UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A search for female sex hormones in salmon embryos of the genus Oncorhynchus Robertson, James Grant 1954

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1954_A1 R6 S3.pdf [ 4.18MB ]
Metadata
JSON: 831-1.0302612.json
JSON-LD: 831-1.0302612-ld.json
RDF/XML (Pretty): 831-1.0302612-rdf.xml
RDF/JSON: 831-1.0302612-rdf.json
Turtle: 831-1.0302612-turtle.txt
N-Triples: 831-1.0302612-rdf-ntriples.txt
Original Record: 831-1.0302612-source.json
Full Text
831-1.0302612-fulltext.txt
Citation
831-1.0302612.ris

Full Text

A SEARCH FOR FEMALE SEX HORMONES IN SALMON EMBRYOS OF THE GENUS ONCORHYNCHUS by SMSm. GRANT ROBERTSON A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS. FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Zoology We accept t h i s thesis as conforming to the standard required from candidates for the degree of DOCTOR OF PHILOSOPHY Members of the Department of Zoology THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1954 ABSTRACT A d i a l y z i n g technique was developed to concentrate an estrogen hormone f r a c t i o n s u i t a b l e for separation by paper p a r t i t i o n chromatography and spectrophotometry assay. Estrogens were not found i n sexually d i f f e r e n t i a t i n g salmon embryos. Small amounts of e s t r i o l , es t radio 1-17>0 and est-rone added to the tissue could not be recovered. However, horse testes assayed by the. same technique- showed the presence of estradiol-17^ and estrone i n concentrations of .097 and .143 mg./kg., respectively. The assay of horse testes was carried out on.90 gram l o t s , whereas the one prev-ious chemical assay was. done on 28,000 grams. I t i s con-cluded that t h i s technique i s very s a t i s f a c t o r y f o r extract-ion of estrogens from animal gonads, but that hormone added to whole salmon embryos i s inactivated by-some unknown system. A p a r t i t i o n technique recently developed by F. M i t c h e l l and R. Davies f o r the extraction of estrogens from.human placentae was s l i g h t l y modified for use with salmon embryos. This method confirmed the negative findings obtained by the d i a l y z i n g technique. On the basis of these experiments, there i s no evid-ence to support the hormonal theory of sex d i f f e r e n t i a t i o n i n f i s h e s . THE UNIVERSITY OF BRITISH COLUMBIA Faculty of Graduate Studies PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of JAMES GRANT ROBERTSON B.Sc. (Manitoba) 1948 M.A. (Brit. Col.) 1951 Monday, July 12, 1954-at 10:00 a.m. in ROOM'403, Applied Science Bldg COMMITTEE IN CHARGE W.A. Clemens, Chairman V.C. Brink I. McT. Cowan M. Darrach-P. Ford 'T, E.S. Goranson B.: Savery A.J. Wood External Examiner - D.R. Idler PUBLISHED PAPERS 1. Smith I n l e t Sockeye. Prog. Reports Pac. Coast. Stat-, i ons F.R.B.C. 75:31-34. 1948. 2. Sockeye F r y Production i n a Small B r i t i s h Columbia Watershed. I b i d . 80:55-57- 1949-3• Sex D i f f e r e n t i a t i o n i n the P a c i f i c Salmon Oncorhynchus keta (Walbaum). Can. Jorn. Z o o l . 31:73-79- 1953-4- The T r o p i c Status of Port John Lake, B r i t i s h Columbia. Journ. F i s h e r i e s Research Board Canada ( i n press) 1954. ABSTRACT A search for female sex hormones in salmon embryos of the genus Oncorhynchus A dialyzing technique was developed to concen-trate an estrogen hormone fraction suitable for separation by paper partition chromatography and spec-trophotometry assay. Estrogens were not found in sexually differentiating salmon embryos- Small amounts of e s t r i o l , estradiol-17B and estrone added to the tissue could not be recovered. However horse testes assayed by the same technique showed estradiol-17B and estrone in concentrations of .097 and .143 mg/kg., respectively. The assay of horse testes was carried out on 90 gram lo t s , whereas the one previous chemical assay was done on 28,000 grams. It i s concluded that this technique i s very satisfactory for extraction of estrogens from mammalian gonads, but that hormone added to whole salmon embryos i s inactivated by some unknown system. A partition technique recently developed .by F. Mitchell from R. Davies for the extraction of estro-gens from human placentae was slightly modified for use with salmon embryos. This method confirmed the negative findings obtained by the dialytic technique. On the basis of these experiments, there i s no evidence to support the hormonal theory of sex d i f -ferentiation in fishes. GRADUATE STUDIES Field of Study: Zoology Experimental Zoology - Dr. W. Hoar Vertebrate Morphogenesis - Dr. P. Ford Biology of Fishes - Dr. w. Hoar Limnology - Dr. p. Larkin Zoological Seminar - Dr. w. Clemens Comparative Physiology - Dr. w. Hoar Histology- - Dr. p. Ford Fisheries Management - Dr. w. Hoar Embryology - Dr. p. Ford Other Studies: General Biochemistry Biochemistry of Steroids Intermediary Metabolism Advanced Cytology Biometry Biochemistry of Cancer -Dr. S. Zbarsky - Dr. M. Darrach -Dr. S. Goranson - Dr. A.H. Hutchin - Dr. V. Brink - Dr. M. Darrach Biochemistry of Antibiotics- Dr. J. Polglaze , i CONTENTS I . INTRODUCTION. ...... * 1 •II . THE DISTRIBUTION OF FEMALE SEX HORMONES........... 6 I I I . MATERIALS. ............. 9 A. B i o l o g i c a l Tissue.......... 9 B. Chemicals. 9 1. ..Hormones 9 2. Solvents...... 10 .... 3. Color reagents. 10 4. Apparatus...... 10 IV. PRELIMINARY CHEMICAL SEPARATION OF AN ESTROGEN FRACTION. . 11 A. p a r t i t i o n Methods..... 11 B. Early attempts to effect an i s o l a t i o n by pap paper chromatography 13 1. Use of Bush*s (1952) paper adsorpt-ion method... ..13 2. Use of Zaffaroni's (1951) paper p a r t i t i o n method • 13 C. Column chromatography 15 Q. D i a l y s i s . . . . . . . . . 17 1. Synthetic system. 18 a) Dialyzing without s t i r r i n g 18 b) Dialyzing with s t i r r i n g . 20 c) Ca l i b r a t i o n curve f o r estimation... E l d) Hormone recoveries i n the syn-t h e t i c system......... 21 2.. B i o l o g i c a l systems 25 V. PAPER PARTITION CHROMATOGRAPHY OF SALMON EXTRACTS. 28 A. Introduction.. 28 B;. Reproduction of ^Axelrod's (1953> method using e s t r i o l , estradiol-17/S and estrone.. 29 1. Procedure. 29 2. Detection of estrogens. 30 i i 3. Positions of the hormones 31 4. El a t i o n of the hormones 33 5. Elimination of background material 33 6. Absorption curves... 33 G. Application to salmon embryo d i a l y s -ates. 35 D. Application to horse testes dialysates 39 -•VI. A-FURTHER SEARCH FOR FEMALE HORMONES IN SALMON EMBRYOS .............. 44 A. The Mitchell-Daviea P a r t i t i o n Method.. 44 B. Experiment 1. Recovery of hormone added to salmon embryos * 47 C. Experiment 2. The f a i l u r e to deteift hormones i n l y o p h i l i z e d salmon embryos 49 1. The estradiol-estrone f r a c t i o n . . . . 49 2. The e s t r i o l f r a c t i o n . . 51 VII i DISCUSSION. 52 VI I I . SUMMARY AND CONCLUSION..,...-.. 62 IX. ACKNOWLEDGMENTS...... ... 64 X. LITERATURE CITED..... i..... 65 i i i LIST OF TABLES, FIGURES, AND FLOW-SHEETS TABLE, I . Recovery of 50 micrograms of estradiol-^17/6 by d i a l y s i s i n a synthetic system...............23 I I . The location of free steroids ( i n centimeters from the s t a r t i n g l i n e ) a f t e r chromatography....32 I I I . Concentration of hormones recovered from dialysates of horse testes ....41 IV. Spectrophotometric assay of the e s t r i o l f r a c t -ion from horse testes.......... ...42 V. Recovery values for hormones added to salmon embryos, Experiment I . . . . . . . . . . 48 VI. Recovery values for Experiment 2 50 FIGURE 1. Ca l i b r a t i o n curve for estradiol-17^ i n a synthetic system.. .22 2. Apparatus used i n d i a l y s i s , . , ......27 3. Absorption spectra showing the elimination of background material (B) from chromatographed estradiol-17^ (,D), e s t r i o l (C), and estrone (E).34 4. Absorption spectra of estradiol-17/6 (A), estrone (B), and e s t r i o l (C), i n concentrated s u l f u r i c acid ,. 36 5. Agent stimulators and i n h i b i t o r s -operating i n sex d i f f e r e n t i a t i o n . (after Witschi, 1950)..,,,., • .54 FLOW-SHEET I , Preliminary p a r t i t i o n system 14 I I . P a r t i t i o n system i n d i a l y s i s of embryos........ .37 I I I . P a r t i t i o n system i n d i a l y s i s of horse testes.,..40 TV, IVa. Mitchell-rDavies p a r t i t i o n system s l i g h t l y modified. 45,46 & SEARCH FOR FEMALE SEX HORMONES LN SALMON. EMBRYOS OF THE GENUS ONCORHYNCHUS By J . G. Robertson I . INTRODUCTION The era of experimental studies on the ro l e of sex hormones i n the embryonic sex d i f f e r e n t i a t i o n of vertebrates commenced with F.R. L i l l i e s * (1917) observ-ations on the freemartin. The freemartin i s a female c a l f that has been transformed into an hermaphrodite. This transformation occurs prenatally, and then only i n the presence of a male twin. I t i s dependent upon a secondary fusion of the two placentae so that blood-borne male hormones can pass from the male to the female twin. There i s no freemartin when the twins are of the same sex. The transformation to the freemartin may be so complete that the female develops a t e s t i s and male ducts, instead of an ovary and female ducts ( W i l l i e r , 1934). The morphological basis of sex d i f f e r e n t i a t i o n i s w e l l worked out. Thus the undifferentiated gonad consists of two d i s t i n c t zones: an inner medulla, representing the t e s t i c u l a r component, and an outer cortex — the o v a r i a l component. Normally only.one of these constituents becomes funct i o n a l , i . e . , forms an ovary or t e s t i s C ^ i t s c h i , 1934). Both constituents may pe r s i s t into adulthood to give a functional hermaphrodite as; i n some cyclostomes (Okkelberg, 1921), or the constituents may not be separable, as i n young eels (D'Ancona, 1947). The gen i t a l t r a c t has two sets of ducts, which i n most vertebrates are simultaneously present and f u l l y formed at some time during development (Burns, 1942, *49). The g e n i t a l tubercle, located i n the urogenital sinus, i s the forerunner of both male and female external g e n i t a l i a . The experimental basis f o r the modus operandi of sex d i f f e r e n t i a t i o n depends upon observations r e s u l t i n g from gonadectomy, graftings of ovarian and t e s t i c u l a r t i s s u e , parabiosis, and ap p l i c a t i o n of sex hormones. Each approach i s designed to determine whether embryonic hormones are es s e n t i a l to the normal or abnormal development of the gonad and i t s accessory sex structures, i . e . reproduce "Nature's experiment** i n the development of the freemartin. Selected examples, of these approaches are outlined here. The removal of the functional l e f t ovary of a chick induced the usually degenerate right ovary to form a t e s t i s producing spermatozoa (Benoit, 1923). A prenatal removal of embryonic rabbit ovaries (Jost, 1947) did not i n h i b i t development of the female ducts. However, i n t e s t i c u l a r c a s t r a t i o n , the embryonic female ducts continued to develop while the male g e n i t a l t r a c t f a i l e d to do so. According to Burns (1949), the anomaly produced by the ovarectomized female rabbit may be the resu l t of interference from maternal hormones. Grafting experiments, i n which pieces of t e s t i s or 3 ovary were inserted on the ehorio-allantoic membrane of a host chick embryo, resulted i n a number of the host embryos being modified i n the expected male or female d i r e c t i o n (Minoura, 1921). These findings could not be confirmed by W i l l i e r (1927, *34). Recently, however, Wolff (1947) effected a p a r t i a l sex reversal i n chicks by implanting t e s t i c u l a r or ovarian rudiments d i r e c t l y into the body cavi t y . Parabiosis, or the. u n i t i n g of whole organisms so that an exchange between t h e i r blood may occur, has resulted i n a duplication of the. freemartin effect i n Amphibia. Thus Burns (1925) joined l a r v a l a x o l o t l s at a time when the gonads were undifferentiated. He found that both members of a p a i r were of the same sex, i n d i c a t i n g that a reversal of sex must have occurred i n a large number of them. Treatment with hormone preparations has generally effected a modification of sex i n vertebrates i n l i n e with the above experiments. In f i s h , Padoa (1939) observed a p a r t i a l feminization of male trout (Salmo irideus) reared i n aquaria containing female hormones. Bullough (1940) showed female minnows (Phoxinus laevis) developed a t e s t i s -l i k e structure a f t e r i n j e c t i o n of male hormone. However, male f i s h injected with female hormone showed an injured, but not transformed, t e s t i s . Berkowitz (1938, *41) founds estrogen caused formation of an ovatestis i n the guppy (Lebistes r e t i o u l a t u s ) . In trout (Salmo t r u t t a ) , treatment with