Fish Physiology and Biochemistry vol. 7 nos 1-4 pp 147-155 (1989) Kugler Publications, Amsterdam/Berkeley
Relationship between metabolic and reproductive hormones in salmonid fish Walton W. Dickhoffl' 3, Liguang Yan l , Erika M. Plisetskaya 2 , Craig V. Sullivanl*, Penny Swanson1 , Akihiko Hara 4 and Melinda G. Bernard', 3 ISchool of Fisheriesand 2 Department of Zoology, University of Washington, Seattle, WA 91895, USA 3 Northwest and Alaska FisheriesCenter, NationalMarine FisheriesService, 2725 Montlake Boulevard East, Seattle, WA 98112, USA 4 Faculty of Fisheries, Hokkaido University, Hakodate, Japan Keywords: reproductive cycle, metabolism, estrogen, vitellogenin, insulin, thyroid hormones, salmon, ovulation, gonadotropin, eggs
Abstract Circulating concentrations of estradiol (E2), vitellogenin (VTG), thyroxine (T4 ), triiodothyronine (T3) and insulin were measured in reproductively maturing four and five year-old Atlantic salmon. Blood samples were collected from the fish in seawater for one year prior to their spawning in November in fresh water. In females, E2 and VTG were low but detectable from December to July, and then increased to peak levels in September and October. Plasma levels of T4 and T3 were relatively constant in winter and spring, and decreased in July. Plasma concentration of T4 increased in November when the fish returned to fresh water. Plasma T 3 levels remained low during the autumn. Both T4 and T3 levels tended to be higher in males than in females during September through November. Plasma insulin concentrations increased during the spring to peak values in May, and then decreased in June and July in fish of both sexes. There was a significant elevation of plasma insulin in males during October, and the levels in males tended to be higher than those found in females during final maturation.
Introduction It is common knowledge among both researchers and fish culturists that the energetic and nutritional requirements of reproductively maturing fish increase to accommodate the process of gametogenesis. Studies of salmonid fish show that restriction of ration or alteration in diet formulation may affect variously, fecundity, egg size, egg fertilizability, survival of offspring and the proportion of maturing adults (Hardy 1985). Unquestionably, the endocrine system plays a pivotal role in the metabolic shifts and reproductive performance of maturing fish. Although our knowledge of the functions of reproductive hormones in maturing salmonids is
expanding, the roles of metabolic hormones in reproduction are comparatively less well known (metabolic hormones are considered in this context to include corticosteroid, thyroid and pancreatic hormones). Theoretically, metabolic hormones could influence reproductive performance by several non-exclusive means. For example, they may regulate anabolic and catabolic metabolism to guarantee that sufficient energy is stored, and then mobilized when needed. In addition, it is possible that metabolic hormones may directly affect the gonad to regulate rates of gametogenesis either in a singular fashion or acting synergistically with reproductive hormones. There is evidence of the importance or possible
* Present address: Department of Zoology, North Carolina State University, Raleigh, NC 27695-7617, USA
148 roles of metabolic hormones in fish reproduction. Activation of the interrenal tissue in maturing salmonids has been clearly demonstrated (Robertson and Wexler 1959; Hane and Robertson 1959, Idler et al. 1959, Phillips et al. 1959; Pickering and Christie 1981; McBride et al. 1986). The elevated blood cortisol is undoubtedly important for mobilization of energy stores (protein catabolism, lipolysis, gluconeogenesis) during final maturation. In some salmonid species, blood concentration of thyroid hormones show complex patterns near the time of spawning (White and Henderson 1977; Osborn et al. 1978; Leatherland and Sonstegard 1980; Pickering and Christie 1981; Sower and Schreck 1982; Biddiscombe and Idler 1983; Ueda et al. 1984; Cyr et al. 1988). There is evidence that thyroid hormones may synergize with gonadotropin to promote ovarian steroidogenesis in goldfish (Hurlburt 1977), carp (Epler and Bieniarz 1983), freshwater perch (Sen and Bhattacharya 1981) and rainbow trout (Cyr and Eales 1988). There appear to be seasonal changes in circulating insulin in fish (Plisetskaya et al. 1976; Murat et al. 1981; Sower et al. 1985; Gutierrez et al. 1987, 1988) that may have some relationship to reproduction either in regard to control of intermediary metabolism or direct action on the gonad analogous to their action in mammals (Poretsky and Kalin 1987). We present here a partial summary of some recent work from our laboratory on the seasonal changes in blood levels of thyroid hormones and insulin in reproductively maturing salmonids, and we suggest possible roles of these hormones in reproduction.
Materials and methods For the analysis of seasonal changes of hormones in maturing adults, virgin Atlantic salmon (Salmo salar) were maintained in floating seawater net-pens at the National Marine Fisheries Service field station at Manchester, WA. The fish were obtained originally as eyed eggs from the U.S. Fish and Wildlife Service (Connecticut River stock 1981 brood year). They were fed three times daily with a standard ration of formula II Oregon Moist Pellets (Moore Clark Inc.). Mean surface water tempera-
tures ranged from 8C (February) to 130 C (August). Two separate groups of fish were sampled. A group of four year-old maturing fish were sampled from August to November, 1986. Mature males and females were transferred to freshwater tanks on Nov. 7 for spawning (between Nov. 10 and Nov. 21, 1986). A second group of fish (five years old) was maintained in seawater for blood sampling from November, 1986 through September, 1987. All fish were individually identified by passive integrating transponder (PIT) tags. Blood samples were taken every three (4 yr olds) or every six (5 yr olds) weeks from fish anesthetized with 0.05 % tricaine methanesulfonate. The plasma fraction was stored in plastic vials at -20°C until assayed. Plasma vitellogenin concentrations were determined by single radial immunodiffusion (Mancini et al. 1965; Hara 1978; Ueda et al. 1984). In this assay, the antiserum raised in rabbits against coho salmon (Oncorhynchus kisutch) egg protein I (Hara and Dickhoff, unpublished) specifically recognizes vitellogenin. Purified Atlantic salmon vitellogenin was used as standard. The minimal detectable plasma concentration of vitellogenin using this assay was at least 100 ng/5 t1d (equivalent to 20 ftg/ml). For estradiol-17/3 (E2 ) determination, plasma was extracted with dichloromethane and extracts were subjected to partition chromatography (Wingfield and Farner 1975). The concentration of E2 was determined by radioimmunoassay (RIA) as described by Sower and Schreck (1982) with slight modification. Plasma thyroxine (T4 ) and triiodothyronine (T3) were determined by RIA as described by Dickhoff et al. (1978, 1982), and plasma insulin concentrations were measured by RIA using anti-coho salmon insulin serum and coho salmon standard and label as described by Plisetskaya et al. (1986). For the study on rainbow trout, virgin 2-year-old female rainbow trout (Salmo gairdneri) were obtained from the University of Washington experimental fish hatchery during their normal spawning season (February, 1987). The fish were checked for maturational state, and those with oocytes in the peripheral germinal vesicle stage were killed and
149 their ovaries were removed for incubation. Groups of about 10 oocytes were dissected from the ovaries. Batches of 30 to 40 oocytes were incubated in flasks in 6 ml of trout balanced saline (Kagawa et al. 1982) either alone or with 0 to 1 tg/ml of a gonadotropin fraction from Sephadex chromatography (SG-G100; Donaldson et al. 1972) and 0 to 500 ng/ml triiodothyronine. Incubations were done in duplicate at 16°C for up to four days in a incubator flushed with 99% 02: 1% CO 2 . At the end of incubation, the oocytes were removed and checked for germinal vesicle breakdown (Jalabert 1976). Incubation medium was centrifuged and supernatants were stored at -80°C until assayed by RIA for E2, androgens (Sower and Schreck 1982) and 17a-203dihydroxy-4-pregnen-3-one (DHP; Scott et al. 1982) concentration.
