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

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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 t h e s i s as conforming to the standard required  from candidates f o r 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 f o r separation by paper p a r t i t i o n chromatography and spectrophotometry  assay.  Estrogens were not found i n s e x u a l l y 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 e s t -  rone added to the t i s s u e could not be recovered.  However,  horse t e s t e s assayed by the. same technique- showed the presence of e s t r a d i o l - 1 7 ^ and estrone i n concentrations of .097 and .143 mg./kg., r e s p e c t i v e l y .  The assay of horse  testes was c a r r i e d out on.90 gram l o t s , whereas the one previous 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 e x t r a c t i o n of estrogens from animal gonads, but that hormone added to whole salmon embryos i s i n a c t i v a t e d by-some unknown system. A p a r t i t i o n technique r e c e n t l y developed by F. M i t c h e l l and R. Davies f o r the e x t r a c t i o n of estrogens from.human placentae was s l i g h t l y modified f o r use w i t h salmon embryos. This method confirmed the negative f i n d i n g s obtained by the d i a l y z i n g technique. On the basis of these experiments, there i s no e v i d ence to support the hormonal theory of sex d i f f e r e n t i a t i o n in fishes.  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. ( B r i t . Col.) 1951 Monday, July 12, 1954at 10:00 a.m. i n ROOM'403, Applied Science Bldg COMMITTEE IN CHARGE W.A. Clemens, Chairman V.C. Brink I. McT. Cowan M. DarrachP. 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. P r o g . R e p o r t s Pac. C o a s t . S t a t - , i o n s F.R.B.C. 75:31-34. 1948.  2.  Sockeye F r y P r o d u c t i o n i n a S m a l l B r i t i s h 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 t h e P a c i f i c Salmon Oncorhynchus k e t a (Walbaum). Can. J o r n . Z o o l . 31:73-79- 1953-  4-  The T r o p i c S t a t u s o f P o r t John L a k e , B r i t i s h Columbia. J o u r n . F i s h e r i e s Research Board Canada ( i n p r e s s ) 1954.  Columbia  ABSTRACT A search f o r female sex hormones in salmon embryos of the genus Oncorhynchus  A dialyzing technique was developed to concentrate an estrogen hormone 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 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 , 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 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 previous chemical assay was done on 28,000 grams. It i s concluded that t h i s technique i s very satisfactory f o r extraction of estrogens from mammalian 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 from 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 t i c technique. On the basis of these experiments, there i s no evidence to support the hormonal theory of sex d i f ferentiation i n fishes.  GRADUATE STUDIES F i e l d of Study:  Zoology  Experimental Zoology Vertebrate Morphogenesis Biology of Fishes Limnology Zoological Seminar Comparative Physiology HistologyFisheries Management Embryology  -  Dr. W. Dr. P. Dr. w. Dr. p. Dr. w. Dr. w. Dr. p. Dr. w. Dr. p.  Hoar Ford Hoar Larkin Clemens Hoar Ford Hoar Ford  Other Studies: General Biochemistry -Dr. Biochemistry of Steroids - Dr. Intermediary Metabolism -Dr. Advanced Cytology - Dr. Biometry - Dr. Biochemistry of Cancer - Dr. Biochemistry of A n t i b i o t i c s - Dr.  S. Zbarsky M. Darrach S. Goranson A.H. Hutchin V. Brink M. Darrach J . Polglaze  , i CONTENTS I. •II. III.  INTRODUCTION. ...... *  1  THE DISTRIBUTION OF FEMALE SEX HORMONES........... 6 MATERIALS. .............  9  A.  B i o l o g i c a l Tissue..........  9  B.  Chemicals.  9  1. ..Hormones 2. Solvents...... .... 3. Color reagents. 4. Apparatus...... IV.  9 10 10 10  PRELIMINARY CHEMICAL SEPARATION OF AN ESTROGEN FRACTION. . A.  p a r t i t i o n Methods.....  B.  E a r l y attempts t o e f f e c t an i s o l a t i o n by pap paper chromatography 13 1. 2.  11  Use of Bush*s (1952) paper adsorption method... ..13 Use of Z a f f a r o n i ' s (1951) paper p a r t i t i o n method • 13  C. Column chromatography Q.  V.  11  Dialysis........  15 .  17  1.  S y n t h e t i c system. 18 a) D i a l y z i n g without s t i r r i n g 18 b) D i a l y z i n g w i t h s t i r r i n g . 20 c) C a l i b r a t i o n curve f o r e s t i m a t i o n . . . E l d) Hormone recoveries i n the synt h e t i c system......... 21  2..  B i o l o g i c a l systems  25  PAPER PARTITION CHROMATOGRAPHY OF SALMON EXTRACTS. 28 A.  Introduction..  B;.  Reproduction of ^Axelrod's (1953> method using e s t r i o l , estradiol-17/S and estrone.. 29 1. 2.  Procedure. Detection of estrogens.  28  29 30  ii  G. D. -•VI.  3. P o s i t i o n s of the hormones 4. E l a t i o n of the hormones 5. E l i m i n a t i o n of background m a t e r i a l 6. Absorption curves... A p p l i c a t i o n t o salmon embryo d i a l y s -  31 33 33 33  ates.  35  A p p l i c a t i o n to horse t e s t e s d i a l y s a t e s 39  A-FURTHER SEARCH FOR FEMALE HORMONES IN SALMON EMBRYOS  ..............  44  A.  The M i t c h e l l - D a v i e a P a r t i t i o n Method.. 44  B.  Experiment 1. Recovery of hormone added to salmon embryos * 47 Experiment 2. The f a i l u r e t o deteift hormones i n l y o p h i l i z e d salmon embryos 49 1. The e s t r a d i o l - e s t r o n e f r a c t i o n . . . . 49  C.  2.  The e s t r i o l f r a c t i o n . .  51  VII i  DISCUSSION.  52  VIII.  SUMMARY AND CONCLUSION..,...-..  62  IX.  ACKNOWLEDGMENTS......  ... 64  X.  LITERATURE CITED.....  i . . . . . 65  iii 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 s y n t h e t i c system...............23 I I . The l o c a t i o n of f r e e s t e r o i d s ( 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 d i a l y s a t e s of horse t e s t e s  ....41  IV. Spectrophotometric assay of the e s t r i o l f r a c t ion from horse t e s t e s . . . . . . . . . . ...42 V. Recovery values f o r hormones added t o salmon embryos, Experiment I . . . . . . . . . . VI. Recovery values f o r Experiment 2 FIGURE  48 50  1. C a l i b r a t i o n curve f o r e s t r a d i o l - 1 7 ^ i n a s y n t h e t i c system.. .22 2. Apparatus used i n d i a l y s i s , . , ......27 3. Absorption spectra showing the e l i m i n a t i o n of background m a t e r i a l (B) from chromatographed e s t r a d i o l - 1 7 ^ (,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 s t i m u l a t o r s 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 . ( a f t e r W i t s c h i , 1950)..,,,., •  .54  FLOW-SHEET I , P r e l i m i n a r y 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 o f 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, I V a . 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  s t u d i e s on the r o 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 w i t h F.R. L i l l i e s * a t i o n s on the f r e e m a r t i n .  (1917) observ-  The freemartin i s a female  c a l f that has been transformed  i n t o an hermaphrodite.  This transformation occurs p r e n a t a l l y , and then only i n the presence o f a male t w i n .  I t i s dependent upon a  secondary f u s i o n of the two placentae so that blood-borne male hormones can pass from the male to the female t w i n . There i s no freemartin when the twins are of the same sex. The transformation t o the freemartin may be so complete t h a t the female develops a t e s t i s and male ducts, i n s t e a d of an ovary and female ducts ( W i l l i e r , 1934). The morphological b a s i s 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 u n d i f f e r e n t i a t e d gonad c o n s i s t s 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 — component.  the o v a r i a l  Normally only.one of these c o n s t i t u e n t s becomes  f u n c t i o n a l , i . e . , forms an ovary o r t e s t i s C ^ i t s c h i , 1934). Both c o n s t i t u e n t s may p e r s i s t i n t o adulthood t o give a f u n c t i o n a l hermaphrodite as; i n some cyclostomes  (Okkelberg,  1921), o r the c o n s t i t u e n t s may not be separable, as i n  young e e l s (D'Ancona, 1947).  The g e n i t a l t r a c t has two s e t s  of ducts, which i n most v e r t e b r a t e s are simultaneously present and f u l l y formed a t some time during development (Burns, 1942, *49). The g e n i t a l t u b e r c l e , l o c a t e d i n the u r o g e n i t a l s i n u s , i s the forerunner o f both male and female external genitalia. The experimental b a s i s 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, g r a f t i n g s of o v a r i a n and t e s t i c u l a r t i s s u e , p a r a b i o s i s , and a p p l i c a t i o n of sex hormones.  Each approach  i s designed t o determine whether embryonic hormones are e s s e n t i a l to the normal or abnormal development of the gonad and i t s accessory sex s t r u c t u r e s , i . e . reproduce "Nature's experiment** i n the development o f the f r e e m a r t i n . S e l e c t e d examples, of these approaches are o u t l i n e d here. The removal of the f u n c t i o n a l l e f t ovary o f a c h i c k induced the u s u a l l y degenerate r i g h t ovary t o form a t e s t i s producing spermatozoa ( B e n o i t , 1923). A p r e n a t a l removal of embryonic r a b b i t o v a r i e s ( J o s t , 1947) d i d 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 r a b b i t may be the r e s u l t of i n t e r f e r e n c e from maternal hormones. G r a f t i n g experiments, i n which pieces of t e s t i s or  3 ovary were i n s e r t e d on the e h o r i o - a l l a n t o i c membrane of a host chick embryo, r e s u l t e d 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 f i n d i n g s could not be confirmed  by W i l l i e r (1927, *34). Recently, however, Wolff (1947) e f f e c t e d a p a r t i a l sex r e v e r s a l 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 i n t o the body cavity. P a r a b i o s i s , 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 r e s u l t e d i n a d u p l i c a t i o n of the. freemartin e f f e c t 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 u n d i f f e r e n t i a t e d .  