involvement of gonadotrophin and steroid hormones in spermiation in the amago salmon, oncorhynchus...

GENERAL AND COMPARATIVE ENDOCRINOLOGY 59, 24-30 (1985)

Involvement of Gonadotrophin and Steroid Hormones in Spermiation in the Amago Salmon, Oncorhynchus rhodurus, and Goldfish,

Carassius aura tus

HIROSHI UEDA,*,~ AKIRA KAMBEGAWA,~ AND YOSHITAKA NAGAHAMA*,*

*Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444, and ,‘Department of Obstetrics und Gynecology, Teikyo University, School of Medicine, Itabashiku, Tokyo 173, Japan

Accepted September 27, 1984

Effects of intraperitoneal injections of chum salmon gonadotrophin (SGA) and various steroid hormones (17a,20J%dihydroxy-4-pregnen-3-one; 17a,ZO@diOHprog, 17a-hydroxyprogesterone, testosterone, 1 I-ketotestosterone) on the induction of in vivo spermiation were examined in nonspermiating amago salmon (Oncorhynchus rhodurus) and goldfish (Carassius auratus). A single injection of SGA to amago salmon and two successive injections to goldfish induced strong spermiation responses. Similarly two successive injections of 17a,20@diOHprog caused precocious spermiation in both species; however, the response to 17o,ZOP-diOHprog was of lesser magnitude than that to SGA. The spermiation response of goldfish to 17u-hydroxyprogesterone was similar to that of 17a,ZOS-diOHprog. Neither testosterone nor 1 I-ketotestosterone were effective in inducing spermiation in amago salmon, but these steroids were found to be slightly effective in goldfish. Effects of a single injection of SGA on changes in serum levels of 17a,20S-diOHprog and 1 I-ketotestosterone were also examined in nonspermiating amago salmon. Serum levels of 17a,20@-diOHprog dramatically increased after treatment and peaked on Day 2 and thereafter declined quickly. Similarly, II-ketotestosterone peaked on Day 2, but the levels remained high throughout the experimental period. Considered together, these findings are consistent with the suggestion that 17a,20@diOHprog is involved in the process of spermiation in teleosts. It is further suggested that this hormone is a testicular steroidal mediator of gonadotrophin- induced spermiation in amago salmon. 0 19X? Academic Press. Inc.

Spermiation generally occurs in male teleosts immediately before or during spawning period, and is prerequisite for successful fertilization. Although the term spermiation is widely used by investigators in fish reproduction, the detailed morpho- logical basis of spermiation in teleosts is not known. In this study, spermiation is defined as the release of hydrated semen, with lower spermatocrit than during intratestic- ular storage, from the genital pore by ap- plying gentle pressure on the abdomen.

’ Present address: Department of Anatomy, Univer- sity of Occupational and Environmental Health, School of Medicine, Yahata-Nishiku, Kitakyushu 807, Japan.

2 To whom requests for reprints should be ad- dressed.

Spermiation has been considered to be under the hormonal control and injection of pituitary extracts or gonadotrophins stim- ulates spermiation in several teleosts (Cle- mens and Grant, 1%5; Yamamoto and Ya- mazaki, 1967; Yamazaki and Donaldson, 1968; 1969; Billard, 1977; Ng and Idler, 1980). Also exogenous application of cer- tain steroids, particularly androgens, can induce precocious spermiation in the goldfish, Carassius auratus (Yamazaki and Donaldson, 1969; Billard, 1976).

Recently, high concentrations of 1701,2Op- dihydroxy-4-pregnen-3-one (17a,20@di- OHprog) have been reported in the plasma or serum of three species of salmonids during the spawning season (amago salmon, Oncorhynchus rhodurus, Ueda et

24 0016~6480/85 $1.50 Copyright Q 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.

ENDOCRINE CONTROL OF TELEOST SPERMIATION 25

al., 1983; chum salmon, Oncorhynchus keta, Ueda et al., 1984; rainbow trout, Salmo gairdneri, Scott and Baynes, 1982). This hormone is known to be the natural maturation-inducing steroid in female salmonids (Nagahama et al., 1984). Although these observations strongly suggest the involvement of 17a,20P-diOHprog in the induction of spermiation in these species, there has been no attempt to induce precocious spermiation in vivo by injection of this hormone.

