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Endogenous testosterone in sockeye salmon (Oncorhynchus nerka) during spawning migration Grajcer, Dov 1961

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ENDOGENOUS TESTOSTERONE IN SOCKETE SALMON (ONCORBTNCHUS NERKA) DURING SPAWNING MIGRATION  by  DOV GRAJCER B.A., University of California at Los Angeles, 1951  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA October, 1961  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study.  I further agree that permission  for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  The University of British Columbia, Vancouver 8, Canada. Date  f  //  ABSTRACT  Testosterone was isolated from both the "free" and conjugated steroid fractions obtained from plasma of spawned male and female sockeye salmon*  Structure of the  steroid was confirmed by several c r i t e r i a including chemical transformation, a sulphuric acid chromogen and infrared spectra*  /  Conjugation with glucuronic acid  /  was established by use of saccharo-l:4-lactone, an inhibitor for ^-glucuronidase* /  i Position through which the conjugation occurs was not established.  In other species  conjugation i s through the 17 /3-hydroxy group rather than the theoretically possible^ 3-enol form i n the ^4—3  ketone*  Testosterone was found i n the conjugated but not i n the "free" form i n testes of migrating 0* nerka* Dehydroepiandrosterone and androsterone, the principal conjugated steroids i n normal human plasma were not detected i n several plasma samples tested*  ACKNOWLDGEMEN  T  This work was made possible through the generous financial assistance of the International Pacific Salmon Commission and the free use of the laboratory f a c i l i t i e s of the Fisheries Research Board Vancouver Technological Station.  In this regard  thanks are due Dr. H.L.A. Tarr, Director of the Vancouver Technological Station and Mr. L.A. Royal. Director of Investigations for the International Pacific Salmon Commission. For his untiring interest, guidance and supervision of this research, the author i s particularly indebted to Dr. J.R. Idler, former Assistant Director of the Vancouver Technological Station, now Director of the Fisheries Research Board Technological Station at Halifax, Nova Scotia. The author also wishes to extend special thanks to Dr. P.A. Larkin, Dr. W.S. Hoar, and Dr. C.C. Lindsey for their encouragement, advice and c r i t i c i s m so generously extended during the preparation of this thesis. To many other associates who i n many ways contributed to the successful completion  of the research, the author extends his grateful appreciation.  iii  TABLE  OP  CONTENTS Page  ABSTRACT. ACKNOWLEDGMENT TABLE OF CONTENTS  i i i i i i  I. II.  INTRODUCTION. . . 1 LITERATURE REVIEW 3 Steroid Conjugates* . • • . • • • • • • • • • • • * • • • • • « • 3 Testosterone* . . . . . . . . . . . .*• • 4 Testosterone Glucuronide Conjugates. . . . . . . . . . . . . . . 4 Biological Effect of Testosterone on Teleost Fish. • • • • • • • 4 Testosterone i n Teleost Fish. * 5 I I I . MATERIALS AND METHODS * 6 Plasma* • • • • • • • » * . . . . . . . . . * . . . . . . . • • 6 Testes Extract. . . . . . . . . . . . . . . . . . 6 Isolation of Conjugated Steroids. . . . . . . . .. • 6 Glucuronides. • 6 Sulphate Conjugates. . . . . . . . . . . . . . . . . . . . . . 7 Residual Conjugates. 7 Column Chromatography. . . . . . . . . . . . . . . . 7 Paper Chromatography. • • • • • • • • • • • • . . . . . . . * • 8 Solvents and Glassware. . . . . . . . . . . . . . . . . . . . . . 8 Sulphuric Acid Chromogens* • • • • • . • • • • • • « • • « . . « 8 Infrared. . 9 Selective Acetylation* • • • • • • • • • « . • • • • . . . . . . 9 Chromic Trioxide Oxidation of 17-0H Group . . . . . . . 9 Deacetylation of Testosterone Acetate. • • • • • • • • • • • • » 9 Oxidation and S p l i t t i n g o f f Side Chain of Carbon 17. * 9 Saccharo-1 s4-Lactone Inhibition of ^-Glucuronidase. . . . . . . 9 Removal of Sacoharo-1:4—Lactone. . . . . . . . . . . . . . . . . 9 IV. RESULTS 11 Column and Paper Chromatography of Plasma Steroids. . . . . . . . 11 Search for Androsterone and Dehydroepiandrosterone. . . . . . . . 13 Evidence for Conjugation of Testosterone as Glucuronide. . . . . 14 Isolation of Testosterone from Testes. . . . . . . . . . . . . . 15 V. DISCUSSION , 17 Absence of Androsterone or Dehydroepiandrosterone Conjugates i n Salmon. . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Testosterone Glucuronide Conjugation. . . . . . . . . . . . . . . 17 Testosterone Conjugates i n Spawned Female Sockeye Plasma. • • .. 17 Low Plasma Testosterone Concentration ("Free"and Conjugated i n Green Females. • • • • • . • • • • • • • • • • • » . . . * • 18 Levels of Testosterone i n Male and Female Plasma During Migration. . . . . . . . . . . . . . . . . . . . . . 18 Testosterone Conjugates i n Plasma and Testes. . . . . . . . . . . 19 VI. LITERATURE CITED 23 ;  - iv -  Page VII.  FIGURES Gradient Elution of Standard Steroids. • • • • • • • • • • • • • • Gradient Elution of the Glucuronide Conjugate Fraction From 650 ml Plasma of Chilko Lake Spawned Male. . . . . . . Sulphuric Acid Chromogen Scanning From 210-600 mu. . . . . . . . . Photograph of Paper Chromatogram of Glucuronide Conjugate Fraction of Spawned Male Plasma. • • • • • • * • « • • • . « • » • « . « . Photograph of Infrared Spectra. . . . . . . . . . . . . . . . . . . Photograph of Infrared Spectra. • • Photograph of Chromatogram. • • • • .......  27 28 29 30 31 32 33  I N T R O D U C T I O N  A number of steroids have been isolated recently from sockeye salmon plasma and their structure established* Cortisol, cortisone and i t s 20^S-dihydroepimer  (D.R, Idler, private  communication), corticosterone, 17-Of-hydroxyprogesterone and i t s 20^^iJbydro epimer, 11-ketotestosterone and adrenosterone have been isolated from the "free" steroid fraction obtained from the plasma of sockeye salmon (Idler et a l * , 1959, 1960, 1960a, 1961j P h i l l i p s et a l . , 1959). To date, no study has been reported concerning the isolation of conjugated steroids from f i s h plasma, although some UDBGA (uridine diphosphate glucuronic acid), one of the chief requirements for glucuronic acid ester i f ication of androgens i n mammalian l i v e r c e l l s (Isselbacher, 1956), has been shown i n spring and coho salmon l i v e r (Tsuyuki et a l . , 1958 and Tsuyuki and Idler, 1961).  Other  substances needed for the glucuronide synthesis which have been shown i n f i s h l i v e r to date are ATP (adenosine triphosphate), glucose—1—phosphate, DIN (diphosphopyridine nucleotide) (H* Tsuyuki, private communication), but an enzyme glucuronosyl transferase found to be present i n mammalian l i v e r c e l l s microsomes, was not yet shown* In normal human plasma, the principal conjugated steroids are found to be androsterone and dehydroepiandrosterone, i n two different modes of conjugation, sulfate and glucuronide (Kellie and Smith, 1957).  The total concentration i n  human peripheral male plasma of dehydroepiandrosterone was found to be 57*5 - 10*5 ug/100 ml (Ortel and Eik-Nes, 1957).  