17α,20β-dihydroxy-4-pregnen-3-one: plasma levels during sexual maturation and in vitro production...

GENERAL AND COMPARATIVE ENDOCRINOLOGY 51, 106-112 (1983)

17a,20p-Dihydroxy-4-pregnen-3-one: Plasma Levels during Sexual Maturation and in Vitro Production by the Testes of Amago Salmon

(Oncorhynchus rhodurus) and Rainbow Trout (S&no gairdneri)

HIROSHI UEDA, GRAHAM YOUNG,’ LAURENCE W. GRIM,* AKIRA KAMBEGAWA,~ AND

YOSHITAKA NAGAHAMA

Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444, Japan; *Marine Sciences Research Laboratory, Memorial University of Newfoundland,

St. John’s, Newfoundland AlC 5S7, Canada; and fTeikoku Hormone Manufacturing Company, Ltd., Kawasaki 213, Japan

Accepted October 8, 1982

The present study investigated seasonal changes in plasma 17a,20l3-dihydroxy-4-pregnen- 3-one (17o,20l3diOHprog) levels in precociously mature and fully grown mature male amago salmon (Oncorhynchus rhodurus) and in vitro 17ol,20@diOHprog production by the testes of amago salmon and rainbow trout (Salmo gairdneri) in response to chum salmon gonadotropin. Plasma 17cY,2OSdiOHprog levels were very low from June to September, and rapidly increased in October at the beginning of the spawning season in both precociously mature and fully grown mature male amago salmon. High levels of 17u,20l%diOHprog were maintained during the period of active spermiation in precociously mature males, and quickly declined in mid-November. Incubation of testicular fragments from spermiating rainbow trout with chum salmon gonadotropin or 17u-hydroxyprogesterone resulted in a highly significant increase in 17u,20@diOHprog levels in the incubation medium. The chum salmon gonadotropin was also effective in stimulating 17c?i,20@-diOHprog production by testicular fragments obtained from amago salmon after the breeding season. These findings are dis- cussed in relation to the possible involvement of 17u,20P-diOHprog in spermiation of sal- monids.

A number of steroids have been isolated and identified from the testes of many teleosts, including the androgens, testosterone, 11-ketotestosterone, and 1 I@hy- droxytestosterone (Ozon, 1972; Kime, 1980). Estradiol-17P and some progesto- gens have also been measured in the plasma and testes of some teleosts (Wingfield and Grimm, 1977; Billard et al., 1978). During spermatogenesis, the major circulating steroids in teleosts are thought to be testosterone and/or 1 1-ketotestosterone. Several studies have suggested a role for androgens in spermatogenesis (Billard et al., 1982; Hunt et al., 1982), and some direct evidence exists for the initiation and mainte-

1 Japan Society for the Promotion of Science-Royal Society (Great Britain) Postdoctoral Fellow.

nance of spermatogenesis in vitro by testosterone in goldfish (Remacle, 1976; De- clercq et al., 1977). However, there is little evidence for the involvement of steroid hormones in spermiation of teleosts.

Since Idler et al. (1960) first isolated 17a,20B - dihydroxy - 4 - pregnen - 3 - one (17ol,20@diOHprog) from female sockeye salmon, this steroid has been shown to be one of the most potent inducers of oocyte maturation in several teleosts (Fostier et aE., 1973; Jalabert, 1976; Goetz and Theofan, 1979; Duffey and Goetz, 1980; Nagahama et al., 1980, 1983a; Sower and Schreck, 1982). Campbell et al. (1980), using a double isotope derivative method, recently identified 17a,20l3-diOHprog in the plasma of sexually mature female and male rainbow trout. The aim of the present study was to

106 0016-6480/83 $1.50 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. -

17a,20g-DIHYDROXY-4-PREGNEN-3-ONE IN MALE SALMON 107

investigate the presence and seasonal changes in plasma 17a,20@diOHprog levels in precociously mature and fully grown mature male amago salmon, Oncorhynchus rhodurus. In addition, in vitro studies were car- ried out to examine whether the testes of rainbow trout, Salmo gairdneri, and amago salmon could produce 17o,20@diOHprog in response to partially purified chum salmon gonadotropin.

