Seasonal patterns of free and conjugated androgens in the brown trout Salmo trutta

GENERAL

AND

Seasonal

COMPARATIVE

Patterns

ENDOCRINOLOGY

48, 222-231 (1982)

of Free and Conjugated Androgens Trout Salmo trutta DAVID E. KIME AND NIGEL

Department

of Zoology,

The University,

Sheffield

in the Brown

J. MANNING SIO 2TN,

United

Kingdom

Accepted November 5, 1981 A radioimmunoassay for the simultaneous determination of testosterone, 1l/3hydroxytestosterone, 1 I-ketotestosterone, and their glucuronides is described. The plasma concentrations of these steroids have been determined throughout the annual reproductive cycle of the male brown trout Salmo trutta. The maximum concentration of testosterone (33.2 f 5.4 nglrnl in late September) precedes that of 11-ketotestosterone (68.7 2 5.5 n&m) which coincides with the period of spermiation in early November. Peak concentrations of testosterone glucuronide (14.5 f 2.7 nglml), 1lp-hydroxytestosterone glucuronide (6.8 -+ 1.6 &ml), 11-ketotestosterone ghrcuronide (5.4 + 0.7 r&ml), and llP-hydroxytestosterone (3.2 1?10.7 &ml) were also found in November. The ratio of free:conjugated testosterone drops significantly between August and December in the male (10.7-0.66) but not in the female (6.8-5.3). Factors such as hormones and mineral ions which may affect glucuronyl transferase activity are discussed, and a possible role for glucuronides in the normal functioning of the reproductive cycle in the trout is suggested.

It is now clearly established that testosterone,l 1l/3-hydroxytestosterone, and 1 lketotestosterone are major products of the teleost testis in vitro (see Kime, 198Oa), but until recently methods have not been available to determine plasma concentrations of these steroids. Double isotope derivative methods, while having the advantage of positive identification of the steroids, are not suited to the routine measurement of large numbers of samples. Radioimmunoassay, although less specific, is more suited to routine analysis and several methods have been reported for the assay of llketotestosterone in teleost plasma (Simp1 Steroid nomenclature: T,testosterone, 17phydroxy-4-androsten-3-one; KT, 11-ketotestosterone, 17P-hydroxy-4-androstene-3,1 I-dione; 1 lPT, 1l/3hydroxytestosterone, llp.l7/3-dihydroxy-4-androsten-3-one; -G, glucuronide; cortisol, llp,l7o, 21-trihydroxy-4-pregne-3,20-dione; cortisone, 17o, 21-dihydroxy-4-pregnene-3,11,20-trione; androstenedione, 4-androstene-3,17-dione; androstenetrione, 4-androstene-3,11,17-trione; 1 lp-hydroxyandrostenedione, 1lp-hydroxy-4-androstene-3,17-dione; dihydrotestosterone, 17@hydroxy-5a-androstan-3-one. 222 0016-6480/82/100222-10$02.00/O Copytiht AU rights

@ 1982 by Academic Press, Inc. of reproduction in any form reserved.

son and Wright, 1977; Sangalang and Freeman, 1977; Sangalang et al., 1978; Idler and Ng, 1979; Ng and Idler, 1980; Scott et al., 1980a). Radioimmunoassay of testosterone is well established in mammals and has also been applied to assay of teleost plasma (Sangalang and Freeman, 1977; Wingfield and Grimm, 1977; Whitehead et al., 1979; Scott et al., 1980a, b), but no radioimmunoassay has so far been reported for 1 l&hydroxytestosterone although this steroid may be the major androgen in some ambisexual fish (Idler et al., 1976). We have recently shown that in some teleosts the formation of glucuronides of testosterone and 1 1-ketotestosterone may be quantitatively important in vitro and have suggested that they may play an important role in the temperature regulation of reproductive development in these species (Kime, 1979; 1980b; Kime and Saksena, 1980). In the Atlantic salmon (Sulmo salur) plasma levels of the glucuronides of testosterone, liphydroxytestosterone and 1l-ketotestosterone were found to be comparable to levels of the unconjugated steroids but the num-

