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UBC Theses and Dissertations

Differentiation of the chick oviduct in vitro Vanderhorst, Elizabeth Wilhelmina Maria 1972

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c> DIFFERENTIATION OF-THE CHICK OVIDUCT i.H V-i-tro by ELIZABETH. W.M. VANDERHORST B.Sc, University of B r i t i s h Columbia, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Poultry Science accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1972. In present ing th i s thes is in pa r t i a l f u l f i lmen t of the requirements fo r an advanced degree at the Un ive rs i t y of B r i t i s h Columbia, I agree that the L ib ra ry sha l l make it f r ee l y ava i l ab le for reference and study. I fu r ther agree that permission for extensive copying of th i s thes i s fo r s cho la r l y purposes may be granted by the Head of my Department or by h is representa t i ves . It is understood that copying or pub l i c a t i on o f th i s thes i s fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Department of The Un ive rs i t y of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT I m m a t u r e c h i c k o v i d u c t s d i f f e r e n t i a t e i n v i v o i n r e s p o n s e t o d a i l y i n j e c t i o n s o f DES. T h i s i s i n d i c a t e d by i n c r e a s e s i n g e n e r a l o v i d u c t p r o t e i n n i t r o g e n : D N A r a t i o s and t h e p r o d u c t i o n o f i m m u n o l o g i c a l l y p r e c i p i t a b l e o v a l b u m i n . T h r e e t y p e s o f o v a l b u m i n a r e p r o d u c e d i n r e s p o n s e t o e s t r o g e n . T h e s e r e s u l t s i n d i c a t e t h a t o v a l b u m i n f r e e o f p h o s p h o r u s a p p e a r s f i r s t i n t h e o v i d u c t t i s s u e i n r e s p o n s e t o DES. The a p p e a r a n c e o f p h o s p h o r u s c o n t a i n i n g o v a l b u m i n f o l l o w s . A t t e m p t s w e r e made t o i n d u c e t h e i n v i t r o d i f f e r e n -t i a t i o n o f o v i d u c t t i s s u e u n d e r a v a r i e t y o f c u l t u r e c o n d i t i o n s . A s t a n d a r d c u l t u r e m e d i a was s u p p l e m e n t e d w i t h DES and s e r u m ( f e t a l c a l f , c o c k e r e l , l a y i n g h e n , o r c h i c k s e r u m ) a s w e l l as s e r u m f r o m DES i n j e c t e d c h i c k s . No c h a n g e s w e r e o b s e r v e d i n t h e t i s s u e h i s t o l o g y n o r i n t h e p r o d u c t i o n o f t h e s p e c i f i c p r o t e i n , o v a l b u m i n , u n d e r t h e a b o v e c o n d i t i o n s . C l o s e l y a s s o c i a t e d t i s s u e s s u c h a s k i d n e y s , o v a r i e s , m e s e n t a r y o r u r e t e r t i s s u e c u l t u r e d w i t h t h e o v i d u c t t i s s u e f a i l e d t o e l i c i t e a n y d e t e c t i b l e m o r p h o l o g i c a l and c h e m i c a l c h a n g e s i n t h e l a t t e r . The a d d i t i o n o f i n s u l i n o r h o m o g e n i z e d p i t u i t a r i e s f r o m DES i n j e c t e d o r n o n - i n j e c t e d c h i c k s was i n e f f e c t i v e a s an i n d u c e r o f d i f f e r e n t i a t i o n i n t h i s s y s t e m . S u b s t i t u t i n g 17 6 - e s t r a d i o l f o r DES a s a n i n d u c e r d i d n o t a l t e r t h e r e s u l t s . • T h u s , w i t h t h e e x c e p t i o n o f e s t r o g e n an y f a c t o r ( s ) n e c e s s a r y f o r t h e i n v i v o d i f f e r e n t i a t i o n o f i m m a t u r e o v i d u c t t i s s u e r e m a i n s unknown a t t h i s t i m e . - I l l -TABLE OF CONTENTS INTRODUCTION LITERATURE REVIEV7 GENERAL MATERIALS AND METHODS A. Culture Methods B. Disc Gel Electrophoresis C. Immunoelectrophoresis D. Sample Preparation; DNA and Protein Nitrogen Analysis E. Histology RESULTS A. Action of DES on chick oviducts i n vivo B. D i f f e r e n t i a t i o n of chick oviducts i n v i t r o with DES in media containing f e t a l c a l f , cockeral, or laying hen serum C. D i f f e r e n t i a t i o n of chick oviducts i n v i t r o with DES i n Medium 199 containing a high l e v e l (60%)of either f e t a l c a l f or laying hen serum. D. D i f f e r e n t i a t i o n of chick oviducts i n v i t r o cultured on Medium 199 containing serum from DES injected chicks E. The maintenance of p a r t i a l l y d i f f e r e n t i a t e d oviducts i n v i t r o employing a medium containing serum from DES injected chicks F. Organogenetic i n t e r a c t i o n and the developing chick oviduct G. The effe c t of i n s u l i n i n addition to DES i n the medium of oviduct organ cultures H. The use of p i t u i t a r y homogenate in Medium 199 with 2 0% estrogenated chick serum i n organ cultures of chick oviducts I. The use of homogenized mature oviducts from laying hens to supplement the culture media i n organ cultures of chick oviducts J. The effe c t of 17g-estradiol on the immature chick oviduct DISCUSSION SUMMARY LITERATURE CITED - i v -APPENDIX 60 Table 1. RF values of densitometry from disc gel electrophoresis of homogenized oviduct samples of chicks treated with DES i n vivo 61 Table 2. RF values of densitometry from disc gel electrophoresis'of homogenized p a r t i a l l y d i f f e r e n t i a t e d oviduct samples cultured with DES serum and control serum as well as non-cultured p a r t i a l l y d i f f e r e n t i a t e d oviducts 63 Table 3. Protein nitrogen/DNA r a t i o s of DES serum cultured p a r t i a l l y d i f f e r e n t i a t e d chick oviducts 67 Formulae for determination of protein nitrogen and DNA 68 LIST OF FIGURES Diagram of incubating chamber Comparison of average oviduct wet weights of DES treated and control chicks Immunoelectrophoresis of homogenized samples of oviducts stimulated with DES i n vivo Disc gel electrophoresis of homogenized samples of oviduct tissues following DES stimulation i n vivo Disc gel electrophoresis and densitometer readings of homogenized samples of oviducts cultured on Medium 19 9 supplemented with f e t a l c a l f serum + DES Immunoelectrophoresis of homogenized samples of oviducts cultured i n f e t a l c a l f serum H i s t o l o g i c a l sections of oviducts cultured i n f e t a l c a l f serum with and without DES Disc gel electrophoresis of homogenized oviducts previously cultured with or without DES serum Disc gel electrophoresis of cultured and non-cultured p a r t i a l l y d i f f e r e n t i a t e d oviduct tissues Immunoelectrophoresis of homogenized samples of oviducts co-cultured with surrounding tissues i n the presence and absence of DES V I LIST OF TABLES Wet weights of tissues and gross weights of DES treated and control chicks Oviduct protein nitrogen/DNA r a t i o s of DES treated and control chicks Protein nitrogen/DNA r a t i o s of DES treated and control cultured oviducts Protein nitrogen/DNA r a t i o s of DES treated and control oviducts cultured on Medium 199 plus 50% f e t a l c a l f and laying hen serum Protein nitrogen/DNA r a t i o s of DES chick serum and control chick serum treated oviducts Protein nitrogen/DNA r a t i o s of DES and control serum treated organ cultures and non-cultured oviduct rudiments Protein nitrogen/DNA r a t i o s of oviducts co-cultured with surrounding tissues i n the presence or absence of DES Protein nitrogen/DNA r a t i o s of DES treated and control oviducts incubated i n medium containing serum - vii -ACKNOWLEDGEMENTS The author wishes to express her sincere appreciation to Dr. R.C. Fitzsimmons who provided super-v i s i o n , suggestions and the f a c i l i t i e s for t h i s study. She would also l i k e to express her appreciation to Dr. P.M. Townsley, Dr. R.J. Hudson and Dr. G. Jain f or t h e i r valuable discussions. Sincere thanks i s also given to Dr. D.B. Bragg, Dr. CR. Krishnamurti and Dr. W.D. K i t t s , the members of the author's committee. Thanks i s extended to Mrs. Enid Stewart who typed t h i s t h e s i s , Miss Janet Gehring for valuable suggestions as to thesis format, and Dr. D. Schaller who did a l l the photography for t h i s presentation. DEDICATION THIS THESIS IS DEDICATED TO MY MOTHER AND FATHER, JACOBA AND IVO VANDERHORST, I INTRODUCTION The action of estrogen on i t s target organ, the uterus i n mammals and the oviduct in b i r d s , has been studied quite extensively in the l a s t decade. In mammals i t has been found that estrogen manifests i t s e l f e a r l i e s t in target tissues by causing hyperemia, histamine release and an accumulation of water, e l e c t r o l y t e s , as well as plasma pro-teins due to an increase i n c a p i l l a r y permeability (33). I t l a t e r binds with receptors