Androsterone has been reported to occur at  concentrations of 18 ug/100 ml and 22*5 ug/100 ml i n the plasma of normal females and males respectively (Migeon, 1955)*  Efforts to establish the presence of  dehydroepiandrosterone and androsterone i n salmon plasma conjugates, either sulfate or glucuronide, by various methods have f a i l e d i n these t r i a l s *  Instead a  conjugated steroid with a ^4-3-ketone moiety, was discovered* This substance was subsequently isolated and identified as testosterone* The fact that testosterone i s found i n female sockeye plasma samples, and that i t has never previously been isolated from any blood sample i n i t s conjugated-  form, provided incentive f o r further research.  Therefore, an attempt was made to  establish the nature of the conjugation, and then the systematic incidence of the substance was explored.  L I T E R A T U R E  REVIEW  Steroid Conjugates The steroid hormones of the adrenal and gonads and their transformation, products are known to be excreted i n significant amounts i n urine or bile as conjugates of glucuronic acid*  Conjugation with glucuronic acid i s a common method by which the  animal renders substances soluble for excretion purposes*  Cohen and Marrian (1936)  identified e s t r i o l glucuronide i n pregnancy urine; Venning and Brown (1936) and Heard et a l * (1944) showed that progesterone i s excreted as pregnanediol glucuronide; Brooksbank and Haslewood (1950) identified androst-16-en-3 - o l glucuronide, and pregnane-3-17-20-triol glucuronide was identified by Mason and Kepler (1945)* Recently testosterone glucuronide was isolated from the urine of a patient receiving large doses of testosterone propionate (Edwards and K e l l i e , 1956), whereas androsterone and etiochblanolone glucuronides were established as major normal constituants i n the human (Kellie and Smith, 1957)* The l i v e r was shown to be a site f o r the biosynthesis of the glucuronides (Fishman and Sie, 1956) and conjugation can also occur i n the human prostate gland (Wotiz, 1954), As early as 1939 Lipschitz and Bueding demonstrated by i n v i t r o studies that the l i v e r was the primary site of glucuronide conjugation i n rats*  They  observed that i f bomeol or menthol were incubated with l i v e r slices containing glycogen, glucuronide synthesis occured.  Mosbach and King (1950); Douglas and  King (1953), as well as others, showed that glucose was transformed to glucuronic acid without any cleavage of the hexose molecule*  Later, Dutton and Storey (1951,  1953) showed that a uridine nucleotide was involved* A complete review on the subject, and glucuronic acid synthesis involving l i v e r microsomes i s given by Isselbacher (1956)*  U n t i l recently, most interest i n the metabolism of glucuronic  acid was directed to i t s functions as a mucopolysaccharide*  Later i t s role i n the  detoxification of alcoholic and phenolic compounds, including the steroids was explored*  Recently, formation of certain steroid glucuronides was suggested to be  a means of preserving the hormone i n passing from i t s place of origin through the circulation to i t s target organs (Fishman, 1951; Fishman and Sie, 1956)*  While  conjugation i s known to reduce the biological a c t i v i t y of some steroids, i t certainly changes the capacity of the l i v e r , prostate, skin and other tissues to oxidize them, and protects them this way from being irreversibly destroyed (Wotiz,, et a l * , 1954).  Testosterone The history of the testicular hormones has been recorded i n several places. The review by Dorfman and Shipley (1956, pp. 2-8) and an a r t i c l e by Roberts and Szego (1955) give a good background. Although the v i r i l i z i n g properties of the t e s t i s have been known for many centuries, i t was not u n t i l 1935 that David, Dingemanse, Freud and Laqueur succeeded i n isolating the testicular hormone i n pure crystalline form, and gave i t the name testosterone.  Evidence for the  secretion of testosterone by the testicular tissue of dogs and human was brought forward by West et a l . (1951, 1951a, 1951b).  The endogenous production of  testosterone i n a normal man was calculated by Fukushima et a l . (1954) to be not more than 36 mg/day and probably even less than 17 mg/day. With the development of new methods, the circulating "free" testosterone i n peripheral blood of normal man and woman was found to be 0.2 - 0.4 ug/100 ml plasma, and O.l/lOO ml respectively (Finkelstein, F o r c h i e l l i and Dorfman, 1961).  Testosterone was also shown recently  i n elasmobranch testes (50 ug/kg of tissue) (Chieffi and Lupo, 1961). Testosterone Glucuronide Conjugates That some tissues are able to conjugate testosterone i s a recent discovery. Fishman and Sie (1955) reported the formation of testosterone glucuronide during the incubation of rat l i v e r slices with testosterone. similar a c t i v i t y of human prostate tissue.  Wotiz et a l . (1956) reported  The conjugated compound was also shown  i n the glucuronide fraction of the urinary steroids after administration of testosterone propionate  (Edwards and K e l l i e , 1956).  Biological Effect of Testosterone on Teleost Fish The biological effect of testosterone i s hard to assess, mainly because response i s both general and varied, and distinction between primary and secondary effects are d i f f i c u l t to separate.  Dorfman and Shipley (1956) l i s t no less than twenty-six  general and specific effects on animals, to wit; Growth of Tissue  Seminal vesicles Prostate Penis Coagulating glands Vas deferentia  Epididymis Increase i n body mass Kidney Muscle Specific hair growth Increase vascularity of organs  Specific Effects Retention Influence Influence Influence Influence  of on on on on  nitrogen creatine metabolism sodium metabolism phosphorus metabolism potassium metabolism  Influence on Enzyme Systems Arginase of kidney Alkaline phosphotase of kidney Succinic dehydrogenase Cholinesterase Coagulating enzyme from coagulating gland Enzyme system of skin tanning due to u l t r a v i o l e t Specific pigment formation i n birds The effect of androgenic hormones on teleost f i s h i s summarized i n several recent literature reviews (Hoar, 1955, 1957, 1957a; Dodd, 1955, 1960; and Aronson, 1959). Most  evidence has been derived from studies involving castration,  transplantation and administration of pure hormone. Major manifestations seem to be i n secondary sex characterization, s t e r i l i z a t i o n i n some cases and even longevity (Robertson, 1961).  In. females of some teleosts, administration of the  hormone over prolonged periods w i l l induce musculanization.  Testosterone also  plays a part in. the behavioral patterns of sexual relationships.  Beach (1948)  discusses potential mechanisms for these actions, and Aronson (1959) summarised the work i n that f i e l d .  Conflicting results by different workers could be  attributed i n part at least to the a b i l i t y of a very small fragment of gonads to maintain normal secondary sex characters and even to regenerate.  Most of the  evidence seem to indicate that courting, spawning and nesting depend, at least p a r t i a l l y , on the testicular hormones. Testosterone i n Teleost Fish Leydig-like c e l l s , have been described f o r different teleosts (Hoar, 1957) including genus Oncorhynchus (Potter and Hoar, 1954; Robertson and Wexler, I960). Salmon gonads were shown to possess androgenic a c t i v i t y (Hazelton and Goodrich, 1937) and this was estimated by using the comb growth of capons assay, (Potter and Hoar, 1954; Tsuyuki and Idler, 1959), but characterization of the androgen responsible has not been affected thus f a r .  