SEASONAL

CHANGES

ber of samples was very small as the method was based on double isotope derivative procedure (Idler et al., 1971). In order to obtain a full profile of circulating androgen levels in teleost fish subjected to both natural seasonal cycles and to artificial temperature and photoperiod regimes we have devised a simultaneous radioimmunoassay procedure for the determination of testosterone, 1 lp-hydroxytestosterone, and 11-ketotestosterone and their glucuronides in teleost plasma. The validation of the method and its application to the measurement of seasonal changes of these hormones in the brown trout Salmo trutta is reported in this paper. MATERIALS

AND METHODS

[1,2,6,7-3H]Testosterone (88.5 Ciimmol) and [l,2,6,7-3H]cortisol (81 Ci/mmol) were obtained from Amersham International. [l,2,6,7-3H]-11Ketotestosterone was prepared from [1,2,6,7JH]cortisol by oxidation with chromium trioxide to androstenetrione followed by partial reduction to llketotestosterone with sodium borohydride. [1,2,6,7-3H]1lo-hydroxytestosterone was prepared by oxidation of [ 1,2,6,7-3H]cortisol to 1I&hydroxyandrostenedione with sodium bismuthate and then partial reduction to 1 lb-hydroxytestosterone with sodium borohydride. For the sodium bismuthate oxidation the procedure of Brooks and Norymberski (1953) was modified. The dried steroid was treated with a suspension of 2.5 mg sodium bismuthate in 100 ~150% aqueous acetic acid for 3 hr at 22”. The product was extracted with 3 ml dichloromethane and washed successively with 1 ml of 1 M sodium bicarbonate solution and twice with 1 ml water before evaporation. The procedures for chromium trioxide oxidation and sodium borohydride reduction were as previously described (Kime, 1978) except that a longer time at a lower temperature was used (10 min at -10”) for the reduction since this gave better yields of the required product. Products were purified by thin-layer chromatography (TLC). Standard steroids were obtained from Steraloids, and /3-glucuronidase (from bovine liver, Type B-l) was obtained from the Sigma Chemical Company. Plastic LP3 assay tubes were purchased from Luckham. All solvents were distilled from calcium hydride before use. Bovine serum albumin (BSA)-saline solution was prepared by dissolving 100 mg BSA in 100 ml 0.9% saline in phosphate buffer, pH 7.3. Sodium azide (1 g/liter) was added to prevent bacterial growth. Dextran-charcoal suspension was made by mixing 750 mg of Norit A charcoal and 750 mg of dextran with 60 Materials.

