIGURE 4: REMOVAL OF OVARIAN SURFACE CELLS BY DISSOCIATIVE ENZYMES 4a. Ovary treated with hyaluronidase followed by trypsin/EDTA then vortex mixed. Note stretches of surface denuded of surface epithelium (arrowheads) with l i t t l e erosion of subsurface stroma. Compare with regions of intact surface epithelium (s.e.). H. and E.. X160. 4b. Ovary treated with collagenase. Note region of surface epithelium loosened from the underlying stroma (arrowhead). Most of the surface epithelium in such ovaries remained in place. H. and E. x200. 4c. Culture of ovarian surface epithelium contaminated by spindle-shaped c e l l s , t y p i c a l of cultures of ovaries treated with combined collagenase, hyaluronidase and vortex mixing (see 4d). Toluidine blue, x120. 4d. Ovary afte r treatment with combined collagenase, hyaluronidase and vortex mixing. Note areas of erosion into underlying stroma and f o l l i c l e s (arrowheads). H. and E. x160. 79 80 mixing tended to increase the y i e l d of c e l l s removed from ovaries treated with, d i s s o c i a t i v e enzymes. The methods resu l t i n g in, impure cultures produced a high y i e l d of c e l l s with much non-ROSE contamination (S(CH)V, SC(TE)V, SHC(TE)V, SHCV) (Figure 4c). H i s t o l o g i c a l examination Of residual ovaries showed large areas denuded of surface epithelium with much erosion into underlying stroma, f o l l i c l e s and corpora lutea (Figure 4d). These treatments also produced much erosion in the h i l a r region of the ovary. Cultures produced by mincing residual ovaries (R(CH)V, RC(TE)V, RHC(TE)V, RHCV) showed reduced ROSE.growth when compared to cultures from ovaries not treated with d i s s o c i a t i v e enzymes. In summary, even the 'good' cultures produced in these t r i a l s were not pure ROSE, and hence were not good enough for v i r a l transformation attempts. vi) Production of Pure F i r s t Passage ROSE C e l l  Cultures Ultimately a very simple method, involving culture of ovarian explants with subsequent subculturing, proved to be the most e f f i c i e n t in producing pure ROSE cultures. When rat ovaries (Figure 5a) were cut and explanted as described in the previous chapter outgrowths of ROSE c e l l s (Figure 5b) 2 to 4 mm wide were produced after 6 to 8 days. The extent of the outgrowth varied with the di f f e r e n t batches of FBS used in the growth medium. In over 80% of the several hundred primary ROSE cultures grown th i s outgrowth ringed the explant (Figure 1). 81 FIGURE 5: CULTURED RAT OVARIAN SURFACE EPITHELIAL CELLS 5a. Section of rat ovary from a 3-month-old animal showing ovarian surface epithelium as a single layer of c e l l s covering the ovary and ranging from squamous to columnar in shape (arrow). H . and E. X145. 5b. ROSE c e l l s in primary culture. Toluidine blue, x145. 5c. A colony of ROSE c e l l s in a f i r s t passage culture showing the morphology of this c e l l type even in small colonies. Note the mitotic figure. Toluidine blue, x145. 5d. A p l a s t i c section cut at right angles to the culture dish through the ovarian explant on the right and the outgrowth of ROSE c e l l s on the l e f t . Note that the surface c e l l s of the outgrowth near the explant are continuous with the surface c e l l s of the explant and that the outgrowth becomes a monolayer at greater distances from the explant ( i n s e t ) . Arrow, a region similar to that shown in Figure 11. Toluidine blue, x285. 82 83 Occasionally primary ROSE cultures were contaminated by s a t e l l i t e colonies of granulosa-like, c e l l s and. areas, of spi.lndl.e-shaped c e l l s . Approximately 50% of over 200 f i r s t passage ROSE cultures set up by thi s method were pure ROSE c e l l in composition as judged by morphological examination. Even very small ROSE c e l l colonies had a morphology which.distinguished them from other: cultured ovarian c e l l types (Figure 5c).. Such pure, f i r s t passage, ROSE cultures were deemed suitable for v i r a l transformation attempts. When ovarian explants and outgrowths were embedded in epon and sectioned' perpendicular to the growth surface, the surface e p i t h e l i a l c e l l s of the explant were seen to be continuous with the surface c e l l s of the outgrowth (Figure 5d). Near the explant there were f i b r o b l a s t -l i k e c e l l s underlying the surface e p i t h e l i a l c e l l s but at greater distances from the explant the outgrowth became a monolayer of ROSE c e l l s . v i i ) Growth Curves For F i r s t Passage ROSE C e l l s Three growth- curves', were-de.termined for.' ROSE e-e>M-s* in - f rrst- . passage (Figure 6). Each of these determinations represents a di f f e r e n t seeding density. The three curves d i f f e r greatly in c e l l density reached at stationary growth, namely 11,500 cells/cm 2 (6a), 35,500 cells/cm 2 (6b), and 4,600 cells/cm 2 (6c). Curves (a) and (b) indicate an increase in c e l l number of approximately 1.5 population doublings whereas in (c) there i s not quite one population doubling. A l l the wells and dishes in these three series were set up 84 FIGURE 6: GROWTH CURVES FOR FIRST PASSAGE ROSE CELLS-35h-30 25 s o (a),(b) in 16 mm wells in 25% FBS/WM O <4H o 2or (c) in 35 mm dishes in 25% FBS/WM o o o Cultures set up on day zero. >-H i—i 2 Q J UJ U 15 10 .CO J L i i 13 DAYS IN CULTURE 85 from 7- to 8-day-old primary cultures. In another series, wells of f i r s t passage ROSE c e l l s , used as controls for. v i r a l transformation experiments, were set up from 6 day old primary cultures at 5,000 cells/well.. These wells were subcultured after 25 days and found to have an average of 102,000 c e l l s / w e l l (over 4 population doublings). These cultures were the or i g i n for a continuous l i n e of c e l l s , ROSE-199, which i s described in section 111.7. From t h i s limited study of. ROSE c e l l growth i t would appear that c e l l density of f i r s t passage ROSE c e l l s at stationary growth depends on c e l l density at subculture and the age of c e l l s in primary culture at the time of subculture. v i i i ) L i f e Span Determination For Cultured ROSE C e l l s Five series of cultures were set up in an attempt to determine the l i f e span of ROSE c e l l s in culture. Four of these series (215, 218, 239 and 241) were set up as described in section I I - 1 - a - v i i i . The f i f t h series, 199, was set up as a control for KiMSV-infected f i r s t passage ROSE cultures. Series 215, derived from primary cultures grown in 25% FBS/WM and passaged in 10% FBS/WM, plated poorly and ceased to grow after the second passage. Series 218, derived from primary cultures grown in 25% FBS/WM and passaged in the same serum concentration ceased growth after the t h i r d passage. Series 239, derived from primary cultures set up in 10% FBS/WM and passaged in medium of the same concentration grew well at a l l passages to become a continuous l i n e , ROSE-239, which was 86 passaged 22 times before being frozen. Series 241, grown only in 25% FBS/WM grew poorly after second passage, and was not. passaged a t h i r d time. Series 199 wells in f i r s t passage were subcultured after 25 days to 4 dishes with 10% FBS/WM and 4 dishes with 25% FBS/WM (approximately 75,000 c e l l s / d i s h ) . C e l l s in the 10% FBS/WM dishes plated very poorly, grew slowly and were not passaged; a second time. C e l l s in 25% FBS/WM dishes flourished and were passaged at approximately weekly intervals, to produce, the, continuous l i n e R O S E - 1 9 9 . These c e l l s grew for 36 passages and over 55 population doublings before being frozen. Morphologic c h a r a c t e r i s t i c s of this l i n e w i l l be discussed in section III-7. The widely variable results of these l i f e span studies do not lend themselves to easy interpretation. Of the five series studied three ceased to grow after two or three passages but the other two became continuous l i n e s . Many more series of cultures would have to be set up to determine i f t h i s incidence of 'spontaneous transformation' of two out of five i s high, that i s , to determine i f cultured ROSE c e l l s readily undergo th i s type of transformation. ix) E ffect of Added E s t r a d i o l on the Growth of  Cultured ROSE C e l l s This experiment was t r i e d to see i f es t r a d i o l had a mitogenic effect on cultured f i r s t passage ROSE c e l l s . A summary of results i s given in Table VI. Cultures treated with added e s t r a d i o l in their medium showed a s t a t i s t i c a l l y . 87 TABLE VI. BINUCLEAr EFFECT OF "E' INDEX OF I ESTRADIOL VI'RST. PASS; ON THE MITOT] VGE. ROSE, CELLS C INDEX AND ; Days 1in Culture Added 2 E s t r a d i o l ^g/dish C e l l s 3 Counted Percent" Mitotic Figures Percent" Binucleate C e l l s • ' 3 '. " 3 3 . o. 10 20 1 605 . 1600 1600 2.62±0.93 3.31 5±0.15 3.63 5±0.75 , 1.56±0.92 4.88 5±1.27 3.50 5±0.76 6 6 6 0 10 20 1830 201 0 1930 1.97±0.5 2.49 5±0.68 2.54 5±0.14 0.93±0.68 0.90±0.44 1.875±0.47 9 9 9 0 1 0 20 1880 1920 2050 2.34+1.05 2..34±0.4 2.24±0. 18" 0.80±0.65 . 1.93 5±0.59 1 .5*1 5 ±0.54 1 - medium was changed on day 5. 2 - growth medium was 25% FBS/WM> 2 ml/dish. 3 - c e l l s from 20 microscope f i e l d s from two dishes were counted for each data point. 4 - the 20 f i e l d s counted for each data point were divided into 4 groups of fi v e f i e l d s for s t a t i s t i c a l analysis. Effors given are standard • deviations. 5 - these data d i f f e r s i g n i f i c a n t l y from control data at a l e v e l of significance equal to 0.05 using an ANOVA test for unpaired independent samples (Larkin, 1976). s i g n i f i c a n t increase in mitotic index- at three:" and4-: six-- days-after subculture when compared with untreated controls. After 9 days in culture there was no difference between treated and control l e v e l s . The index of binucleate c e l l s was shown s t a t i s t i c a l l y to be s i g n i f i c a n t l y higher than controls on a l l days tested (3, 6 and 9). These results suggest that e s t r a d i o l has a mitogenic effect on cultured ROSE c e l l s . Would steroids, p a r t i c u l a r l y the estrogens-present in* the'-f e t a l bovine serum in the growth medium render these results 88 inconclusive? According to a published report (Esber et a l . , 1973) commercially available f e t a l boy in e sera, c.onta.in , 1.8.0. to 360 . pg/ml of estrogens (estrone, e s t r a d i o l and e s t r i o l , but. mainly e s t r a d i o l , 160-250 pg/ml). In the experiment at question here the. 2 ml of 25% FBS/WM per dish would contain 0.5 ml of FBS with approximately 90-180 pg. The quantities of est r a d i o l added to each dish in thi s experiment were 10 and 2 0 u g , or 5x10" times as much as the amount l i k e l y to be found in the serum. Hence results of thi s experiment are probably a good indication that e s t r a d i o l has a mitogenic effect on cultured ROSE'cells. A trul y d e f i n i t i v e test of estrogen influence on ROSE c e l l s would require use of steroid free medium for the growth of these c e l l s . b) Peritoneal C e l l and Muscle Fascia Fibroblast Culture Primary peritoneal cultures a r i s i n g from transparent explants of mesentery from the small intestine contained both, e p i t h e l i o i d and f i b r o b l a s t - l i k e c e l l s . The e p i t h e l i o i d c e l l s were l i k e l y mesothelial i n origd>n (Figure 7'a-)- and the fibroblast-1ike c e l l s probably f i b r o b l a s t s and adipocytes (Figure 7b). C e l l outgrowth from explants was luxuriant; large sheets of c e l l s were produced within one week. Of the enzyme treatments used on pieces of mesentery from the small intestine, collagenase released a mixture of e p i t h e l i o i d and f i b r o b l a s t -l i k e c e l l s , hyaluronidase released very few c e l l s , and trypsin/EDTA released high yi e l d s of mainly e p i t h e l i o i d c e l l s . Muscle fascia f i b r o b l a s t s grew as spindle-shaped c e l l s (Figure 8). C e l l outgrowth from f a s c i a l explants in 25% FBS/WM was slow 89 FIGURE 7: CULTURE MORPHOLOGY OF PERITONEAL CELLS 7a. E p i t h e l i o i d c e l l s growing from explants of tissue taken from the mesentery of the small intestine of 3-month-old female rats. These c e l l s are probably mesothelial in o r i g i n . Toluidine blue, X180. 7b. Spindle-shaped, fibroblast-1ike c e l l s derived from mesenteric explants. These c e l l s are l i k e l y f i b r o b l a s t s or adipocytes. Toluidine blue, xl80. FIGURE 8: CULTURE MORPHOLOGY OF MUSCLE FASCIA FIBROBLASTS Spindle-shaped c e l l s growing from fibrous tissue removed from regions between layers of abdominal muscle. Toluidine blue, X180. 90 91 for 4 to 5 days, but increased rapidly after a week. F i r s t passage muscle fascia f i b r o b l a s t s grew well, in, 10%, FBS/WM. 2. HISTOCHEMISTRY a) Dehydrogenase Test ing of Ovarian Cryostat Sect ions Histochemical data for ovarian cryostat sections are summarized in Table VII.. When 8? frozen sections of whole rat ovary were tested for A5-3p-HSDH a c t i v i t y , with DHEA as substrate, heavy deposits of dark blue formazan were found only in the corpora l u t e a and theca interna. The surface epithelium, granulosa c e l l s and stromal areas were negative (Figure 9a). Sections tested for 17p-HSDH a c t i v i t y using either e s t r a d i o l or testosterone as substrate showed marked deposits of formazan in the surface epithelium. The rest of the ovary, with the exception of a few small formazan patches in the corpora lutea, tested negative for t h i s enzyme (Figure 9b). Lactate dehydrogenase a c t i v i t y was - found' in. most ovarian tissues by t h i s histochemical test (Figure 10a). Formazan deposits appeared in the surface epithelium within 10 minutes of incubation. The stain in this tissue tended to be more intense than in the other ovarian tissues, but the difference in intensity diminished with longer incubation times. 92 TABLE VII. HI SECTIONS STOCHEMICAL I )ATA FOR OVAI UAN CRY0STA1 ? c e l l type A5-3/J-HSDH a c t i v i t y 1 17 0-HSDH Act i v i t y 2 LDH . Act i v i ty 3 L i p i d By ORO" surface e p i t h e l i a l . granulosa l u t e a l thecal stromal vascular smooth muscle vascular endothelial control s e c t i o n 7 ++ ++ • + • ++ 5 + + + + • +/-.++ + n.a. 8 1 - dehydroepiandrosterone (DHEA). as substrate. 2 - e s t r a d i o l or testosterone as substrate. 3 - sodium lactate as substrate. 4 - o i l red 0.. 5 - the surface epithelium stained more quickly and intensely than other c e l l types. 6 - there were small patches of positive c e l l s in an otherwise negative tissue. 7 - for a l l c e l l types. 8 - not applicable. b) L i p i d as Determined by O i l Red 0 _in Frozen Sections The results of ORO staining are summarized in Table VII. Corpora lutea and theca interna were very intensely stained for l i p i d by th i s test. Granulosa c e l l s and surface e p i t h e l i a l c e l l s showed some c e l l s p o s i t i v e for l i p i d (Figure 10b). c) Dehydrogenase Testing of Cultured C e l l s These histochemical data are summarized in Table VIII. Primary cultures of ROSE c e l l s tested negative for A5-30-HSDH a c t i v i t y (Figure 9c) and posit i v e for 17^-HSDH (Figure 9d), the same as in cryostat sections. C e l l s in mixed ovarian cultures 93 FIGURE 9: HYDROXYSTEROID DEHYDROGENASE HISTOCHEMISTRY OF THE RAT OVARIAN SURFACE EPITHELIUM 9a. An 8 »im cryostat section of rat ovary with formazan deposits in the surface epithelium (A) and in small patches of the corpora lutea ( A ) , indicating 170-HSDH a c t i v i t y in these c e l l s . Substrate e s t r a d i o l ; counterstain Safranin 0. xi 50. 9b. An 8 »im cryostat section of rat ovary with heavy formazan deposits in theca interna (A) and corpora lutea (arrow), indicating A5-30-HSDH a c t i v i t y in these tissues. Note that the surface epithelium (A) and granulosa c e l l s (O) test negative for this enzyme. Substrate dehydroepiandrosterone; counterstain Safranin 0. x150. 9c. A primary culture of ROSE c e l l s showing formazan deposits indicating 17^-HSDH a c t i v i t y in these c e l l s . Substrate e s t r a d i o l , xl70. 9d. A primary culture of ROSE c e l l s lacking formazan deposits after incubation with A5-3p-HSDH testing solution, showing these c e l l s to be histochemically negative for t h i s enzyme. Substrate dehydroepiandrosterone, X170. FIGURE 10: LACTATE DEHYDROGENASE AND LIPID HISTOCHEMISTRY OF THE RAT OVARIAN SURFACE EPITHELIUM 10a. An 8 iim cryostat section of rat ovary with formazan deposits in the surface epithelium indicating LDH a c t i v i t y in t h i s tissue (arrowhead). Other ovarian tissues also exhibit LDH a c t i v i t y . Substrate sodium lactate; counterstain Safranin 0, X160. 10b. An 8 »/m cryostat section of rat ovary showing high concentrations of l i p i d droplets in corpus luteum (c) and i n t e r s t i t i a l tissues (i) and a few l i p i d droplets in the surface epithelium (s.e.), as shown by O i l Red 0 staining. Haemotoxylin counterstain, x160. 10c. A primary culture of ROSE c e l l s showing c e l l s with formazan deposits varying from s l i g h t to dense, indicating d i f f e r e n t l e v e l s of LDH a c t i v i t y . In the course of testing, some c e l l s have l i f t e d off and many remaining ones have been rounded up. Substrate sodium lactate, x160. I0d. A primary culture of ROSE c e l l s with varying amounts of cytoplasmic l i p i d as shown by O i l Red 0 staining. Cultured ROSE c e l l s show a higher cytoplasmic l i p i d content than those in vivo. x400. 94 96 TABLE VIII. HISTOCHEMICi ^L DATA FOR C 3ULTURED CELI ,S c e l l type 1 A5-30-HSDH a c t i v i t y 2 1 70-HSDH A c t i v i t y 3 LDH A c t i v i t y 4 l i p i d By ORO5 ROSE granulosa l u t e a l stromal peritoneal muscle fascia f i b r o b l a s t s V - 7 + + _ 8 + V - 7 + • . + 6 + + +/-+ +/-+/-. +/-• ++ . +/-+/-1 - of ovarian o r i g i n unless otherwise stated. 2 - DHEA as substrate. 3 - e s t r a d i o l as substrate. 4 - sodium lactate as substrate. 5 - O i l Red 0 6 - many c e l l s showed very intense staining and others a low l e v e l . 7 - granulosa c e l l s tend to l u t e i n i z e in culture (Channing, 1969a; Stadnicka, 1976). In t h i s study granulosa c e l l s in mixed c e l l ovarian cultures showed some posit i v e c e l l s for both enzymes. 8 - from Slavinski et a l . , 1974. tentatively i d e n t i f i e d as l u t e a l c e l l s stained intensely and quickly (usually within 45 minutes) for A5-3p-HSDH and 170-HSDH. Ce l l s in mixed ovarian culture thought to be granulosa c e l l s showed the occasional positive c e l l for both enzymes. Granulosa c e l l s tend to l u t e i n i z e in culture, that is they become positive for A5-3/3-HSDH (Channing, 1969a) and I7p-HSDH (Stadnicka, 1 976). In Table IX the results of histochemical testing for the two steroid dehydrogenases in ovarian c e l l s both in cryostat sections and in culture are compared. Published data from other authors are also l i s t e d to complete the comparison." From th i s table i t i s readily seen that the ovarian surface epithelium has a d i s t i n c t set of results compared to other 97 TABLE IX. A COMPARISON OF ACTIVITIES OF HYDROXYSTEROID DEHYDROGENASES IN OVARIAN CRYOSTAT SECTIONS AND CULTURED CELLS 1 C e l l Type Enzyme A c t i v i t y A5-30-HSDH 170-HSDH cryostat section culture cryostat section culture ROSE granulosa l u t e a l thecal stromal + + + 2 3 + + 5 + + 2 + + 5 1 - data are from this study unle,ss otherwise stated. 2 - Stadnicka, 1976. 3 - Fischer and Kahn, 1972. 4 - there were small patches of formazan deposit in an otherwise negative tissue. 5 - Stadnicka and Stoklosowa, 1976. ovarian c e l l types. Peritoneal c e l l s and muscle fascia f i b r o b l a s t s were negative for both these enzymes. When Fischer's- method^ (Fischer, et. a-L.., 1972;). was used- to-test ovarian and peritoneal c e l l s for A5-30-HSDH a c t i v i t y , a l l c e l l s tested showed some staining, even control cultures incubated without steroid substrate. This procedure was judged a poor one, probably due to the very high concentrations of reagents used. A l l c e l l types tended to have some degree of LDH a c t i v i t y . Cultures of ROSE c e l l s showed intense staining' in many cells-with other c e l l s scarcely stained (Figure 10c). 98 d) O i l Red 0 Staining for L i p i d in Cultured C e l l s These data are also given in Table VIII.. Primary ROSE c e l l cultures showed some l i p i d in most c e l l s by t h i s test (Figure 1ud)i Luteal c e l l s were very heavily stained, exhibiting many large droplets in every c e l l . Peritoneal c e l l s and muscle fascia f i b r o b l a s t s showed staining in some c e l l s while others were negative. 