male or with female hormone gave c o n f l i c t i n g r e s u l t s 4 i n that s i m i l a r modifications occurred with either hormone i n either sex (Ashby, 1952)• I t i s i n t e r e s t i n g that at l e a s t i n one plant (Melandrium; dioeclum), mammalian sex hormones shifted the-normal and intersexual flowers to the male or female side i n accord with the hormone applied (Love and Love, 1940, *45). According to Burns (1949), the c o l l e c t i v e r e s u l t s show that hormones function as sex d i f f e r e n t i a t i n g p r i n c i p l e s , and are i d e n t i c a l with those of adult animals. This thesis has also been developed or supported by Dantchakoff (1950), Wolff (1950), l i t s c h i (1950, *51), and Jost (1953). I t i s opposed by Moore (1947, t50), p r i n c i p a l l y on the grounds that there i s no evidence for the secretion of sex hormones at a time when the gonads are sexually d i f f e r e n t i a t i n g . In the animal kingdom, the chemical identi t y , o f the natural sex hormones has been worked out only f o r the mammals. The female hormones have-been found to possess the steroid r i n g system, as do the 'testicular and adrenocortical hormones. They d i f f e r by vi r t u e of t h e i r phenolic character and poss-ession of fewer carbon atoms. The present, study attempts to evaluate the problem by supplying chemical evidence for the embryonic secretion of female sax hormones. Any such evaluation should consider the interference from hormones of maternal o r i g i n . This i n t e r -ference would not be expected i n animals whose embryonic development occurs outside of the body of the mother, which i s the case i n most f i s h . Since the histogenesis of sex 5 d i f f e r e n t i a t i o n has been worked out for the chum salmon Oncorhynchus keta, (Robertson, 1953), the same species, was selected f o r t h i s investigation* 6 I I . THE DISTRIBUTION OF FEMALE SEX HORMONES In the few i s o l a t i o n studies attempted f o r female hormones, large amounts of source material, were used. Thus MaeCorquodale, Thayer, and Doisy (1936) aspirated 400 l i t r e s of f o l l i c u l a r f l u i d from the graafian f o l l i c l e s of four tons of sow ovaries. A f t e r processing t h i s . f l u i d , they obtained 12 milligrams of estradiol-17^. Human and mare pregnancy urine have been the chief source for the i s o l a t i o n of the remaining natural, estrogens. These are estrone, e s t r i o l , estradiol-17«c, e q u i l i n , equilenin, and. h i p p u l i n . Because of the expense and hazards encountered i n processing tissues f o r an i s o l a t i o n of t h e i r hormones, most present-day research i s carried out e n t i r e l y on urine. The r e l a t i v e s i m p l i c i t y of t h i s medium and the development of microchemical methods for i t s assay have resulted i n an extensive l i t e r a t u r e , none of which i s conclusive (Heard and Saffron, 1949). The methods dp not appear to be applicable to extracts of blood, body tissues, feces or the urine of men (pincus, 1948). However a counterrrcurrent d i s t r i b u t i o n method developed by Engel et a l (1950) and Engel (1950) f o r urine analyses has been help-f u l to a study of the human placenta (Diczfalusy, 1953). I t i s not su r p r i s i n g to f i n d , therefore, the continued use of bioassay methods... Their advantages for detecting estrogenic substances i n microgram quantities from r e l a t i v e l y crude fra c t i o n s has greatly outweighed t h e i r disadvantages. Two of these are the d i f f e r e n t i a l a c t i v i t y of hormones and 7 •the presence of suppressing or augmenting substances i n the f r a c t i o n s being assayed (Emmens, 1950; Cohen and Bates, 1952), The evidence f o r the presence of female hormones e l s e -where i n vertebrates: i s dependent upon the extraction of a phenol f r a c t i o n that w i l l induce vaginal c o r n i f i c a t i o n i n the spayed mouse or rat (bioassays). I t should be noted that t h i s estrogen a c t i v i t y can, also be effected by petroleum and l i g n i t e (Doisy, 1934) and a variety of synthetic substances (Emmens, 1950) such as s t i l b e s t e r o l . However, the i s o l a t i o n of estrone from palm kernel extracts, and e s t r i o l from female willow flowers (see Fieser and Fieser, 1949) suggests a wide-spread occurrence of mammalian hormones, Fe l l n e r (.1925) was the f i r s t to investigate female hormones i n f i s h . He obtained an ovarian extract from an unstated species that would enlarge the rabbit uterus. He states that the aphrodisiac nature of caviar i s probably due to the presence of the female hormones indicated by his study, Weismann et a l (1937) extracted a phenolic f r a c t i o n from * swordfish (Xiphlas gladius) ovaries which gave an estrus response i n r a t s . The neutral male f r a c t i o n a r i s i n g from h i s extraction procedure was used as a control. I t did not give an estrus response. Donahue (1941) made an alcohol extract of the ovaries from winter flounders. After further process-ing, the extract was estimated to contain l e s s than ten rat u n i t s of estrogen. This i s equivalent to less than one microgram of pure estrone. The only other evidence f o r female hormones i n f i s h 8 appears to be that given by B r u l l and Ouypers (1954). They cautiously claim to have made a chemical detection of f o l l i c u l i n (estrone) and other "phenolsteroids" i n an extract of 420 ml, of urine obtained from 25 Lophius p i s c a t o r i u s . On the basis of the studies discussed there can be l i t t l e doubt that the estrogens-are present i n adult females of a wide variety of f i s h , i f not a l l of them. 9 I I I . MATERIALS A. B i o l o g i c a l Tissue Chum salmon (Oneorhynchus keta) and sockeye salmon (0,. nerka) were spawned at Cultus lake i n the autumns of 1952 and 1953 and incubated i n the University of B r i t i s h Columbia's f i s h hatchery. The chum salmon were removed fo r study at two stages i n the spring of those years. The f i r s t stage, taken three weeks before hatching, represented the period of germ c e l l m u l t i p l i c a t i o n ; the second stage was taken at a time when the sexes were well d i f f e r e n t i a t e d (yolk just absorbed, two months post-hatching) (Robertson, 1953). No e f f o r t was made to c o l l e c t the sockeye i n stages since they were being used as test material. A l l f i s h subsisted s o l e l y on t h e i r yolk sacs. The f i s h were used i n whole because removal of the embryonic gonads i s impractical. The Alsask Processors Limited, Edmonton, Alberta, deep-froze horse testes and air-expressed them i n dry i c e to the Vancouver Air p o r t . They were used immediately upon a r r i v a l . A second l o t of testes were obtained from Mr. Fos Hoy, Newton, B.G. B. Chemicals 1. Hormones. The c r y s t a l l i n e hormones used i n t h i s i n v e s t -i g a t i o n were supplied by the Ciba Company Limited and Ayerst, McKenna and Harrison Limited, Montreal, Canada. I t i s a pleasure to acknowledge t h e i r generosity ? The i d e n t i t y and '1. The nomenclature followed i s that of Fieser and Fieser (1949). 10 pu r i t y of these hormones were determined on a Fisher micro-melting point apparatus af t e r drying over phosphorus pent-oxide. The melting point ranges found (set i n parentheses) were i n v i r t u a l agreement with those of e s t r i o l (280-283.5°C,), estradiol-17^ (174-175°C.) and estrone (258-264°C.)• 2. Solvents. A l l solvents used were of reagent grades The hydrocarbons such as petroleum ether, benzene and methyl-cyclohexane were washed with concentrated s u l f u r i c acid and r e - d i s t i l l e d . (The petroleum ether was collected at 34-54°0>). Their derivatives (chloroform, O-dichlorobenzene, nitrobenzene and dichloromethane) were r e d i s t i l l e d . Commercial grade 95$ and absolute alcohol were r e d i s t i l l e d except when used i n making up color reagents. Here the absolute grade was refluxed i n 2,4-dinitrophenylhydrazine and t r i p l e d i s t i l l e d (Lappen and Clark, 1951). Methanol, r e d i s t i l l e d , was used i n preference to ethanol. Mormal butyl alcohol was r e d i s t i l l e d . Formamide was made ammonia free by standing over concentrated s u l f u r i c a c i d . Ether was used when peroxide free to 10% acid potassium iodide. Ethyl acetate was p u r i f i e d as outlined by Vogel (1952). 3. (Solor reagents. These were made up and used as described by Axelrod (1953), Rosenkrantz (1953), and M i t c h e l l and Davies (1954). 4. Apparatus. Ground glass equipment was used with water l u b r i c a t i o n . D i s t i l l a t i o n s i n vacuo were serviced by a water pump. The chromatographic apparatus consisted of 12 x 24 inch Corning glass cylinders and standard solvent tray assemblies (Cave and Company Limited). 11 IV. PRELIMINARY CHEMICAL SEPARATION OF AN ESTROGEN FRACTION A. P a r t i t i o n Methods. Estrogens may be removed from tissues by a var i e t y of solvents such as ethyl acetate (Kurzrok and Ratner, 1932), ethyl alcohol (West erf eld'; Doisy, et a l , 1938), n-butanol ('Venning et a l , 1937), chloroform (Weismann et a l , 1937), and acetone (Szego and Roberts, 1946). I t appears, as the F i e s e r , s (1949) state,-any organic solvent i s suita b l e . However where the estrogens are to be removed as a group Mathers (1942) points out that neither benzene or toluene are suitable f o r extraction of e s t r i o l , nor are the petroleum ethers useful f o r any estrogen. N-butanol i s a powerful extractor for both the free and conjugated estrogens, but i s not widely used (Emmens, 1950) perhaps because of i t s high b o i l i n g point (118°C.) and d i f f i c u l t i e s encountered i n maintaining i t f r e e of b u t y r i c a c i d . The choice of solvent depends larg e l y on the medium being extracted. For ti s s u e s , water miscible solvents such as alcohol are used* The present study on salmon embryos began with ethyl acetate i n order to keep f a t contaminants at a minimum l e v e l (Deuel, 1950). Subsequent steps i n the p u r i f i c a t i o n process showed that ethyl acetate extracted powerful emulsifying agents (mainly phospholipids) which interfered at a l k a l i p a r t i t i o n l e v e l s by forming emulsions. These could not be broken by s a l t i n g out (NaCl), centrifugation (4,000 r.p.m., 12 4- hour minimal), cr standing at 5°C. f o r as much as a week. This d i f f i c u l t y was lar g e l y overcome by extracting with acetone and removing the phospholipids. This was done by adding a few drops of ethanol saturated with MgCXg (Bush, 1952) to the acetone extract, followed by f i l t r a t i o n at -5°C. (Mortin et a l , 1952).. Subsequent p a r t i t i o n with an a l k a l i (M-KOH) gave an emulsion, but t h i s could be broken by centrifuging at 2500 r.p.m. f o r 15 minutes, Centrifugation, a wasteful process, was replaced by a gentle r o l l i n g movement of the separatory funnel containing the immiscible phases, for a minimum of 10 minutes, on each extraction. I t i s important to point out that while the p a r t i t i o n system may e f f e c t i v e l y overcome the emulsion problem, i t may not e f f e c t i v e l y remove an estrogen f r a c t i o n . A study of p a r t i t i o n c o e f f i c i e n t s of estrogens i n synthetic systems tabled by Bachman and P e t i t (1941), Mathers (1942), Friedgood and Garst (1950), and Engel (1950) indicated that the p a r t i t i o n of Friedgood and Garst (carbon tetrachloride:ether (18:1)-N-K0H) would e f f e c t i v e l y place the estrogens i n the a l k a l i phase. Moreover, comparatively strong polar substances that contamin-ate the estrogen f r a c t i o n could be washed out i n an ether extract with 9$ NaHCOg,in a butanol extract with »3M NagCOs, or a combination of these. Accordingly, Flow-sheet I was formulated and the estrogen f r a c t i o n s at the l e v e l s indicated were analysed by paper chromatography. At t h i s time no paper methods had 13 been devised that would effect a quantitative separation of, the female hormones. Since the present hormone fractions were not used i n subsequent studies, t h e i r further treatment i s included here. The p r i n c i p l e s of paper chromatography are outlined i n Section ".TV, page 28. B, Early Attempts to Effect An I s o l a t i o n By Paper chromato-graphy. 1. Use of Bush's (1952} paper adsorption method. S t r i p s (2 x 32 cm.).were impregnated with aluminum sulfa t e and spotted with salmon extracts (Flow-sheet I) to which female hormones were added. In mobile phase systems consisting of benzene-chloroform mixtures, i n 3:1, 2:1 and 1:1 r a t i o s (by volume) and benzene-acetone (19:1) no steroid zones were i d e n t i f i e d by the iodine reaction. The large amount of extraneous "biological material hindered r e s o l -ution. I t may be noted that M i t c h e l l and Davies (1954) did not f i n d Bush ,s method h e l p f u l to t h e i r studies of placental extracts. 