in the cytoplasm of c e l l s and i s then transferred into the nucleus where i t i s found bound to smaller p a r t i c l e s . The hormone has been found to bind with the chromatin by displacing histones (34). F i n a l l y , an increase i n RNA and protein synthesis occurs. P r a c t i c a l l y a l l of t h i s knowledge has been obtained from experiments involving i n vivo and i n v i t r o homogenate preparations. Studying undifferentiated target tissues i n a culture system to determine the requirements for complete estrogen response has received l i t t l e attention. The culture of completely undifferentiated u t e r i and oviducts and subsequent development i n response to estrogen added to the culture medium could provide a very useful t o o l for studying estrogen's mode of action. The com-plex environment of target organs i n i n t a c t animals as well as the unnatural conditions of tissue homogenates would be a l l e v i a t e d . The purpose of t h i s study was to culture u n d i f f e r -entiated oviducts i n v i t r o and test various parameters which may be necessary for estrogen induction of protein synthesis LITERATURE REVIEW D i f f e r e n t i a t i o n of the chick oviduct i s c h a r a c t e r -i z e d m o r p h o l o g i c a l l y by the development of s p e c i f i c c e l l u l a r s t r u c t u r e s and b i o c h e m i c a l l y by the synthesis of s p e c i f i c p r o t e i n s . Kohler et_ a l . (18) found that f o l l o w i n g the i n j e c t i o n of 3-day o l d chicks with 5 mg d i e t h y l s t i l b e s t e r o l (DES) d a i l y , three d i s t i n c t types of e p i t h e l i a l c e l l s appeared. These c e l l s d i f f e r e n t i a t e d from the homogenous appearing p r i m i t i v e c e l l s of the immature oviduct mucosa. This l a y e r of c e l l s c o n s i s t s of p s e u d o s t r a t i f i e d columnar e p i t h e l i u m r e s t i n g upon a compact stroma of polygonal c e l l s . The t u b u l a r gland c e l l s and the goblet c e l l s produce the s p e c i f i c p r o t e i n s , ovalbumin and a v i d i n , r e s p e c t i v e l y . As the t u b u l a r c e l l s form i n response t o estrogen, organized bundles of o f i l a m e n t s 40-50 A i n diameter appear at the l u m i n a l end of the c e l l s . These s t r u c t u r e s are not present i n u n d i f f e r e n -t i a t e d o v i d u c t s and they may be important agents i n morpho-genesis because of t h e i r c o n t r a c t i l e p r o p e r t i e s (36). A v i d i n , the b i o t i n b i n d i n g p r o t e i n , has been noted to appear i n the estrogen s t i m u l a t e d c h i c k oviduct a f t e r a s i n g l e dose of progesterone (13, 22). The t h i r d type of c e l l formed i n the developing oviduct are c i l i a t e d c e l l s concerned w i t h m o t i l i t y . Terenius (35) has found evidence f o r the e x i s t e n c e of estrogen b i n d i n g s i t e s i n t a r g e t t i s s u e s . In_ v i t r o o v i d u c t s from 7 to 10 day o l d c h i c k s take up 1 7 3 - e s t r a d i o l s p e c i f i c a l l y and t h i s reveals a s i m i l a r comparison to mammalian estrogen target tissues where estrogen has been found to be taken up by p a r t i c l e s in the cytoplasm of r a t , c a l f , goat and mouse u t e r i , (7, 37). Marked perivascular edema i s an i n i t i a l response of the immature oviduct stroma to DES and t h i s i s accompanied by i n t e r s t i t i a l migration of mononuclear c e l l s . Mitosis occurs within 24 hours and tubular gland c e l l s begin to develop at the 2nd, 3rd and 4th day of treatment. Fine apic a l granules appear i n the e p i t h e l i a l c e l l s at day 2 and ovalbumin can be detected immuno-chemically by day 3 at which time a new species of nuclear RNA ha.s been i d e n t i f i e d . Ovalbumin granules form within the condensing vacuoles i n the g o l g i zone and are released into the lumen of the gland a c i n i at about day 6 of treatment (17). Increased high molecular weight nuclear RNA and cytoplasmic transfer RNA have been found i n oviduct tissues following estrogen administration to immature chicks (6, 2 3). DES also causes a prolonged stimulation of oviduct nuclear . polymerase, the maximum rate of enzyme increase being between 12 and 24 hours post i n j e c t i o n . This has been interpreted as r e f l e c t i n g an a l t e r a t i o n i n nuclear t r a n s c r i p t i o n involving genes governing the synthesis of ovalbumin, lysozyme and numerous other proteins (24). I t has been shown by means of the nearest-neighbour analysis that estrogen acts to a l t e r t r a n s c r i p t i o n and increase DNA-chrc>matin template a c t i v i t y a f t e r 3 days of DES treatment but more d e f i n i t e l y by day 6 of steroid induced d i f f e r e n t i a t i o n (25). The steroid promotes the synthesis of d i f f e r e n t types of RNA that arise during oviduct development and are not present i n the undifferen-t i a t e d gland. The population of hybridizable RNA from unstimu lated and DES stimulated animals d i f f e r s . However, i t has bee pointed out that caution must be exercised i n equating hybridizable RNA with mRNA for there i s no evidence that these same RNA species are subsequently translated (12, 26). An increasing number of hormones have been found to be mediated by c y c l i c adenosine 3', 5'-monophosphate (29). This, compound, cAMP, has been shown to increase i n u t e r i of ovariectomized rats following an intravenous i n j e c t i o n of e s t r a d i o l (10). Furthermore, i t was found that uterine amino acid uptake was stimulated. However, Rosenfeld et a l . (31) have found that adenyl cyclase was not stimulated i n chick oviduct or rat prostate by estrogen or testosterone, suggest-ing that growth and d i f f e r e n t i a t i o n of these target tissues are not mediated by cAMP. Oka et a l . (21) have shown that estrogen and progesterone are s y n e r g i s t i c i n the production of lysozyme and ovalbumin. Progesterone antagonizes the formation of tubular gland c e l l s induced by estrogen. A two to three f o l d increase i n oviduct glycogen has been found i n addition to the synthesis of s p e c i f i c protein when 17S-estradiol i s administered to immature chicks. A l l f i v e anatomical parts of the oviduct, (infundibulum, magnum, isthmus, and uterovagina) have the same glycogen response. There i s no change i n oviduct glucose, whereas, in the rat increases i n uterine glycogen are pa r a l l e l e d by increases in uterine glucose. However, in the rabbit uterine glycogen synthesis i s not pa r a l l e l e d by increased glucose l e v e l s . Glycogen synthesis i n both the chick and the rabbit are not dependent on r i s i n g glucose l e v e l s (2). t A few studies involving tissue culture of chick oviducts have been reported. O'Malley and Kohler (27) examined 6 week DES treated monolayer oviduct cultures obtained from DES treated chicks for ovalbumin induction. A small amount of ovalbumin synthesis observed during the control period was followed by an increase of immunologically pr e c i p i t a b l e ovalbumin a f t e r the introduction of DES into the c e l l cultures. A peak was reached on the 10th day a f t e r which a gradual decline to the base l i n e occurred even though exposure to DES was continued. They also found that when DES was added to oviduct monolayer cultures obtained from 14'\day-old non-injected chicks, the predominantly f i b r o b l a s t i c population of c e l l s transformed into e p i t h e l i a l c e l l s . The :morphological changes to an epithel o i d type of c e l l appeared to be r e v e r s i b l e , suggesting the c e l l s had not undergone spontaneous or v i r a l induced transformation associated with change i n c e l l morphology. Although DNA synthesis did occur a f t e r an i n i t i a l lag phase, there was no report of i n i t i a t i o n of ovalbumin synthesis. A confluent layer of f i b r o b l a s t l i k e c e l l s appeared to be necessary for the above e f f e c t . DES added at the time of i n i t i a t i o n of the cultures did not appear to increase the growth of e p i t h e l o i d c e l l s (19). Cohen et a l . (4) found an elevation of ornithine carboxylase enzyme when oviducts from immature chicks were incubated as organ cultures i n a medium containing chick serum and 5 ug DES/ml culture medium. The stimulation which occurred may favour an early step i n estrogen action. However, ovalbumin synthesis was not detected. The complete d i f f e r e n t i a t i o n of the undifferentiated chick oviduct