3. BIOCHEMICAL DETERMINATION OF HYDROXYSTEROID DEHYDROGENASE ENZYME ACTIVITY IN CULTURED CELLS a) 1 4C-Pregnenolone Incubations A summary of the main chromatographic results from these incubations i s given in Table X. Chromatographic positions of various steroids used as markers in TLC and paper chromatography are found in a c a l i b r a t i o n table in Appendix IV. When ROSE c e l l s were incubated with 1"C-pregnenolone and the medium extract analysed by TLC, seven radioactive bands other than 1"C-pregnenolone were found. One band was at the l e v e l of progesterone and another at a l e v e l similar to that for the radioinert steroids 17c-hydroxypregnenolone, 20a-dihydropregnenolone, 17a~hydroxyprogesterone and 20a-dihydroprogesterone. These four steroids were not well separated on TLC plates using system I I I . The remaining f i v e were very faint and located near the o r i g i n . Two of these f a i n t bands were also i n - the blank incubation. Peritoneal c e l l s 99 TABLE X. < SUMMARY OF CHROMATOGI tAPHIC RESULTS C e l l Type 1 1"C-pregnenolone 2 1 "C-estradiol 3 TLC paper TLC paper ROSE peritoneal . MFF blanks" + • + + + + + + + 1 - a l l c e l l s incubated in f i r s t passage. 2 - + indicates, radioactive material running with progesterone. 3 - + indicates radioactive material running with estrone. 4 - medium incubated with radioactive steroid. but no c e l l s . incubated with 1"C-pregnenolone showed an. almost i d e n t i c a l pattern of radioactive bands on TLC system I I I . Muscle fascia f i b r o b l a s t s have been shown previously to be negative for A5-30-HSDH a c t i v i t y (Slavinski et a l . , 1974). Analysis of medium extracts from ROSE c e l l 1"C-pregnenolone incubations by paper chromatography (system I) showed three minor radioactive bands near- the? origin,. None- of these coincided with the positions of radioinert steroids 17c-hydroxyprogesterone, 20c-dihydroprogesterone or 20a-dihydropregnenolone. The band nearest the o r i g i n was at the le v e l of 17o-hydroxypregnenolone. The runoff material from these chromatograms contained radioactive material which, when tested on TLC system III, showed two radioactive regions, one coinciding with progesterone-and the other running ahead of progesterone. There was only a 100 very low l e v e l of radioactive material in the runoff from the 1 "C-pregnenolone blank, none of which ran wi/th progesterone on TLC system 111. Peritoneal c e l l incubations showed a pattern of radioactive products similar to that of ROSE c e l l s when analysed in the same way. b) 1 "C-Estradiol Incubations A summary of chromatographic results from these incubations is given in Table X. When medium extracts from 1"C-estradiol incubations were analysed by TLC a major radioactive band was found at the estrone l e v e l in both ROSE and peritoneal extracts, but not in MFF extracts or blank incubations. Four faint bands of radioactive material were found near the o r i g i n in TLC's of a l l incubations analysed, those with c e l l s as well as blanks. Hence there must be enzymes or reagents in the medium a l t e r i n g the 1 " C - e s t r a d i o l . On paper chromatograms (system II) the e s t r a d i o l and estrone regions were well separated (see Appendix IV). Chromatograms of 1 " C - e s t r a d i o l incubation medium extracts run for both ROSE and peritoneal c e l l s showed radioactive material running with estrone, but no such material was found in chromatograms of MFF or blank incubations. A faint region of r a d i o a c t i v i t y was noted at the o r i g i n for a l l c e l l types and for the blanks. Radioactive regions at the l e v e l of estrone in system II paper chromatograms were eluted, dried, redissolved in 1 ml of ethanol, and 20 y l aliquots were counted in a s c i n t i l l a t i o n 101 counter. Runoffs from system I paper chromatograms suspected of TABLE XI. RADIOACTIVITY .IN PROGESTERONE AND ESTRONE REGIONS OF PAPER CHROMATOGRAMS C e l l Type ROSE ROSE Peritoneal Peritoneal Incubations 1 Number Type T o t a l 2 Extract (cpm) Estrone 3 Region (cpm) Progesterone" Runoff (cpm) Percentage Of Total. Extract 1 « 7 C-preg 2.2x106 1 1 1"C-E. 2.1x1O6 1.3x105 1 Hr-C preg 7.1X10 5 1"C-E-6.6x105 1.9x10" 3.0x10" 1 .4 6.2 3.6x10" 5. 1 2.9 1 - in 1"C-pregnenolone incubations approximately 400,000 cpm was used per incubation. In almost a l l 1"C-est r a d i o l incubations 200,000 cpm was used inadvertently. 2 - these figures are derived from radioactive counts of whole medium extracts before chromatography. 3 - t h i s material contains at least two radioactive compounds' as- shown by rechromastographd-ngr eluantS" on TLC plates. 4 - these runoffs contain at least two radioactive compounds as shown by chromatographing on TLC system I I I . containing 1"C-progesterone were also counted. These data are given in Table XI. 102 c) R e c r y s t a l l i z a t i o n Of Radioactive Estrone To Constant  Specific A c t i v i t y Radioactive material, eluted from the estrone regions of two chromatograms of medium extracts from ROSE c e l l incubations (a t o t a l of. 1.8X106 c e l l s ) was pooled and r e c r y s t a l l i z e d with radioinert estrone. When thi s sample (54,200 cpm)' was r e c r y s t a l l i z e d with estrone (26.4 mg) constant s p e c i f i c a c t i v i t y (CSA) was reached after six r e c r y s t a l l i z a t i o n s . The data are given in Table XII. TABLE XII. SPECIFIC ACTIVITIES 1 FOR CRYSTALS AND MOTHER LIQUORS FROM RECRYSTALLIZATIONS Estrone ML2 Sample SA3 cpm/mg XL" Sample SA cpm/mg ML 3 21 50 XL 3 1 504 ML 4 1433 XL 4 1705 ML 5 1815 XL 5 1 576 ML 6 1 629 XL 6 1617 1 - masses determined by GLC, r a d i o a c t i v i t y by-s c i n t i l l a t i o n counter. 2 - mother liquor . 3 - s p e c i f i c a c t i v i t y . 4 - c r y s t a l . The average s p e c i f i c a c t i v i t y (SA) for c r y s t a l s (XL) 4, 5 and 6 was 1632 cpm/mg. The SA of the three c r y s t a l s d i f f e r e d from t h i s mean by 4.5%, 3.4% and 1% respectively. The SA of the mother liquors (ML) approached the SA of the c r y s t a l s as the cry s t a l s became purer. The average SA for ML's 4, 5 and 6 was 103 1626 cpm/mg. Hence constant s p e c i f i c a c t i v i t y was achieved and the presence of 1 4C-estrone in the. sample confirmed. Based, on the average SA of 1632 cpm/mg and 26.4 mg of radioinert estrone, the o r i g i n a l radioactive specimen must have contained 43,100 cpm of 1"C-estrone with some 11,000 cpm of other 1"C compounds.. Thus 7% of the t o t a l extract chromatographed (632,000 cpm) was reclaimed as 1 uC-estrone. It i s thus confirmed that cultured ROSE c e l l s can express 17^-HSDH a c t i v i t y and do convert e s t r a d i o l to estrone as was indicated histochemically. 4. ELECTRON MICROSCOPY OF CULTURED ROSE CELLS Cultured ROSE c e l l s were examined u l t r a s t r u c t u r a l l y in primary culture in areas near the explant and in areas of monolayer growth distant from the explant. In regions of the outgrowth near the explant ROSE c e l l s had a low cuboidal shape with numerous m i c r o v i l l i , underlying basal lamina, apical junctions resembling junctional complexes, large nuclei, RER and perinuclear filaments. (Figure. 11a). En* monolayer' regions; or primary cultures ROSE c e l l s were near squamous in shape and had api c a l junctions resembling junctional complexes. No basal lamina could be detected but there were traces of fine f i b r i l l a r or amorphous material below the basal c e l l surfaces. C e l l s in monolayer regions tended to overlap one another with long processes and there were a few m i c r o v i l l i varying greatly in length. In Figure 5d i s seen the location of a region similar to that examined in Figure 11a. 104 FIGURE 11: ULTRASTRUCTURE OF ROSE CELLS IN CULTURE 11a. An electron micrograph of ROSE c e l l s in primary culture selected from a region in the outgrowth near the explant similar to that indicated by an arrow in Figure 5d. Note the ap i c a l junctions ( O , basal lamina (arrow), m i c r o v i l l i and perinuclear filaments ( f ) . Underlying c e l l s (s) are l i k e l y stromal f i b r o b l a s t s . x7,000. 11b. An enlargement of part of figure 11a to show d e t a i l s of an i n t e r c e l l u l a r contact. Note the i n t e r d i g i t a t ion of l a t e r a l c e l l membranes, the ap i c a l junction, and the m i c r o v i l l i . x24,000. 11c. An enlargement of part of figure 11a to show the well-defined basal lamina ( A) . x14,000. 105 106 This u l t r a s t r u c t u r a l picture of the ovarian surface epithelium In v i t r o correlates, well with published reports of the ovarian surface epithelium i_n s i t u from several mammalian species (Anderson et a l . , 1976; Donaldson, 1976; Papadaki and Beilby, 1971; Weakley, 1969; Wischnitzer, 1965). This u l t r a s t r u c t u r a l evidence, along with the preceding morphological and histochemical data, substantiates the claim that these c e l l s are of ovarian surface e p i t h e l i a l o r i g i n . 5. TRANSFORMATION OF CULTURED CELLS BY THE KIRSTEN MURINE  SARCOMA VIRUS a) Transformation of C e l l s by KiMSV Table XIII l i s t s the names and origins of KiMSV transformed l i n e s produced in th i s study. Mixed c e l l ovarian cultures were the o r i g i n of three transformed l i n e s . Complete morphological transformation became evident at 3 to 4 weeks after i n f e c t i o n . Primary ROSE, c e l l cultures - infected.«'wit"h--KiMSV:: showed- froc-i-- of transformed c e l l s ( r e f r a c t i l e , spindle-shaped c e l l s which tended to p i l e up) within one week (Figure 12a) and showed obvious morphological transformation within a month. However due to underlying fi b r o b l a s t s in regions of the outgrowth near the explant the c e l l s of o r i g i n of these transformed lines was not ce r t a i n . Sixteen morphologically pure, f i r s t passage' ROSE1 cuiture's1" were infected with KiMSV. A l l cultures showed small f o c i of 107 TABLE XIII. SUM* 1ARY OF KIMS\ J TRANSFORMEI ) CELL LINES Culture Type Number of Cultures . Infected . Transformed Lines Produced Names of Transformed Lines primary ovarian (mixed) primary ROSE pure f i r s t -passage ROSE primary peritoneal • 6 16 6 3 3 3 4 V54,V551,V56 V193,V194,V196 V197-10B,V197-13A, V197-15A VP6,VP55, VP197,VP198 1 - this l i n e grew poorly after storage under l i q u i d nitrogen and was discarded. transformed c e l l s after one week. Only three became f u l l y morphologically transformed (Figure 12b,c,d), l i n e V197-15A at one week, and lines V197-10B and V197-13A at four weeks. The remaining 13 wells reverted in appearance to that of uninfected wells after 4 to 8 weeks. Primary cultures of peritoneal c e l l s were generally readily transformed by KiMSV. Foci of transformed c e l l s appeared in a l l cultures infected within a week and four of these six cultures became f u l l y morphologically transformed in 10 days to 4 weeks. The morphology of a l l these ovarian and peritoneal l i n e s was quite diverse, varying from spindle-shaped to rounded c e l l s . A l l cultures exhibited much p i l i n g up of c e l l s . With the exception of l i n e V194, derived from a primary ROSE culture, a l l li n e s grew well on 10% FBS/WM and reached c e l l densities of 3 to 5 m i l l i o n c e l l s per flask compared to 1 m i l l i o n or fewer for the 108 FIGURE 12: VIRAL TRANSFORMATION OF CULTURED ROSE CELLS 12a. A f i r s t passage culture of ROSE c e l l s one week after infection with KiMSV. Note the f o c i of rounded-up and spindle-shaped c e l l s which have p i l e d up. These f o c i are indicative of morphological transformation by the v i r u s . Toluidine blue, X150. 12b,c,d. Cultures of three l i n e s of transformed ROSE c e l l s , V197-10B, V197-13A and V197-15A, at f i f t h passage afte r transformation. Toluidine blue, X150. 109 110 non-transformed c e l l s . Line V194 required 25% FBS/WM for rep l i c a t i o n and c e l l attachment. The three l i n e s derived from pure, f i r s t passage ROSE cultures d i f f e r e d greatly from each other in morphology. C e l l s of l i n e V197-15A were very spindle shaped (Figure I2d)., Line V197-10B, composed mainly of rounded c e l l s , formed many spherical structures at near regular intervals under crowded conditions (Figure 12b). Line V197-13A had mainly rounded up c e l l s with many large, spread out cells, (Figure 12c). The difference between these morphologies and that of the untransformed ROSE culture was most s t r i k i n g (Figure 5b). b) Histochemical Testing of KiMSV-Transformed C e l l s Histochemical data for KiMSV-transformed c e l l s are summarized in Table XIV. A l l li n e s derived from pure, f i r s t passage ROSE c e l l s were posit i v e for 17p-HSDH a c t i v i t y and showed faint A5-30-HSDH a c t i v i t y with not a l l c e l l s stained for the l a t t e r (Figure 13). The strong positive reactions for li n e V197-10B occurred only after . the 10th passage, with e a r l i e r passages being d e f i n i t e l y negative. In some control cultures there were some positive c e l l s indicative of non-specific dehydrogenase staining; t h i s was especially evident in l i n e V197-10B. Line V54, derived from a mixed c e l l ovarian culture, showed some I7p-HSDH a c t i v i t y . The other four ovarian l i n e s were a l l negative for t h i s enzyme. Surprisingly, peritoneal l i n e VP197 was positive for both the enzymes". The other peritoneal l i n e s were negative for both enzymes as were TABLE XIV. SUMMARY OF HISTOCHEMICAL DATA FOR KIMSV-. TRANSFORMED C : E L L S A5-3 0-HSDH2 170-HSDH3 C e l l Line 1 Act i v i t y A c t i v i t y L i p i d ORO" V1 97-1 OB5 v - ++ ++ V1 97.-1 3A +/- + . . + V197-15A +/- + + V193 - -V194 - ++ V196 - - v -V54 +/- + V56 - +/-VP6 - - +/-VP55 - - v -VP.1 97 6 . +/-• + / . -VP1 98 — — +/-1 - see table XIII for sources of c e l l l i n e s . 2 - DHEA as substrate. 3 - e s t r a d i o l as substrate. 4 - O i l Red 0 test done on glutaraldehyde or formaldehyde fixed cultures. 5 - th i s l i n e showed positive hydroxysteroid dehydrogenase tests only aft e r the 10th passage. 6 - the posit i v e results for th i s are surprising in the l i g h t of the negative results for the untransformed c e l l s and the other transformed peritoneal l i n e s . untransformed peritoneal cultures. A l l c e l l s tested by O i l Red 0 showed the presence of l i p i d to some degree from s l i g h t to very heavy (V194). 1 1 2 FIGURE 13: HYDROXYSTEROID DEHYDROGENASE HISTOCHEMISTRY OF KIMSV-TRANSFORMED ROSE CELLS 13a,b,c. Heavy to li g h t formazan deposits in most c e l l s in cultures of transformed ROSE c e l l s , indicating f a i r l y strong 17*-HSDH a c t i v i t y in l i n e s V197-10B, V197-13A and V197-15A. Substrate e s t r a d i o l . a,b X120, CX110. 13d,e,f. Deposits of formazan in many c e l l s in cultures of lines V197-10B, V197-13A and V197-15A of transformed ROSE c e l l s , indicating A5-3p-HSDH a c t i v i t y . Substrate, dehydroepiandrosterone. d,e x120, f X110. I3g,h,i. Control cultures of li n e s V197-10B, V197-13A and V197-15A incubated with dehydrogenase testing solution without substrates, showing formazan deposits in some c e l l s (g) indicating some nonspecific dehydrogenase a c t i v i t y . g,h X120, i x110. 113 ; . 114 c) Tumourigenesis Test ing of Transformed C e l l s i) Gross Observations on Result ing Tumours: A l l eight ovarian l i n e s and four peritoneal l i n e s of KiMSV--.' transformed c e l l s at early passages (3 to 11) were tumourigenic in immunosuppressed 4 week old female rats. Table XV presents a summary of gross observations on. the resulting tumours at subcutaneous s i t e s , and Table XVI a similar summary for intraperitoneal tumours. . Tumours at subcutaneous s i t e s were generally evident by palpation at 3 to 5 days after i n j e c t i o n . Tumours resulted from a l l i n j e c t i o n s . Most tumours grew quickly and some reached r e l a t i v e l y enormous sizes by two weeks (V196 in p a r t i c u l a r ) . Animals were s a c r i f i c e d at 5 to 14 days and tumours fixed. Two l i n e s produced invasive tumours in which the body wall was invaded (V54, V196). Most tumours were grossly 'healthy' in appearance without obvious signs of necrosis. Subcutaneous tissues in the region of tumours tended to be more vascular than that in other locations. Tumours tended to be pale in colour but some had small hemorrhagic blotches on their surface and others were frankly hemorrhagic in appearance and bled on cutting. Eight tumours were firm to hard and four were soft as judged by ease of cutting with a s c a l p e l . Tumours from the three l i n e s of pure ROSE c e l l o r i g i n resembled one another somewhat, but not s t r i k i n g l y so in comparison to the other ovarian and peritoneal l i n e s . 11 5 TABLE XV. GROSS OBSERVATIONS ON TUMOURS PRODUCED BY KIMSV-TRANSFORMED CELLS AT SUBCUTANEOUS, INJECTION SITES.1 K i l l e d Volume" Colour 5 Necr- Skin 6 I nva-Line Pass 2 Days 3 (cm3) Texture osis vase. s i on 7 V1.97-1 0B(6) 13 3.8 P B F - + • -V197-13AU) 6-10 1.1 P He F - + V197-15A(4) • 10 0.5 P B Ha - ' •+/~ -V193(4) 6-10 6.8 He F - . ++ -V194(3,4) 14 0.5 P F - v - - -V196(5,6) 14 13.5 P S - + + ++9 V54(5) 7-12 1.5 He F - + + ++ V56(5) 5-12 0.1 P S + -VP6(8-11) 9 0.4 P S +/-. + -VP55(5) 5-12 2.4 He S +/- + -VP197(3) 7 0.3 P F" - -VP198(3) 6 0.4 B F — + — 1 2 3 4 5 6 -7 -8 -9 -3-5X10 6 c e l l s per i n j e c t i o n . numbers in ( ) are passages after transformation, most tumours were f i r s t evident at 3-5 days after i n j e c t i o n . size averaged over a l l s.c. tumours of a given line, P=pale, B=blotched (small s u p e r f i c i a l hemorrhage), He=hemorrhagic, F=firm, Ha=hard, S=soft. increased size and density of blood vessels in skin close to tumour. obvious adhesion to or penetration into the skin or body wall. Two of four s.c. tumours adhered to the body wall. These tumours were hemorrhagic at the centre. One of these tumours penetrated the body wall, entered- the peritoneal, cavity/-- and* adhered*- t'o^the! ovarian mesentery. A l l KiMSV-transformed c e l l l i n e s tested by intraperitoneal inj e c t i o n produced tumours. Most injections resulted in intraperitoneal tumour growth. Table XVI summarizes gross observations on intraperitoneal tumour growth. Most intraperitoneally injected animals exhibited evidence of tumour development within one week of i n j e c t i o n . These 1 16 A s c i t e s 3 Line Pass 2 K i l l e d vol(ml) Volume" nodules 5 days Nature (cm3) V197-10B(6) 7 3 B+ 2.0 BW D G I MS OM S . VI97-13A(4) 6,7, 5 B 3.8 BW D G I K L MS OM V197-15A(4) 7 1 B 1.4 BW G I S V193(4) 6, 8 7 2 B 1 .0 BW D G OM V194(3,4) 7 2 B. 1 .3 D G I L MS6 V196(5,6) 7 1 S 3.5 D G I L OM6 V54(5-9) 5,87 2 B- 2.5 G I MC OM S V56(8) 1 4 1 S 0.2 BW D G VP6( 1 1 ) 5,14 1 B 3.0 BW D L VP55(5,10) 5,9 1 B 6.0 N+ VP197(3) 6 1 B 1 .5 D G L OM VP 198(3) 6,7 3 B 0.3 BW D G I K MS OM TABLE XVI. GROSS OBSERVATIONS ON TUMOURS PRODUCED BY KIMSV-TRANSFORMED CELLS INJECTED INTRAPERITONEALLY1 1 - 3-5xl0 6 c e l l s per i n j e c t i o n . 2 - numbers in parentheses are passages after transformation. 3 - B = bloody, B+ = very bloody, B- = s l i g h t l y bloody, S = serous or straw coloured. 4 - size of tumour averaged over a l l tumours from a given l i n e developing in the greater omentum. 5 - nodules varied in size but with the exception of tumours in the greater omentum most nodules were smaller than 5x5x5 mm. Letters give the location of nodules: BW = body wall, D = diaphragm, G = greater omentum, I = on body wall at inje c t i o n s i t e , K = kidney, L = l i v e r , MC = mesentery of the large i n t e s t i n e , MS- -^mesentery of the ! small intestine, N+ = many small nodules studding most peritoneal surfaces with p e a r l - l i k e tumours loose in ascites f l u i d , OM = ovarian mesentery, S = small inte s t i n e . 6 - tumour specimens growing in the greater omentum were adherent to l i v e r and loops of small bowel. 