Shift In Steroidogenesis Research Articles

Luteinizing Hormone Surge-Induced Krüppel-like Factor 4 Inhibits Cyp17A1 Expression in Preovulatory Granulosa Cells.

Previous in vivo and in vitro studies have demonstrated a dramatic up-regulation of Krüppel-like factor 4 (Klf4) in rat preovulatory granulosa cells (GCs) after LH/hCG treatment and its role in regulating Cyp19A1 expression during the luteal shift in steroidogenesis. In this study, we examined whether Klf4 also mediates the LH-induced repression of Cyp17A1 expression in primary rat preovulatory GCs. In response to LH treatment of GCs in vitro, Cyp17A1 expression declined to less than half of its initial value by 1 h, remaining low for 24 h of culture. Overexpression of Klf4 decreased basal and Sf1-induced Cyp17A1 expressions and increased progesterone secretion. Reduction of endogenous Klf4 by siRNA elevated basal Cyp17A1 expression but did not affect LH-stimulated progesterone production. Overexpression of Klf4 also significantly attenuated Sf1-induced Cyp17A1 promoter activity. On the other hand, mutation of the conserved Sp1/Klf binding motif in the promoter revealed that this motif is not required for Klf4-mediated repression. Taken together, these data indicate that the Cyp17A1 gene may be one of the downstream targets of Klf4, which is induced by LH in preovulatory GCs. This information may help in identifying potential targets for preventing the molecular changes occurring in hyperandrogenic disorders.

Steroidogenesis and its regulation in teleost-a review.

Steroid hormones modulate several important biological processes like metabolism, stress response, and reproduction. Steroidogenesis drives reproductive function wherein development and differentiation of undifferentiated gonads into testis or ovary, and their growth and maturation, are regulated. Steroidogenesis occurs in gonadal and non-gonadal tissues like head kidney, liver, intestine, and adipose tissue in teleosts. This process is regulated differently through multi-level modulation of promoter motif transcription factor regulation of steroidogenic enzyme genes to ultimately control enzyme activity and turnover. In view of this, understanding teleostean steroidogenesis provides major inputs for technological innovation of pisciculture. Unlike higher vertebrates, steroidal intermediates and shift in steroidogenesis is critical for gamete maturation in teleosts, more essentially oogenesis. Considering these characteristics, this review highlights the promoter regulation of steroidogenic enzyme genes by several transcription factors that are involved in teleostean steroidogenesis. It also addresses different methodologies involved in promoter regulation studies together with glucocorticoids and androgen relationship with reference to teleosts.

Stress and sex: does cortisol mediate sex change in fish?

Cortisol is the main glucocorticoid (GC) in fish and the hormone most directly associated with stress. Recent research suggests that this hormone may act as a key factor linking social environmental stimuli and the onset of sex change by initiating a shift in steroidogenesis from estrogens to androgens. For many teleost fish, sex change occurs as a usual part of the life cycle. Changing sex is known to enhance the lifetime reproductive success of these fish and the modifications involved (behavioral, gonadal and morphological) are well studied. However, the exact mechanism behind the transduction of the environmental signals into the molecular cascade that underlies this singular process remains largely unknown. We here synthesize current knowledge regarding the role of cortisol in teleost sex change with a focus on two well-described transformations: temperature-induced masculinization and socially regulated sex change. Three non-mutually exclusive pathways are considered when describing the potential role of cortisol in mediating teleost sex change: cross-talk between GC and androgen pathways, inhibition of aromatase expression and upregulation of amh (the gene encoding anti-Müllerian hormone). We anticipate that understanding the role of cortisol in the initial stages of sex change will further improve our understanding of sex determination and differentiation across vertebrates, and may lead to new tools to control fish sex ratios in aquaculture.