The present study was therefore under- taken to compare the effects of 17a,20@ diOHprog with those of other steroid hormones and gonadotrophin on the induction of in vivo spermiation in two species of teleosts, the amago salmon and goldfish. In addition, serum levels of 17a,20@di- OHprog and 1 I-ketotestosterone, a potent androgenic steroid in male teleosts, were monitored during the gonadotrophin-induced spermiation in amago salmon in an attempt to better understand the hormonal mechanisms involved in spermiation.

MATERIALS AND METHODS Animals. On September 13, 1982, a total of 45

hatchery-reared male amago salmon (1 year old, about 130-2.50 g) were obtained from the Gifu Prefectural Fisheries Experimental Station and transported to the National Institute for Basic Biology where the fish were kept in fresh water tanks of 250-liter capacity at 12°C under natural light conditions. They were about 1 month prior to natural spawning period and showed no signs of spenniation. Goldfish, weighing 8.5-17.2 g, were purchased from a commercial fish supplier in Okazaki on January 10, 1983; they were about 2 months before natural spawning period and did not show any signs of spermiation. They were kept in sep- arate glass aquaria with 5 liters of constantly aerated fresh water at 12°C under artificial photoperiod of 12 hr light and 12 hr darkness. Neither the amago salmon nor the goldfish were fed during the experimental period.

Experiments. Thirty-one amago salmon were sepa- rated into five groups, each consisting of four to seven males, and were injected intraperitoneally with various hormones (1 pg/g body wt) or saline (0.6% NaCl). One group (control) received a single injection of 0.3 ml saline. The second group received a single injection

of salmon gonadotrophin (SGA; Syndel Laboratories, Vancouver, Canada). The remaining three groups received two successive daily injections of one of three steroids (17o,ZOB-diOHprog, testosterone, or 1 l-ketotestosterone; Sigma, St. Louis, MO.). SGA was dis- solved directly in saline. Stock solutions of various steroids in ethanol were diluted in saline to give final concentrations containing less than 1% ethanol.

Forty-nine goldfish were divided into six groups, each consisting of seven to nine males. Each group received two successive daily injections of a single hormone (testosterone, 11-ketotestosterone, 17a-hydroxyprogesterone, 17o,2OB-diOHprog, or SGA; 1 kg/ g body wt) or saline.

Fourteen amago salmon were divided into two groups of seven fish, and marked individually by tin- clipping. One group received a single intraperitoneal injection of SGA (1 pg/g body wt), and the second group (control) received a single saline injection. Fish were anesthetized lightly and bled successively at 2, 4, and 6 days after injection through the caudal vas- culature with a l-ml syringe.

Radioimmunoassay. Serum concentrations of 17a,20l3-diOHprog were measured by radioimmunoassay, previously developed for this steroid (Young et al., 1983). Full details of the radioimmunoassay for 1 I-ketotestosterone and its validation will be pub- lished separately. The basic procedure followed the method of Kagawa et al. (1982), using [l,Z3Hlll-ketotestosterone (Amersham International) and an antiserum against 11-ketotestosterone-3-(O-carboxyme- thyl)-oxime-bovine serum albumin (BSA) at 100,000 dilution. The antiserum specificity is: 1 l-ketotestosterone, 100%; ll@-hydroxytestosterone, 5.5%; testosterone, 1.9%; most of the large range of testicular steroids and corticosteroids, < 1 .O%. Comparison of sam- ples assayed with or without chromatography on Sephadex LH-20 columns revealed no significant difference in values and the present study assayed sam- ples without chromatography (recovery 91.4 -+ 3.2%). The intraassay variance was 9.5%. The correlation coefftcient for observed and expected values showed no significant difference. For practical purpose, values less than 30 pg/ml were considered nondetectable.