7 - at 8 days after injection two animals died from their tumours (1 from each l i n e ) . animals became thin and lethargic, and had bloated abdomens. A few animals died of their tumours at 8 days. On autopsy, tumour growth was seen to be mainly in the greater omentum, with some li n e s producing quite large masses (e.g. V196). There were • • • • 1 1 7 usually also smaller nodules studding peritoneal surfaces, in mesenteries and on i n t e s t i n a l walls, on the diaphragm, liver, and kidney. A l l l i n e s produced ascites f l u i d , most of which was bloody in nature. Volumes varied from less than 1 ml to over 7 ml in d i f f e r e n t animals. As with the subcutaneous tumours, the three l i n e s derived from pure ROSE cultures resembled one another but not s t r i k i n g l y so when compared with the other ovarian and peritoneal l i n e s . i i ) Histopathology of Tumours Histopathologically a l l tumours produced were sarcomatous in nature. A summary of the histopathology of these tumours i s given in Table XVII. Tumours from the three l i n e s derived from pure ROSE c e l l s were highly c e l l u l a r and poorly d i f f e r e n t i a t e d with spindle-shaped c e l l s having vacuolated cytoplasm (Figure 14a,b,c). These tumours resemble the highly c e l l u l a r endometrioid stromal sarcoma, a rare form of ovarian cancer (Figure 14e) (Kao et a l . , 1978; Russell, P., 1979). This tumour of the ovary i s termed an endometrioid stromal sarcoma because i t resembles the endometrial stromal sarcoma of the uterus (Figure 14d). Tumours produced by other ovarian and peritoneal lines tended to be fibrosarcomatous in nature, with the exception of tumours of peritoneal l i n e VP55 which were c l a s s i f i e d as malignant mesotheliomas (Figure I5e,f). Tumours of ovarian l i n e V54 had a pronounced storiform 1 18 TABLE XVII. HISTOPATHOLOGY OF TUMOURS DERIVED FROM KIMSV-TRANSFORMED CELLS c e l l Line origi n histopathology V197-1 OB V197-13A .VI97-15A V1 93 V1 94 V1 96 V54 V56 VP6 VP 5 5 VP1 97 VP1 98 pure ROSE pure ROSE pure ROSE primary ROSE primary .ROSE primary ROSE mixed c e l l ovary mixed c e l l ovary mixed peritoneal mixed peritoneal mixed peritoneal mixed peritoneal endometrioid stromal sarcoma endometrioid, stromal sarcoma endometrioid stromal sarcoma f ibrosarcoma (spindle-shaped c e l l s ) f ibrosarcoma ( e p i t h e l i o i d c e l l s ) fibrosarcoma (with c a l c i f i e d bodies and giant c e l l s ) fibrosarcoma resembling malignant fibrous histiocytoma low grade sarcoma f ibrosarcoma malignant mesothelioma fibrosarcoma non-specific sarcoma (mixed spindle-shaped and e p i t h e l i o i d c e l l s ) pattern (Figure 15a) which has been seen in certain rare thecomas of the human ovary. However thecomas are considered benign growths (Scully, 1977b) and the V54 tumours were aggressively malignant, with pleomorphic c e l l s and many mitoses. The V54 tumours also strongly resembled the malignant fibrous histiocytoma, a tumour which has been reported in the human ovary (Ueda e a l . , 1977). Areas of tumours of ovarian l i n e V193 had a storiform pattern not as pronounced as that of V54; there were also fibrosarcomatous regions of p a r a l l e l spindle-shaped c e l l s (Figure 15b). Tumours of ovarian l i n e V194 (Figure 15c) were fibrosarcomatous and had plumper-, more epithelioid" c e l l s ' with a vaguely storiform pattern. The highly malignant tumours 119 FIGURE 14: HISTOPATHOLOGY OF TUMOURS FROM KIMSV-TRANSFORMED ROSE CELLS 14a,b,c. Histology of tumours formed by the l i n e s V197-10B, V197-13A and V197-15A. Note the highly c e l l u l a r nature of the tumours, the large pleomorphic nuclei and the vacuolated cytoplasm. Note the resemblance to endometrial and endometroid stromal sarcomas of the human uterus and ovary (Figure 14d,e). These rat tumours are of a higher grade of malignancy than the human tumours shown. H. and E . x360. 14d. Histology of a human endometrial stromal sarcoma of the uterus. H. and E . - x400. 14e. Histology of a human endometrioid stromal sarcoma of the ovary. H. and E. x400. 14d,e. Micrographs are of c l i n i c a l specimens provided by Dr. P. B. Clement, Dept. of Pathology, Vancouver General Hospital. FIGURE 15: HISTOPATHOLOGY OF TUMOURS FROM KIMSV TRANSFORMED CELL LINES OF OVARIAN AND PERITONEAL ORIGIN 15a. Histology of tumour from ovarian l i n e V54 demonstrating the storiform pattern of f i b r o b l a s t - l i k e c e l l s seen in malignant fibrous histiocytomas and in certain rare thecomas of the human ovary. H. an& E . x240. 15b. Tumour of the ovarian l i n e V193 composed of spindle-shaped c e l l s with a fibrosarcomatous histology. H. and E. x240. 15c. Tumour of the ovarian l i n e V194 composed of plump, e p i t h e l i o i d c e l l s with a fibrosarcomatous histology. H. and E . x240. 15d. Tumour of the peritoneal l i n e VP198 showing a non-s p e c i f i c sarcomatous histopathology of mixed spindle-shaped and e p i t h e l i o i d c e l l s with marked edema. H. and E . x240. I5e. Nodules of intraperitoneal tumour derived from peritoneal l i n e VP55, found loose in a s c i t i c f l u i d . H. and E . x55. 15f. A higher magnification of part of figure 15e showing tumour to be a malignant mesothelioma. C e l l s in the central regions of nodules are more e p i t h e l i o i d whereas those at the edges are more spindle-shaped. The blood vessel seen in the lower nodule shows that at least some of the nodules loose in the peritoneal f l u i d were once attached to the peritoneal surface. H. and E . x240. 120 121 122 FIGURE 16: ULTRASTRUCTURE OF TUMOURS DERIVED FROM KIMSV-TRANSFORMED ROSE CELLS 16a. An electron micrograph of a V197-10B tumour showing abundant RER (R) and extensive e x t r a c e l l u l a r filaments ( f ) . x9,500. 16b. Electron micrograph of a V197-13A tumour showing very c l o s e l y spaced c e l l s w i t h ^ m i c r o v i l l i (A) and SER (S). Note the prominent nucleolus (^) and i n t r a c e l l u l a r filaments (F). A simple desmosome-like junction found occasionally in thi s tumour is shown in the inset. x9,500, inset x45,000. 16c. Electron micrograph of a V197-15A tumour i l l u s t r a t i n g a c e l l with well-developed SER and numerous Golgi complexes ( A ) . Note the long c e l l processes, s l i g h t amorphous e x t r a c e l l u l a r material and i n t e r c e l l u l a r filaments in cross-section (F). X15,500. FIGURE 17: HISTOPATHOLOGY OF A TUMOUR ARISING IN A RAT INJECTED WITH R O S E - 1 9 9 CELLS 17a,b. Histopathology of a tumour a r i s i n g subcutaneously 11 months after injection with 3-5 m i l l i o n ROSE-199 c e l l s . This tumour was very fibrous (b) with small, more c e l l u l a r regions (a). The tumour was diagnosed as a low grade fibrosarcoma or a tumour of mesothelial o r i g i n . H. and E. -x440. FIGURE 18: EVIDENCE FOR VIRAL TRANSFORMATION OF CULTURED ROSE CELLS 18a. An electron micrograph showing p a r t i c l e s resembling C-type retroviruses budding from a c e l l of l i n e Vl97-a5A and in e x t r a c e l l u l a r spaces. x69,000. 18b. Foci of spindle-shaped, r e t r a c t i l e , transformed c e l l s seen in a monolayer of NRK c e l l s one week after medium ( m i l l i p o r e - f i l t e r e d ) from a KiMSV-transformed ROSE c e l l l i n e was added. Toluidine blue, X180. 123 124 . • 125 of ovarian l i n e V196 resembled those of l i n e V54 but also had c a l c i f i e d bodies, (not true psammoma. bodies since, they were not laminated) and giant c e l l s of the foreign-body type. Ovarian l i n e V56 produced very low grade sarcomas with the main part of the "tumours" being granulation tissue.. Peritoneal l i n e s VP6 and VP197 produced fibrosarcomas with areas having vaguely storiform patterns of spindle-shaped c e l l s . Peritoneal l i n e VP198 (Figure 15d) produced edematous tumours having a non-specific sarcomatous histology of mixed spindle-shaped and e p i t h e l i o i d c e l l s . Tumours of l i n e VP55 were malignant mesotheliomas and intraperitoneally produced large numbers of small nodules studding peritoneal surfaces and tiny p e a r l - l i k e clumps of c e l l s free in peritoneal f l u i d (Figure 15e,f). i i i ) Ultrastructure of Tumours Derived from KiMSV- transf ormed ROSE Ce l l s U l t r a s t r u c t u r a l l y , tumours produced by a l l three transformed ROSE li n e s (V197-1 OB, V197-13A, V197-15A) appeared as clusters and cords of c e l l s with shapes ranging from elongated to polygonal (Figure 16). C e l l s were partly surrounded by f i b r i l l a r e x t r a c e l l u l a r material which was most abundantly and consistently present in tumours of l i n e V197-10B. Basement membrane was not seen in tumours from any of these three l i n e s . M i c r o v i l l i were, numerous in lines V197-13A and V197-T5A. Simple desmosome-like junctions associated' with cytoplasmic filaments were seen in V197-13A tumours (Figure 126 16b). In terms of cytoplasmic c h a r a c t e r i s t i c s a l l three l i n e s were heterogeneous. They contained some., c e l l s where the predominant secretory organelle was rough endoplasmic reticulum (RER), and others where short tubules and vesi c l e s of SER and Golgi complexes were abundant. C e l l s with much and often d i l a t e d RER were most common in V197-10B tumours (Figure 16a), while c e l l s with prominent SER were more common in tumours of the other two l i n e s (Figure 16c). Occasional l i p i d droplets were seen in a l l tumours, most often in tumours of l i n e V197-10B, the same li n e exhibiting heavy o i l red 0 staining in culture. Tumours of a l l l i n e s contained many round to oval mitochondria with lamellar c r i s t a e . Nuclei were pleomorphic, frequently had prominent n u c l e o l i , and occasionally were very irregular in shape. In tumours of a l l these l i n e s p a r t i c l e s resembling type C retroviruses were occasionally seen either budding from c e l l surfaces or free in i n t e r c e l l u l a r spaces, iv) Negative Control Tests for Tumourigenesis As negative controls- for tumour i genesis? 12: immunosuppressed rats were injected either subcutaneously (s.c.) or i n t r a p e r i t o n a l l y (i.p.) with c e l l s of the continuous l i n e ROSE-199. Each injection contained 3 to 5 m i l l i o n c e l l s at passages 18, 19 or 25. There was no evidence of tumours of any kind in the f i r s t three months after i n j e c t i o n when animals were frequently palpated for tumour development. However, three tumours were discovered at 10, 11 and 12 months. One tumour, measuring 15x15x10 mm was discovered at 11 months at the s.c. inje c t i o n s i t e just anterior to the l e f t haunch. J 127 Histopathologically this tumour resembled either a low grade fibrosarcoma or a benign tumour of the. pleura, (Dalton. et a l . , 1979) (Figure 17). It i s possible that this tumour could have developed from the injected c e l l s , but an incidence of 1 in 12 is hardly conclusive. Another tumour developing in an s.c. injected . rat was discovered / at 10 months on the right underside under the most posterior nipple. It measured 20x20x10 mm and was c l a s s i f i e d as an undifferentiated carcinoma (Figure 21b). It seems unlikely that t h i s tumour could have resulted from an injection given s.c. just anterior to the l e f t haunch. The t h i r d tumour, measuring 30x30x20 mm arose in an i.p. injected animal and was located on the upper chest, just posterior to the neck. It was classed as a fibroadenoma of the mammary gland, a tumour a r i s i n g in 16% of aging female Fischer rats (Goodman, 1979). There was no sign of tumour intraperitoneally. in th i s rat. The remaining 9 animals were either k i l l e d at 6 to 8 months due to infections (3 animals) or at 12 months. None of these nine showed any evidence ;of tumour development in subcutaneous or intraperitoneal regions, d) Evidence for V i r a l Transformation of Cultured C e l l s In electron micrographs from the three transformed ROSE lines V197-10B, V197-13A and V197-15A, p a r t i c l e s resembling type C retroviruses were frequently seen either budding from c e l l surfaces or in i n t e r c e l l u l a r spaces (Figure 18a). When one-day-old medium1 from" these three' transformed' ROSE lin e s was passed through a m i l l i p o r e f i l t e r onto subconfluent 1 28 NRK c e l l s , f o c i of transformed NRK c e l l s were seen within one week (Figure 18b). When, rapidly growing cultures of the transformed ROSE li n e s were incubated with 5- 3H-uridine, l a b e l l e d p a r t i c l e s . w e r e produced in the medium which banded in sucrose density gradients at densities t y p i c a l of KiMSV (1.15 to 1.17 g/ml). This range of densities was determined previously (Auersperg et a l . , 1977). 6. AUTORADIOGRAPHIC INVESTIGATION OF TISSUES AND CULTURED  CELLS FOR THE PRESENCE OF ESTROGEN RECEPTOR ACTIVITY' a) Autoradiography Of Sections The results of autoradiography done on sections was not quantified by grain counting. Sections of whole ovary treated autoradiographically showed heavy densities of s i l v e r grains in the surface epithelium, corpora lutea, theca interna and granulosa c e l l s with stroma underlying the ovarian surface epithelium less heavily la b e l l e d (Figure 19a). Labelling tended to be mainly cytoplasmic. In sections of uterus, e p i t h e l i a l c e l l s of the uterine lumen and glands were r e l a t i v e l y densely l a b e l l e d with the stroma and smooth muscle less heavily l a b e l l e d . Scattered eosinophils in the uterine stroma were extremely densely l a b e l l e d (Figure 19b). Sections of skel e t a l muscle were labe l l e d more heavily than expected. Tissue-free areas of la b e l l e d s l i d e s showed a considerable density of grains. This high background was" l i k e l y due to binding of radioactive e s t r a d i o l by albumin in the adhesive used to bind 129 FIGURE 19: AUTORADIOGRAPHY OF SECTIONS OF OVARY AND UTERUS 19a. A 5 >im section of rat ovary ( l e f t ) and ovarian bursa (right) processed autoradiographically for the detection of estrogen receptors. Note the heavy, mainly cytoplasmic concentration of s i l v e r grains in most tissues of the ovary, namely, the surface epithelium (s.e.) and a corpus luteum (c). Stroma underlying the surface epithelium i s less heavily l a b e l l e d . The ovarian bursa, a pouch of peritoneal tissue surrounding the ovary i s much less l a b e l l e d . Labelled with 2,4,6,7- 3H-estradiol. Exposure 3 weeks. .H... and.E. x600. 19b. A 5 urn section of rat uterus processed autoradiographically for the detection of estrogen receptors. Note the mainly cytoplasmic deposition of s i l v e r grains over stromal (s) and glandular (g) c e l l s . Note the densely l a b e l l e d eosinophils in the stroma (e). Labelled with 3 H - e s t r a d i o l . Exposure 3 weeks. H. and E. x400. FIGURE 20: AUTORADIOGRAMS OF CULTURED ROSE CELLS 20a. An autoradiogram of ROSE c e l l s in primary culture l a b e l l e d with 3H-estradiol after ethanol f i x a t i o n . Note the dense l a b e l l i n g of the c e l l s . Note the high background l a b e l l i n g due probably to the binding of 3H-estradiol to serum proteins bound to the p l a s t i c growth surface. Exposure 3 weeks. Toluidine blue and eosin. x350. 20b. An autoradiogram of a confluent culture of ROSE c e l l s l a b e l l e d l i v e with 3H-estradiol, washed in unlabelled medium for a 45 minute chase, then freeze-dried. Note the mainly cytoplasmic d i s t r i b u t i o n of grains. Exposure 2 weeks. Toluidine blue and eosin. x500. 20c. An autoradiogram of ROSE c e l l s l a b e l l e d l i v e with 3H-e s t r a d i o l , washed in unlabelled medium for a chase of 6.5 hours then freeze-dried. Note the markedly increased nuclear l a b e l l i n g of these c e l l s when compared with c e l l s in Figure 20b. Exposure 2 weeks. Toluidine blue and eosin. x400. 130 131 sections to the glass. Unlabelled control s l i d e s showed very few grains except for dense grains over areas containing red blood c e l l s . This positive chemographic effect could possibly be due to the reduction of s i l v e r ions in the emulsion by ferrous ions in the haemoglobin. There was no evidence of negative chemography, that i s , fading of the image, in s l i d e s exposed to l i g h t then kept in the dark for three weeks before development. When sections la b e l l e d with t r i t i a t e d e s t r a d i o l were washed in PBS containing 4 >#g/ml of radioinert e s t r a d i o l or estrone there was a very large drop in grain densities in a l l labelled tissues compared with control s l i d e s . When the PBS for washing contained radioinert progesterone or testosterone there was a lesser drop in grain densities. These grain densities were not analysed quantitatively by grain counting. This effect suggests that the estrogen a f f i n i t y exhibited by these tissues i s composed of a s p e c i f i c a f f i n i t y for estrogens plus a non-s p e c i f i c a f f i n i t y for steroids;, b) Autoradiography of Cultured ROSE C e l l s Autoradiograms of ROSE c e l l s prepared either from c e l l s l a b e l l e d after ethanol f i x a t i o n or l a b e l l e d l i v e and then freeze-dried showed obvious l a b e l l i n g of c e l l s . In both methods there was a r e l a t i v e l y high number of s i l v e r grains in c e l l - f r e e areas, usually about 1/5 to 1/4 that in areas covered with c e l l s . This was l i k e l y due to binding- of tTitrated' e s t r a d i o l to" proteins in the serum in the growth medium which were in turn 1 32 bound to the p l a s t i c coverslips. There were l i k e l y serum proteins bound to c e l l surfaces as well,. It is. d i f f i c u l t to see how proper correction could be made for t h i s error. However, such bound steroid would show no evidence of translocation from cytoplasm to nucleus in l i v e - l a b e l l e d cells.. Most of the data presented in t h i s section were not corrected for this type of background. Unlabelled control sections had very few grains either over the c e l l s or the p l a s t i c surfaces (approximately 50 grains per half microscope f i e l d at x25 objective). Ethanol f i x a t i o n caused considerable shrinkage of c e l l s (Figure 20a). Freeze-drying caused a d i s t o r t i o n of morphology but nuclei were generally distinguishable from cytoplasm with suitable staining (Figure 20b). There was much less shrinkage in freeze-dried c e l l s . In Table XVIII a comparison i s given for ROSE c e l l s l a b e l l e d after ethanol f i x a t i o n and c e l l s l a b e l l e d l i v e , with respect to s i l v e r grains per c e l l and r a t i o of nuclear to cytoplasmic grains. C e l l s l a b e l l e d after- ethanol' f i x a t i o n - had' a s i g n i f i c a n t l y greater number of grains per c e l l than c e l l s l a b e l l e d l i v e . In c e l l s l a b e l l e d l i v e the r a t i o of nuclear grains to cytoplasmic grains was very s i g n i f i c a n t l y greater than for c e l l s l a b e l l e d after ethanol f i x a t i o n . Data for each method shown in Table XVIII were obtained by counting whole or half f i e l d s with the x25 objective (one whole and two half f i e l d s per method). A Z-test for equality of proportions (Lindgren and Berry, 1981) indicated that the pr o b a b i l i t y of equality of the r a t i o s N/C for the two methods i s extremely small (much less 133 TABLE XVI CELLS LAI II . COMPAI 3ELLED AFTEI II SON OF / * FIXATIO* iUTORADIOC I; WITH, THC JRAMS OF . ROE )SE LABELLEI ; E ) LIVE method1 c e l l s 2 counted N 3 C grains 5 per c e l l ; N / C ethanol fixed l i v e -l a b e l l e d 382 390 1487 837 8743 3278 26.8 10.6 0.17 0.26 1 - 3 weeks exposure, i d e n t i c a l evelopment times. 2 - c e l l s were counted using the x25 objective with a grid in the eyepiece. For each method two half f i e l d s of intermediate sized c e l l s and one whole f i e l d of large, spread out c e l l s was counted. Similar f i e l d s were chosen for both methods. 3 - t o t a l number of nuclear grains. 4 - t o t a l number of cytoplasmic grains. 5 - calculated by dividing t o t a l grains by t o t a l number of c e l l s . than .001). The greater proportion of nuclear grains in the l i v e - l a b e l l e d c e l l s argues for translocation of label from cytoplasm to nucleus. In both autoradiographic methods used the number of grains per c e l l increased^ as-' the-- cel.1. a-rea- (as: measured- by- microscope/ i . e . in a two dimensional plane) increased (Figure 21). In autoradiograms c e l l areas were c l a s s i f i e d to the nearest 0.5 grid square and grains counted in nucleus and cytoplasm. Nuclear areas were estimated to the nearest 0.1 grid square. Ten c e l l s were analysed for each data point. Data were corrected for background l a b e l l i n g due to serum proteins binding t r i t i a t e d e s t r a d i o l . For c e l l s l a b e l l e d after ethanol f i x a t i o n the number of grains per c e l l increased f i v e f o l d for a f i v e f o l d increase in c e l l area. The r a t i o N/C of nuclear to cytoplasmic 50 r-40 GRAINS PER CELL AND N/C VERSUS CELL AREAA FOR ROSE CELLS LABELLED AFTER ETHANOL FIXATION 0- grains per c e l l •- N/C 1- at 4 weeks exposure 30 20 10 I I J 0 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 CELL AREA (100 ym )• GRAINS PER CELL AND N/C VERSUS CELL AREA FOR ROSE CELLS LABELLED LIVE 1.25 T75 2.0 2.5 O 375 470 4.5 5.0 575 CELL AREA (100 ym 2) 135 grains changed very l i t t l e with a s l i g h t drop detectable over the range of c e l l u l a r areas studied. On the other hand,, for c e l l s l a b e l l e d l i v e , the number of grains per c e l l only doubled as the area quadrupled, but the r a t i o N/C dropped markedly. This reduced proportion of nuclear to cytoplasmic grains i s what would be expected due to the greater average .distance steroid receptor complexes would have to tr a v e l through the cytoplasm to get to the nucleus in larger c e l l s . When ROSE c e l l s were., la b e l l e d l i v e with a. one hour pulse of. t r i t i a t e d e s t r a d i o l and then l e f t in radioinert medium for chases of 0.75 to 6.5 hours, the r a t i o N/C in the resulting autoradiograms was highest for the longest chase times. (Table XIX). Each data point in Table XIX i s based on grain counts TABLE XIX. ESTRADIOL h PULSE-CH/ MOVEMENT lb iSE ANAI I LIVE-I ,YSIS OE J A B E L L E I - TRITIATED ) ROSE CELLS > 1 Chase Time (hrs) C e l l s 2 Counted N 3 c« Grain s-Per C e l l N/C 0.75 2.5 4.5 6.5 456 397 731 389 1 042 580 1 643 21 35 3828 3094 3585 2909 10.6 9.3 7.2 13.0 0.27 0.19 0.46 0.73 1 - two weeks exposure time, i d e n t i c a l development and f i x a t i o n conditions for a l l autoradiograms in t h i s table 2 - three microscope f i e l d s (3 half f i e l d s or two half f i e l d s and one whole f i e l d ) of c e l l s counted per chase 3 - t o t a l nuclear grains counted 4 - t o t a l cytoplasmic grains-' counted 1 36 from three microscope f i e l d s (wholes and halves) per chase. In Figure 20b an autoradiogram of a chase, freeze-dried a f t e r 0.75 hour shows s i l v e r grains mainly in the cytoplasm. Figure 20c, an autoradiogram of a 6.5 hour chase has a high proportion of c e l l s with predominantly nuclear grains. This is further evidence that the radioactive steroid, was translocated from cytoplasm to nucleus and that r a d i o a c t i v i t y became more concentrated in the nucleus with time. TABLE XX. STEROID C :OMPETITIO* I IN ROSE CELLS Method 1 S t e r o i d 2 Added C e l l s 3 Counted Total" Grains Grains Per C e l l % Drop In Grains ethanol f ixed none E 2 Prog 298 333 307 1 0,030 6,696 8,427 33.7 20. 