40
40
-4
D
E
a E2 *-VTG
3
303 3
.f 20
W 10
I
I Dec Jan Feb
I I I Mar Apr May Jun Jul Aug Sep
O
Oct Nov
Fig. 1. Plasma concentrations of estradiol (E2) and vitellogenin (VTG) in 4-year-old (July to November) and 5-year-old (November to July) female Atlantic salmon. Samples were taken from November 1986 to October 1987 from fish in seawater net-pens. Samples taken during November 1987 were from fish in fresh water. All fish ovulated between Nov. 10 to Nov. 20, 1987. Symbols represent mean values of 7 to 19 fish; brackets indicate + one standard error.
20
Results Atlantic salmon The changes in plasma concentrations of estradiol (E2) and vitellogenin (VTG) of the Atlantic salmon females are shown in Fig. 1. The data from the two groups of fish are combined and presented together to facilitate comparisons. Both VTG and E 2 were detectable in the plasma of females, but not males, throughout the sampling period. They increased to levels that were significantly higher (ANOVA, p < 0.05) than initial levels by August (E2) or September (VTG). Both VTG and E2 decreased significantly during the period of ovulation in November. Blood plasma concentrations of both thyroxine (T4) and triiodothyronine (T3 ) were determined in male and female Atlantic salmon during the year prior to maturation (Fig. 2). Plasma T4 levels in males remained at around 10 ng/ml from November 1986 to July 1987, and then decreased in August. Plasma T4 increased in September when the males began maturing as indicated by elevated plasma testosterone and 11-oxotestosterone (data not shown). The highest plasma T4 levels in males were observed in fish in fresh water (November 1987). During the early part of the year, plasma T4 tended to be higher in females compared to males, but
E
'9di0
Dec Jan
Feb Mar Apr May Jun
Jul Aug Sep
Oct Nov
Fig. 2. Plasma concentrations of thyroxine (T4 ) in 4- and 5-year-old female Atlantic salmon as in Fig. 1.
the differences were not statistically significant. Peak T4 levels in previtellogenic females occurred in early July. T4 levels declined in females in August and then increased in November when the fish were returned to fresh water. Plasma T 3 levels in male and female fish were similar from November 1986 through June 1987 (Fig. 3). At various times during the spring, plasma T 3 was significantly elevated from initial levels. Plasma T3 remained elevated in males, but declined in females, during the period from June to August. In both males and females T3 concentrations were at their lowest levels during maturation in the autumn. However, the sexual difference (higher T3 in males compared to females) was maintained throughout maturation. Insulin levels in the plasma were similar and con-
150 - Male 10E
8
I 624-
1_4;~4 0
I
I
I
Dec Jan
I
I
I
I
Feb Mar Apr May Jun
I
I
I
Jul Aug Sep
l
Oct
i
Nov
Fig. 3. Plasma concentrations of triiodothyronine (T3 ) in 4- and 5-year-old female Atlantic salmon as in Fig. 1. 40
e Male + Female
E . 30
S
20
._ a
lO
O' Nov
l
l Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct
Nov
Fig. 4. Plasma concentrations of insulin in 4- and 5-year-old female Atlantic salmon as in Fig. 1.
stant in males and females from November 1986 through February 1987 (Fig. 4). Plasma insulin increased during the spring to reach peak levels by the beginning of May, and then declined to basal levels by July. Mean concentration of insulin at the peak was higher in females compared to males, but the difference was not statistically significant. During maturation, mean plasma insulin was always higher in males compared to females, and a significant increase was observed in males, but not in females, during October.
Rainbow trout The changes of thyroid hormones that are often observed in maturing salmonids inspired a test of their possible role in final maturation of the ovary. Results from the incubation of oocytes with or without varying concentrations of T3 or SG-G100 are shown in Table 1. Germinal vesicle breakdown (GVBD) was not observed spontaneously or in response to the gonadotropin preparation (SG-G 100) at concentrations of 0.1 and 1 ng/ml. The SG-G100
was effective in stimulating GVBD at concentrations of 10, 100 and 1,000 ng/ml. At the 10 ng/ml concentration of SG-G100, significantly greater GVBD was observed when T3 was present in the medium at concentrations of 5 or 50 ng/ml compared to T 3 concentrations of 0 or 0.5 ng/ml. The combination of T3 at 500 ng/ml and SG-G100 at 10 ng/ml significantly reduced GVBD compared to SG-G100 alone. No effect of T3 was observed at higher or lower concentrations of SG-G100. The effect of the combinations of SG-G100 and T3 on steroidogenesis agreed with results of GVBD (Table 1). The concentrations of E 2 in the medium were lower and the concentrations of androgen were higher in those incubations which contained concentrations of SG-G100 that were effective in inducing GVBD (Table 1). The levels of 17a,203dihydroxy-4-pregnen-3-one (DHP) were low or nondetectable in medium from oocyte incubations in which GVBD was not observed, i.e., those containing 1 ng/ml SG-G100 or less. The calculated secretion rate of DHP was above 10 pg/oocyte/hr for incubations containing 10 ng/ml of SG-G100, and above 100 pg/oocyte/hr for the higher concentrations of SG-G100. There was a significant linear relationship (P < 0.01) between the DHP secretion rate and T3 concentration for incubations containing SG-G100 at 10 ng/ml, suggesting that T3 acted through stimulation of DHP secretion.
Discussion The analysis of seasonal changes in plasma levels of thyroid hormones and insulin in adult Atlantic salmon indicate that these hormones are elevated in both males and females during the spring before the onset of reproductive maturation. Plasma concentrations of the hormones decrease during the summer just prior to exogenous vitellogenesis in females. Sexual differences in the plasma concentrations of the hormones are apparent at some times during final maturation in the autumn. Changes in thyroid hormones during maturation have been observed in studies of several species of salmonids. Such studies have been conducted on wild, migrating and captive stocks of fish. In Great
151 Table 1. Percent germinal vesicle breakdown and follicular secretion rate of estradiol, androgen and DHP (parentheses) without triiodothyronine (T3) for 72 hours. T3 Concentration (ng/ml) SG-G100 (ng/ml)
0
0.5
5
50
500
0
0 (2.3, 56, 0.2) 0 (2.4, 54, 0.5) 0 (2.1, 101, 1.0) 13 + 10 (1.1, 376, 18.7) 55 + 14 (1.1, 641, 254.4) 57 + 18 (1.0, 424, 211.6)
0 (3.0, 45, ND) 0 (1.7, 40, 0.1) 0 (2.3, 87, ND) 18+13 (1.6, 430, 15.7) 64 + 16 (1.3, 645, 247.1) 61 + 14 (1.0, 536, 372.3)
0 (2.7, 31, ND) 0 (2.4, 46, ND) 0 (1.6, 62, ND) *32± 16 (1.2, 396, 38.5) 60+ 15 (1.3, 849, 321.6) 58 + 15 (1.0, 530, 323.0)
0 (2.9, 47, ND) 0 (3.1, 64, ND) 0 (2.6, 47, 0.8) *38 + 14 (1.1, 377, 35.8) 55 15 (1.2, 696, 319.6) 50 + 14 (1.1, 607, 315.2)
0 (2.7, 40, ND) 0 (2.3, 56, ND) 0 (1.9, 150, ND) *3+6 (1.4, 347, 10.5) 62 + 19 (1.1, 736, 272.5) 43 20 (1.3, 1074, 246.1)
0.1 1 10 100 1000
2, Asterisks indicate values which are significantly different from control values (without T3; X p < 0.05); follicular secretion rate is expressed as pg/oocyte/h; steroid assays were run in duplicate. ND = nondetectable.