Spermiation. Spermiation responses to various hormone treatments were checked every day by the application of gentle pressure on the abdomen three times. The spermiation response was graded into three categories according to Yamazaki and Donaldson (1968): -, no semen appears after three successive strippings; + , semen appears at the first stripping, but only a small amount of semen appears at the third time; + + , large amounts of semen appear even at the third stripping. The gonadosomatic index (GSI) was calculated as total gonad wt/body wt multipled by 100. When enough volume of semen was collected, the

26 UEDA, KAMBEGAWA, AND NAGAHAMA

spermatocrit was measured in capillaries after centrif- ugation at 12,000 rpm for 10 min. The water content of testes was estimated by heating testes at 110°C for 18 hr and was calculated as (wet testis weight - dry testis weight)/wet testis weight multipled by 100.

Staristics. All data were expressed as the means k SEM. Differences were calculated by analysis of variance followed by Student-Newman-Keuls’ multiple range test.

RESULTS

Effects of Various Hormone Treatments on the Induction of Spermiation

Amago salmon. Spermiation responses of amago salmon to hormonal treatments are shown in Table 1. Spermiation occurred in all fish which received either a single injection of SGA or two successive injections of 17a,20@diOHprog; however, the response to 17a,20@diOHprog was of lesser magnitude than that to SGA. The GSI value of SGA-injected fish was significantly higher than that of control fish (P < 0.05). Neither 11-ketotestosterone nor testosterone was effective in inducing spermiation. Similarly, none of the six saline-injected controls showed spermiation response.

Goldfish. The spermiation response of goldfish is shown in Table 2. Seven out of the eight SGA-injected goldfish showed spermiation response. Four out of the seven responding fish showed a strong response ( + +). Two successive injections of SGA resulted in an increase in the values of GSI and water content (P < 0.05). Injec- tion of 17cu,20@diOHprog induced spermiation in eight out of nine fish, although a strong response was observed in only one fish. Similar effects were seen in fish injected with 17a-hydroxyprogesterone. Two out of the seven 1 l-ketotestosterone-in- jetted and one out of the nine testosterone- injected fish showed spermiation responses, but the responses were relatively weak. No spermiation was observed in the control fish.


N N N N N N

Time Course Changes in Serum Steroid Hormone Levels of Amago Salmon after SGA Injection

Time course changes in serum 17a,20@ diOHprog and 1 1-ketotestosterone levels after the saline or SGA injection are presented in Fig. 1. All seven SGA-injected fish showed strong spermiation response on Day 2, and the response continued until Day 6. Spermatocrit values of the SGA-in- jetted fish were 13.0 of: 2.3% on Day 2 and 11.2 ? 3.3% on Day 6. None of the saline- injected fish showed any sign of spermiation throughout the experimental period. Testicular water content was not significantly different between the SGA-injected (81.4 + 0.9%) and saline-injected (79.9 * 0.6%) groups on Day 6.

Serum levels of 1 1-ketotestosterone and 17o,20P-diOHprog on Day 0 were 17.7 + 1.3 and 1.0 ? 0.2 rig/ml, respectively (Fig. 1). SGA injection resulted in significant increases in both 17o,2O@diOHprog and ll- ketotestosterone levels. A surge in serum 17q20P-diOHprog (65.7 -+ 5.5 rig/ml) was observed on Day 2, and thereafter the levels rapidly fell to 22.8 +- 4.4 rig/ml on Day 4 and to 6.0 + 1.1 rig/ml on Day 6 when

Xl0 f .a0 ,b z .60 P e .40 “t 3 P

.20 2 t = i 0 2 4 6

Days alter inje3ion

FIG. 1. Influence of a single injection of chum salmon gonadotrophin (SGA) on serum concentrations of 1 I-ketotestosterone and 17a,20@-dihydroxy-4- pregnen-3-one in nonspermiating amago salmon.


the levels were not significantly different from those of the saline-injected group. Similarly, serum 1 1-ketotestosterone levels peaked on Day 2 (85.6 + 9.2 &ml), but remained at high levels throughout the re- mainder of the experimental period. Saline injection had no significant effect on serum 1 1-ketotestosterone or 17cw,ZOl3-diOHprog levels over the course of the experimental period.