IN

TROUT

ANDROGENS

223

ml of buffered saline. A scintillation fluid containing 14 g of 2,5-diphenyloxazole (PPO) and 1.2 g of 1,4-di-[2(S-phenyloxazolyl)]benzene (POPCIP) in 1.6 liters of toluene and 1 liter of 2ethoxyethanol was used. Fish. Hatchery-reared brown trout, Salmo irutta, were kept in outdoor tanks at the Windemere laboratories of the Freshwater Biological Association. Each tank (1500-liter capacity) was supplied with a constant flow of lake water. At monthly intervals fish were removed from each tank, stunned by a blow to the head, and a blood sample taken from the caudal vessels by means of a heparinized syringe. The blood was centrifuged and the plasma kept frozen at -20” until required for assay. The fish used in these experiments were 2 years old at the beginning of the experiment. The samples for the last month (April) were from a later hatching and were 2 years old. Five to eight fish were used for each monthly sample. Preparation of antisera. The 3-(U-carboxymethyl) oxime-BSA conjugates of 11-ketotestosterone and 1lP-hydroxytestosterone were prepared according to the procedure of Simpson and Wright (1977). Testosterone-17/3-hemisuccinate-BSA conjugate was purchased from Steraloids. The antisera were raised in rabbits with the kind help of Dr. Patricia M. Ingleton in this Department using the procedure previously described (Hargreaves and Ball, 1977). Testosterone antiserum was used at a dilution of 1:9000, llketotestosterone and ll@hydroxytestosterone antisera were used at dilutions of l:lO,OOO in BSA-saline. Chromatography. Thin-layer chromatography was carried out using 20-cm2 preprepared plastic sheets of silica gel (Merck Kieselgel 60F 254) divided into 2.5-cm lanes. The sheets were prewashed by an overnight continuous development in distilled methanol. Androgens were separated by development in chloroform-methanol (95:5). Radioactive areas were detected using a Packard Model 7220 radiochromatogram scanner. The zones corresponding to the radioactive peaks were scraped into plastic LP3 tubes with a scalpel blade, agitated with 1 ml BSA-saline solution, and centrifuged. The supematant was frozen until required for assay. Extraction of plasma. To 100 ~1 of plasma was added 100 ~1 each of [1,2,6,7-3H]testosterone, [l,2,6,7-3H]ll-ketotestosterone, and [l,2,6,7-3H]1 l/3hydroxytestosterone solutions (0.1 @i/ml in acetate buffer, pH 4.8). Each radioactive solution (100 ~1) was also pipetted into a counting vial to give dpm added. The plasma plus radioactive tracers was extracted three times with 3 ml distilled dichloromethane, and the combined extracts evaporated to give the free steroid fraction. To the aqueous residue was added 50 ~1 g-glucuronidase solution (8 mgfml in 0.1 M acetate buffer, pH 4.8) and the mixture incubated overnight. A further 50 ~1 enzyme solution was added and the incubation continued for a further 48 hr. Tritiated steroids were added as above and the steroid moieties of the

224

KIME

AND

hydrolysed glucuronides extracted with dichloromethane. Both the free and glucuronide fractions were chromatographed on TLC and the three radioactive peaks corresponding to testosterone (R, 0.54), llketotestosterone (R, 0.29), and 1lp-hydroxytestosterone (& 0.15) eluted with BSA-saline. Each eluate (100 ml) was taken for scintillation counting to measure recovery. Assay of steroids. Each of the three androgens, testosterone, 11-ketotestosterone, and 1lp-hydroxytestosterone, was assayed by the same general procedure. Tubes for the standard curve were prepared by drying a series of known weights of the steroid into plastic LP3 tubes to give suitable points on a standard curve of 0 to 400 pg. BSA-saline (100 ~1) was added to each standard tube. For the plasma androgen samples, 100 ~1 of the TLC eluate was pipetted into LP3 tubes. If high values were expected, smaller aliquots were used and the volume made up to 100 ~1 with BSA-saline. All samples and standards were assayed in duplicate. To each tube was added 100 ~1 of the appropriate tritiated steroid in BSA-saline, (approx 10,000 dpm), the tube vortexed, and allowed to equilibrate at room temperature for 30 min. The appropriate antiserum (100 ~1) was added, vortexed, and left at room temperature for 30 mitt, then transferred to ice for 10 min. Dextran-charcoal suspension (100 ~1) was added, vortexed, left in ice for 25 min, and then centrifuged for 20 min at 2000 rpm. Aliquots (100 ~1) from each tube were pipetted into counting vials and 10 ml ethoxyethanol scintillant added. Samples were counted for 10 min in a Kontron MR 300 scintillation counter fitted with dpm correction. A standard curve of dpm vs picograms steroid was constructed, from which the nanograms per milliliter of steroid in plasma could be calculated after correction for recovery.

