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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 in 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 // A B S T R A C T 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 criteria 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 is through the 17 /3-hydroxy group rather than the theoretically possible^ 3-enol form in the ^4—3 ketone* Testosterone was found in the conjugated but not in the "free" form in testes of migrating 0* nerka* Dehydroepiandrosterone and androsterone, the principal conjugated steroids in normal human plasma were not detected in several plasma samples tested* A C K N O W L D G E M E N 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 is 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 criticism so generously extended during the preparation of this thesis. To many other associates who in many ways contributed to the successful completion of the research, the author extends his grateful appreciation. i i i T A B L E OP C O N T E N T S Page ABSTRACT. i ACKNOWLEDGMENT i i TABLE OF CONTENTS i i i I. INTRODUCTION. . . 1 II. LITERATURE REVIEW 3 Steroid Conjugates* . • • . • • • • • • • • • • • * • • • • • « • 3 Testosterone* . . . . . . . . . . . . * • • 4 Testosterone Glucuronide Conjugates. . . . . . . . . . . . . . . 4 Biological Effect of Testosterone on Teleost Fish. • • • • • • • 4 Testosterone in Teleost Fish. * 5 III. 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 Splitting off 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 in Salmon. . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Testosterone Glucuronide Conjugation. . . . . . . . . . . . . . . 17 Testosterone Conjugates in Spawned Female Sockeye Plasma. • • .. 17 Low Plasma Testosterone Concentration ("Free"and Conjugated in Green Females. • • • • • . • • • • • • • • • • • » . . . * • 18 Levels of Testosterone in Male and Female Plasma During Migration. . . . . . . . . . . . . . . . . . . . . . 18 Testosterone Conjugates in Plasma and Testes. . . . . . . . . . . 19 VI. LITERATURE CITED 23 - i v -Page VII. FIGURES Gradient Elution of Standard Steroids. • • • • • • • • • • • • • • 27 Gradient Elution of the Glucuronide Conjugate Fraction From 650 ml Plasma of Chilko Lake Spawned Male. . . . . . . 28 Sulphuric Acid Chromogen Scanning From 210-600 mu. . . . . . . . . 29 Photograph of Paper Chromatogram of Glucuronide Conjugate Fraction of Spawned Male Plasma. • • • • • • * • « • • • . « • » • « . « . 30 Photograph of Infrared Spectra. . . . . . . . . . . . . . . . . . . 31 Photograph of Infrared Spectra. • • 32 Photograph of Chromatogram. • • • • . . . . . . . 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 Phillips et a l . , 1959). To date, no study has been reported concerning the isolation of conjugated steroids from fish plasma, although some UDBGA (uridine diphosphate glucuronic acid), one of the chief requirements for glucuronic acid ester i f ication of androgens in mammalian liver cells (Isselbacher, 1956), has been shown in spring and coho salmon liver (Tsuyuki et a l . , 1958 and Tsuyuki and Idler, 1961). Other substances needed for the glucuronide synthesis which have been shown in fish liver to date are ATP (adenosine triphosphate), glucose—1—phosphate, DIN (diphospho-pyridine nucleotide) (H* Tsuyuki, private communication), but an enzyme glucuronosyl transferase found to be present in mammalian liver cells microsomes, was not yet shown* In normal human plasma, the principal conjugated steroids are found to be androsterone and dehydroepiandrosterone, in two different modes of conjugation, sulfate and glucuronide (Kellie and Smith, 1957). The total concentration in 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 in the plasma of normal females and males respectively (Migeon, 1955)* Efforts to establish the presence of dehydroepiandrosterone and androsterone in salmon plasma conjugates, either sulfate or glucuronide, by various methods have failed in 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 is found in female sockeye plasma samples, and that i t has never previously been isolated from any blood sample in i t s conjugated-form, provided incentive for 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 R E V I E W Steroid Conjugates The steroid hormones of the adrenal and gonads and their transformation, products are known to be excreted in significant amounts in urine or bile as conjugates of glucuronic acid* Conjugation with glucuronic acid is a common method by which the animal renders substances soluble for excretion purposes* Cohen and Marrian (1936) identified estriol glucuronide in pregnancy urine; Venning and Brown (1936) and Heard et a l * (1944) showed that progesterone is excreted as pregnanediol glucuronide; Brooksbank and Haslewood (1950) identified androst-16-en-3 -ol 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 Kellie, 1956), whereas androsterone and etiochblanolone glucuronides were established as major normal constituants in the human (Kellie and Smith, 1957)* The liver was shown to be a site for the biosynthesis of the glucuronides (Fishman and Sie, 1956) and conjugation can also occur in the human prostate gland (Wotiz, 1954), As early as 1939 Lipschitz and Bueding demonstrated by in vitro studies that the liver was the primary site of glucuronide conjugation in rats* They observed that i f bomeol or menthol were incubated with liver 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 liver microsomes is given by Isselbacher (1956)* Until recently, most interest in the metabolism of glucuronic acid was directed to i t s functions as a mucopolysaccharide* Later i t s role in 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 