M A T E R I A L S  AND  METHODS  Plasma Blood was obtained from spawned Chilko lake male and female sockeye salmon i n September, 1959, spawned Cultus lake male and females on November 28, 1960, and Siwash Bridge females (green) on August 28, 1959,  Pish were bled by severing the  caudal artery and precautions were taken to exclude slime and excreta*  The  heparinized blood was c h i l l e d on ice, centrifuged promptly at 4,000 - 5,000 r.p.m. and the plasma stored i n polyetheline containers on dry ice and later at -35°C. The "free" steroids were extracted from the alkalized plasma three times with 2*5 volumes of dichloromethane or ethyl acetate as previously described (Idler et a l * , I960), Testes Extract Testes (l200g) were extracted from 0* nerka caught about 3 weeks pre-spawned at Weaver Creek, B.C., I960*  These were kept on dry ice and further cooled (liquid  nitrogen), then crushed to a fine powder*  The powder was treated with four volumes  of 95$ ETOH (ethanol) and l e f t on the shaker for 3 days* (centrifuged at 5860 x gravity)* ETOH extract was pooled*  The ETOH was pressed out  This procedure was repeated three times and the  The extract was treated further i n the same manner as the  deproteinized plasma* Isolation of Conjugated Steroids Grlucuronides*  The plasma residue from the extraction of the free steroids  was treated with 2*5 volumes of ethanol to precipitate the proteins and the precipitate was removed by centrifugation at 9,000 xg*  The precipitate was washed  with 70$ ETOH and the washings were added to the supernate* was carried out at 0°C*  The entire procedure  The supernate was evaporated i n vacuo at ca 30°C using a  f l a s h evaporator, the receiver of which was cooled i n a dry ice-acetone mixture* The residue was suspended i n a volume of water equivalent to that of the plasma and extracted three times with dichloromethane to remove any steroids which might have been released from the protein*  Dichloromethane was used as a preservative*  Versene  (5 mg/100 ml) was added and the aqueous phase was adjusted to pH 4.5 and treated with^-glucuronidase (110 mg/100 ml), at 37°C for 48 hours.  The pH was then  adjusted to 3*5 and the hydrolysis was continued for an additional 24 hours.  Prior  to use the^-glucuronidase was suspended i n water and extracted three times with 2 volumes of dichloromethane.  The steroids obtained by this procedure w i l l be  referred to as glucuronide conjugated steroids*  The steroids freed by the  glucuronidase treatment were removed by three extractions with The dichloromethane following:  dichloromethane*  extract was washed three times with 0*1 volumes of each of the  5% sodium bicarbonate, 0*1 N acetic acid and water*  The organic solvent  was removed i n vacuo as described above and the residue was partitioned between 70$ methanol and hexane. The methanol was removed by flash evaporation, the residue taken up i n water and the glucuronide conjugated steroids extracted with dichloromethane* Sulphate Conjugates* The aqueous phase from the extraction of the glucuronide conjugated steroids was made 2 N with respect to H^SO^ and 1 volume of ethyl acetate was added (Burstein and Lieberman, 1958)*  The two phases were thoroughly  equilibrated i n a separatory funnel for several minutes and the ethyl acetate phase incubated at 37°G for 48 hours*  The ethyl acetate was washed with sodium  bicarbonate, 0*1 N acetic acid and water*  This fraction w i l l be referred to as  the sulfate conjugated steroids* Residual Conjugates*  The ^ 4 P h H  2  a s e  from the liberation of the sulfate  conjugates was neutralized with concentrated NaOH to a pH of 0*8 and the solution was continuously extracted with ether for 48 hours*  A small amount of water was  added to the ether (to prevent the r i s k of a high acid concentration when the volume of the solution was reduced) and the ether was removed at room temperature using a f l a s h evaporator as described above*  More water was added to the aqueous suspension  of the residue which was then extracted with ethyl acetate and washed with sodium bicarbonate, acetic acid and water*  The substances released during this treatment  w i l l be referred to as the "pH 0*8" conjugated steroids* Column Chromatography 3 Neutral Woelm  a c t i v i t y 1 alumina was converted to a c t i v i t y 3 by shaking 15 gms  with 0*9 ml of water for one hour*  A slurry of the adsorbent i n benzene was poured  into a 1-rcm. internal diameter column and the column of adsorbent was adjusted to ca 20 cm by gently tapping the wall of the column*  Gradient elution was carried out  as described by Lakshmanan and Lieberman (1954) and the reservoirs contained Afo ethanol i n benzene, and benzene respectively*  The column was operated at a flow  rate of ca 1*3 ml/min. Eighty 10-ml fractions were collected, and then 4$ ETOH i n benzene was used to elute residual steroids* Suitable aliquots of the residues representing the various steroid conjugate  fractions were leached at room temperature with several small volumes of acetone and the acetone removed with a stream of nitrogen.  The residue was dissolved i n a  small amount of benzene and applied to the adsorbent. A glass-wool plug was placed on top of the adsorbent i n order to minimize plugging by insoluble material* The solvent was removed from the individual or pooled fractions with the aid of a stream of nitrogen and a water bath maintained at 59°C.  The  17-oxygenated  steroids were determined by the Bongiovanni modification of the Zimmermann reaction (1957).  In this method N-benzyl trimethylammonium methoxide i s substituted for KOH  (potassium hydroxide).  The reaction was carried out at 4-8°C f o r five hours.  max. was 515 mu and O.D.  The  (optical density) values were also read at 435 and 595 mu,  and Allan's correction was applied.  When the Zimmermann reagent was used as a spray  for paper chromato grams, one part of 40fe N-benzyl trimethylammonium methoxide was mixed with one volume of ETOH and 2 volumes of a 2$ ethanolic solution of metadinitrobenzene purified as described by Callow et a l . (1938). developed f i r s t at room temperature and then with heat.  The colour was  The method was sufficiently  sensitive to detect 0.6 ug of dehydroepiandrosterone which had been run as a spot i n the haptane—80$ methanol solvent pair. Paper Chromatography Paper was washed i n a Soxhlet apparatus for long periods with acetic acid, ammonium hydroxide and methanol as described by Idler et a l . (1959). When infrared spectra were to be determined the paper was washed, by descending  chromatography,  with hexane followed by methanol. Hexane-propylene glycol (50$ i n methanol) (HP-50), heptane:benzene 1*1-70$ methanol (HM-70), toluene-70% methanol (TM-70) and heptane-80$ methanol (BM-80) were used as solvent systems* Solvents and Glassware Dichloromethane was freshly d i s t i l l e d and stabilized with methanol*  Methanol  was added periodically during the removal of dichloromethane from steroid extracts* A l l other solvents were d i s t i l l e d *  Glassware was washed with acid-chromic oxide  cleaning solution followed by water, versene i n aqueous methanol and d i s t i l l e d water* Sulphuric Acid Chromogens Sulphuric acid (36N) was added to the steroid to give a steroid concentration of ca 10 ug/ml*  The spectra of the chromogens were determined after 2 hours by  scanning from 210-600 mu, with the a i d of a Beckman DK-1  spectrophotometer*  Infrared Infrared spectra were recorded with a Beckman IR-4 double-beam spectrophotometer equipped with a beam condensing system* 