TABLE CROSS-REACTIONS

OF STEROIDS

WITH AND

ANTISERA

RESULTS Validation of the Assay Standard curves. A plot of dpm bound vs picograms of steroid was used as a standard curve. The percent binding for 0 pg steroid was 63.7 2 0.4 (SE) (n = 8), 63.5 + 0.2, and 53.5 f 0.3 for testosterone, ll-ketotestosterone, and 1 I@hydroxytestosterone, respectively. The dpm bound dropped to 50% of the zero value on addition of 54, 48, and 30 pg of the three steroids, respectively. Specificity. Cross-reactions of a number of steroids with each of the three antisera are shown in Table 1. These values are expressed both as the conventional 50% level (ratio of the mass of the androgen to the mass of steroid which is required to decrease the bound label to half of its value at 0 pg), and at the 100 pg level (the diminution in the proportion of bound label produced by 100 pg of steroid relative to that produced by 100 pg of the androgen). Simpson and Wright (1977) have shown that the values obtained by the two methods may give very different results, the commonly quoted value at 50% level often giving very optimistic values. Our results (Table 1) confirm this finding and show the characteristics of our 11-ketotestosterone antise-

1

TO TESTOSTERONE-17~-BS&~l-KETOTESTOSTERONE-3-BSA

lip-HYDROXYTESTOSTERONE-3-B&4

1lPT antiserum 1l@Hydroxytestosterone 1I-Ketotestosterone Testosterone Androstenedione Androstenetrione 1l/%Hydroxyandrostenedione Dihydrotestosterone Cortisol Cortisone

MANNING

100 41.2 20.7 31.8 41.4 52.1 3.6 4.4

(loo) (4.6) (1.4)

11-KT antiserum 20.1 100 20.6 76.9 30.1 52.0 3.3 1.3

(2.2) (loo) (1.3)

T antiserum

4

48.9 (13.8) 47.7 (12.2) 100 (loo) 100 (100) 100 55.4 (4.0) 1.9 1.2

0.15 0.29 0.54 0.75 0.75 0.53 0.62 0.10 0.21

Note. Cross-reactivities are measured at the lOO-pg level; figures in parentheses indicate cross-reaction at the 50% level.

SEASONAL

CHANGES

rum to be comparable to that of Simpson and Wright. Of the major cross-reacting steroids, dihydrotestosterone, androstenedione, and androstenetrione all have greater mobility than testosterone on TLC. Only 1 lp-hydroxyandrostenedione which has a significant cross-reaction with testosterone fails to separate from it on TLC. Since our in vitro experiments showed rapid conversion of 17-keto to 17P-hydroxy (Kime, 1979) and the levels of 1 I@-hydroxytestosterone are very low in the current work, we would not anticipate the presence of high levels of 1lp-hydroxyandrostenedione. Sensitivity. Since high levels of the androgens were found in most samples and ample plasma was available we have made no attempt to optimize sensitivity. The limit of detectable androgen in the present work was estimated to be about 0.5 n&ml. Water blanks carried through the entire chromatography and assay procedure gave values indistinguishable from zero on the standard curve. Recovery. The recovery of tritiated androgen through the extraction and chromatographic steps was 1 l/3-hydroxytestosterone, 63.5 rt: 1.4 (SE)%; ll-ketotestosterone, 60.9 ? 1.9%; and testosterone, 47.4 + 4.3%. The lower recovery of testosterone was probably due to the elution procedure using BSA-saline which would be expected to be less efficient for nonpolar steroids. Although higher recovery is obtained by elution with methanol, the BSAsaline extraction is preferred in the current procedure for its speed and convenience since sensitivity is not a limiting factor. Precision. The intraassay variation was determined by five replicate assays of the same plasma sample. This gave values of 40.7 -C 1.2 (SE) @ml (coefficient of variation, CV 6.7%) for testosterone, 51.3 + 0.24 (CV 1.1%) for 11-ketotestosterone, and 2.16 i 0.16 r&ml (CV 16.6%) for liphydroxytestosterone. Accuracy. To test the accuracy of the assay, known weights of testosterone, ll-

