Identification of maturation-inducing steroid in a freshwater perch Anabas testudineus and differential responses of intact follicles and denuded oocytes to cyclic AMP in oocyte maturation

Postvitellogenic follicles of freshwater perch Anabas testudineus incubated with [(3)H]pregnenolone as exogenous precursor produced several metabolites, including 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) and 5 beta-pregnane-3 alpha, 17 alpha,20 beta-triol (5 beta-3 alpha,17 alpha,20 beta-P). These were identified by chromatography, microchemical reactions, and crystallization to constant specific activity. Following stimulation with fish (perch) pituitary extract (FPE) there was significant high production of DHP and 5 beta-3 alpha,17 alpha,20 beta-P, concomitant with a high percentage of germinal vesicle breakdown (GVBD). Inhibitor of steroidogenesis (trilostane) and inhibitors of protein synthesis (cycloheximide and actinomycin-D) completely blocked FPE-induced pregnenolone metabolism and oocyte maturation. The effectiveness of various C(21) steroids in inducing GVBD was examined. Results indicate that DHP was the most potent inducer of GVBD than other structurally related C(21) steroids. In intact follicles, FPE-stimulated production of DHP was shown to be mediated through the adenylate cyclase-cAMP pathway. Addition of IBMX or forskolin, which increases the endogenous cAMP level, as well as directly supplementing dbcAMP to the incubation medium, had no inhibitory effect on DHP-induced GVBD in the intact follicles. But all these agents were shown to inhibit GVBD in fully denuded oocytes. This study provides evidence that DHP, produced by postvitellogenic follicles through the adenylate cyclase-cAMP pathway, is the maturation-inducing steroid in freshwater perch and that the role played by cAMP in the induction of GVBD in intact follicles is different from that in the denuded oocytes. J. Exp. Zool. 287:294-303, 2000.     

Induction of maturation of Atlantic croaker oocytes by 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one in vitro: consideration of some biological and experimental variables

We characterized the in vitro control of germinal vesicle breakdown (GVBD) by 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) in intact ovarian follicles of gonadotropin-primed Atlantic croaker. 20 beta-S-induced GVBD was determined in relation to ovarian (oocyte) morphology, duration of incubation, steroid metabolism, and interaction with other steroids. The rate of GVBD in vitro in the absence of exogenous steroid was positively correlated with initial stage of ovarian morphological development. Maximal responsiveness to 20 beta-S was seen in ovaries with oocytes showing the first signs of morphological maturation. Dose-response experiments with 20 beta-S and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P) over a range of incubation times yielded similar results for both steroids, suggesting that conversion of 17 alpha,20 beta-P to 20 beta-S is not required for 17 alpha,20 beta-P-induced GVBD. The ED50 of these steroids markedly decreased with increasing incubation times. Comparisons between patterns of follicular transformation of various radiolabelled steroids to 20 beta-S and their respective activities (using unlabelled steroids) in the GVBD bioassay suggested that, in addition to 17 alpha,20 beta-P, progesterone has some intrinsic maturational activity. However, the maturational effects of 11-deoxycortisol and pregnenolone may be explained by their conversion to 20 beta-S. For the first time in any vertebrate, we showed that the proposed maturation-inducing steroid (20 beta-S) is not significantly transformed to any extractable, potentially active metabolite by intact, maturing ovarian follicles. These findings strongly suggest that 20 beta-S is the terminal product of the MIS biosynthetic pathway in Atlantic croaker ovaries. Estradiol had no acute effects on 20 beta-S-induced GVBD. However, testosterone decreased and cortisol augmented the maturational activity of 20 beta-S. Excess progesterone reduced the activity of a maximally effective dose of 20 beta-S, but pregnenolone was without effect. The effects of these steroids on 20 beta-S-induced GVBD are discussed in relation to their possible interactions with 20 beta-S at the MIS receptor level.     