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 r e v e r s a l of sex must have occurred i n a large number of them. Treatment with hormone preparations  has generally  e f f e c t e d a m o d i f i c a t i o n of sex i n vertebrates i n l i n e w i t h the above experiments.  I n f i s h , Padoa (1939) observed a  p a r t i a l f e m i n i z a t i o n of male t r o u t (Salmo i r i d e u s ) reared i n aquaria containing female hormones.  Bullough (1940)  showed female minnows (Phoxinus l a e v i s ) developed a t e s t i s l i k e s t r u c t u r e a f t e r i n j e c t i o n of male hormone.  However,  male f i s h i n j e c t e d w i t h female hormone showed an i n j u r e d , but not transformed, t e s t i s .  Berkowitz (1938, *41) founds  estrogen caused formation of an o v a t e s t i s i n the guppy ( L e b i s t e s r e t i o u l a t u s ) . I n trout (Salmo t r u t t a ) , treatment w i t h male or w i t h female hormone gave c o n f l i c t i n g r e s u l t s  4 i n t h a t s i m i l a r m o d i f i c a t i o n s occurred w i t h e i t h e r hormone i n e i t h e r sex (Ashby, 1952)• I t i s i n t e r e s t i n g that a t l e a s t i n one p l a n t (Melandrium; dioeclum), mammalian sex hormones s h i f t e d the-normal and i n t e r s e x u a l f l o w e r s t o the male or female side i n accord w i t h the hormone a p p l i e d (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 f u n c t i o n 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 w i t h those of adult animals.  This t h e s i s  has a l s o been developed or supported by Dantchakoff (1950), Wolff (1950), l i t s c h i (1950, *51), and J o s t (1953).  It i s  opposed by Moore (1947, 5 0 ) , p r i n c i p a l l y on the grounds that t  there i s no evidence f o r the s e c r e t i o n of sex hormones at a time when the gonads are s e x u a l l y d i f f e r e n t i a t i n g . I n the animal kingdom, t h e chemical i d e n t i t y , o f the n a t u r a l sex hormones has been worked out only f o r the mammals. The female hormones have-been found to possess the s t e r o i d r i n g system, as do the ' t e s t i c u l a r and a d r e n o c o r t i c a l hormones. They d i f f e r by v i r t u e of t h e i r p h e n o l i c character and possession of fewer carbon atoms. The present, study attempts to evaluate the problem by supplying chemical evidence f o r the embryonic s e c r e t i o n of female sax hormones.  Any such evaluation should consider the  i n t e r f e r e n c e 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 h i s t o g e n e s i s of sex  5 d i f f e r e n t i a t i o n has been worked out f o r the chum salmon Oncorhynchus k e t a , (Robertson, 1953), the same species, was selected f o r t h i s investigation*  6 II.  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, l a r g e amounts of source material, were used.  Thus  MaeCorquodale, Thayer, and Doisy (1936) a s p i r a t e d 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 g r a a f i a n f o l l i c l e s of four tons of sow o v a r i e s .  A f t e r processing t h i s . f l u i d , they obtained  12 m i l l i g r a m s of e s t r a d i o l - 1 7 ^ .  Human and mare pregnancy  u r i n e have been the c h i e f source f o r 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 , e q u i l e n i n , and. h i p p u l i n .  Because  of the expense and hazards encountered i n p r o c e s s i n g t i s s u e s 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 c a r r i e d out e n t i r e l y on u r i n e .  The r e l a t i v e  simplicity  of t h i s medium and the development of microchemical methods f o r i t s assay have r e s u l t e d i n an extensive l i t e r a t u r e , none of which i s conclusive (Heard and S a f f r o n , 1949).  The methods  dp not appear to be a p p l i c a b l e to e x t r a c t s of blood, body t i s s u e s , feces or the u r i n e 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 h e l p f u l to a study of the human placenta ( D i c z f a l u s y , 1953). I t i s not s u r p r i s i n g to f i n d , t h e r e f o r e , the continued use of bioassay methods... Their advantages f o r d e t e c t i n g estrogenic substances i n microgram q u a n t i t i e s from r e l a t i v e l y crude f r a c t i o n s has g r e a t l y 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 e x t r a c t i o n of a phenol f r a c t i o n that w i l l induce v a g i n a l c o r n i f i c a t i o n i n the spayed mouse or r a t ( b i o a s s a y s ) .  I t should be noted that t h i s  estrogen a c t i v i t y can, a l s o be e f f e c t e d by petroleum and l i g n i t e (Doisy, 1934) (Emmens, 1950)  and a v a r i e t y of s y n t h e t i c substances  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 k e r n e l e x t r a c t s , and e s t r i o l from female w i l l o w flowers (see F i e s e r and F i e s e r , 1949)  suggests a wide-  spread occurrence of mammalian hormones, F e l l n e r (.1925) was the f i r s t to i n v e s t i g a t e female hormones i n f i s h .  He obtained an ovarian e x t r a c t from an  unstated species that would enlarge the r a b b i t uterus. s t a t e s that the aphrodisiac nature of c a v i a r i s probably  He due  to the presence of the female hormones i n d i c a t e d by h i s study, Weismann et a l (1937) extracted a p h e n o l i c f r a c t i o n from * swordfish (Xiphlas gladius) ovaries which gave an e s t r u s response i n r a t s .  The n e u t r a l male f r a c t i o n a r i s i n g from h i s  e x t r a c t i o n procedure was used as a c o n t r o l . I t d i d not give an e s t r u s response.  Donahue (1941) made an a l c o h o l e x t r a c t  of the ovaries from winter f l o u n d e r s .  A f t e r f u r t h e r process-  i n g , the e x t r a c t was estimated to contain l e s s than ten r a t u n i t s of estrogen.  This i s equivalent to l e s s 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  c a u t i o u s l y claim to have made a chemical d e t e c t i o n of f o l l i c u l i n (estrone) and other "phenolsteroids" i n an e x t r a c t of 420 m l , of u r i n e obtained from 25 Lophius p i s c a t o r i u s . On the b a s i s of the studies discussed there can be l i t t l e doubt that the estrogens-are of a wide v a r i e t y of  present i n adult females  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 a t C u l t u s l a k e i n the autumns of and  1953  and  incubated  f i s h hatchery.  The  i n the U n i v e r s i t y of B r i t i s h  The  three weeks b e f o r e h a t c h i n g , represented  the sexes were w e l l d i f f e r e n t i a t e d months post-hatching)  (Robertson,  to c o l l e c t the sockeye i n stages  first  stage,  two  taken  the p e r i o d of germ  c e l l m u l t i p l i c a t i o n ; the second stage was  The  Columbia's  chum salmon were removed f o r study a t  stages i n the s p r i n g of those y e a r s .  as t e s t m a t e r i a l .  1952  taken at a time when  ( y o l k j u s t absorbed, 1953).  No  e f f o r t was  two made  s i n c e they were b e i n g used  A l l f i s h s u b s i s t e d s o l e l y on t h e i r y o l k s a c s .  f i s h were used i n whole because removal of the  embryonic gonads i s i m p r a c t i c a l . The A l s a s k P r o c e s s o r s L i m i t e d , Edmonton, A l b e r t a , deepf r o z e horse t e s t e s  and a i r - e x p r e s s e d them i n dry i c e to the  Vancouver A i r p o r t .  They were used immediately upon a r r i v a l .  A second l o t of t e s t e s were obtained from Mr. Newton,  Fos  Hoy,  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 s u p p l i e d by the Ciba Company L i m i t e d and McKenna and H a r r i s o n L i m i t e d , M o n t r e a l , p l e a s u r e t o acknowledge t h e i r g e n e r o s i t y  '1.  Ayerst,  Canada. I t i s a ?  The  identity  and  The nomenclature f o l l o w e d i s t h a t of F i e s e r and F i e s e r (1949).  10 p u r i t y of these hormones were determined on a F i s h e r m i c r o m e l t i n g point apparatus a f t e r d r y i n g over phosphorus pentoxide.  The m e l t i n g point ranges found ( s e t i n parentheses)  were i n v i r t u a l agreement w i t h those of e s t r i o l (280-283.5°C,), e s t r a d i o l - 1 7 ^ (174-175°C.) and estrone (258-264°C.)• 2. S o l v e n t s . A l l solvents used were of reagent grades  The  hydrocarbons such as petroleum e t h e r , benzene and methylcyclohexane were washed w i t h concentrated s u l f u r i c a c i d and re-distilled. 54°0>).  (The petroleum ether was c o l l e c t e d a t 34-  Their d e r i v a t i v e s (chloroform, O-dichlorobenzene,  nitrobenzene and dichloromethane) were r e d i s t i l l e d .  Commercial  grade 95$ and absolute a l c o h o l were r e d i s t i l l e d except when used i n making up c o l o r reagents. Here the absolute grade was r e f l u x e d i n 2,4-dinitrophenylhydrazine and t r i p l e d i s t i l l e d (Lappen and C l a r k , 1951). preference t o ethanol.  Methanol, r e d i s t i l l e d , was used i n  Mormal b u t y l a l c o h o l was r e d i s t i l l e d .  Formamide was made ammonia f r e e by standing over concentrated sulfuric acid.  Ether was used when peroxide f r e e t o 10% a c i d  potassium i o d i d e .  E t h y l acetate was p u r i f i e d as o u t l i n e d 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.  lubrication.  Ground glass equipment was used w i t h water  D i s t i l l a t i o n s i n vacuo were s e r v i c e d by a water  pump. The chromatographic apparatus consisted of 12 x 24 inch Corning glass c y l i n d e r s and standard solvent t r a y assemblies (Cave and Company L i m i t e d ) .  11  IV. A.  PRELIMINARY CHEMICAL SEPARATION OF AN ESTROGEN FRACTION P a r t i t i o n Methods. Estrogens may be removed from t i s s u e s by a v a r i e t y of  s o l v e n t s such as e t h y l acetate (Kurzrok and Ratner, 1932), e t h y l a l c o h o l (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 s u i t a b l e . ,  However where the estrogens are t o be removed as a group Mathers (1942) p o i n t s out that n e i t h e r benzene or toluene are s u i t a b l e f o r e x t r a c t i o n of e s t r i o l , nor are the petroleum ethers u s e f u l f o r any estrogen.  