2. Use of Zaf f aroni*s (1951) paper p a r t i t i o n method. Zaffaroni (-1951, *53) impregnated f i l t e r paper s t r i p s with propylene g l y c o l or fromamide i n various concentrations to serve as stationary phases for the resolution of adrenal hormones. Development of, the chromatograms was carried out i n mobile phases consisting of hexane, benzene and toluene. Attempts were made to use these systems for salmon extracts containing,female hormones (Flow-sheet I ) . .Since Bush (1952) 14 FLOW SHEET I ( 1 ) Lyophilized embryos ( 5 0 grams) Extracted with acetone (2 x 1 0 0 , 2 x 5 0 ml.) in a 12 hour period.. ( 3 ) residue discarded. ( 5 ) residue discarded. ( 7 ) NaHCOj phase Discarded after washing once with 2 5 ml. HOH which is added to KOH phase. (9) CGl^:Et20 phase Discarded after washing once with 2 5 ml. HOH which is added to KOH phase. (11) aqueous phase discarded. (2) acetone extract A few drops MgCl2 in methanol (saturated) added, cooled to -5°C. and f i l t e r -ed at -5°C. (k) acetone filtrate Evaporated in vacuo and residue transferred with Et20 (3 x 2 5 ml.) to separatory funnel. Et^O wash-ed with % NaHCOj (2 x 25 ml.). (6) Et20 phase Evaporated in vacuo and CCl^tEt^jO (18 t l , 7 6 ml.)added and extracted with N-KOH (5 x 35 ml.). (8) KOH phase Brought to pH 3 - 4 (HC1) and extracted with Et^ jO ( 4 x 80 ml. )« (10) EtgO phase Washed with HOH (2 x 2 5 ml.) which is discarded.. EtgO is evaporated to dryness in vacuo. (12) estrogen fraction Transferred in methanol to pyrex tubes for chromatography. 15 was detecting adrenal steroid zones on paper according to t h e i r property of absorbing u l t r a - v i o l e t l i g h t at a wave-length of 240 ny., the p r i n c i p l e was used here for female hormones, These absorb u l t r a - v i o l e t l i g h t at 280 . In the absence of a f i l t e r system d e l i v e r i n g t h i s wavelength, a min-e r a l lamp with a strong emission band at 226 uy. was used. I t was observed t h i s l i g h t source caused fluorescence •('as-opposed to absorption) of female .hormones i n concentration of the order of 100 micrograms per square centimeter on wet paper. Application of t h i s method to the detection of the hormones on paper f a i l e d because i n t e r f e r i n g substances of salmon o r i g i n fluoresced much better. The conundrum a r i s i n g from these early attempts to sep-arate the female hormones on paper was solved by L.R. Axelrod (1953)* G. Column chromatography. Some carotenoids are found i n a l k a l i f r a c t i o n s of f i s h o r i g i n (Bailey, Carter, and Swain, 1952). Since t h i s f r a c t i o n also contains the estrogens,, an attempt was made to eliminate the carotenoids by column chromatography. Stimmel (1946) successfully extracted the estrogen present i n l a t e preg-nancy urine by using an alumina column. According to the discussion of a paper presented by Bauld (1952) at least two independent investigators (Swyer and Bates) were not able to duplicate Stimmels(1946) r e s u l t s , nor apparently, could Stimmel duplicate h i s own findings with another batch of alumina. Bates, however, was successful when Stimmel*s o r i g i n a l alumina and apparatus were used, Bitman and Sykes (1953) demonstrated that an a l k a l i n e c e l i t e column would remove estrogens i n a synthetic system, This method f a i l e d i n the presence of chemical contaminants of b i o l o g i c a l o r i g i n when attempted by M i t c h e l l and Davies (1954), An a l k a l i n e c e l i t e column of d i f f e r e n t preparation i s being used by Heard (1954) to e f f e c t p u r i f i c a t i o n of estrone i n pregnant mare urine. The present study used Sammuels (1949) method as a work-ing basis. Alumina of 80-200 mesh size was made more r e -tentive by heating at approximately 500°C. f o r 4 hours i n covered crucibles; between 2 bunsen burners. Glass tubing (1 x 30 cm.) was plugged at one end with 3 cm, of f i n e glass wool, A glass rod flattened at the t i p just s u f f i c i e n t that i t would f i t the tubing, served to tamp the alumina gently into the column over a distance of 20 cm. The open end of the column was then f i t t e d with a 50 ml, capacity dropping funnel which delivered successive volumes of el u t i n g solvents. The column was washed once with 50 ml, of petroleum ether before use. An extract was prepared from salmon embryos by evaporat-ing an ether extract (Flow-sheet I , step 3) and suspending the residue i n petroleum ether. After adding the suspension to the uppermost surface of the alumina., successive 50 ml. volumes of petroleum ether, 10$ chloroform i n petroleum ether, I? 25% chloroform i n petroleum ether and f i n a l l y 50% chloroform i n petroleum ether,, were run through the column at a flow rate of approximately 1 ml. per minute. The column was kept wet with the solvents at a l l times. According to Sammuels, estrogens should he present i n the f i n a l volume* Under the conditions of t h i s experiment, as much as 300 micrograms of added estrogen could not be detected by the Kober (1931) t e s t . I t was observed that at least two "carotenoids* remained on the column. They were located as zones immediate-l y below the column head. The f i r s t of the zones was red i n color and the second, yellow. Their combined distances amounted to approximately 3 centimeters of the 20 centimeter column. Varying the nature of the e l u t i n g solvents by s u b s t i t u -t i o n of s i m i l a r series of benzene-ether and benzene-methanol combinations did not a l t e r the p i c t u r e . The substitution of the alumina by a s i l i c a - c e l i t e s l u r r y (2:1 by weight) i n petroleum ether also f a i l e d to give a Kober detectable estrogen f r a c t i o n . The large amount of residue obtained from every f r a c t i o n eluted from the columns showed that better p u r i f i c a t i o n methods were required to effect a recovery of estrogens. Hb attempt was made to use these f r a c t i o n s i n paper p a r t i t i o n systems* D. D i a l y s i s . Szego and Roberts (1946) and Roberts and Szego (.1946) 18 were the f i r s t investigators, to use the p r i n c i p l e of d i a l y s i s (Thomas, 1934) to recover a steroid hormone f r a c t i o n . Using bioassay measurements they showed that the sodium s a l t s of e s t r i o l , estradiol-^17/3, and estrone dialyzed into water and that the method provided a means of demonstrating estrogenic a c t i v i t y i n blood plasma. These results contrasted thos© of Rakoff, Paschkis, and Cantarow (1943), who found that estrogens i n pregnancy serum would not pass through a collodion membrane by u l t r a f i l t r a t i o n . Zaffaroni and Burton (.1953) and Zaffaroni (1953) showed that adrenal hormones would dialyze independent-l y of one another into a 40$ methanol solution from c i t r a t e d blood. The e f f i c i e n c y of t h e i r method was greatly increased by adding chloroform which continuously extracted the hor-mones as they diffused into the outer system. The conditions f o r optimum d i a l y s i s of female hormones were therefore investigated. Since Szego and Roberts had d i f f i c u l t y i n d i a l y z i n g free e s t r a d i o l - ! ? ^ f or bioassay, t h i s hormone was used as a standard i n the chemical assay to be described. 1. Synthetic system, a ) Dialyzing without s t i r r i n g . Visking sausage casing (diameter 4,2 cm,) was cut into 30 cm. s t r i p s and placed i n 3 l i t r e s of water saturated with the solvents used, i n d i a l y s i s . After £ - 1 hour the s t r i p s were removed and double-knotted at one end. These were washed twice by f i l l i n g and emptying the bag with the s o l u -t i o n already used. Two washes were made with water, the 19 second wash being used to check for leaks. Each bag was f i l l e d with 10 ml. of Gothlin's saline s o l u t i o n , 1 ml. methanol containing 50 /^g. of hormone, and 40 ml, of water. The contents were sealed by a double knot at the open end of the casing. The r e s u l t i n g d i a l y z i n g bag was immersed i n 250 ml, of water, a volume which i s 5 times greater than that inside the bag* This r a t i o was suggested by Szego and Roberts (1946). Twenty-five ml, of extracting solvent were then added. Butanol was used exclusively at pH values greater than 7, since chloroform may not remove the hormones i n t h i s range. Afte r 7 days (168 hours) the d i a l y z i n g bag was removed and rinsed with 10-15- ml. of water into the dialy s a t e . This was neutralized with HC1 (pH 4-5) on alkacid test ribbon and extracted with chloroform (.4sx 75 ml.). The contents of the d i a l y z i n g bag were emptied into a beaker and the inner casing w a l l rinsed with 10-15 ml* of water into the same beaker. This residuum was treated as above, except that 4 x 25. ml, volumes of chloroform were used to extract the aqueous phase. After evaporating the chloroform i n vacuo, the residue was transferred i n methanol (3 x 5 ml,) by a medicinal dropper to a pyrex tube. Washings were used. The methanol was evaporated i n a water bath at 75°C. and the residue dried over phosphorus pentoxide. The hormone content was then assayed by the Kober test as modified by Venning et a l , (1937) except that the reagents were used i n reduced volumes to make 20 a f i n a l volume of 5 ml. The o p t i c a l density at 522 m/t/. was read on a Bookman model DtT spectrophotometer f i t t e d with a tungsten l i g h t source, b) D i a l y z i n g with s t i r r i n g . The Visking sausage casing was cut into 50 cm. s t r i p s and washed i n the manner described except that the casing was double-knotted at a s i n g l e locus. I t was then made 39 cm. long from knot to open end and fi x e d with linen thread (40 guage). to the rimmed end of a cut pyrex tube (1.3 x 2.4 cm.) f i t t e d into a bored rubber stopper. This d i a l y z i n g bag was placed inan r tExax n 1000 ml. graduate cylinder containing 500 ml. of water. The synthetic medium, consisting of 25 ml. of Gothlin's saline and 155 ml of water, was poured into the casing using a glass funnel. E s t r a d i o l was added by pipe t t e . The outside medium was then made up to volume (350 ml. of water and. 50 ml. of extracting solvent). A glass rod flattened to a button shape at the t i p , and reaching to within 1 cm. of the bottom of the bag served to s t i r the contents, and thereby decrease the time required for d i a l y s i s . The rod was driven by a power s t i r r e r set at i t s slowest speed. After 48 hours s t i r r i n g the casing was removed and rinsed on the outside with approximately 20 ml. of water into the cylinder containing the dialysate. I f butanol had been added to the outside medium, the volume was reduced to 500 ml* i i i vacuo a f t e r bringing the aqueous medium to pE 4-5. This procedure removes most of the butanol as a water azeotrope. The remaining f r a c t i o n was extracted with 4 x 100 ml. of chloroform. 21 The residuum i n the d i a l y z i n g bag was removed with washing and extracted with 4 x 50 ml. of chloroform, c) C a l i b r a t i o n curve f o r estimation. High values obtained i n the f i r s t group of experiments (numbers 1, 6, 7, 8, 11, Table I) showed the necessity f o r blanks prepared from dummy runs. A c a l i b r a t i o n curve was therefore prepared by po.oling chloroform extracts of d i a l -ysatea i n 6 dummy runs. After evaporating the chloroform, the residue was dissolved i n methanol and divided into 6 portions. Different amounts of hormone were added to 5 of these portions, the s i x t h serving as a blank; A Kober reaction was carried out on a l l tubes. The c a l i b r a t i o n curve obtained i s shown i n figure 1. d) • Hormone recoveries i n the synthetic system. The r e s u l t s from t h i r t y experiments (Table I) showed considerable v a r i a t i o n at s i m i l a r pH values and through-out the pH range attempted. This v a r i a t i o n i s d i r e c t l y the res u l t of the heterogeneity appearing i n blanks run through-out the experiments, and also, therefore, i n the hormone runs themselves. Thus a blank of Kober reagents gave the following values for blanks prepared from dummy runs: pH System Op t i c a l Density ca 14.0 Na 2GQ 3 - butanol .033 ca 6.8 HOH - butanol .278 ca 6.8 HOH - butanol • - - - , .038 ca 6.8 HOH - chloroform ,185 ca 6,8 water ,046 '4.0 HOI - butanol ,126 2 2 . M I C R O G R A M S Figure 1. Calibration curve for estradiol-17^ in a synthetic system© TABLE I . RECOVERY OF 50 MICROGRAMS OF ESTRADIOL^!?/* BY DIALYSIS IN A SYNTHETICAL SYSTEM* No. of Duration of Micrograms, Ratio Percent Experiment External Solvent D i a l y s i s recovered recovered out Days In Out In/out 1 ,3M NasC03 (pH ca 14) 7/ 14.4 73.9 11.300 147.8 s 2 *• Butanol 7 9,9 14.2 1.430 28.4 3 7 3.5 45.9 13.100 91.8 4 2 14.0 64.4 4.600 128.8 5 2 7.0 32.8 4,665 65.6 6 NagCOs.NaHCOs |pH 10.1) 7 9.7 53.4 5.505 106.8* *• Butanol 7 H3B03:Na0H (pH 9,2) ? 