Lakes coho salmon (Oncorhynchus kisutch), Leatherland and Sonstegard (1980) found higher plasma levels of T4 and T3 in spring before maturation compared to those found during maturation in autumn. Decreases in plasma T4 and/or T3 have been reported during the seawater to freshwater transition, or during upstream migration and maturation of wild coho salmon (Sower and Schreck 1982), sockeye salmon (0. nerka; Biddiscombe and Idler 1983) and chum salmon (0. keta; Ueda et al. 1984). Decreases in T4 and T3 as spawning approached were reported for non-anadromous brook trout (Salvelinusfontinalis;White and Henderson 1977) and rainbow trout (Salmo gairdneri; Osborn et al. 1978). In captive salmonids, an increase in T4 during the time of ovulation has been reported for brown trout (Salmo trutta; Pickering and Christie 1981). In rainbow trout on regulated photoperiods, Cyr et al. (1988) found high plasma levels of T4 and T3 in the previtellogenic phase; both hormones decreased as maturation proceeded, and then increased soon after spawning. Our results with Atlantic salmon agree with most published studies on other salmonids in that thyroid hormones are elevated before vitellogenesis and then decrease around the time when E 2 and vitellogenin levels are known to rise. Likewise, our finding of a
decrease in plasma T3 during ovulation and spermiation is in agreement with published reports on other species. In contrast, there appears to be some variability in reports on changes in T4 during spawning. Our finding of an increase in T4 during spawning of Atlantic salmon agrees with the report on the congeneric brown trout (Pickering and Christie 1981). A significant elevation in T4 during spermiation was also reported for sockeye salmon (Biddiscombe and Idler 1983). Comparison of these results are complicated by several factors including different species and stock, feeding or fasting status, and length of time in fresh water (migratory fish), among others. Sexual differences in thyroid hormone levels have been reported for maturing fish in several instances. Our finding of generally higher T4 and T3 in maturing Atlantic salmon males compared to females is similar to that reported for coho salmon (Sower and Schreck 1982) and sockeye salmon (Biddiscombe and Idler 1983). In contrast, T4 was found to be higher in maturing female brown trout (Pickering and Christie 1981). Ueda et al. (1984) found no difference in T4 and T3 comparing maturing male and female chum salmon. The basis for this sexual difference or lack of it is not apparent. It has been suggested that E 2 may be responsible
152 for low T 3 in maturing female salmonids, since E2 administration depresses plasma T3 levels in rainbow trout (Leatherland 1985; MacLatchy et al. 1986), and reduces the activity of deiodinase in rainbow trout hepatocytes (Shields and Eales 1986). However, the disagreement in the literature regarding sexual differences in thyroid hormone levels suggests that this mechanism may not be operable in all salmonid species, or it may be over-ridden by other factors in some situations. A role for thyroid hormones in final maturation of salmonids is clearly suggested by the results of our in vitro study on germinal vesicle breakdown. The combination of SG-G100 at 10 ng/ml and T3 at 5 or 50 ng/ml resulted in a higher incidence of GVBD in incubated oocytes when compared with SG-G100 alone. This concentration of SG-G100 and the lower concentration of T 3 are near the physiological range of plasma concentrations observed during final maturation (Fostier et al. 1978; Scott et al. 1983; Dye et al. 1986). The fact that T3 alone had no effect on GVBD or steroid production indicates a synergistic role of thyroid hormones in ovulation. Our data also suggests that this synergistic effect of T 3 can be surmounted by concentrations of SG-G100 as high as 100 ng/ml. In other words the synergistic action of T3 on ovulation could be made physiologically insignificant by a large preovulatory surge of gonadotropin. Nevertheless, the possibility of a contributing role of thyroid hormones in spawning cannot be ignored. A synergistic effect of thyroid hormones acting in conjunction with gonadotropin to induce ovulation has been reported for goldfish (Carassiusauratus; Hurlburt 1977) and carp (Cyprinus carpio; Epler and Bieniarz 1983). Thyroid hormones may help oocytes of sturgeon (Acipenser stellatus)retain the capacity to respond to gonadotropin under adverse conditions of temperature (Dettlaff and Davydova 1979). Cyr and Eales (1988) have demonstrated a synergistic effect of T 3 on gonadotropininduced E2 production by rainbow trout ovaries in vitro. Furthermore, putative thyroid hormone receptors have been identified in a freshwater perch (Anabas testudineus; Chakraborti et al. 1986). Thus, evidence for a role of thyroid hormones in
several phases of fish reproduction is accumulating. The significant elevation in plasma insulin that we observed in Atlantic salmon during the spring prior to onset of maturation can be compared with similar changes reported for scorpion fish (Scorpaena; Plisetskaya et al. 1976) and sea bass (Dicentrarchus labrax; Gutierrez et al. 1987). Plasma insulin was higher before than during spawning of scorpion fish. Using an RIA based on mammalian insulin, Gutierrez et al. reported an increase in insulin in sea bass plasma during the prespawning period; plasma insulin decreased during spawning. In contrast to these observations in sea bass, in Atlantic salmon plasma insulin decreased before the increases in E 2 and vitellogenin and did not show an additional decrease during spawning. A vernal surge in plasma insulin may be an annual event in salmonids since it has also been observed in juvenile coho salmon prior to the parr to smolt transformation (Plisetskaya et al. 1988). In Atlantic salmon, the generally higher mean level of plasma insulin in males compared to females is similar to the sexual difference in plasma insulin observed in pink salmon (0. gorbuscha) during their spawning migration (Plisetskaya et al. 1987). However, the results from the study on pink salmon insulin and our study differ in that during the up-river migration of pink salmon there was a significant transient elevation of insulin in females, whereas we observed a significant transient elevation of insulin in males transferred to fresh water. The higher levels of insulin in the Atlantic salmon compared to the pink salmon may have been due to the fact that the Atlantics were fed throughout their captivity in net-pens whereas the pink salmon had ceased feeding during their upstream migration (McBride et al. 1986). The patterns of change in plasma concentration of metabolic hormones invite speculation on their possible function either in controlling reproduction or the energetics of maturation. It has been shown that insulin may promote the uptake of vitellogenin into growing oocytes of rainbow trout in vitro (Tyler et al. 1987). Although plasma insulin levels in the Atlantic salmon females tended to be lower than in males and insulin was at its highest levels before vitellogenesis, it is possible that normal levels
153 of insulin are necessary for many aspects of reproduction. The surge of plasma insulin in the spring may function to increase metabolic stores in anticipation of maturation. Energy stored as lipid or protein are needed for maintenance of activity and gametogenesis when the salmon stop feeding near the time of spawning. During maturation there is a switch from somatic to gonadal growth. Somatic weight of maturing coho salmon has been shown to stop increasing in July-August, and decrease thereafter to end up with a 37%7o loss (dry weight) at spawning in December (Hardy et al. 1984). Growth of the ovary continues while somatic growth has stopped. Even after feeding has ceased, the ovary continues to incorporate protein and lipid. Therefore, the nutrients deposited in the ovary must originate in maternal tissues. It has been estimated that 7% to 8°7% of the lipid loss from somatic tissues is transferred to the ovaries in maturing coho (Hardy et al. 1984) and sockeye (Idler and Bitners 1960) salmon. In Atlantic salmon, the high levels of plasma insulin in April and May may increase protein and lipid storage, whereas the decreasing plasma insulin in July along with maintained high levels of thyroid hormones will promote lipolysis. The reason for the relatively higher levels of plasma insulin and thyroid hormones in males compared to females during final maturation is not entirely clear. We could speculate that these two hormones may be acting to guard against early depletion of energy reserves in males. The need for maintaining energy reserves in males may be illustrated by the mating process. When female salmon arrive on the spawning grounds, they dig a redd, and then release all of their eggs at once. In contrast, male salmon must be on the spawning grounds when the females arrive. The males defend their territory by displays and fights with other males. They may spawn with one female, and then remain on the grounds for another reproductive opportunity.