1 27.5 0 40.3 18.6 1 i ve-la b e l l e d none E 2 prog 393 349 351 7,339 2,679 3,507 18.6 7.7 10.0 0 58.6 46.2 1 - three weeks exposure. Data represent two separate experiments. Developing and f i x a t i o n times were constant for each experment.-. 2 - Radioinert steroids, e s t r a d i o l (E 2) or progesterone were added at 2 »q/ml (600x concentration of 3H-E 2 used for l a b e l l i n g ) . 3 - Three half microscope f i e l d s (at x25) of similar sized c e l l s were counted for each treatment and control. A l l nine f i e l d s in each experiment (ethanol fixed or l i v e - l a b e l l e d ) were as similar as possible. 4 - These data are corrected for background due to binding of 3H-E 2 to serum proteins bound to coverslips. Grains were counted for half f i e l d s of c e l l - f r e e areas of coverslips and an average background value subtracted from counts for half f i e l d s containing c e l l s . 137 Table XX summarizes the results of steroi d competition experiments in which radioinert steroids were included with t r i t i a t e d e s t r a d i o l in the l a b e l l i n g of ROSE c e l l cultures. In a l l treatments evaluated in th i s table similar f i e l d s of similar sized c e l l s were counted. This was essential since, as shown, previously, the number of grains per c e l l varies with c e l l area. In ethanol-fixed cultures l a b e l l e d with t r i t i a t e d e s t r a d i o l in PBS and washed with PBS containing 2 »q of radioinert e s t r a d i o l per ml, the number of grains, per c e l l was 4.0.3%. lower than for control c e l l s washed only in PBS. If the PBS contained progesterone the drop was 18.6%, s i g n i f i c a n t l y d i f f e r e n t from both controls and the e s t r a d i o l treated cultures. From these data i t appears that estrogen a f f i n i t y in ethanol fixed ROSE c e l l s i s composed of a s p e c i f i c a f f i n i t y for estrogen (estradiol) and a non-specific a f f i n i t y for other steroids. In l i v e - l a b e l l e d c e l l s in which the l a b e l l i n g medium contained both radioactive and radioinert e s t r a d i o l the number of grains dropped by 59.3% from control l e v e l . However, the addition of 2 nq/ml of progesterone to the l a b e l l i n g medium caused a drop in grain count of 46.2% from control l e v e l . Both treatments result in large, s i g n i f i c a n t drops from control l e v e l , and the two treatment results were somewhat less, but s t i l l s i g n i f i c a n t l y , d i f f e r e n t from each other. This result suggests that progesterone might act on l i v i n g ROSE c e l l s to reduce estrogen a f f i n i t y . In retrospect', progesterone' was- not' a good choice of steroid to show non-competition-with radioactive 138 e s t r a d i o l . 7. CONTINUOUS CELL LINES ROSE-199 AND ROSE-239 In the course of experiments performed to determine the l i f e span of ROSE c e l l s , two continuous lines arose, namely ROSE-199 and ROSE-239. a) ROSE-199 Continuous c e l l l i n e ROSE-199 had a morphology in subconfluent culture indistinguishable from that of pure, f i r s t passage ROSE c e l l s (Figure 22a). In cultures after the 19th passage, crowded cultures formed ridged structures (Figure 22b). Crowded cultures also produced large numbers of f l o a t i n g c e l l s in singles and clumps. These f l o a t i n g c e l l s were readily cultured when transferred to fresh culture vessels, and had ROSE morphology. In Figure 23 the c e l l densities reached at each passage are graphed. Passages 2 to 6 were not counted at subculture. Passage 1 cultures were in three 16 mm wells which were pooled on subculture and seeded into four 35 mm dishes, a d i l u t i o n of 1:6. Passages 2 to 18 were subcultured at approximately weekly intervals at 1:2 d i l u t i o n s . At passage 19, three dishes were subcultured to four 25 cm2 flasks, a d i l u t i o n of 1:3. Passages 20 to 35 were subcultured at weekly intervals at 1:2 d i l u t i o n . C e l l densities graphed were those reached at the time of subculture. At passage 20 the c e l l density began to r i s e sharply. After reaching' a peak of over 400,000 cells/cm 2' the c e l l density began to f a l l gradually. This l i n e of c e l l s 139 FIGURE 22: CULTURE MORPHOLOGY OF ROSE-199 CELLS 22a. A 3-day-old subconfluent culture of ROSE-199 c e l l s at the 30th passage. Note the mitotic figures and the d i s t i n c t i v e morphology of the small colonies. These c e l l s have the morphology of ROSE c e l l s at f i r s t passage. Toluidine blue. x160. 22b. A 30-day-old culture of ROSE-199 c e l l s (30th passage) which has formed complex ridged and multilayered structures. Note the mitotic figures s t i l l present in this crowded culture. Cultures such as this one exfoliate viable c e l l s into the culture medium. Toluidine blue. X160. 22c. A p l a s t i c section of a f l o a t i n g sheet of ROSE-199 c e l l s at the 20th passage. This culture was six weeks old. Note the p a p i l l a r y structures and areas of c e l l u l a r s t r a t i f i c a t i o n . The thick a c e l l u l a r layer separating the two layers of c e l l s i s composed almost e n t i r e l y of collagen f i b r e s (see Figure 25). Note the resemblance of structures in t h i s section to those seen in Figure 22d. Toluidine blue. x300. 22d. A pa r a f f i n section of a human ovarian tumour c l a s s i f i e d as a serous p a p i l l a r y cystadenoma of borderline malignancy. Micrograph of a c l i n i c a l specimen provided by Dr. P. B. Clement, Department of Pathology, Vancouver General Hospital. H. and E. x360. PASSAGES - 142 was frozen every 5th passage from the 21st to the 36th passage. By the 36th passage over 55 population doublings, had., been achieved. In order to determine saturation densities reached by ROSE-199 c e l l s , cultures at the 25th passage were set up at 10,000 cells/cm 2 in 35 mm dishes in both .10% FBS/WM (Figure 24a) and 25% FBS/WM (Figure 24b). Both curves had a lag period of 5 days followed by a rapid increase in c e l l numbers between days 5 and 10. From day 10 onward cultures, were, confluent and produced ridges and fl o a t i n g c e l l s . The 10% FBS/WM series exhibited a short period of stationary growth (days 10 to 14) followed by a second sharp r i s e to approximately double the c e l l number. A seemingly permanent stationary growth period was reached which lasted three weeks. These cultures exhibited many mitotic figures and fl o a t i n g c e l l s however. The 25% FBS/WM curve was not as regular but reached a c e l l density at the end of the series very close to that of the 10% series (144,000 cells/cm 2 compared with 170,000). These d e n s i t i e s - represent 4''-popuiatdon. doublings from seeding d e n s i t i e s . These c e l l densities were much lower than those reached for passages 24 to 26 grown in flasks (approximately 300,000 c e l l s / c m 2 ) . Cultures of ROSE-199 c e l l s at the 20th and later passages, i f allowed to age 2 to 4 weeks after confluence was attained, developed ridged structures and appeared multilayered when viewed with an inverted mic roscope-. In* such'a culture" the" c e l l sheet floated off the growth surface. This sheet was embedded 1 4 3 144 in epon and sectioned perpendicular to the c e l l layers. In thick epon sections stained with toluidine blue and examined by l i g h t microscopy, the sheet of c e l l s was seen to be composed of. two c e l l layers separated by a thick a c e l l u l a r layer (Figure 22c). One layer was simple and composed of flattened c e l l s . The other layer was s t r a t i f i e d in places, forming p a p i l l a r y structures. This second layer was composed of c e l l s ranging from squamous to low columnar in shape. The histology of the p a p i l l a r y layer resembles that seen in human ovarian serous p a p i l l a r y cystadenomas of borderline malignancy (Figure 22d) (Czernobilsky, 1977; Russell and Merkur, 1979). When these c e l l layers were examined by electron microscopy both c e l l layers had i n t e r c e l l u l a r junctions resembling junctional complexes. The thick a c e l l u l a r layer was f u l l of coarse f i b r e s of collagen showing cross s t r i a t i o n s t y p i c a l of connective tissue collagen (Figure 25). Nuclei were frequently irregular in shape. Ce l l s of l i n e ROSE-199 at passages 18, 19 and 25 were used as negative controls for tumourigenesis for v i r a l l y transformed l i n e s . Of the 12 rats injected either subcutaneously or i n t r a p e r i t o n e a l l y with ROSE-199 c e l l s (3-5X10 6 c e l l s per injection) only one subcutaneously injected animal developed a tumour at the i n j e c t i o n s i t e . The histology of t h i s tumour, discovered 11 months after i n j e c t i o n , is shown in Figure 17a. It was extremely fibrous and hard with scattered c e l l s and' c e l l c l u s t e r s . Histopathologically t h i s tumour resembled a low grade 145 FIGURE 25: ULTRASTRUCTURE OF MULTILAYERED STRUCTURES FORMED BY ROSE-199 CELLS IN VITRO 25a. An electron micrograph of a multilayered structure formed in a 6-week-old culture of ROSE-199 c e l l s at the 20th passage. Note the i n t e r c e l l u l a r junctions between c e l l s of both c e l l sheets ( O . Note the complex i n t e r d i g i t a t i o n of c e l l s in the upper part of the micrograph and the large b i z a r r e l y shaped nucleus in the bottom. Note also the Golgi complexes, numerous ve s i c l e s , abundant i n t e r c e l l u l a r collagen, and the lack of basal lamina. x15,600. 25b. An electron micrograph of another region of the structure seen in Figure 25a. Note the complex i n t e r c e l l u l a r junction resembling junctional complexes t y p i c a l of e p i t h e l i a l c e l l s , along with abundant i n t e r c e l l u l a r , s t r i a t e d collagen f i b r e s t y p i c a l of stromal c e l l s . x15,600. 25c. A higher magnification of Figure 25b showing d e t a i l s of the i n t e r c e l l u l a r junction. x42,000. 25d. Collagen fibres in the space between the two c e l l u l a r layers. x34,000. 25e. Cytoplasmic organelles. x60,000. 146 147 1 48 fibrosarcoma or tumour of mesothelial o r i g i n (Dalton et a l . , 1979) . b) ROSE-2 39 Continuous c e l l l i n e ROSE-239 arose from primary ROSE cultures set up in 10% FBS/WM and subcultured only in th i s serum concentration. The morphology of ROSE-239 c e l l cultures was indistinguishable from that of f i r s t passage ROSE c e l l s . C e l l densities for this l i n e at certain passages are given in Table XXI. Cultures were grown in 35 mm dishes after the f i r s t TABLE XXI. CELL DENSITIES OF ROSE-239 CULTURES Passage 1 C e l l Density 2 (cells/cm 2) 1 34,000 3 38,500 5 , 41,300 7 61 ,300 ' 8 117,000 9 129,000 1 2. 1 1 5 v 000-1 5 125,000 20 130,000 1 - passage 1 was grown in 16 mm wells. Passages 2-22 were grown in 35 mm dishes. Dilution at subculture was 1 to 2 except for passage 2 where d i l u t i o n was 1 to 2.5. 2 - densities were determined from c e l l counts at the time of subculture. passage, and subcultured at weekly intervals with a 1 to 2 d i l u t i o n . C e l l densities became" f'airly Constant' a t 1 15,000-130,000 cells/cm 2 after the 7th passage. In some cultures allowed to age for 6 weeks a. noticed, but there was no sign l i n e was frozen at the 16th and 149 small amount of ridging was of multila.yering of cells.. This. 22nd passages. 150 CHAPTER IV. DISCUSSION 1. IDENTIFICATION OF RAT OVARIAN SURFACE EPITHELIAL CELLS IN CULTURE The f i r s t and most essential phase of thi s project was confirmation of the identity of the rat ovarian surface, epithelium in culture. Morphologically, cultured ROSE c e l l s d i f f e r e d from other cultured ovarian c e l l types portrayed in the literature.. Granulosa c e l l s from preovulatory f o l l i c l e s , rat (Fischer and Kahn, 1972), equine, human (Channing, 1969a,b) and porcine (Stadnicka, 1976) grow as e p i t h e l i o i d sheets of c e l l s which lu t e i n i z e d after 3 to 4 days in culture, that i s , they undergo hypertrophy and acquire many l i p i d droplets and A5-3p-HSDH a c t i v i t y . The morphology of cultured ROSE c e l l s was obviously d i s t i n c t from that of c e l l s in these reports and of rat granulosa c e l l s grown in thi s laboratory by a modification of the method of Redmond (1970) (Figure 2). Cultured ROSE c e l l s showed no tendency to l u t e i n i z e in culture. According to Channing granulosa c e l l s from small f o l l i c l e s grow as s t e l l a t e fibroblast-1ike colonies without evidence of l u t e i n i z a t i o n . Luteal c e l l s grow as large e p i t h e l i o i d c e l l s with abundant l i p i d in the cytoplasm (Gospodarowicz and Gospodarowicz, 1972b; Stadnicka, 1977), a morphology quite d i s t i n c t from that of ROSE 151 c e l l s . Other cultured c e l l types grow only as f i b r o b l a s t - l i k e c e l l s , namely thecal (Channing., 1969a, b; Stadnicka. and. Stoklosowa, 1976; Stadnicka, 1977) and stromal (Channing, I969a,b), and hence bore no resemblance to the orderly e p i t h e l i o i d morphology of cultured ROSE c e l l s . Long, in 1940, claimed to have cultured the mouse "germinal" epithelium. Cultures depicted in Long's paper strongly resemble ROSE c e l l s . These c e l l s were most l i k e l y mouse ovarian surface e p i t h e l i a l c e l l s . However, Long supports his claim that the c e l l s were of "undoubted" germinal e p i t h e l i a l o r i g i n by the presence of oocytes in the cultures. In a very brief 1952 report containing no micrographs Scott described the growth of sheets of e p i t h e l i a l c e l l s from pieces of rat ovary. She made no attempt to i d e n t i f y the c e l l s even t e n t a t i v e l y . Some of the most convincing evidence presented in the i d e n t i f i c a t i o n of cultured ROSE c e l l s were sections of ovarian explants and c e l l u l a r outgrowths showing the surface epithelium of- the ovarian explant continuous--with the- surface.* cells?-of- the-outgrowth. In regions near the explant f i b r o b l a s t - l i k e c e l l s underlaid the surface c e l l s , but at greater distances the outgrowth became a monolayer of surface c e l l s only (Figure 5d). The fact that outgrowths of ROSE c e l l s ringed the explant also supports the claim of their surface e p i t h e l i a l o r i g i n . This growth pattern would exclude the p o s s i b i l i t y that these sheets of c e l l s might be vascular endothelial in o r i g i n . Cultured vascular endothelial c e l l s have a morphology similar to that of ROSE c e l l s (Gimbrone, 1976). 152 Histochemically, with respect to a c t i v i t y of the enzymes A5-3p-HSDH and 17p-HSDH, the surface epithelium of the rat ovary is d i s t i n c t from other ovarian c e l l types both in cryostat section and in culture. Rat ovarian surface epithelium tested negative for A5-30-HSDH a c t i v i t y and positive for 170-HSDH both in culture and in frozen sections. As reported in the l i t e r a t u r e , cultured granulosa, l u t e a l and thecal c e l l s are histochemically positive for A5-3p-HSDH (Fischer et a l . , 1972; Gospodarowicz et a i . , 1972; Stadnicka et a!., 1976). In cryostat sections corpora lutea, theca interna and i n t e r s t i t i a l c e l l s are histochemically p o s i t i v e for »«5-3^-HSDH in many species including the rat (Adams and Auersperg, 1981a; B a i l l i e , 1966; Bjersing, 1967; Davies et a l . , 1966; Deane et a l . , 1962; Levy et a l . , 1959). Cultured granulosa and thecal c e l l s have been reported positive for 17^-HSDH (Fischer and Kahn, 1972; Stadnicka, 1976, 1977). In cryostat sections the surface epithelium of ovaries of rat and rabbit ( B a i l l i e , 1966), mouse (Hart et a l . , 1966) and human (Blaustein and Lee, 1979) was shown to be positive for 17^-HSDH with the rest of the ovary generally negative. Davies (1966) and Pearson (1959) claimed that the entire ovary was negative, for this enzyme. The ovarian surface epithelium s i t u has been examined u l t r a s t r u c t u r a l l y for several species, mouse, rat, rabbit, hamster and human (Anderson et a l . , 1976; Blaustein and Lee, 1979; Donaldson, 1976; Papadaki and Beilby, 1971; Weakley, 1969; Wischnitzer, 1965). From these reports the ovarian surface 1 53 epithelium in s i t u i s seeen to be a simple epithelium resting on a well defined basal lamina with c e l l s , ranging from, squamous to., low columnar in shape. C e l l s are joined with apical junctional complexes consisting of focal tight junctions, gap junctions and desmosomes with p l i c a t i o n s of the l a t e r a l membranes below the junctional complex. Apical surfaces are covered with numerous m i c r o v i l l i and occasionally an isolated c i l i u m . A l l surfaces show marked endocytic and exocytic behavior with numerous coated and uncoated v e s i c l e s . Internally c e l l s have large irregular nuclei, many mitochondria with lamellar c r i s t a e , perinuclear microfilaments, abundant RER, large Golgi complexes, and some l i p i d . The u l t r a s t r u c t u r a l picture of cultured c e l l s claimed to be of rat ovarian surface e p i t h e l i a l o r i g i n i s consistent with published c h a r a c t e r i s t i c s of the ovarian surface epithelium in  s i t u . No other ovarian c e l l type has been reported as exhibiting tight junctions, although endothelial c e l l s might be expected to be so joined. Granulosa c e l l s are joined by gap junctions and desmosomes with no observed tight junctions ( A l b e r t i n i and Anderson, 1974). Luteal c e l l s i_n vivo have c h a r a c t e r i s t i c s t y p i c a l of steroid-secreting c e l l s , namely mitochondria with tubular c r i s t a e , abundant smooth endoplasmic reticulum and much l i p i d (McKerns, 1969). There are no reports on the ultrastructure of cultured l u t e a l c e l l s . Cultured ROSE c e l l s are u l t r a s t r u c t u r a l l y distinguished from granulosa and l u t e a l c e l l s by the presence of tight junctions and the absence of mitochondria with tubular c r i s t a e . Hence i t has been demonstrated by morphological, 154 histochemical and u l t r a s t r u c t u r a l means that the cultured c e l l s named ROSE c e l l s are in fact ovarian s u r f a c e , e p i t h e l i a l , in, o r i g i n . 2 A COMPARISON OF GROWTH CURVES FOR DIFFERENT OVARIAN  CULTURES In the course of thi s study four types of non-virus-infected ovarian c e l l cultures were analysed for growth c h a r a c t e r i s t i c s . The culture types were mixed c e l l cultures from whole ovary, ovarian c e l l l i n e 125 (a continuous l i n e from mixed c e l l c ultures), f i r s t passage ROSE c e l l s and ROSE 199 c e l l s (a continuous l i n e of ROSE c e l l o r i g i n ) . The two growth curves for mixed c e l l cultures, although seeded at the same c e l l density, had widely d i f f e r e n t slopes in the growth portion of the curves, and reached densities at stationary growth d i f f e r i n g by a factor of two. This lack of re p r o d u c i b i l i t y was l i k e l y due to the heterogeneity of c e l l populations for these cultures. Growth curves for ovarian l i n e 125 were very similar to each other, probably r e f l e c t i n g the homogeneity of the c e l l population of thi s l i n e . Although the rate of growth for li n e 125 was approximately three times that for the f i r s t passage cultures, the c e l l density reached at stationary growth was similar to the higher density reached by the f i r s t passage mixed cultures (26-28,000' c e l l s / c m 2 ) . Growth curves determined for f i r s t passage ROSE c e l l 155 cultures demonstrated that the rate of growth and c e l l density at stationary growth depend on seeding density and age of c e l l s in primary culture at the time of subculture. The highest densities reached for these cultures were similar to those for mixed c e l l cultures and l i n e 125. A l l three culture types so far discussed grew as c e l l u l a r monolayers. There are no reports of c e l l densities attained by other ovarian c e l l types in culture. Under similar culture conditions muscle fascia f i b r o b l a s t s from adult male Fischer rats reached densities of 45-50,000 cells/cm 2', and adrenocortical cultures reached densities of 40,000 cells/cm 2 (Slavinski et a l . , 1974). D i f f e r i n g greatly from the above three culture types were c e l l s of the l i n e ROSE 199. These c e l l s grew, as multilayered structures after two to four weeks in culture, and even very crowded Cultures had mitotic figures. Such cultures produced many exfoliated viable c e l l s . C e l l s of the 25th passage, after a lag of fiv e days, grew at a rate over seven times the highest rate attained by fir s t * passage* ROS& cells-; At stationary growth-, these c e l l s reached densities more than five times those reached by the other non-virus-infected ovarian culture types. C e l l densities reached by ROSE 199 c e l l s were similar to those reached by KiMSV-transformed ovarian c e l l cultures (200,000 cells/cm 2) and by KiMSV-transformed adrenocortical c e l l s (Auersperg et a l . , 1977). This discussion serves to bring"' these' disparate observations together for the sake of comparison. It i s 156 d i f f i c u l t to draw any conclusions from them other than to say that c e l l densities reached by f i r s t passage ovarian cultures were similar to those reached by other early passage rat c e l l s in culture, and that densities reached by ROSE 199 c e l l s were commensurate with those of KiMSV-transformed c e l l s . 