Cloning and expression analysis of the cytochrome P450c17s enzymes during the reproductive cycle in ovoviviparous Korean rockfish (Sebastes schlegeli)

Cytochrome P450c17 (CYP17, 17α-hydroxylase/17, 20-lyase) plays a critical role in the production of androgens and estrogens in vertebrates. We isolated the full length cDNAs of P450c17-I and P450c17-II from Sebastes schlegeli. The cDNA sequences of P450c17-I and P450c17-II encoded 515 and 533 amino acid residues respectively. The putative P450c17-I and P450c17-II enzymes of Korean rockfish share high sequence identity with that of Japanese flounder (92% and 81%) respectively. Our current study describes that P450c17s of Korean rockfish are mainly expressed in gonads, head kidney and kidney by RT-PCR. Quantitative real-time PCR showed that the expression patterns of Korean rockfish P450c17s were developmental stage-dependency. In addition, the testosterone (T) and gonadosomatic index (GSI) levels further support the important role of P450c17-I during shift in steroidogenesis. Taken together, this study provides information about the Korean rockfish P450c17s characterization and mRNA expression as such helps in further understanding of its function in gonadal development.

Molecular mechanism of P450c17-II (17, 20-lyase) regulating gonad development in femaleCynoglossus semilaevis

Cytochrome P450c17 (CYP17, 17α-hydroxylase/17,20-lyase) is a critical enzyme in the production of androgens and estrogens in vertebrates. A 2102 bp full-length cDNA of P450c17-II (CYP17A2) has been isolated from the ovary of half-smooth tongue sole, Cynoglossus semilaevis which encodes 524 amino acids. The putative P450c17-II enzyme shares higher sequence identity with those of teleosts than with P450c17-I of vertebrate. The similarity between the two types of tongue sole P450c17 was 48%.Semi-quantitative RT-PCR analysis of spatial expression showed the enzyme was specifically expressed in the ovary and the head kidney. However, temporal expression shows that P450c17-II can be found in the brain. Furthermore, temporal expression pattern of P450c17-II in ovary and brain revealed developmental stage-dependency, and ovary P450c17-II expressed remarkably throughout the whole reproductive cycle. Otherwise, the expression pattern of P450c17-II in head kidney indicated negative ovary development-dependence. In addition, combined with our data on P450c17-I, T and E2 levels, the results further endorse the critical role of P450c17-II during shift in steroidogenesis, suggesting that P450c17-I and -II may act together to this physiological process. Based on the present study, we indicate an important role for P450c17-II during ovarian development.

Aromatase Is Expressed and Active in Growing Oocytes of Two Non-Mammalian Vertebrates (Xenopus laevis and Oncorhynchus mykiss).

Numerous reports have previously demonstrated that ovarian follicles synthesize the three major classes of sex steroids (androgens, estrogens, and progestins) in amphibians and fish. Precisely, during late oogenesis, a shift in steroidogenesis occurs from estradiol-17β (E2) to maturation-inducing steroid production by the ovarian follicles. While most studies have focused on the steroidogenic capacity of somatic follicular layers, the oocyte compartment has received far less attention. However, a previous study identified cyp17 activity exclusively in Xenopus laevis oocytes, suggesting that Xenopus oocytes may produce androgens during late oogenesis and particularly during oocyte maturation. Somatic cells within the ovarian follicle synthesize E2 mainly during oocyte growth, also called vitellogenesis. The aim of the present study was to determine if the enzyme responsible for E2 synthesis, the aromatase, was also expressed in Xenopus oocyte during late vitellogenesis. Moreover in order to further investigate whether oocyte contribution to E2 synthesis was a mechanism shared by evolutionary distant non-mammalian vertebrates, we performed similar experiments in a teleost fish, the rainbow trout (Oncorhynchus mykiss) in which follicular steroidogenesis has been thoroughly documented. Using In Situ Hybridization, quantitative RT-PCR and aromatase activity measurements, we demonstrate that ovarian aromatase is not only expressed and active in the follicular cells but also in vitellogenic oocytes of both species. Moreover oocyte aromatase expression and activity decreased during late oogenesis concomitantly with the trend observed in surrounding follicular layers. Nevertheless we identified species features: Aromatase expression and activity measured in Xenopus vitellogenic oocytes correspond to 25% of that in the corresponding whole follicle versus 3% only in rainbow trout oocytes. This lower contribution of the rainbow trout germinal compartment to follicular aromatase activity may reflect existing biological differences between Xenopus and trout which exhibit distinct ovarian development types (asynchronous vs group-synchronous, respectively). Thus, E2 synthesized by the Xenopus oocyte may be directly used or stored by the oocyte itself and would not influence the development of other oocytes. In rainbow trout, as all vitellogenic oocytes develop simultaneously, it is hypothesized that E2 produced by germ and somatic cells is secreted and will control the whole ovarian development. Discrepancies of oocyte contribution between both species may also result from the different methodologies used to collect oocyte content. Altogether our results suggest that oocyte synthesize E2 during its growth in both species. Nevertheless the exact function of E2 production by the oocyte remains to be clarified. Supported by ANR-08-GENM-033 OSCILE. (platform)