11.5 64.8 5.630 129.6* * Butanol 8 9% NaHC03 CpH ca 8.2) 7 5.8 66.1 11.397 132.2* 9 f. Butanol 7 3.5 13.4 3.828 26.8 10 7 3.0 36.2 12.067 72.4 11 HOH (pH ca 6,8) 7 4.5 59.3 13.178 118.6* 12 * Butanol 7 7.7 33.9 4.403 67.8 13 7 — 47.2 _ 94.4 14 7 — 27.0 54.0 15 7 1.3 _ _ _ 16 2 4.9 49.0 0.100 98.0 17 2 - 13.5 ' - 27,0 18 HOH *• Chloroform 7 4.8 27.6 5.750 55.2 19 7 4.2 25.9 6.167 51.8 TABLE I continued No. of Experiment External Solvent Duration of Di a l y s i s Micrograms recovered Ratio Percent recovered out Bays In Out In/Out 20 21 HOE «- Chloroform: Ether 2 2 4.0 11.2 34.7 46.0 8.675 4.107 69.4 92.0 22 23 HOH (pH ca 6.8) 7 ? 4.0 7.7 22.0 14.8 5.500 1.922 44.0 29.6 24 25 Nasci trate:NaOH:EC1 (pH 5.2) ¥ Butanol 7 7 13.4 7,2 35.8 18.3 2.672 2.542 71.6 36.6 26 K phthalate:HG1 (pH 4.1) * Butanol 7 3.4 30.0 8.824 60.0 27 28 HC1 CpH 4.0) ¥ Chloroform: Ether 2 2 9.8 9,0 24.3 41.7 2.479 4,547 48.6 83; 4 29H 30** HOH * chloroform 7 7 1.4 1.3 10.8 11,9 7.714 9,154 21.6 23;8 * Blank of chemical reagents only E s t r i o l 25 Ihere the hutanol system could serve as a blank, the chloro-form system gave o p t i c a l densities of •060, .££3, and 0. S i m i l a r l y , when the chloroform system served as a hlank, the hutanol systems gave densities of •210, 0, 0, .083, and .017. Differences between s t i r r e d and unstirred blanks were ..068 {against a s t i r r e d blank),and »017 and .083 (against.an unstirred blank). In face.of such an impasse the synthetic runs were discontinued and salmon material introduced. 2. B i o l o g i c a l Systems. The rather consistent values obtained i n the chloro-form-water and chloroform:ether-water systems (experiments 18-21, Table I) suggested the use of t h i s system with s a l -mon mat e r i a l . Since the carbonate-butanol system (experi-ments 2-5, Table I) appeared to give higher values, i t was also included i n the t e s t . I t may be noted, however that a 7 day run i n the two systems and i n the absence of d i a l y z -ing casing favored the chloroform system. Here a 71$ recovery was made from 50 //g. e s t r a d i o l as compared to 56$ from butanol. Both systems were run i n volume r a t i o s suggested by s 2 . Ether was added to chloroform i n view of experiments 29, 30, (Table I) which show chloroform does not readi l y take up e s t r i o l . 26 Zaffaroni (1953) f o r the adrenal steroids. Thais 60 g. of minced salmon embryos i n 120 ml. of water were dialyzed under s t i r r i n g . I t was observed that the butanol became yellow with impurities whereas the chloroform-ether system remained c o l o r l e s s . The procedure f i n a l l y adopted was as follows. The casing was washed i n a water-95$ ethanol solution C2:l) and f i t t e d to the apparatus described on page 20 and then 100 ml. of chloroform:ether (20;1) were placed in-a graduated cylinder and made up to 850 ml. with water. The tissue to be dialyzed was put through a meat grinder while i n the frozen state. S i x t y grams of minced tissue was mixed i n 120 ml. of water and placed i n the di a l y z i n g bag which was then s t i r r e d for 48 hours at room temperature, Figure 2, After t h i s time the di a l y z i n g bag was removed, rinsed, and discarded. The d i a l -ysate was brought to pH 4-5 and transferred to a separatory funnel. The chloroform:ether already present was used to make an i n i t i a l extraction. The p a r t i t i o n method tiuen used i s described i n Flow-sheet I I . The extra volume, of added ether was used to prevent loss of e s t r i o l when a washing was made with sodium bicarbonate. The estrogen f r a c t i o n obtained by t h i s method was analyzed by paper chromatography (page 35)* Figure 2. Apparatus used i n d i a l y s i s 28 V. PAPER PARTITION CHROMATOGRAPHY OF SALMON EXTRACTS A* Introduction. Paper chromatography i s a microchemical method f o r i s o l a t i n g the components of a complex mixture. I t consists of placing the mixture to be chromatographed on s t r i p s of f i l t e r paper at a measured distance from one end. This end i s immersed i n a solvent (the mobile phase) which d i f f -uses down the paper. In most steroid hormone studies the paper i s previously impregnated with a highly polar solvent or s a l t (the stationary phase). The chemical compounds mak-ing up the residue then move at d i f f e r e n t rates down the paper according to t h e i r molecular differences. The process i s regarded as a large number of consecutive p a r t i t i o n s of the substances being chromatographed between two phases (Cassidy, 1948). In t h i s respect paper chromatography i s not essent-i a l l y d i f f e r e n t from ordinary chemical p a r t i t i o n methods. Since the success of these chemical methods depend upon.the concentration of the substances being p u r i f i e d to the con-taminants present ( o i l s e t c ) (Engel, 1950) i t must also follow that these conditions are l i m i t i n g factors i n paper chromatography. Hence only l i m i t e d amounts of material can be resolved i n a paper system. As these amounts are made small (portioning into l o t s , spreading the spot on larger widths of paper) the a b i l i t y to detect the substances being p u r i f i e d becomes a l i m i t i n g factor ( d i l u t i o n f a c t o r ) . I t i s because of these p r i n c i p l e s that preliminary p u r i f i c a t i o n methods were used (Section IV). 29 B. Reproduction of Axelrod's (1955) Method Using E s t r i o l , Estradiol-17jg t and Estrone, 1) Procedure. Since t h i s method i s b r i e f l y outlined by Axelrod £1953), i t i s enlarged upon here to show how i t was used, and to confirm the technique, A s t a r t i n g l i n e was ruled (2-H pencil) 11 cm, from the edge of a 5 x 46 cm. s t r i p of Whatman No. 1 f i l t e r paper* Tlie a t r i p was used as such or divided into 4 columns of 1 centimeter diameter beginning 2 centimeters above the s t a r t -ing l i n e (Burton et a l , 1951). After being washed i n water 12-24 hours, the s t r i p was transferred to 95$ ethyl alcohol f or 24 hours. I t was dried and impregnated with 50$ formamide i n methanol (Y/V) and blotted between f i l t e r paper previously cleaned. The methanol was evaporated i n a i r and !; the s t r i p l a i d on a sheet of glass so that the ruled s t a r t i n g l i n e f e l l over a glass gate 1 cm, high. The gate was con-structed of 1 cm. glass rods bent to form a rectangle 4 x 10 cm. The paper s t r i p was covered below the gate with the f i l t e r paper used to blot i t . A glass box placed (open end down) below the s t a r t i n g l i n e and on the covering paper served to steady the hand when hormone fr a c t i o n s were applied to the s t a r t i n g l i n e by a 1 or 4 m i c r o l i t r e pipette. A stream of a i r , directed frequently on the s t a r t i n g l i n e , helped to prevent enlargement of the spot. The paper s t r i p was trans-ferred to tanks for development of the ehromatogram. The tanks contained 250-300 ml. of the developing solvent which permeated a f i l t e r paper l i n i n g . A further 30 100 ml, of the developing solvent, saturated with the s t a t -ionary phase, was added to troughs, suspending the paper s t r i p into the- tank. Sealing was effected by a glass cover made a i r - t i g h t with a paste of starch i n glyc e r o l and a heavy weight. Three tanks were used and for each, the condition of the developing solvent was maintained by replacement* The methylcyclohexane-formamide system (hereafter referred to as tank I) served to make 24 hour runs at 20±2°C, on paper just spotted. This removes i n e r t impurities.from the hor-mone f r a c t i o n s . Chromatograms containing estradiol-17£ and estrone were then transferred f o r a 12 hour run i n the Or-dichlorobenzene-formamide system (hereafter referred to as tank I I ) , Paper containing the e s t r i o l f r a c t i o n was transferred- from tank, I f o r resolution in.a. dichloromethane-formamide system (tank I I I ) . The running time here was 10 hours. On removal, the chromatograms were dried by a warm a i r fan (30°C,) approximately 10 hours and then by a cold fan (room temperature) for 10-20 hours. The chromatograms were considered dry, when samples of the paper from above the o r i g i n did not char_..in. 15% fuming s u l f u r i c a c i d . This i s a very important test and devised i n the present i n v e s t i g -ation to overcome negative r e s u l t s obtained with, color indicators: used when the paper only appeared to be dry. 2) Detection of estrogens, Axelrod l i s t s 7 color reagents for the detection of 31 the hormones. Of these, 15$ fuming s u l f u r i c a c i d , benzoyl chiloride-zince chloride, and ni t r o u s acid-mercuric n i t r a t e (Millon's reagent modified) give eonsis.tant r e s u l t s (and were therefore used i n t h i s study). The phenolsulfonate-phosphoric acid test was not attempted. Rosenkrantz (1953) antimony t r i c h l o r i d e i n nitrobenzene was found very useful. The most sensitive i n d i c a t o r was F o l i n and Ciocalteau's phenol test as modified f o r paper chromatography by M i t c h e l l and Davies (1954). This test was not available u n t i l very l a t e i n these studies. 3) P o s i t i o n of the hormones. The positions of e s t r i o l , estradiol-17yS and estrone, and a mixture of these are shown (Table II) af t e r 24 hours i n tank I . . I t i s clear that these hormones do not stay exactly on the s t a r t i n g l i n e as claimed by Axelrod. Attempts to adjust t h i s observation by chromatography i n petroleum ether, cyclohexane, and cyclohexane-methylcyclohexane (1:1, -Y/V) did not a l t e r the pict u r e . I d e n t i c a l positions also were found when undiluted formamide was used as the s t a t i o n -ary phase. Observations made on a mixed ehromatogram carried over and developed i n tank I I (12 hours) showed estradio 1-17/5 and estrone to move i n accord with migration rates expected of t h i s system. However e s t r i o l did not remain stationary as suggested by Axelrod. E s t r i o l transferred from tank I to tank I I I for development gave the re s u l t s defined by Axelrod. 32 Table I I . The location of free steroids ( i n centimeters from the s t a r t i n g l i n e ) a f t e r chromatography. Development i n FOrmamide-methyl-eyclohexane (Tank I) Fo rmami de-O-dic h l o r o - " benzene ^ (Tank I I ) * Dichloro-methane ^ (Tank III}3™ Starti n g l i n e E s t r i o l Estradiol-17^ Estrone Mixture. Time (hours) 0.0-G.5 em. • 1.3- 4.3 cm. 1.4- 4.7 cm. 1.4-4.6 cm. 1.3-4.8 cm. 24 8.0-12.1 em. 14.5-19.5 cm. 34.4-40.2 cm. 12 3.7-6.9 cm. 10 f i x t u r e of hormones, aft e r development i n Tank I . ^ E s t r i o l a f t e r development i n Tank I . 33 4) E l u t i o n of the hormones. After detection of the spots on measured 1-5 mm. s t r i p s cut from the ehromatogram, the hormones remaining on the paper were eluted with methanol (2 x 5 ml., 2 x 1 hour) into pyrex tubes. The eluates were transferred with a medicinal dropper to sintered glass f i l t e r s (medium porosity) and the f i l t r a t e s collected i n pyrex tubes. Washings were made throughout t h i s transfer. The methanol was concentrated to 5 ml. and the amount of hormone determined in. a Beck-man DU spectrophotometer having an u l t r a v i o l e t light.source. Blanks and standards were prepared i n the same way. 5) Elimination of background material. Despite previous washing of the f i l t e r paper, impun-i t i e s of paper o r i g i n were present i n the hormone eluates. These interfer r e d with o p t i c a l density values. This d i f f -i c u l t y was overcome by preparing a blank from the paper being ehromatographed (Axelrod, 1953). The results, obtained i n the present study are shown i n figure 3. A small v a r i a t i o n at the absorption maximum i s apparent (curves C, D, E). This i s the r e s u l t of the wide margin of error introduced by using small amounts of hormone (10 yc/g./ml.), According to Eriedgood and Garst (1950) u l t r a -v i o l e t measurements should not be attempted when hormone concentrations are less than 15 //g./ml. 6) Absorption Curves. Umberger and Curtis (1948) used a s u l f u r i c acid r e -action to i d e n t i f y i n d i v i d u a l estrogens. S p e c i f i c absorpt-ion curves were shown for each estrogen when measured i n the from chromatographed estradiol-17^ (D), estriol (C), and estrone (E)« Curve A is estradiol-17B against pure methanol. Curve B is a prepared blank against pure methanol* 35 400-550 m^ . range. Axelrod modified the method by eliminating the heat period of 12 minutes. The hormones were stood i n s u l f u r i c acid for one and three quarter hours (minimum) i n the dark. The absorption curves were then determined at wave-lengths from 220-600 m//. The present r e s u l t s from chromatographed estrogens are shown i n Figure 4. E s t r i o l gave a sharp peak at 310 myt/., i n agreement with reported values. However a., .small peak heretofore recorded at 452 m//. was evident at 440 myu. i n t h i s study. Estradiol-17p gave absorption maxima at 310, 370, 430, and 452 m^ . Only the peak of 310 agrees with Axelrod's r e s u l t . However those at 430 and 452 were obtained by Umberger and C u r t i s . Estrone showed maxima at 300 and 450 i n agreement with previous findings. Since i t i s w e l l known that both the brand and s p e c i f i c gravity of s u l f u r i c acid as well as the concent-rations of hormone affect the findings (Umberger and C u r t i s , 1948) i t i s concluded that serviceable r e s u l t s were obtained from the 6 //g./ml. (as opposed to Axelrod's 60-80../'g./ml.) • C. Application to salmon Embryo Dialysates* Minced salmon embryo (60 g.) containing 100 y g . quantities of added e s t r i o l , estradiol-17^, and estrone was dialyzed and the extract (Flow-sheet II) examined chrom-atographically. After 24 hours development i n tank I , a yellow zone of impurities occupied a p o s i t i o n s i m i l a r to a mixture of estrogens i n a pure system. Color tests made 56 .15 O" > co Z .lOO« LU Q _ J < y a O 050-.OOO l 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 MILLIMICRONS Figure 4. Absorption spectra of estradiol-17fJ (A), estrone (B), and e s t r i o l (C), i n concentrated sulfuric acid© 37 FLOW SHEET IX (1) dialysate Brought to pH 4^5 and extracted with CHCI3 . Et 2G {20 : 1) (6 x 100 ml,). I ~ (3) aqueous: phase discarded. ('6) aqueous phase discarded. (2) CHCI3 , E t 2 0 100 ml, EtgO added and washed with 9% .NaHC0 3 (2 x 100 ml.) and then with HOH (2 x 100 ml.). (4) CHCI3 , EtgO Evapo ra t ed t o d ryn ess i n vacuo. (5) t o t a l estrogen f r a c t i o n Transferred i n methanol to pyrex tubes f o r chromatography. 