3. FURTHER CHARACTERIZATION OF CULTURED ROSE CELLS Certain aspects of the behavior of the ovarian surface epithelium, both j_n vivo in the l i t e r a t u r e and in. v i t r o in t h i s study, suggest that i t i s an estrogen target tissue. As early as 1942 i t was noted that estrone, d i r e c t l y applied (injected into the ovarian bursal space) to the rat ovary caused a great increase in mitoses in the ovarian surface epithelium (Stein and Alle n , 1942). The same researchers noted an increase in mitoses, 24 hours after ovulation, in the ovarian surface epithelium adjacent to the ovulation s i t e . They speculated that t h i s l o c a l stimulation was due to the liquor f o l l i c u l i bathing the ovarian surface after ovulation-. Such' a- mi*t*otdc~ increase-could also be due to a wound healing response independent of estrogen influence. Certainly the rapid and abundant outgrowth of ROSE c e l l s from ovarian explants noted in my study suggests a healing response. However, the rate and amount of outgrowth varied with the batch of f e t a l bovine serum (FBS) used. Batches of commercial FBS do d i f f e r greatly with respect to concentration of estrogens and other steroids (Esber et a l . , 1973). These sera are such complex, irreproducible mixtures of b i o l o g i c a l molecules that i t would be d i f f i c u l t to pinpoint 157 precise factors responsible for this difference in growth.of ROSE c e l l s . In this, study s i g n i f i c a n t increases in the proportion of mitotic figures and binucleate c e l l s were noted in ROSE cultures grown in medium supplemented with e s t r a d i o l . It would seem from the l i t e r a t u r e and this project that estrogens do have a mitogenic effect on ovarian surface e p i t h e l i a l c e l l s . The ovarian surface epithelium exhibits 170-HSDH enzyme a c t i v i t y in several species, including rat and human, as previously noted, and i s present in cultured ROSE c e l l s . This enzyme, which is in fact an oxidoreductase, catalyses the interconversion between e s t r a d i o l (E 2) and estrone (E,) as well as that between testosterone and androstenedione. Biochemical determinations done on cultured ROSE c e l l s confirmed the presence of 170-HSDH a c t i v i t y in these c e l l s , but did not exclude the reverse reaction. Which reaction i s preferred in cultured ROSE c e l l s could be determined by the use of double l a b e l l i n g (for example, 1 4C-E 2 and 3H-E!) on c e l l s cultured in estrogen free: medium. The enzyme 170-hydroxysteroid oxidoreductase i s frequently found in estrogen target tissues, for example the uterus of several species ( E i l e t z et a l . , 1980; Gurpide and Marks, 1981; King et a l . , 1.981; Kreitmann et a l . , 1979; Wahawisan et a l . , 1980), normal and neoplastic human mammary gland (Pollow et a l . , 1977), placenta (Bitar, 1979; S t r i c k l e r , 1980), and neural tissue (Reddy, 1979). In uterus the- highest l e v e l of170-HSDH i s found -histochemically in the epithelium of the luminal 158 endometrium and glands, and to a much less extent in the stroma and smooth muscle (Patinawin et a l . , 1980). In a l l species reported above except rat (Wahawisan et a l . , 1980) the uterine enzyme p r e f e r e n t i a l l y converted E 2 to E,, and i t s a c t i v i t y was increased by progesterone or a r t i f i c i a l progestins. In neural tissue the p r e f e r e n t i a l d i r e c t i o n was.:-. E, to E 2 . Thus 170-oxidoreductase appears to regulate estrogen metabolism. When acting as a dehydrogenase (oxidizing agent; E 2 to E,) i t decreases the effect of estrogens, but when acting as a reductase (E, to E 2) i t increases the estrogenic e f f e c t , since e s t r a d i o l i s the most potent of the natural estrogens. It i s interesting to note that neoplastic breast tissue has a generally lower 170-HSDH a c t i v i t y than does normal breast tissue. Thus neoplastic breast tissue i s perhaps more susceptible to estrogenic stimulation than is normal ti s s u e . The ovarian surface epithelium i s derived from the coelomic epithelium and arises very near the s i t e of invagination of the Mullerian Duct. The epithelia- of Mullerian Duct derivatives-(oviduct, endometrium, cervix) are a l l estrogen target tissues, with oviduct and endometrial e p i t h e l i a reported p o s i t i v e for 170-HSDH a c t i v i t y . Most of the ovarian cancers generally thought to be derived from the ovarian surface epithelium exhibit h i s t o l o g i c and secretory c h a r a c t e r i s t i c s of the Mullerian Duct-derived tissues. There is no report of any attempts to determine 170-HSDH a c t i v i t y in any ovarian tumours. This enzyme has been histochemically detected in frozen sections of human ovarian surface epithelium but not in the peritoneal 159 mesothelium (Blaustein and Lee, 1979). This result has been confirmed in this study for cultured ROSE c e l l s and peritoneal c e l l s . Histochemical examination for this enzyme, of c e l l s e xfoliated into the peritoneal cavity might well detect ovarian surface, e p i t h e l i a l c e l l s . Such a test could provide valuable c y t o l o g i c a l information toward early detection of ovarian cancer. U n t i l now cy t o l o g i c a l examination of peritoneal f l u i d s , using conventional morphological markers, has detected cases of advanced ovarian cancer, but has f a i l e d as a screening test for early cases (Jones et a l . , 1981; Bush, 197'9; Graham, 1968). By autoradiographic means I have produced evidence that cultured ROSE c e l l s exhibit estrogen receptor-like a c t i v i t y . This evidence consists of data indicating translocation of t r i t i a t e d e s t r a d i o l from cytoplasm to nucleus in l i v e c e l l s , and competition of radioinert e s t r a d i o l with t r i t i a t e d e s t r a d i o l for estrogen binding s i t e s in ROSE c e l l s . These results support the hypothesis that the ovarian surface epithelium is an estrogen target tissue. Biochemical analyses of homogenates of ROSE c e l l s would have to be done to determine other c h a r a c t e r i s t i c s of their putative estrogen receptors. Such c h a r a c t e r i s t i c s include sedimentation constant, Stokes radius, molecular weight, concentration in the cytoplasm, and binding constant. These c h a r a c t e r i s t i c s have been well documented for estrogen target tissues such as uterus and mammary gland (Rochefort et a l . , 1980; Puca, 1970). Such determinations would require about one 160 gram of tissue, that i s about, one b i l l i o n c e l l s , or 25,000 primary ROSE cultures. A l t e r n a t i v e l y a. continuous l i n e such as ROSE-199 could be used, but the amount of tissue culturing required would s t i l l be very great. Estrogen receptors as well as receptors for other steroids (progesterone, androgens, glucocorticoids) have been detected in homogenates of whole ovary and of ovarian cancers derived from the surface epithelium ( G a l l i et a l . , 1981; Hamilton et a l . , 1981; Holt et a l . , 19.8.1; Janne et a l . , 1980; Jacobs et a l . , 1980). Prior to thi s study there had been no report of estrogen receptor-like a c t i v i t y in the normal ovarian surface e p i t h e l i a l c e l l s (Adams and Auersperg, 1981b). It seems l i k e l y that the ovarian surface epithelium is an estrogen target tissue. This claim i s supported by experiments showing that e s t r a d i o l has a mitogenic' e f f e c t on cultured ROSE c e l l s , that ROSE c e l l s have 170-HSDH a c t i v i t y , and that these c e l l s have estrogen receptor-l i k e a c t i v i t y . An interesting observation arose from steroid competition autoradiographic experiments on l i v e ROSE c e l l s incubated with 3H-E 2 and radioinert progesterone. There was a very marked drop in l a b e l l i n g similar to that occuring when radioinert e s t r a d i o l was used. It has been stated that progesterone has no a f f i n i t y for the estrogen receptor (Rochefort et a l . , 1980). Another report indicates that progesterone acts on rat uterus in vivo to reduce the a f f i n i t y of estrogen target tissues for estrogens 161 (Okulicz et al..,: 1981). This antiestrogenic effect of progesterone i s not thought to involve competition.... The. effect seems to be a progesterone-induced loss of nuclear estrogen receptors which occurs rapidly (within four hours) after administration of the steroid. Perhaps progesterone produced i t s "pseudo-competitive" effect on ROSE c e l l s in a similar way. This result suggests that ROSE c e l l s may be progesterone target c e l l s . Autoradiographic tests using t r i t i a t e d progesterone could readily be done to investigate t h i s p o s s i b i l i t y . It has been suggested that ovarian cancers of surface e p i t h e l i a l o r i g i n which contain estrogen receptors might be susceptible to the sort of hormonal therapy currently used for cancers of the breast and endometrium ( G a l l i et a l . , 1981). Detection of estrogen receptors, either by autoradiography or fluorescence l a b e l l i n g (Barrows et a l . , 1980), could be used in cases of ovarian and other,cancers where there i s i n s u f f i c i e n t tissue for a biochemical determination. Such techniques could also be used to follow patients after/ s-urg.e;r.y--foT.-s*r.gns- of. recurrence. Autoradiographic detection of estrogen receptors along with histochemical testing for 17.S-HSDH a c t i v i t y has potential use for the i d e n t i f i c a t i o n of ovarian surface e p i t h e l i a l c e l l s , normal and neoplastic, in the peritoneal cavity. These markers might, in conjunction with conventional c y t o l o g i c a l markers, lead to e a r l i e r detection of ovarian cancer. 162 4. AN EVALUATION OF AUTORADIOGRAPHIC TECHNIQUES Sections of rat ovary lab e l l e d after ethanol f i x a t i o n and examined autoradiographically for estrogen receptors according to U r i e l ' s method (Uriel et a l . , 1973) were very heavily lab e l l e d in most tissues. The stroma underlying the surface epithelium was the least heavily l a b e l l e d . By autoradiography of tissues la b e l l e d _in vivo Stumpf detected estrogen receptor a c t i v i t y in granulosa, thecal and l u t e a l c e l l s of rat ovary with l i t t l e l a b e l l i n g of ova and stroma (Stumpf, 1969). He, made no mention of the status of the surface epithelium. Sections of uterus la b e l l e d after ethanol f i x a t i o n presented a picture of c e l l a f f i n i t y for estrogens very similar to those done by in  vivo l a b e l l i n g (Jensen et a l . , 1973; Tchernitchin, 1979), and by l a b e l l i n g of ethanol-fixed sections (Uriel et a l . , 1973). U r i e l ' s method of iri v i t r o l a b e l l i n g of fixed sections requires very much less radioactive material than does i_n vivo l a b e l l i n g , and far shorter exposure times than those used by Jensen and Stumpf. Sections- did not -lend- themselves as readii !y to-analysis^ as did cultured c e l l s . The autoradiographic preparation of cultured c e l l s l a b e l l e d after ethanol f i x a t i o n was based on U r i e l ' s method for sections. The method employed for l i v e - l a b e l l e d c e l l s was new (Adams and Auersperg, 1981b). Previously reported methods for l i v e -l a b e l l e d c e l l s involved either laborious dry mounting techniques with six months exposure (Weiller et a l . , 1974, based on Stumpf and Roth, 1969), or techniques using l i p i d solvents and epon 163 embedding after l a b e l l i n g (Richter and McGaughey, 1979). This l a t t e r technique, using l i p i d solvents.would leach, out steroids. The method used in the present study was technically simple, r e l a t i v e l y inexpensive and, autoradiographically speaking, quick (at one to fiv e weeks exposure). In cells, labelled after ethanol f i x a t i o n the amount of label taken up was a function of the actual number of estrogen binding molecules in the c e l l . T r i t i a t e d e s t r a d i o l entered both cytoplasmic and nuclear compartments through membranes damaged by ethanol f i x a t i o n . On the other hand, in c e l l s l a b e l l e d l i v e , any label reaching the nucleus must have f i r s t passed through the cytoplasm. The t o t a l uptake of label by l i v e c e l l s represented the amount of t r i t i a t e d steroid l e f t in the c e l l as a result of steroid entering and unbound steroid leaving the c e l l during the l a b e l l i n g period. When the two methods were compared with respect to grains per c e l l and r a t i o of nuclear to cytoplasmic grains, l i v e - l a b e l l e d c e l l s had fewer grains per c e l l but a signif-icantl-y greater- ratio--of-• nucl.ea.-r to cytoplasmic grains. This i s good evidence that there was translocation of label from cytoplasm to nucleus in l i v e - l a b e l l e d c e l l s . The pulse-chase experiment and analysis of nuclear to cytoplasmic r a t i o versus c e l l area further reinforced t h i s interpretation. In other target tissues e s t r a d i o l i s retained in the nucleus for about 24 hours (J. Anderson et a l . , 1975). If long enough chase times were used in this study label would start to leave the nucleus. 164 In autoradiographic studies for estrogen receptors using a one hour in vivo l a b e l l i n g period s i l v e r grains are mainly nuclear (Stumpf et a l . , 1969). In the present study l i v e l a b e l l i n g of cultured ROSE c e l l s resulted in predominantly cytoplasmic grains. This result probably r e f l e c t s the altered geometry of cultured c e l l s which are flattened and more spread out in comparison to c e l l s i_n vivo. It was shown that radioinert e s t r a d i o l competed with t r i t i a t e d . e s t r a d i o l for estrogen binding s i t e s in both l i v e -l a b e l l e d ROSE c e l l s and those l a b e l l e d after ethanol f i x a t i o n . Perhaps a better "competition" could have been achieved in ethanol-fixed c e l l s i f radioinert and t r i t i a t e d e s t r a d i o l were combined in the l a b e l l i n g medium. The methods developed in t h i s study for autoradiographic investigation of estrogen a f f i n i t y in cultured c e l l s have considerable potential for the analysis of factors e f f e c t i n g the transport of steroid receptors to, and their retention in, the nucleus. 5. DEVELOPMENT OF MODELS FOR OVARIAN CANCER 165 a) Transformation Of ROSE C e l l s With Kirsten Murine Sarcoma  Virus The rat ovarian surface epithelium was f a i r l y readily transformed in culture by the Kirsten murine sarcoma virus (KiMSV). Three transformed l i n e s resulted from 16 pure ROSE cultures infected with this v i r u s . On transformation ROSE c e l l s in a l l three l i n e s retained a d i f f e r e n t i a t e d c e l l marker, namely 17 0-HSDH a c t i v i t y , and showed a low le v e l of A5-3p-HSDH a c t i v i t y . H i s t o l o g i c a l l y , tumours of these three lines resembled endometrioid stromal sarcoma, a rare form of human ovarian cancer (Kao et a l . , 1978; Russell, 1979). A l l three l i n e s derived from v i r a l transformation of pure ROSE cultures were "producer" c e l l s , that i s , they produced i n f e c t i v e v i r u s . C e l l l i n e s such as these would be expected to change with time, subculturing, freezing and regrowing. Over the ten or so passages undergone by these c e l l s no s t r i k i n g change was noted in the morphology of each l i n e . Line V197-1 OB did, however, display a negative 17 J S - H S D H reaction u n t i l the eleventh passage when i t tested strongly po s i t i v e for this enzyme and less so for A5-3p-HSDH. There i s no ready explanation for this change. Even cloning of these l i n e s would not produce homogeneous populations of c e l l s . A type of in  v i t r o c e l l u l a r evolution would produce d i f f e r e n t variants. It seems that one cannot dip one's pipette into the same c e l l l i n e twice. Signs exhibited by rats injected intraperitoneally with 166 transformed ovarian and peritoneal c e l l s resembled signs seen in patients with advanced ovarian cancer, that i s , cachexia, bowel obstruction, laboured breathing (Fuller et a l . , 1979). These signs are not s p e c i f i c for c e l l s of ovarian or peritoneal o r i g i n , but are seen also in rats injected i n t r a p e r i t o n e a l l y with KiMSV-transformed adrenocortical c e l l s and other a s c i t e s -producing c e l l s . This c l i n i c a l picture was caused by widespread intraperitoneal tumour growth with a s c i t e s . In vivo the Kirsten virus p r e f e r e n t i a l l y transforms c e l l s of mesodermal o r i g i n , producing sarcomas. This virus readily transformed steroid-producing c e l l s of adrenocortical o r i g i n a f t e r short-term culture. Transformed adrenocortical c e l l s produced carcinomas, both well d i f f e r e n t i a t e d and anaplastic, and sarcomas (Auersperg et a l . , 1981). The mesodermal or i g i n of these d i f f e r e n t i a t e d c e l l s of the adrenal cortex probably accounts for their ready transformation by KiMSV, although KiMSV has . transformed c e l l l i n e s derived from rat l i v e r (endodermal) (Rhim et a l . , 1977). When cultured rat granulosa c e l l s were infected with KiMSV a low incidence of transiently transformed c e l l s resulted (Harrison and Auersperg, 1981). It is interesting to note that, in humans, cancers a r i s i n g from granulosa c e l l s are much less frequent (5% to 10%) than those a r i s i n g from the surface epithelium (85% to 90%). Perhaps t h i s large difference in rates of malignant transformation between these two tissues i s r e f l e c t e d in the difference of s u s c e p t i b i l i t y of these two rat tissues to v i r a l transformation in v i t r o . 