IN

TROUT

225

ANDROGENS

ketotestosterone, and 1 lfi-hydroxytestosterone (200-1800 pg) were added to lOO~1 samples of plasma obtained from female trout with fully regressed ovaries. The plasma was then assayed in the normal manner and regression lines calculated for picograms of steroid measured versus picograms of steroid added. The regression lines for the three steroids were y = 0.944~ + 17.7 (r = 0.998) for testosterone, y = 0.949~ + 44.8 (r = 0.998) for ll-ketotestosterone, and y = 0.934 x - 14.8 (r = 0.999) for 1 Ifi-hydroxytestosterone. The amount of steroid was thus very closely correlated with the steroid added. Seasonal

changes

in androgen

levels.

The seasonal changes in plasma concentrations of the three major steroids, llketotestosterone, testosterone, and testosterone glucuronide, are shown in Fig. 1, and those of the minor plasma steroids, 1 I-ketotestosterone glucuronide, 1 l/3hydroxytestosterone, and 1 lp-hydroxytestosterone glucuronide, are shown in Fig. 2. Seasonal changes in biological (gonadosomatic index (GSI) and body weight) and environmental (daylength and water temperature) parameters are also indicated in Fig. 1. Table 2 shows the values obtained for the female fish captured during the months of August to November. These results are fully discussed in the following section. DISCUSSION

During the last decade in vitro studies on a large number of teleost species have clearly shown that 1 l-oxygenated androgens are formed in high yield by the testis. The product varies considerably, but this may in most cases be more dependent upon the substrate and the incubation conditions than upon the species. The products most commonly formed are lip-hydroxy and 11-keto derivatives of both testosterone and androstenedione (see review by Kime, 1980a). In vitro results, while indicating the presence of enzyme systems, do not necessarily reflect the nature of the actual

KIME

AND

MANNING

450

-

-i t 250

. Seasonal changes in the major plasma androgens, body weight, gonadosomatic index, daylength, and water temperature for male brown trout. Vertical bars indicate f SE. sp, sperm&ion; H, KT; A, T; A, T-G.

steroids secreted in vivo. These can be determined only by measurement of the circulating plasma steroid concentrations, usually either by a specific double-isotope method or by the less specific, but much more rapid, radioimmunoassay procedure. Testosterone radioimmunoassays are commonly used in mammalian studies and these have been applied to a number of teleost

species to measure “androgen” levels (Schreck er al., 1972, 1974; Sanchez-Rodriguez et al., 1978; Wingfield and Grimm, 1977). These assays often use an antisera with high cross-reaction to 1 l-ketotestosterone which may give misleading results if the two androgens show peaks at different times. This can lead to rather curious results as in those described by Bonnin

SEASONAL

J

J’A.S-0

CHANGES

N-D

J

2. Seasonal changes in llp-hydroxytestosterone, 1Ifi-hydroxytestosterone glucuronide, and 1lketotestosterone glucuronide in the male brown trout. n , KT-G; l , ll@T; A, 11/3T-G. FIG.

(1979) in Gobius niger where a double peak of “testosterone” was observed. More recently, a number of authors have reported the preparation of antisera for 1 Iketotestosterone (Sangalang and Freeman, 1977; Simpson and Wright, 1977; Idler and Ng, 1979; Scott et al., 1980a). No antiserum has previously been reported for liphydroxytestosterone although Idler et al. (1976), using a double-isotope derivative assay method, showed it to be present in levels of 7 to 14 @ml in three species of ambisexual fish even though 1l-ketotestosterone was not detectable. Studies of seasonal changes in the plasma androgen profile in teleosts are rare. While