Biosynthesis of steroids in ovarian follicles of red seabream,Pagrus major (Sparidae,Teleostei) during final oocyte maturation and the relative effectiveness of steroid metabolites for germinal vesicle breakdown in vitro

The steroid synthesis pathway in the ovarian follicles of the red seabream during final oocyte maturation (FOM) was investigated by incubating intact follicles with different radioactively labeled steroid precursors. During FOM, the steroidogenic shift from estradiol-17beta to 20 beta-hydroxylated progestin production occurred mainly due to a combination of inactivation of C 1720-lyase and activation of 20 beta-hydroxysteroid dehydrogenase. Of the steroids produced, 1720 beta-dihydroxy-4-pregnen-3-one (1720 beta-P) and 1720 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) exhibited the greatest effect on germinal vesicle breakdown (GVBD) in vitro. 1720 beta-P was further converted to its 5 beta-reduced form, 1720 beta-dihydroxy-5 beta-pregnan-3-one (1720 beta-P-5 beta), which had lower GVBD activity, suggesting that 5 beta-reduction plays a role in the inactivation of the maturation-inducing ability of 1720 beta-P. In contrast, no 5 beta-reduced metabolite of 20 beta-S was found. Serum levels of 1720 beta-P and 20 beta-S, measured by ELISA, showed that circulating levels of both progestins increased during FOM, and 20 beta-S levels were approximately twice as high as 1720 beta-P levels. This study clarified the complete steroidogenesis pathway during FOM in red seabream ovarian follicles and showed that two 20 beta-hydroxylated progestins, 1720 beta-P and 20 beta-S, act as maturation-inducing hormones in this species. The catabolites of these two progestins and their physiological roles in reproduction are also discussed.     

Steroidogenesis in Fundulus heteroclitus. IV. Dichotomous effects of a phorbol ester on ovarian steroid production and oocyte maturation.

The possible role of protein kinase C (PKC) activation in mediating the stimulatory actions of a Fundulus pituitary extract (FPE) on ovarian steroidogenesis and oocyte maturation was investigated. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), alone slightly increased basal 17 alpha-hydroxy,20 beta-dihydroprogesterone (DHP) and 17 beta-estradiol (E2) synthesis and significantly stimulated germinal vesicle breakdown (GVBD). Addition of FPE promoted synthesis of DHP, testosterone (T), and E2, and initiated GVBD. Phorbol ester inhibited FPE-induced steroidogenesis but increased the number of oocytes that underwent GVBD. Phorbol ester also markedly impeded induction of steroidogenesis by dibutyryl cAMP and differentially affected the conversion of 25-hydroxycholesterol, pregnenolone, or progesterone to DHP, T, and E2: DHP production was not affected; T production diminished; and E2 synthesis increased (T aromatization also increased). These results suggest an inhibitory role for the PKC pathway on FPE-induced ovarian steroid production, with PMA appearing to affect various steroidogenic steps. The stimulatory action of PMA on oocyte maturation seems to be independent of follicular steroid production since aminoglutethimide, an inhibitor of steroidogenesis, did not block PMA-induced GVBD. Moreover, PMA had a marked stimulatory effect on GVBD in denuded oocytes. Thus, in contrast to the inhibitory role found for the PKC pathway on ovarian follicular steroidogenesis, activation of PKC in the oocyte may serve as a signal-transducing mechanism leading to GVBD.     

Regulation of ovarian steroidogenesis in vitro by follicle-stimulating hormone and luteinizing hormone during sexual maturation in salmonid fish

The regulation of ovarian steroidogenesis in vitro by coho salmon FSH and LH was investigated in intact coho salmon follicles and isolated follicular layers at various stages of oocyte maturation, from late vitellogenesis until the completion of germinal vesicle breakdown (GVBD). In granulosa layers from all stages, LH, but not FSH, stimulated 17alpha,20beta-dihydroxy-4-pregnen-3-one (17, 20beta-P) production. In theca-interstitial layers from all stages, FSH and LH stimulated steroid production, LH being more potent than FSH. The basal steroid output of intact follicles was significantly lower than that of isolated follicular layers, and their response to FSH and LH also differed. First, the intact follicles produced 17alpha-hydroxyprogesterone in response to FSH during the central germinal vesicle stage while theca-interstitial layers did not. Second, estradiol-17beta production was not inhibited by LH during final oocyte maturation in intact follicles, as observed for granulosa layers. Our results indicate that LH is the determining factor regulating the production of the maturation-inducing steroid, 17,20beta-P, and the induction of GVBD in the salmonid ovary. In summary, we have provided evidence for maturation-associated changes in the effects of FSH and LH in the salmonid ovary, which further supports the hypothesis that FSH and LH have distinct functions in the teleost ovary.     