38 at t h i s stage did not indicate that hormones were present on either side of the yellow zone. A s i m i l a r run was made on a second l o t except.that i t was carried over for further development i n tank I I . The yellow zone noted previously had migrated 2-3 cm. more down the paper. Color tests made on sample s t r i p s f a i l e d to indicate any one of the hormones. In another run areas before, a f t e r , and including, the yellow zone were eluted and examined spectrophotometrically at 270-300 mp. with negative r e s u l t s . These were confirmed by the Kober test made on the eluates. This f a i l u r e suggested that larger amounts of hormone would have to be added i f a recovery was to be effected. However, since the present l e v e l s being used were.5 times greater than any known source of female hormone, further recovery experiments were regarded as being of no value to the present study. On t h i s basis i t may also be considered that d i a l y s i s i s not an effe c t i v e method for a chemical assay of hormones i n . b i o l o g i c a l t i s s u e . This generalization, however, i s unwarranted- i n view of the organ systems ( l i v e r , kidney, blood etc.) present i n the f i s h embryos. Such syst-ems i n mammals are believed to contain substances which destroy or inactivate as much as 90% of added.estrogen (Pearlman, 1948; Paschkis and Rakoff, 1950; Fishman, 1951). Accordingly the method was evaluated by assaying e s t r a d i o l -17£ and estrone present i n horse testes, . ' 39 D. Application to Horse Testes Dialysates. Horse testes were selected for assay p a r t l y because of the large amounts of estradiol-17/& and estrone known to be present, and p a r t l y because only one chemical assay had been made previously ( B e a l l , 1940). Like most i s o l a t i o n studies of hormone i n t i s s u e , B e a l l used a large amount of testes (28 kg.) from which the hormones were extracted with 280 l i t r e s of alcohol. After p u r i f i c a t i o n he obtained 210 //g. of estradiol-17^ and 360 yfg. of estrone per kilogram of t i s s u e . These values suggested that the d i a l y z i n g technique would y i e l d r e s u l t s on approximately 100 g. of horse testes. Three 60 g. l o t s of minced testes from 3 d i f f e r e n t horses were dialyzed and estracted for separation by paper chromatography (Flow-sheet I I I ) * The estradiol-estrone f r a c t i o n was portioned into two l o t s from 20 ml. of methanol. One l o t was tested by the Eober reaction (which was positive) and the other, ehrom-atographed. A sample s t r i p from"the ehromatogram was tested for the presence of hormones by the Mitchell-Davies (1954) phenolic t e s t , now a v a i l a b l e . Two zones reacted to the t e s t . They did not occupy pos i t i o n s , however, s i m i l a r to pure estradiol-17^ and estrone run i n p a r a l l e l . I t i s w e l l known that b i o l o g i c a l impurities a l t e r migration rates (Rosenkrantz, 1953) and i t i s presumed that t h i s explanation of sobserved r e s u l t s applied» The zones remaining on. the ehromatogram were eluted and assayed spectrophotometrically for the presence of the 40 FLOW SHEET I I I (1) 3 x 60 g. minced horse testes. (.2) dialysate Brought to pH 4-5 and extracted with CHCl 3.Eta0 (.20:1) (6 x 100 ml.). I (4) aqueous phase discarded, I (3) CHCl 3:Et20 ' 100 ml. EtgO added and washed with 9% NaHC03 (2 x 100 ml.) and then With HOH (2 x 100 ml.), (6) aqueous phases discarded, (5) CHCl3:EtgO Pooled. In vacuo to dry: ness; 2 ml. ethanol and 250 ml. benzene added ( i n ordler) • Extracted with EtgO (4 x 200 ml.), (9) aqueous phase Extracted with EtgO (4 x 150 ml.). (7) benzene phase Evaporated i n vacuo to dryness. (10) aqueous phase (11) Et g o (8) e s t r a d i o l and estrone discarded i n vacuo f r a c t i o n to dryness Transferred i n methanol to pyrex tube for chroma to graphy. (12) e s t r i o l f r a c t i o n Transferred to pyrex tube i n methanol f o r chromatography. 41 phenol group (present i n a l l female hormones and i n many-other substances) already indicated by the Mitchell-Davies • t e s t . No such group was observed. These contradictions, suggested that background material obscured the absorption peak expected at 280 m/s. A Kober test was therefore applied to each eluate. Each gave a clear, deep pink color, a p o s i t i v e reaction. The concentrations of the Kober p o s i t i v e material i n the eluates were measured against the chromatographed blank, estradiol-17^, and estrone by the method of Yenning et a l (1937), The r e s u l t s are shown i n Table..Ill, . Small amounts of hormone ( i n ord.er...of 10 yyg./90 grams of testes), were successfully extracted* Table I I I . Concentration of hormones recovered from dialysates of horse testes. Materials Wave] Length Difference 522-420 420 522 Blank E s t r a d i o l standard .089 .718 .629 52.1 Estrone standard .036 .585 .549 . 52.1 Estradiol,, horse. .209 .316 .107 . .. . 8.862/90 g. Estrone horse .211 .348 . .137 12.999/90 g. The p o s s i b i l i t y remained that the substances i d e n t i f i e d as estradiol-17^ and estrone i n these horse testes might be male hormone despite the chemical differences between male and female hormones.- - Hence androsterone and testosterone were chromatographed i n concentrations (20 /Jg.) twice that of the estrogens recovered. No trace was found by the Mitchell-Davies t e s t . A Kober test made on 20 /^g. amounts of c r y s t a l l i n e 42 androsterone and testosterone gave pale orange and v i o l e t colors, respectively. The pink color of the estrogens did not, therefore, develop. In resume, the evidence i d e n t i f y i n g estradlol-17^ and estrone i n the present study i s : 1. Both hormones were isola t e d from horse testes by B e a l l (1940). a. Both hormones were located on a paper p a r t i t i o n system designed for female hormones. 3. The Mitehell-Davies test (1954) requires a phenol group i f the substances under test are i n micro-quantities. The female hormones recovered s a t i s -f i e d these conditions. 4. The Kober test (Kober, 1931; Brown, 1953; Bauld, 1954) requires an oxygen group at the 17 p o s i t i o n and an available H at the 16 po s i t i o n (Bates, 1952.)., Such positions are t y p i c a l of sex hormones. 5. Male hormones did not give the Kober color s . The t o t a l e s t r i o l f r a c t i o n from 180 g, of horse testes was run on a 1 cm. s t r i p (page 29). After development i n tanks I and I I I , a Mitchell-Davies-rpositive zone.was evident close to the s t a r t i n g l i n e . The zone however did not give a Kober color. Furthermore, measurements were not possible against a blank and control run of e s t r i o l (Table I V), Table IV. Spectrophotometry assay of the e s t r i o l f r a c t i o n from horse testes. Materials Wavelength Difference 522-420 Amount 420 522 Blank mm mm E s t r i o l standard .013 .348 .335 • 22,9 "Mi t che11-Davi es" 0 0 Zone 43 Thus,, no e s t r i o l was found, an observation i n accord with Beall*s (1940) r e s u l t s . F i n a l l y , the entire- experiment was repeated on a si m i l a r amount of horse testes. These were from a d i f f e r e n t source, and of much smaller s i z e (approximately 150 g, per testi c l e - as" compared to 330 g. i n the previous experiment). In t h i s test the dialysate extract was separated into male and female f r a c t i o n s using the KOH/CCl^EtgO p a r t i t i o n (Flow-sheet I ) . The estrogen portion, however, was not separated into estradiol-estrone and e s t r i o l f r a c t i o n s . The male f r a c t i o n did not respond to Kober*s t e s t . The entire female f r a c t i o n from 180 g, of minced ti s s u e was chromatographed (as compared with 90 g. previous^-ly); • Only the e s t r a d i o l zone was located by the phenol t e s t , and t h i s appeared to be i n much lower concentration than found e a r l i e r . I t was eluted and rechromatographed f o r further p u r i f i c a t i o n i n order to obtain i t s absorption curve i n s u l f u r i c a cid. The material could not, however, be located a f t e r t h i s procedure. This f a i l u r e to reproduce the previous r e s u l t s could have been caused by: 1. The smaller testes from probably younger horses, 2. Storage period of the testes which was at least two w.eeks longer than those f i r s t used. For placentae,. M i t c h e l l and Davies (1954) point out processing should occur within 30 minutes of deli v e r y , 3. Estrone may not have been present i n the testes. For example, Diczfalusy (1953) found e s t r a d i o l -Vip i n only 2 of 6 placentae examined. -44 VI. A 'FURTHER SEARCH FOR FEMALE HORMONES IN SALMON EMBRYOS. A. The Mitchell-Davies P a r t i t i o n Method. Because, of the apparent or possible destruction of hormones when salmon embryos were being dialyzed, use was made of a technique recently developed by M i t c h e l l and Davies (1954) f o r the recovery of estrogens i n placentae. This method, based on previous studies by Engel (1950), offered the further advantage of recovering conjugated and protein-bound estrogens. Their paper p a r t i t i o n analysis was not attempted i n view, of Axelrod*s paper method already worked out. Their method as applied to salmon embryos i s shown i n Flow-sheets IV and IVa. I t w i l l be noticed that an extra step i s included just p r i o r to the a l k a l i p a r t i t i o n (step 20). This was found necessary because of the emulsion formed when a 10 ml. aliquot of EtgjQrCC^ was partitioned with 5 ml. of N-KOH (experiment 2). Moreover, the N-NaOH used by M i t c h e l l and Davies was replaced by N-KOH i n order to effect a better p a r t i t i o n (Friedgood and Garst, 1950). The extraction of the hormones from an a l k a l i n e solution at pH 9 (Engel, 1950) i s the most outstanding feature of the method. . This was done with, a Beckman pH meter i n the present experiments, but "hydrion" paper was used by Engel and presumably by M i t c h e l l and Davies. Under these conditions the entire procedure was carried through without interference by emulsions. h5 FLOW SHEET IV (1) Minced embryos (kOO grams) (Lyophilized embryos (220 grams) Extracted with 8 $ (V/V) ethanol (2 x k50 ml.) and 95% (V/T) ethanol (2 x kOO ml..). (2) ethanol extract Cooled at k°C. 12 hours, filtered at k°C (3) residue Extracted with n-butanol (3 x k00 ml.), (7) residue fraction A Pooled with fraction B. (k) ethanol extract L _ (5) butanol extract , I (6) Combined and evapor-ated to dryness in vacuo. Suspended in kOO ml. waterj ex-tracted with h x 200 ml. EtgO. (8 ) residue fraction B Fraction A added and made up to 700 ml. with HOH and 700 ml, 5% (W/V) NaOH added. Stood at room temperature 2k hours and made acid with concentrated HgS0ho Divided into two portions and each extracted with k x 200 ml. EtgO. Combine EtgO. |  (9) aqueous phase (10) EtgO extract Brought to k25 ml. "free" with HOHj: boiled j 75 ml., concentrated HC1 addedj refluxed kO minutes; cooled}; Extract with EtgO (k x 200 ml. ). estrogen fraction (1). (11) EtgO (lk) aqueous extract fraction "protein'* discarded, bound estrogen fraction (3). (13) aqueous phase discarded. (12) EtgO extract "Conjugated11 estrogen fraction (2). Estrogen fractions 1,2, 3 treated as outlined in Flow sheet IVa 1*6 FLOW SHEET IVa (15) Fractions 1, 2, 3 combined and evapor-ated in vacuo to 800 mlj: washed with 9$ (W/V) MaHCO^  (3 x 80 ml.). I 1 (17) aqueous phase (16) Et20 extract discarded. Removed EtgP in vacuo. 