DISCUSSION

Treatment of intact nonspermiating amago salmon and goldfish with partially purified chum salmon gonadotrophin was very effective in inducing precocious spermiation. This confirms earlier reports that injections of pituitary extracts or various gonadotrophins stimulate spermiation in several teleosts (Billard et al., 1982). In both amago salmon and goldfish, gonadotrophin injection increased both GSI and testicular water content. This GSI increase seems more likely to result from increase in testicular water content. In amago salmon, gonadotrophin also decreased spermatocrit. These results together suggest that gonadotrophin-induced spermiation in these species involves testicular hy- dration and resultant thinning of semen.

It is generally assumed that, in teleosts, exogenous gonadotrophin does not act directly to induce spermiation, but works in concert with testicular somatic elements to stimulate the production of steroidal mediator(s). In this connection, it is of great interest to note that in amago salmon treatment of chum salmon gonadotrophin caused a dramatic increase in serum 17o,20@diOHprog levels, providing evidence to suggest that 17cll,2O@diOHprog is a steroidal mediator of gonadotrophin-induced spermiation.

There is now much evidence to suggest that 17a,20@diOHprog is the natural steroidal mediator of final oocyte maturation in female salmonid species (Fostier et al., 1983; Goetz, 1983; Nagahama et al., 1984).

However, up to recent years there has been no suggestion as to its function in males. In our previous studies with amago salmon, plasma levels of 17ol,20@diOHprog were found to be very low from June to Sep- tember but rapidly increased in October, concomitant with the period of spermiation (Ueda et al., 1983). The close association between high blood levels of 17o,20P-di- OHprog and spermiation has also been reported in rainbow trout (Scott and Baynes, 1982) and chum salmon (Ueda et al., 1984). Based on these results, we have suggested that 17a,20@diOHprog is involved in the process of spermiation in salmonids (Na- gahama et al., 1984). This suggestion is strongly supported by the present study, demonstrating that injections of 1701,2Op- diOHprog were effective in inducing in vivo spermiation in both amago salmon and goldfish. This is the first evidence showing the ability of 17or,20@diOHprog to induce spermiation. Scott and Baynes (1982) reported that injections of 17ol,20@diOHprog into spermiating rainbow trout significantly raised the K+/Na+ ratio of the seminal fluid. It has been reported that in hypophysectomized goldfish, progesterone is more effective in maintaining spermiation than testosterone or methyltestosterone (Bil- lard, 1976). In the present study, however, the total volume of semen was not measured. Nevertheless, it is evident from our results on the spermiation assay that the volume of semen collected in males injected with 17a,20@diOHprog was smaller than that with SGA. Scott and Baynes (1982), using spermiating rainbow trout, reported no effect of injections of 17a,20@ diOHprog on the volume of milt or the sperm count. Thus, the possibility remains that some other steroids besides 17a,20@ diOHprog may also have acted as a testicular mediator(s) of gonadotrophin-induced spermiation in amago salmon.

In the present study with goldfish, 17~ hydroxyprogesterone was found to be effective, almost equally with 17a,20&di-


OHprog, in inducing spermiation. Our in vitro studies (Ueda ef al., 1983) and those of others (Arai and Tamaoki, 1967) have shown that 17cu-hydroxyprogesterone can be converted to 17a,20@-diOHprog by testicular tissue of rainbow trout. It is therefore possible that the activity of 17a-hydroxyprogesterone is due in part to its con- version to 17ol,20P-diOHprog rather than through direct stimulation.

In the present study, treatment with ll- ketotestosterone or testosterone was found to be slightly effective in inducing spermiation in goldfish, but responses of amago salmon to these hormones were completely negative. At this time, it is not known whether the differences in spermiation response to androgen treatment observed between amago salmon and goldfish represent species specificities in the steroid hormone spermiation responses. Androgens have been reported to be potent spermiation in- ducers in goldfish (Yamazaki and Don- aldson, 1969; Billard et al., 1982). Among them, 11-ketotestosterone was found to be the most effective steroid (Yamazaki and Donaldson, 1969). However, the spermiation response of goldfish to androgens obtained in the present study appears to be much weaker than those reported by Ya- mazaki and Donaldson (1969). Possibly the differences in the preexisting conditions of the testes may explain these differences in responses. Another consideration is the dose of the hormones used in the present study. The use of a single dose of 1 pg for each steroid may not be adequate to deter- mine the relative potency of the hormones in the induction of spermiation.