MATERIALS AND METHODS Animals. Amago salmon were obtained from the Gifu

Prefectural Fisheries Experimental Station. For the investigation of changes in plasma 17n,20BdiOHprog levels, 6- to 13-month-old precociously mature males (5-9 fish per sampling) ancl immature males (2-3 fish) and 2-year-old fully grown mature males (3-4 fish) were sampled monthly except in October, when weekly samples were taken. Testicular development in male amago salmon began in early summer (June) and dra- matic increases in gonadosomatic indices (GSI, gonad weight/body weight x 10(J) occurred during the month of September. Active spermiation began in early Oc- tober and continued into early November. For the in vitro experiment sexually regressed (“spent”) precocious male parr (2 fish) were sampled in later De- cember 1981. Spermiating rainbow trout were collected from the Samegai Trout Hatchery, Shiga Pre- fecture, in mid-December 1981. Plasma samples for radioimmunoassay (RIA) of plasma 17d,20B-di- OHprog were collected from the caudal vasculature.

In vitro steroid production. Fish were killed by de- capitation and their testes were carefully removed and transferred to large glass petri dishes containing ice- cold Hepes-NaOH-buffered trout balanced salt solu- tion (Ringer, pH 7.5; Kagawa et al., 1982). Testes were minced with scissors into small pieces and incubated in plastic tissue culture dishes (Costar) containing 1 ml Ringer (several testicular fragments per dish) with various concentrations of chum salmon gonadotropin preparation (SGA, Syndel Lab. Ltd., Canada) and 17~ hydroxyprogesterone (Sigma). In all assays, controls consisted of samples of testicular fragments incubated in Ringer. Three replicates were made for controls and each dose of SGA and 170c-hydroxyprogesterone. The incubates were maintained for 18 hr in a humidified incubator in an atmosphere of 100% air at 1.5”. Fol- lowing incubation, the incubation medium and testicular fragments were collected and were stored at - 20” until assayed for 17o,20@diOHprog and protein, respectively.

Radioimmunoassay. Full details of the radioimmunoassay for 17u,20B-diOHprog and its validation will

be published separately. A brief description is given in Young et al. (1983).

Protein assay. The protein content of the testicular fragments was determined using a Bio-Rad protein assay kit. Testicular fragments were homogenized in I ml Ringer and centrifuged at 10,000 rpm for 10 min, and the supernatant was assayed for protein.

Statistics. All data were expressed as the mean t SE. Differences were calculated by analysis of vari- ance followed by Student-Newman-Keuls’ multiple range test.

RESULTS

1. Changes in Plasma 17cq2OfS diOHprog Concentrations

a. Precociously mature male. Changes in the GSI, plasma gonadotropin (GtH), and androgen concentrations of precociously mature male and sexually immature male amago salmon par-r have been reported pre- viously (Ueda et al., 1983).

From June to September, plasma 1701,2@3- diOHprog levels were extremely low in both precociously maturing males and immature males of the same age (Fig. 1). A shBrp increase in plasma 17a,20l3-diOWprog occurred in precocious males collected’ in early October, and high levels of about 5-g ng/ ml were maintained until early November. The levels showed a distinct drop on No- vember 14, and remained low in December and January.

b. Fully grown mature male. Changes rn the GSI values in 2-year-old fully grown sexually maturing male amago salmon during July to October are shown in Fig. 2. As in the case of precociously mature fish, plasma 17cx,20@diOHprog levels were low from June to September (Fig,. 3). Plasma 17ol,20@diOHprog levels showed a con- spicuous increase to approximately 8 rig/ml on October 24. As in other ~~~~~~~~‘c~~s species, spawning is followed by the so- called “programmed death.”

2. In vitro 17cx,20@diOHprog Production by Testes

a. Rainbow trout. At the peak of the spawning season, the average plasma

108 UEDA ET AL.

1 2.

I - “...“a....“~

June July Aug. Sept. Oct. Nov. Dec. Jan.

FIG. 1. Changes in plasma 17q20P-dihydroxy-4-pregnen-3-one levels during precocious sexual maturation in male amago salmon. Precocious male parr (0); immature male smolt (0). The vertical bars represent the mean f SE.