The shift from C-19 to C-21 steroid synthesis in spawning male common carp, Cyprinus carpio, is regulated by the inhibition of androgen production by progestogens produced by spermatozoa

There is a rapid shift in the steroidogenic pathway from androgen to progestogen production in spawning male common carp, Cyprinus carpio. Experiments were conducted to determine the mechanism regulating this shift using in vitro cultures of testicular fragments and isolated sperm of spermiating male carp. The levels of 11-ketotestosterone (11-KT) continually increased for 48 h with or without gonadotropin (GtH) stimulation, suggesting that 11-KT is the principal androgen produced by carp testes. Ovine prolactin (oPRL) enhanced GtH-stimulated 11-KT production, but by itself had no effect. Gonadotropin, carp pituitary extract, and pregnenolone all enhanced the production of 11-KT, testosterone (T), and 17 alpha-hydroxyprogesterone (17-P) in a dose-dependent manner. No 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) was detected in response to any of these agents; 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P) was not measured. Both 17,20 beta-P and 17,20 alpha-P inhibited 11-KT production in a dose-dependent manner in the presence of either GtH, 17-P, or T. Isolated sperm and testicular fragment preparations both produced 17,20 beta-P and approximately tenfold more 17,20 alpha-P when incubated with 17-P. Only testicular fragments, however, produced 11-KT. We conclude that androgen synthesis occurs only within somatic cells of common carp testes. GtH, and perhaps PRL, stimulates the production of steroid precursors that, under normal physiological conditions, are metabolized to androgens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oocyte maturation-inducing steroids in protandrous black porgy, Acanthopagrus schlegeli

The study objectives aimed to investigate the maturation-inducing steroid (MIS) in marine protandrous black porgy, Acanthopagrus schlegeli. The characteristics of oocyte maturation were also described. Females were injected with two successive doses of LHRH analog (LHRH-A, 10 and 50 microg/kg of fish). The ovarian tissue was obtained at 6-h intervals for in vitro oocyte maturation. Both 17,20 beta-dihydroxy-4-pregnen-3-one (DHP) and 17,20 beta,21-trihydorxy-4-pregnen-3-one (20 beta-S) were the most effective steroids to induce in vitro maturation (e.g. germinal vesicle breakdown, GVBD) in oocytes cultured for either 24 h or 1 min. 20 beta-S had a better potency than DHP in inducing oocyte maturation. 17-hydroxyprogesterone, 11-deoxycortisol, and 20 beta-21-dihydroxy-4-pregnen-3-one also significantly induced oocyte maturation at high concentrations. The process of oocyte maturation (after the injection of LHRH analog) was founded to be divided into four stages: hormone-insensitive stage (insensitive to gonadotropin and MIS); MIS-insensitive (respond to gonadotropin, but not MIS); MIS-sensitive (respond to MIS); and spontaneous stage (GVBD in the hormone-free condition), respectively. Cycloheximide blocked GVBD at the MIS-insensitive stage, control (hormone-free), and hormone-induced GVBD at the MIS-sensitive stage in a dose-dependent effect.     

Activation of both Mos and Cdc25 is required for G2-M transition in perch oocyte

Resumption of meiosis from diplotene arrest during the first meiotic prophase in vertebrate oocytes is universally controlled by MPF, a heterodimer of Cdk1 and cyclin B. Activation of MPF depends on the withdrawal of Cdk1 inhibition by Wee1/Myt1 kinase on the one hand and the activation of Cdk1 by Cdc25 phosphatase on the other. It is relevant to know whether both these pathways are necessary to rescue diplotene arrest or if either one of them is sufficient. In MIH (17alpha, 20beta dihydroxy-4-pregnen-3-one) incubated perch (Anabas testudineus) oocytes we have examined these possibilities. Perch oocyte extract following MIH incubation showed a significant increase in Myt1 phosphorylation from 12 to 16 hr indicating its progressive deactivation. MIH induced Mos expression markedly increased at 16 hr effecting 95% GVBD. Cycloheximide inhibited MIH induced Mos expression and its phosphorylation, which in turn reduced Myt1 phosphorylation and GVBD. Myt1 phosphorylation was blocked in Mos immunodepleted oocytes. All these suggest the involvement of Mos in Myt1 phosphorylation. Oocytes incubated in MIH for 16 hr activated Cdc25, but such activation could not rescue the inhibition of GVBD due to Myt1 in Mos immunodepleted oocytes. Blocking Cdc25 with an antisense oligo significantly inhibited GVBD even though Myt1 remained deactivated during this period. Taken together, our findings indicate that MIH requires both pathways for perch oocyte maturation: the expression and activation of Mos, which is linked to Myt1 deactivation on the one hand, and the activation of Cdc25 on the other, as blocking either pathway compromised G2-M transition in perch oocytes.     