in 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 is known to reduce the biological activity of some steroids, i t certainly changes the capacity of the liver, 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 in several places. The review by Dorfman and Shipley (1956, pp. 2-8) and an article by Roberts and Szego (1955) give a good background. Although the v i r i l i z i n g properties of the testis have been known for many centuries, i t was not until 1935 that David, Dingemanse, Freud and Laqueur succeeded in isolating the testicular hormone in 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 in 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 in 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, Forchielli and Dorfman, 1961). Testosterone was also shown recently in elasmobranch testes (50 ug/kg of tissue) (Chieffi and Lupo, 1961). Testosterone Glucuronide Conjugates That some tissues are able to conjugate testosterone is a recent discovery. Fishman and Sie (1955) reported the formation of testosterone glucuronide during the incubation of rat liver slices with testosterone. Wotiz et a l . (1956) reported similar activity of human prostate tissue. The conjugated compound was also shown in the glucuronide fraction of the urinary steroids after administration of testosterone propionate (Edwards and Kellie, 1956). Biological Effect of Testosterone on Teleost Fish The biological effect of testosterone is hard to assess, mainly because response is 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 in body mass Kidney Muscle Specific hair growth Increase vascularity of organs Specific Effects Retention of nitrogen Influence on creatine metabolism Influence on sodium metabolism Influence on phosphorus metabolism Influence on 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 ultra violet Specific pigment formation in birds The effect of androgenic hormones on teleost fish is summarized in 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 in secondary sex characterization, sterilization in some cases and even longevity (Robertson, 1961). In. females of some teleosts, administration of the hormone over prolonged periods will 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 in that f i e l d . Conflicting results by different workers could be attributed in part at least to the ability 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 partially, on the testicular hormones. Testosterone in Teleost Fish Leydig-like cells, have been described for different teleosts (Hoar, 1957) including genus Oncorhynchus (Potter and Hoar, 1954; Robertson and Wexler, I960). Salmon gonads were shown to possess androgenic activity (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 far. M A T E R I A L S A N D M E T H O D S Plasma Blood was obtained from spawned Chilko lake male and female sockeye salmon in 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 chilled on ice, centrifuged promptly at 4,000 - 5,000 r.p.m. and the plasma stored in 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* The ETOH was pressed out (centrifuged at 5860 x gravity)* This procedure was repeated three times and the ETOH extract was pooled* The extract was treated further in 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* The entire procedure was carried out at 0°C* The supernate was evaporated in vacuo at ca 30°C using a flash evaporator, the receiver of which was cooled in a dry ice-acetone mixture* The residue was suspended in 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 in water and extracted three times with 2 volumes of dichloromethane. The steroids obtained by this procedure will be referred to as glucuronide conjugated steroids* The steroids freed by the glucuronidase treatment were removed by three extractions with dichloromethane* The dichloromethane extract was washed three times with 0*1 volumes of each of the following: 5% sodium bicarbonate, 0*1 N acetic acid and water* The organic solvent was removed in 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 in 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 in 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 H 2 ^ 4 P h 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 risk of a high acid concentration when the volume of the solution was reduced) and the ether was removed at room temperature using a flash 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 will be referred to as the "pH 0*8" conjugated steroids* Column Chromatography 3 Neutral Woelm activity 1 alumina was converted to activity 3 by shaking 15 gms with 0*9 ml of water for one hour* A slurry of the adsorbent in 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 in 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 in 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 in a small amount of benzene and applied to the adsorbent. A glass-wool plug was placed on top of the adsorbent in 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 is substituted for KOH (potassium hydroxide). The reaction was carried out at 4-8°C for five hours. The max. was 515 mu and O.D. (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 chroma to 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). The colour was developed f i r s t at room temperature and then with heat. The method was sufficiently sensitive to detect 0.6 ug of dehydroepiandrosterone which had been run as a spot in the haptane—80$ methanol solvent pair. Paper Chromatography Paper was washed in 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$ in 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 distilled* Glassware was washed with acid-chromic oxide cleaning solution followed by water, versene in aqueous methanol and di 