N-butanol i s a  powerful e x t r a c t o r f o r both the f r e e 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 l a r g e l y on the medium being e x t r a c t e d . F o r t i s s u e s , water m i s c i b l e solvents such as a l c o h o l are used* The present study on salmon embryos began w i t h e t h y l acetate i n order t o 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 t h a t e t h y l acetate extracted powerful e m u l s i f y i n g agents (mainly phospholipids) which i n t e r f e r e d 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), c e n t r i f u g a t i o n (4,000 r.p.m.,  12 4- hour minimal), c r standing a t 5°C. f o r as much as a week. T h i s d i f f i c u l t y was l a r g e l y overcome by e x t r a c t i n g with acetone and removing the phospholipids.  This was done by  adding a few drops of ethanol saturated w i t h MgCXg (Bush, 1952)  to the acetone e x t r a c t , 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 w i t h an  a l k a l i (M-KOH) gave an emulsion, but t h i s could be broken by c e n t r i f u g i n g at 2500 r.p.m. f o r 15 minutes, C e n t r i f u g a t i o n , a w a s t e f u l process, was replaced by a gentle r o l l i n g movement of the separatory f u n n e l containing the immiscible phases, f o r a minimum of 10 minutes, on each e x t r a c t i o n . 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 s y n t h e t i c systems tabled by Bachman and P e t i t (1941), Mathers (1942), Friedgood  and  Garst (1950), and Engel (1950) i n d i c a t e d that the p a r t i t i o n of Friedgood and Garst (carbon t e t r a c h l o r i d e : e t h e r (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 p o l a r substances that contaminate the estrogen f r a c t i o n could be washed out i n an ether e x t r a c t w i t h 9$ NaHCOg,in a butanol e x t r a c t w i t h »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 i n d i c a t e d were analysed by paper chromatography.  At t h i s time no paper methods had  13 been devised t h a t would e f f e c t a q u a n t i t a t i v e separation of, the female hormones.  Since the present hormone f r a c t i o n s were  not used i n subsequent s t u d i e s , t h e i r f u r t h e r treatment i s included  here.  The p r i n c i p l e s of paper chromatography are  o u t l i n e d i n S e c t i o n ".TV, page 28. B,  E a r l y Attempts to E f f e c t An I s o l a t i o n By Paper chromatography.  1.  Use of Bush's (1952} paper adsorption method. S t r i p s (2 x 32 cm.).were impregnated with aluminum  s u l f a t e and spotted w i t h salmon e x t r a c t s (Flow-sheet I) t o which female hormones were added.  I n mobile phase systems  c o n s i s t i n g 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 s t e r o i d zones were i d e n t i f i e d by the i o d i n e r e a c t i o n .  The l a r g e  amount of extraneous " b i o l o g i c a l m a t e r i a l hindered r e s o l ution.  I t may be noted that M i t c h e l l and Davies (1954) d i d  not f i n d Bush s method h e l p f u l to t h e i r studies of p l a c e n t a l ,  extracts. 2.  Use of Zaf f a r o n i * s (1951) paper p a r t i t i o n method. Z a f f a r o n i (-1951, *53) impregnated f i l t e r paper s t r i p s  w i t h propylene g l y c o l or fromamide i n various concentrations to serve as s t a t i o n a r y phases f o r the r e s o l u t i o n of adrenal hormones.  Development of, the chromatograms was c a r r i e d out i n  mobile phases c o n s i s t i n g of hexane, benzene and toluene. Attempts were made to use these systems f o r salmon e x t r a c t s 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.) i n a 1 2 hour period.. ( 3 ) residue discarded.  (2) acetone extract A few drops MgCl2 i n methanol (saturated) added, cooled to -5°C. and f i l t e r ed at -5°C.  ( 5 ) residue discarded.  (k) acetone f i l t r a t e Evaporated i n vacuo and residue transferred with Et20 (3 x 2 5 ml.) to separatory funnel. Et^O washed with % NaHCOj ( 2 x 2 5 ml.).  ( 7 ) NaHCOj phase Discarded after washing once with 2 5 ml. HOH which is added to KOH phase.  (6) Et20 phase Evaporated i n vacuo and CCl^tEt^jO (18 t l , 7 6 ml.)added and extracted with N-KOH ( 5 x 35 ml.).  (9) CGl^:Et20 phase Discarded after washing once with 2 5 ml. HOH which i s added to KOH phase.  (8) KOH phase Brought to pH 3 - 4 (HC1) and extracted with Et^jO ( 4 x 80 ml. )«  (11) aqueous phase discarded.  (10) EtgO phase Washed with HOH (2 x 2 5 ml.) which i s discarded.. EtgO i s evaporated to dryness i n vacuo. (12) estrogen fraction Transferred i n methanol to pyrex tubes for chromatography.  15 was d e t e c t i n g adrenal s t e r o i d zones on paper according t o t h e i r property of absorbing u l t r a - v i o l e t l i g h t a t a wavelength of 240 ny., the p r i n c i p l e was used here f o r female hormones,  These absorb u l t r a - v i o l e t l i g h t a t 280  . I n 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 mine r a l lamp w i t h 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 •('asopposed t o absorption) of female .hormones i n concentration of the order of 100 micrograms per square centimeter on wet paper.  A p p l i c a t i o n of t h i s method t o the d e t e c t i o n o f the  hormones on paper f a i l e d because i n t e r f e r i n g substances o f salmon o r i g i n f l u o r e s c e d much b e t t e r . The conundrum a r i s i n g from these e a r l y attempts t o separate 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 ( B a i l e y , C a r t e r , and Swain, 1952).  Since t h i s f r a c t i o n  a l s o contains the estrogens,, an attempt was made t o e l i m i n a t e the carotenoids by column chromatography.  Stimmel (1946)  s u c c e s s f u l l y extracted the estrogen present i n l a t e pregnancy u r i n e by using an alumina column.  According t o the  d i s c u s s i o n of a paper presented by Bauld (1952) a t l e a s t two independent i n v e s t i g a t o r s (Swyer and Bates) were not able t o d u p l i c a t e Stimmels(1946) r e s u l t s , nor apparently, could Stimmel  d u p l i c a t e h i s own f i n d i n g s w i t h another batch of alumina. Bates, however, was s u c c e s s f u l 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 s y n t h e t i c 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 u r i n e . The present study used Sammuels (1949) method as a working basis.  Alumina of 80-200 mesh s i z e was made more r e -  t e n t i v e by h e a t i n g 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 w i t h 3 cm, of f i n e g l a s s wool,  A g l a s s rod f l a t t e n e d at the t i p j u s t 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 i n t o the column over a distance of 20 cm.  The open end of  the column was then f i t t e d w i t h a 50 m l , capacity dropping f u n n e l which d e l i v e r e d successive volumes of e l u t i n g s o l v e n t s . The column was washed once w i t h 50 ml, of petroleum ether before use. An e x t r a c t was prepared from salmon embryos by evaporati n g an ether extract (Flow-sheet I , step 3) and suspending the residue i n petroleum e t h e r . A f t e r adding the suspension to the uppermost surface of the alumina., successive 50 ml. volumes of petroleum e t h e r , 10$ chloroform i n petroleum e t h e r ,  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 r a t e of approximately 1 ml. per minute. wet w i t h the s o l v e n t s at a l l times.  The column was  kept  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) test.  I t was observed that at l e a s t 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  c o l o r and the second, y e l l o w .  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 s o l v e n t s by s u b s t i t u t i o n of s i m i l a r s e r i e s of benzene-ether and benzene-methanol combinations d i d not a l t e r the p i c t u r e .  The s u b s t i t u t i o n 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 a l s o f a i l e d to give a Kober detectable estrogen f r a c t i o n . The l a r g e amount of residue obtained from every f r a c t i o n eluted from the columns showed that b e t t e r p u r i f i c a t i o n methods were required to e f f e c t 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.  Dialysis. Szego and Roberts (1946) and Roberts and Szego (.1946)  18 were the f i r s t i n v e s t i g a t o r s , 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 s t e r o i d 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 d i a l y z e d i n t o water and that the method provided a means of demonstrating estrogenic a c t i v i t y i n blood plasma.  These r e s u l t s contrasted thos© of  Rakoff, P a s c h k i s , and Cantarow (1943), who found that estrogens i n pregnancy serum would not pass through a c o l l o d i o n membrane by u l t r a f i l t r a t i o n .  Z a f f a r o n i and Burton (.1953) and Z a f f a r o n i  (1953) showed that adrenal hormones would d i a l y z e  independent-  l y of one another i n t o a 40$ methanol s o l u t i o n 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 g r e a t l y increased  by adding chloroform which continuously extracted the h o r mones as they d i f f u s e d i n t o the outer system. The c o n d i t i o n s f o r optimum d i a l y s i s of female hormones were therefore i n v e s t i g a t e d . 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 f r e e e s t r a d i o l - ! ? ^ f o r bioassay, t h i s hormone was used as a standard i n the chemical assay to be described. 1. a  )  S y n t h e t i c system, D i a l y z i n g without s t i r r i n g . V i s k i n g sausage casing (diameter 4,2 cm,) was cut i n t o  30 cm. s t r i p s and placed i n 3 l i t r e s of water saturated w i t h the s o l v e n t s used, i n d i a l y s i s .  A f t e r £ - 1 hour the s t r i p s  were removed and double-knotted a t one end. These were washed twice by f i l l i n g and emptying the bag w i t h the s o l u t i o n already used.  Two washes were made w i t h water, the  19 second wash being used to check f o r l e a k s .  Each bag was  f i l l e d w i t h 10 ml. of Gothlin's s a l i n e s o l u t i o n , 1 ml. methanol c o n t a i n i n g 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 m l , of water, a volume which i s 5 times greater than t h a t i n s i d e the bag* Roberts (1946). then added.  T h i s r a t i o was suggested by Szego and  Twenty-five m l , of e x t r a c t i n g solvent were  Butanol was used e x c l u s i v e l y at pH values  greater than 7, since chloroform may not remove the hormones i n t h i s range.  A f t e r 7 days (168 hours) the d i a l y z i n g bag  was removed and r i n s e d w i t h 10-15- ml. of water i n t o the dialysate.  