2 was spread over an area of ca 0*5 inch  Potassium bromide (5-10 mg infrared grade) i n a mortar warmed on a micro heater to a  temperature sufficient to v o l a t i l i z e dichloromethane*  The steroid (ca 20-80 ug)  was taken up i n 0*05-0*1 ml of dichloromethane and dispersed on the KBr (potassium bromide) which was then gently mixed with a pestle*  The pellet was pressed i n the  usual manner* Selective Acetylation The steroid (20-40 ug) was incubated overnight with 0*1 ml of 20/1 mixture of acetic-anhydride, and dry pyridine (Vogel, 1957)*  The reaction was stopped with  0*4 ml ET0H by incubating for one hour at 37°C and the solvent was removed under nitrogen*  This procedure acetylated testosterone but not 17<*-hydroxyprogesterone »  Chromic Trioxide Oxidation of 17-OH Group The steroid was dissolved i n 2*0 ml g l a c i a l acetic acid and 2*0 ml of  1*2$  chromic trioxide i n 60% acetic acid and stirred for about 30 min* (reaction can be followed by measuring a rapid increase i n absorption at 568 mu)*  Excess chromic  trioxide was destroyed with a few drops of saturated NafiSO^ solution (Kupfer et a l * , I960). Deacetylation of Testosterone Acetate The steroid was dissolved i n 0*5 ml of NaOH and 1*5 ml of methanol and incubated for 45 min* at 37°C*  Four volumes of water were added and the steroid extracted with  ethyl acetate i n the usual manner* Oxidation and S p l i t t i n g o f f Side Chain at Carbon 17 Two mg of steroid were dissolved i n 10 ml of 50$ acetic acid i n a titerlenmeyer* The solution was shaken with one gram NaBi03 (sodium bismuthate) for one hour*  A  suitable aliquote was f i l t e r e d through Whatman No* 1 paper and extracted with 20 ml dichloromethane*  The dichloromethane extract was washed with 5% sodium bicarbonate,  0*1 N acetic acid, and water, then f i l t e r e d through glass wool and concentrated under a flow of nitrogen* Saccharo-l;4—lactone Inhibition of ^-Glucuronidase |  Two ml of inhibitor at a concentration of 0,5 mg/ml of water were used f o r each 50 mg of enzyme i n the preparation (Lewy, 1952)* Removal of Saccharo-lt4—lactone The plasma sample, to which inhibitor had been added, was taken to dryness by  - 10 -  lyophilization.  The residue was suspended i n water and passed through a column  of Amberlite—400 (OH) analytical grade ion exchange resin* measured 1*8 x 12 cm*  The resin column  The resin bed was washed with water to remove the  glueosaccharo-l:4-lactone and f i n a l l y with 5$ acetic acid to elute any weakly acidic steroid conjugates*  - 11  R E S U L T S  Column and Paper Chromatography of Plasma Steroids The glucuronide conjugate fraction from 650 ml of Chilko Lake spawned male plasma was chromatographed by gradient elution*  Several appropriate standards  (Fig* l ) were treated i n the same manner and quantitatively determined with the Zimmermann reagent* While i t must be remembered that a l l  Zimmermann-positive  steroids are not equally chromogenic, the results can be expressed as dehydroepiandrosterone equivalents, on which basis tubes 2-4 contained approximately 60 ug: 5-10, 35 ug: 14-15, 15 ug: 19, 10 ug and tubes 25-39, 100 ug*  Tubes 2-4 of the 4%  ethanol—benzene eluate also contained a small amount of Zimmermann-positive material* In this work the main concern was with the material i n tubes 25-39 which produced a Zimmermann chromogen reaching peak intensity (visual) i n ca 3 hours (Fig* 2). A peak intensity i n 3 hours would indicate a 3 rather than a 17 carbon oxygenation (keto), (Wilson, 1954), The glucuronide conjugate fraction from 860 ml of Chilko Lake spawned male plasma was chromatographed as above and the contents of tubes 25-39 were pooled and a sulphuric acid chromogen was run on one half of the residue remaining after removal of the solvent*  The chromogen had an absorption maximum at 308 mu and a  shoulder at 410 mu (Fig* 3)*  There were 60 ug of testosterone equivalents i n these  tubes calculated on the intensity of the absorption at 308 mu*  The remainder of the  residue was applied to paper as a spot and chromatographed i n the HM-80 solvent system for 3 hours*  A substance which absorbed u l t r a v i o l e t l i g h t , was observed at  4*1-5*6 cm corresponding to testosterone and 17<3(-hydroxyprogesterone standards (Fig, 4)*  The chromatogram-was sprayed with the Zimmermann reagent but no chromogens  developed u n t i l after the paper was heated*  These experiments exhausted the supply  of this fraction from male plasma* The glucuronide conjugated fraction from 410 ml of plasma taken from Chilko Lake females was chromatographed using gradient elution*  The residue from the  contents of tubes 25-39 was chromatographed i n the HM-80 solvent system for 2*5 hours and a UV-absorbing material was present which had a mobility similar to the material obtained from male plasma* max* i n methanol at 238 mu* system for 15 hours*  The substance was eluted from the paper and i t had a A The substance was re-chromatographed i n the same solvent  The ultraviolet-absorbing material from plasma and testosterone  - 12 -  both had R. = 0.93 cm/hr, while the R. of 17Ct-hydroxyprogesterone and 4 ll^-hydroxy-/\-8ndrostane-3.17-dione  were 0.75 and 0.27 cm/hr, respectively.  The plasma steroid was recovered from the paper, acetylated and chromatographed for two hours i n the HM-80 solvent system.  The plasma substance and testosterone  acetate both had R^=0.79. The acetylation was essentially quantitative for both substances since no testosterone was detected after acetylation. More plasma was processed and the steroid-acetate from a total of 525 ml was taken for the determination of the infrared spectrum (Fig. 5). The steroid-acetate recovered from the KBr pellet was equivalent to 2.5 ug/l00 ml of plasma. The steroid-acetate from plasma and testosterone acetate were hydrolyzed with NaOH and methanol and the steroids treated with chromic trioxide. The steroids were recovered from the oxidation mixtures and chromatographed along with 4 -androsten-3, 17-dione i n the HM-80 solvent for 2 hours.  A l l steroids had  R^=0.30 and were detected both by their absorption of ultraviolet l i g h t and with the Zimmermann reagent. Plasma (180 ml) obtained from Cultus Lake females was extracted with ethyl acetate and the glucuronide conjugate fraction obtained.  The residue was  chromatographed on paper for 24 hours using the HM-80 solvent system, and the region corresponding to testosterone eluted and re-chromatographed i n the same manner. Testosterone was determined quantitatively by ultraviolet absorption employing Allan's correction. The material from plasma exhibited maximum absorption at 238 mu and gave 00 (optical density) readings at 220, 240 and 260 of 0.460, 0.870 and 0.291, respectively. There were 7.6 ug of testosterone released by^-glucuronidase from 100 ml of plasma based on the recovery of authentic testosterone through the procedure. The "free" steroid fraction was prepared from 660 ml of plasma obtained from spawned Chilko Lake females.  The residue was chromatographed i n the HP—30 solvent  system and the area of paper which would contain 17 0(-hydroxyprogesterone  equivalents  as determined by ultraviolet absorption employing Allan's correction. The residue was acetylated and chromatographed i n the HBM-70 solvent system. There were two substances from the plasma strip which absorbed ultraviolet l i g h t . (R,£ = 0.6) had the mobility of 17 (X-hydroxyprogesterone corresponded to testosterone acetate.  