i n a tissue culture system has not yet been accomplished. The object of t h i s study was to culture undifferentiated oviduct tissue in v i t r o and attempt to obtain estrogen induced d i f f e r e n t i a t i o n as evidenced by the produc-t i o n of the s p e c i f i c protein, ovalbumin. GENERAL MATERIALS AND METHODS A population of bred White Leghorn chickens was the parent stock for the chicks used i n these experiments. Oviducts from chicks ranging i n ages from 3 days to 3 weeks were used for i n v i t r o culture work. In the experiments where chick serum was used to supplement Medium 199, chicks of the same age as those supplying the oviducts for culture were bled. Tissue culture Medium 199 and f e t a l c a l f serum were obtained from Microbiological Associates; d i e t h y l s t i l b e s t e r o l (DES) from Merk and Co.; 17B-estradiol from Sigma Chemical Co.; rabbit antiovalbumin serum from N u t r i t i o n a l Biochemical Co.; and agar from Microbiological Associates. A. Culture Methods DES was prepared for i n j e c t i o n by di s s o l v i n g 2.5 gm of the powder i n 17 ml ninety-five percent ethanol a f t e r which 33 ml of peanut o i l was added. The injected dose for a l l injected chicks was 5 mg (0.1 ml of the DES s o l u t i o n ) . When DES was added to the culture medium 5 pg was dissolved i n 2 0 ml of ethanol and 1 ml of t h i s solution was added to 50 ml culture medium giving a concentration of 5 ug DES per ml of medium. When 178-estradiol was used, 1.2 mg of the hormone was.dissolved i n 10 ml ethanol and 0.25 ml of the solution was added to 30 ml culture medium giving a concen-t r a t i o n of 1 ug per ml of medium. Oviducts were removed from DES injected or non-injected chicks under s t e r i l e conditions. Tissues were washed i n 2 changes of culture Medium 199 before being placed on s t a i n l e s s s t e e l grids i n disposable Flacon p l a s t i c organ culture dishes containing the media. Five mis chick Ringer solution was applied to the absorbent disc around the centre well to provide a humid atmosphere i n the dishes. Sixteen of the culture dishes were placed i n a p l a s t i c chamber containing a small amount of water. The chamber was gassed continually with 95% oxygen - 5% carbon dioxide during the incubation period of 6 to 7 days. The gas flow was 1.5 cubic feet per hour and the incubation temperature of the chamber was 37.5 + 0.5°C. The culture medium was changed every 2 or 3 days. The antibotics used in.the culture medium were p e n i c i l l i n and streptomycin. A solution consisting of 0.12 gm p e n i c i l l i n - K and 13.20 gm streptomycin sulfate dissolved i n 200 ml of d i s t i l l e d water was s t e r i l i z e d by m i l l i p o r e f i l t r a t i o n (0.45 u pore size) and stored frozen i n small v i a l s . One-tenth ml for every 10 ml of medium was used. Blood was obtained from the branchial vein of one year old hens and cockerels. Young female chicks (ranging i n age from 17 to 25 days) were bled from the jugular vein. Blood was incubated for one hour at 37.5 + 0.5°C af t e r which the serum was coll e c t e d and m i l l i -pore f i l t e r e d . Adult chicken serum was always prepared 9. gas exhaust gas input e l e c t r i c i a n s tape to seal chamber p l a s t i c chamber inverted c e t r i plates t o support culture dishes water -i FIGURE 1. D i a g r a m o f i n c u b a t i n g c h a m b e r . 10. the day before an experiment was i n i t i a t e d i n a large enough quantity to supply the entire experiment; whereas , chick serum was prepared on the day of culture and on each day of medium change. Cultures were terminated by homogenizing each oviduct i n 0.1ml phosphate buffer i n 2 ml conical centrifuge tubes with a small glass rod. They were stored at -10°C u n t i l analyzed. The buffer consisted of 0.07 M KC1 , 0.004 M MgCl 2 and .02 M phosphate buffer (pH 7.1) (28). B. Disc Gel Electrophoresis The procedure followed and reagents used for disc gel electrophoresis were the same as those of Davis (5). The gel tubes throughout t h i s work were.0.5 cm in diameter and 6.2 cm long. Eight gels were run per reservoir in a current of 40 milliamperes for about one hour. The oviduct samples (about 10 mg each) were run i n duplicate. The ovalbumin standard was prepared from fresh egg white as described by Kekwick and Cannan (16). The procedure used for i t s p u r i f i c a t i o n was the batch technique and column ion-exchange with carboxymethylcellulose (30). Approximately 10 ug of p u r i f i e d ovalbumin was applied to each standard gel. The gels were scanned on a densicord for i d e n t i f i c a t i o n of proteins i n the homogenate samples. C. Immunoelectrophoresis Standard macro-immunoelectrophoresis was carried out on 2 by 3 inch glass plates using a 2% agar i n sodium barbitol-hydrochloric acid buffer (pH 8.3). The ingredients are the same as outlined by Hudson et a l . (14). To each s l i d e , duplicate 10 yg aliquots of sample and a single 5 yg aliquot of standard ovalbumin were applied. The slide s were placed i n a current of 20 milliamperes for 45 to 90 minutes. 0.12 ml of rabbit antiovalbumin serum was applied to each of the two troughs per s l i d e and incubated at 21°C for 12 hours. D. ' Sample Preparation; DNA and Protein Nitrogen Analysis DNA determination was a modification of Schneider et a l . (32) using diphenylamine to develop the color. The protein nitrogen analysis was a modification of B r i t t and Herrmanns procedure (1). After the desired aliquots of the oviducts homogenate had been removed for disc gel and Immunoelectro-phoresis, the samples were prepared for DNA and protein nitrogen analysis. Oviduct samples were washed in 1'ml 95% ethanol, centrifuged at 2000 r.p.m. and decanted. Fat was removed by adding 1 ml of chloroform-ethanol (1:3) and incubating samples at 70°C i n a water bath for 15 minutes. After centrifugation and decanting the supernate, excess chloroform was removed by a wash with ethanol followed by a cold t r i c h l o r o a c e t i c acid wash to remove RNA. The DNA i n the samples was extracted by incubating the p r e c i p i t a t e at 90°C in two changes of t r i c h l o r o a c e t i c acid ( f i r s t 1 ml and then 0.5 ml). The two DNA extracts were combined and stored at 8°C u n t i l DNA analysis. To the remaining p r e c i p i t a t e 0.02 ml of selenium catalyst and 0.2 ml H 2S0 4 (3 parts H 20: 2 parts concentrated H0SO. ) were added. Samoles were incubated overnight i n a 9 6°C oven and then were placed i n a sand bath at 100°C. The temperature was gradually raised to 280°C and samples were digested overnight. Those samples which turned turbid were cleared by the addition of one or more drops of h^C^ s a f t e r which further incubation of one hour was necessary. One ml of d i s t i l l e d water was added to the samples and suitable aliquots (0.2, 0.1 or 0.05 ml) of the samples were analyzed, with 5 ml di l u t e d Nesslers solution (40 parts water to 12 parts Nesslers). Quanti-tati o n of protein nitrogen was done c o l o r i m e t r i c a l l y on a Beckman spectrophotometer at 500 X. T r i p l i c a t e nitrogen standards of 20, 30 and 50 mg/ml were used to estimate the unknowns. DNA was quantitated c o l o r i m e t r i c a l l y with diphenylamine. One ml of the t r i c h l o r o a c e t i c acid extract was mixed with 2 ml of diphenylamine solution (a fresh solution containing 100 mg diphenylamine per 10 ml acid solution (400 parts g l a c i a l acetic acid: 11 parts concen-trated H-SO.)) and incubated i n a b o i l i n g water bath for 13. 10 minutes. Reading was done on the ice cooled samples at 60 0 X on a Beckman spectrophotometer within one hour a f t e r the incubation period. T r i p l i c a t e one ml samples of each DMA standard at 20, 50 and 100 yg DNA/ml were prepared and treated i n the same manner as the t r i c h l o r o a c e t i c acid extracts. The formulae for ca l c u l a t i n g the amount of protein nitrogen and of DNA i n each sample i s given in Appendix V. E. Histology A standard technique for preparing the tissues and for t h e i r subsequent staining was used as outlined by Lynch et a l . (20). The thickness of the sections was 8 my and the stain used as the hematoxylin-eosin s t a i n . E r l i c k s hematoxylin solution was prepared i n accord with Fords' method (8). 