T h i s was n e u t r a l i z e d w i t h HC1 (pH 4-5) on a l k a c i d  t e s t ribbon and extracted with chloroform (.4 x 75 m l . ) . s  The contents of the d i a l y z i n g bag were emptied i n t o a beaker and the inner casing w a l l r i n s e d w i t h 10-15 ml* of water i n t o the same beaker.  This residuum was treated as  above, except that 4 x 25. m l , volumes of chloroform were used to e x t r a c t the aqueous phase. A f t e r evaporating the chloroform i n vacuo, the residue was t r a n s f e r r e d i n methanol (3 x 5 ml,) by a m e d i c i n a l dropper to a pyrex tube.  Washings were used.  evaporated i n a water bath a t 75°C.  The methanol was and the residue d r i e d  over phosphorus pentoxide. The hormone content was then assayed by the Kober t e s t 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 w i t h a tungsten l i g h t b)  source,  D i a l y z i n g with s t i r r i n g . The V i s k i n g sausage casing was cut i n t o 50 cm. s t r i p s  and washed i n the manner described except that the casing was double-knotted  at a single locus.  I t was then made 39  cm. long from knot t o open end and f i x e d w i t h l i n e n thread (40 guage). t o the rimmed end o f a cut pyrex tube (1.3 x 2.4 cm.) f i t t e d i n t o a bored rubber stopper.  This d i a l y z i n g bag  was placed inan Exax 1000 ml. graduate c y l i n d e r containing rt  500 ml. o f water.  n  The s y n t h e t i c medium, c o n s i s t i n g of 25  ml. of G o t h l i n ' s s a l i n e and 155 ml of water, was poured i n t o the casing using a g l a s s f u n n e l . pipette.  E s t r a d i o l was added by  The outside medium was then made up t o volume (350  ml. of water and. 50 ml. of e x t r a c t i n g s o l v e n t ) . A g l a s s rod f l a t t e n e d t o a button shape at the t i p , and reaching t o w i t h i n 1 cm. o f the bottom of the bag served t o s t i r the contents, and thereby decrease the time required f o r d i a l y s i s .  The rod  was d r i v e n by a power s t i r r e r set a t i t s slowest speed. A f t e r 48 hours s t i r r i n g the casing was removed and r i n s e d on the outside w i t h approximately  20 ml. of water  i n t o the c y l i n d e r containing the d i a l y s a t e . I f butanol had been added t o the outside medium, the volume was reduced t o 500 ml* i i i vacuo a f t e r b r i n g i n g 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 w i t h 4 x 100  ml. of chloroform.  21 The residuum i n the d i a l y z i n g bag was removed w i t h washing and extracted w i t h 4 x 50 ml. of chloroform, c)  C a l i b r a t i o n curve f o r e s t i m a t i o n . High values obtained i n the f i r s t group of experiments  (numbers 1, 6, 7, 8, 11, Table I) showed the n e c e s s i t y 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 e x t r a c t s o f d i a l ysatea i n 6 dummy runs.  A f t e r evaporating the chloroform,  the residue was d i s s o l v e d i n methanol and d i v i d e d i n t o 6 portions.  D i f f e r e n t amounts o f hormone were added t o 5 of  these p o r t i o n s , the s i x t h s e r v i n g as a blank; r e a c t i o n was c a r r i e d out on a l l tubes.  A Kober  The c a l i b r a t i o n  curve  obtained i s shown i n f i g u r e 1. d) • Hormone r e c o v e r i e s i n the s y n t h e t i c 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 throughout 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  r e s u l t of the heterogeneity appearing i n blanks run throughout the experiments, and a l s o , t h e r e f o r e , i n the hormone runs themselves.  Thus a blank of Kober reagents gave the  f o l l o w i n g values f o r blanks prepared from dummy runs: pH ca 14.0 ca 6.8 ca 6.8 ca 6.8 ca 6,8 '4.0  System Na GQ HOH HOH HOH water HOI 2  3  butanol butanol butanol chloroform butanol  O p t i c a l Density  • ---  .033 .278 , .038 ,185 ,046 ,126  22.  MICROGRAMS Figure 1. Calibration curve for estradiol-17^ i n a synthetic system©  TABLE I . RECOVERY OF 50 MICROGRAMS OF ESTRADIOL^!?/* BY DIALYSIS IN A SYNTHETICAL SYSTEM* No. of Experiment  E x t e r n a l Solvent  Micrograms, recovered  Duration of Dialysis Days  In  Out  Ratio  Percent recovered out  In/out  1 2 3 4 5  ,3M NasC03 (pH ca 14) *• Butanol  7/ 7 7 2 2  14.4 9,9 3.5 14.0 7.0  73.9 14.2 45.9 64.4 32.8  11.300 1.430 13.100 4.600 4,665  147.8 28.4 91.8 128.8 65.6  6  NagCOs.NaHCOs |pH  7  9.7  53.4  5.505  106.8*  7  H3B03:Na0H (pH 9,2) * Butanol  ?  11.5  64.8  5.630  129.6*  8 9 10  9% NaHC03 CpH ca 8.2) f. Butanol  7 7 7  5.8 3.5 3.0  66.1 13.4 36.2  11.397 3.828 12.067  132.2* 26.8 72.4  11 12 13 14 15 16 17  HOH (pH ca 6,8) * Butanol  7 7 7 7 7 2 2  4.5 7.7  59.3 33.9 47.2 27.0  13.178 4.403  118.6* 67.8 94.4 54.0  -  49.0 13.5  0.100 '-  98.0 27,0  18 19  HOH *• Chloroform  7 7  4.8 4.2  27.6 25.9  5.750 6.167  55.2 51.8  *• Butanol  10.1)  —  —  1.3 4.9  _  _  _  _  s  TABLE I continued No. of Experiment  E x t e r n a l Solvent  Micrograms recovered  Duration o f Dialysis Bays  In  Ratio  Out  In/Out  Percent recovered 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  HOH * chloroform  7 7  1.4 1.3  10.8 11,9  7.714 9,154  21.6 23;8  H  29  30**  * Blank o f chemical reagents only Estriol  25  Ihere the hutanol system could serve as a blank, the c h l o r o form system gave o p t i c a l d e n s i t i e s 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 d e n s i t i e s of •210, 0, 0, .083, and .017. D i f f e r e n c e s between s t i r r e d and u n s t i r r e d blanks were ..068 {against a s t i r r e d blank),and »017 and .083 (against.an unstirred blank).  I n face.of such an impasse the s y n t h e t i c  runs were discontinued and salmon m a t e r i a l introduced. 2.  B i o l o g i c a l Systems. The r a t h e r c o n s i s t e n t values obtained i n the c h l o r o -  form-water and chloroform:ether-water systems (experiments 18-21, Table I ) suggested the use of t h i s system mon m a t e r i a l .  with s a l -  Since the carbonate-butanol system ( e x p e r i -  ments 2-5, Table I ) appeared t o give higher values, i t was a l s o 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 i n g 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 t o 56$ from butanol. Both systems were run i n volume r a t i o s suggested by  s  2.  Ether was added t o chloroform i n view o f experiments 29, 30, (Table I ) which show chloroform does not r e a d i l y take up e s t r i o l .  26 Z a f f a r o n i (1953) f o r the adrenal s t e r o i d s .  Thais 60 g. of  minced salmon embryos i n 120 m l . of water were d i a l y z e d under s t i r r i n g .  I t was observed that the butanol became  yellow w i t h i m p u r i t i e s 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 f o l l o w s .  The  casing was washed i n a water-95$ ethanol s o l u t i o n C2:l) and f i t t e d t o t h e apparatus described on page 20 and then 100 m l . of chloroform:ether (20;1) were placed i n - a graduated c y l i n d e r and made up t o 850 ml. w i t h water.  The t i s s u e t o be d i a l y z e d  was put through a meat grinder while i n the f r o z e n s t a t e . S i x t y grams of minced t i s s u e was mixed i n 120 ml. of water and placed i n the d i a l y z i n g bag which was then s t i r r e d f o r 48 hours a t room temperature, Figure 2, A f t e r t h i s time the d i a l y z i n g bag was removed, r i n s e d , and discarded.  The d i a l -  ysate was brought t o pH 4-5 and t r a n s f e r r e d t o a separatory funnel.  The chloroform:ether already present was used t o  make an i n i t i a l e x t r a c t i o n .  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 t o prevent l o s s of e s t r i o l when a washing was made w i t h 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. A*  PAPER PARTITION CHROMATOGRAPHY OF SALMON EXTRACTS 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 p l a c i n g 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 s t e r o i d hormone s t u d i e s the  paper i s p r e v i o u s l y impregnated w i t h a h i g h l y p o l a r solvent or s a l t (the s t a t i o n a r y phase).  The chemical compounds mak-  i n g up the residue then move at d i f f e r e n t r a t e s down the paper according to t h e i r molecular d i f f e r e n c e s .  The process  i s regarded as a l a r g e number of consecutive p a r t i t i o n s of the substances being chromatographed between two phases (Cassidy, 1948).  I n 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 contaminants present ( o i l s e t c ) (Engel, 1950) i t must a l s o f o l l o w that these c o n d i t i o n s are l i m i t i n g f a c t o r s i n paper chromatography.  Hence only l i m i t e d amounts of m a t e r i a l can  be r e s o l v e d i n a paper system.  As these amounts are made s m a l l  ( p o r t i o n i n g i n t o l o t s , spreading the spot on l a r g e r 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 f a c t o r ( 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 p r e l i m i n a r y p u r i f i c a t i o n methods were used (Section I V ) .  29 B.  Reproduction of Axelrod's (1955) Method Using E s t r i o l , Estradiol-17jg and Estrone, t  1) Procedure. Since t h i s method i s b r i e f l y o u t l i n e d 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 r u l e d (2-H p e n c i l ) 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 d i v i d e d i n t o 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).  A f t e r being washed i n  water 12-24 hours, the s t r i p was t r a n s f e r r e d to 95$ e t h y l a l c o h o l f o r 24 hours.  I t was d r i e d and impregnated w i t h 50$  formamide i n methanol (Y/V) and b l o t t e d between f i l t e r paper p r e v i o u s l y 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 g l a s s so that the r u l e d s t a r t i n g l i n e f e l l over a g l a s s gate 1 cm, h i g h .  The gate was con-  s t r u c t e d of 1 cm. g l a s s 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 b l o t i t . A g l a s s 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 f r a c t i o n s were a p p l i e d 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 p i p e t t e .  A stream of  a i r , d i r e c t e d f r e q u e n t l y 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 t r a n s -  f e r r e d to tanks f o r 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 s o l v e n t , saturated w i t h the s t a t ionary phase, was added t o troughs, suspending the paper s t r i p i n t o the- tank.  