167 The ovarian surface epithelium, is, of, mesenchymal, origin.. On malignant transformation in humans i t i s claimed, on h i s t o l o g i c a l evidence, that t h i s tissue produces the common e p i t h e l i a l ovarian cancers, most of which exhibit h i s t o l o g i c and secretory c h a r a c t e r i s t i c s of tissues of the Fallopian tube, endometrium and cervix (Scully, 1977a; Woodruff and Ju l i a n , 1970). This theory for the histogenesis of these tumours i s supported by the fact that the ovarian surface epithelium and the Mullerian Duct epithelium are both derived from the embryonic coelomic epithelium, and their s i t e s of or i g i n l i e close together in the embryo. Hence i t i s not d i f f i c u l t to conceive that, on transformation, t h i s mesenchymally derived, multipotent epithelium could express stromal c h a r a c t e r i s t i c s , in par t i c u l a r those of the stroma of Mullerian Duct-derived tissues. The endometrioid stromal sarcoma i s included in the "Common E p i t h e l i a l Tumours" category of the World Health Organization c l a s s i f i c a t i o n of ovarian tumours (Scully, 1977a) although i t i s generally considered to be derived from the ovarian stroma. In l i g h t of the results achieved here, perhaps the o r i g i n of t h i s type of ovarian cancer should be reconsidered. There have been no reports describing the ultrastructure of endometrioid stromal sarcomas of the ovary. The tumours produced in thi s study were d i f f i c u l t to c l a s s i f y u l t r a s t r u c t u r a l l y because of the heterogeneity of their c e l l u l a r composition. Cords of oval to elongate c e l l s with extensive 168 RER, surrounded by f i b r i l l a r e x t r a c e l l u l a r material, were in keeping with a c l a s s i f i c a t i o n of sarcoma. Such c e l l s predominated in l i n e V197-1 OB. A l l three l i n e s , especially V197-13A and Vl97-I5a, also contained c e l l s where the predominant SER and Golgi complexes resembled the ultrastructure of certain functional carcinomas of steroid secreting . tissues (Valente et a l . , 1978). Perhaps t h i s ultrastructure is associated with the histochemically demonstrated a b i l i t y of these c e l l s to metabolize certain steroids. Line V197-13A has filament-associated desmosbme-like junctions suggestive of an e p i t h e l i a l character. On the u l t r a s t r u c t u r a l level these tumours have both sarcomatous and carcinomatous regions. The results of th i s study do not indicate that the common di f f e r e n t i a t e d e p i t h e l i a l ovarian cancers can be induced by C-type retroviruses alone. However, Rapp and Todaro (1980) have derived a carcinoma inducing variant of a C-type mouse retrovirus which, when injected intraperitoneally into neonate Swiss mice, produced a low but s i g n i f i c a n t incidence- ((18' in- 5-3-0')' of ovarian carcinomas resembling those found in humans. Such in  vivo work cannot prove the c e l l of o r i g i n of such tumours. It would be very interesting to infect cultured mouse ovarian surface e p i t h e l i a l c e l l s with t h i s variant virus and test r e s u l t i n g transformed c e l l s for tumourigenicity. There is a report that human ovarian carcinomas and adenocarcinomas of surface e p i t h e l i a l o r i g i n contain p a r t i c u l a t e DNA polymerase' with properties s p e c i f i c for the reverse transcriptase of C-type retroviruses (Gerard et a l . , 1978). These, along with other 169 evidence suggestive of a v i r a l etiology (Lingeman, 1974; McGowan et a l . , 1979; Menczer et a l . , 1979).,. urge, that the involvement of viruses in the induction of ovarian cancer, perhaps in conjunction with other carcinogens, should be investigated further. b.) Continuous Line ROSE-199, A Tumour In V i t r o Continuous l i n e ROSE-199 i s perhaps the single most interesting result of this whole project. This l i n e produced, in a culture flask, complex p a p i l l a r y structures, resembling ovarian serous p a p i l l a r y cystadenomas of borderline malignancy (Czernobilsky, 1977; Russell, 1979) (Figure 22). Recall that primary ovarian cancers of borderline malignancy can metastatize throughout the peritoneal cavity but do not invade underlying stroma. The ROSE-199 p a p i l l a r y structures were in one layer of a complex arrangement consisting of two c e l l u l a r layers separated by a thick layer of collagen (connective tissue type by u l t r a s t r u c t u r a l c r i t e r i a ) . There was no sign of basement membrane. The appearance of ROSE-199 c e l l s was at a l l times e p i t h e l i a l with subconflent cultures being indistinguishable morphologically from f i r s t passage ROSE cultures. As cultures of ROSE-199 c e l l s became crowded they began to form ridges and to s p l i t off viable c e l l s from the growth surface. These flo a t i n g c e l l s grew readily when transferred to a fresh culture vessel. This behavior" resembles' the predominant- mode of' spread' of ovarian cancers, that i s , by seeding into the peritoneal 170 f l u i d malignant c e l l s which develop into metastatic nodules on peritoneal surfaces. As controls for the v i r a l l y transformed ROSE l i n e s , aliquots of ROSE-199 c e l l s were injected into immunosuppressed rats either subcutaneously or in t r a p e r i t o n e a l l y . A tumour arose at t h e i n j e c t i o n s i t e i n one s.c. injected rat. This tumour, according to the opinions of three pathologists,.resembled either a low grade fibrosarcoma or a benign tumour of the pleura. These benign tumours of the pleura are thought by some to be mesothelial in . o r i g i n , and by others to be of submesothelial o r i g i n (Dalton et a l . , 1979). According to Goodman (1979) aging female Fischer 344 rats (over 1700 studied) have a 0.3% chance of spontaneously developing a fibrosarcoma subcutis, and a 0.2% chance of developing a tumour of mesothelial o r i g i n in any body cavity. The chances of developing a mesothelial tumour subcutis would be n i l i f the peritoneal cavity was negative for such growth. There i s the interesting p o s s i b i l i t y that t h i s tumour might have been of: ROSE-199 o r i g i n . A response of one tumour in twelve rats i s not a convincing sample, but i t is suggestive that ROSE-199 c e l l s might be marginally tumourigenic. The tumour i_n vivo i s quite di f f e r e n t from the structures developed i_n v i t r o , although both contain much collagen. A much larger sample of animals and possibly d i f f e r e n t inocculation methods would have to be used to provide a d e f i n i t i v e answer. Thus, ROSE-199-cells, derived from the ovarian surface 171 epithelium, a tissue of e p i t h e l i a l morphology but mesenchymal o r i g i n , maintain their e p i t h e l i a l appearance, but. express, the stromal c h a r a c t e r i s t i c s of abundant collagen production. The tumour-like behavior, both h i s t o l o g i c and metastatic, that these c e l l s exhibit in v i t r o reinforces the hypothesis (Woodruff and Jul i a n , 1970; Scully, 1977a) that the common e p i t h e l i a l tumours of the ovary are derived from the ovarian surface epithelium. It i s generally held that the stromal component of ovarian cancers i s contributed, by the ovarian stroma. Could tumour stroma of the common e p i t h e l i a l ovarian tumours also be derived from the multipotent surface epithelium? This interesting p o s s i b i l i t y i s supported by the results of thi s study, namely the production of a stromal sarcoma by v i r a l l y transformed ovarian surface e p i t h e l i a l c e l l s , and by collagen production by a.line of spontaneously transformed surface e p i t h e l i a l c e l l s . 6. CONCLUSION After much has been said, and some of i t done, what response can be given to the questions raised in the Introduction? Are the common e p i t h e l i a l ovarian cancers in humans derived from the ovarian surface epithelium? This study, although using c e l l s of rat o r i g i n , does indicate that i t i s the ovarian surface epithelium which undergoes neoplastic transformation to 172 produce the common e p i t h e l i a l tumours of the ovary. C e l l s of li n e ROSE-199, of ovarian surface e p i t h e l i a l , o r i g i n , produced, structures i_n v i t r o resembling the ovarian, serous p a p i l l a r y cystadenoma of borderline malignancy. V i r a l l y transformed rat ovarian surface e p i t h e l i a l c e l l s produced, in rats, tumours resembling the endometrioid stromal sarcoma, a rare human ovarian cancer. Both these malignancies are l i s t e d as "common e p i t h e l i a l tumours" in the World Health Organization's c l a s s i f i c a t i o n of ovarian tumours. Some would claim that the endometrioid stromal sarcoma was never said to be derived from ovarian surface epithelium, but was c l a s s i f i e d with these tumours for convenience sake. Thus the answer to the question asked i s po s i t i v e , even i f q u a l i f i e d . What i s i t about the ovarian surface epithelium that makes i t so susceptible to malignant transformation? This i s a very large question to which are given a few small answers. This study has shown that the rat ovarian surface epithelium i s susceptible to malignant' transformation-, by a"-C-type 5-retrovirus. C e l l s of ovarian surface e p i t h e l i a l o r i g i n , while retaining e p i t h e l i a l morphology, can exhibit stromal c h a r a c t e r i s t i c s such as abundant collagen production. Perhaps the potential of this tissue to express both e p i t h e l i a l and stromal c h a r a c t e r i s t i c s i s a contributing factor to i t s ready neoplastic transformation. That the ovarian surface epithelium i s in fact an estrogen target tissue seems f a i r l y c e r t a i n from the evidence presented here. That these c e l l s have estrogen receptor-like a c t i v i t y , f 1 73 that they exhibit 17^-HSDH a c t i v i t y , and that they respond to es t r a d i o l by an increase in mitosis has been, shown. It appears that the surface epithelium of the ovary should, l i k e l y be considered along with other estrogen target tissues (mammary, uterine) which readily undergo transformation and account for such large numbers of human malignancies. The a v a i l a b i l i t y of cultured ovarian surface e p i t h e l i a l c e l l s allows the investigation of the growth, behavior, structure and function of these interesting c e l l s . From such cultures have come two markers, namely estrogen receptors and 17»s-HSDH a c t i v i t y , with potential for the cy t o l o g i c a l i d e n t i f i c a t i o n of ovarian surface e p i t h e l i a l c e l l s exfoliated into the peritoneal cavity. These markers, along with others yet to be discovered, might lead to screening tests for ovarian cancer at an early stage. The culture of pure rat ovarian surface epithelium, as established and investigated in this work, could be readily used to study chemical, physical and v i r a l carcinogenesis in this tissue to develop experimental models of ovarian cancers a r i s i n g in the ovarian surface epithelium. 1 74 CHAPTER V. SUMMARY In the course of this work I have cultured rat ovarian surface e p i t h e l i a l (ROSE) c e l l s in pure form, and have confirmed their i d e n t i t y by histochemical, morphological and u l t r a s t r u c t u r a l c r i t e r i a . These c e l l s , both in cryostat sections and in culture, were found to be histochemically posi t i v e for the enzyme 17p-hydroxysteroid dehydrogenase (.17 0-HSDH) , an enzyme ch a r a c t e r i s t i c of estrogen target tissues. This enzyme a c t i v i t y was confirmed biochemically. Cultured ROSE c e l l s were found to be histochemically negative for the enzyme A5-3p-hydroxysteroid dehydrogenase, an enzyme c h a r a c t e r i s t i c of steroid synthesizing tissues. These c e l l s stained intensely for the enzyme lactate dehydrogenase and had a moderate amount of cytoplasmic l i p i d as shown by o i l red 0. U l t r a s t r u c t u r a l l y , cultured ROSE c e l l s were seen to be e p i t h e l i a l with apical junctional complexes, basal lamina and numerous m i c r o v i l l i . They had large nuclei, copious ve s i c l e s , abundant rough endoplasmic reticulum, Golgi complexes, oval to elongate mitochondria with lamellar c r i s t a e and bundles of perinuclear filaments. By autoradiographic techniques the presence of estrogen receptor-like a c t i v i t y was demonstrated in cultured ROSE c e l l s by evidence of translocation of labe l l e d e s t r a d i o l from cytoplasm to nucleus, and of estrogen s p e c i f i c binding. 175 Est r a d i o l was shown to be mitogenic for cultured f i r s t passage ROSE c e l l s . Cultured ROSE c e l l s were shown to be transformable by a C-type retrovirus, the Kirsten murine sarcoma virus, with retention of a d i f f e r e n t i a t e d c e l l marker, namely 17.0-HSDH a c t i v i t y . V i r a l l y transformed ROSE c e l l s produced tumours in immunosuppressed rats. These tumours resembled the endometrioid stromal sarcoma, a rare form of ovarian cancer in humans. 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Leipzig as referred to in Anderson et a l . 1976. Weakley, B. S. D i f f e r e n t i a t i o n of the surface epithelium of the hamster ovary: an electron microscopic study. J. Anat. 105:129-147, 1969. Weiller, S., C. le Goascogne, E-E. Baulieu. Radio-autographie des hormones steroides appliquee aux c e l l u l e s en culture. C. R. Acad. A c i . Paris, Serie D. 278*: 769-771 , 1974-.. Wilson, J. E. Adenocarcinomata in hens kept in constant environment. Poult. S c i . 37:1253, 1958. Wischnitzer, S. The ultrastructure of the germinal epithelium of the mouse ovary. J. Morphol. 117:387-400, 1965. Woodruff, J. D., C. G. J u l i a n . Explorations into the genesis of ovarian malignancy. Int. J. Gynaecol. Obstet. 8:587-592, 1970. Witschi, E. Migration of the germ c e l l s of human embryos from the yolk sac to the primitive gonadal folds. Carnegie Inst. Wash. Pub. Contrib. Embryol. 32:67-80, 1948. Wyke, J. Strategies of v i r a l oncogenesis. Nature 290:629-630, 1981 . 187 Zuckerman, S. The number of oocytes in the mature ovary. Recent Progress in Hormone Research 6:63-109, 1951 . 188 APPENDIX I. SOURCES OF MATERIALS Except as noted below, materials were obtained from the following sources: BDH BDH Chemicals, Vancouver, Canada BECTON Becton Dickinson, Los Angeles, Ca. CORNING Corning Glassworks, Corning N. Y. EMS Electron Microscopy Sciences, Fort Washington, Pa. FISHER Fisher S c i e n t i f i c Company, Fairlawn, N. J . GIBCO Grand Island B i o l o g i c a l Company, Grand Island, N. Y. JBEM J. B. EM Services, Dorval Quebec KODAK Eastman Kodak, Rochester, N. Y. LUX Lux S c i e n t i f i c , Newbury Park, Ca. PIERCE Pierce Chemical Co., Rockford, 111. SIGMA Sigma Chemical Co., St. Louis, Mo. STERALOIDS Steraloids, Wilton, N. H. Sources of materials are l i s t e d under the following headings: c e l l culture, histology, histochemistry, electron microscopy, st e r o i d biochemistry, v i r a l transformation, and autoradiography. 1. CELL CULTURE ANIMALS - Fischer 344 rats bred at the Zoology Department vivarium, U. B. C. ANAESTHETICS Diethyl ether, anaesthetic grade, Mallinckrodt Inc. S c i e n t i f i c Products D i v i s i o n , St. Louis, Mo. Chloroform, BDH CULTURE WARE Dishes, (35 mm, disposable- p l a s t i c ) , CORNING', LUX,' GTBCO' (Nunc) Flasks, (disposable p l a s t i c ) , 189 25 cm2 growth surface, CORNING 75 mm2 growth surface, Nunc, GIBCO Coverslips, 25 mm, c i r c u l a r p l a s t i c for tissue culture, Thermanox, LUX Wells, disposable p l a s t i c , trays of 24, 16 mm wells, Linbro S c i e n t i f i c Co., New Haven Conn. Grids, s t a i n l e s s s t e e l , triangular, 15 mm, for organ culture, Falcon P l a s t i c s , BECTON ENZYMES AND MEDIA FOR CELL DISSOCIATION Collagenase, l y o p h i l i z e d powder, type I, from Clostridium  histolyticum, SIGMA Ethylene diamine tetraacetic. acid (EDTA,). N u t r i t i o n a l Biochemicals Corp., Cleveland, Ohio Hanks Balanced Salt Solution (BSS), Ca + + , Mg + + free, 10X concentration, GIBCO Hyaluronidase, l y o p h i l i z e d powder, type II, from sheep testes, SIGMA Trypsin, in s t e r i l e v i a l s of 20 ml 2.5% trypsin in saline, GIBCO FILTERS FOR FILTER STERILIZATION M i l l i p o r e Corp., Bedford, Mass. For large volumes, pore size 0.22 t> in assemblies for small volumes, Swl-nnex-1;3: f i l t e r / units.,, pore size 0.22 » or 0.45 ? GROWTH MEDIUM COMPONENTS Waymouth medium, MB 752/1, (with L-glutamine, without sodium bicarbonate), GIBCO A n t i b i o t i c s , penicillin/streptomycin, in v i a l s with 4 ml aqueous solution, 25,000 I.U. P e n i c i l l i n G per ml and 25,000 (sq streptomycin per ml, Microbiological Associates, Walkersvilie, Md. Serum, Fetal Bovine Serum (FBS), mycoplasma tested and virus screened, GIBCO Sodium bicarbonate, c e r t i f i e d ACS, powder, FISHER 2 HISTOLOGY 190 Ethanol, FISHER Formaldehyde, 37% solution, FISHER Phosphates for buffer, NaHjPO^.HjO and Na 2HPO„.7H 20, FISHER Eosin, c e r t i f i e d b i o l o g i c a l stain, FISHER Toluidine Blue 0, c e r t i f i e d b i o l o g i c a l stain, FISHER 3^ HISTOCHEMISTRY Aqueous mounting medium Farrant's, BDH, Poole, England Aquamount, kindly provided by the Zoology histology laboratory (U. B. C) Haemotoxylin, FISHER Nicotinamide, KODAK 0-Nicotinamide adenine dinucleotide (0-NAD), grade III, SIGMA Nitro Blue Tetrazolium, grade III, SIGMA N,N-dimethyl formamide, FISHER O i l Red 0, SIGMA Safranin 0, c e r t i f i e d b i o l o g i c a l stain, FISHER Steroids, dehydroepiandrosterone, e s t r a d i o l , testosterone, STERALOIDS 4_^  ELECTRON MICROSCOPY FIXATIVES Glutaraldehyde, 10 ml ampules, 25% aqueous glutaraldehyde, JBEM Osmium tetroxide, 5 ml ampules, 4% aqueous 0s0 4, JBEM EMBEDDING MATERIALS Epon 812 resin, #JBS-030, JBEM Dodecenyl succinic anhydride (DDSA), EMS' Nadic methyl anhydride (NMA), EMS 191 2,4,6-tri(dimethylaminomethyl)phenol (DMP), FISHER SOLUTES FOR BUFFER SOLUTIONS Glucose, FISHER Sodium hydroxide, FISHER NaH 2PO„.H 20, FISHER STAINS Lead c i t r a t e , Polysciences Inc., Warrington, Pa. Uranyl acetate, Polysciences Inc. HARDWARE Grids, copper, 200 mesh Pelco, Ted' Pella inc., Tustin, Ca. Knives Glass (25x6.35x400 mm pieces), LKB Produkter-AB, Sweden Diamond, Dupont Canada'ltd., Vancouver, Canada 5 STEROID BIOCHEMISTRY * CHROMATOGRAPHY SUPPLIES A) GAS LIQUID CHROMATOGRAPHY (GLC) Column packing Chromosorb, W(HP), column packing, 100/120 mesh, PIERCE OV225 l i q u i d phase for estrogen determinations, PIERCE 3% Densil 200, on chromosorb W(HP) in 2 f t column, for progesterone determination, Western Chromatography Supplies, New Westminster, B.C. Chemicals BSTFA (N,0-Bis(trimethyl s i l y 1 ) - t r i f l u o r o acetamide) in 1 gm ampules, PIERCE Pyridine, s i l y l a t i o n grade in 50 ml hypovials, PIERCE Reacti v i a l s , (0.3 ml and 1.0 ml), thick-walled glass with conical i n t e r i o r , t e f l o n - l i n e d capsv, to carry out s i l y l a t i o n reaction, PIERCE B) PAPER AND THIN LAYER CHROMATOGRAPHY 192 Chromatography paper, 46x57 cm Whatman #1, VWR S c i e n t i f i c Inc., Seattle, Wash. TLC plates s i l i c a gel, without fluorescent 'indicator, KODAK Phase separation paper, Whatman PS, VWR S c i e n t i f i c Inc. S c i n t i l l a t o r , Omnifluor, New England Nuclear, Boston,. Mass. SOLVENTS, ( a l l glass d i s t i l l e d by supplier 1 or in thi s laboratory 2) Acetone V, BDH Benzene 1, FISHER Chloroform 1, BDH Ethanol 2, FISHER Ethyl acetate 2, Baker Chemical Co., Phil l i p s b u r g , N.J. Hexane 1, BDH L i g r o i n e 1 , KODAK Methylene d i c h l o r i d e 1 , BDH Toluene, 'spectranalysed', FISHER STEROIDS a) radioactive, New England Nuclear, Boston, Mass. 1 " C - e s t r a d i o l , 57 mCi/m mol purchased in 0.05 mCi lo t s (o.24 mg in 2.5 ml benzene/ethanol 9:1), prepared from 19-Nortestosterone acetate 1"C-pregnenolone, 55.7 mCi/m mol, purchased in 0.05 mCi lots (0.28 mg in 2.5 ml benzene/ethanol 9:1), prepared from 1 uC-progesterone b) radioinert A l l radioinert steroids used in this study, STERALOIDS X-RAY AUTORADIOGRAPHY SUPPLIES X-ray film, Kodak, high speed, no screen 14"X17", NS5T, KODAK Developer, Kodak l i q u i d X-ray developer, KODAK Fixer, Kodak rapid f i x e r , KODAK Safe l i g h t , Kodak Wratten Series 6B, KODAK 6^ VIRAL TRANSFORMATION KIRSTEN MURINE SARCOMA VIRUS (KiMSV) Harvested from supernatant media from KiMSV transformed rat / 1 93 kidney c e l l l i n e NRK 58967 o r i g i n a l l y obtained from V. Klement. (Roy-Burman and Klement, 1975). NORMAL .RAT KIDNEY (NRK) CELLS A continuous l i n e of c e l l s derived from rat kidney, obtained from V. Klement. (Due-Nguyen et a l , 1966) 7^ AUTORADIOGRAPHY Emulsion, Kodak NTB-3, Nuclear Track Emulsion, KODAK Developer, Kodak D19, KODAK Fixer, Kodak, KODAK Grid, for microscope eyepiece, Graticules Ltd., Tonbridge, Kent, England. Microscope s l i d e s , Clay Adams Div., BECTON Safe l i g h t , Wratten O.C series, KODAK Slide mailer tubes, 1.5x3x8 cm, p l a s t i c , Evergreen P l a s t i c s , Hong Kong Tissue culture coverslips, ( p l a s t i c , 25 mm), Thermanox, LUX T r i t i a t e d e s t r a d i o l , 2,4,6,7- 3H-estradiol, 101.7 Ci/m mol, .. New England Nuclear, Boston, Mass. 1 94 APPENDIX II. PREPARATION OF SOLUTIONS This appendix . contains preparation procedures and directions for the following: a) media and enzyme solutions for c e l l culture, bj cryopreservation of cultured c e l l s under l i q u i d nitrogen, c) solutions for histology, d) solutions for histochemistry, e) solutions for electron microscopy. Sources of materials are given in Appendix I. a) MEDIA AND ENZYME SOLUTIONS FOR CELL CULTURE 1. WAYMOUTH MEDIUM WITH PENICILLIN AND STREPTOMYCIN One pack (13.8 gm) of Waymouth medium powder (with L-glutamine and without sodium bicarbonate) i s dissolved in one l i t r e of d i s t i l l e d water. 2.24 gm of sodium bicarbonate is added and dissolved by swirling. 4.00 ml of penicillin/streptomycin solution (25,000 I.U. P e n i c i l l i n G per ml, 25,000 nq streptomycin per ml) are added, producing medium which has 100 I.U./ml p e n i c i l l i n G and 100 Mg/ml streptomycin. In a hood the medium i s s t e r i l i z e d by passage through a mill i p o r e f i l t e r . After f i l t r a t i o n , the medium i s drained into two s t e r i l e 500 ml bottles and capped with s t e r i l e caps. 2. ADDITION OF FETAL BOVINE SERUM TO WAYMOUTH MEDIUM The serum is stored frozen and after thawing must be swirled to mix. Serum i s added to Waymouth medium, measured in percentage by volume. If the pH of the resulting medium i s out of the desired 7.2 to 7.4 range, 1.0 N NaOH i s added dropwise to reach the proper pH. 3. TRYPSIN SOLUTION FOR CELL DISSOCIATION The 20 ml of 2.5% trypsin in s t e r i l e saline i s thawed and removed from the v i a l by s t e r i l e syringe and di l u t e d to 400 ml with Hanks BSS (1X concentration, C a + + , Mg + +-free) to obtain a f i n a l concentration of 0.125% trypsin. The pH should be 1 95 adjusted to the 7.2 to 7.4 range with a f i l t e r s t e r i l i z e d solution of sodium bicarbonate. Trypsin solution i s stored frozen in 100 ml bottles at -20°C. 4. COLLAGENASE SOLUTION FOR CELL DISSOCIATION FROM WHOLE OVARIES : Collagenase powder is dissolved in 5% FBS/WM to produce a solution 0.1 % in collagenase. The solution i s f i l t e r s t e r i l i z e d before use. 5. HYALURONIDASE SOLUTION FOR CELL DISSOCIATION FROM WHOLE OVARIES Hyaluronidase powder i s dissolved in sodium bicarbonate buffered Hanks BSS to make a solution 0.1% in hyaluronidase. The solution i s f i l t e r s t e r i l i z e d before use. 6. 