Steroidogenesis in ovarian follicles of chub mackerel, Scomber japonicus

We incubated different radiolabeled steroid precursors with intact chub mackerel ovarian follicles to clarify the synthetic pathways of steroid hormones during vitellogenesis and following final oocyte maturation (FOM). During vitellogenesis, estradiol-17beta (E2) was synthesized from pregnenolone via 17-hydroxypregnenolone, 17-hydroxyprogesterone, androstenedione, and testosterone. The physiological significance of the intermediate metabolites of E2 in the ovarian follicles was examined by comparing follicular steroidogenesis between gonochoric and hermaphroditic fish species. After vitellogenesis, the steroidogenic pathway shifted from E2 to maturation-inducing hormone (MIH) production owing to the inactivation of 17,20-lyase and the activation of 20 beta-hydroxysteroid dehydrogenase. Of the new steroids produced during FOM, 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) was most effective at inducing germinal vesicle breakdown in vitro. Circulating levels of 17,20beta-P increased specifically around the time of germinal vesicle migration, while another FOM-specific 20beta-hydroxylated progestin, 17,20beta,21-trihydroxy-4-pregnen-3-one, was present at consistently low levels during FOM. These results indicate that 17,20beta-P is the MIH of chub mackerel.     

Shift in Steroidogenesis in the Ovarian Follicles of the Goldfish (Carassius auratus) during Gonadotropin-Induced Oocyte Maturation

Both partially purified chum salmon gonadotropin and 17α-hydroxyprogesterone stimulated in vitro production of testosterone by postvitellogenic follicles of goldfish ( Carassius auratus ). Chum salmon gonadotropin further enhanced the conversion of exogenously supplied 17α-hydroxyprogesterone to 17α, 20β-dihydroxy-4-pregnen-3-one. The increased medium concentrations of 17α, 20β-dihydroxy-4-pregnen-3-one were associated with the induction of final oocyte maturation.
The capacity of postvitellogenic follicles to produce steroids in response to exogenous 17α-hydroxyprogesterone was examined in females at various stages of final oocyte maturation following the administration of human chorionic gonadotropin in vivo combined with elevation of holding temperature. The maximum production of testosterone in response to 17α-hydroxyprogesterone was obtained in follicles from initial controls. In contrast, 17α 20β-diOHprog production was very low in initial controls and markedly increased during oocyte maturation (3–6 hr following injection), followed by a significant decrease in follicles collected at 15 hr. Estradiol-17β production by the follicles was very low at any stages of gonadotropin-induced oocyte maturation. These results suggest that gonadotropin-induced shift in the biosynthetic pathway in the follicle from the secretion of predominantly testosterone to 17α, 20β-dihydroxy-4-pregnen-3-one secretion is a prerequisite step for the induction of oocyte maturation in goldfish.     

Cdc2-cyclin B-induced G2 to M transition in perch oocyte is dependent on Cdc25

The G2 to M phase transition in perch oocytes is regulated by maturation promoting factor (MPF), a complex of Cdc2 and cyclin B. In Anabas testudineus, a fresh water perch, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, the maturation inducing hormone (MIH), induced complete germinal vesicle breakdown (GVBD) of oocytes at 21 h. An unusual cyclin, p30 cyclin B, has been identified in oocyte extract using both monoclonal and polyclonal antibodies. Surprisingly, Cdc2 could not be identified, although a Northern blot with Cdc2 cDNA demonstrated expression of the gene. Purification of MPF through an immunoaffinity column followed by SDS-PAGE showed three proteins, Cdc2, cyclin B, and a 20 kDa fragment, indicating earlier failure in immunodetection may be due to the interference by this fragment. In uninduced oocytes, p30 cyclin B was present, and its expression was increased by MIH. MIH increased p30 cyclin B accumulation at 3 h, a high level which was maintained between 9 and 21 h, but an effective increase in GVBD and H1 kinase activation could only be observed between 15 and 21 h. This delay in active MPF formation was found to be related to the activation of Cdc25, phosphorylation of which was detected at 12 h, and a substantial increase occurred during 15-18 h. Sodium orthovanadate, a tyrosine phosphatase inhibitor, inhibited H1 kinase activity and GVBD, suggesting the requirement of Cdc25 activity in MPF activation. Our results show occurrence of pre-MPF in uninduced oocytes and its conversion to active MPF requires dephosphorylation by Cdc25, the existence of which has not yet been shown in fish.     