One substance  and the other (R^ = 0.88)  Visual observation suggested that two  substances were present i n comparable quantity. The infrared spectra of the substance identified as testosterone and standards are shown i n F i g . 5.  - 13 -  Search for Androsterone and Dehydroepiandrosterone Several attempts were made to obtain evidence f o r the occurrence of dehydroepiandrosterone (DBA.) or androsterone i n salmon plasma*  The glucuronide  and the sulphuric acid conjugated fractions from 218 ml of plasma from spawned Chilko Lake females were chromatographed as streaks i n the HP-50 solvent system* The areas of the paper corresponding to .DHA and androsterone were eluted and the residues chromatographed as spots*  No Zimmermann-positive material was detected*  The glucuronide conjugate fraction from 136 ml of plasma taken from spawned Chilko Lake males was treated i n a similar manner and again no material with the mobility of either DHA or androsterone was detected*  The sulphuric acid fractions from 544  and 136 ml of plasma also contained no detectable DHA or androsterone*  The "pH 0*8'*  fraction from 136 ml and the sulphuric acid fraction from 47 ml of the same plasma also showed no evidence of DHA or androsterone*  The glucuronide conjugate fraction  of the plasma from 412 ml of spawned Chilko Lake females was chromatographed by gradient elution.  The contents of the tubes, which should have contained DHA and  androsterone had they been present, were chromatographed i n the HN-80 solvent system and no Ultraviolet-absorbing or Zimmermann-positive substances were detected. The sulphuric acid conjugate from the plasma (870 ml) of spawned Chilko Lake females was chromatographed on a 15-cm s t r i p of paper i n the HP-50 solvent system.  The  Zimmermann reaction was carried out on 10$ of the eluate from paper strips taken from the areas which would be occupied by DHA or androsterone and no detectable chromogen was produced*  The remainder of the material with the mobility of  androsterone and 25$ and 10$ respectively of the material corresponding i n chromatographic mobility with DHA and testosterone were re-chromatographed as spots i n the same solvent system and no Zimmermann-positive material was detected. The glucuronic conjugate and sulphuric conjugate fractions from 224 ml and 560 ml of plasma, respectively, taken from feamles captured at Siwash Bridge were chromatographed by gradient elution*  The Zimmermann reaction was carried out on the contents of tubes  1-80 and only tube 2 contained a significant amount of chromogenic material*  Another  aliquot equivalent to 47 ml of plasma was chromatographed i n HP-50 alongside 17c£-hydroxyprogesterone, dehydroepiandrosterone, androsterone and etiocholanolone* There was a faint Zimmermann-positive spot with the mobility of  17#-hydroxyprogesterone  (or testosterone) and nothing corresponding to the other reference substances*  The  "pH 0.8" conjugates prepared from 170 ml of plasma taken from spawned Chilko Lake  - 14 -  males were subjected to column chromatography.  Only a trace of Zimmermann-positive  material was detected. Evidence for Conjugation of Testosterone as Glucuronide Blood from spawned male sockeye salmon, taken at Cultus Lake i n November, 1960, was processed to obtain the deproteinized plasma.  Two portions each equivalent  to 480 ml of plasma were taken. One was treated with^-glucuronidase (525 mg) and incubated at 37°C for 24 hours under the conditions described.  The other aliquot  was treated i n an identical manner except that 45 mg of glucosaccharo-1:4—lactone was added prior to incubation. The incubation mixtures were extracted with methylene chloride which was then washed with 0.05 N NaOH and 0.1 N acetic acid and the residue after removal of the solvent was partitioned between hexane and 70$ aqueous methanol.  The methanol-water  phase was taken to dryness i n a flash evaporator at room temperature and the residue suspended i n acetone and f i l t e r e d *  The acetone soluble material was chromatographed  on Whatman No* 1 paper, thoroughly washed as described, i n the HM-80 solvent system for 16 hours. Testosterone, 17 X-hydroxyprogesterone and 11-ketotestosterone were £  included as reference standards. An ultraviolet-absorbing substance with the mobility of testosterone was solvent extracted from the incubate prepared without the addition of the enzyme inhibitor but no UV-absorbing substance could be detected from the sample to which the inhibitor was added.  The UV-absorbing substance was eluted from  the paper and exhibited a maximum absorption at 240 mu equivalent to 39.4 ug/480 ml of plasma of testosterone equivalent calculated on the OD readings at 220, 240 and 260 mu and employing Allen's correction.  The UV-absorbing substance was acetylated and  chromatographed by descending chromatography i n HM-80$ methanol for 2 hours.  Authentic  testosterone acetate and the UV-absorbing substance from the plasma both had an B  f  = 0.80.  The acetylated UV-absorbing substance from the plasma was incorporated  into a KBr pellet and the infrared spectrum was the same as that of authentic testosterone acetate. Tie hydrolysis of the plasma samples to which no inhibitor was added was continued for an additional 24 hours and no additional quantity of testosterone could be detected. The plasma sample, to which inhibitor had been added, was taken to dryness by lyophilization*  The residue was washed by the use of an Amber1ite-400 column.  acetic acid eluate was taken to dryness on a flash evaporator at ca 20C. V  The  The  residue was taken up i n water and the pH adjusted to 4.5 with NaOH. The hydrolysis  - 15 -  procedure witbyy-glucuronidase was repeated.  The steroid residue obtained by-  extraction with methylene chloride was chromatographed on paper i n the HM-80 solvent system for 19 hours.  The chromatogram was sprayed with the Zimmeimnn-Bongiovanni  (1957) reagent and a Zimmermann-positive substance with the chromatographic  mobility  of testosterone was present from the plasma sample. Isolation of Testosterone from Testes Twelve hundred grams of testes were extracted from 0, nerka caught at Weaver Creek, B.C., i n 1960,  These were kept i n dry ice and further cooled (liquid N),  then crushed to a fine powder. The powder was treated with 4 volumes of 95$ ETOH and l e f t on the shaker for 3 days.  The ETOH was pressed out (centrifuged at 5860 x g ) .  This procedure was repeated three times and the ETOH extract was pooled. The protein residue fraction "A" was made up to 1200 ml with water.  The pH  mobility of testosterone could be shown. The 95$ ETOH pooled extract was concentrated i n vacuo on a flash evaporator employing a receiver c h i l l e d i n dry ice and acetone.  The residue was dissolved i n  1500 ml of water and extracted three times with equal volumes of dichloromethane. The combined extract fraction "B" ("free * steroids) was washed with 0,05N NaOH and 1  0,1 N  acetic acid and the residue after removal of solvent was partitioned between  hexane and 70$ aqueous methanol (this procedure was repeated twice). The aqueous methanol fraction was taken to dryness at room temperature i n a flash evaporator and the residue suspended i n acetone and f i l t e r e d .  The acetone soluble material was  chromatographed as two parallel spots, representing 10$ and 90$ of the t o t a l , along with authentic testosterone as a standard.  