Acknowledgements This work was supported by grants from the National Science Foundation (DCB-8615521), the
U.S. Department of Agriculture (S/E-Aq) under agreement # 85-CRSR-2-2603 and Washington Sea Grant Project R/A 49. We thank F. William Waknitz, Conrad V.W. Mahnken, Tom Flagg and Earl F. Prentice of the National Marine Fisheries Service for their assistance in this work.
References cited Biddiscombe, S. and Idler, D.R. 1983. Plasma levels of thyroid hormones in sockeye salmon (Oncorhynchus nerka) decrease before spawning. Gen. Comp. Endocrinol. 52: 467-470. Chakraborti, P., Maitra, G. and Bhattacharya, S. 1986. Binding of thyroid hormone to isolated ovarian nuclei from a freshwater perch, Anabas testudineus. Gen. Comp. Endocrinol. 62: 239-246. Cyr, D.G., Bromage, N.R., Duston, J. and Eales, J.G. 1988. Seasonal patterns in serum levels of thyroid hormones and sex steroids in relation to photoperiod-induced changes in spawning time in rainbow trout, Salmo gairdneri.Gen. Comp. Endocrinol. 69: 217-225. Cyr, D.G. and Eales, J.G. 1988. In vitro effects of thyroid hormones on gonadotropin-induced estradiol-170 secretion by ovarian follicles of rainbow trout, Salmo gairdneri. Gen. Comp. Endocrinol. 69: 80-87. Dettlaff, T.A. and Davydova, S.I. 1979. Differential sensitivity of cells of follicular epithelium and oocytes in the stellate sturgeon to unfavorable conditions, and correlating influence of triiodothyronine. Gen. Comp. Endocrinol. 39: 236-243. Dickhoff, W.W., Folmar, L.C. and Gorbman, A. 1978. Changes in plasma thyroxine during smoltification of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 36: 229-232. Dickhoff, W.W., Folmar, L.C., Mighell, J.L. and Mahnken, C.V.W. 1982. Plasma thyroid hormones during smoltification of yearling and underyearling coho salmon and yearling chinook salmon and steelhead trout. Aquaculture 28: 39-48. Donaldson, E.M., Yamazaki, F., Dye, H.M. and Philleo, W.W. 1972. Preparation of gonadotropin from salmon (Oncorhynchus tshawytscha) pituitary glands. Gen. Comp. Endocrinol. 18: 469-481. Dye, H.M., Sumpter, J.P., Fagerlund, U.H.M. and Donaldson, E.M. 1986. Changes in reproductive parameters during the spawning migration of pink salmon, Oncorhynchus gorbuscha (Walbaum). J. Fish Biol. 29: 167-176. Epler, P. and Bieniarz, K. 1983. Role of triiodothyronine and some gonadotrophic and steroid hormones on the maturation of carp (Cyprinus carpio L.) oocytes in vitro. Reprod. Nutr. Dev. 23: 1011-1017. Fostier, A., Weil, C., Terqui, M., Breton, B. and Jalabert, B. 1978. Plasma estradiol-173 and gonadotropins during ovulation in rainbow trout (Salmo gairdneriR.). Ann. Biol. Anim. Biochem. Biophys. 18: 929-936.
154 Gutierrez, J., Fernandez, J., Carillo, M., Zanuy, S. and Planas, J. 1987. Annual cycle of plasma insulin and glucose of sea bass, Dicentrarchus labrax L. Fish Physiol. Biochem. 4: 137-141. Hane, S. and Robertson, O.H. 1959. Changes in plasma 17-hydroxycorticosteroids accompanying sexual maturation and spawning of the Pacific salmon (Oncorhynchus tshawytscha) and rainbow trout (Salmo gairdneri). Proc. Nat. Acad. Sci. 45: 886-893. Hara, A. 1978. Sexual differences in serum proteins of chum salmon and the purification of female-specific serum proteins. Bull. Jap. Soc. Sci. Fish. 44: 689-693. Hardy, R.W., Shearer, K.D. and King, I.B. 1984. Proximate and elemental composition of developing eggs and maternal soma of pen-reared coho salmon (Oncorhynchus kisutch ) fed production and trace element fortified diets. Aquaculture 42: 147-165. Hardy, R.W. 1985. Salmonid broodstock nutrition. In Salmonid Reproduction. pp. 98-108. Edited by R.N. Iwamoto and S.A. Sower. Washington Sea Grant Program, Seattle. Hurlburt, M.E. 1977. Role of the thyroid gland in ovarian maturation of the goldfish Carassiusauratus L. Can. J. Zool. 55: 1906-1913. Idler, D.R., Ronald, A.P. and Schmidt, P.J. 1959. Biochemical studies on sockeye salmon during spawning migration. VII. Steroid hormone in plasma. Can. J. Biochem. Physiol. 37: 1227-1238. Idler, D.R. and Bitners, 1. 1960. Biochemical studies on sockeye salmon during spawning migration. IX. Fat, protein and water in the major internal organs and cholesterol in the liver and gonads of the standard fish. J. Fish. Res. Bd. Can. 17: 113-122. Jalabert, B. 1976. In vitro oocyte maturation and ovulation in rainbow trout (Salmo gairdneri), northern pike (Esox lucius) and goldfish (Carassiusauratus). J. Fish. Res. Bd. Can. 33: 974-988. Kagawa, B., Young, G. and Nagahama, Y. 1982. Estradiol-173 production in isolated amago salmon (Oncorhynchus rhodurus) ovarian follicles and its stimulation by gonadotropins. Gen. Comp. Endocrinol. 47: 361-365. Leatherland, J.F. 1985. Effects of 170-estradiol and methyl testosterone on the activity of the thyroid gland in rainbow trout, Salmo gairdneriRichardson. Gen. Comp. Endocrinol. 60: 343-352. Leatherland, J.F. and Sonstegard, R.A. 1980. Seasonal changes in thyroid hyperplasia serum thyroid hormone and lipid concentrations, and pituitary gland structure in Lake Ontario coho salmon, Oncorhynchus kisutch Walbaum and a comparison with coho salmon from Lakes Michigan and Erie. J. Fish Biol. 16: 539-562. MacLatchy, D.L., Cyr, D.G. and Eales, J.G. 1986. Estradiol173 depresses T4 to T3 conversion in rainbow trout. Am. Zool. 26: 106A (Abstract). Mancini, G., Varbona, A.O. and Neremans, J.F. 1965. Immunochemical quantitation of antigens by single radial im-