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 aid 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* Potassium bromide (5-10 mg infrared grade) 2 was spread over an area of ca 0*5 inch in a mortar warmed on a micro heater to a temperature sufficient to volatilize dichloromethane* The steroid (ca 20-80 ug) was taken up in 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 in 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 in 2*0 ml glacial acetic acid and 2*0 ml of 1*2$ chromic trioxide in 60% acetic acid and stirred for about 30 min* (reaction can be followed by measuring a rapid increase in 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 in 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 in the usual manner* Oxidation and Splitting off Side Chain at Carbon 17 Two mg of steroid were dissolved in 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 filtered 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 filtered 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 for each 50 mg of enzyme in 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 in water and passed through a column of Amberlite—400 (OH) analytical grade ion exchange resin* The resin column measured 1*8 x 12 cm* The resin bed was washed with water to remove the glueosaccharo-l:4-lactone and finally 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 in 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 dehydroepi-androsterone 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 in tubes 25-39 which produced a Zimmermann chromogen reaching peak intensity (visual) in ca 3 hours (Fig* 2). A peak intensity in 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 in 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 in the HM-80 solvent system for 3 hours* A substance which absorbed ultraviolet light, 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 until 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 in 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* The substance was eluted from the paper and i t had a A max* in methanol at 238 mu* The substance was re-chromatographed in the same solvent system for 15 hours* 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 in 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 in the HM-80 solvent for 2 hours. A l l steroids had R^ =0.30 and were detected both by their absorption of ultraviolet light 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 in 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 in 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 in the HBM-70 solvent system. There were two substances from the plasma strip which absorbed ultraviolet light. One substance (R,£ = 0.6) had the mobility of 17 (X-hydroxyprogesterone and the other (R^ = 0.88) corresponded to testosterone acetate. Visual observation suggested that two substances were present in comparable quantity. The infrared spectra of the substance identified as testosterone and standards are shown in Fig. 5. - 13 -Search for Androsterone and Dehydroepiandrosterone Several attempts were made to obtain evidence for the occurrence of dehydroepiandrosterone (DBA.) or androsterone in salmon plasma* The glucuronide and the sulphuric acid conjugated fractions from 218 ml of plasma from spawned Chilko Lake females were chromatographed as streaks in 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 in 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 in 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 strip of paper in 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 in chromatographic mobility with DHA and testosterone were re-chromatographed as spots in 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 in 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 in 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 in 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 in a flash evaporator at room temperature and the residue suspended in acetone and filtered* The acetone soluble material was chromatographed on Whatman No* 1 paper, thoroughly washed as described, in 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 in 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. The acetic acid eluate was taken to dryness on a flash evaporator at ca 20 VC. The residue was taken up in 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 in the HM-80 solvent system for 19 hours. The chromatogram was sprayed with the Zimmeimnn-Bongiovanni 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., in 1960, These were kept in 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 in vacuo on a flash evaporator employing a receiver chilled in dry ice and acetone. The residue was dissolved in 1500 ml of water and extracted three times with equal volumes of dichloromethane. The combined extract fraction "B" ("free1* steroids) was washed with 0,05N NaOH and 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 in a flash evaporator and the residue suspended in acetone and filtered. The acetone soluble material was chromatographed as two parallel spots, representing 10$ and 90$ of the total, along with authentic testosterone as a standard. Whatman No, 1 paper, thoroughly washed in 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 in 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 in the HM-80 solvent (1957) reagent and a Zimmermann-positive substance with the chromatographic mobility - 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 in 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 in the area of testosterone could be shown* KBr pellets of testosterone acetate and testes conjugated steroid (Fraction "G") after acetylation were dissolved in 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 