17a,20&diOHprog level of spermiating tropin (0.01, 0.1, 1 pgiml) and 17a-hy- rainbow trout (n = 5) was 15.85 k 6.27 ng/ droxyprogesterone (0.01, 0.1 tJ,g/ml). The ml. Testicular fragments from a spermiating accumulation of 17cq20@diOHprog in media male were incubated in Ringer for 18 hr in is shown in Fig. 4. The concentration of the continuous presence or absence of var- 17a,20@diOHprog released into the me- ious concentrations of chum salmon gonado-

1 JUIW July Aug. Sept. Oct.

July Aug. Sept. Oct. FIG. 3. Changes in plasma 1701,20@lihydroxy-4-

FIG 2. Changes in the gonadosomatic index during pregnen-3-one levels during sexual maturation in fully sexual maturation in fully grown male amago salmon. grown male amago salmon. The vertical bars represent The vertical bars represent the mean +- SE. the mean -C SE.

17u,20@-DIHYDROXY-4-PREGNEN-3-ONE IN MALE SALMON 109

I %A 17a-OH-Prog

FIG. 4. Effects of chum salmon gonadotropin (SGA) and 17a-hydroxyprogesterone (17o-OH-Prog) on 17o,2Op-dihydroxy-4-pregnen-3-one production by rainbow trout testes. Testicular fragments were incubated in Ringer alone (R, dark bar) or Ringer with various doses of SGA (0.01-l &ml) or 17a-OH-Prog (0.01 and 0.1 pg/ml) for 18 hr. The vertical bars represent the mean I SE of the three replicates.

dium was significantly stimulated (P < 0.01) by chum salmon gonadotropin in a dose- dependent manner. A similar significant increase occurred after incubation of testicular fragments with 17a-hydroxyprogesterone. Over 300 pg/Fg of protein was produced at the highest dose levels of chum salmon gonadotropin and 17cx-hydroxyprogesterone.

b. Amago salmon. The effect of chum salmon gonadotropin on 17a,20lSdiOHprog production was assessed during an 1%hr incubation using testicular fragments from a fully spent (no sperm produced when man- ually stripped) and a partially spent (some

sperm produced when stripped) precocious male parr in December. Histological ex- amination of the testes of the fully spent fish showed an almost complete absence of degenerated sperm. The testes of the partially spent fish contained large amounts of degenerated sperm.

As in the previous experiment, chum salmon gonadotropin at concentrations of 0.1 and 1 yg/ml significantly (P < 0.05 and P < 0.01, respectively) enhanced 17cu,20~- diOHprog production in partially spent and fully spent precocious males. The testes of partially spent precocious males, however, were about 10 times more responsive in terms of 17a,20@diOHprog production, producing over 50 pglbg of protein (Fig. 5).

DISCUSSION

The present study clearly shows that in precocious and fully grown male amago salmon plasma 17cx,2O@diOHprog levels remain low during the major part of the development of the testes (June to Sep- tember) and reach a peak in October, coin- ciding with the spawning (spermiation) period. The concentration of 176~,20,%di- OHprog in plasma is almost exactly the same (about 8 r&ml) in both groups of males. A similar pattern of occurrence of 17cx,2O@diOHprog in plasma has also been found in female amago salmon, although the peak concentration, which was found at oocyte maturation, was much higher (over 70 ngiml; Young et al., 1983).

In our previous study on the precocious male amago salmon parr, elevated plasma GtH levels are maintained during the period of active spermiation, suggesting an impor- tant role for GtH in spermiation (Ueda et al., 1983). Although the mode of action of GtH in spermiation remains unclear in teleosts, it is generally accepted that GtII in- teracts with steroidogenic tissues to stimulate the synthesis and secretion of steroids. It seems unlikely that androgen is the major testicular steroid maintaining spermiation in the precocious male amago

O-

0.