Endocrine control of diurnal oocyte maturation in the kyusen wrasse,Halichoeres poecilopterus

The present study examined diurnal cycles of oocyte development and maturation in the kyusen wrasse, Halichoeres poecilopterus, and investigated the sensitivity of oocytes to maturation-inducing hormone (MIH) and gonadotropic hormone (GTH). Female fish were sampled at fixed intervals throughout the day, revealing that final oocyte maturation and ovulation were completed by 6:00 hr, and that spawning occurred daily between 6:00 and 9:00 hr. In vitro experiments showed that the steroids 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) and 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) were equally potent and highly effective inducers of germinal vesicle breakdown (GVBD) in kyusen wrasse oocytes. Additionally, circulating levels of 17,20beta-P and 20beta-S increased around the time of GVBD and ovulation, suggesting that 17,20beta-P and 20beta-S act as MIHs in the kyusen wrasse. Moreover, in vitro experiments clearly showed that kyusen wrasse oocytes had a daily developmental cycle of GTH and MIH sensitivity, and that oocytes that completed vitellogenesis acquired GTH-induced maturational competence. An endogenous GTH surge likely occurs between 12:00 and 15:00 hr, and this daily pre-maturational GTH surge probably controls the diurnal maturation cycles of kyusen wrasse oocytes.     

Steroidogenic and maturation-inducing potency of native gonadotropic hormones in female chub mackerel, Scomber japonicus

ABSTRACT: BACKGROUND: The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are produced in the pituitary gland and regulates gametogenesis through production of gonadal steroids. However, respective roles of two GtHs in the teleosts are still incompletely characterized due to technical difficulties in the purification of native GtHs. METHODS: Native FSH and LH were purified from the pituitaries of adult chub mackerel, Scomber japonicus by anion-exchange chromatography and immunoblotting using specific antisera. The steroidogenic potency of the intact chub mackerel FSH (cmFSH) and LH (cmLH) were evaluated in mid- and late-vitellogenic stage follicles by measuring the level of gonadal steroids, estradiol-17beta (Epsilon2) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In addition, we evaluated the maturation-inducing potency of the GtHs on same stage follicles. RESULTS: Both cmFSH and cmLH significantly stimulated E2 production in mid-vitellogenic stage follicles. In contrast, only LH significantly stimulated the production of 17,20beta-P in late-vitellogenic stage follicles. Similarly, cmLH induced final oocyte maturation (FOM) in late-vitellogenic stage follicles. CONCLUSIONS: Present results indicate that both FSH and LH may regulate vitellogenic processes, whereas only LH initiates FOM in chub mackerel.     

Role of inhibin and activin in the modulation of gonadotropin- and steroid-induced oocyte maturation in the teleost Fundulus heteroclitus

Background  

Activin and inhibin are glycoproteins structurally related to the transforming growth factor-beta superfamily. These peptides were first described as factors that regulate the follicle-stimulating hormone (FSH) at the pituitary level. The possible role of inhibin and activin, at the ovarian level, in mediating the stimulatory actions of a Fundulus pituitary extract (FPE) and 17alpha,20beta-dihydroprogesterone (DHP) on oocyte maturation was investigated in this study.     

Oocyte maturation in the amago salmon (Oncorhynchus rhodurus): in vitro effects of salmon gonadotropin, steroids, and cyanoketone (an inhibitor of 3 beta-hydroxy-delta 5-steroid dehydrogenase)