14. RESULTS A. Action of DES on Chick Oviducts i n vivo The purpose of the f i r s t experiment was to induce young chick oviducts to d i f f e r e n t i a t e i n vivo and to measure the magnitude of th i s response i n terms of organ wet weight and the synthesis of t o t a l protein, DMA, and ovalbumin. Procedures: • Four day old chicks were injected d a i l y with 5 mg DES and were s a c r i f i c e d e v e r y 3 o r 4 d a y s . A g r o u p of chicks of the same hatch injected with 0.1 ml of c a r r i e r solution without DES supplied control oviducts. Control and experimental oviducts were stored i n 0.1 ml and 0.3 ml buffer respectively. The weights o f oviducts, l i v e r s and chicks were recorded (Table 1). The r e s u l t s of t h i s experiment indicate that DES causes d i f f e r e n t i a t i o n of immature chick oviducts. A marked'increase i n wet weight of oviducts did occur a f t e r 3 da i l y i n j e c t i o n s of DES. No such increase was observed i n the controls where oviduct weights remained below 0.1 gm (Figure 2). Injecting 5 mg DES d a i l y into immature p u l l e t s does have a detrimental effect on t h e i r growth rate. After 14 such i n j e c t i o n s chicks showed an average body weight of 112 gm, whereas the control chicks weighed an average of 168 gm. After 14 DES in j e c t i o n s the l i v e r weight i s an average of 7.0 gm and i s extremely f a t t y i n appearance i n contrast to the l i v e r s of control chicks which averaged 5.2 gm and remained red i n color. 15. TABLE 1. WET WEIGHTS OF TISSUES AND GROSS WEIGHTS OF DES TREATED AND CONTROL CHICKS'. Treatment Age of Chicks at S a c r i f i c e i n Days 8^  11 15 18 OVIDUCT DES Injected .43+.13 Control Injected .01+0 Weight Grams 1.40+.05 . 01+0 1.71+.16 .03+.01 2.54+.13 .05+.01 LIVER DES Injected Control Iniected 6.75+.74 4. 53+.52 7.00+.99 5.23+.19 CHICK DES Injected Control Injected 98.00+8.09 112.56+8.12 147.00+2.89 168.00+9.60 a. The 8-day old chicks had been injected 4 times. The 18-day old chicks had been injected 14 times. b. Each value represents the average weight of 3 chicks + standard deviation of the mean. T h e r e was an i n c r e a s e i n g e n e r a l o v i d u c t p r o t e i n s y n t h e s i s as i n d i c a t e d by p r o t e i n n i t r o g e n / D N A r a t i o s ( T a b l e 2 ) . The a v e r a g e r a t i o f o r o v i d u c t s a m p l e s f r o m 4 day i n j e c t e d c h i c k s was 1.88 i n c o n t r a s t t o 0.52 f o r t h e c o n t r o l . T h i s v a l u e i n c r e a s e s s h a r p l y t o 3.64 a f t e r 14 i n j e c t i o n s . No c h a n g e was o b s e r v e d i n t h e c o n t r o l v a l u e s . The a p p e a r a n c e o f i m m u n o l o g i c a l l y p r e c i p i t a b l e o v a l b u m i n o c c u r s a f t e r o n l y one i n j e c t i o n o f DES as s e e n i n F i g u r e 3. Rhodes e t a l . ( 2 7 ) f o u n d by p u r i f i c a t i o n o f egg w h i t e t h a t 3 t y p e s o f o v a l b u m i n c o u l d be s e p a r a t e d by t h e u s e o f c e l l u l o s e i o n e x c h a n g e r e s i n w i t h ammonium a c e t a t e b u f f e r s . Y e t on f i l t e r p a p e r e l e c t r o p h o r e s i s t h e c o m p o n e n t s a p p e a r e d as one homogeneous b a n d . C o n f i r m a t i o n o f t h e i d e n t i t y o f t h e f r a c t i o n s was o b t a i n e d f r o m p h o s p h o r u s a n a l y s i s . The m o l a r r a t i o o f p h o s p h o r u s t o p r o t e i n was 1.99 f o r t h e f i r s t f r a c t i o n a n d 0.93 f o r t h e s e c o n d . The f i r s t f r a c t i o n was e l u t e d a t pH 4.55, t h e s e c o n d a t pH 4.7 5 and t h e t h i r d a t pH 5. T h e s e r e s u l t s i n d i c a t e t h a t t h e d i f f e r e n c e b e t w e e n t h e t h r e e o v a l b u m i n s i s i n t h e i r p h o s p h o r u s c o n t e n t . The f i r s t o v a l b u m i n ( A l ) c o n t a i n s 2 atoms o f p h o s p h o r u s p e r p r o t e i n m o l e c u l e ; t h e s e c o n d ( A 2 ) c o n t a i n s one a tom o f p h o s p h o r u s p e r p r o t e i n m o l e c u l e ; and t h e t h i r d ( A 3 ) c o n t a i n s no p h o s p h o r u s . The r a t e o f e l e c t r o p h o r e t i c m i g r a t i o n i n d i s c g e l e l e c t r o p h o r e s i s d e p e n d s on t h e d e g r e e o f i o n i z a t i o n and A l w o u l d m i g r a t e t h r o u g h t h e g e l f i r s t , A2 s e c o n d and A3 l a s t . Rhodes e t a l . ( 2 7 ) a l s o f o u n d t h a t t h e amount o f t h e 17. three types of ovalbumin d i f f e r e d . A l was the most concen-trated and A3 the least concentrated. The gels of DES treated oviducts in t h i s experiment revealed the three types of ovalbumin (Figure 4). I t was found that during the 14 day i n j e c t i o n period ovalbumin type A3 appeared f i r s t followed by types A2 and A l i n that order (Figure 4). I n i t i a l l y , then, the non-phosphorylated form of ovalbumin appears as a r e s u l t of DES treatment. Gels of control oviduct samples did not contain the three ovalbumins. There were two f a i n t bands, one of which migrated at about the same rate as A3. Immunoelectro-phoresis indicated that t h i s was not ovalbumin. The other band migrated at a much slower rate and was probably con-albumin which can be eluted by c e l l u l o s e exchange at pH 6 (Rhodes et a l . (27)). 18. 3.00 2.75 2.50 Age of Chicks , Days FIGURE 2. Comparison of average oviduct wet weights of DES treated and control chicks. TABLE 2. OVIDUCT PROTEIN MITROGEN/DNA RATIOS OF DES TREATED AND CONTROL CHICKS. Treatment 3 Age of Chicks at S a c r i f i c e i n Davs 8 11 15 " 18 PN/DNA R a t i o s 5 DES Injected 1.88+.11 2.67+.14 3.00+.23 3.64+.07 Control Injected .52+.43 .52+.37 .54+.06 a. The 8-day old chicks had been injected 4 times The 18-day old chicks had been injected 14 times. b. Each value represents the mean r a t i o of 3 oviducts + standard deviation of the mean. 20 FIGURE 3. Immunoelectrophoresis of homogenized samples of oviducts stimulated with DES i n vivo, a. Represents a 14-day old oviduct exposed to one i n j e c t i o n and c. represents 15-day old oviduct exposed to two i n j e c t i o n s ; b. indicates standard ovalbumin. FIGURE 4. Disc gel electrophoresis of homogenized samples of oviduct tissues following DES stimulation in vivo. B. D i f f e r e n t i a t i o n o f C h i c k O v i d u c t s i n v i t r o w i t h DES i n M e d i a C o n t a i n i n g F e t a l C a l f , C o c k e r e l , o r L a y i n g Hen Serum. S e r a f r o m f e t a l c a l f , c o c k e r e l , and t h e l a y i n g hen w e re u s e d i n t h e c u l t u r e medium t o d e t e r m i n e i f t h e y c o n t a i n e d s u b s t a n c e s w h i c h a r e n e c e s s a r y f o r d i f f e r e n -t i a t i o n o f i m m a t u r e c h i c k o v i d u c t s . P r o c e d u r e s : T h r e e d u p l i c a t e d o r g a n c u l t u r e e x p e r i m e n t s w e re p e r f o r m e d u s i n g u n d i f f e r e n t i a t e d o v i d u c t s f r o m n o n - i n j e c t e d 3 day o l d c h i c k s . The e x p e r i m e n t s w e re i d e n t i c a l w i t h t h e e x c e p t i o n o f t h e s u p p l e m e n t e d s e r u m . F e t a l c a l f , c o c k e r e l , and l a y i n g h e n s e r a w e r e u s e d i n t h e f i r s t , s e c o n d and t h i r d e x p e r i m e n t s r e s p e c t i v e l y . O v i d u c t s were i n c u b a t e d a t 37.5°C i n 80% Medium 199 a n d 20% s e r u m. E a c h e x p e r i m e n t c o n s i s t e d o f t w o g r o u p s o f o v i d u c t s c u l t u r e d i n t h e p r e s e n c e o r a b s e n c e o f DES (5 y g DES/ml c u l t u r e m e d i a ) . A f t e r s e v e n d a y s o f i n c u b a t i o n t h e c u l t u r e s w e r e t e r m i n a t e d and s t o r e d a t -10°C u n t i l a n a l y s i s by d i s c g e l e l e c t r o p h o r e s i s and DNA and p r o t e i n n i t r o g e n q u a n t i t a t i o n . D i s c g e l e l e c t r o p h o r e s i s o f t i s s u e h o m o g e n a t e s f r o m o v i d u c t s c u l t u r e d on Medium 199 s e p p l e m e n t e d w i t h f e t a l c a l f s e r u m r e v e a l e d no i n c r e a s e i n p r o t e i n s y n t h e s i s . D e n s i t o m e t e r r e a d i n g s o f t h e s e g e l s ( F i g u r e 5b) i n d i c a t e d t h a t l i t t l e o r no p r o t e i n s y n t h e s i s o c c u r r e d d u r i n g t h e t r e a t m e n t . G e l s and d e n s i t o m e t e r r e a d i n g s o f o v i d u c t s c u l t u r e d on Medium 199 w i t h c o c k e r e l and l a y i n g h e n s e r u m r e v e a l e d t h e same r e s u l t s . The o v e r a l l averages of protein nitrogen/DNA .ratios between DES and control treatments did not d i f f e appreciably for tissues cultured i n medium supplemented with f e t a l c a l f or cockeral serum (Table 3). 