Cloning and expression of P450c17-I (17α-hydroxylase/17,20-lyase) in brain and ovary during gonad development in Cynoglossus semilaevis

Cytochrome P450c17 (CYP17, 17a-hydroxylase/17,20-lyase) is a critical enzyme in the production of androgens and estrogens in vertebrates. A 2,469bp full length cDNA of P450c17-I (CYP17A1) has been isolated from the ovary of half-smooth tongue sole, Cynoglossus semilaevis which encodes 509 amino acids. Additionally, a relatively shorter cDNA (1,742bp), a likely result of polyadenylation, was also found. The putative P450c17-I enzyme shares high sequence identity with that of the fathead minnow (73%), zebrafish (71%), the Japanese eel (70%), catfish (70%), tilapia (79%), three-spined stickleback (81%), medaka (79%), dogfish (60%), chicken (65%), rat (47%), and human (49%). Semi-quantitative RT-PCR analysis of spatial expression showed the enzyme was predominantly expressed in the ovaries and the brain. P450c17-I was also detected in the stomach, intestine, gill, spleen, kidney, and head kidney, albeit weakly. Further examination of temporal expression pattern of P450c17-I in ovary and brain revealed developmental stage-dependency. In addition to this our data on T and E2 levels further endorse the critical role of P450c17-I during shift in steroidogenesis. Based on the present study we indicate an important role for P450c17-I during ovarian development. However, further studies are needed at transcriptional regulation level for deeper insights into the physiological functions of P450c17-I.

Ca2+-Calmodulin regulation of testicular androgen production in Mozambique tilapia (Oreochromis mossambicus)

The Ca2+-Calmodulin (CaM) signaling pathway has previously been shown to be involved in the regulation of teleost fish ovarian steroidogenesis. However, a putative role of CaM in testicular steroidogenesis and potential targets has not been examined. To examine whether basal steroidogenesis is modulated by Ca2+ and CaM levels in the testis of Mozambique tilapia (Oreochromis mossambicus) we have incubated testicular fragments in vitro under different conditions and analyzed steroid output. Calcium-free medium with or without EGTA did not affect testicular basal 11-ketotestosterone (11-KT) and testosterone (T) secretion. However, addition of 80μM the CaM inhibitor W7 significantly reduced basal 11-KT, T and androstenedione secretion. Interestingly, the decreased androgen production by 80μM of W7 was accompanied by increased 11-desoxicortisol output and by the activation of cortisol synthesis in the testis, the latter undetected in untreated tissues. However, production of 17,20α-dihydroxy-4-pregnen-3-one was unaltered by W7. This suggests that C17,20 desmolase, 21-hydroxylase and possibly 11β-hydroxysteroid dehydrogenase are targets for CaM. In addition, androgen production was also found to be regulated by the level of cAMP since incubations with forskolin (FK) significantly increased 11-KT and T output. A cross-talk between the cAMP and Ca2+-CaM signaling pathways was detected since W7 administration also decreased FK stimulated androgen production. Altogether, these data show that both basal and cAMP stimulated androgen levels were modulated by intracellular Ca2+-dependent CaM and that possibly Ca2+-CaM determines the shift in steroidogenesis from C21 steroids to androgens.