Whatman No, 1 paper, thoroughly washed  i n the HM-80 solvent system for 16 hours was used, as previously described. No UV-absorbing  substances with the mobility of testosterone could be detected i n either  the 10$ or 90$ portions.  The area from the 90$ portion parallel i n mobility to  testosterone was cut out and eluted with methanol for further studies. The rest of the paper was sprayed with the Zimmermann-Bongiovanni spray but no Zimmermann-positive spot could be detected. The aqueous residue fraction "C" (conjugated steroids) pH was dropped to 4,5 with 1,0 N H^SO^ and incubated with^-glucuronidase as previously described. Extraction with a volume of dichloromethane  (three times) was performed after 48 hours. The  combined extract was treated as fraction B, Chromatography i n the HM-80 solvent  - 16 ~  system for 12 hours showed a U.V.-absorbing area with the mobility of testosterone* The U,V.-absorbing substance of fraction "C" was eluted from the paper and exhibited a maximum absorption at 240 mu equivalent to 39 ug/l200 g of testes of testosterone-equivalent calculated on the 0,D. reading at 220, 240, and 260 mu and employing Allen's correction* This was evaluated as equal to 61 ug/kg*  Testes of  conjugated testosterone calculated from recovery of testosterone through the same procedure* Fraction "C" as well as Fraction "B" eluate with the mobility of testosterone were concentrated under N and then acetylated with acetic-anhydride pyridine mixture* Resultant compounds were chromatographed for 2 hours i n the BM-80 system and scanned for U*V* absorption,)  The material from Fraction "C" which had the mobility of  testosterone after acetylation had the mobility of testosterone acetate*  No substance  was detected from the acetylated area of Fraction "B" which would have contained testosterone had i t been present*  Testosterone acetate as well as the testes  acetylated steroid were eluted with methanol and.scanned for the infrared (Fig* 6), The rest of chromatogram was sprayed with the Zimmermann-Bongiovanni and no definite positive spot i n the area of testosterone could be shown* KBr pellets of testosterone acetate and testes conjugated steroid (Fraction "G") after acetylation were dissolved i n H^O and extracted with dichloromethane* The dichloromethane was evaporated under N^ and the residues deacetylated and then oxidized with GrO^. The oxidized testosterone from testosterone acetate, oxidized testosterone from Fraction "C" as well as oxidized testosterone and authentic^4-androstene-3,  17-dione  were chromatogramed on Whatman No* 1 paper i n the BM-80 solvent system f o r two hours* U*V* scanning as well as the Zimme rmann-Bongiovanni spray revealed spots with identical mobilities of R. = 13*6/36*5 = 0.37 (Fig, 7)*  D I S C U S S I O N  Absence of Androsterone or Dehydroepiandrosterone Conjugates i n Salmon The f a i l u r e , i n the present study, to detect either DHA or androsterone i n any of several plasma samples i s of interest since both these steroids are present i n r e l a t i v e l y high concentration i n peripheral human plasma*  Dehydroepiandrosterone  has been isolated and positively identified i n human plasma*  I t i s reported to  occur at a concentration of 57*5 - 10*5 ug/100 ml (Ortel and Eik-Nes, 1958)* Androsterone has been reported to occur at a concentration of 18 ug/100 ml and 22,5 ug/100 ml i n the plasma of normal females and males respectively (Migeon and Plager, 1955)* Testosterone Glucuronide  Conjugation  The f i r s t demonstration of a steroid conjugated at a position other than the C3 hydroxyl group was the isolation of testosterone-ljy^-glucuronide from the urine of a patient receiving large doses of testosterone propionate (Edwards and K e l l i e , 1956)*  Testosterone has been shown to be converted ( i n low yield) by  rat l i v e r slices (Fishman and Sie, 1956) and by surviving human prostate tissue (Wotiz et a l . , 1956) i n v i t r o , to a substance positively identified as testosterone17/^-glucuronide  (Wotiz et a l * , 1958)*  Unconjugated testosterone has recently been  isolated from human systemic blood following the administration of human chorionic gonadotropin  (Ortel and Eik-Nes, 1959)*  Isolation of testosterone conjugate from  the blood has not been previously reported* Testosterone Conjugates i n Spawned Female Sockeye Plasma In the present work there were 7*6 ug/100 ml of testosterone i n the glucuronide fraction of plasma taken from spawned Cultus Lake females* 2*5 ug/100 ml of testosterone were released by^-glucuonidase taken from spawned Chilko Lake females*  In another experiment from 525 ml of plasma  The latter material had been taken through  two paper chromatograms, one column chromatogram, acetylation and recovery from a KBr p e l l e t .  The recovery from each paper chromatogram was estimated from the recovery  of pure steroids to be ca 85$*  As no testosterone was detected on the paper  chromatogram following acetylation by this method, the recovery was estimated at ca 95$,  The recovery from the KBr pellet and the column chromatography can only  be estimated at ca 85$, There were therefore ca 5*1 ug of testosterone released from each 100 ml of plasma by ^-glucuronidase.  This estimated level of testosterone i n  •* 18 *•*  the spawned Chilko Lake females was comparable to the level accurately determined i n the glucuronide fraction of plasma obtained from Gultus Lake spawned females. Low Plasma Testosterone Concentration ("free" and conjugated) i n Green Females The plasma samples obtained from females captured at Siwash Bridge, approximately three weeks prior to spawning, contained no detectable Zimmermann positive substance i n tubes 4-80 following column chromatography.  I t must be remembered that the  Zimmermann reaction as used i n this study i s quite ineffective i n 17-hydroxy steroids and the intensity of the color produced with 3-4teto steroids reaches a maximum i n 3 hours, and has decreased i n intensity by the time the tubes are read at the end of 5 hours, the optimum time for 17-keto steroids.  Significant amounts of steroids  with a ^4—3 ketone structure were not expected to occur as conjugates at the time these chromatograms were run, and testosterone, unless present at a concentration of ca 2 ug/lOO ml, would probably not have been detected.  However, testosterone should  have been detected by paper chromatography i f i t had been present i n significant quantities.  I t has been shown i n a separate investigation that these plasma samples  contained only 3.8 ug/lOO ml of "free" 17- OC-hydroxyprogesterone plus testosterone. Unlike the plasma of green f i s h the plasma from spawned Chilko Lake females contained 7.6 ug/100 ml of testosterone i n the glucuronide fraction; 7.8 ug/lOO ml of testosterone and 9.8 ng/100 ml of 17-c(-hydroxyprogesterone i n the "free" steroid fraction.  The  steroids were determined following paper chromatography i n heptane-80% methanol solvent system f o r 16 hours (Schmidt and Idler, 1961).  It can be concluded that  testosterone was present at a considerably higher concentration i n both the glucuronide and "free" fractions of spawned females than i n the corresponding fractions prepared from the plasma of females captured prior to a r r i v a l i n the spawning area. Levels of Testosterone i n Male and Female Plasma During Migration There were 13.7 ug of testosterone released by/^-glucuronidase from 100 ml of spawned male plasma (Table l ) .  The "free" testosterone content of the spawned male  sample was found to be 1,7 ug/100 ml plasma.  