In amago salmon, treatment of gonadotrophin caused a quick increase in serum levels of 1 I-ketotestosterone, and high levels of this steroid were maintained for 6 days. However, positive spermiation response observed after gonadotrophin treatment does not seem to be associated with this increase, since injections of 1 l-ketotestosterone or testosterone were not effective

in inducing spermiation. Nonetheless, it is possible that 11-ketotestosterone plays a role in enhancing or facilitating the efficacy of gonadotrophin to induce spermiation.

Temporal changes in serum levels differ between 11-ketotestosterone and 17a,20@ diOHprog; in the former case, levels are sustained at an increased level, while in the latter case, a short peak occurs and disap- pears. Our recent data (Ueda et al., unpub- lished data) on changes in serum ll-ketotestosterone levels during sexual maturation in male amago salmon showed that levels of this hormone in mid-September were relatively high (26 rig/ml). In contrast, the plasma levels of 17o,20l%diOHprog during this period were found to be almost nondetectable (Ueda et al., 1983). In fact, in the present study, initial levels of ll-ketotestosterone (18 &ml) were much higher than those of 17a,20@diOHprog (1 &ml). These results suggest that the testis at this stage has a capacity for nearly exclusive 1 l- ketotestosterone production. Thus, observed differences in time course changes of steroid hormone levels after gonadotrophin treatment may be related to the pre- dominance of the biosynthetic pathway for 11-ketotestosterone at this stage.

In summary, the present study with nonspermiating amago salmon and goldfish clearly demonstrates that chum salmon gonadotrophin or 17a,20l%diOHprog induces precocious spermiation l-2 months prior to the normal spermiation period. Further- more, treatment with chum salmon gonadotrophin quickly and dramatically increases serum levels of 17a,20@-diOHprog. These results strongly suggest that 17o,20@di- OHprog acts as a testicular mediator of gonadotrophin-induced spermiation in amago salmon. However, the results obtained in the present study, based on in viva experiments with intact fish, should be viewed with caution. The possibility remains that the secretion of endogenous steroid hormones resulting from exogenous application of hormones may have contributed to


the observed responses. The development salmon (Oncorhynchus rhodurus) ovarian folli-

of an in vitro assay for spermiation would cles: Role of the thecal and granulosa cells. Gen.

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here, and to further understand the hor- Nagahama, Y., Young, G., Ueda, H., Kagawa, H., and

Adachi. S. (1985). Endocrine control of final aa- , monal mechanisms involved in the sper- mete maturation in salmonids. In “Proceedings, miation in teleosts. 1st Int. Symp. on Salmonid Reprod.,” in press.

Ng, T. B., and Idler, D. R. (1980). Gonadotropic reg- ulation of androgen production in flounder and

ACKNOWLEDGMENTS We thank Dr. C. W. Walker for reading the manu-

script and Mr. F. Tashiro, Director of the Gifu Prefec- tural Fisheries Experimental Station, for providing amago salmon. This research has been supported in part by Grant-in-Aid for Special Project Research (Project 58119008) and Scientific Research from the Ministry of Education, Science, and Culture.

salmonids. Gen. Comp. Endocrinol. 42, 25-38. Scott, A. P., and Baynes, S. M. (1982). Plasma levels

of sex steroids in relation to ovulation and spermiation in rainbow trout (Sa/mo gairdneri). In “Reproductive Physiology” (C. J. J. Richter and H. J. Th. Goes, eds.), pp. 103-106. Center for Agricultural Publishing and Documentation, Wag- eningen, The Netherlands.

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involvement of gonadotrophin and steroid hormones in spermiation in the amago salmon, oncorhynchus...

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