0.125% TRYPSIN AND 0.02% EDTA SOLUTIONS FOR CELL DISSOCIATION FROM WHOLE OVARIES Powdered EDTA (ethylene diamine tetraacetic acid) is dissolved in 0.125% trypsin solution (Hanks BSS, C a + + , Mg + +-free) to produce a solution 0.02% in EDTA. This solution i s s t e r i l i z e d by f i l t r a t i o n . b) CRYOPRESERVATION OF CULTURED CELLS UNDER LIQUID NITROGEN Ce l l s freeze best when they are growing rapidly. After t r y p s i n i z i n g , c e l l s are centrifuged and resuspended in 8-10% dimethylsulfoxide and 90-92% growth medium (usually 10% FBS/WM) at 5x10s to 106 c e l l s per ml. Approximately 1.0 ml of c e l l suspension i s added to each freezing ampule. Ampules are frozen at 1°C per minute to -20°C (or as indicated in freezing i n s t r u c t i o n s ) , then- stored- at liquid- nitrogen: temperatures'." The tank used for freezing should be at least two thirds f u l l of l i q u i d nitrogen and should not have been opened e a r l i e r on the same day. To thaw frozen c e l l s for culture, the frozen v i a l i s put in warm water (40°C) to thaw rapidly. The v i a l i s wiped with alcohol before opening, and the c e l l s transferred to a centrifuge tube. One ml of medium is added and the c e l l s allowed to stand for five minutes, then two ml of medium is added and the c e l l s l e f t for fi v e minutes in this d i l u t e d freezing medium. The suspension i s centrifuged for 4-5 minutes at 900-1,000 rpm, the supernatant i s removed, the c e l l s are then taken up in growth medium and transferred to a culture flask. c) SOLUTIONS FOR HISTOLOGY 1 . EOSIN SOLUTION 196 Powdered eosin is dissolved in d i s t i l l e d water to produce a solution 0.5% or 1% in eosin. 2. HAEMATOXYLIN This solution was received already prepared from h i s t o l o g i s t s in the Departments of Zoology and Anatomy. Preparation procedures can be found in most standard texts on h i s t o l o g i c a l procedures (Culling, 1974). 3. TOLUIDINE BLUE . Powdered Toluidine Blue 0 i s dissolved in aqueous solution to produce a 2% solution. This stain preparation i s f i l t e r e d before each use. 4. PHOSPHATE BUFFER (0.1 M) Two solutions, A and B, are prepared. Solution A is 0.1 M in disodium hydrogen phosphate (Na2HPO.,). Solution B i s 0.1 M Prepare ition of 0.1 M PI losphate Buffer pH Solution A ml Solution B ml 7.1 7.2 7.3 7.4 7.5 7.6 7.7 66.6 72.0 76.8 80.8 84. 1 87.0 89.4 33.4 28.0 23.2 19.2 15.9 13.0 10.6 in sodium dihydrogen phosphate (NaH 2PO„). These two sa l t s sometimes come as hydrates. In order to produce a 0.1 M phosphate buffer of a given pH, solutions A and B are combined as indicated in the accompanying table (Gurr, 1960). 5. PHOSPHATE BUFFERED 10% FORMALIN Concentrated formaldehyde solution ("formalin") which i s approximately 37% formaldehyde by weight is di l u t e d tenfold with 0.1 M phosphate buffer (pH 7.2-7.4) to produce a 10% formalin solution (3.7% formaldehyde). 6. PHOSPHATE BUFFERED SALINE (PBS) To each 100 ml of 0.1 M phosphate buffer i s added 0.9 gm sodium chloride. -197 7. SCOTT'S TAP WATER SUBSTITUTE This c e l l s . It solution was is an aqueous used to "blue" haemotoxylin stained solution of 0.2% NaHC03 and 2% MgSO«. d) SOLUTIONS FOR HISTOCHEMISTRY 1. DEHYDROGENASE TESTING SOLUTION ACCORDING TO LEVY'S METHOD. This recipe i s s l i g h t l y modified from that published by Levy (Levy et a l , 1959). In Levy's report, propylene g l y c o l . Hydroxysteroid Dehydrogenase Solution for Cryostat Sections Component Concentrat ion Amount F i n a l 2 Concentration S t e r o i d 3 NNDMF 4Nitrd Blue Tetrazolium 5 Nicotinamide 5 NAD5 Phosphate Buffer 1 mg/ml 1.6 mg/ml 3 mg/ml 0.1 M pH 7.1-7. 1 5 0 5 0 mg ml ml ml ml 20.0 ml 0.03 mg/ml 7% by vol 0.14 mg/ml 0.16 mg/ml 0.34 mg/ml 0.06 M 1. A l l component solutions should be at 37°C before combining with the steroid (dissolved in NNDMF) to minimize p r e c i p i t a t i o n of the steroid . 2. F i n a l concentration in testing solution. 3. Steroid i s dissolved f i r s t in the NNDMF. For A5-3p-HSDH, dehydroepiandroste-rone is used as substrate. For-I7p-HSDH, es t r a d i o l or testosterone are used as substrates. For hydroxysteroid dehydrogenase testing of cultures, the concentration of steroid i s doubled. For LDH testing, steroid and NNDMF are omitted and replaced by 50 mM sodium lactate (0.25 ml of 60% sodium lactate syrup for 32.5 ml of LDH testing solution). 4. This i s the only a l t e r a t i o n in Levy's method. NNDMF i s used to dissolve the steroid in the aqueous testing solut ion. 5. These components are dissolved i n d i v i d u a l l y in d i s t i l l e d water in the concentrations shown. These solutions may be stored at -20°C. was used as the steroid solvent instead of N,N-dimethyl formamide (NNDMF). The individual components of t h i s testing solution are prepared and combined according to the accompanying 198 table to make up 35 ml of testing solution. This testing solution i s usually made up fresh on the day needed and i s not kept over either in l i q u i d or frozen form. 2. DEHYDROGENASE TESTING SOLUTION ACCORDING TO FISCHER'S METHOD In Fischer's method (Fischer et a l , 1972) the components of a testing solution for hydroxysteroid dehydrogenases, and their f i n a l concentrations, were given as follows: n i t r o blue tetrazolium 0.5 mg/ml (in 0.1 M phosphate buffer, pH 7.5), NAD (or NADP) 2 mg/ml, steroid substrate 0.25 mg/ml. No mention i s made of a steroid solvent so NNDMF was used at 1.5 ml per 20 ml of dehydrogenase solution. Also, nicotinamide was not mentioned in Fischer's paper. 3. OIL RED 0 Stock solution is composed of O i l Red Q (0.25-0.5%) in isopropyl alcohol. This solution i s stable at room temperature for months. See Appendix III for use. 4. SAFRANIN 0 This' pink dye was used as a counterstain for formazan deposits in dehydrogenase tested cryostat sections. It i s usually prepared as a 1% aqueous solution. e) SOLUTIONS FOR ELECTRON MICORSCOPY 1. MILLONIG'S PHOSPHATE BUFFER The various Millonig's buffers used in preparation of tissues for electron microscopy are made up by combining three solutions, A, B and C in various proportions: Solution A - 4.52% NaH2POa.H20 Solution B - 5.04% NaOH Solution C - 5.4% glucose. For Millonig's buffer for washing and rinsing the following combination i s used: Solution A - 83 ml Solution B - 17 ml d i s t i l l e d water - 100 ml. (At t h i s point the pH should be 7.3-7.4.) Solution C - 20 ml. For Millonig's buffer for glutaraldehyde ( i . e . without glucose) the following combination i s used: 199 Solution A - 83 ml Solution B - 17 ml d i s t i l l e d water - 100 ml To prepare 2.5%.glutaraldehyde in Millonig's buffer, 10 ml of 25% aqueous glutaraldehyde (in a sealed ampule) i s diluted to 100 ml with Millonig's buffer (without glucose). For Millonig's buffer for osmium tetroxide (OsOa) Solutions A, B and C are combined as follows: Solution A - 83 ml Solution B - 17 ml Solution C - 10 ml. Stock solutions of Os0 4 are usually 2% in d i s t i l l e d water. This i s prepared by d i l u t i o n of 5 ml of 4% OsOa aqueous (in ampules) to 10 ml with d i s t i l l e d water. The stock solution is stable for short period's i f kept at 4°C in a l i g h t - t i g h t bottle. The stock solution is d i l u t e d just before use by addition of an equal volume of Millonig's buffer for osmium tetroxide, to form a 1% solution. This 1% buffered solution is not as stable as the 2% stock solution. 2. PREPARATION OF EPON EMBEDDING MATERIAL Two solutions, A and B, are prepared as follows with thorough mixing: Solution A: epon 812 resin - 62 ml dodecenyl succinic anhydride - 100 ml Solution B: epon 812 resin - 100 ml nadic methyl anhydride- - 89=- mk.<-These solutions may be stored, t i g h t l y covered, under re f r i g e r a t i o n for long periods. For use, solutions A and B are combined at room temperature in the proportion A:B equals 1:1.3. The greater the proportion of A, the softer the block; the greater the proportion of B, the harder the block. Just before use the accelerator, 2,4,6tri(dimethylaminomethyl) phenol (DMP-30), i s added in the proportion 1 to 2% by volume. This i s thoroughly mixed to insure uniform hardening of the block. 3. LEAD CITRATE STAIN To 10 ml of d i s t i l l e d water 0.1 ml of 10 N NaOH (carbonate free) i s added. Lead c i t r a t e (0.01 to 0.04 gm) i s added. This is shaken vigorously to' dis'solve. Any'persistent- sediment- i-s-removed by centrifugation. The lead c i t r a t e solution i s stored at 4°C. 200 4. URANYL ACETATE STAIN To 25 ml of 70% aqueous ethanol, 1.75 gm of uranyl acetate is added with prolonged shaking. This solution is saturated with respect to uranyl acetate. The stain i s stored at 4°C in a brown bottle. 201 APPENDIX III . PROCEDURES FOR HISTOLOGY, HISTOCHEMISTRY AND ELECTRON MICROSCOPY In th i s appendix procedures are l i s t e d under the headings: histology, histochemistry, electron microscopy. The preparation of solutions used can be found in Appendix II; sources of materials are given in Appendix I. a) HISTOLOGY 1. ETHANOL.FIXATION OF CULTURED CELLS Cultures were rinsed in Hanks BSS or Waymouth medium to remove traces of serum. Cultures were then l e f t in 95% ethanol for 5 minutes or more. C e l l s were usually stained in toluidine blue according to the next paragraph. 2. TOLUIDINE BLUE STAINING' OF ETHANOL FIXED' CELLS Cultures in 95% ethanol were brought to water via a graduated ethanol/water series (a few minutes in each of 90% ethanol, 70% ethanol, 40% ethanol, water). C e l l s were then stained for approximately 1 minute with 2% aqueous toluidine blue solution which had been f i l t e r e d immediately before use. After thorough rinsing with water cultures were a i r dried. If c e l l s were overstained cultures were rinsed with 90% ethanol, and i f understained the staining was repeated. For viewing, cultures in p l a s t i c dishes were mounted with immersion o i l and the c o v e r s l i p rimmed with clear n a i l p o l i s h . 3. FORMALIN FIXATION OF TISSUE SPECIMENS Tissue specimens for l i g h t microscopy were fixed in phosphate buffered 10% formalin at least overnight. Tumour specimens were often stored in this- solution for long-periods. For storage, fresh f i x a t i v e solution was added after the f i x a t i o n period. Dehydration and embedding were done routinely by the h i s t o l o g i s t in the Zoology department. Dehydration was by a graded alcohol series, clearing by xylene, and embedding in wax in an automated tissue processer. b) HISTOCHEMISTRY 1. TESTING FOR ACTIVITY OF DEHYDROGENASE ENZYMES Dehydrogenase testing solutions "were prepared as outlined in Appendix I I . In the case of the hydroxy steroid' dehydrogenases A5-3p-HSDH and 17p-HSDH i t was important to have the aqueous solutions at 37°C before combining with the steroid dissolved in NNDMF to minimize p r e c i p i t a t i o n of the steroid. 202 To test tissue sections by t h i s method, tissue specimens were placed on cryostat. adhesive on a cryostat chuck and frozen in l i q u i d nitrogen immediately after removal from the animal. Tissue was sectioned at 8-10 M in a cryostat at -20°C. Sections were picked up on coverslips kept at room temperature. The sections were then returned to the refrigerated cabinet of the cryostat and allowed to dry for about 30 minutes. Sections were rinsed in 0.1 M phosphate buffer (pH 7.2-7.4) in small staining jars (Columbia jars) for 5 minutes at 37°C. Coverslips were then transfered to the dehydrogenase testing solution or to control solutions lacking only substrate, and were incubated at 37°C. For the enzymes tested 15 to 45 minutes was ample incubation time. Lactate dehydrogenase testing was usually complete at 15-30 min while the hydroxysteroid dehydrogenases took 30-45 min. After incubation, sections were fixed in 10% formalin in 50% ethanol for 15 minutes and then counterstained b r i e f l y (less than a minute) in 1% aqueous safranin 0. Coverslips were mounted to microscope slides using an aqueous mounting medium. As these preparations are not stable over long periods they were assessed by microscope and photographed soon after mounting. Cultured c e l l s in p l a s t i c dishes were • tested for dehydrogenase a c t i v i t y in an unfixed condition or after freezing at -20°C. It was very important to rinse cultures thoroughly with phosphate buffer to remove traces of serum in the growth medium. Fetal bovine serum is r i c h in many steroids, especially estrogens. This could lead to false p o s i t i v e r e s u l t s . Explants were also removed from cultures before testing. The testing solution for hydroxysteroid dehydrogenases in culture contained double the steroid concentration used for testing sections. Incubation was at 37°C. For A5-30-HSDH testing, cultured l u t e a l c e l l s showed dark staining, usually within an hour. However, 170-HSDH testing of* cultured: ROSE ceiis- gene'rally took 2:. hours. After incubation, c e l l s were rinsed with buffer and then fixed in phosphate buffered 10% formalin. C e l l s were mounted with an aqueous mounting medium and assessed soon aft e r mounting. Any storage was at 4°C. 2. OIL RED 0 FOR LIPID IN SECTIONS AND CULTURED CELLS, WITH HAEMOTOXYLIN COUNTERSTAIN This procedure is a modification of L i l l i e and Ashburn's isopropanol o i l red 0 method as outlined in Cu l l i n g (p.361, C u l l i n g , 1974). Immediately before use 6 ml of stock o i l red 0 solution (Appendix II) was diluted with 4 ml of d i s t i l l e d water, allowed to stand for 5 to 10 minutes, and then filtered'. This solution" i s not stable for more than an hour or two, and should not be used after a pre c i p i t a t e forms. Cultures to be tested were either frozen and thawed rapidly three times or were . fixed in 203 phosphate buffered 10% formalin. Cryostat sections of tissue were tested without further preparation. Sections or c e l l s were incubated with the o i l red 0 stain for 10 tq 15 minutes at room temperature. The stain was drained off and the c e l l s rinsed quickly (a few seconds only) in 60% isopropyl alcohol, then flooded with water. At th i s point cultures and sections were counterstained with haematoxylin for approximately two minutes. After rinsing in tap water c e l l s were allowed to "blue" in Scott's tap water, substitute for a few minutes and rinsed again. If the c e l l s were overstained they were rinsed for a few seconds in 1% HC1 to remove stain and then blued in Scott's. If the c e l l s were not stained enough they were restained. Preparations were then rinsed, drained well, mounted with an. aqueous mounting medium, and assessed promptly. c) ELECTRON MICROSCOPY 1. THE FIXATION, DEHYDRATION AND EMBEDDING OF CULTURED CELLS FOR ELECTRON MICROSCOPY Cultured c e l l s were prepared for electron microscopy according to the following schedule: C e l l s were rinsed gently with Waymouth medium.• Cultures were fixed in 2.5% glutaraldehyde in Millonig's buffer for one hour at 4°C. Cultures were rinsed with Millonig's buffer 2 or 3 times for 5 minutes each rinse. (N.B. Cultures could be stored at th i s point at 4°C.) Ce l l s were post fixed in 1% OsO„ in Millonig's buffer for 15 to 30 minutes at 4°C. Cultures were rinsed with Millonig's buffer twice for 5 minutes each rinse. C e l l s were next dehydrated in a graded ethanol then embedded in epon as follows: serles and 50% ethanol 70% ethanol 90% ethanol 100% ethanol 100% ethanol 100% ethanol ethanol/epon 3:1 ethanol/epon 1:3 epon fresh epon same epon 5 minutes 5 minutes 5 minutes 30 minutes 30 minutes 30 minutes 1 hour 1 hour 1 hour overnight 24 hours 4°C 4°C room room room room room room room' 37°C 56°C temperature temperature temperature temperature temperature temperature temperature 204 A l l dehydrating steps, and embedding steps done at room temperature were done with gentle shaking. 2. THE FIXATION, DEHYDRATION AND EMBEDDING OF SOLID TISSUE FOR ELECTRON MICROSCOPY Small pieces of tissue, about a millimeter in diameter, were fixed, dehydrated and embedded in epon according to the following schedule: Tissue was fxed overnight in 2.5% glutaraldehyde in Millonig's buffer at 4°C. Tissue was rinsed in Millonig's buffer twice at 30 minutes each rinse. Specimens were post fixed in 1% OsO„ for one hour at 4°C and then given two rinses in Millonig's.buffer for 30 minutes, each rinse. Dehydration of tissues was achieved by a graded ethanol series followed by propylene oxide and then embedding in epon as follows: ( a l l dehydrating steps and the f i r s t embedding step were done with gentle shaking) 50% ethanol 10 minutes 4°C 70% ethanol 10 minutes 4°C 90% ethanol 10 minutes 4°C 100% ethanol 1 15 minutes room temperature 100% ethanol 30 minutes room temperature ethanol/propylene oxide 2 1:1 30 minutes room temperature propylene oxide 2 30 minutes toom temperature propylene oxide/epon 1 : 1 1 hour room temperature propylene oxide/epon 1:3 1 hour room temperature epon 1 hour room temperature fresh epon overn ight-" 37 °C same epon 24 hours 56°C 1 - oven dried glass and plasticware only from th i s step onwards 1-2 - great care must be taken not to allow c e l l s to dry out during these steps. 3. STAINING OF THIN EPON SECTIONS FOR ELECTRON MICROSCOPY For staining thin (50-100 nm) epon sections mounted on carbon coated copper grids, covered glass Petrie dishes lined with a layer of paraf f i n were used. Drops of the stain (uranyl acetate and lead c i t r a t e in separate dishes) were placed on the pa r a f f i n surface. In the dish with the lead c i t r a t e p e l l e t s of sodium hydroxide were placed at least 5 minutes before the staining period to absorb carbon dioxide in order to minimize the production of lead carbonate. To stain, grids were placed section side down on the drops of s t a i n . Depending on section thickness and the intensity of staining desired, sections were 205 generally stained for 10 minutes in the uranyl acetate solution, rinsed thoroughly in d i s t i l l e d water (30 dips), stained 4 to 6 minutes in the lead c i t r a t e solution,. and. r.insed (30 dips).. 4. STAINING OF THICK EPON SECTIONS FOR LIGHT MICROSCOPY Thick epon sections (0.5-1 n) were placed on a drop of water on a glass microscope s l i d e , and the s l i d e was heated on a hot plate (125-150°C) to evaporate the water and f i x the sections to the s l i d e . Sections, were then stained b r i e f l y (about 30 seconds) with heating in 1% toluidine blue in 1% sodium borate solution and then rinsed thoroughly. 206 APPENDIX IV. TECHNIQUES FOR STEROID BIOCHEMISTRY This appendix is organized, under the following headings,:. a) s c i n t i l l a t i o n counting, b) paper chromatography, c) thin layer chromatography, d) lo c a t i o n of spots on chromatograms e) c a l i b r a t i o n chromatogramfor steroids used f) X-ray f i l m autoradiography g) gas l i q u i d chromatography. The sources of materials are found in Appendix I. a) SCINTILLATION COUNTING For most of this study (everything but the determination of s p e c i f i c a c t i v i t i e s of the progesterone XL and ML specimens) a Searle Mark II model #6847 s c i n t i l l a t i o n counter from Nuclear Chicago was used. For steroid specimens the s c i n t i l l a t i o n c o c k t a i l used was Omnifluor in toluene (4 gm Omnifluor to 1 l i t r e toluene). Samples to be counted were added to 10 ml of thi s c o c k t a i l and thoroughly mixed. For background counts s c i n t i l l a t i o n v i a l s containing 10 ml of c o c k t a i l only were placed before and after the radioactive v i a l s in the counter. In general samples having fewer than 5,000 counts per minute were counted for 20 minutes and those with more than 5,000 cpm for 10 minutes. Previous tests, done in th i s lab showed quenching to be negligible' with^ the- steroid samples used-. Solvents in samples greater than a few nl were dried down before c o c k t a i l was added. b) PAPER CHROMATOGRAPHY For most paper chromatograms done the paper (Whatman #1 chromatography paper in 46x57 cm sheets) was cut according to the accompanying diagram. The paper was soaked in a solution of propylene glycol/methanol (1:1) and then well blotted between sheets of chromatography paper. Propylene glycol was the stationary phase in a l l paper chromatograms done. Extracts were quickly applied to the orig i n with one s t r i p used for radioinert marker steroids only. The mobile phases used were l i g r o i n e (System I) and benzene/hexane 1:1 (System I I ) . System' I was generally used to separate the products of 1"C-progesterone incubations, and System II those of 1 " C - e s t r a d i o l incubations. 