R 0.01 01 I pg R 0.0 I.1 I pg

SGA GA

UEDA ET AL.

ment. After incubation of testicular fragments with [14C]testosterone for 18 hr at 15”, 11-ketotestosterone was identified as the major metabolite. Using 17Lu-[W]hydroxyprogesterone as substrate, however, 17cx,2O@diOHprog was the major metabolite (Ueda, unpublished data). Similarly, 17cr,2OP-diOHprog has been identified as a major metabolite of pregnenolone or progesterone in cell-free homogenates (Arai and Tamaoki, 1967) or chopped tissue (Kime, 1979) of rainbow trout testes.

Fully spent precocious Partmlly spent precocious mature male mature male

FIG. 5. Effects of chum salmon gonadotropin (SGA) on 17a,20@dihydroxy-4-pregnen-3-one production by amago salmon testes. Testicular fragments from fully and partially spent fish were incubated in Ringer alone (R, dark bar) or Ringer with various doses of SGA (0.01-l pg/ml) for 18 hr. The vertical bars represent the mean I SE of the three replicates.

salmon parr (Ueda et al., 1983). In many species of teleosts high plasma levels of 1 l- ketotestosterone occur during the reproductive cycle, but Hunt et al. (1982) recently showed that both testosterone and 1 I-ketotestosterone sharply decline in plasma of Atlantic salmon at the onset of spermiation. These results may suggest that this sharp decline in plasma androgens is involved to some extent in the initiation of spermiation. Although seasonal changes in plasma 1 1-ketotestosterone levels have not yet been analysed in amago salmon, a pre- liminary thin-layer chromatography study has been performed using rainbow trout testes obtained at the same time as those used in this study for the in vitro experi-

The physiological significance of the sharp increase in plasma 17a,20P-diOHprog levels at the time of spermiation is not yet known. There is increasing evidence to indicate that 17a,20@diOHprog has a major role in the induction of oocyte maturation in several species of teleost (Tamaoki et al., 1983). It has been reported that in hypophysecto- mized goldfish, progesterone is more effective in maintaining spermiation than testosterone or methyltestosterone (Billard, 1976). A common feature of oocyte maturation and spermiation in teleosts may be the process of hydration. Hirose (1976) demonstrated that in the oocytes of medaka and ayu, a substantial increase in size due to water uptake (hydration) occurs during oocyte maturation. Gonadotropin appeared to effect water uptake, possibly by a direct action on the oocyte. Similarly, in male carp and rainbow trout spermiation involves a hormone-dependent (pituitary-induced) thinning, or hydration, of the semen (Clemens and Grant, 1965). Billard et al. (1982) suggested that this hydration of the semen, by increasing interlobular pressure, allows the sperm to migrate to the vas deferens where they are stored. Considering the probable similarities of the hydration process in both sexes, it is worthwhile speculating that the observed increase in plasma 17o,20l%di- OHprog levels prior to and during the spermiation period may point to an involvement of this steroid, either directly or indirectly, in sperm fluidity.

The present paper describes for the first

1701,20~-DIHYDROXY-4-PREGNEN-3-ONE IN MALE SALMON 111

time the stimulation by gonadotropin of 17q20l3-diOHprog production by the teleost testis in vitro. Additionally, the con- version of exogenous 17cx-hydroxyprogesterone to 17at,20@-diOHprog by testicular tissues in vitro in the absence of gonadotropin was clearly demonstrated. In female rainbow trout (Fostier et al., 1981) and fe- ‘male amago salmon (Young et al., 1983), in vitro studies have shown that the ovarian follicles are the source of this steroid, although the precise cellular site of synthesis in the follicle remains to be confirmed (Na- gahama et al., 1983b). The cellular site of synthesis of 17cq20P-diOHprog in the testes has not yet been investigated.

The somatic elements of the testis, such as the interstitial cells and/or the Sertoli cells (lobule boundary cells) are obvious candi- dates for the site of biosynthesis. Histolog- ically, Sertoli cells became hypertrophied at the time of active spermiation in precocious male amago salmon and remained in this condition after the completion of the spawning season when the cells contained degenerated sperm (Ueda et al., 1983). At this time, chum salmon gonadotropin can still stimulate in vitro 17cx,20@diOHprog production.