our work was in progress, Scott et al. (198Oa) described the seasonal changes in testosterone and 11-ketotestosterone in the rainbow trout Salmo gairdneri. These authors showed that the peak of testosterone appeared approximately 2 months before that of 11-ketotestosterone which corresponded to the period of maximum spermiation. Using a double-isotope derivative assay Sangalang and Freeman (1974) showed a very similar effect in the brook trout Salvelinus fontinalis, although results were based on single assays of pooled plasma. The peak of testosterone preceded that of 11-ketotestosterone by about 4 weeks, and again spermiation coincided with the peak of II-ketotestosterone. Stuart-Kregor et al. (1981) showed that plasma levels of both testosterone and llketotestosterone were higher in mature Atlantic salmon parr (stages V and VI) than in fish at earlier stages of maturity. Recently we have described the effect of temperature on in vitro steroidogenesis in the rainbow trout Salmo gairdneri and the goldfish Carassius auratus (Kime, 1979, 1980b; Kime and Saksena, 1980) and shown that at elevated temperatures steroid glucuronides may be quantitatively the most important product of the testis in vitro. We have suggested that these glucuronides may play an important role in the reproductive physiology of some species of teleost fish, but few data are available on their plasma concentrations in vivo or on their relation to reproductive development. Idler et al. (1971) has shown that in the Atlantic salmon Salmo salar concentrations of gluc-

TABLE PLASMA

ANDROGENS

2

IN FEMALE

Plasma concentration Date

KT

T

August 27 September 25 November 5

0.45 t 0.3

20.3 k 5.9 57.1 2 6.5 77.6 k 13.0

0.81 + 0.13 1.40 f 0.24

227

IN TROUT ANDROGENS

BROWN

TROUT

(@ml) 2 SE KT-G 0.6 ? 0.1 0.6 2 0.2 0.6 2 0.2

T-G 3.4 f 0.9 7.7 f 1.0 14.6 f 2.2

T/T-G 6.8 * 1.5 7.6 k 0.8 5.3 ‘- 0.4

228

KIME

AND

uronides of testosterone, 1l-ketotestosterone, and 1 l@hydroxytestosterone were comparable to those of the unconjugated steroid in both peripheral and testicular plasma, and Bonnin (1979) found that in Gobius niger testosterone glucuronide formation was significant only toward the end of the “testosterone” peak. In order to further elucidate the role of glucuronides in the reproductive physiology of teleost fishes and to examine the seasonal changes in the individual androgens we have devised a simple and rapid radioimmunoassay for the simultaneous measurement of testosterone, 1l-ketotestosterone, and 1 l/3-hydroxytestosterone and their glucuronides. Details of the method and its validation are given above. The results of monthly sampling of brown trout Salmo trutta from the hatchery at Windemere are depicted graphically in Figs. 1 and 2, with the monthly changes in gonadosomatic index and body weight. Changes in daylength and water temperature are also indicated. The two major products were testosterone and 11-ketotestosterone which showed maxima of 33.2 + 5.4 (SE) and 68.7 & 5.5 (SE) @ml, respectively. Although these values are considerably lower than those reported by Scott et al. (1980) for the rainbow trout, they fall within the range of values reported by other authors for teleost fish. Although testosterone levels show a maximum in late September, the fall to early November is not significant. llKetotestosterone, however, shows a very clear maximum in early November which corresponds to the period at which all fish were spermiating. Without more frequent sampling it is not possible to define the maximum for either hormone, but indications are that the testosterone maximum precedes that of 11-ketotestosterone which is in agreement with that found in the rainbow trout (Scott et al., 1980a) and the brook trout (Sangalang and Freeman, 1974). Little is known of the different biological roles of these two androgens but