A role for cytochrome P450 17α-hydroxylase/c17-20 lyase during shift in steroidogenesis occurring in ovarian follicles prior to oocyte maturation

Cytochrome P450 17α-hydroxylase/c17-20 lyase (P450c17) is regarded as one of the key enzymes involved in the steroidogenic shift that occurs prior to oocyte maturation in teleosts. Role of P450c17 in the shift in steroidogenesis during oocyte maturation is a contentious issue even after identification of a novel type of P450c17 that lacks lyase activity. To understand the role of P450c17 in steroidogenic shift explicitly, a full length cDNA encoding p450c17 from ovary of air-breathing catfish, Clarias gariepinus was cloned. p450c17 transiently expressed in COS-7 cells converted progesterone to androstenedione through 17α-hydroxyprogesterone and catfish p450c17 was found to be expressed ubiquitously with relatively higher levels in gonads, brain, kidney and gills. Immunocytochemical analysis revealed the presence of P450C17 in follicular layer of ovarian follicle, interstitial cells and spermatocytes of testis. p450c17 expression and ratio of lyase to hydroxylase was high in preparatory and pre-spawning phases of ovary and low in spawning phase. Expression of p450c17 correlated well with testicular recrudescence with maximum expression in preparatory and spawning phases. Neither protein expression nor lyase/hydroxylase activity changed significantly during hCG-induced oocyte maturation, in vitro and in vivo though mRNA levels increased. These results tend to suggest that the ovarian follicles attains capacity to produce maximum precursor steroid levels before spawning that might contribute to the shift in steroidogenesis.

Expression of 20β-hydroxysteroid dehydrogenase and P450 17α-hydroxylase/c17–20 lyase during hCG-induced in vitro oocyte maturation in snake head murrel Channa striatus

Partial cDNAs encoding carbonyl reductase like 20beta-hydroxysteroid dehydrogenase (20beta-HSD) and P450 17alpha-hydroxylase/c17-20 lyase (CYP17) were isolated from the ovary of snake head murrel and they exhibited high sequence identity to the Nile tilapia and rainbow trout, respectively. A low transcript level of both 20beta-HSD and CYP17 were detected in pre-vitellogenic follicles, while the transcript level was high in full-grown immature follicles. In hCG-induced in vitro oocyte maturation, we found a significant increase in 20beta-HSD transcript level after 2 h. The CYP17 transcripts also showed a considerable increase following hCG-induction compared to saline-treated controls. On the other hand, Western blot analysis demonstrated no significant change in the CYP17 protein level during hCG-induced in vitro oocyte maturation. Taken together, we suggest that in addition to 20beta-HSD, the CYP17 might have a role in the shift in steroidogenesis during meiotic maturation of snake head murrel.

Oxytocin inhibits LH-stimulated production of androstenedione by bovine theca cells

Oxytocin secretion by bovine granulosa cells increases dramatically after the LH/FSH surge. We have shown that oxytocin stimulates progesterone secretion and inhibits FSH-stimulated estradiol secretion in vitro by granulosa cells from bovine preovulatory follicles obtained before the LH/FSH surge. To determine if oxytocin regulates LH-stimulated steroid production by bovine theca interna cells, theca cells were isolated from preovulatory follicles obtained before the LH surge and were cultured for 4 days in the presence or absence of LH (2 or 4 ng/ml), without or with graded doses of oxytocin (125–1000 ng/ml). LH increased accumulation of androstenedione and progesterone. Oxytocin inhibited LH-stimulated androstenedione production, but had no effect on LH-stimulated progesterone production by cultured theca interna. The next objective was to determine if oxytocin regulates LH-stimulated steroidogenesis by modulating the levels of mRNA for steroidogenic enzymes and/or Steroidogenic Acute Regulatory protein (StAR). Low doses of LH alone increased the levels of mRNA for P450 17α-hydroxylase (17α-OH), 3β-hydroxysteroid dehydrogenase (3β-HSD) and cytochrome P450 side-chain cleavage, but not for StAR. In contrast, the effects of oxytocin on LH-stimulated androstenedione production were not associated with changes in the levels of mRNA for steroidogenic enzymes or StAR. These results suggest that oxytocin may play a paracrine role in regulating the follicular/luteal phase shift in steroidogenesis by decreasing androstenedione secretion by theca cells of the ovulatory follicle and that this effect is not mediated by changes in the levels of mRNA for steroidogenic enzymes and StAR.