munodiffusion. Immunochem. 2: 235-254. McBride, J.R., Fagerlund, U.H.M., Dye, H.M. and Bagshaw, J. 1986. Changes in structure of tissues and in plasma cortisol during the spawning migration of pink salmon, Oncorhynchus gorbuscha (Walbaum). J. Fish Biol. 29: 153-166. Murat, J.C., Plisetskaya, E. and Woo, N.Y.S. 1981. Endocrine control of nutrition in cyclostomes and fish. Comp. Biochem. Physiol. 68A: 149-158. Osborn, R.H., Simpson, T.H. and Youngson, A.F. 1978. Seasonal and diurnal rhythms of thyroidal status in the rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 12: 531-540. Phillips, J.G., Holmes, W.N. and Bondy, P.K. 1959. Adrenocorticosteroids in salmon plasma (Oncorhynchus nerka). Endocrinology 65: 811-818. Pickering, A.D. and Christie, P. 1981. Changes in the concentrations of plasma cortisol and thyroxine during sexual maturation of the hatchery-reared brown trout, Salmo trutta L. Gen. Comp. Endocrinol. 44: 487-496. Plisetskaya, E.M., Leibush, B.N. and Bondareva, V. 1976. The secretion of insulin and its role in cyclostomes and fishes. In The Evolution of Pancreatic Islet. pp. 251-269. Edited by A. Grillo, L. Leibson and A. Epple. Pergamon Press, Oxford. Plisetskaya, E.M., Dickhoff, W.W., Paquette, T.L. and Gorbman, A. 1986. The assay of salmon insulin by homologous radioimmunoassay. Fish Physiol. Biochem. 1: 37-43. Plisetskaya, E.M., Donaldson, E.M. and Dye, H.M. 1987. Plasma insulin levels during the spawning migration of the pink salmon, Oncorhynchus gorbuscha. J. Fish Biol. 31: 21-26. Plisetskaya, E.M., Swanson, P., Bernard, M.G. and Dickhoff, W.W. 1988. Insulin in coho salmon (Oncorhynchus kisutch) during the parr to smolt transformation. Aquaculture 72: 151-164. Poretsky, L. and Kalin, M.F. 1987. The gonadotropic function of insulin. Endocr. Rev. 8: 132-141. Robertson, O.H. and Wexler, B.C. 1959. Hyperplasia of the adrenal cortical tissue in Pacific salmon (Genus Oncorhynchus) and rainbow trout (Salmo gairdneri) accompanying sexual maturation and spawning. Endocrinology 65: 225-238. Sen, S. and Bhattacharya, S. 1981. Role of thyroxine and gonadotropins on the mobilization of ovarian cholesterol in a teleost Anabas testudineus (Bloch). Ind. J. Exp. Biol. 19: 408-412. Scott, A.P. Sheldrick, E.L. and Flint, A.P.F. 1982. Measurement of 17a-20$-dihydroxy-4-pregnen-3-one in plasma of trout (Salmo gairdneri Richardson): Seasonal changes and response to salmon pituitary extract. Gen. Comp. Endocrinol. 46: 444-451. Scott, A.P., Sumpter, J.P. and Hardiman, P.A. 1983. Hormone changes during ovulation in the rainbow trout (Salmo gairdneri Richardson). Gen. Comp. Endocrinol. 49: 128134. Shields, C.A. and Eales, J.G. 1986. Thyroxine 5'-monodeiodinase activity in hepatocytes of rainbow trout, Salmo gairdneri:Distribution, effects of starvation, and exogenous
155 inhibition. Gen. Comp. Endocrinol. 63: 334-343. Sower, S.A. and Schreck, C.B. 1982. Steroid and thyroid hormones during sexual maturation of coho salmon (Oncorhynchus kisutch) in seawater or fresh water. Gen. Comp. Endocrinol. 47: 42-53. Sower, S.A., Plisetskaya, E.M. and Gorbman, A. 1985. Changes in plasma steroid and thyroid hormones and insulin during final maturation and spawning of the sea lamprey Petromyzon marinus. Gen. Comp. Endocrinol. 58: 259-269. Tyler, C., Sumpter, J. and Bromage, N. 1987. The hormonal control of vitellogenin uptake into cultured oocytes of the
rainbow trout. Proc. Third Int. Symp. Reprod. Physiol. Fish. p. 142, St. John's, Newfoundland. Ueda, H., Hiroi, O., Hara, A., Yamauchi, K. and Nagahama, Y. 1984. Changes in serum concentrations of steroid hormones, thyroxine, and vitellogenin during spawning migration of the chum salmon, Oncorhynchus keta. Gen. Comp. Endocrinol. 53: 203-211. White, B.A. and Henderson, N.E. 1977. Annual variations in the circulating levels of thyroid hormones in the brook trout, Salvelinus fontinalis, as measured by radioimmunoassay. Can. J. Zool. 55: 475-481.
Relationship between metabolic and reproductive hormones in salmonid fish Walton W. Dickhoffl' 3, Liguang Yan l , Erika M. Plisetskaya 2 , Craig V. Sullivanl*, Penny Swanson1 , Akihiko Hara 4 and Melinda G. Bernard', 3 ISchool of Fisheriesand 2 Department of Zoology, University of Washington, Seattle, WA 91895, USA 3 Northwest and Alaska FisheriesCenter, NationalMarine FisheriesService, 2725 Montlake Boulevard East, Seattle, WA 98112, USA 4 Faculty of Fisheries, Hokkaido University, Hakodate, Japan Keywords: reproductive cycle, metabolism, estrogen, vitellogenin, insulin, thyroid hormones, salmon, ovulation, gonadotropin, eggs
Abstract Circulating concentrations of estradiol (E2), vitellogenin (VTG), thyroxine (T4 ), triiodothyronine (T3) and insulin were measured in reproductively maturing four and five year-old Atlantic salmon. Blood samples were collected from the fish in seawater for one year prior to their spawning in November in fresh water. In females, E2 and VTG were low but detectable from December to July, and then increased to peak levels in September and October. Plasma levels of T4 and T3 were relatively constant in winter and spring, and decreased in July. Plasma concentration of T4 increased in November when the fish returned to fresh water. Plasma T 3 levels remained low during the autumn. Both T4 and T3 levels tended to be higher in males than in females during September through November. Plasma insulin concentrations increased during the spring to peak values in May, and then decreased in June and July in fish of both sexes. There was a significant elevation of plasma insulin in males during October, and the levels in males tended to be higher than those found in females during final maturation.