In conclusion, this study demonstrates that peak plasma 17a,20@-diOHprog levels coincide with the period of spermiation in amago salmon males and that the production of this steroid by the testes of amago salmon and rainbow trout can be stimulated in vitro by gonadotropin. The possibility that this steroid has a hormonal function in male teleosts is now under investigation.

ACKNOWLEDGMENTS We wish to express thanks to Mr. S. Adachi for his

excellent technical assistance and Mr. F. Tashiro and the staff of the Gifu Prefectural Fisheries Experi- mental Station for providing amago salmon and laboratory space. We also wish to thank Dr. B. Tamaoki and Dr. H. Kagawa for their helpful suggestions. This study was supported by a grant-in-aid for special project research on marine biological processes (No. 521906) from the Ministry of Education, Science and Culture, of Japan.

REFERENCES

Arai, R., and Tamaoki, B. (1967). Steroid biosynthesis in vitro by testes of rainbow trout. Salmo gairdneri. Gen. Comp. Endocrinol. 8, 305-313.

Billard, R. (1976). Induction of sperm release in the goldfish by some steroids. IRCS Libr. Compend. 4, 42.

Billard, R., Breton, B., Fostier, A., Jalabert, B., and Weil, C. (1978). Endocrine control of the teleost reproductive cycle and its relation to external fac- tors: Salmonid and cyprinid model. In “Compar- ative Endocrinology” (P. J. Gaillard and H. II. Boer, eds.), pp. 37-48. Elsevier, Amsterdam/New York.

Billard, R., Fostier, A., Weil, C., and Breton, B. (1982). The endocrine control of spermatogenesis in teleost fish. Canad. J. Fish. Aquat. Sci. 39, 65-79.

Campbell, C. M., Fostier, A., Jalabert, B., and Bus- cott, B. (1980). Identification and quantification of steroids in the serum of rainbow trout during spermiation and oocyte maturation. 1. Endocrinol. 85, 371-378.

Clemens, H. P, and Grant, F. B. (1965). The seminal thinning response of carp (Cyprinus carpio) and rainbow trout (Salmo gairdneri) after injections of pituitary extracts. Copeia 2, 174-177,

Declercq, L., Remacle, C., and Demal, J. (1977). Go- nads of Carassius aurabus (teleosts) in organ culture: A new technique and the effects of testosterone. Zn Vitro 13, 85-90.

Duffey, R. J., and Goetz, F. W. (1980). The in vitro effects of 17e-hydroxp2OB-dihydroprogesterone on germinal vesicle breakdown in brook trout (Salvelinus fontinalis) oocytes. Gen. Comp. En- docrinol. 41, 563-565.

Fostier, A., Jalabert B., Campbell, C., Terqui, M., and Breton, B. (1981). Cinetique de liberation in vitro de 17cu hydroxy-20B dihydroprogesterone par des follicules de Truite arc-en-ciel, Salvo ghirdPze.+. C. R. Acad. Sci., Ser. III 292, 777-780.

Fostier, A., Jalabert, B., and Terqui, M. (1973). Action predomenante d’un derive hydroxyle de la progesterone sur la maturation in vitro des ovocytes de la Truite arc-en-ciel, Salmo gairdEerii. C. R. Acad. Sci., Ser. D 277, 421-424.

Goetz, E W., and Theofan, G. (1979). ftz vitro s~knu- lation of germinal vesicle breakdown and ovulation of yellow perch (Perca Jluvescens) oocytes. Effects of 17ol-hydroxy-20p-dihydroprogesterone and prostaglandins. Gen. Comp. Endocyinol. 37, 273-285.

Hirose, K. (1976). Endocrine control of ovulation in medaka (Oryzias latipes) and ayu (Piecoglossus altiveiis). J. Fish. Res. Board Canad. 33, 989- 994.

Hunt, S. M. V., Simpson, T. H., and Wright, R. S. (1982). Seasonal changes in the levels of ll-oxo- testosterone and testosterone in the serum of male

112 UEDA r- ‘- 01 AL.

salmon Salmo s&r L., and their relationship to growth and maturation cycle. J. Fish Biol. 20, 105-119.