MANNING

the seasonal changes in the three species of salmonid indicate that testosterone may be involved in spermiogenesis whereas 1 lketotestosterone is more involved in the initiation of spermiation. Further support for this is indicated by recent work of Fostier et al. (1981), in which it was shown that the sperm volume was related to the plasma levels of 11-ketotestosterone in the rainbow trout. Plasma levels of 1 lp-hydroxytestosterone in the brown trout were very low, reaching a maximum of only 3 rig/ml in early November corresponding to the peak in 1 lketotestosterone and spermiation. This value is comparable to that found in spermiating rainbow trout by double-isotope methods (Campbell et al., 1980). One of the main purposes of our work was to investigate the role of glucuronides during the seasonal reproductive cycle. The major glucuronide was that of testosterone (Fig. 1) which reached a maximum of 14.5 + 2.7 @ml in early November. Glucuronides of 11-ketotestosterone and 1 lp-hydroxytestosterone also peaked in early November with values of 5.4 t 0.7 and 6.8 2 1.6 ng/ ml, respectively. It is interesting to note that at all times concentrations of lip-hydroxytestosterone glucuronide were higher than that of the free steroid, whereas only a small proportion of 11-ketotestosterone was present as the glucuronide. As with our earlier in vitro studies it is clear that the major glucuronide is that of testosterone. Plasma steroid levels reflect a steady state situation and a balance between secretion and excretion rates. In mammals glucuronidation is considered to be a hepatic detoxifying mechanism which facilitates the excretion of steroids by converting them into water soluble and more readily excretable products. In teleost fish where glucuronidation may be either hepatic or testicular a similar difference in excretion rates of free and conjugate might also be expected. From the observed plasma steroid levels it is therefore probable that the actual testicular secretion of glucuro-

SEASONAL

CHANGES

nides may in fact exceed that of free steroids, and investigations are currently in progress to examine the dynamics of steroid production and excretion. It is thus possible that small changes in glucuronyl transferase activity may result in relatively large changes in plasma free steroid concentrations, and the increase in conjugate formation from the end of September at the expense of free steroid secretion may precipitate the fall in free testosterone levels. In our earlier in vitro studies we demonstrated an increase in glucuronide formation with temperature and in vivo we have shown that the failure of rainbow trout to spermiate at elevated temperatures may be related to a fall in 1 I-ketotestosterone levels as a result of increased glucuronide secretion (Kime and Manning, 1981). The lack of a seasonal correlation between glucuronide formation and water temperature, however, indicates that in the normal trout other factors may be responsible for the stimulation of glucuronide synthesis. In a recent extensive review of the literature of glucuronidation, Dutton (1980) has described a large number of factors which may affect the activity of the glucuronyl transferase enzyme in mammalian liver. Of major relevance to possible environmental and endocrine influences in teleost fish are stimulation of glucuronide formation by magnesium and calcium ions, the latter being of particular relevance in female teleosts in which plasma calcium is greatly elevated during vitellogenesis (Scott et al., 1980b). Storey (1950) has shown that in mouse liver glucuronyl transferase activity is strongly stimulated by bicarbonate and carbon dioxide, and the influence of sex and sex hormones on hepatic glucuronide formation has been discussed (Schriefers, 1967; Ghraf et al., 1973). In their study of the seasonal changes in androgens in the brook trout, Sangalang and Freeman (1974) showed that sublethal concentrations of cadmium could greatly perturb the patterns of both androgens and that the higher con-

IN TROUT

ANDROGENS

229

ASONDJ FIG. 3. Seasonal changes in free:conjugated ratio in the male brown trout. A, Total; W, KT/KT-G; +, T/T-G.

centrations in the treated fish failed to fall in the normal way following spermiation. Since cadmium is a potent inhibitor of glucuronide synthesis in pig kidney (Rao et at., 1976) it is possible that some, at least, of this perturbation of steroid levels may be a result of inhibition of glucuronide synthesis in vivo. Many fish, while appearing perfectly healthy, fail to breed in captivity and it is possible that this may in some cases be due to changes in environmental factors which are not readily detectable. The role of glucuronides, already implicated in the inhibition of trout spermiation at high temperature (Kime and Manning, 1981) may be of even greater importance in the normal functioning of the reproductive cycle. We are currently investigating factors which may influence the synthesis of these compounds. It is apparent from Fig. 3 that a