800 ml. acetone ana a few drops MgClg in alcohol (saturated) added. Cooled to ^ 0°C. and filtered at this temperature. (19) residue discarded. (21) EtgOtCClj, discarded. , x 1 ( 1 8 ) acetone filtrate Evaporated in vacuo; a solution of 600 ml. EtgO and 30 ml. CCL. is added. Extracted with N-KOH (6 x 100 ml.). | (23) aqueous phase discarded. 7 ~ I (20) aqueous phase Washed once with 100 ml. EtgO which is discarded. Aqueous phase brought to exactly pH 9 with HgSO^  using 6 gr« NagCO* as a buffer. Extracted with EtgO \6 x 200 ml.). (27) aqueous phase Extracted with EtgO (5 x 150 ml.). (22) Et 2 0 extract In vacuo to dryness} h ml., ethanol and 500 ml. benzene added (in order)* Extracted with HOH (5 x 150 ml.). (2k) benzene phase Evaporated in vacuo just sufficient to remove benzene (no odor). (28) aqueous (29) EtgO phase (25) phase Evaporated to discarded.. dryness and treated as in fraction (k) except that .k times the volumes of methanol and petroleum ether are used. (30) estriol fraction (5) (26) Transferred in methanol to pyrex tube for chromatography. residue Dissolved in 25 ml. methanol and washed with 25 ml. petroleum ether% ether fraction back-extracted with 3 x 25 ml. 90# methanol and the pooled methanol evaporated to dryness in vacuo. Estradiol-estrone fraction (k) Transferred in methanol to pyrex tube for chromatography. 47 B. Experiment 1, Recovery of Hormone Added to Salmon Embryos. 400 g, of minced salmon embryos were used to test the magnitude of recovery. M i t c h e l l and Davies (1953) obtained average recoveries i n placentae of 27% ( e s t r i o l ) and 13$ ( e s t r a d i o l and estrone). Accordingly, i t was estimated that 200 g. each of added e s t r i o l , estradiol-17^ and estrone would provide, s u f f i c i e n t hormone f o r assay a f t e r a paper p a r t i t i o n separation. The hormones were added to the salmon at the 80$ ethanol l e v e l ( M i t c h e l l and Davies, 1954) and processed. At the stage (step 6) where the material i s brought to near dryness, i t was accidently charred, r e s u l t i n g i n an unknown loss of hormone and other chemicals of tissue o r i g i n . The damage done was quite evident at the a l k a l i p a r t i t i o n since an acetone step was not needed to avoid an emulsion. I t became apparent that there would be i n s u f f i c i e n t hormone to carry out the remaining steps. Rather than d i s -continue the labor already involved another 100 ^g. of each hormone was added (ether phase, step 20). The e s t r i o l and estradiols-estrone f r a c t i o n s were dried over phosphorus pentoxide and chromatographed. Because of the large amount of residue i n the l a t t e r f r a c t i o n , only half of i t s volume was used. The developed chromatograms showed amber zones just o f f the s t a r t i n g l i n e of both e s t r i o l and estradiol-estrone f r a c t i o n s . Sample s t r i p s were recovered from each chromatogram and examined f o r hormones by the Mitchell-Davies phenol t e s t . 48 Two zones appeared i n addition to the amber zones previously-noted. I t was evident that e s t r i o l and estradiol-17/? did not advance much beyond t h e i r respective amber zones. The area marking the advance, was eluted f o r assay. Contrary to these fi n d i n g s , estrone was w e l l i s o l a t e d . I t also was eluted f o r assay (Table V). . Table J . Recovery values f o r hormones added to salmon embryos, Experiment 1. M a t e r i a l /'g. Wavelength Difference Recoveries added P. 420 5J*Si 522-420 ( d i l u t i o n correction) Blank E s t r a d i o l standard — .036 . 585. .549- 52.1 Estrone standard — .089 .718 .629 52.1 — E s t r a d i o l f embryo 300 .266 .332 .066 5.5 11,0 3.66 Estrone embryo 300 .094 .309 .215 30.4 40.8 13.60 Blank — — — — — — E s t r i o l standard — .013 .348. .335 22.9 — E s t r i o l • embryo 300 .016 .111 .095 6.5 2.16 I t w i l l be reca l l e d that t h i s experiment was charred at a c r i t i c a l stage and that an extra 100 jug. of each hormone was added l a t e i n the experiment. Accordingly the percent recoveries of 3.66 and 13.60 for e s t r a d i o l and estrone, r e -spectively, have l i t t l e meaning. The small recovery made of e s t r a d i o l when compared to estrone was caused by the f a i l u r e 49 of e s t r a d i o l to separate completely from adjacent impur-i t i e s (the amber zone). An i n d i c a t i o n of the interference i s shown by the high, density value of e s t r a d i o l when com-pared to estrone at 420 m^ , (Table IV). This explanation i s also applicable to the e s t r i o l recovery. A l l recoveries indicate a great loss i n processing the 100 ^g. quantities of each hormone added to step 20 (Flow-sheet IVa). 0. Experiment 2, The F a i l u r e to Dectect Hormones i n 220 g. of Lyophilyzed Salmon Embryos. This experiment d i f f e r e d from the preceding one i n that no hormone was added, charring did not occur, and acetone was necessary for a clean a l k a l i p a r t i t i o n (.Blow-sheet IV, IVa). The residue representing the estradiolrrestrone f r a c t i o n was enormously large compared with that found i n the previous experiment. A s i m i l a r f r a c t i o n but of placental o r i g i n weighed 2-10 mg. ( M i t c h e l l and Davies, 1954). In the present case i t was at least of 1 ml. volume a f t e r drying over phos-phorus pentoxide. The e s t r i o l f r a c t i o n was minute and not noticeably larger than that found i n experiment 1. 1. The estradiol-estrone f r a c t i o n . This f r a c t i o n was made up to 10 ml. i n methanol and two I ml. aliquots were taken for chromatography. Forty micrograms each, of eastradiol-17^ and estrone were added to aliquot 1, Both aliquots were then chromatographed on separate s t r i p s . The chromatograms showed the same amber zones found for t h i s f r a c t i o n i n experiment 1. The Mitchell-Davies test 50 on sample s t r i p s showed an additional 2 zones only f o r a l -iquot 1 which contained the added hormones. The amber zones from both papers, and.the hormone zones were eluted and Kober tested. Only the hormone zones gave a p o s i t i v e react-i o n . The spectrophotometer readings are shown i n Table VI. Table VI. Recovery values f o r Experiment 2. Material Wavelength Difference 522-420 Recovery 420 522 $ recovery Blank. „» E s t r a d i o l standard Lost i n ma l i p u l a t i o n Estrone standard .095 .699 .604 50.0 — Amber zone, -aliquot 1 1.170 .860 — — — Amber zone, aliquot, 2^ .655 .509 — — — 40 g. E s t r a d i o l * .245 .344 .099 8.20 20.5 40 g. Estrone* ;194 .480 .286 23.65 59.1 t £ Added to residue being chromatographed. Aliquot 1 containing 40 //g. each of added estradiol-17 and estrone gave percent recoveries of 20.5 and 59.1, respect-i v e l y . These values are i n accord with the i n t e r f e r i n g mat-e r i a l s whose density values are shown at wavelength. 420, Table VI. The o p t i c a l densities obtained from the eluted amber zone showed an absence of hormone. The amber zone of aliquot 2 o r i g i n was also Kober negative and no hormone could be detected spectrophoto-m e t r i c a l l y . No other-zones, appeared i n t h i s a l i q u o t . The recovery obtained from 40 ^g. of added e s t r a d i o l i n aliquot 1, suggested that 20 //g. would be the minimum l i m i t f or assay. I f t h i s were so, then 10 x 20 yt/g. would be required 51 fo r an assay of the t o t a l estradiol-estrone f r a c t i o n . I f the Mitchell-Davies average recovery value of 13$ from the estradiol-17^ added to placentae could he extrapolated to f i s h embryos, then the amount of free e s t r a d i o l needed f o r a chemical assay of these embryos, would be 200 x 100/13, 1. e. 3.75 mg../kg. This value i s at least 10 times greater than any other value reported f or b i o l o g i c a l t i s s u e . Accord-i n g l y , i t indicates the. extreme improbability of detecting hormones i n salmon embryos. 2. The e s t r i o l f r a c t i o n . A chromatogram of t h i s f r a c t i o n showed a single zone, the amber zone. This was eluted and found to be Kober negative. . The r e s u l t s show that the hormones under study by t h i s method, could be recovered o n l y i n those fractions containing added hormone. At the hormone le v e l s required to make t h i s detection, .there was an absence of these hormones i n salmon embryos. 52 VII. DISCUSSION The basic pattern of the undifferentiated gonad i n vertebrates consists of an inner medullary ( t e s t i c u l a r ) zone and an outer c o r t i c a l ( o v a r i a l zone). Germ c e l l s migrate into these areas a f t e r which they may be induced to form male; or female c e l l s i n t h e i r respective areas, and i r r e s p e c t i v e -l y of t h e i r genetic c o n s t i t u t i o n . Thus, i n Amphibia, the cortex appears before the medulla i n the undifferentiated gonad. Germ c e l l s migrating into t h i s area become female c e l l s . Later the medullary area d i f f e r e n t i a t e s and germ c e l l s migrating into t h i s area become male c e l l s . Both male and female c e l l s are therefore present at the same time. Further d i f f e r e n t i a t i o n r e s u l t s i n a loss of one or the other of these areas. When t h i s occurs the i n i t i a l hermaphrodite becomes male or female. The sex reversal effected by high temperature rearings and parabiotic studies of frogs (Witschi, 1929, *34), provided evidence that inductor substances were ess e n t i a l components of the d i f f e r e n t areas. Witschi c a l l e d these inductors medullarin and c o r t i c i n . C o r t i c i n suppresses the action of medullarin and stimulates c o r t i c a l d i f f e r e n t i a t i o n so that female c e l l s form. S i m i l a r l y medullarin opposes the action of c o r t i c i n and induces- the formation of male c e l l s . Since both inductors are present simultaneously, the genetic constitution normally determines the outcome of sex. Thus i n a genetically determined female, c o r t i c i n normally would 53 have mere inductive force than medullarin. Recently (Witschi, 1950) restated h i s theory to conform to a hormonal theory of sex d i f f e r e n t i a t i o n (Figure 5). Apparently Witschi does not i d e n t i f y the inductor substances with sex hormones. Burns (1949) i n s i s t s that inductors and hormones are i d e n t i c a l , since the d i f f e r e n t i a t i o n effect ascribed to the inductors may also be closely simulated with hormones. Moore (1947) however, found that androgen and estrogen treatment Of young opossum did not modify the ovary or testes, nor did ovarectomy prevent the d i f f e r e n t i a t i o n of the g e n i t a l t r a c t . From t h i s , h i s many other experiments on the opossum, and an evaluation of the l i t e r a t u r e , Moore (.1947) concluded that **the most acceptable evidence for the control of sex d i f f e r -e n t i a t i o n i n vertebrates rests upon the concept of the opera-t i o n of genetic factors'*, while admitting (Moore, 1950) "that no one has provided a more acceptable explanation (for the freemartin) than that o r i g i n a l l y given (by L i l l i e , 1917)'*. Moore*s arguments are based p r i n c i p a l l y on the widespread f a i l u r e to obtain an experimental reversal of sex i n the gonads of young mammals, and the lack of evidence for a hormone secretion from f o e t a l gonads, consistent with t h e i r d i f f e r e n t i a t i o n and that of the gen i t a l t r a c t . However, Burns. (1.950). found that female hormone would.induce a sex reversal (indicated by c o r t i c a l d i f f e r e n t i a t i o n ) i n the t e s t i s of the opossum. Castration and grafting experiments on the rabbit and the r a t , respectively, lead J"ost (1950, '53) to 54 Sec. Sex Char. F. Gynogen Hormones Teatost erone >COflTEX—&^*Ovagenesis /is i -,m E s t r a d i o l >ME&LLA—SkL^Spermatogenesia Androgen Hormones Sec. Sex Char. M. Figure 5. Agent stimulators- >and i n h i b i t o r s - — > operating i n sex d i f f e r e n t i a t i o n . (Witschi, 1950). o, cortesin; m, medullarin. 