Introduction It is common knowledge among both researchers and fish culturists that the energetic and nutritional requirements of reproductively maturing fish increase to accommodate the process of gametogenesis. Studies of salmonid fish show that restriction of ration or alteration in diet formulation may affect variously, fecundity, egg size, egg fertilizability, survival of offspring and the proportion of maturing adults (Hardy 1985). Unquestionably, the endocrine system plays a pivotal role in the metabolic shifts and reproductive performance of maturing fish. Although our knowledge of the functions of reproductive hormones in maturing salmonids is
expanding, the roles of metabolic hormones in reproduction are comparatively less well known (metabolic hormones are considered in this context to include corticosteroid, thyroid and pancreatic hormones). Theoretically, metabolic hormones could influence reproductive performance by several non-exclusive means. For example, they may regulate anabolic and catabolic metabolism to guarantee that sufficient energy is stored, and then mobilized when needed. In addition, it is possible that metabolic hormones may directly affect the gonad to regulate rates of gametogenesis either in a singular fashion or acting synergistically with reproductive hormones. There is evidence of the importance or possible
* Present address: Department of Zoology, North Carolina State University, Raleigh, NC 27695-7617, USA
148 roles of metabolic hormones in fish reproduction. Activation of the interrenal tissue in maturing salmonids has been clearly demonstrated (Robertson and Wexler 1959; Hane and Robertson 1959, Idler et al. 1959, Phillips et al. 1959; Pickering and Christie 1981; McBride et al. 1986). The elevated blood cortisol is undoubtedly important for mobilization of energy stores (protein catabolism, lipolysis, gluconeogenesis) during final maturation. In some salmonid species, blood concentration of thyroid hormones show complex patterns near the time of spawning (White and Henderson 1977; Osborn et al. 1978; Leatherland and Sonstegard 1980; Pickering and Christie 1981; Sower and Schreck 1982; Biddiscombe and Idler 1983; Ueda et al. 1984; Cyr et al. 1988). There is evidence that thyroid hormones may synergize with gonadotropin to promote ovarian steroidogenesis in goldfish (Hurlburt 1977), carp (Epler and Bieniarz 1983), freshwater perch (Sen and Bhattacharya 1981) and rainbow trout (Cyr and Eales 1988). There appear to be seasonal changes in circulating insulin in fish (Plisetskaya et al. 1976; Murat et al. 1981; Sower et al. 1985; Gutierrez et al. 1987, 1988) that may have some relationship to reproduction either in regard to control of intermediary metabolism or direct action on the gonad analogous to their action in mammals (Poretsky and Kalin 1987). We present here a partial summary of some recent work from our laboratory on the seasonal changes in blood levels of thyroid hormones and insulin in reproductively maturing salmonids, and we suggest possible roles of these hormones in reproduction.
Materials and methods For the analysis of seasonal changes of hormones in maturing adults, virgin Atlantic salmon (Salmo salar) were maintained in floating seawater net-pens at the National Marine Fisheries Service field station at Manchester, WA. The fish were obtained originally as eyed eggs from the U.S. Fish and Wildlife Service (Connecticut River stock 1981 brood year). They were fed three times daily with a standard ration of formula II Oregon Moist Pellets (Moore Clark Inc.). Mean surface water tempera-
tures ranged from 8C (February) to 130 C (August). Two separate groups of fish were sampled. A group of four year-old maturing fish were sampled from August to November, 1986. Mature males and females were transferred to freshwater tanks on Nov. 7 for spawning (between Nov. 10 and Nov. 21, 1986). A second group of fish (five years old) was maintained in seawater for blood sampling from November, 1986 through September, 1987. All fish were individually identified by passive integrating transponder (PIT) tags. Blood samples were taken every three (4 yr olds) or every six (5 yr olds) weeks from fish anesthetized with 0.05 % tricaine methanesulfonate. The plasma fraction was stored in plastic vials at -20°C until assayed. Plasma vitellogenin concentrations were determined by single radial immunodiffusion (Mancini et al. 1965; Hara 1978; Ueda et al. 1984). In this assay, the antiserum raised in rabbits against coho salmon (Oncorhynchus kisutch) egg protein I (Hara and Dickhoff, unpublished) specifically recognizes vitellogenin. Purified Atlantic salmon vitellogenin was used as standard. The minimal detectable plasma concentration of vitellogenin using this assay was at least 100 ng/5 t1d (equivalent to 20 ftg/ml). For estradiol-17/3 (E2 ) determination, plasma was extracted with dichloromethane and extracts were subjected to partition chromatography (Wingfield and Farner 1975). The concentration of E2 was determined by radioimmunoassay (RIA) as described by Sower and Schreck (1982) with slight modification. Plasma thyroxine (T4 ) and triiodothyronine (T3) were determined by RIA as described by Dickhoff et al. (1978, 1982), and plasma insulin concentrations were measured by RIA using anti-coho salmon insulin serum and coho salmon standard and label as described by Plisetskaya et al. (1986). For the study on rainbow trout, virgin 2-year-old female rainbow trout (Salmo gairdneri) were obtained from the University of Washington experimental fish hatchery during their normal spawning season (February, 1987). The fish were checked for maturational state, and those with oocytes in the peripheral germinal vesicle stage were killed and
149 their ovaries were removed for incubation. Groups of about 10 oocytes were dissected from the ovaries. Batches of 30 to 40 oocytes were incubated in flasks in 6 ml of trout balanced saline (Kagawa et al. 1982) either alone or with 0 to 1 tg/ml of a gonadotropin fraction from Sephadex chromatography (SG-G100; Donaldson et al. 1972) and 0 to 500 ng/ml triiodothyronine. Incubations were done in duplicate at 16°C for up to four days in a incubator flushed with 99% 02: 1% CO 2 . At the end of incubation, the oocytes were removed and checked for germinal vesicle breakdown (Jalabert 1976). Incubation medium was centrifuged and supernatants were stored at -80°C until assayed by RIA for E2, androgens (Sower and Schreck 1982) and 17a-203dihydroxy-4-pregnen-3-one (DHP; Scott et al. 1982) concentration.
40
40
-4
D
E
a E2 *-VTG
3
303 3
.f 20
W 10
I
I Dec Jan Feb
I I I Mar Apr May Jun Jul Aug Sep
O
Oct Nov
Fig. 1. Plasma concentrations of estradiol (E2) and vitellogenin (VTG) in 4-year-old (July to November) and 5-year-old (November to July) female Atlantic salmon. Samples were taken from November 1986 to October 1987 from fish in seawater net-pens. Samples taken during November 1987 were from fish in fresh water. All fish ovulated between Nov. 10 to Nov. 20, 1987. Symbols represent mean values of 7 to 19 fish; brackets indicate + one standard error.