S e a l i n g was e f f e c t e d by a g l a s s cover made  a i r - t i g h t w i t h a paste of s t a r c h i n g l y c e r o l and a heavy weight. Three tanks were used and f o r each, the c o n d i t i o n of the developing solvent was maintained by replacement* The methylcyclohexane-formamide  system (hereafter r e f e r r e d t o  as tank I ) served to make 24 hour runs a t 20±2°C, on paper j u s t spotted. This removes i n e r t i m p u r i t i e s . f r o m the hormone f r a c t i o n s .  Chromatograms c o n t a i n i n g estradiol-17£  and estrone were then t r a n s f e r r e d f o r a 12 hour run i n the Or-dichlorobenzene-formamide as tank I I ) ,  system (hereafter r e f e r r e d to  Paper c o n t a i n i n g the e s t r i o l f r a c t i o n was  transferred- from tank, I f o r r e s o l u t i o n in.a. dichloromethaneformamide system (tank I I I ) .  The running time here was 10  hours. On removal, the chromatograms were d r i e d by a warm a i r f a n (30°C,) approximately 10 hours and then by a cold f a n (room temperature) f o r 10-20 hours.  The chromatograms  were considered dry, when samples of the paper from above the o r i g i n d i d not char_..in. 15% fuming s u l f u r i c a c i d .  This i s  a very important t e s t and devised i n the present i n v e s t i g a t i o n t o overcome negative r e s u l t s obtained with, c o l o r indicators: used when the paper only appeared t o be d r y . 2)  D e t e c t i o n of estrogens, Axelrod l i s t s 7 c o l o r reagents f o r the d e t e c t i o n of  31 the hormones.  Of these, 15$ fuming s u l f u r i c a c i d , benzoyl  chiloride-zince c h l o r i d e , and n i t r o u s acid-mercuric n i t r a t e ( M i l l o n ' s reagent modified) g i v e eonsis.tant r e s u l t s (and were therefore used i n t h i s study).  The p h e n o l s u l f o n a t e -  phosphoric a c i d t e s t was not attempted.  Rosenkrantz (1953)  antimony t r i c h l o r i d e i n nitrobenzene was found very u s e f u l . The most s e n s i t i v e i n d i c a t o r was F o l i n and Ciocalteau's phenol t e s t as modified f o r paper chromatography by M i t c h e l l and Davies (1954).  This t e s t was not a v a i l a b l e u n t i l very  l a t e i n these s t u d i e s . 3)  P o s i t i o n of the hormones. The p o s i t i o n s of e s t r i o l , estradiol-17yS and estrone,  and a mixture of these are shown (Table I I ) a f t e r 24 hours i n tank I . . I t i s c l e a r that these hormones do not stay e x a c t l y on the s t a r t i n g l i n e as claimed by A x e l r o d . to adjust t h i s observation by chromatography  Attempts  i n petroleum  ether, cyclohexane, and cyclohexane-methylcyclohexane (1:1, -Y/V) d i d not a l t e r the p i c t u r e . were found when undiluted  Identical positions also  formamide was used as the s t a t i o n -  ary phase. Observations made on a mixed ehromatogram c a r r i e d over and developed i n tank I I (12 hours) showed e s t r a d i o 1-17/5 and estrone t o move i n accord with m i g r a t i o n r a t e s expected of t h i s system.  However e s t r i o l d i d not remain s t a t i o n a r y as suggested  by A x e l r o d . E s t r i o l t r a n s f e r r e d from tank I t o tank I I I f o r development gave the r e s u l t s defined by A x e l r o d .  32  Table I I .  The l o c a t i o n of f r e e s t e r o i d s ( 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  Starting l i n e Estriol Estradiol-17^ Estrone Mixture. Time (hours)  FOrmamide-methyleyclohexane (Tank I ) 0.0-G.5 1.3-4.3 1.4- 4.7 1.4-4.6 1.3-4.8 24  em. • cm. cm. cm. cm.  Fo rmami de-O- D i c h l o r o d i c h l o r o - " methane ^ benzene ^ (Tank III} ™ (Tank I I ) * 3  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, a f t 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. A f t e r d e t e c t i o n 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 w i t h methanol (2 x 5 ml., 2 x 1 i n t o pyrex tubes.  hour)  The eluates were t r a n s f e r r e d w i t h a  m e d i c i n a l dropper to s i n t e r e d g l a s s f i l t e r s (medium p o r o s i t y ) and the f i l t r a t e s c o l l e c t e d i n pyrex tubes. made throughout t h i s transfer.  Washings were  The methanol was concentrated  to 5 ml. and the amount of hormone determined in. a Beckman DU spectrophotometer having an u l t r a v i o l e t l i g h t . s o u r c e . Blanks and standards were prepared i n the same way. 5)  E l i m i n a t i o n of background  material.  Despite previous washing of the f i l t e r paper, impuni t i e s of paper o r i g i n were present i n the hormone e l u a t e s . These i n t e r f e r r e d w i t h o p t i c a l d e n s i t y 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 f i g u r e 3. A small v a r i a t i o n at the absorption maximum i s apparent (curves C, D, E ) .  T h i s i s the r e s u l t of the wide  margin of e r r o r introduced by using s m a l l amounts o f 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 l e s s than 15 //g./ml. 6)  Absorption Curves. Umberger and C u r t i s (1948) used a s u l f u r i c a c i d r e -  a c t i o n 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-  i o n curves were shown f o r each estrogen when measured i n the  from chromatographed estradiol-17^ (D), estriol (C), and estrone (E)« Curve A i s estradiol-17B against pure methanol. Curve B i s a prepared blank against pure methanol*  35 400-550 m^.  range.  Axelrod modified the method by e l i m i n a t i n g  the heat period of 12 minutes.  The hormones were stood i n  s u l f u r i c a c i d f o r 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 w i t h reported values.  However a., .small peak  heretofore recorded at 452 m//. was evident at 440 myu. i n t h i s study.  E s t r a d i o l - 1 7 p gave absorption maxima at 310,  430, and 452 m^. result.  370,  Only the peak of 310 agrees w i t h Axelrod's  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 w i t h previous f i n d i n g s . Since i t i s w e l l known that both the brand and s p e c i f i c g r a v i t y of s u l f u r i c a c i d as w e l l as the  concent-  r a t i o n s of hormone a f f e c t the f i n d i n g s (Umberger and C u r t i s , 1948)  i t i s concluded that s e r v i c e a b l e r e s u l t s were obtained  from the 6 //g./ml. (as opposed to Axelrod's 60-80../'g./ml.) • C.  A p p l i c a t i o n to salmon Embryo D i a l y s a t e s * Minced salmon embryo (60 g.) c o n t a i n i n g 100 y g .  q u a n t i t i e s of added e s t r i o l , e s t r a d i o l - 1 7 ^ , and estrone  was  d i a l y z e d and the e x t r a c t (Flow-sheet I I ) examined chromatographically.  A f t e r 24 hours development i n tank I ,  a yellow zone of i m p u r i t i e s 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 t e s t s made  56  .15 O"  > co Z .lOO« LU Q _J <  y  a O  050-  .OOO l 200  300  400  500  600  MILLIMICRONS Figure 4. Absorption spectra of estradiol-17fJ (A), estrone (B), and e s t r i o l (C), i n concentrated s u l f u r i c acid©  37  FLOW SHEET IX (1)  dialysate Brought to pH 4^5 and extracted w i t h CHCI3 . E t G {20 : 1) (6 x 2  I (3) aqueous: phase discarded.  ~  (2)  100  ml,).  CHCI3 , E t 0 2  100 m l , EtgO added and washed w i t h 9% .NaHC0 (2 x 100 ml.) and then w i t h HOH (2 x 100 m l . ) . 3  ('6) aqueous phase discarded.  (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 d i d not i n d i c a t e that hormones were present on e i t h e r 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 c a r r i e d over f o r f u r t h e r development i n tank I I . The yellow zone noted p r e v i o u s l y had migrated 2-3 cm. more down the paper.  Color t e s t s made on sample s t r i p s f a i l e d t o  i n d i c a t e any one of the hormones.  I n another run areas  before, a f t e r , and i n c l u d i n g , the yellow zone were e l u t e d and examined s p e c t r o p h o t o m e t r i c a l l y at 270-300 mp. w i t h negative r e s u l t s .  These were confirmed by the Kober t e s t  made on the e l u a t e s . This f a i l u r e suggested that l a r g e r amounts of hormone would have t o be added i f a recovery was t o be e f f e c t e d . However, since the present l e v e l s being used were.5 times greater than any known source o f female hormone, f u r t h e r recovery experiments were regarded as being of no value to the present study.  On t h i s b a s i s i t may a l s o be considered  that d i a l y s i s i s not an e f f e c t i v e method f o r a chemical assay of hormones i n . b i o l o g i c a l t i s s u e .  This g e n e r a l i z a t i o n ,  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 s y s t -  ems i n mammals are b e l i e v e d t o contain substances  which  destroy or i n a c t i v a t e 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 t e s t e s ,  . '  39 D.  A p p l i c a t i o n to Horse Testes D i a l y s a t e s . Horse t e s t e s were s e l e c t e d f o r assay p a r t l y because  of the l a r g e 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 p r e v i o u s l y ( B e a l l , 1940).  L i k e most i s o l a t i o n  s t u d i e s of hormone i n t i s s u e , B e a l l used a large amount of t e s t e s (28 kg.) from which the hormones were extracted w i t h 280 l i t r e s of a l c o h o l .  A f t e r p u r i f i c a t i o n he obtained  210  //g. of e s t r a d i o l - 1 7 ^ and 360 yfg. of estrone per kilogram of tissue.  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 t e s t e s . Three 60 g. l o t s of minced t e s t e s from 3 d i f f e r e n t horses were d i a l y z e d and estracted f o r separation by paper chromatography (Flow-sheet I I I ) * The e s t r a d i o l - e s t r o n e f r a c t i o n was portioned i n t o two l o t s from 20 ml. of methanol.  One  l o t was t e s t e d by the  Eober r e a c t i o n (which was p o s i t i v e ) and the other, ehromatographed.  A sample s t r i p from"the ehromatogram was  tested  f o r the presence of hormones by the M i t c h e l l - D a v i e s (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 d i d not occupy p o s i t i o n s , however, s i m i l a r t o pure e s t r a d i o l - 1 7 ^ 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 i m p u r i t i e s a l t e r migration r a t e s (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 e l u t e d  and assayed spectrophotometrically  f o r the presence of the  40  FLOW SHEET I I I (1) 3 x 60 g. minced horse t e s t e s . (.2) d i a l y s a t e Brought t o pH 4-5 and extracted w i t h CHCl .Eta0 (.20:1) (6 x 100 m l . ) . 