2 4 . FIGURE 5a. Disc gel electrophoresis and densitometer readings of homogenized samples of oviducts cultured on Medium 19 9 supplemented with f e t a l c a l f serum + DES. Gel 1 was prepared from oviduct cultured with DES and Gel 2 was prepared from control oviduct. Gel 3 i s that of the standard ovalbumin. 25. FIGURE 5b. Disc gel electrophoresis and densitometer reading cf homogenized samples of oviducts cultured on Medium 199 supplemented with f e t a l c a l f serum + DES. Densitometer reading of gel prepared from oviduct cultured on Medium containing DES. 26 . FIGURE 5c. Disc g e l e l e c t r o p h o r e s i s and densitometer readings of homogenized samples of oviducts c u l t u r e d on Medium 199 supplemented w i t h f e t a l c a l f serum + DES. Densitometer read-i n g o f g e l prepared from standard ovalbumin. TABLE 3. PROTEIN NITROGEN/DNA RATIOS OF DES TREATED AND CONTROL CULTURED OVIDUCTS. a PN/DNA Ratios Serum DES Control b Fetal c a l f 7.19 + .86 (8) 7.29 + .59 (6) Cockerel 7.98 +1.02 (5) 7.66 +1.62 (4) a. Each value represents mean r a t i o + standard deviation of mean. b. Number of determinations used for c a l c u l a t i n g each value 2 8 . C. D i f f e r e n t i a t i o n of Chick Oviducts in v i t r o with DES i n Medium 19 9 Containing a High Level (50%) of e i t h e r Fetal Calf Serum or Laying Hen Serum. Various serum supplements as 2 0% of the culture medium did not r e s u l t i n d i f f e r e n t i a t i o n of oviducts treated with DES i n the previous experiment. The purpose of t h i s experiment was to determine i f a higher l e v e l of serum would be more e f f e c t i v e . Procedures: Three day old chick oviducts were cultured i n Medium 199 containing 50% serum. Of the 20 oviducts cultured 10 were incubated with f e t a l c a l f serum and 10 with laying hen serum. Five cultures from each group were exposed to 5 ug DES per ml of'culture medium, the remaining ones serving as the control. The incubation period was 7 days. Cultures were terminated and anlyzed by Immunoelectrophoresis and disc gel electrophoresis and one culture of each treatment was used for h i s t o l o g i c a l preparations. Undifferentiated oviduct cultured i n Medium 199 containing high l e v e l s of either f e t a l c a l f or laying hen serum did not d i f f e r e n t i a t e when exposed to DES as indicated by both disc gel and Immunoelectrophoresis. Disc gel electrophoresis indicated that no increase i n amount or types of protein synthesis occurred during the culture period under the influence of DES. Immunoelectrophoresis did not reveal the synthesis of the s p e c i f i c protein ovalbumin (Figure 6). Protein nitrogen/DNA r a t i o s , however, did reveal a s l i g h t but i n s i g n i f i c a n t increase i n t o t a l protein synthesis i n DES treated cultures (Table 4). H i s t o l o g i c a l sections of tissues cultured with both f e t a l c a l f and laying hen serum are shown i n Figure There was no apparent difference i n the morphology of DES treated oviducts compared with the controls. 30 . FIGURE 6. Immunoelectrophoresis of homogenized samples of oviducts c u l t u r e d i n f e t a l c a l f serum. S e c t i o n a. of s l i d e was a p p l i e d w i t h oviduct sample th a t had been c u l t u r e d w i t h DES, while S e c t i o n c. was a p p l i e d w i t h the c o n t r o l . S e c t ion b. contains p r e c i p i t i n bands of standard ovalbumin. TABLE 4. PROTEIN NITROGEN/DNA RATIOS OF DES TREATED AND CONTROL OVIDUCTS CULTURED ON MEDIUM 19 9 PLUS 5 0% FETAL CALF AND LAYING HEN SERUM. a PN/DNA Ratios Serum DES Control Fetal c a l f 1.46 + .13 j.1.34 + .07 Laying hen. 1.71 + .15 1.44 + .13 a. Each value represents the mean r a t i o of 4 cultures + standard deviation of the mean. 3 2 . FIGURE 7a. H i s t o l o g i c a l sections of oviducts cultured i n f e t a l c a l f serum with and without DES. Oviduct cultured with DES; magnification i s 40X. 33 . FIGURE 7b. H i s t o l o g i c a l s e c t i o n s of o v i d u c t s c u l t u r e d i n f e t a l c a l f serum wi t h and without DES. Oviduct c u l t u r e d with DES; m a g n i f i c a t i o n i s 100X. 34 . FIGURE 7c. H i s t o l o g i c a l sections of oviducts cultured i n f e t a l c a l f serum with and without DES. Oviduct cultured without DES; magnification i s 40X. D. D i f f e r e n t i a t i o n o f C h i c k O v i d u c t s i n v i t r o c u l t u r e d on Medium 199 c o n t a i n i n g Serum f r o m DES I n j e c t e d C h i c k s . D u p l i c a t e e x p e r i m e n t s (A and B) w e r e c o n d u c t e d o v e r t i m e t o t e s t w h e t h e r s e r u m o b t a i n e d f r o m DES i n j e c t e d c h i c k s t h e same age as t h o s e w h i c h d o n a t e d t h e o v i d u c t s w o u l d p r o m o t e e s t r o g e n d e p e n d i n g d i f f e r e n t i a t i o n i n c u l t u r e s . P r o c e d u r e s : The f i r s t o f two t r e a t m e n t s c o n s i s -t e d o f c u l t u r i n g o v i d u c t s f r o m 17 day o l d c h i c k s on 80% Medium 199 and 20% c h i c k s e r u m . T h i s s e r u m was o b t a i n e d f r o m 17 day o l d c h i c k s t h a t h a d b e e n i n j e c t e d w i t h 5 ug DES 4 h o u r s b e f o r e b l e e d i n g . The c o n t r o l g r o u p was t h e same e x c e p t t h e c h i c k s b l e d f o r s e r u m h a d n o t b e e n t r e a t e d w i t h DES. N o n - c u l t u r e d c o n t r o l o v i d u c t s o f 1 7 , 19 and 21 day o l d c h i c k s w e r e a n a l y z e d as w e l l . D i s c g e l e l e c t r o p h o r e s i s a n d d e n s i t o m e t r y a n a l y s i s r e v e a l e d no n o t i c e a b l e i n c r e a s e i n p r o t e i n s y n t h e s i s by t h e o v i d u c t s c u l t u r e d w i t h DES s e r u m ( F i g u r e 8) One b a n d c o n s i s t e n t l y a p p e a r s i n a l l g e l s w i t h an R f v a l u e c l o s e t o t h a t o f A3 i n t h e s t a n d a r d g e l s . T h i s i s n o t an o v a l b u m i n b and as p r e v i o u s l y t e s t e d by I m m u n o e l e c t r o p h o r e s i s The p r o t e i n n i t r o g e n / D N A r a t i o s o f t h e DES s e r u m t r e a t e d o v i d u c t s a p p e a r e d s l i g h t l y h i g h e r t h a n t h e c o n t r o l ( T a b l e 5 ) . 3 6 mmm a. b. e. f. FIGURE 8. Disc gel electrophoresis of homogenized oviducts previously cultured with or without DES serum. The gels are as follows: a. DES serum treatment b. Control serum treatment c. Non-cultured control oviduct chick from 17 day old d. Non-cultured control oviduct chick from 19 day old e. Non-cultured control oviduct chick from 21 day old f. Standard ovalbumin TABLE 5. PROTEIN NITROGEN/DNA RATIOS OF DES CHICK SERUM AND CONTROL CHICK SERUM TREATED OVIDUCTS. a PN/DNA Ratios DES Serum Control Serum Experiment A 0.98 + .11 0.88 + .07 Experiment B 1.21 + .13 1.14 + .15 a. Each value represents the mean r a t i o of 6 cultures + standard deviation of the mean. 38 . E. The Maintenance of P a r t i a l l y D i f f e r e n t i a t e d Oviducts i n v i t r o Employing a Medium Containing Serum from DES Injected Chicks. P a r t i a l l y d i f f e r e n t i a t e d oviducts obtained from 17 day old chicks were cultured on a medium containing serum from previously injected chicks of the same age to determine i f t h e i r d i f f e r e n t i a t e d state would be maintained. Procedures: Chicks were injected with DES on the 12th, 14th and 15th day of age. The p a r t i a l l y d i f f e r e n t i a t e d oviducts were removed on the 17th day and the magnum portion was cut into 2 mm pieces, each oviduct y i e l d i n g about 5 or 6 pieces. Two pieces of each oviduct were cultured separately i n Medium 199 containing 20% chick serum (the chicks had been injected with 5 ug DES four hours p r i o r to bleeding). Two more pieces were cultured i n Medium 199 containing 20% chick serum obtained from control chicks. The remaining portions of each oviduct served as non-cultured controls. The tissues were cultured 7 days and the culture medium was changed every 3 days. The cultures were terminated and analyzed as described i n the Methods Section. Disc gel electrophoresis and densitometry indicated that two of three ovalbumins (A2 and A3) were present i n both'the DES and non DES cultured tissues as well as the non-cultured tissues (Figure 9). (See Appendix Table 2 for a complete record of Rf values). The protein nitrogen/DNA r a t i o s of the f i r s t t r i a l shows that there was more protein i n the non-cultured control oviducts than the cultured ones (Table 6). There was s l i g h t l y less protein i n the DES serum treated cultures than the cultured controls; although the data varies i n the two t r i a l s DES does not appear to further stimulate the oviduct tissues i n v i t r o . Appendix Table 3 shows separate readings of oviduct tissues from each chick for the various treatments. 