20
Results Atlantic salmon The changes in plasma concentrations of estradiol (E2) and vitellogenin (VTG) of the Atlantic salmon females are shown in Fig. 1. The data from the two groups of fish are combined and presented together to facilitate comparisons. Both VTG and E 2 were detectable in the plasma of females, but not males, throughout the sampling period. They increased to levels that were significantly higher (ANOVA, p < 0.05) than initial levels by August (E2) or September (VTG). Both VTG and E2 decreased significantly during the period of ovulation in November. Blood plasma concentrations of both thyroxine (T4) and triiodothyronine (T3 ) were determined in male and female Atlantic salmon during the year prior to maturation (Fig. 2). Plasma T4 levels in males remained at around 10 ng/ml from November 1986 to July 1987, and then decreased in August. Plasma T4 increased in September when the males began maturing as indicated by elevated plasma testosterone and 11-oxotestosterone (data not shown). The highest plasma T4 levels in males were observed in fish in fresh water (November 1987). During the early part of the year, plasma T4 tended to be higher in females compared to males, but
E
'9di0
Dec Jan
Feb Mar Apr May Jun
Jul Aug Sep
Oct Nov
Fig. 2. Plasma concentrations of thyroxine (T4 ) in 4- and 5-year-old female Atlantic salmon as in Fig. 1.
the differences were not statistically significant. Peak T4 levels in previtellogenic females occurred in early July. T4 levels declined in females in August and then increased in November when the fish were returned to fresh water. Plasma T 3 levels in male and female fish were similar from November 1986 through June 1987 (Fig. 3). At various times during the spring, plasma T 3 was significantly elevated from initial levels. Plasma T3 remained elevated in males, but declined in females, during the period from June to August. In both males and females T3 concentrations were at their lowest levels during maturation in the autumn. However, the sexual difference (higher T3 in males compared to females) was maintained throughout maturation. Insulin levels in the plasma were similar and con-
150 - Male 10E
8
I 624-
1_4;~4 0
I
I
I
Dec Jan
I
I
I
I
Feb Mar Apr May Jun
I
I
I
Jul Aug Sep
l
Oct
i
Nov
Fig. 3. Plasma concentrations of triiodothyronine (T3 ) in 4- and 5-year-old female Atlantic salmon as in Fig. 1. 40
e Male + Female
E . 30
S
20
._ a
lO
O' Nov
l
l Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct
Nov
Fig. 4. Plasma concentrations of insulin in 4- and 5-year-old female Atlantic salmon as in Fig. 1.
stant in males and females from November 1986 through February 1987 (Fig. 4). Plasma insulin increased during the spring to reach peak levels by the beginning of May, and then declined to basal levels by July. Mean concentration of insulin at the peak was higher in females compared to males, but the difference was not statistically significant. During maturation, mean plasma insulin was always higher in males compared to females, and a significant increase was observed in males, but not in females, during October.
Rainbow trout The changes of thyroid hormones that are often observed in maturing salmonids inspired a test of their possible role in final maturation of the ovary. Results from the incubation of oocytes with or without varying concentrations of T3 or SG-G100 are shown in Table 1. Germinal vesicle breakdown (GVBD) was not observed spontaneously or in response to the gonadotropin preparation (SG-G 100) at concentrations of 0.1 and 1 ng/ml. The SG-G100
was effective in stimulating GVBD at concentrations of 10, 100 and 1,000 ng/ml. At the 10 ng/ml concentration of SG-G100, significantly greater GVBD was observed when T3 was present in the medium at concentrations of 5 or 50 ng/ml compared to T 3 concentrations of 0 or 0.5 ng/ml. The combination of T3 at 500 ng/ml and SG-G100 at 10 ng/ml significantly reduced GVBD compared to SG-G100 alone. No effect of T3 was observed at higher or lower concentrations of SG-G100. The effect of the combinations of SG-G100 and T3 on steroidogenesis agreed with results of GVBD (Table 1). The concentrations of E 2 in the medium were lower and the concentrations of androgen were higher in those incubations which contained concentrations of SG-G100 that were effective in inducing GVBD (Table 1). The levels of 17a,203dihydroxy-4-pregnen-3-one (DHP) were low or nondetectable in medium from oocyte incubations in which GVBD was not observed, i.e., those containing 1 ng/ml SG-G100 or less. The calculated secretion rate of DHP was above 10 pg/oocyte/hr for incubations containing 10 ng/ml of SG-G100, and above 100 pg/oocyte/hr for the higher concentrations of SG-G100. There was a significant linear relationship (P < 0.01) between the DHP secretion rate and T3 concentration for incubations containing SG-G100 at 10 ng/ml, suggesting that T3 acted through stimulation of DHP secretion.
Discussion The analysis of seasonal changes in plasma levels of thyroid hormones and insulin in adult Atlantic salmon indicate that these hormones are elevated in both males and females during the spring before the onset of reproductive maturation. Plasma concentrations of the hormones decrease during the summer just prior to exogenous vitellogenesis in females. Sexual differences in the plasma concentrations of the hormones are apparent at some times during final maturation in the autumn. Changes in thyroid hormones during maturation have been observed in studies of several species of salmonids. Such studies have been conducted on wild, migrating and captive stocks of fish. In Great
151 Table 1. Percent germinal vesicle breakdown and follicular secretion rate of estradiol, androgen and DHP (parentheses) without triiodothyronine (T3) for 72 hours. T3 Concentration (ng/ml) SG-G100 (ng/ml)
0
0.5
5
50
500
0
0 (2.3, 56, 0.2) 0 (2.4, 54, 0.5) 0 (2.1, 101, 1.0) 13 + 10 (1.1, 376, 18.7) 55 + 14 (1.1, 641, 254.4) 57 + 18 (1.0, 424, 211.6)
0 (3.0, 45, ND) 0 (1.7, 40, 0.1) 0 (2.3, 87, ND) 18+13 (1.6, 430, 15.7) 64 + 16 (1.3, 645, 247.1) 61 + 14 (1.0, 536, 372.3)
0 (2.7, 31, ND) 0 (2.4, 46, ND) 0 (1.6, 62, ND) *32± 16 (1.2, 396, 38.5) 60+ 15 (1.3, 849, 321.6) 58 + 15 (1.0, 530, 323.0)
0 (2.9, 47, ND) 0 (3.1, 64, ND) 0 (2.6, 47, 0.8) *38 + 14 (1.1, 377, 35.8) 55 15 (1.2, 696, 319.6) 50 + 14 (1.1, 607, 315.2)
0 (2.7, 40, ND) 0 (2.3, 56, ND) 0 (1.9, 150, ND) *3+6 (1.4, 347, 10.5) 62 + 19 (1.1, 736, 272.5) 43 20 (1.3, 1074, 246.1)
0.1 1 10 100 1000
2, Asterisks indicate values which are significantly different from control values (without T3; X p < 0.05); follicular secretion rate is expressed as pg/oocyte/h; steroid assays were run in duplicate. ND = nondetectable.