The effect of partially purified chinook salmon gonadotropin (SG-G100) and a number of steroids on the induction of germinal vesicle breakdown (GVBD) in amago salmon (Oncorhynchus rhodurus) oocytes (with intact follicle layers) was investigated in vitro. SG-G100 was effective only at the highest concentration tested (1 microgram/ml). 17 alpha,20 beta-Dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) was the most potent maturation-inducing steroid tested, followed by 17 alpha-hydroxyprogesterone. Testosterone or deoxycorticosterone (DOC) enhanced the rate of GVBD in response to SG-G100. DOC also enhanced the response to 17 alpha,20 beta-diOHprog but testosterone was without effect, suggesting that DOC has a direct action on the oocyte while testosterone probably acts at the level of the follicle. Estradiol-17 beta had no effect on GVBD in response to SG-G100 or 17 alpha,20 beta-diOHprog. The action of SG-G100 was shown to be dependent on the synthesis of a second delta 4 steroidal mediator of maturation since cyanoketone, a specific inhibitor of 3 beta-hydroxy-delta 5-steroid dehydrogenase, completely abolished the maturational effects of the gonadotropin and pregnenolone but not delta 4 steroids. Radioimmunoassay of media in which oocytes were induced to mature in vitro with SG-G100 revealed significantly elevated levels of progesterone and 17 alpha,20 beta-diOHprog. Estradiol-17 beta levels, high in control media, were only elevated twofold by SG-G100. Levels of the two progestogens were extremely low or nondetectable in media in which oocytes were incubated with cyanoketone, while estradiol-17 beta levels remained high. These results are discussed in relation to other evidence indicating that 17 alpha,20 beta-diOHprog is the naturally occurring maturation-inducing steroid of amago salmon. The role of other steroid hormones, particularly the possible involvement of corticosteroids, in the control of final oocyte maturation in teleosts is explored.     

Physiological effects of the lunar cycle on the spawning of a coral reef fish,Abudefduf Vaigiensis: in vivo and in vitro trait

The aim of the present study was to investigate the lunar cycle effects of the spawning of Audefduf vaigiensis through in vivo and in vitro analysis. For this purpose, the indices of GSI, serum levels of sex steroids, including testosterone (T), 17α-hydroxyprogesterone (OHP), 17α, 20β-dihydroxyprogesterone (DHP), and 11-keto-testosterone (11-KT) as well as the germinal vesicle breakdown (GVBD) were measured. The sampling pattern was weekly, based on the moon cycles as the new moon (NM), the first quarter (FQ), the full moon (FM), and the last quarter (LQ). In females, the highest in vivo values of the GSI index were obtained in FQ and LQ, and in males, this value was significantly higher in LQ than NM. The highest in vivo level of OHP in females was observed in FQ, whereas in males was obtained in FM. In both sexes, the in vivo serum levels of DHP were obtained in LQ. In males, the level of 11-KT were at the peak in NM. In vitro analysis showed the highest rate of GVBD in LQ. Moreover, the in vitro levels of T, OHP, and DHP were significantly higher in LQ compare to NM in both sexes. However, in males, the in vitro levels of 11-KT was significantly higher in NM than LQ. These cyclical changes obtained from in vivo plasma steroid hormones and in vitro data on GVBD suggested that lunar periodicity is a major external regulator that synchronized ovarian and testicular activity of A. vaigiensis with emphasis on spawning phenomenon.

     

Effect of Actinomycin D and Cycloheximide on Gonadotropin-Induced 17α, 20β-Dihydroxy-4-pregnen-3-one Production by Intact Ovarian Follicles and Granulosa Cells of the Amago Salmon,Oncorhynchus rhodurus*

The effect of actinomycin D and cycloheximide on gonadotropin (partially purified chum salmon gonadotropin, SGA)-induced 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-diOHprog, a maturation-inducing steroid in amago salmon) production was examined in intact ovarian follicles and granulosa cells of postvitellogenic amago salmon, Oncorhynchus rhodurus. Both actinomycin D and cycloheximide blocked gonadotropin-induced 17α, 20β-diOHprog production by intact follicles. In contrast, gonadotropin-induced 17α-hydroxyprogesterone production by intact follicles was not abolished by actinomycin D, but was abolished by cycloheximide, suggesting that postvitellogenic amago salmon ovarian follicles already contain the RNAs necessary for the synthesis of 17α-hydroxyprogesterone. In isolated granulosa cells, chum salmon gonadotropin was able to stimulate 17α, 20β-diOHprog production only when a precursor, 17α-hydroxyprogesterone was provided in the incubation medium, indicating that gonadotropin acts directly on granulosa cells to enhance the activity of 20β-hydroxysteroid dehyrogenase (20β-HSD). Total inhibition of 20β-HSD enhancement in granulosa cells, judged by 17α, 20β-diOHprog production, was achieved when actinomycin D was added between 1 hr before the start of incubation with 17α-hydroxyprogesterone and gonadotropin to 6 hr after. With cycloheximide total inhibition was observed when added in the period of 1 hr before to 9 hr after the start of the incubation. These results suggest that chum salmon gonadotropin acts on granulosa cells to enhance the de novo synthesis of 20β-HSD by a mechanism involving RNA synthesis.