40. a. b. c. d. FIGURE 9. Disc gel electrophoresis of cultured and non-cultured p a r t i a l l y d i f f e r e n t i a t e d oviduct tissues. The gels are as follows: a. DES serum culture treatment b. Control serum culture treatment c. Non-cultured control d. Standard ovalbumin H I TABLE 6. PROTEIN NITROGEN/DNA RATIOS OF DES AND CONTROL SERUM TREATED ORGAN CULTURES AND NON CULTURED OVIDUCT RUDIMENTS. a PN/DNA Ratios DES Serum Control Serum Non Cultured T r i a l 1 1.50 + .21 ( 6 ) b 2.07 + .25 (6) 3.3 + .16 (10) T r i a l 2 2.01 + .08 (6) 2.07 + .32 (7) 1.6 + .16 (13) a. Each value represents the mean r a t i o + standard deviation of the mean. b. Number of determinations used for c a l c u l a t i n g each value. F. Organogeretic Interaction and the Developing Chick Oviduct Tissue interactions are v i t a l i n the d i f f e r e n t i a -t i o n of many c e l l types. The following experiment was carried out to determine i f the tissues of the mesentery, ureter, kidney or ovary provide i n vivo some inducing stimulus necessary for i n i t i a t i o n of d i f f e r e n t i a t i o n of immature chick oviducts. Procedures: Each 4 day old chick oviduct was cultured along with 2 mm pieces of kidney, the mesentery and ureter as w e l l as a 1 mm ovary rudiment. The oviduct, ureter, kidney and ovary are found i n close opposition to each other i n vivo, being held together by mesentery and the complete unit was kept i n t a c t as possible upon dissection. The tissues were cultured i n Medium 199 containing 20% f e t a l c a l f serum. Twelve cultures were set up, 6 of which had DES (10 ug/ml culture medium) added. Medium was changed every 2 days and the cultures were terminated a f t e r 7 days for analysis as described i n the Methods Section. The data i n the above experiment shows no i n d i c a -t i o n of there being any i n t e r a c t i o n between the immature oviduct and surrounding tissues. Disc gel electrophoresis and densitomer readings revealed no new protein synthesis. No ovalbumin synthesis could be detected by Immunoelectro-phoresis (Figure 10) and protein nitrogen/DNA r a t i o s were s i m i l a r (see Table 7). The morphology of the cultured tissues were s i m i l a r to those shown i n Figure 7, with no apparent differences due to the treatments. 43 . FIGURE 10. Immunoelectrophoresis of homogenized samples of oviducts co-cultured with surrounding tissues i n the presence and absence of DES. Section a. was applied with oviduct sample treated with DES, while section c. was applied with the control oviduct sample. Section b. contains p r e c i p i t i n bands of standard ovalbumin. TABLE 7. PROTEIN NITROGEN/DNA RATIOS OF OVIDUCTS CO-CULTURED WITH SURROUNDING TISSUES IN THE PRESENCE OR ABSENCE OF DES. PN/DNA Ratios DES Control 1.66 + .26 1.58 + .11 a. Each value represents the r a t i o of 3 cultures + standard deviation of the mean. G. The Effect of I n s u l i n i n Addition to DES i n the Medium of Oviduct Organ Cultures. Ceriani (3) has found that i n s u l i n i s a require-ment for.the d i f f e r e n t i a t i o n of mouse mammary gland i n v i t r o i n response to p r o l a c t i n , aldosterone, and progesterone. Because Medium 199 does not contain i n s u l i n , i n s u l i n was added to the culture medium along with DES to determine i f i t i s necessary i n the culture system for DES-induced d i f f e r e n t i a t i o n . Procedures: Five-day old chicks were s a c r i f i c e d and oviducts were cultured on Medium 199 containing 20% f e t a l c a l f serum and 10 ug DES ml.of culture medium. I n s u l i n (0.05 mg/ml) was added to both control and experimental cultures. The incubation period was 6 days and the media was changed every 2 days. The cultures were terminated and analyzed as described i n the Methods Section. This experiment reveals no evidence that i n s u l i n was stimulatory to the tissues under the culture conditions. The protein nitrogen/DNA r a t i o s were s i m i l a r for both the control and treated oviducts (Table 8). Disc gel e l e c t r o -phoresis and densitometer readings did not reveal new protein synthesis. Ovalbumin synthesis was not detected i n these cultures by Immunoelectrophoresis. H i s t o l o g i c a l sections were s i m i l a r to those shown i n Figure 7. There was no apparent change i n morphology of DES treated tissues when compared to the control t i s s u e s . TABLE 8. PROTEIN NITROGEN/DNA RATIOS OF DES TREATED AND CONTROL OVIDUCTS INCUBATED IN MEDIUM CONTAINING INSULIN PN/DNA R a t i o s a DES Control 1.37 + .13 ( 2 ) b 1.29 + .14 (3) a. Each value represents the r a t i o of 3 cultures + standard deviation of the mean. b. Number of determinations used for c a l c u l a t i n g each value. 47 . H. The Use of P i t u i t a r y Homogenate i n Medium 19 9 with 2 0% Estrogenated Chick Serum i n Organ Cultures of Chick Oviducts. Factor(s) to date have not been i s o l a t e d from the p i t u i t a r y that stimulate d i f f e r e n t i a t i o n of oviducts. The object of t h i s experiment was to determine i f homogenized p i t u i t a r i e s from DES and non-DES injected chicks woudl help i n i t i a t e d i f f e r e n t i a t i o n . Procedures: Ten day old chicks were injected with DES four hours p r i o r to t h e i r s a c r i f i c e . The chicks were bled for serum and t h e i r p i t u i t a r i e s were removed. Oviducts from control chicks were put i n culture with minced p i t u i t a r i e s and serum from DES treated chicks (20% i n Medium 199). One h a l f of the cultures had control p i t u i t a r i e s and the remainder had p i t u i t a r i e s from DES injected chicks. The medium was changed every 2 days and the cultures were terminated a f t e r 7 days and tested for ovalbumin with Immunoelectrophoresis. P i t u i t a r i e s from either DES injected or control chicks did not enhance d i f f e r e n t i a t i o n oviducts i n v i t r o . Immunoelectrophoresis r e s u l t s were s i m i l a r to the preceding two experiments (Figure 9) i n which no ovalbumin was detected. I . The Use of Homogenized Mature Oviducts from Laying Hens to Supplement the Culture Medium i n Organ Cultures of Chick Oviducts. Homogenized laying hen oviduct was used as a supplement i n Medium 199 with 20% f e t a l c a l f serum and 10 ug DES per ml of culture medium to test for possible factors i n the mature oviduct which would, along with the DES, i n i t i a t e d i f f e r e n t i a t i o n of chick oviducts. The magnum portion of an oviduct from a mature hen was removed under s t e r i l e conditions and homogenized i n 5 ml Medium 199 at room temperature. Ten 3-day-old chicks were s a c r i f i c e d and oviducts were put into culture dishes with Medium 199 containing 20% f e t a l c a l f serum. One-tenth ml mature oviduct homogenate was added to each of the cultures. Five of the cultures were treated with DES and the remainder served as controls. The medium was changed once at 3 days and the cultures were terminated a f t e r 6 days of incubation. The cultures were examined for morphological indications of d i f f e r e n t i a t i o n , by means of the "h i s t o l o g i c a l procedure. H i s t o l o g i c a l sections of the cultured tissues treated with DES did not indicate any apparent morphological development over control tissues. A l l the prepared sections were s i m i l a r to those shown i n Figure 7. J . The Effect of 17 3-estradiol on the Immature Chick Oviduct. The object of t h i s experiment was to determine i f the natural hormone would i n i t i a t e d i f f e r e n t i a t i o n that DES had not been able to mimic i n v i t r o . Procedures: Four-day old chicks were s a c r i f i c e d and oviducts were cultured i n Medium 199 containing 20% f e t a l c a l f serum with 1 ug/ml 173-estradiol. The incubation period was 6 days and the medium was changed every 2 days. This experiment revealed no evidence that 173-e s t r a d i o l was able to induce d i f f e r e n t i a t i o n of the cultured oviduct t i s s u e s . Ovalbumin synthesis was not detected by Immunoelectrophoresis. These r e s u l t s were s i m i l a r to those of preceding experiments where DES was added to the media. DISCUSSION Fetal c a l f serum has been used as a supplement to Medium 199 by O'Malley et a l . (27) i n 6 week monolayer cultures started from p a r t i a l l y d i f f e r e n t i a t e