3  I  I (4) aqueous phase discarded,  (3) CHCl :Et20 ' 100 ml. EtgO added and washed with 9% NaHC0 (2 x 100 ml.) and then With HOH (2 x 100 ml.), 3  3  (6) aqueous phases discarded,  (5) CHCl :EtgO Pooled. I n vacuo t o dry: ness; 2 ml. ethanol and 250 ml. benzene added ( i n ordler) • Extracted w i t h EtgO (4 x 200 ml.),  (9) aqueous phase Extracted w i t h EtgO (4 x 150 m l . ) .  (7) benzene phase Evaporated i n vacuo to dryness.  3  (10) aqueous phase (11) E t o (8) e s t r a d i o l and estrone discarded i n vacuo fraction to dryness Transferred i n methanol to pyrex tube f o r chroma to graphy. g  (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 manyother substances) already i n d i c a t e d by the M i t c h e l l - D a v i e s • test.  No such group was observed.  These c o n t r a d i c t i o n s ,  suggested that background m a t e r i a l obscured the absorption peak expected at 280 m/s. to each e l u a t e .  A Kober t e s t was t h e r e f o r e applied  Each gave a c l e a r , deep pink c o l o r , a  positive reaction. The concentrations of the Kober p o s i t i v e m a t e r i a l i n the eluates were measured against the chromatographed blank, e s t r a d i o l - 1 7 ^ , and estrone by the method of Yenning et a l (1937),  The r e s u l t s are shown i n T a b l e . . I l l , . Small amounts  of hormone ( i n ord.er...of 10 yyg./90 grams of testes), were s u c c e s s f u l l y extracted* Table I I I . Concentration of hormones recovered from d i a l y s a t e s of horse t e s t e s . Materials Blank E s t r a d i o l standard Estrone standard Estradiol,, horse. Estrone horse  Wave]Length 420 522 .089 .036 .209 .211  .718 .585 .316 .348  Difference 522-420 .629 .549 . .107 . ... . . .137  52.1 52.1 8.862/90 g. 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 e s t r a d i o l - 1 7 ^ and estrone i n these horse t e s t e s might be male hormone despite the chemical d i f f e r e n c e s between male and female hormones.- - Hence androsterone and testosterone were chromatographed i n concentrations estrogens recovered. test.  (20 /Jg.) twice that of the  No trace was found by the M i t c h e l l - D a v i e s  A Kober t e s t made on 20 /^g. amounts of c r y s t a l l i n e  42 androsterone and testosterone colors, respectively.  gave p a l e orange and v i o l e t  The pink c o l o r of the estrogens d i d  not, t h e r e f o r e , develop. I n resume, the evidence i d e n t i f y i n g e s t r a d l o l - 1 7 ^ and estrone i n the present study i s : 1.  Both hormones were i s o l a t e d from horse t e s t e s 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 f o r female hormones.  3.  The Mitehell-Davies t e s t (1954) requires a phenol group i f the substances under t e s t are i n microq u a n t i t i e s . The female hormones recovered s a t i s f i e d these c o n d i t i o n s .  4.  The Kober t e s t (Kober, 1931; Brown, 1953; Bauld, 1954) r e q u i r e s an oxygen group at the 17 p o s i t i o n and an a v a i l a b l e H at the 16 p o s i t i o n (Bates, 1952.)., Such p o s i t i o n s are t y p i c a l of sex hormones.  5. Male hormones d i d not give the Kober c o l o r 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 t e s t e s was run on a 1 cm. s t r i p  (page 29).  A f t e r development i n  tanks I and I I I , a Mitchell-Davies-rpositive zone.was evident close t o the s t a r t i n g l i n e . Kober c o l o r .  The zone however d i d not give a  Furthermore, measurements were not p o s s i b l e  against a blank and c o n t r o l 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 t e s t e s . Materials Blank E s t r i o l standard "Mi t che11-Davi es" Zone  Wavelength 420 522 .013 0  Difference 522-420  mm  mm  .348 0  .335  Amount  • 22,9  43 Thus,, no e s t r i o l was found, an observation i n accord w i t h B e a l l * s (1940) r e s u l t s . F i n a l l y , the entire- experiment was repeated on a s i m i l a r amount o f horse t e s t e s .  These were from a d i f f e r e n t  source, and of much s m a l l e r s i z e (approximately 150 g, per t e s t i c l e - as" compared to 330 g. i n the previous experiment). I n t h i s t e s t the d i a l y s a t e e x t r a c t was separated i n t o 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 p o r t i o n , however, was not  separated i n t o e s t r a d i o l - e s t r o n e 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 d i d not respond to Kober*s t e s t . The e n t i r e female f r a c t i o n from 180 g, o f minced t i s s u e was chromatographed (as compared w i t h 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 f u r t h e r p u r i f i c a t i o n i n order to o b t a i n i t s absorption curve i n s u l f u r i c a c i d .  The m a t e r i a l 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 t e s t e s from probably younger horses,  2.  Storage p e r i o d of the t e s t e s which was a t l e a s t two w.eeks longer than those f i r s t used. F o r placentae,. M i t c h e l l and Davies (1954) p o i n t out processing should occur w i t h i n 30 minutes o f delivery,  3.  Estrone may not have been present i n the t e s t e s . For example, D i c z f a l u s y (1953) found e s t r a d i o l Vip i n only 2 of 6 placentae examined.  -44 VI. A.  A 'FURTHER SEARCH FOR FEMALE HORMONES I N SALMON EMBRYOS. The M i t c h e l l - D a v i e s P a r t i t i o n Method. Because, of the apparent o r p o s s i b l e d e s t r u c t i o n o f  hormones when salmon embryos were being d i a l y z e d , use was made o f a technique r e c e n t l y 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 s t u d i e s by Engel (1950), o f f e r e d the f u r t h e r advantage of r e c o v e r i n g conjugated and protein-bound estrogens.  Their paper p a r t i t i o n a n a l y s i s  was not attempted i n view, o f Axelrod*s paper method already worked out. Their method as a p p l i e d t o salmon embryos i s shown i n Flow-sheets I V and IVa. I t w i l l be n o t i c e d that an extra step i s i n c l u d e d j u s t p r i o r t o 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 m l . a l i q u o t of EtgjQrCC^ was p a r t i t i o n e d w i t h 5 m l . o f 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 t o e f f e c t a b e t t e r p a r t i t i o n (Friedgood and Garst, 1950).  The  e x t r a c t i o n of the hormones from an a l k a l i n e s o l u t i o n a t pH 9 (Engel, 1950) i s the most outstanding f e a t u r e of the method. . This was done with, a Beckman pH meter i n the present experiments, but " h y d r i o n " paper was used by Engel and presumably by M i t c h e l l and Davies.  Under these c o n d i t i o n s  the e n t i r e procedure was c a r r i e d through without i n t e r f e r e n c e 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  (k) ethanol (7) residue (5) butanol fraction A extract extract Pooled with L_ , I fraction B. (6) Combined and evaporated to dryness i n vacuo. Suspended i n kOO ml. waterj extracted 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 HgS0h Divided into two portions and each extracted with k x 200 ml. EtgO. Combine EtgO. |  (9) aqueous phase (10) Brought to k25 ml. with HOHj: boiled j 75 ml., concentrated HC1 addedj refluxed kO minutes; cooled}; Extract with EtgO (k x 200 ml. ). (13) aqueous phase discarded.  Extracted with n-butanol (3 x k00 ml.),  EtgO extract "free" estrogen fraction (1).  o  (11) EtgO (lk) aqueous extract fraction "protein'* discarded, bound estrogen fraction (3).  (12) EtgO extract "Conjugated estrogen fraction (2).  11  Estrogen fractions 1,2, 3 treated as outlined i n Flow sheet IVa  1*6 FLOW SHEET IVa (15) Fractions 1, 2, 3 combined and evaporated i n 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 i n vacuo. 800 ml. acetone ana a few drops MgClg in alcohol (saturated) added. Cooled to ^ 0°C. and filtered at this temperature. , 1 (19) residue ( 1 8 ) acetone f i l t r a t e discarded. Evaporated i n vacuo; a solution of 600 ml. EtgO and 30 ml. CCL. i s added. Extracted with N-KOH (6 x 100 ml.). x  (21) EtgOtCClj, 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.).  (23) aqueous phase discarded.  (22) E t 0 extract In vacuo to dryness} h ml., ethanol and 500 ml. benzene added (in order)* Extracted with HOH (5 x 150 ml.).  (27) aqueous phase Extracted with EtgO (5 x 150 ml.).  (2k) benzene phase Evaporated i n vacuo just sufficient to remove benzene (no odor).  2  (28) aqueous (29) EtgO phase (25) phase Evaporated to discarded.. dryness and treated as i n fraction (k) except that .k times the volumes of methanol and petroleum ether are used. (30) e s t r i o l fraction (5) Transferred i n methanol to pyrex tube for chromatography.  residue Dissolved i n 25 ml. methanol and washed with 25 ml. petroleum ether% ether fraction backextracted with 3 x 25 ml. 90# methanol and the pooled methanol evaporated to dryness i n vacuo.  (26) Estradiol-estrone fraction (k) Transferred i n 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 t e s t the magnitude of recovery.  M i t c h e l l and Davies (1953) obtained  average r e c o v e r i e s i n placentae of 27% ( e s t r i o l ) and ( e s t r a d i o l and e s t r o n e ) . 200  13$  A c c o r d i n g l y , i t was estimated that  g. each of added e s t r i o l , e s t r a d i o l - 1 7 ^ 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) processed.  and  At the stage (step 6) where the m a t e r i a l i s  brought to near dryness, i t was a c c i d e n t l y charred, r e s u l t i n g i n an unknown l o s s of hormone and other chemicals of t i s s u e origin.  The damage done was q u i t e 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 l a b o r 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 d r i e d 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 h a l f of i t s volume was used. The developed chromatograms showed amber zones j u s t 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 e s t r a d i o l - e s t r o n e fractions.  Sample s t r i p s were recovered from each chromatogram  and examined f o r hormones by the M i t c h e l l - D a v i e s phenol t e s t .  48 Two zones appeared i n a d d i t i o n t o the amber zones previouslynoted.  