230

KIME

AND

major shift occurs in the ratio of free to conjugated steroids during the later stages of gonadal recrudescence. A possible implication of androgen is suggested by the figures for female brown trout (Table 2) which we obtained in August to November in which the T:TG ratio is significantly higher than in the male. The steroid levels in the female confirm the finding of Scott et al. (1980b) that although 1 I-ketotestosterone levels are very low, testosterone levels are in fact higher in the female than the male. The role of this high plasma testosterone in the female remains unclear. The present work has described and validated a method for the study of androgen profiles during the reproductive cycle of teleost fish and has applied this to the annual cycle of the brown trout. While implicating glucuronide synthesis in this cycle, the factors which control its formation remain unclear. Studies to elucidate these factors are now in progress in our laboratory. ACKNOWLEDGMENTS We express our gratitude to Dr. A. J. Pickering and Mr. T. G. Pottinger of the Freshwater Biological Association for making their facilities available to us and for their help in obtaining plasma samples. This work was carried out under a grant from the Science Research Council (GRIA94843) to D. E. Kime.

REFERENCES Bonnin, J. P. (1979). Variations saisonnieres de la testosterone plasmatique chez un poisson teleostean, Gobius niger L. C.R. hebd. Seances Acad. Sci., Paris 288D, 627-630. Brooks, C. J. W., and Norymberski, J. K. (1953). The oxidation of corticosteroids with sodium bismuthate. Biochem. J. 55, 371-378. Campbell, C. M., Fostier, A., JaIabert, B., and Truscott, B. (1980). Identification and quantification of steroids in the serum of rainbow trout during spermiation and oocyte maturation. J. Endocrinol.

gonadotropin during the beginning of spermiation in rainbow trout (Snlmo gairdneri, R.). Gen. Camp.

and Fla. and and

Endocrinol.

46, 428-434.

Ghraf, R., Hoff, H-G., Lax, E. R., and Schriefers, H. (1973). The S/3-metabolites of testosterone; mode and sex-specificity of their formation in the rat liver. Actu Endocrinol. (Copenhagen) 73,577-W. Hargreaves, G., and Ball, J. N. (1977). Cortisol in Poecilia latipinna: Its identification and the validation of methods for its determination in plasma. Steroids 30, 303-314. Idler, D. R., and Ng, T. B. (1979). Studies on two types of gonadotropins from both salmon and carp pituitaries. Gen. Comp. Endocrinol. 38,421-440. Idler, D. R., Horne, D. A., and Sangalang, G. B. (1971). Identification and quantification of the major androgens in testicular and peripheral plasma of Atlantic salmon (Salmo salar) during sexual maturation. Gen. Comp. Endocrinol. 16, 257-267. Idler, D. R., Reinboth, R., Walsh, .I. M., and Truscott, B. (1976). A comparison of ll-hydroxytestosterone and 11-ketotestosterone in blood of ambisexual and gonochoristic teleosts. Gen. Comp. Endocrinol. 30, 517-521. Kime, D. E. (1978). Steroid biosynthesis by the testes ofthe dogfish Scyliorhinus caniculus. Gen. Comp. Endocrinol. 34, 6- 17. Kime, D. E. (1979). The effect of temperature on the testicular steroidogenic enzymes of the rainbow trout, Salmo gairdneri, Gen. Comp. Endocrinol. 39, 290-296. Kime, D. E. (1980a). Comparative aspects oftesticular androgen biosynthesis in nonmammalian vertebrates. In “Steroids and Their Mechanism of Action in Nonmammalian Vertebrates” (G. Delrio and J. Brachet, eds.), pp. 17-31. Raven Press, New York. Kime, D. E. (1980b). Androgen biosynthesis by testes of the goldfish (Carassius aurutus) in v&o-The effect of temperature on the formation of steroid glucuronides. Gen. Comp. Endocrinol. 41, 164- 172. Kime, D. E., and Manning, N. J. (1981). The effect of temperature on androgen biosynthesis in the rainbow trout, Salmo gairdneri, in vivo. Abstr. 3rd Symp.

on Fish

Physiology,

Bangor,

September

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