d oviducts of DES injected chicks. These c e l l s did respond by synthesizing ovalbumin when DES was added to the culture system. There-fore, Medium 199 plus f e t a l c a l f serum does supply the required nutrients for oviduct c e l l response to DES i f the c e l l s or t h e i r progenates have been previously exposed to DES in vivo. However, the re s u l t s of t h i s study indicate that t h i s medium i s inadequate for the in v i t r o d i f f e r e n t i a -t i o n of untreated oviduct c e l l s . Chick, cockerel or laying hen serum did not i n i t i a t e d i f f e r e n t i a t i o n . There are numerous blood c o n s t i t u -ents in the laying, hen that are affected by the i n vivo l e v e l of estrogen. There i s an increase in c i r c u l a t i n g l i p i d s , c h o l e s t e r o l , proteins, globulins, albumins, plasma, i r o n , vitamins ( i . e . vitamin A, b i o t i n and r i b o f l a v i n ) , manganese and protein bound calcium. When only DES i s present in the culture system, the conditions under which the oviduct responds are very abnormal. In the culture system, there i s no increase i n the above factors as t h e i r increase i s dependent on other parts of the chicken's body. For example, the action of estrogen on the hen's l i v e r i s responsible for lipemia and i t s action on bone i s responsible for calcemia. The addition of laving hen serum to the culture svstem should then furnish many nutrients not present i n f e t a l c a l f , chick or cockerel serum. However, the absence of p o s i t i v e data leads one to suggest that factors other than those prsent i n t h i s type of serum are involved i n the i n i t i a t i o n of d i f f e r e n t i a t i o n . There was no confirmation that there are age dependent factors necessary for induction of d i f f e r e n t i a t i o n i n serum of young injected chicks. Once a hen has come into lay and the reproductive processes have been i n i t i a t e d , a factor or complex of factors may no longer be necessary to maintain the d i f f e r e n t i a t e d oviduct. O'Malley et a l . (27) found that d i f f e r e n t i a t e d oviduct from chicks injected 12 times and then incubated i n monolayer cultures kept t h e i r a b i l i t y to respond to estrogen even a f t e r 6 weeks i n culture. Once the c e l l s were induced to d i f f e r e n t i a t e they retained the capacity to respond to DES over a considerable length of time. Serum from young injected chicks did not reveal the presence of a short l i v e d i n i t i a t i n g factor. When-serum obtained from chicks injected only once was added to the culture medium without further addition of DES to the medium there may not have been an adequate amount of protein bound DES i n the serum. However, Jensen et a l . (15) found that 2 hours a f t e r the i n j e c t i o n of rats with t r i t i a t e d estrone, t h e i r u t e r i contained t r i t i a t e d e s t r a d i o l i n one tenth of the amount for the e s t r a d i o l control (estrone must f i r s t be oxidized to e s t r a d i o l to be b i o l o g i c a l l y a c t i v e ) . The maximum percentage dose of e s t r a d i o l i n the uterus from subcutaneous o i l i n j e c t i o n of estrone was found 4 hours a f t e r the i n j e c t i o n . Therefore, i f chicks were injected with DES 4- hours p r i o r to being bled, there must be an adequate amount of protein bound DES i n the serum providing that DES acts i n the same manner as estrone. In the l a s t decade considerable progress has been made toward understanding interactions between components of developing organ rudiments. Organ rudiments can be cultured separately or i n combination. Failure of rudiments to continue developemnt i n i s o l a t i o n and renewal of develop-ment on recombination i s a test for developmental i n t e r -action. When tissues undergo a developmental change follow-ing the association, the in t e r a c t i o n i s often r e c i p r o c a l . The period of dependency on induction i s of measurable duration. Rudiments cultured alone may lose the a b i l i t y to interact with other tissues. In contrast, components separated following a s u f f i c i e n t period of association can continue development independently. In a few instances active c e l l extracts have been prepared from tissues that are respondible for continuing development of neighbouring tissues (11). I t was thought that perhaps, i n the f i r s t steps of d i f f e r e n t i a t i o n with DES, the oviduct rudiment may require a factor or complex from tissues i n i t s close proximity ( i . e . kidney, ovary, ureter, or surrounding mesentery). These r e s u l t s show no i n d i c a t i o n of there being any i n t e r a c t i o n between the immature oviduct and surrounding tissues. I n s u l i n has been shown by Ceriani (3) to be a requirement for the development of new born rat mammary glan tissue i n organ culture. I n s u l i n , p r o l a c t i n , aldosterone and progesterone i n the culture medium cause the synthesis of a casein l i k e material. Without i n s u l i n the other hormones are incapable of causing t h i s response. I n s u l i n i s known to affect u t i l i z a t i o n of glucose by glyconeogenesis and other pathways. Pancreatectomy i n mammals i n h i b i t s the u t i l i z a t i o n of glucose (9). Medium 199 used i n a l l the preceding experiments contains no i n s u l i n , although some media such as Trowell T8 medium do contain 6 0 mg per l i t e r . These re s u l t s reveal no evidence that i n s u l i n i s involved i n the induction of oviduct rudiments. P i t u i t a r i e s from DES or non-DES treated chicks did not enhance d i f f e r e n t i a t i o n of immature oviducts when added to a culture system i n which medium 19 9 was supple-mented with 20% serum from DES treated chicks. There would be no FSH production by p i t u i t a r i e s of eit h e r injected or non-injected chicks. A high l e v e l of the injected c i r c u l a -t i n g hormone would i n h i b i t any FSH production i n chicks. FSH i s not produced by p i t u i t a r i e s of immature non-injected chicks. In addition to FSH, the p i t u i t a r y may produce a fa c t o r , h i therto unidentified that could a s s i s t DES i n the d i f f e r e n t i a t i o n of the immature oviduct. The re s u l t s of t h i s study do not reveal the presence of an i n i t i a t i n g factor necessary for the d i f f e r e n t i a t i o n of the immature chick oviduct. I t can be concluded that there are no factors in the p i t u i t a r i e s either stimulated with DES or not previously stimulated with DES which could i n i t i a t e the d i f f e r e n t i a t i o n process along with DES of the immature cultured chick oviduct. H i s t o l o g i c a l sections do not reveal any apparent development of the immature oviducts cultured with mature hen oviduct homogenate and DES. There are no factors evident i n the mature oviduct which could help i n i t i a t e the d i f f e r e n t i a t i o n process. Although DES mimics the action of estrogen i t s chemical structure i s very d i f f e r e n t from that of a l l the estrogenic hormones. No positive r e s u l t s were obtained when 178-estradiol was added to organ cultures of oviducts on Medium 199 supplemented with 20% f e t a l c a l f serum. This suggests that 178-estradiol, although a natural compound, does not i n i t i a t e d i f f e r e n t i a t i o n better than DES under the conditions of these experiments. 55 . SUMMARY Immature chick oviducts d i f f e r e n t i a t e i n vivo i n response to d a i l y i n j e c t i o n s of DES. This i s indicated by increases i n general oviduct protein nitrogen:DNA r a t i o s and the production of immunologically p r e c i p i t a b l e ovalbumin. Three types of ovalbumin are produced i n response to estrogen. These re s u l t s indicate that ovalbumin free of phosphorus appears f i r s t i n the oviduct tissue i n response to DES. The appearance of phosphorus containing ovalbumin f ollov7s. Attempts were made to induce the i n v i t r o d i f f e r e n -t i a t i o n of oviduct tissue under a variety of culture conditions. A standard culture media was supplemented with DES and serum ( f e t a l c a l f , cockerel, laying hen, or chick serum) as we l l as serum from DES injected chicks. No changes were observed i n the tissue histology nor i n the production of the s p e c i f i c protein, ovalbumin, under the above conditions. Closely associated tissues such as kidneys, ovaries, mesentary or ureter tissue cultured with the oviduct tissue f a i l e d to e l i c i t e any detectible morphological and chemical changes i n the l a t t e r . The addition of i n s u l i n or homogenized p i t u i t a r i e s from DES injected or non-injected chicks was i n e f f e c t i v e as an inducer of d i f f e r e n t i a t i o n i n t h i s system. Substituting 17 3-estradiol for DES as an inducer did not a l t e r the r e s u l t s . Thus, with the exception of estrogen any factor(s) necessary for the i n vivo d i f f e r e n t i a t i o n of immature oviduct tissue remains unknown at t h i s time. 56. 