I t was evident that e s t r i o l and estradiol-17/? d i d  not advance much beyond t h e i r r e s p e c t i v e amber zones. area marking the advance, was eluted f o r assay. these f i n d i n g s , estrone was w e l l i s o l a t e d .  The  Contrary to  I t a l s o was  eluted f o r assay (Table V ) . . Table J . Recovery values f o r hormones added to salmon embryos, Experiment 1. Material  Recoveries  /'g. Wavelength Difference P. added 420 5J*Si 522-420  Blank Estradiol standard — Estrone standard — Estradiol embryo 300 Estrone embryo 300 — Blank Estriol standard — Estriol • embryo 300  (dilution correction)  .036  . 585. .549-  52.1  .089  .718  .629  52.1  .266  .332  .066  5.5  11,0  3.66 13.60  .094  .309  .215  — f  —  —  —  —  30.4  40.8  .013  .348.  .335  22.9  —  .016  .111  .095  6.5  —  2.16  I t w i l l be r e c a 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  r e c o v e r i e s of 3.66 and 13.60 f o r e s t r a d i o l and estrone, r e s p e c t i v e l y , have l i t t l e meaning.  The small recovery made of  e s t r a d i o l when compared t o 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 impuri t i e s (the amber zone).  An i n d i c a t i o n of the i n t e r f e r e n c e  i s shown by the high, d e n s i t y value of e s t r a d i o l when compared to estrone at 420 m^,  (Table I V ) .  This explanation  i s a l s o a p p l i c a b l e to the e s t r i o l recovery.  A l l recoveries  i n d i c a t e a great l o s s i n processing the 100 ^ g . q u a n t i t i e s of each hormone added to step 20 (Flow-sheet I V a ) . 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, c h a r r i n g d i d not occur, and acetone was necessary f o r a clean a l k a l i p a r t i t i o n (.Blowsheet IV, I V a ) . The residue r e p r e s e n t i n g the estradiolrrestrone f r a c t i o n was enormously l a r g e compared w i t h 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 p l a c e n t a l o r i g i n  weighed 2-10 mg.  ( M i t c h e l l and Davies, 1954). I n the present  case i t was at l e a s t of 1 ml. volume a f t e r d r y i n g over phosphorus pentoxide.  The e s t r i o l f r a c t i o n was minute and not  n o t i c e a b l y l a r g e r than that found i n experiment 1.  1.  The e s t r a d i o l - e s t r o n e 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. a l i q u o t s were taken f o r chromatography. F o r t y micrograms each, of e a s t r a d i o l - 1 7 ^ and estrone were added to a l i q u o t 1,  Both a l i q u o t s were then chromatographed on  separate s t r i p s . The chromatograms showed the same amber zones found f o r t h i s f r a c t i o n i n experiment 1.  The M i t c h e l l - D a v i e s t e s t  50 on sample s t r i p s showed an a d d i t i o n a l 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 e l u t e d and Kober t e s t e d . Only the hormone zones gave a p o s i t i v e r e a c t ion.  The spectrophotometer  Table V I . Material  readings are shown i n Table V I .  Recovery values f o r Experiment 2. Wavelength 420  522  Difference  Recovery  522-420  „» 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 Amber zone, — aliquot 1 1.170 .860 Amber zone, — a l i q u o t , 2^ .655 .509 40 g. E s t r a d i o l * .245 .344 .099 40 g. Estrone* ;194 .480 .286  $ recovery  50.0  —  —  —  —  —  -  t  8.20 23.65  20.5 59.1  £ Added to residue being chromatographed. A l i q u o t 1 c o n t a i n i n g 40 //g. each of added e s t r a d i o l - 1 7 and estrone gave percent r e c o v e r i e s of 20.5 and 59.1, r e s p e c t ively.  These values are i n accord w i t h 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 V I .  The o p t i c a l d e n s i t i e s obtained from the e l u t e d  amber zone showed an absence of hormone. The amber zone of a l i q u o t 2 o r i g i n was a l s o Kober negative and no hormone could be detected spectrophotometrically.  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 a l i q u o t 1, suggested that 20 //g. would be the minimum l i m i t f o r assay.  I f t h i s were so, then 10 x 20 yt/g. would be required  51 f o r an assay of the t o t a l e s t r a d i o l - e s t r o n e f r a c t i o n .  If  the M i t c h e l l - D a v i e s average recovery value of 13$ from the e s t r a d i o l - 1 7 ^ added to placentae could he extrapolated to f i s h embryos, then the amount of f r e e 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 a t l e a s t 10 times greater than any other value reported f o r b i o l o g i c a l t i s s u e . Accordi n g l y , i t i n d i c a t e s the. extreme i m p r o b a b i l i t y of d e t e c t i n g 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 s i n g l e zone, the amber zone. T h i s 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 f r a c t i o n s c o n t a i n i n g added hormone.  At the hormone l e v e l s r e q u i r e d  to make t h i s d e t e c t i o n , .there was an absence of these hormones i n salmon embryos.  52 VII.  DISCUSSION  The b a s i c p a t t e r n of the u n d i f f e r e n t i a t e d gonad i n vertebrates c o n s i s t s 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  i n t o 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 r e s p e c t i v e 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 u n d i f f e r e n t i a t e d gonad.  Germ c e l l s m i g r a t i n g i n t o t h i s area become female  cells.  L a t e r the medullary area d i f f e r e n t i a t e s and germ  c e l l s m i g r a t i n g i n t o t h i s area become male c e l l s .  Both male  and female c e l l s are t h e r e f o r e 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 l o s s 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 r e v e r s a l effected by high temperature r e a r i n g s and p a r a b i o t i c s t u d i e s of frogs ( W i t s c h i , 1929, *34), provided evidence that inductor substances were e s s e n t i a l components of the d i f f e r e n t areas. m e d u l l a r i n and c o r t i c i n .  W i t s c h i c a l l e d these i n d u c t o r s C o r t i c i n suppresses the a c t i o n of  m e d u l l a r i n and s t i m u l a t e s 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 m e d u l l a r i n opposes the a c t i o n  of c o r t i c i n and induces- the formation of male c e l l s .  Since  both i n d u c t o r s are present simultaneously, the genetic c o n s t i t u t i o n normally determines the outcome of sex.  Thus  i n a g e n e t i c a l l y determined female, c o r t i c i n normally would  53  have mere i n d u c t i v e force than m e d u l l a r i n . 1950)  Recently ( W i t s c h i ,  r e s t a t e d h i s theory to conform to a hormonal theory o f  sex d i f f e r e n t i a t i o n (Figure 5 ) .  Apparently W i t s c h i does not  i d e n t i f y the inductor substances w i t h 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 e f f e c t ascribed to the inductors may a l s o be c l o s e l y simulated w i t h hormones. Moore (1947) however, found that androgen and estrogen treatment Of young opossum d i d not modify the ovary o r t e s t e s , nor d i d 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 e v a l u a t i o n of the l i t e r a t u r e , Moore (.1947) concluded that **the most acceptable evidence f o r the c o n t r o l of sex d i f f e r e n t i a t i o n i n vertebrates r e s t s upon t h e concept o f the operat i o n of genetic factors'*, while admitting (Moore, 1950) "that no one has provided a more acceptable explanation ( f o r 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 r e v e r s a l of sex i n the gonads of young mammals, and the lack of evidence f o r a hormone s e c r e t i o n 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 g e n i t a l t r a c t .  However,  Burns. (1.950). found that female hormone would.induce a sex r e v e r s a l ( i n d i c a t e d 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.  C a s t r a t i o n and g r a f t i n g experiments on the  r a b b i t and the r a t , r e s p e c t i v e l y , lead J"ost (1950, '53) to  54  Sec.  Sex Char. F.  Gynogen Hormones Teatost erone  >COflTEX—&^*Ovagenesis /is  i  Estradiol  -  ,m >ME&LLA—SkL^Spermatogenesia Androgen Hormones Sec.  Figure 5.  Sex Char. M.  Agent s t i m u l a t o r s >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 . ( W i t s c h i , 1950). o, c o r t e s i n ; m, m e d u l l a r i n .  55 a s c r i b e to the f o e t a l t e s t i s the r o l e of androgenic hormone production, and that these androgens d i r e c t e d sex d i f f e r e n t i ation. The o b j e c t i v e of the present study was to show the presence of female sex hormones i n salmon embryos. of the suggestiveness  Because  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 t h a t a p o s i t i v e assay would provide the evidence that sex hormones were the f a c t o r s 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 u n d i f f e r - . e n t i a t e d 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 o f mammals. The r e s u l t s , discussed p r o g r e s s i v e l y 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 a t that point where the phenolic  estrogen  f r a c t i o n i s separated from the n e u t r a l 'steroids. The emulsions, which occur a t t h i s a l k a l i p a r t i t i o n have been an obstacle even f o r the i n i t i a l e x t r a c t i o n of urine (Bachman and P e t t i t , 1941).  G e n t r i f u g a t i o n has l a r g e l y overcome t h i s  d i f f i c u l t y but,, as suggested by D i c z f a l u s y (1953),  micro-  gram q u a n t i t i e s o f estrogen might be discarded, i n m i c e l l e s formed from some impurity a t the interphase between immiscible solvents.  I t i s not s u r p r i s i n g to f i n d t h e r e f o r e , that an  e x t r a c t i o n procedure such as that worked out i n a s y n t h e t i c system (Friedgood and Garst, 1951) has no r e a l a p p l i c a t i o n  56 i n the presence of b i o l o g i c a l l y complex m a t e r i a l such as salmon embryos with t h e i r attached y o l k sacs,  Centrifugation  and other methods used to break emulsions, d i d not e f f e c t a separation between a carbon t e t r a c h l o r i d e : partition,  ether •- N-KOH  This problem was overcome by removing.most o f the  emulsion-promoting contaminants, such as the p h o s p h o l i p i d s , with MgCl  2  and c h i l l e d acetone.  As thorough as may have been the e x t r a c t i o n 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 m a t e r i a l .  