207 o r i g i n 20 cm. 10 cm. 47 cm. Chromatograms were run in large (30x30x60 cm), a i r - t i g h t glass tanks equiped with trough supports and troughs for the mobile phase. Mobile phase was poured into the tank to a depth of about 1.5 cm. The atmosphere in the tanks . should be saturated with mobile phase before a chromatogram i s started. The chromatogram was folded about 3 cm above the o r i g i n and secured in the trough in the tank. Beakers were set under each point of the chromatogram to catch any runoff. Mobile phase was poured into the trough, the tank covered with a weighted glass cover, and the edges of the tank sealed with s i l i c o n e grease. For best results the tank should be kept at f a i r l y constant temperature during the run. After the run the chromatogram was hung in a fume hood to dry. c) THIN LAYER CHROMATOGRAPHY (TLC) Before use, TLC plates were washed with either chloroform/methanol (1:1) or benzene/ethyl acetate (3:1), and allowed to dry thoroughly before spotting. Plates were spotted with extracts at 2 cm from the bottom, clamped between the glass sheets of the TLC apparatus, and set in a trough . containing mobile phase (System I I I , benzene/ethyl acetate 3:1) to a depth of 1 cm. The chromatogram was allowed to run u n t i l the solvent front reached the top of the TLC plate, usually 1.5 to 2 hours. The plate was then allowed to dry throughly in a fume hood. d) LOCATION OF SPOTS ON CHROMATOGRAMS 1. ULTRAVIOLET ABSORPTION In a dark room dry chromatograms were examined with a UV lamp (short wave length UV). Protective glasses were worn during t h i s procedure. Areas on chromatograms appearing darker than the surrounding paper were outlined in p e n c i l . Steroids containing the 3-keto A4 structure (e.g. progesterone) or a saturated A ring (the estrogens) absorb UV radiation and were hence posit i v e for t h i s t e s t . Steroids containing the 3-hydroxy A5 structure (e.g. pregnenolone) do not absorb UV and were thus 208 negative for this test. 2. PHOSPHOMOLYBDIC ACID (PMA) TEST, Most steroids containing a hydroxyl group turn blue when treated with an alcoholic solution of PMA. Progesterone and A4-androstenedione were the only steroids in thi s study which were negative for the PMA test. This test was carr i e d out in a fume hood. Dry chromatograms (usually only the side s t r i p s containing radioinert steroids) were thoroughly sprayed with the yellow PMA solution (8% PMA in ethanol) but not to the point that the solution ran on the paper. The paper was then placed in an oven at about 65°C. When blue spots began to appear the paper was removed and the location of the spots recorded. With time the entire paper turns blue; hence results must be recorded promptly. In this test the PMA'crosslinks steroids containing hydroxyl groups thus a l t e r i n g the steroid i r r e v e r s i b l y . Hence thi s reagent must never be applied to chromatograms to be used for elution and i d e n t i f i c a t i o n of steroids by r e c r y s t a l l i z a t i o n . e) CALIBRATION CHROMATOGRAMS FOR STEROIDS USED In an attempt to ide n t i f y radioactive products of 1 "C-pregnenolone and 1"C-estradiol incubations with c e l l s , many other steroids were run in chromatograms for reference. The result of c a l i b r a t i o n chromatograms are given in the table on the next page. The paper chromatograms were run for 22.5 hours and the TLC plates for 1 hour and 50 minutes. Numbers given in the table are distances in cm from the or i g i n to the centre of the spot. In paper chromatograms spots some distance from the or i g i n spread over as much as 10 cm. In paper chromatograms 100 Mg samples of steroids were used, and in TLC's 50 »g samples. f) X-RAY FILM AUTORADIOGRAPHY Radioactive regions on chromatograms were located by X-ray fi l m autoradiography. Thoroughly dry chromatograms were stapled to X-ray fi l m and exposed in a l i g h t - t i g h t cassette for 4 to 5 days. A l l darkroom work using X-ray fi l m was done using a Kodak safe l i g h t f i l t e r , Wratten Series 6B, only. X-ray films were developed in Kodak X-ray developer for 5 minutes, placed in a stop bath of water a c i d i f i e d with g l a c i a l acetic acid (1-2 ml of g l a c i a l acetic acid in a large t r a y f u l l of water) for 1 minute, and fixed in Kodak rapid f i x e r for 8 minutes. Film was then rinsed for a few hours in running water and allowed to dri p dry. Chromatograms were lined up with the fil m (matching staple holes) and radioactive regions were outlined in p e n c i l . 209 C A L I B R A T I O N C H R O M A T O G F tAMS F O R S T E : R O I D . S T A N D A F IDS  1 P A P E R P A P E R T L C S T E R O I D S Y S T E M S Y S T E M S Y S T E M UV5 P M A 6 I 2 ( C M ) I I 3 ( C M ) I I I " ( C M ) pregnenolone 13.5 25 6.0 + progesterone runof f runoff 7.3 + — 17a-hydroxy-pregnenolone 0 1-5 •; . 3.4 —' + 17a-hydroxy-progesterone 1.5 •11.5 3.8 + + 20a-dihydro-pregnenolone 1.3 4.0 3.4 — + 20o-dihydro-progesterone 5.5 20.5 3.7 + + estrone 0.5 6.5 9.9 + 7 + e s t r a d i o l 0 0.8 5.7" + + e s t r i o l 0.5 0.3 0.5 + ^  + androstenedione. - - 5.7 + -D H E A - - 5.0 - + testosterone 2.5 11.0 3.1 + + 1 - numbers given are distances in cm between or i g i n and centre of spot 2 - l i g r o i n e i s mobile phase - running time 22 .5 hours at room temperature 3 - benzene/hexane 1:1 i s mobile phase - running time was 22.5 hours at room temperature 4 - mobile phase was benzene/ethyl acetate 3: 1 - running time was 1 hour 50 minutes 5 - a plus sign s i g n i f i e s absorption of UV radiation 6 - a plus sign s i g n i f i e s development of a blue spot 7 - these steroids had a s l i g h t UV absorbance g) GAS LIQUID CHROMATOGRAPHY DETERMINATION OF SPECIFIC ACTIVITY FOR ESTROGEN SPECIMENS The s p e c i f i c a c t i v i t y (cpm/mg) for estr a d i o l (E 2) and estrone (E,) c r y s t a l s and mother liquors was determined using a Hewlett Packard GLC model #5830A (in the laboratory of K. McErlane, Faculty of Pharmaceutical Sciences, U.B.C.). A 6 foot glass column, o.d. 0.25 inch, packed with 3% OV225 on Chromosorb W(HP) 100/120 mesh was used in the analysis. The column was packed and conditioned according to the procedure outlined in a Government of Canada technical b u l l e t i n (McErlane, 1977). The main points in this procedure are summarized here. This procedure was also c a r r i e d out' in Dir. K. McErlane's laboratory. The glass column was thoroughly cleaned with chromic acid 210 (30 minutes), rinsed consecutively with d i s t i l l e d water, methanol and acetone, and a i r dried. The column was f i l l e d with a solution of 5% dimethyl-chlorosilane in.toluene, l e f t to stand 30 minutes, and then thoroughly rinsed with methanol and acetone and dried as before. This procedure s i l y l a t e s active hydrogen si t e s in glass column to prevent interference with s i l y l a t e d steroids. Columnn packing material was prepared as follows. A solution of 0.45 gm of OV-225 (methyl phenyl cyanopropyl s i l i c o n e ) in 30 ml chloroform was applied uniformly with a Pasteur pipette in a series of contiguous streaks, to 15 gm of support phase (Chromosorb W(HP) 100/120 mesh) spread uniformly over the bottom of a 5.5 cm diameter p e t r i dish. The barely moist cake of support was transferred to a f l u i d i z e r (HI-EFF Applied Science Laboratories Inc., State College, Pa.) through which clean, f i l t e r e d , o i l - f r e e nitrogen was flowing. The f l u i d i z e r was placed on a hot plate at 150-170°C and the support phase allowed to tumble u n t i l a l l the chloroform was gone. This procedure causes the support phase p a r t i c l e s to become uniformly coated in the l i q u i d phase and removes undersized p a r t i c l e s . The column was packed with support (about 5.3 gm) by applying vacuum to one end (with a glass wool pledget in the tube end) and adding enough support at the other end to f i l l 6 inches of the tube at a time. The column was inserted in the gas chromatograph such that the di r e c t i o n of the c a r r i e r gas flow was the same as the d i r e c t i o n of the packing. Column conditioning was carried out i n i t i a l l y with the detector end of the column disconnected. With the c a r r i e r gas flowing the oven temperature was increased from room temperature to 175°C at 3°/minute, and t h i s temperature was maintained for 16 hours. The oven temperature was then raised to 235°C at 3°/minute and held constant for 24 hours. The detector end of the column was then connected and- conditioning continued at 235°C u n t i l a stable baseline was achieved. This could take several days. Columns prepared as above had higher resolving power than those subjected to less rigorous treatment (McErlane, 1977). Column conditions for these E,/E2 runs were as follows: column temperature 225°C, injec t i o n port 240°C, detector (flame ionization detector) 240°C, c a r r i e r flow rate 41 ml/min (helium c a r r i e r gas). A l l Ei/E2 specimens were derivatized before use to their corresponding trimethyl s i l y l ethers. These trimethyl s i l y l groups are added at each hydroxyl group. In a R e a c t i - v i a l the radioactive specimen i s thoroughly dried down at 40°C under nitrogen along with the internal standard. For estrone unknowns es t r a d i o l was used as internal standard.. Usually 100 jig of internal standard was used since the unknown was approximately 21 1 100 »q also. To the dry v i a l were added 50 »/l of s i l y l a t i o n grade dry pyridine and 100 I>1 of the s i l y l a t i n g agent, BSTFA. The v i a l was capped with i t s teflon l i n e d cap and heated at 60°C for at least 10 minutes. For analysis 1-2 »il' aliquots were CALIBRATION CURVE FOR HP-GLC MODEL #5830A FOR TRIMETHYLSILYL DERIVATIVES OF ESTRADIOL (E 2) AND ESTRONE (El) IN PYRIDINE injected by m i c r o l i t r e syringe into the GLC. Retention times for the estrogen were very uniform from day to day with estrone at 17 minutes and e s t r a d i o l at 7 minutes. A response curve was prepared by i n j e c t i n g E^/E2 specimens of known proportionality by weight and determining the r a t i o of their areas on the GLC printouts. These printouts contained the percentage of t o t a l area occupied by each peak; hence determination of ratios of areas was s i m p l i f i e d . The response curve i s given in the accompanying graph. 212 APPENDIX V. TECHNIQUES INVOLVED WITH KIRSTEN VIRUS This appendix is organized under the following headings: a) containment precautions for work with KiMSV b) c o l l e c t i o n and concentration of the Kirsten virus c) KiMSV focus forming assay d) i r r a d i a t i o n of animals for tumourigenesis testing. e) t r i t i a t e d uridine incorporation and sucrose density gradient analysis The o r i g i n a l source for NRK c e l l s and KiMSV transformed NRK c e l l s is given in Appendix I. Virus used for this . study was taken from frozen stock derived from the o r i g i n a l l o t . a) CONTAINMENT PRECAUTIONS FOR WORK WITH KiMSV For the duration of this study both tissue culture and animal work was done at the 'C containment l e v e l as outlined in MRC Guidelines (MRC, 1979). Since that work was done the Kirsten virus has been downgraded to 'B' l e v e l . Tissue culture was carr i e d out in a v e r t i c a l laminar flow hood (Bio Gard model B40-002, Baker Co., Sanford, Maine) in Medical Block B, U.B.C. Virus infected rats were kept in cages housed in a v e r t i c a l laminar flow hood (Bio Gard model . B40-112) in the Zoology vivarium. Both of these rooms were kept under negative pressure and exhausted d i r e c t l y to the outside. Both Bio Gard hoods were equiped with HEPA (high e f f i c i e n c y p a r t i c l e attrapment) f i l t e r s to prevent virus p a r t i c l e s from passing into the room. These f a c i l i t i e s were tested and; regularly inspected by the U.B.C. Biohazards Committee. b) COLLECTION AND CONCENTRATION OF THE KIRSTEN VIRUS Cultures of KiMSV transformed NRK c e l l s derived from ampules of frozen c e l l s were grown in 25 cm2 flasks in 5% FBS/WM u n t i l crowded. Each culture was then subcultured to four 75 cm2 f l a s k s . Supernatant medium was harvested from subconfluent cultures. This supernatant was centrifuged at 5,200 rpm for 30 minutes at 4°C to get r i d of f l o a t i n g c e l l s . The preparation was then stored frozen at -60°C. The virus was concentrated by centrifugation of 500 ml of supernatant for one hour at 25,000 rpm at 4°C in polycarbonate' tubes with screw caps. It was essential that the centrifuge tubes be c a r e f u l l y balanced. The h a l f - l i f e of the virus i s short (approximately 1 hour) at room temperature; hence wherever 213 possible the preparations were kept on ice. Inside the laminar flow hood, the s u p e r n a t a n t was ca r e f u l l y poured o f f . The pel l e t in each tube was resuspended in ,2 ml of cold (4°C) 5% FBS/WM (heat treated FBS). Suspensions from several tubes were pooled and di l u t e d to 50 ml with 5% FBS/WM (heat treated FBS). This preparation was taken up in 20 ml syringes and passed through m i l l i p o r e f i l t e r s (0.45 M) into p l a s t i c freezing ampules. F i l t r a t i o n i s essential to remove any transformed NRK c e l l s . The ampules were immediately frozen and kept under l i q u i d nitrogen. c) KiMSV FOCUS FORMING ASSAY Cultures of non virus-transformed NRK c e l l s were grown to 60-80% confluence. Virus was added to dishes in five d i f f e r e n t concentrations: concentrate and 0.1, 0.01, 0.001 and 0.0001 d i l u t i o n s . Cultures were monitored for the development of fo c i of transformed c e l l s . About a week after infection foc i were counted in suitable dishes (usually the "second d i l u t i o n " dishes) and the number of focus forming units (FFU) per ml in the o r i g i n a l concentrate determined. d) IRRADIATION OF ANIMALS FOR TUMOURIGENESIS TESTING Animals used for tumourigenesis testing were female Fischer rats 4 to 5 weeks old. They were subjected to a whole body dose of 400 rads from a 280 kV X-ray source at the Cancer Control Agency of B r i t i s h Columbia. Rats were placed in a 20x20x9 cm box with no l i d . The 20x20 cm plexiglass cone of the radiation unit was placed over the box, 2.5 cm from the rats. Time, required for the 400 rad dose was 8 minutes. T h i r t y - s i x hours after i r r a d i a t i o n rats were injected with v i r a l l y transformed c e l l s . e) TRITIATED URIDINE INCORPORATION AND SUCROSE DENSITY GRADIENT  ANALYSIS This procedure was done by J . B. Hudson, Department of Medical Microbiology, U.B.C. Subconfluent cultures of v i r a l l y transformed c e l l s in 75 cm2 flasks were incubated in 10 ml of medium (Minimal Essential Medium) containing 100 nCi of 5-( 3H)-uridine for 16 hours. The medium was removed and centrifuged at 10,000 rpm for 10 minutes at 4°C to remove c e l l u l a r debris. The supernatant was centrifuged for 2 hours at 20,000 rpm at 4°C to p e l l e t the viru s . The p e l l e t s were resuspended in 0.5 ml PBS. From each p e l l e t suspension 0.3 ml was layered onto 20-60% sucrose gradients in PBS and centrifuged at 30,000 rpm for 17 hours at 4°C. Fractions were c o l l e c t e d onto 3 mm paper s t r i p s 214 for t r i t i u m counting (10 drops per f r a c t i o n ) . Every f i f t h f r a c t i o n , 3 drops were coll e c t e d separately for re f r a c t i v e index measurement and correlated with: the, sucrose density in gm/ml. From previous work the density of the KiMSV in sucrose i s 1.15 to 1.17 gm/ml. 215 APPENDIX VI. AUTORADIOGRAPHIC DETECTION OF ESTROGEN RECEPTORS Appendix VI is organized under the: following headings:.' a) preparation of tissue specimens for autoradiography b) preparation of cultured c e l l s for autoradiography. c) coating s l i d e s and coverslips with autoradiographic emulsion d) development of autoradiograms. Sources of materials are found in Appendix I. a) PREPARATION OF TISSUE SPECIMENS FOR AUTORADIOGRAPHY Tissue specimens to be examined autoradiographically for estrogen receptors were fixed in either absolute ethanol or in 2.5% phosphate buffered glutaraldehyde at 4°C overnight. Ethanol fixed tissue was cleared in xylene and embedded in p a r a f f i n . Glutaraldehyde fixed tissue was dehydrated in a graded ethanol series as per routine h i s t o l o g i c a l procedure, cleared in xylene, and embedded in p a r a f f i n . Wax blocks were trimmed and sectioned at 5 v. Sections were floated on 4% formalin solution on one end of s l i d e s coated with albumin adhesive. Slides were warmed at 40°C on a warming tray to evaporate the water and were l e f t overnight at 41°C in an oven to secure the sections to the glass. Within 48 hours of sectioning, sections were rehydrated to PBS and l a b e l l e d for 30 minutes in PBS containing 1 >/Ci/ml of 2,4,6,7- 3H-estradiol ( 3H-E 2). For l a b e l l i n g purposes, p l a s t i c s l i d e mailing tubes make good containers for the s l i d e s and radioactive solution. After l a b e l l i n g ^ sect ions, were rinsed- in. PBS for two 20 minute periods with gentle a g i t a t i o n . Slides were then rinsed b r i e f l y in d i s t i l l e d water and allowed to a i r dry. b) PREPARATION OF CULTURED CELLS FOR AUTORADIOGRAPHY 1. CELLS LABELLED AFTER ETHANOL FIXATION Explants were removed and cultures on p l a s t i c coverslips were rinsed in PBS for 20 minutes to remove traces of serum. In order to be able to t e l l readily on which side of the cove r s l i p the c e l l s were located, each cover s l i p was notched asymetrically as shown in the accompanying diagram. With the sharp notch in the orientation shown the c e l l s were on the top face. This marking of coverslips greatly helped- during darkroom- procedures. C e l l s were fixed in absolute ethanol for 20 minutes at 4°C. Cultures were rehydrated to PBS for one minute. At t h i s point 216 c e l l s were la b e l l e d for 30 minutes at room temperature in PBS J notched V / p l a s t i c \^ y c o v e r s l i p containing 1 nCi/ml of 3H-E 2. After l a b e l l i n g cultures were rinsed in unlabelled PBS for two 20 minute periods. Coverslips were b r i e f l y dipped in water and allowed to a i r dry. 2. CELLS LABELLED LIVE THEN FREEZE DRIED Living c e l l s were rinsed with two changes of serum-free Waymouth medium for 10 minute periods at 37°C to remove traces of serum. C e l l s were then incubated for one hour at 37°C with Waymouth medium containing 1 »iCi/ml of 3H-E 2. Incubation was in a C0 2 incubator (5% C0 2, 95% a i r , humidified atmosphere). Labelled c e l l s were rinsed twice with Waymouth medium for 20 minutes each time. Following a brief dip in d i s t i l l e d water, c e l l s were immediately frozen in a desiccator at -20°C. c \ COATING SLIDES AND COVERSLIPS WITH AUTORADIOGRAPHIC EMULSION The autoradiographic emulsion was removed from r e f r i g e r a t i o n and allowed to reach room temperature. Any freeze-dried cultures on coverslips were kept in their desiccator u n t i l room temperature was reached to prevent condensation on the c e l l s . The emulsion was weighed and dil u t e d (under safe l i g h t - Kodak Wratten OC series) one to one with pre-weighed d i s t i l l e d water at 60-70°C. Usually 8 gm of water and 8 gm of emulsion were s u f f i c i e n t for the experiments done (approximately 50 sl i d e s or coverslips per experiment). After d i l u t i o n the emulsion was mixed by s t i r r i n g and was poured into a p l a s t i c s l i d e mailer tube kept warm in water at 50-60°C. Slide mailing tubes were found to be very convenient containers for this d i l u t e d emulsion. Dry coverslips or s l i d e s were grasped firmly by forceps and dipped in the emulsion, then allowed to drain dry at room temperature for 30 minutes. During th i s time emulsion coated s l i d e s were loosely covered with sheets of aluminium f o i l to minimize fogging from the safe l i g h t . Slides and coverslips were then stored in l i g h t - t i g h t boxes at 4°C for 1 to 5 weeks. Coverslips were placed c e l l side up on glass s l i d e s . d) DEVELOPMENT OF AUTORADIOGRAMS After 1 to 5 weeks exposure autoradiograms were developed 217 at 15°C in Kodak D19' (diluted 1:1 with water just before use) for 4 to 6 minutes. Jars with developer were placed in a water bath at 15°C. Slides and coverslips. were rinsed for on eminute in water then fixed in Kodak developer for 7.minutes. This developing procedure is adapted from that of Leighton (Leighton, 1980). After f i x a t i o n s lides and coverslips were rinsed for at least 5 minutes in running water. As controls for positive chemography and fogging of emulsion from radioactive sources other than the 3H-E 2 used for l a b e l l i n g , unlabelled slides were exposed and developed under the same conditions as labe l l e d s l i d e s . As controls for negative chemography, a few la b e l l e d s l i d e s were exposed to li g h t after coating with emulsion, and were then placed in l i g h t - t i g h t boxes and exposed and developed as previously outlined. This l a t t e r control i s to detect fading of images during the exposure period in the dark. 

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