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The Role of Chromatin in Estrogen Action i n the Uterus. I, The Control of Template Capacity and Chemical Composition and the Binding of H E s t r a d i o l 176. Proc. Nat. Acad. S c i . U.S.A., 60: 1410-1417. 35. Terenius, L. 1969. Oestrogen Binding Sites in the Chicken Oviduct Similar to those of the Mouse • Uterus. Acta. Endocrinol., 60: 79-90. 36. Wrenn, J.T. and Wessels, N.K. 19 70. Cytochalasin B: Effect upon Microfilaments Involved i n Morpho-genesis of Estrogen Induced Glands of Oviducts. Proc. Nat. Acad. S c i . U.S.A., 66: 904-9 08. 37. Zimmering, P.E., Kahn , I. and Lieberman, S. 1970. Es t r a d i o l and Progesterone Binding to a Fraction of Ovine Endometrial Cytoplasm. Ciochem. , 9: 2498-2506. 60. A P P E N D I X TABLE 1. 61. RF VALUES OF DENSITOMETRY FROM DISC GEL ELECTRO-PHORESIS OF HOMOGENIZED OVIDUCT SAMPLES OF CHICKS TREATED WITH DES In Vivo Control Oviducts A3 Peaks A2 A l Experi-mental Oviducts A3 Peaks A2 A l Standard . 69 . 77 .83 Standard . 70 .77 .83 3d 1 .71 h .72 3 d l ' .70 h .71 3d 0 . 72 J2 .73 Standard .69 .76 .82 h .76 .85 Standard .68 .78 .83 Standard .74 .83 .90 3d 3 . 71 3 d 3 .72 I J 1 .68 .73 .80 17d 1 .66 I Z 1 .71 .78 17d 1 .68 I I 2 .65 .70 .72 17d 2 . 68 I I 2 .66 .76 . 82 17d 0 .64 J I 3 .66 . 71 .77 Standard . 68 .76 . 81 I J 3 .69 .72 .79 Standard .69 .77 . 82 Standard .68 .75 . 81 17d 3 .72 I I X 1 .68 .73 .82 17d 3 .71 I1J1 .67 .73 .79 Standard .70 .78 .85 I I I 2 .65 .71 .79 17d 4 .68 I I I 2 .65 .71 .78 17d 4 .72 I I X 3 .65 .70 17d 5 . 69 I I I 3 .68 .74 .85 17d 5 .75 Standard .66 . 74 .82 Standard .70 .79 .85 I V 1 . 65 .73 . 80 19d 1 .76 I V2 .71 .72 .81 19d x .73 i v 2 .69 .71 .73 19d 2 .73 I V 3 .68 .73 .81 19d 2 .72 I V 3 .69 .72 . 80 Standard .70 .79 . 84 21d r .72 21d 1 .72 2 1 d 2 .74 21d 2 . 72 KEY TO TABLE 1 Roman numerals i n d i c a t e the treatments as f o l l o w s : Treatment Age of oviduct Number of DES i n days before I n j e c t i o n s s a c r i f i c e I 4 4 I I 11 7 I I I 15 11 IV 18 14 Subs c r i p t s ( i . e . 1^, 1^ e t c . ) . The roman numeral represents the treatment number and d i f f e r e n t sub-s c r i p t s represent the d i f f e r e n t c h i c k s that donated oviduct sample i n a treatment. I d e n t i c a l s u b s c r i p t s represent d u p l i c a t e d i s c gels of samples obtained from the same chi c k . The c o n t r o l oviducts are l a b e l l e d with the age of the oviduct i n days. The s u b s c r i p t s represent the c h i c k s t h a t donated the oviduct sample. I d e n t i c a l s u b s c r i p t s represent d u p l i -cate d i s c g e l s of samples obtained from the same c o n t r o l c h i c k . Standard r e f e r s t o the standard prepared ovalbumin. One o r two standard d i s c gels were run per r e s e r v o i r . TABLE 2 RF VALUES OF DENSITOMETRY FROM DISC GEL ELECTROPHORESIS OF HOMOGENIZED PARTIALLY DIFFERENTIATED OVIDUCT SAMPLES CULTURED WITH DES SERUM AND CONTROL SERUM AS WELL AS NON CULTURED PARTIALLY DIFFERENTIATED OVIDUCTS TREATMENTS 1. Oviducts + . 2. Oviducts + 3. Non-Cultured 4. Non-Cultured,Non-DES Serum Control Serum Injected Controls Injected Controls Chick No. A3 Peaks A2 A l Chick No. . A3 Peaks A2 A l Chick No. A3 Peaks A2 A l Chick No. A3 Peaks A2 A l Standard .68 .78 .84 Standard .70 . 77 .84 Standard .68 .76 . 81 .69 .76 .82 Standard Standard .70 .76 . 84 Standard . 69 .77 . 82 .68 .76 .82 III . 70 .75 . 81 II .72 I .72 . 81 3d^ .76 III . 72 .75 . 82 II .71 I .67 .73 . 84 3 d l .72 V . 71 . 80 IV . 80 I .68 .74 . 82 Standard .69 .77 .83 IV .75 I .67 .73 .81 3 d2 . 71 IV .73 Standard .73 . 81 .87 3d 2 .70 IV .70 Standard .70 .79 . 84 3d 3 .72 Standard .68 .75 .82 Standard .68 .78 .84 II .68 .73 . 85 Standard .69 .76 . 82 II .75 . 79 . 87 Standard . 69 .78 . 83 IV .77 .72 II . 65 . 74 .81 3d-4 .71 IV .75 . 80 .72 II .71 . 74 . 8 3 3d 4 .72 II .65 .69 .75 .75 III . 66 . 71 . 80 17d 1 .66 II .70 .75 .82 Standard .73 . 86 III . 61 . 67 .73 17d 1 .68 CO TABLE 2 ( C o n t i n u e d ) TREATMENTS 1. O v i d u c t s + 2. O v i d u c t s + 3. N o n - C u l t u r e d 4. N o n - C u l t u r e d , N o n -DES Serum C o n t r o l Serum I n j e c t e d C o n t r o l s I n j e c t e d C o n t r o l s P e a k s P e a k s P e a k s P e a k s C h i c k A A A C h i c k A A A C h i c k A A A C h i c k A A A No. A 3 A 2 A l No. A 3 A 2 A l No. A 3 A 2 A l No. A 3 A 2 A l I I I .74 .83 S t a n d a r d .70 .77 .82 1 7 d 2 .68 I I I . 71 .76 . 84 I I I . 74 . 81 S t a n d a r d .69 . 77 .83 1 7 d 2 .64 S t a n d a r d .73 .81 .86 I I I .74 .82 IV .73 .77 .83 S t a n d a r d .68 S t a n d a r d S t a n d a r d I V .75 . 86 S t a n d a r d .69 . 74 . 82 S t a n d a r d .69 .76 . 82 VI .70 .76 . 82 .72 . 76 . 84 V .68 .76 . 82 V I .71 .76 . 84 . 71 .76 . 85 V . 72 .80 .72 . 74 . 84 . 76 .85 V .73 .81 .73 .76 .86 V .67 .72 .80 S t a n d a r d .68 . 77 . 83 S t a n d a r d .70. 76 .83 .69 .72 .82 .72 . 80 .70 .75 . 82 .65 .73 .71 .81 .73 .83 cn 4r TABLE 2 ( C o n t i n u e d ) TREATMENTS O v i d u c t + DES Serum O v i d u c t s + C o n t r o l Serum N o n - C u l t u r e d I n j e c t e d C o n t r o l s N o n - C u l t u r e d , N o n -I n j e c t e d C o n t r o l s C h i c k No. P e a k s A 3 A 2 A i C h i c k No. P e a k s A 3 A 2 A i C h i c k NO. P e a k s A 3 A 2 A l C h i c k No. P e a k s A 3 A 2 A l S t a n d a r d .69 .76 .83 S t a n d a r d .68 .74 . 81 S t a n d a r d .65 .72 . 78 I . 69 I .69 I . 71 •I .68 I .74 I .70 I I . 70 I I . 70 S t a n d a r d . 71 . 79 . 59 I I .70 I I . 71 I I .70 S t a n d a r d .69 .75 .81 S t a n d a r d .67 .76 .80 I I .70 I I I . 80 I I I . 69 I I I .70 I I I . 67 I I I . 67 I I I . 71 IV . 72 IV . 67 S t a n d a r d .72 . 81 .88 IV . 68 S t a n d a r d .68 .76 .93 IV . 71 S t a n d a r d .68 .81 .87 V .67 I V . 67 V .71 V . 78 V .78 V .68 V I .73 V .73 V I . 68 V I .67 S t a n d a r d . 69 .76 .83 S t a n d a r d .65 .72 .78 V I .70 .69 V I .83 V I I . 71 V I I . 70 < cn cn TABLE 2 ( C o n t i n u e d ) a) E a c h Roman n u m e r a l r e p r e s e n t s one c h i c k w h i c h d o n a t e d 1 o r 2 p i e c e s o f o v i d u c t t o e a c h o f t h e f i r s t t h r e e t r e a t m e n t s . b) The n o n - c u l t u r e d , n o n - d i f f e r e n t u a t e d o v i d u c t s a r e l a b e l l e d w i t h t h e age o f t h e o v i d u c t i n d a y s . I d e n t i a l s u b s c r i p t s r e p r e s e n t d u p l i c a t e d i s c g e l s o f s a m p l e s o b t a i n e d f r o m t h e same c h i c k . c ) S t a n d a r d r e f e r s t o t h e s t a n d a r d p r e p a r e d o v a l b u m i n . One o r two s t a n d a r d d i s c g e l s w e r e r u n p e r r e s e r v o i r . cn cn 67. TABLE 3 PROTEIN NITROGEN/DNA RATIOS OF DES SERUM CULTURED PARTIALLY DIFFERENTIATED CHICK OVIDUCTS DES-Serum Control-Serum Non-Cultured Controls Ch i ck PN/DNA Chick PN/DNA Chick PN/DNA No. No. No. I 1.2 8 I 1.19 I 2 . 48 I I I 2.69 I I 1.65 I I 1.85 I I 3.62 I I I I I I 3 .82 I I I 1.46 I I I 2.56 I I I 3 . 82 I I I 1.56 I I I I I I 3.57 IV .2.51 IV 2.02 IV 2.77 IV IV 2.93 IV 3 .33 V 0.94 V V 3.87 V + .48 V V 3.05 1.5 + .21 2 .07 + .25 3 .3 + .16 I I I 1.29 I 2.0 I I . 87 I I 2. 32 I I 1.85 I I 1.24 I I I I I I 1. 87 I I I 2. 04 I I I 1. 57 I I I 1.7 5 I I I I I I I I I 2 .72 IV 2 .10 IV 3.43 IV 2.03 IV IV IV 2.18 V 1.71 V 1. 94 V 1. 31 V V V 1.95 VI 1.92 VI 2. 39 VI 1.08 VI VI VI 1. 89 VII VII 2 . 64 VII . 65 2.01 + .08 2 .07 + .32 1 .6 + .16 T r i a l 1 AVERAGE T r i a l 2 AVERAGE Each c h i c k donated 2 pieces of p a r t i a l l y d i f f e r e n t i a t e d oviduct toward each treatment. Chicks were l a b e l l e d w i t h Roman numerals. 68 FORMULAE FOR DETERMINATION OF PROTEIN NITROGEN AND DNA. 1. Protein Nitrogen 10 O.D. observed av.O.D. 10 yg N of v o l u m e X experimental X f a Q t o r 10 s amp1e s average protein bound nitrogen in sample volume factor was (1 i f .2 ml) (2 i f .1 ml) - (4 i f .05 ml) 2. DNA JLO av.O.D. 10 yg DNA A n ~ , _^ „ O.D. of sample ^ i 5 observed 10 amount of DNA i n sample, 

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