Separation of 100 ^ g . each of female  hormones added to t h i s mass was not p o s s i b l e 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.  I n view of f a i l u r e s (Bauld, 1952)  experienced w i t h 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 t o be u n s a t i s f a c t o r y f o r c o l o r d e t e c t i o n (Kober, 1931) o f 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 , d i d not a l t e r the f i n d i n g s .  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 o r e x t r a c t i n g estrogens, f a i l e d when used w i t h b i o l o g i c a l m a t e r i a l ( M i t c h e l l and Davies, 1954), The success obtained by Z a f f a r o n i (1S53) i n d i a l y z i n g ..  57 a d r e n o c o r t i c a l 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 p r e v i o u s l y 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 u n c e r t a i n t y that .this method  was s u i t e d to a chemical assay prompted a study of the condit i o n s necessary f o r a maximum y i e l d of estrogens.  The r e s u l t s  from a s y n t h e t i c system were d i f f i c u l t to i n t e r p r e t because, apparently, of the heterogeneity of the Y i s k i n g d i a l y z i n g casing.  This heterogeneity i n t e r f e r r e d w i t h o p t i c a l density  values obtained i n both blank and hormone, d i a l y s a t e s .  How-  ever a choice seemed evident between the butanol-.3M ETagOOg system and the chloroform:ether-water system.  By a t e s t 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 o f f e r e d the advantage of a cleaner e x t r a c t , and was therefore adopted. A f t e r f u r t h e r p u r i f i c a t i o n of the e x t r a c t a paper p a r t i t i o n a n a l y s i s ( A x e l r o d , 1953) was c a r r i e d out on the residue.  The r e s u l t s showed t h a t 100 ^/g. each of added  hormones could not, be detected on the paper ehromatogram or e m p i r i c a l 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 t i s s u e sources ( B e a l l , 1940), d i a l y s i s of salmon embryos d i d not appear to warrant f u r t h e r 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 d e s t r u c t i o n 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 b i n d i n g of the hormones t o salmon p r o t e i n s (Eik-Hes et a l , 1954),  However, f o r blood plasma, d i a l y s i s i s be-  l i e v e d to release 65$ of the p r o t e i n 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 t e s t e s .  I n 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., r e s p e c t i v e l y .  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 s i o l o g i c a l v a r i a t i o n such as  t h a t known to occur i n placentae ( D i c z f a l u s y , 1953) may have a f f e c t e d the determination.  The i n t e n s i t y of the Kober  c o l o r d i d not suggest t h a t much i n t e r f e r e n c e would be e f f e c t e d by the c o r r e c t i o n readings made at .420 mjx, (Yenning, et a l , 1937), : This p o s s i b i l i t y d i d not prove t© be so, e s p e c i a l l y i n the case of e s t r a d i o l .  Thus b e t t e r r e s u l t s might have  been obtained by a p p l i c a t i o n of other c o l o r m e t r i c procedures (Haslewood, 1950) f o r a Kober quantation. E s t r i o l , which has been i s o l a t e d only from the human placenta (Pincus and Pearlman, 1943), was not found i n the horse t e s t e s . An attempt to d u p l i c a t e these r e s u l t s on a second l o t of t e s t e s was u n s u c c e s s f u l . Here, the e n t i r e 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 chromatographed.  E s t r a d i o l was l o c a t e d by the phenol t e s t ( M i t c h e l l  and Davies, 1954), but.estrone d i d not appear.  The s m a l l amount  of e s t r a d i o l , suggested by the c o l o r r e a c t i o n , was eluted and rechromatographed i n order to p u r i f y i t f o r a determination of i t s absorption curve i n s u l f u r i c a c i d . located on a second ehromatogram.  I t could not be  These r e s u l t s  suggested  that storage of the t e s t e s may have destroyed some of the hormones ( M i t c h e l l and Davies,(1954) recommend processing p l a c e n t a l t i s s u e immediately on d e l i v e r y from the mother) or t h a t , i n the ease of estrone, i t was not present.  Thus  D i c z f a l u s y (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 f o r recovering conjugated and protein-bound estrogen f r a c t i o n s , and because of an apparent d e s t r u c t i o n of hormone added to salmon embryos being d i a l y z e d , a r e c e n t l y developed  procedure  f o r e x t r a c t i o n 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 s p o i l e d by charring.  The remaining experiment  showed the d i f f i c u l t i e s •;  p r e v i o u s l y 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 w i t h t h e i r p o s s i b l e hormone content.  This, content however was not evident when  the e s t r a d i o l - e s t r o n e f r a c t i o n was chromatographed i n f r a c t i o n s  . 60 of .1 volume.  This volume, represented  the maximum load the  chromatography paper could handle and be consistent w i t h a s t a r t i n g l i n e spot no wider than .5 cm. The e s t r i o l f r a c t i o n was e s t r i o l was  (Axelrod, 1953).  chromatographed i n t o t o . No  found.  A t e s t recovery was made on an a l i q u o t 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$ f o r estradiol-17/6 added to placentae, i t was estimated  that the amount of e s t r a d i o l  needed f o r chemical assay of salmon embryos was 3.75 mg./kg. tissue.  The magnitude of t h i s quantity w i t h 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 i m p r o b a b i l i t y of d e t e c t i n g hormones i n salmon embryos. I t must be concluded, t h e r e f o r e , that u n t i l more s e n s i t i v e chemical methods are developed, d i r e c t 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 r e f i n e d by f i r s t i n a c t i v a t i n g the enzymes or b a c t e r i a that appear to destroy the added hormone.  This might be e f f e c t e d by a  p r e l i m i n a r y heating of the t i s s u e i n a s a l i n e s o l u t i o n , or b e t t e r , by d i a l y z i n g near 0°G.  The p o o l i n g of e x t r a c t s of  d i a l y s a t e s would compensate f o r the comparatively  small  amount of s t a r t i n g m a t e r i a l that can be managed by t h i s method.  The d i a l y s a t e too, may  be more p r o f i t a b l y t r e a t e d  by a p r e l i m i n a r y enzyme (such as glucuronidase) h y d r o l y s i s of i t s r e s i d u e .  or a c i d  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 o f f e r s any  61 advantage because of the l a r g e amount of m a t e r i a l i n the f i n a l r e s i d u e . However, the M i t c h e l l - D a v i e s paper p a r t i t i o n technique (not used i n t h i s study) appears to be e a s i e r and quicker than that of Axelrod's. The chemical methods a p p l i e d to salmon embryos do not represent a l l of the techniques a v a i l a b l e f o r microchemical s t u d i e s .  Methods i n v o l v i n g counter-current d i s -  t r i b u t i o n , paper e l e c t r o p h o r e s i s , polarography, and f l u o r imetry are being worked out, and while these are s t i l l u n s a t i s f a c t o r y f o r most purposes, t h e i r f u r t h e r refinement may make p o s s i b l e a more p r o f i t a b l e study of hormone s e c r e t i o n i n embryos.  62 VIII.  SUMMARY AND CONCLUSION  The problem of sex hormone s e c r e t i o n during embryogeneses of v e r t e b r a t e animals was approached by a chemical search; f o r female hormones i n salmon embryos. A f t e r p r e l i m i n a r y experimentation, a d i a l y z i n g t e c h nique was developed to concentrate an estrogen f r a c t i o n s u i t a b l e 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  s e x u a l l y 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 t i s s u e could not be recovered.  However horse t e s t e s assayed by the  same technique showed the presence of estradiol-17/* and estrone i n concentrations of .097 and .143 mg./kg., r e s p e c t ively.  The assay of horse t e s t e s was c a r r i e d 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 o r e x t r a c t i o n of estrogen from animal gonads but hormone added to whole salmon embryos i s i n a c t i v a t e d by some unknown system. A p a r t i t i o n technique r e c e n t l y developed by F. M i t c h e l l and R. Davies f o r the e x t r a c t i o n of estrogens from human placentae was s l i g h t l y modified f o r use w i t h salmon embryos. T h i s method confirmed the negative f i n d i n g s obtained by the dialyzing  technique.  On the. b a s i s of these experiments on f i s h , no evidence could be obtained i n support of the hormonal theory of sex  63 differentiation.  T h i s i s not a withdrawal from support of  the concept, hut r a t h e r i n d i c a t e s that a good d e a l of refinement i n chemical assay procedure w i l l he necessary before a proper c o n s i d e r a t i o n 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 c a r r y out t h i s research provided by Dr. W.A. Clemens, D i r e c t o r of the F i s h e r i e s I n s t i t u t e , and former Head o f the Department of Zoology, the U n i v e r s i t y of B r i t i s h Columbia, The program was, developed under the s u p e r v i s i o n o f Dr. W.S, Hoar, P r o f e s s o r of F i s h e r i e s and Zoology, and suggestions by, Dr. M. Darrach, Head of the Department of Biochemistry, the U n i v e r s i t y o f B r i t i s h Columbia.  I am  deeply g r a t e f u l to them f o r t h e i r h e l p . I wish t o thank Dr. K. C a r t e r , D i r e c t o r o f the P a c i f i c F i s h e r i e s Experimental S t a t i o n , the F i s h e r i e s Research -Board of Canada, f o r making a v a i l a b l e 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 o f that s t a t i o n , f o r h i s suggestion o f the s i l i c a - e e l i t e column used i n the p r e l i m i n a r y experiments. Dr. A.W. Matthews, Dean of the F a c u l t y of pharmacy, k i n d l y allowed me t o use t h e i r Beckman DU spectrophotometer. M i s s M. Nagai, Miss D. Timberley, and Mr. A. Beach a s s i s t e d w i t h the typing and p r e p a r a t i o n of the i l l u s t r a t i o n s .  .65 ,LITERATURE CITED 1.  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