in the BM-80 solvent system for 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 in Salmon The failure, in the present study, to detect either DHA or androsterone in any of several plasma samples is of interest since both these steroids are present in relatively high concentration in peripheral human plasma* Dehydroepiandrosterone has been isolated and positively identified in human plasma* It is 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 in 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 Kellie, 1956)* Testosterone has been shown to be converted (in low yield) by rat liver slices (Fishman and Sie, 1956) and by surviving human prostate tissue (Wotiz et a l . , 1956) in vitro, to a substance positively identified as testosterone-17/^-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 in Spawned Female Sockeye Plasma In the present work there were 7*6 ug/100 ml of testosterone in the glucuronide fraction of plasma taken from spawned Cultus Lake females* In another experiment 2*5 ug/100 ml of testosterone were released by^-glucuonidase from 525 ml of plasma taken from spawned Chilko Lake females* The latter material had been taken through two paper chromatograms, one column chromatogram, acetylation and recovery from a KBr pellet. 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 in •* 18 *•* the spawned Chilko Lake females was comparable to the level accurately determined in the glucuronide fraction of plasma obtained from Gultus Lake spawned females. Low Plasma Testosterone Concentration ("free" and conjugated) in Green Females The plasma samples obtained from females captured at Siwash Bridge, approximately three weeks prior to spawning, contained no detectable Zimmermann positive substance in tubes 4-80 following column chromatography. It must be remembered that the Zimmermann reaction as used in this study is quite ineffective in 17-hydroxy steroids and the intensity of the color produced with 3-4teto steroids reaches a maximum in 3 hours, and has decreased in 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 in significant quantities. It has been shown in 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 fish the plasma from spawned Chilko Lake females contained 7.6 ug/100 ml of testosterone in the glucuronide fraction; 7.8 ug/lOO ml of testosterone and 9.8 ng/100 ml of 17-c(-hydroxyprogesterone in the "free" steroid fraction. The steroids were determined following paper chromatography in heptane-80% methanol solvent system for 16 hours (Schmidt and Idler, 1961). It can be concluded that testosterone was present at a considerably higher concentration in both the glucuronide and "free" fractions of spawned females than in the corresponding fractions prepared from the plasma of females captured prior to arrival in the spawning area. Levels of Testosterone in 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 total testosterone content of the plasma was therefore similar in the sexes but the hormone i s present to a much greater extent in the conjugated form in the male. In the female plasma "free" testosterone reached a peak concentration during the spawning period, and there was a marked drop in concentration just after spawning. If i t is accepted that "free" testosterone is the biologically active form of the hormone, then i t would be logical to assume that this compound is involved in the - 19 -Table 1. Testosterone in the Plasma of Sockeye Salmon (ug/100 ml). PLASMA FRACTION GREEN RIPE POST SPAWNING (but migrating) Conjugated Testosterone *i None - - 13.7 7.6 (Cultus race) "Free" Testosterone None 4.8 14.0 1.7 7.8 (Cultus race) "Free" Testosterone None >2.0 - - 1.0 7.6 (Chilko race) Total Plasma Testosterone in Cultus Lake Fish 15.4 15.4 events leading to, and/or the act of spawning i t s e l f . Paul A. Wright (1961), recently discovered that ovulation could be induced in Rana pipiens in vitro with both progesterone and testosterone but not estrogenic compounds like estrone, estradiol benzoate and diethylstilbesterol. This might parallel the mechanism in P_. nerka. His work indicates that the most efficient steroid i s progesterone. Testosterone's action is less rapid, and causes fewer eggs to be released. However, work done on migrating female salmon did not show any progesterone in the plasma (Idler et a l . , 1959). The absence of progesterone must be taken with caution since i t was not specifically looked for, and could have been missed. The presence of a metabolite of progesterone, 17-<¥*<hydroxyprogesterone (Idler et a l . , 1959; Grajcer and Idler, 1961), in the plasma in large quantities emphasizes this possibility. Testosterone Conjugates in 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 in the testes of spawning sockeye represents a distinct deviation from the known testosterone picture in mammalian blood and testes. Fi r s t l y the concentration of the hormone is many times greater than that found in normal human plasma (Finkelstein et a l . , 1961), secondly, testosterone has not previously been found in the conjugated form in testis tissue. The existence of testosterone in the spawning salmon testes in the form of a glucuronide conjugate could be accounted for in one of three waysi « 20 -a) The conjugated testosterone is a metabolite of another substance, and has no physiological activity of i t s own* b) The glucuronide conjugated form is the active form in 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 activity of that organ (Potter and Hoar, 1954j Tsuyuki and Idler, 1959), and therefore possibility (a) is unlikely* Possibility (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 in testosterone esters produces an androgen having a more intense and prolonged action (tested on seminal vesicle and prostate gland of castrated rats)* This effect is d i f f i c u l t to evaluate however, since the assaying technique employs the biological responses of tissues* It is not yet possible to determine whether the esterified compound is more effective because i t has a higher biological activity or because i t is better protected until i t reaches the target area* This last possibility coincides with (c) (Fishman and Sie, 1956)* The suggestion that testosterone i s produced in the testis, and then transported by the blood in the conjugated form to target area, is plausible; however, the very low concentration of the "free" hormone at a l l times in the male plasma seems to refute this argument in the male* It is 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 in 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 activity of testosterone as assayed by the chick comb growth test (Idler et a l . , 1961). 11-Ketotestosterone is 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 is a precursor in vivo of 11-Ketotestosterone (D.R. Idler, Private communication). However, this does not adequately explain the very high concentration of conjugated testosterone in the spawning male* In summary five points should be considered: a) Progesterone is apparently not a major steroid in ripe female sockeye plasma, although 17- oC-hydroxyprogesterone, a metabolite of progesterone, is present in considerable concentration* * 21 -b) Testosterone is found in substantial quantities in post-spawned female plasma, occuring in equal portions in the "free" and the conjugated forms, c) Concentration of the "free" testosterone in the female reaches a peak at spawning time, and declines later, d) In contrast to the female plasma, the analysis of male sockeye showed 90$ of the testosterone in the conjugated form, e) It was shown that testosterone occurs in the testes in 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 in the plasma of a spawning female sockeye was shown to be considerably higher than that reported in any other species. To date including values for the plasma of hilus-cell tumor in the human female where a concentration of 2,0 ug/100 ml of plasma is attained and adrenal adenoma were l»3ug/l00 ml of plasma is recorded (Finkelstein et a l , , 1961), In this report testosterone was found in the glucuronide fraction of testes, and for the f i r s t time testosterone was identified conclusively in a teleost fish (Chieffi and Lupo recently identified testosterone in an elasmobranch testes, 1961), At this point, adequate background material concerning the metabolism of the above compounds is lacking. Any attempt to explain their functional significance would therefore be highly speculative. To attain an understanding of the function(s) of testosterone, in the migrating salmon (both male and female) some knowledge of the following points would be essential: 1, Establishing whether testosterone or testosterone glucuronide is the active form (or each one on a different site). Progress in this direction could be made by applying testosterone glucuronide to a known site of action of testosterone activity like the capon comb; both "free" testosterone and testosterone-glucuronide with saccharo—ls 4—lactone (^-glucuronidase inhibitor) could be used as controls, 2, To ascertain the activity in f i s h , a standard test could be devised, such as male colour development. Since testosterone is capable of inducing ovulation in the amphibians (Wright, 1961} Shapiro, 1936; Ramaswami and Lakshman, 1958) the same might be tried on salmon ovaries* 3, Should "free" testosterone be found to be the active form in the female, then an assay for £-glucuronidase activity in migrating female ovaries would be advisable. M 22 4* The site of the biological synthesis of testosterone and i t s glucuronide conjugate in the migrating salmon should be sought* Testosterone has been found in gonads and adrenals of many higher vertebrates (Dorfman and Shipley, 1956) as well as in testes of elasmobranchs (Chieffi and Lupo, 1961). For completeness, the adrenals of both male and female fish should be assayed, as well as the ovaries* This will only prove the presence or absence of the hormone* To show the biosynthesis tissue culture technique and the appropriate precursors would be used* If testosterone i s shown to possess some biological activity in 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 for testosterone (both for the "free" and conjugated forms) in female plasma and ovaries could be attempted* The assay should be done on three groups of fish; one group of fish which matured in sexual confinement, a second which matured with a male in a separate glass partition (within sight), and a third which matured with males in the same tank* As a group these experiments would yield 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 in fish 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. 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Acta, 22: 266-268. Wotiz, HJH., H.-G. Sie and W.H. Fishman. 1958. Characterization of Biosynthetic Testosterone Glucuronic Acid.. J . Biol. Chem. 232: 723-727. Wright, P.A. 1961. Induction of Ovulation in Vitro in 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 50 ug B. Denydroepiandrosterone 60 ug C. Epiandrosterone 100 ug D. Androsterone 70 ug E. Etiocholaneolame 200 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 e Sulphuric acid chromogen of pooled tubes 25-39 from glucuronide conjugate fraction of 860 ml plasma of Chilko Lake spawned male» 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 DOUBLE-BEAM 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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