Lakes coho salmon (Oncorhynchus kisutch), Leatherland and Sonstegard (1980) found higher plasma levels of T4 and T3 in spring before maturation compared to those found during maturation in autumn. Decreases in plasma T4 and/or T3 have been reported during the seawater to freshwater transition, or during upstream migration and maturation of wild coho salmon (Sower and Schreck 1982), sockeye salmon (0. nerka; Biddiscombe and Idler 1983) and chum salmon (0. keta; Ueda et al. 1984). Decreases in T4 and T3 as spawning approached were reported for non-anadromous brook trout (Salvelinusfontinalis;White and Henderson 1977) and rainbow trout (Salmo gairdneri; Osborn et al. 1978). In captive salmonids, an increase in T4 during the time of ovulation has been reported for brown trout (Salmo trutta; Pickering and Christie 1981). In rainbow trout on regulated photoperiods, Cyr et al. (1988) found high plasma levels of T4 and T3 in the previtellogenic phase; both hormones decreased as maturation proceeded, and then increased soon after spawning. Our results with Atlantic salmon agree with most published studies on other salmonids in that thyroid hormones are elevated before vitellogenesis and then decrease around the time when E 2 and vitellogenin levels are known to rise. Likewise, our finding of a
decrease in plasma T3 during ovulation and spermiation is in agreement with published reports on other species. In contrast, there appears to be some variability in reports on changes in T4 during spawning. Our finding of an increase in T4 during spawning of Atlantic salmon agrees with the report on the congeneric brown trout (Pickering and Christie 1981). A significant elevation in T4 during spermiation was also reported for sockeye salmon (Biddiscombe and Idler 1983). Comparison of these results are complicated by several factors including different species and stock, feeding or fasting status, and length of time in fresh water (migratory fish), among others. Sexual differences in thyroid hormone levels have been reported for maturing fish in several instances. Our finding of generally higher T4 and T3 in maturing Atlantic salmon males compared to females is similar to that reported for coho salmon (Sower and Schreck 1982) and sockeye salmon (Biddiscombe and Idler 1983). In contrast, T4 was found to be higher in maturing female brown trout (Pickering and Christie 1981). Ueda et al. (1984) found no difference in T4 and T3 comparing maturing male and female chum salmon. The basis for this sexual difference or lack of it is not apparent. It has been suggested that E 2 may be responsible
152 for low T 3 in maturing female salmonids, since E2 administration depresses plasma T3 levels in rainbow trout (Leatherland 1985; MacLatchy et al. 1986), and reduces the activity of deiodinase in rainbow trout hepatocytes (Shields and Eales 1986). However, the disagreement in the literature regarding sexual differences in thyroid hormone levels suggests that this mechanism may not be operable in all salmonid species, or it may be over-ridden by other factors in some situations. A role for thyroid hormones in final maturation of salmonids is clearly suggested by the results of our in vitro study on germinal vesicle breakdown. The combination of SG-G100 at 10 ng/ml and T3 at 5 or 50 ng/ml resulted in a higher incidence of GVBD in incubated oocytes when compared with SG-G100 alone. This concentration of SG-G100 and the lower concentration of T 3 are near the physiological range of plasma concentrations observed during final maturation (Fostier et al. 1978; Scott et al. 1983; Dye et al. 1986). The fact that T3 alone had no effect on GVBD or steroid production indicates a synergistic role of thyroid hormones in ovulation. Our data also suggests that this synergistic effect of T 3 can be surmounted by concentrations of SG-G100 as high as 100 ng/ml. In other words the synergistic action of T3 on ovulation could be made physiologically insignificant by a large preovulatory surge of gonadotropin. Nevertheless, the possibility of a contributing role of thyroid hormones in spawning cannot be ignored. A synergistic effect of thyroid hormones acting in conjunction with gonadotropin to induce ovulation has been reported for goldfish (Carassiusauratus; Hurlburt 1977) and carp (Cyprinus carpio; Epler and Bieniarz 1983). Thyroid hormones may help oocytes of sturgeon (Acipenser stellatus)retain the capacity to respond to gonadotropin under adverse conditions of temperature (Dettlaff and Davydova 1979). Cyr and Eales (1988) have demonstrated a synergistic effect of T 3 on gonadotropininduced E2 production by rainbow trout ovaries in vitro. Furthermore, putative thyroid hormone receptors have been identified in a freshwater perch (Anabas testudineus; Chakraborti et al. 1986). Thus, evidence for a role of thyroid hormones in
several phases of fish reproduction is accumulating. The significant elevation in plasma insulin that we observed in Atlantic salmon during the spring prior to onset of maturation can be compared with similar changes reported for scorpion fish (Scorpaena; Plisetskaya et al. 1976) and sea bass (Dicentrarchus labrax; Gutierrez et al. 1987). Plasma insulin was higher before than during spawning of scorpion fish. Using an RIA based on mammalian insulin, Gutierrez et al. reported an increase in insulin in sea bass plasma during the prespawning period; plasma insulin decreased during spawning. In contrast to these observations in sea bass, in Atlantic salmon plasma insulin decreased before the increases in E 2 and vitellogenin and did not show an additional decrease during spawning. A vernal surge in plasma insulin may be an annual event in salmonids since it has also been observed in juvenile coho salmon prior to the parr to smolt transformation (Plisetskaya et al. 1988). In Atlantic salmon, the generally higher mean level of plasma insulin in males compared to females is similar to the sexual difference in plasma insulin observed in pink salmon (0. gorbuscha) during their spawning migration (Plisetskaya et al. 1987). However, the results from the study on pink salmon insulin and our study differ in that during the up-river migration of pink salmon there was a significant transient elevation of insulin in females, whereas we observed a significant transient elevation of insulin in males transferred to fresh water. The higher levels of insulin in the Atlantic salmon compared to the pink salmon may have been due to the fact that the Atlantics were fed throughout their captivity in net-pens whereas the pink salmon had ceased feeding during their upstream migration (McBride et al. 1986). The patterns of change in plasma concentration of metabolic hormones invite speculation on their possible function either in controlling reproduction or the energetics of maturation. It has been shown that insulin may promote the uptake of vitellogenin into growing oocytes of rainbow trout in vitro (Tyler et al. 1987). Although plasma insulin levels in the Atlantic salmon females tended to be lower than in males and insulin was at its highest levels before vitellogenesis, it is possible that normal levels
153 of insulin are necessary for many aspects of reproduction. The surge of plasma insulin in the spring may function to increase metabolic stores in anticipation of maturation. Energy stored as lipid or protein are needed for maintenance of activity and gametogenesis when the salmon stop feeding near the time of spawning. During maturation there is a switch from somatic to gonadal growth. Somatic weight of maturing coho salmon has been shown to stop increasing in July-August, and decrease thereafter to end up with a 37%7o loss (dry weight) at spawning in December (Hardy et al. 1984). Growth of the ovary continues while somatic growth has stopped. Even after feeding has ceased, the ovary continues to incorporate protein and lipid. Therefore, the nutrients deposited in the ovary must originate in maternal tissues. It has been estimated that 7% to 8°7% of the lipid loss from somatic tissues is transferred to the ovaries in maturing coho (Hardy et al. 1984) and sockeye (Idler and Bitners 1960) salmon. In Atlantic salmon, the high levels of plasma insulin in April and May may increase protein and lipid storage, whereas the decreasing plasma insulin in July along with maintained high levels of thyroid hormones will promote lipolysis. The reason for the relatively higher levels of plasma insulin and thyroid hormones in males compared to females during final maturation is not entirely clear. We could speculate that these two hormones may be acting to guard against early depletion of energy reserves in males. The need for maintaining energy reserves in males may be illustrated by the mating process. When female salmon arrive on the spawning grounds, they dig a redd, and then release all of their eggs at once. In contrast, male salmon must be on the spawning grounds when the females arrive. The males defend their territory by displays and fights with other males. They may spawn with one female, and then remain on the grounds for another reproductive opportunity.
Acknowledgements This work was supported by grants from the National Science Foundation (DCB-8615521), the
U.S. Department of Agriculture (S/E-Aq) under agreement # 85-CRSR-2-2603 and Washington Sea Grant Project R/A 49. We thank F. William Waknitz, Conrad V.W. Mahnken, Tom Flagg and Earl F. Prentice of the National Marine Fisheries Service for their assistance in this work.
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