The t o t a l testosterone content of the  plasma was therefore similar i n the sexes but the hormone i s present to a much greater extent i n the conjugated form i n the male. In the female plasma "free" testosterone reached a peak concentration during the spawning period, and there was a marked drop i n concentration just after spawning. If i t i s accepted that "free" testosterone i s the biologically active form of the hormone, then i t would be logical to assume that this compound i s involved i n the  - 19 -  Table 1.  Testosterone i n the Plasma of Sockeye Salmon (ug/100 ml).  PLASMA FRACTION  Conjugated Testosterone (Cultus race)  GREEN (but migrating)  *i  "Free" Testosterone (Cultus race)  None  "Free" Testosterone (Chilko race)  None  None  RIPE  4.8  >2.0  -  -  13.7  7.6  1.7  7.8  1.0  7.6  15.4  15.4  14.0 -  Total Plasma Testosterone i n Cultus Lake Fish events leading to, and/or the act of spawning i t s e l f .  POST SPAWNING  Paul A. Wright (1961),  recently discovered that ovulation could be induced i n Rana pipiens i n v i t r o with both progesterone and testosterone but not estrogenic compounds like estrone, estradiol benzoate and diethylstilbesterol. P_. nerka.  This might p a r a l l e l the mechanism i n  His work indicates that the most e f f i c i e n t steroid i s progesterone.  Testosterone's action i s less rapid, and causes fewer eggs to be released.  However,  work done on migrating female salmon did not show any progesterone i n the plasma (Idler et a l . , 1959).  The absence of progesterone must be taken with caution since  i t was not s p e c i f i c a l l y looked f o r , and could have been missed.  The presence of a  metabolite of progesterone, 17-<¥*<hydroxyprogesterone (Idler et a l . , 1959; Grajcer and Idler, 1961), i n the plasma i n large quantities emphasizes this p o s s i b i l i t y . Testosterone Conjugates i n Plasma and Testes In this work the release by^-glucuronidase of 13.7 ug of testosterone from 100 ml of male plasma, and the discovery of testosterone glucuronide as the major androgenic component i n the testes of spawning sockeye represents a distinct deviation from the known testosterone picture i n mammalian blood and testes.  F i r s t l y the concentration  of the hormone i s many times greater than that found i n normal human plasma (Finkelstein et a l . , 1961), secondly, testosterone has not previously been found i n the conjugated form i n t e s t i s tissue. The existence of testosterone i n the spawning salmon testes i n the form of a glucuronide conjugate could be accounted f o r i n one of three waysi  « 20 -  a)  The conjugated testosterone i s a metabolite of another substance, and has no physiological activity of i t s own*  b)  The glucuronide conjugated form i s the active form i n the male 0* nerka.  c)  The conjugation preserves the hormone which might be released for action at a target area elsewhere* Sixty-one ug testosterone/kg of testes tissue probably accounts for the total  androgenic a c t i v i t y of that organ (Potter and Hoar, 1954j Tsuyuki and Idler, 1959), and therefore p o s s i b i l i t y (a) i s unlikely*  P o s s i b i l i t y (b) cannot be challenged at  this stage especially since the work of Ott et a l * (1952) showed that the introduction of a cyclic grouping on the propienyl group i n testosterone esters produces an androgen having a more intense and prolonged action (tested on seminal vesicle and prostate gland of castrated rats)*  This effect i s d i f f i c u l t to evaluate however,  since the assaying technique employs the biological responses of tissues*  I t i s not  yet possible to determine whether the esterified compound i s more effective because i t has a higher biological a c t i v i t y or because i t i s better protected u n t i l i t reaches the target area*  This l a s t p o s s i b i l i t y coincides with (c) (Fishman and Sie, 1956)*  The suggestion that testosterone i s produced i n the t e s t i s , and then transported by the blood i n the conjugated form to target area, i s plausible; however, the very low concentration of the "free" hormone at a l l times i n the male plasma seems to refute this argument i n the male*  I t i s possible that a quick metabolism of testosterone  occurs, and that the subsequent compound i s the active hormone*  11-JKe to testosterone  which has been shown to occur at high concentration i n the plasma of the post spawned male sockeye salmon could be such a compound (Idler et a l * , I960)*  This substance  was shown to have 58$ of the androgenic a c t i v i t y of testosterone as assayed by the chick comb growth test (Idler et a l . , 1961).  11-Ketotestosterone i s capable of  bringing about a rapid development of the secondary sex characteristics of the male salmon (Idler et a l * , In Press) and C^-testosterone i s a precursor i n vivo of 11-Ketotestosterone  (D.R. Idler, Private communication).  However, this does not  adequately explain the very high concentration of conjugated testosterone i n the spawning male* In summary f i v e points should be considered: a)  Progesterone i s apparently not a major steroid i n ripe female sockeye plasma, although 17- oC-hydroxyprogesterone, a metabolite of progesterone, i s present i n considerable concentration*  * 21 -  b)  Testosterone i s found i n substantial quantities i n post-spawned female plasma, occuring i n equal portions i n the "free" and the conjugated forms,  c)  Concentration of the "free" testosterone i n the female reaches a peak at spawning time, and declines l a t e r ,  d)  In contrast to the female plasma, the analysis of male sockeye showed 90$ of the testosterone i n the conjugated form,  e)  I t was shown that testosterone occurs i n the testes i n the conjugated form. In this work testosterone was isolated for the f i r s t time from the glucuronide  fraction of plasma. The concentrations of testosterone i n the plasma of a spawning female sockeye was shown to be considerably higher than that reported i n any other species.  To date  including values for the plasma of h i l u s - c e l l tumor i n the human female where a concentration of 2,0 ug/100 ml of plasma i s attained and adrenal adenoma were l»3ug/l00 ml of plasma i s recorded (Finkelstein et a l , , 1961), In this report testosterone was found i n the glucuronide fraction of testes, and for the f i r s t time testosterone was identified conclusively i n a teleost f i s h (Chieffi and Lupo recently identified testosterone i n an elasmobranch testes, 1961), At t h i s point, adequate background material concerning the metabolism of the above compounds i s lacking. Any attempt to explain their functional significance would therefore be highly speculative. To attain an understanding  of the function(s) of testosterone, i n the migrating  salmon (both male and female) some knowledge of the following points would be essential: 1,  Establishing whether testosterone or testosterone glucuronide i s the active form  (or each one on a different s i t e ) .  Progress i n this direction could be made by  applying testosterone glucuronide to a known site of action of testosterone a c t i v i t y l i k e the capon comb; both "free" testosterone and testosterone-glucuronide with saccharo—ls 4—lactone (^-glucuronidase 2,  inhibitor) could be used as controls,  To ascertain the a c t i v i t y i n f i s h , a standard test could be devised, such as  male colour development.  