55 ascribe to the f o e t a l t e s t i s the role of androgenic hormone production, and that these androgens directed sex d i f f e r e n t i -a t i o n . The objective of the present study was to show the presence of female sex hormones i n salmon embryos. Because of the suggestiveness of the l i t e r a t u r e , on sex d i f f e r e n t i a -t i o n , i t appeared that a pos i t i v e assay would provide the evidence that sex hormones were the factors of sex d i f f e r -e n t i a t i o n , i f the hormone could be shown i n the undiffer-. entiated gonad. An approach was made by using, chemical assay methods based on the recovery of added mammalian hormones. This would provide proof both f o r the f r a c t i o n a t i o n procedures being used and the s i m i l a r i t y of f i s h hormones to those of mammals. The r e s u l t s , discussed progressively i n respect to the l i t e r a t u r e and methods being attempted, showed great d i f f i c u l t i e s at that point where the phenolic estrogen f r a c t i o n i s separated from the neutral 'steroids. The emul-sions, which occur at t h i s a l k a l i p a r t i t i o n have been an obstacle even for the i n i t i a l extraction of urine (Bachman and P e t t i t , 1941). Gentrifugation has larg e l y overcome t h i s d i f f i c u l t y but,, as suggested by Diczfalusy (1953), micro-gram quantities of estrogen might be discarded, i n micelles formed from some impurity at the interphase between immiscible solvents. I t i s not surprising to f i n d therefore, that an extraction procedure such as that worked out i n a synthetic system (Friedgood and Garst, 1951) has no r e a l application 56 i n the presence of b i o l o g i c a l l y complex material such as salmon embryos with t h e i r attached yolk sacs, Centrifugation and other methods used to break emulsions, did not effect a separation between a carbon tetrachloride: ether •- N-KOH p a r t i t i o n , This problem was overcome by removing.most of the emulsion-promoting contaminants, such as the phospholipids, with MgCl 2 and c h i l l e d acetone. As thorough as may have been the extraction procedures i n the p a r t i t i o n systems used to concentrate an estrogen f r a c t i o n , t h i s f r a c t i o n nonetheless was made up of a large mass of material. Separation of 100 ^ g. each of female hormones added to t h i s mass was not possible by the paper adsorption method of Bush (1952), Attempts were made to concentrate an estrogen f r a c t i o n by column chromatography. In view of f a i l u r e s (Bauld, 1952) experienced with an aluminum column method (Stimmel, 1946) t h i s procedure, was not attempted, A method suggested by Samuels (1947) was used and was found to be unsatisfactory for color detection (Kober, 1931) of comparatively large amounts of added estrogen. Replacement of the solvent systems, and/or of the alumina by a s i l i c a - c e l i t e s l u r r y , did not a l t e r the findings. I t may be noted that an a l k a l i n e -c e l i t e column developed by Bitmsn and Sykes (1953) f or extract-ing estrogens, f a i l e d when used with b i o l o g i c a l material ( M i t c h e l l and Davies, 1954), The success obtained by Zaffaroni (1S53) i n d i a l y z i n g .. 57 adrenocortical hormones suggested that d i a l y s i s would e f f e c t i v e l y concentrate, a female hormone f r a c t i o n . Moreover Szego and Roberts (1946) and Roberts and Szego (1946) had previously used t h i s p r i n c i p l e to demonstrate estrogen a c t i v i t y i n blood plasma. The uncertainty that .this method was suited to a chemical assay prompted a study of the condi-tions necessary for a maximum y i e l d of estrogens. The r e s u l t s from a synthetic system were d i f f i c u l t to interpret because, apparently, of the heterogeneity of the Yisking d i a l y z i n g casing. This heterogeneity i n t e r f e r r e d with o p t i c a l density values obtained i n both blank and hormone, dialysates. How-ever a choice seemed evident between the butanol-.3M ETagOOg system and the chloroform:ether-water system. By a test run on b i o l o g i c a l m a t e r i a l , the l a t t e r system offered the advantage of a cleaner extract, and was therefore adopted. After further p u r i f i c a t i o n of the extract a paper p a r t i t i o n analysis (Axelrod, 1953) was carried out on the residue. The r e s u l t s showed that 100 ^/g. each of added hormones could not, be detected on the paper ehromatogram or empirical eluates cut from i t . Since the concentration of the added hormones represented 5 times the values reported as detectable by ...other methods f o r estrogens from other tissue sources ( B e a l l , 1940), d i a l y s i s of salmon embryos did not appear to warrant further study. The apparent f a i l u r e could be the r e s u l t of i n a e t i v a t i o n or destruction of the hormone by unknown i n a c t i v a t i n g systems (Fishman,. 1951) or psrhaps 58 by a binding of the hormones to salmon proteins (Eik-Hes et a l , 1954), However, for blood plasma, d i a l y s i s i s be-lieved to release 65$ of the protein bound hormone (Szego and Roberts, 1946)* Accordingly, the d i a l y t i c method developed was evaluated by assaying the estrogen content of horse testes. In the one previous chemical assay, B e a l l (1940) i s o l a t e d c r y s t a l l i n e estradiol-17/6 (.210 mg./kg,) and estrone (.360 mg/kg.) from 28,000 grams of t i s s u e . The values obtained from a 90 g. l o t i n the present study showed e s t r a d i o l and estrone i n concentrations of ,097 and ,143 mg./kg., respectively. The smaller values obtained here f o r e s t r a d i o l and estrone may be more apparent than r e a l . P h y siological v a r i a t i o n such as that known to occur i n placentae (Diczfalusy, 1953) may have affected the determination. The i n t e n s i t y of the Kober color did not suggest that much interference would be effected by the correction readings made at .420 mjx, (Yenning, et a l , 1937), : This p o s s i b i l i t y did not prove t© be so, especially i n the case of e s t r a d i o l . Thus better r e s u l t s might have been obtained by application of other colormetric procedures (Haslewood, 1950) f o r a Kober quantation. E s t r i o l , which has been isolat e d only from the human placenta (Pincus and Pearlman, 1943), was not found i n the horse testes. An attempt to duplicate these r e s u l t s on a second l o t of testes was unsuccessful. Here, the entire e s t r a d i o l -59 estrone f r a c t i o n from a 180 g. l o t of t i s s u e was chromato-graphed. E s t r a d i o l was located by the phenol test ( M i t c h e l l and Davies, 1954), but.estrone did not appear. The small amount of e s t r a d i o l , suggested by the color reaction, was eluted and rechromatographed i n order to purify i t for a determination of i t s absorption curve i n s u l f u r i c a c i d . I t could not be located on a second ehromatogram. These r e s u l t s suggested that storage of the testes may have destroyed some of the hormones ( M i t c h e l l and Davies,(1954) recommend processing placental t i s s u e immediately on delivery from the mother) or that, i n the ease of estrone, i t was not present. Thus Diczfalusy (1953) found e s t r a d i o l i n only two of s i x placentae examined. Since the t e s t i c l e s were apparently from younger horses, t h i s too, may have influenced the r e s u l t s . Since the d i a l y t i c methods used were not adapted for recovering conjugated and protein-bound estrogen f r a c t i o n s , and because of an apparent destruction of hormone added to salmon embryos being dialyzed, a recently developed procedure for extraction of hormones from placentae ( M i t c h e l l and Davies, (1954) was attempted. A recovery experiment from 200 ^ g , each, of the hormones added to minced t i s s u e , was spoiled by charring. The remaining experiment showed the d i f f i c u l t i e s •; previously noted i n p a r t i t i o n analyses, v i z . , the f i n a l f r a c t i o n s were enormous i n comparison with t h e i r possible hormone content. This, content however was not evident when the estradiol-estrone f r a c t i o n was chromatographed i n fractions . 60 of .1 volume. This volume, represented the maximum load the chromatography paper could handle and be consistent with a s t a r t i n g l i n e spot no wider than .5 cm. (Axelrod, 1953). The e s t r i o l f r a c t i o n was chromatographed i n toto. No e s t r i o l was found. A test recovery was made on an aliquot of the estradiol-estrone f r a c t i o n . By using M i t c h e l l and Davies* (1954) average recovery value of 13$ for estradiol-17/6 added to placentae, i t was estimated that the amount of e s t r a d i o l needed for chemical assay of salmon embryos was 3.75 mg./kg. ti s s u e . The magnitude of t h i s quantity with respect to values found i n b i o l o g i c a l t i s s u e , suggested the extreme improbability of detecting hormones i n salmon embryos. I t must be concluded, therefore, that u n t i l more sensitive chemical methods are developed, direct support of the hormone theory of sex d i f f e r e n t i a t i o n cannot be found i n f i s h . The d i a l y z i n g method developed here could be refined by f i r s t i n a c t i v a t i n g the enzymes or bacteria that appear to destroy the added hormone. This might be effected by a preliminary heating of the tissue i n a saline s o l u t i o n , or better, by d i a l y z i n g near 0°G. The pooling of extracts of dialysates would compensate for the comparatively small amount of s t a r t i n g material that can be managed by t h i s method. The dialysate too, may be more p r o f i t a b l y treated by a preliminary enzyme (such as glucuronidase) or acid hydrolysis of i t s residue. I t does not seem at a l l probable that the p a r t i t i o n method of M i t c h e l l and Davies of f e r s any 61 advantage because of the large amount of material i n the f i n a l residue. However, the Mitchell-Davies paper p a r t i t i o n technique (not used i n t h i s study) appears to be easier and quicker than that of Axelrod's. The chemical methods applied to salmon embryos do not represent a l l of the techniques available for micro-chemical studies. Methods involving counter-current d i s -t r i b u t i o n , paper electrophoresis, polarography, and f l u o r -imetry are being worked out, and while these are s t i l l unsatisfactory for most purposes, t h e i r further refinement may make possible a more p r o f i t a b l e study of hormone secretion i n embryos. 62 V I I I . SUMMARY AND CONCLUSION The problem of sex hormone secretion during embryo-geneses of vertebrate animals was approached by a chemical search; f o r female hormones i n salmon embryos. After preliminary experimentation, a d i a l y z i n g tech-nique was developed to concentrate an estrogen f r a c t i o n suitable f o r separation by paper p a r t i t i o n chromatography and spectrophotometrie assay. Estrogens were not found i n sexually d i f f e r e n t i a t i n g salmon embryos. Small amounts of e s t r i o l , estradio1-17p and estrone added to the tissue could not be recovered. However horse testes assayed by the same technique showed the presence of estradiol-17/* and estrone i n concentrations of .097 and .143 mg./kg., respect-i v e l y . The assay of horse testes was carried out on 90 gram l o t s , whereas the one previous chemical assay was done on ..." 28,000 grams. I t i s concluded that t h i s technique i s very s a t i s f a c t o r y f or extraction of estrogen from animal gonads but hormone added to whole salmon embryos i s inactivated by some unknown system. A p a r t i t i o n technique recently developed by F. M i t c h e l l and R. Davies for the extraction of estrogens from human placentae was s l i g h t l y modified for use with salmon embryos. This method confirmed the negative findings obtained by the d i a l y z i n g technique. On the. basis of these experiments on f i s h , no evidence could be obtained i n support of the hormonal theory of sex 63 d i f f e r e n t i a t i o n . This i s not a withdrawal from support of the concept, hut rather indicates that a good deal of refinement i n chemical assay procedure w i l l he necessary before a proper consideration of the problem can be given* 64 AC3CN0WLEP-GMENTS' The author g r a t e f u l l y acknowledges the opportunity to carry out t h i s research provided by Dr. W.A. Clemens, Director of the Fisheries I n s t i t u t e , and former Head of the Department of Zoology, the University of B r i t i s h Columbia, The program was, developed under the supervision of Dr. W.S, Hoar, Professor of Fish e r i e s and Zoology, and suggestions by, Dr. M. Darrach, Head of the Department of Biochemistry, the University of B r i t i s h Columbia. I am deeply g r a t e f u l to them for t h e i r help. I wish to thank Dr. K. Carter, Director of the P a c i f i c Fisheries Experimental Station, the Fisheries Research -Board of Canada, for making available t h e i r l y o p h i l i z i n g apparatus, and Dr. D» I d l e r of that s t a t i o n , for h i s suggestion of the s i l i c a - e e l i t e column used i n the preliminary experiments. Dr. A.W. Matthews, Dean of the Faculty of pharmacy, kindly allowed me to use t h e i r Beckman DU spectrophotometer. Miss M. Nagai, Miss D. Timberley, and Mr. A. Beach assisted with the typing and preparation of the i l l u s t r a t i o n s . .65 ,LITERATURE CITED 1. Ashby, K.R. 1952, The development of the reproductive system of Salmo t r u t t a under normal conditions and under the influence of administered s t e r o l hor-mones, Revista d i B i o l o g i a , 44:1-19, 2. Axelrod, L.R. 1953, The. quantitative separation of estrogens by paper p a r t i t i o n chromatography, J . B i o l , Chem., 201:59-69. 