Idler, D. R., Fagerlund, U. H. M., and Ronald, A. P. (1960). Isolation of pregn-Gene-1701,20/3-dial-3-one from plasma of Pacific salmon (Oncorhynchus nerka). Biochem. Biophys. Res. Commun. 2, 133- 137.

Jalabert, B. (1976). In vitro oocyte maturation and ovulation in rainbow trout (Salmo gairdnen?, northern pike (Esox lucius), and goldfish (Caras- sius auratus). J. Fish. Res. Board Canad. 33, 974- 988.

Kagawa, H., Young, G., and Nagahama, Y. (1982). Estmdiol-17/3 production in isolated amago salmon (Oncorhynchus rhodurus) ovarian follicles and its stimulation by gonadotropin. Gen. Comp. Endo- crinol. 47, 361-365.

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. (1980). Comparative aspects of testicular androgen biosynthesis in nonmammalian vertebrates. In “Steroids and Their Mechanism of Ac- tion in Nonmammalian Vertebrates” (G. Dehio, ed.), pp. 17-31. Raven Press, New York.

Nagahama, Y., Kagawa, H., and Tashiro, E (1980). The in vitro effects of various gonadotropins and steroid hormones on oocyte maturation in amago salmon Oncorhynchus rhodurus and rainbow trout Salmo gairdneri. Bull. Japan. Sot. Sci. Fish. 46, 1097-l 102.

Nagahama, Y., Hirose, K., Young, G., Adachi, S., Suzuki, K., and Tamaoki, B. (1983a). Relative in vitro effectiveness of 17u,20/3-dihydroxy-4- pregnen-3-one and other pregnene derivatives on germinal vesicle breakdown in oocytes of ayu (Plecoglossus altivelis), amago salmon (Onco- rhynchus rhodurus), rainbow trout (Satmo gaird-

nerii and goldfish (Curassius nurutus). Gen. Comp. Endocrinol. 51, 15-23.

Nagahama, Y., Young, G., and Kagawa, H. (1983b). Steroidogenesis in the amago salmon (Oncorhyn- thus rhodurus) ovarian follicle: A two cell-type model. In Proceedings, 9th Int. Symp. on Comp. Endocrinol. ,” in press.

Ozon, R. (1972). Androgens in fishes, amphibians, reptiles and birds. In “Steroids in Nonmammalian Vertebrates” (D. R. Idler, ed.), pp. 328-389. Ac- ademic Press, New York/London.

Remacle, C. (1976). Actions hormonales sur les cel- lules germinales males de Carussius aurafus L. en culture organotypique. Gen. Comp. Endocrinol. 29, 480-491.

Sower, S. A., and S&reck, C. B. (1982). In vitro induction of final oocyte maturation of oocytes in coho salmon. Trans. Amer. Fish. Sot. 111, 399- 403.

Tamaoki, B., Suzuki, K., Hirose, K., and Nagahama, Y. (1983). Biosynthesis of 17a,20P-dihydroxy-4- pregnen-3-one, an inducer for germinal vesicle breakdown of fish oocytes by teleostean ovaries. Zn “Proceedings, 9th Int. Symp. on Comp. En- docrinol. ,” in press.

Ueda, H., Nagahama, Y., Tashiro, F., and Crim, L. W. (1983). Some endocrine aspects of precocious sexual maturation in the amago salmon, Onco- rhynchus rhodurus. Bull. Japan. Sot. Sci. Fish., in press.

Wingfield, J. W., and Grimm, A. S. (1977). Seasonal changes in plasma cortisol, testosterone and oes- tradiol-17P in the plaice, Pleuronectes platessa L. Gen. Comp. Endocrinol. 31, l-11.

Young, G., Crim, L. W., Kagawa, H., Kambegawa, A., and Nagahama, Y. (1983). Plasma 17a,20@- dihydroxy-4-pregnen-3-one levels during sexual maturation of amago salmon (Oncorhynchus rhodurus): Correlation with plasma gonadotropin and in vitro production by ovarian follicles. Gen. Comp. Endocrinol. 51, 96-105.

17α,20β-dihydroxy-4-pregnen-3-one: plasma levels during sexual maturation and in vitro production...

Documents