Since testosterone i s capable of inducing ovulation i n the  amphibians (Wright, 1961}  Shapiro, 1936; Ramaswami and Lakshman, 1958) the same might  be t r i e d on salmon ovaries* 3,  Should "free" testosterone be found to be the active form i n the female, then  an assay for £-glucuronidase a c t i v i t y i n migrating female ovaries would be advisable.  M  4*  22  The site of the biological synthesis of testosterone and i t s glucuronide conjugate  i n the migrating salmon should be sought*  Testosterone has been found i n gonads and  adrenals of many higher vertebrates (Dorfman and Shipley, 1956) as well as i n testes of elasmobranchs (Chieffi and Lupo, 1961).  For completeness, the adrenals of both  male and female f i s h should be assayed, as well as the ovaries* prove the presence or absence of the hormone*  This w i l l only  To show the biosynthesis tissue culture  technique and the appropriate precursors would be used* If testosterone i s shown to possess some biological a c t i v i t y i n female sockeye then experiments could be designed to test the male sockeye as a possible exogenous source of hormone for the female* 5*  An assay f o r testosterone (both for the "free" and conjugated forms) i n female  plasma and ovaries could be attempted*  The assay should be done on three groups of  f i s h ; one group of f i s h which matured i n sexual confinement, a second which matured with a male i n a separate glass partition (within sight), and a t h i r d which matured with males i n the same tank* As a group these experiments would y i e l d information concerning the active form of the hormone, the physiological site of i t s action, i t s probable site of synthesis (in male and female) and i t s possible interaction between the sexes*  Only then  could the function of the hormone i n f i s h be inferred with some accuracy*  - 23 -  L I T E R A T U R E  C I T E D  Aronson, L.R. 1959. Hormones and Reproductive Behaviort Some Fhylogenetic Considerations. Comparative Endocrinology. John Wiley & Sons, Inc., New York, pp. 746. Beyler, A.L. and CM. Szego. 1954. Correlation of Ovarian Cholesterol Levels with Changes i n ^-Glucuronidase A c t i v i t y of Reproductive Tract During the Estrous Cycle of Pregnancy. Endocrinology 54: 323-333. Bongiovanni, A.M., W.R. Eberlein and P.Z. Thomas. 1957. Use of an Organic Base i n the Zimmermann Reaction. J . C l i n . Endocrinol, and Metab. 37: 331-332. Brooksbank, B.W.L. and Haslewood, G.A.D. 1950. The Nature of Pregnandiol-like Glucuronide. Biochem. J . 47: 36-43. Burstein, S. and S. Lieberman. 1958. Hydrolysis of Ketosteroid Hydrogen Sulfates by Solvolysis Procedures. J . B i o l . Chem. 233: 331-335. Callow, N.H., R.K. Callow and C.W. Emmens. 1938. Colorimetric Determination of Substances Containing the Grouping -CH CO- i n Urine Extracts as an Indication of Androgen Content. Biochem. J . 32: 1312-1331. 2  C h i e f f i , G. and C. Lupo. 1961. Identification of Estradiol-17 , Testosterone and i t s Precursors from Scylliorhinus s t e l l a r i e s Testes. Nature 190: 169-170. Cohen, S.L. and G.F. Marian. 1936. The Isolation and Identification of a Combined Form of Oestriol i n Human Pregnancy Urine. Biochem. J . 30: 57. Dodd, J.M. 1955. The Hormones of Sex and Reproduction and Their Effects i n Fish and Lower Chordates. Mem. Soc. Endocrinol. 4: 166-187. Dodd, J.M, 1960. Marshall's Physiology of Reproduction. Vol. 1, part 2, pp. 417-558, Spottiswoode, Ballantyne & Co. Ltd., London, 878 pp. Dorfman, R.I. and R.A. Shipley. 1956. Androgens. John Wiley & Sons, Inc., New York. Chapman & Hall Ltd., London, 590 pp. 14 Douglas, J.F. and C.G. King. 1952. The Conversion of C Labelled Glucose to Glucuronic Acid i n the Guinea Pig. J . B i o l . Chem. 202: 265-871. Dutton, G.J. and J.D.E. Storey. Biochem. J . 48: xxix.  1951. Glucuronide Synthesis i n Liver Homogenates.  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B i o l . Chem. 206: 863-874. Grajcer, D. and D.R. Idler. Testosterone, Conjugated and "Free" i n the Blood of Spawned Fraser River Sockeye Salmon (Oncorhynchus nerka). J . Can. Biochem. Physiol. In Press. Hazelton, L.W. and F.J. Goodrich. 1937. Note on the Presence of Male Sex Hormone i n Fish Testes. J . Amer. Pharm. Assoc., 26: 420-421. Heard, R.D.H., M.M. Hoffman and G.E. Mack. 1944. The Structure of Pregnandiol Glucuronide from Human Pregnancy Urine. J . B i o l . Chem. 155: 607-614. Hoar, W.S.  1955»  Reproduction i n Teleost F i s h .  Mem. Soc. Endocrinol. 4: 5-24.  Hoar, W.S. 1957. Endocrine Organs. 245-258. The Physiology of Fishes. I. Margaret E. Brown. Academic Press Inc., New York, pp. 1-447. Hoar, W.S. 1957a. The Gonads and Reproduction. 287-321. The Physiology of Fishes. I . Margaret E. Brown. Academic Press Inc., New York, pp. 1-447. Idler, D.R., A.P. Ronald and P.J. Schmidt. 1959. Steroid Hormones i n Plasma. Can. J . Biochem. and Physiol. 37_: 1227-1238. 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Chromogenic Values of Various Ketosteroids i n a Micro Modification of the Zimmermann Reaction: Comparison with the Macro Procedure. Arch. Biochem. and Biophys. 52} 217-235, Wotiz, H.H., H.M. Lemon and A. Voulgaropoulos. Human Tissue S l i c e s . J . B i o l . Chem. 209:  1954. Metabolism of Testosterone by 437-445.  Wotiz, H.H., B.S. Ziskind, H.H. Lemon and M. Gut. Biochem. Biophys. Acta, 22: 266-268.  1956. Studies i n Steroid Metabolism.  Wotiz, HJH., H.-G. Sie and W.H. Fishman. 1958. Characterization of Biosynthetic Testosterone Glucuronic Acid.. J . B i o l . Chem. 232: 723-727. Wright, P.A. 1961. Induction of Ovulation i n Vitro i n Rana Pipiens with Steroids. Gen. Comp. Endocrinol. It 20-23.  - 27 -  FIGURE 1. GRADIENT ELUTION OF STANDARD STEROIDS (Lakshmanan, T.K. and S„ Lieberman, 1954). A« y3-Etiocholaneolane B.  50 ug  Denydroepiandrosterone  C. Epiandrosterone D.  Androsterone  E.  Etiocholaneolame  100 ug  70 ug 200 ug  60 ug  •o <0  - 28 -  FIGURE 2. GRADIENT ELUTION (Lakshmanan, T.K. and S. Lieberman, 1954), OF THE GLUCURONIDE CONJUGATE FRACTION FROM 650 ml PLASMA OF CEjLKO LAKE SPAWNED MALE.  A,  Unknown  B»  Cholesterol area  C.  Testosterone area  D«  4% Ethanol-Benzene eluate  FIGURE 3.  SULPHURIC ACID CHROMOGEN SCANNING FROM 210-600 mu (SCANNING WITH THE  AID OF BECKMAN KD-1 SPECTROPHOTOMETER).  A*  Authentic testosterone sulphuric acid chromogen  B  Sulphuric acid chromogen of pooled tubes 25-39 from glucuronide  e  fraction of 860 ml plasma of Chilko Lake spawned male»  conjugate  WAVE  LENGTH  IN  mu  - 30 -  FIGURE 4 .  PHOTOGRAPH OF PAPER CHROMATOGRAM OF GLUCURONIDE CONJUGATE FRACTION  (TUBES 25-39) OF SPAWNED MALE PLASMA (CENTER)« 17 o<0H progesterone (left) Epiandrosterone (right)  -31 -  FIGURE 5. PHOTOGRAPH OF INFRARED SPECTRA (RECORDED WITH A BECKMAN IR-4 DOUBLEBEAM SPECTROPHOTOMETER EQUIPPED WITH A BEAM CONDENSING SYSTEM). Testosterone acetate standard (bottom) Acetylated steroid of glucuronide conjugate fraction from Chilko Lake female plasma (top) Acetylated steroid of "free" fraction from Chilko Lake female plasma (center)  - 32 -  FIGURE 6.  PHOTOGRAPH OF INFRARED SPECTRA  Testosterone acetate standard (bottom) Acetylated steroid from glucuronide conjugate fraction of salmon testes (top)  - 33 -  FIGURE 7. PHOTOGRAPH OP CHROMATOGRAM  A*  Oxidized testosterone from testosterone acetate  B>  Oxidized steroid from glucuronide conjugate of salmon testes 4  C»  /\ -androsterone-3, 17-dione  D, Oxidized testosterone  


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