3. Bachman, C. and P e t t i t , D.S. 1941, A procedure f o r the estimation of the estrogens of pregnancy urine, J . B i o l , Chem, 138:689-704, 4. Bailey, B.E., Carter, N.M., and Swain, L.A, 1952, Marine o i l s with p a r t i c u l a r reference to those of Canada. Fi s h . Res, Bd. Canada. B u l l , 89. 413 pp, 5. Bauld, W.S.. 1952, The extraction and p u r i f i c a t i o n of urinary ©estrogens, Ciba Foundation Colloquia on Endocrinology. 2:72-83. 6. Bauld, W.S. 1954. Some errors i n the colorimetric estima-ti o n of o e s t r i o l , oestrone and oestradiol by the Kober reaction. Biochem. Journ, 56:426-434. 7. B e a l l , D. 1940. The i s o l a t i o n o f ^ - o e s t r a d i o l and oestrone from horse testes. Biochem. Journ, 34:1293-1298. 8. Bates, R.W. 1952.. Chemistry and estimation of urinary oestrogens, Ciba Foundation Colloquoia on Endo-crinology. 2:58-71. 9. Benoit, J . 1923. Cited from Moore, C.R. 1947. 10. Berkowitz, P. 1938, The effects of estrogenic substances i n the. Lebistes r e t i c u l a t u s . Anat. Rec. .71:161-175. 11. • 1941. The effects of estrogenic substances i n the f i s h Lebistes r e t i c u l a t u s . J . E x p t l . Zool. 87:223-243. 12. Bitman, J , and Sykes, J.F. 1953, Chromatographic separa-t i o n of estrone, e s t r a d i o l and e s t r i o l . Science. 117:356-358. 13. Brown, J.B, 1952. Mechanism of the Kober reaction. Ciba Foundation Colloquia on Endocrinology. 2:132-125. 66 14. B r u l l , L. and Cuypers, Y. 1954. Quolques charaeterist-igues de Lophius piscatorius L.. Arch. Internat. P h y s i o l . 162:70-75. 15. Bullough, W.S. 1940. A study of sex reversal i n the minnow (Phoxinus l a e v i s , L.}. J . I x p t l . Zool. 85:475-501. 16. Burns, R.K. 1925. Cited from W i l l i e r , 1934. 17* ;~ 1942. Hormones and the growth of the parts of the gen i t a l apparatus i n mammalian embryos. Cold' Spring Harbour Symposia on Quantitative Biology. 10:27-33. 18. . 1949. Hormones and the d i f f e r e n t i a t i o n of sex. B i o l . Prog. 1:233-266. 19. Burton, R.B., Zaff a r o n i , A. , and Keutman, E.H. 1951. Paper chromatography of steroids. I I . Corticosteroids and related compounds. J . B i o l . Chem. 188:763-772. 20. Bush, I.E. 1952, Methods of paper chromatography of steroids applicable to the study of steroids i n mammalian blood and tissues. Biochem. Journ. 50:370-378. 21. Cassidy, H.G. 1948. Chromatography. Annals New York Acad. S c i . 49:141-326. 22. Cohen, H. and Bates, R.W, 1952, The demonstration of a non-estrogenie uterine stimulatory-and estrogen augmentry substance i n pregnant mares* urine. Endocrinology. 50:5-15. 23. D'Ancona, U, 1947. Somatic influences on the sexual d i f f e r e n t i a t i o n of germ c e l l s . Proc, 6th. I n t e r -nat. Congr. Exp.. Cytology. 571-577. 24. Dantchakoff, ¥. 1950. La d i f f e r e n c i a t i o n du sexe chez le s vertebres. Arch. Anat. Micr. et, Morph. EKp. 39:367r394. 25. Deuel, G. 1951. L i p i d s : t h e i r chemistry and biochem-i s t r y . Inter-science Pub. Inc. N.Y. 1:1-982. 26. B i c z f a l u s y , C. 1953. Chorionic gonadotrophin and oes-trogens i n the human placenta. Acta Endocrin-o l o g i c a l Supplement 12:1-161, 27. Boisy, E.A. 1934. Biochemistry of the f o l l i c u l a r hor^ mono, t h e e l i n . In Sex and Internal Secretions. (Edited by E. Allen).. Chapt. X. William and Wilkins, Co., Baltimore. 951 pp* 67. 28. Boisy, E.A. 1941. S o l u b i l i t y of estrogens. J• B i o l . Chem. 138:238-285. 29. Bonahue, J.K. 1941. Occurrence of estrogens i n the ov-ari e s of the winter flounder. Endcrinology. 28:519-520. -30. Eik-Nes, K., Schellman, J.A,, Lumry, R., and Samuels, L..T, 1954, The binding of steroids to protein. I.. S o l u b i l i t y determinations.J. B i o l . Chem. 206:411-419, 31. Emmens, C.W. 1950. Estrogens. In Hormone Assay. (Edited by C,W. Emmens), Acad. Press, Inc. K.Y. 556 pp. 32. Engel, L.L., Slaunwhite, W.R., Carter, P,, and lathan-soni I . I . 1950. The separation of natural estro-gens; by countercurrent d i s t r i b u t i o n . J . B i o l . Chem. 185:255-261. 33. Engel, L.L. 1950. The chemical estimation of steroid metabolites. Rec. Prog. Horm. Res. 5:335-379* 34. F e l l n e r , 0.0. 1925. Tiber das vorkommen des flemininen sexual-lipoids i n vogeleiern and den e l e r -stocken der f i s c h e . Klnische.lfbchenschrift. 4:1651-1652. 35. Fieser, L.E., and Fieser, H, 1949. natural products related to phenanthrene. Monograph 70. Reinhold Publish. Corp. K.Y, 704 pp, 36. Fishman, W,H, 1951. Relationship between estrogens and enzyme a c t i v i t y , Y i t , and Horm, 9:213-236, 37. Friedgood, H.B., and Garst, J.B. 1950. The identifrc-r ation and quantitative microdetermination of estrogens by u l t r a v i o l e t absorption spectro-photometry, Rec. Progi Horm. Res. 2:31-78. 38. Haslewood, G.A.B. 1950. Chemical assay of estrogens and pregnanediol. In Hormone Assay. (Ed. by C.W. Emmens). Acad. Press Inc. N.Y. 556 pp. 39. - Heard, R.B.H. 1949. The metabolism of estrogens. Rec. •Prog.- Horm. Res. 4:25-42. 40. Heard, R.B.H. and Saffron, J.C* 1949. The metabolism of estrogens. Rec. Prog. Horm. Res. 4:43-63, 41. Heard, R.B.H., and 0*Bonnell, Y.J. 1954. Biogenesis of the estrogens. The f a i l u r e of cholesterol-4-G^ 4 to give r i s e to estrone i n the pregnant mare. Endocrinology 54:209-215. 68. 42. Jost, A. 1947. The age factor i n the castration of male rabbit foetuses. Proc. Soc. E x p t l . B i o l , and Med. 66:302-303. 43. . 1950. Sur l e controle hormonal de l a d i f f e r e n -c i a t i o n sexuelle du l a p i n . Arch. Anat, Micr. et.Morph. Exp. 39:215-246. 4 4» 1953. Problems of f o e t a l endocrinology: the gonadal and hypophysial hormones. Rec. Prog. Horm. Res, 8:379-418. 45. Kober, S. 1931. Eine kolorimetrische bestimmung des brunsthormons. (menformon}:. Biochem. Z. 239:209-212. 46. Kurzrok, R., and Ratner,-S. 1932. The r e l a t i o n of amen-orrhea accompanied by g e n i t a l hypoplasia to the f o l l i c u l a r hormone i n the urine. Am. J.. Obst. and Gynec. 23:689-694, 47. Lappin, Gf.R., and Clark, L.C. 1951. Colorimetric method for determination of traces of carbonyl compounds. Anal. Chem. 23:541-542, 48. L i l l i e , F.R. 1917, The freemartin: a study of the action of sex hormones i n the f o e t a l l i f e of c a t t l e . Journ. E x p t l , Zool. 23:371-452, 49. Love, A,, and Love, D. 1940, '45. Cited from K.Y. Thim-"ann, Chapt. I I I . In Pincus-Thimann's, The Horm-ones. Acad, Press Inc. K.Y. I , 886pp, 1948, 50. MacGorquodale, D.I., Thayer, S.A., and Doisy, E.A. 1936. The i s o l a t i o n of the p r i n c i p a l estrogenic sub-' stance of liq u o r f o l l i c u l i . J . B i o l . Chem. 115:435-448. 51. Mather, A. 1942. D i s t r i b u t i o n of estrogens between immis-c i b l e solvents. J . B i o l , Chem. 144:617-623. 52. Minoura, T. 1921. A study of t e s t i s and ovary grafts on the hen*s egg and t h e i r effects on the embryo. J". E x p t l . Zool, 33:1-61. 53. - M itchell, F.L., and Davies, R.E."1954. The i s o l a t i o n and estimation of the steroid•estrogens i n placenta t i s s u e . Biochem, Journ. 56:690-698.. 54. Moore, CR, 1947. ^Embryonic sex hormones and sexual d i f f e r e n t i a t i o n . Charles C. Thomas, Publisher. S p r i n g f i e l d , I l l i n o i s . 81 pp. 69 .55. Moore, G.R. 1950. Studies on sex hormones and sexual d i f f e r e n t i a t i o n i n mammals. Arch. Anat. Micr, et Morph. Exp. 39:484-498, 56. Mortin, R.A., Glover, M., and Rosen, D.G. 1952, Ast-axanthin, cholesterol and l i p i n s i n developing salmon eggs. Biochem. Journ. 50:425-429. 57. Okkelberg, P. 1921, The early history of the gem c e l l s i n the brook lamprey, Entosphenus w i l d e r i (Gage), up to and including the period of sex d i f f e r e n t -i a t i o n . J . Morph. 35:1-152. 58. -Padoa, E:. 1939. observations u l t e r i e u r e s sur l a d i f f -erentiation du sexe, normale et modifie par l*ad-m i n i s t r a t i o n d'hormone f o l l i c u l a i r e , chez l a t r u i t e i r i d i e (Salmo i r i d e u s ) . Bio. Morphosis. Basle. 1:337-354. 59. Paschkis, K.E., and Rakoff, A.E. 1950. Some aspects of the physiology of estrogenic hormones. Rec. Prog. Horm. Res. 5:115-150. 60. Pearlman,. W.H. 1948. The chemistry and metabolism of the estrogens.. In Pincus-Thimann*s, The Hormones. Ghapt. X. Acad. Press Inc. ©86 pp. 61. Pincus, G. and Pearlman, W.H. 1943. The intermediate metabolism of the sex hormones*,Yit. and Horm. 1:293-343. 62. Pincus, G. 1948. Assay of ovarian hormones,. In Pincus-Thimann's, The Hormones. 1:333-349. Acad. Press Inc. N.Y. 63. Rakoff, A.E., Paschkis, K.E., and Gantarow, A. 1943. Conjugated ^estrogens i n human pregnancy serum. Am. J . Obst. and Gyn. 46:856-860. 64. Roberts, S., and Szego, GUM. 1946. The nature of c i r c -u l a t i n g estrogens; lipoprotein-bound estrogen i n human plasma.. Endocrinology. 39:183-187. 65. Robertson, JUG. 1953, Sex d i f f e r e n t i a t i o n i n the P a c i f i c salmon Oncoryhnchus keta (Walbaum). Can. Journ. Zool. 31:73-79. 66. Rosenkrantz, H. 1953. An antimony t r i c h l o r i d e reagent s u i t -able for the detection and estimation on non-keton-i c steroids. Arch. Biochem, and Biophysics, 44;1-8. 67. Samuels, L.T. 1949. The metabolism of androgens by tissues. Rec. Prog. Horm. Res. 4:65-83. 70. 68. Stimmel, B.F. 1946. The fr a c t i o n a t i o n and photometric estimation of the estrogens i n human preg-nancy urine. J . Biol,Chem. 162:99-109. 69. Szego, CM., and Roberts, S. 1946. Nature of c i r c u l -ating estrogens. Procl Soc. E x p t l . B i o l , and Med. 61:161-164. 70. Szego, CM., and Samuels, L.T. 1943. Colorimetric determination of estrogens: A method for the determination of t o t a l estrone and e s t r a d i o l from tissue sources. J . B i o l . Chem. 151:587-598. 71. Thomas, A,W. 1934. C o l l o i d Chemistry. McG-raw H i l l Book Company, Inc. N.Y. pp 68-97, 72. Umberger, E.J., and C u r t i s , J.M. 1948, The use of the s u l f u r i c acid reaction for the estimation of «cand n e s t r a d i o l s and of estrone and e q u i l i n i n binary mixtures i n pure solutions. J . B i o l . Chem, 178:275-287. 73. Yenning, E.Y>, Evelyn, K,E., Harkness, E.'Y., and Brown, J.S.L. 1937. The determination of e s t r i n i n urine with the photoelectric colorimeter. J*. B i o l . Chem, 120:225-237. 74. Yogel, A.I. 1951, P r a c t i c a l Organic Chemistry including q u a l i t a t i v e organic analysis. Longmans, Green, and .Co. 1033 pp. 75. Weisman, A.I., Mishkind, D.I., K l i n e r , A.S., and Coates, C.W. 1937. Estrogenic hormones i n the ovaries of swordfish. Endocrinology. 21:413-414, 76. Westerfeld, W.W., Thayer, S.A., MacCorquodale, D.W., and Doisy, E.A, 1938, The ketonic estrogen of sow ovaries. J . B i o l . Chem. 126:181-194, 77. W i l l i e r , B.H, 1927..Cited from W i l l i e r , B.H. 1934. 78. . . . 1934. Embryological foundation of sex i n vertebrates. In sex and i n t e r n a l secretions. (Edited by E. A l l e n ) , Williams and Wilk i n Co. Baltimore. 951 ppp 79. Witschi, E. 1929. Sex reversal i n female tadpoles of Rana sy l v a t i c a following the application of high temperature. J.Exptl. Zool. 52:267-280. 80. . 1934. Sex deviations, inversions and para-b i o s i s . In Sex and Internal Secretions. (Edited by E, A l l e n ) , William and Wilkins Go. 951 pp. 71 81. Witschi, E. 1942. Hormonal regulation of development of sex i n lower vertebrates. Gold Spring Harbor Symposia on Quantitative Biology. 10:145-151, 82. . , 1950. Genetique et physiologie de l a d i f f -erenciation du sexe. Arch. Anat, Micr. et Morph, Exp. 39:215-246. 83. _. • 1951. Gonad development and function. Rec. Prog. Horm. Res.. 6:1-23. 84. Wolff, E. 1947. Recherches sur l ' i n t e r s e x u a l i t e exper-imentale produite par l a methode des greffes de gonades a l'embryon de poulet. Arch. Anat. Micr. et Morph. Exp, 36:69-90. 85. » 1950. Le role des hormones embryonnaires dan l a d i f f e r e n e i a t i o n sexuelle des oiseaux. Arch. Anat. Micr. et Morph. Exp, 39:426-450.. 86. Zaffaroni, A., and Burton, R.B. 1951. I d e n t i f i c a t i o n of corticosteroids of beef adrenal extract by paper chromatography. J . B i o l . Chem. 193:749-767, 87. . . . 1953. Corticosteroids present i n adrenal veing blood of dogs. Arch. Biochem. and Biophysicsi 42:1-6. 88. Zaffaroni, A. 1953. Micromethods for the analysis of adrenocortical steroids. Rec. Prog. Horm. Res. 8:51-56. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0302612/manifest

Comment

Related Items