In vitro steroid production by isolated ovarian follicles of the striped trumpeter

Ovarian follicles from striped trumpeter Latris lineata were incubated in L15 medium alone, or medium supplemented with gonadotropin (GtH) preparations (human chorionic GtH, carp maturational GtH or partially purified salmon GtH), testosterone (T) or 17‐hydroxyprogesterone (17P). Levels of oestrone (E1), 17β‐oestradiol (E2), T, and 17,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20βP) in the medium after incubation were measured by radioimmunoassay. Basal production of E2 was high from previtellogenic follicles, whereas little T was produced. Both T and E2 production increased in response to treatment with GtH or steroid precursors. Vitellogenic follicles showed basal production of both T and E2, and T but not E2 levels generally increased in response to hormone treatment. Preparations containing follicles nearing final maturation showed low basal production of E2 but high production of T. Treatment with steroids resulted in little change in E2 but often very large increases in T production, whereas GtH stimulated lesser increases. 17,20βP production was detectable from incubations of maturing follicles from two out of five fish, and in those two incubations, increased in response to treatment with 17P. E1 was not detectable in any incubations. The results indicate that there is a shift in steroidogenesis from E2 to T production during oocyte development, and provide further evidence that steroid biosynthesis in non‐salmonids is principally regulated by substrate availability.

Differential effects of oxytocin on steroid production by bovine granulosa cells

Oxytocin (OT) and its mRNA are expressed at very low levels in granulosa cells from bovine preovulatory follicles isolated before the LH FSH surge and increase dramatically between the surge and ovulation. We have shown previously that OT stimulates progesterone secretion by granulosa cells obtained before, but not after the gonadotropin surge, suggesting that OT may be involved in the follicular/luteal phase shift in steroidogenesis from estradiol/androgen to progesterone. One objective of this study was to determine if OT affects estradiol as well as progesterone production by utilizing culture conditions that maintain estradiol secretion in vitro. A second objective was to determine if OT regulates steroidogenesis by effects on the levels of mRNA for the steroidogenic enzymes involved in progesterone synthesis, cytochrome P450 side-chain cleavage (P450 scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD), or in estradiol production, cytochrome P450 aromatase (P450 arom). Granulosa cells were isolated from bovine preovulatory follicles and cultured for 3 days with or without OT in medium supplemented with either insulin (1 μg/ml) + 1% fetal calf serum (FCS), which maintains basal estradiol secretion, or low doses of FSH (1 and 2 ng/ml) + 1% FCS, a culture condition that maximizes effects of FSH on estradiol secretion. After the first day of culture, OT stimulated progesterone ( P < 0.01) and inhibited estradiol production ( P < 0.01) in both control and FSH-treated cultures. In contrast, OT had only a small stimulatory effect on the levels of mRNA for P450 scc and 3β-HSD and no effect on mRNA for P450 arom. These findings suggest that: (1) OT plays an autocrine role in regulating the follicular luteal phase shift in steroidogenesis by both increasing progesterone and inhibiting estradiol production and (2) the differential effects of OT on steroid production are not mediated primarily by effects on levels of mRNA for steroidogenic enzymes.

Isolation and expression of rainbow trout (Oncorhynchus mykiss) ovarian cDNA encoding 3β-hydroxysteroid dehydrogenase/Δ5−4-isomerase

A distinct shift in steroidogenesis from testosterone to 17α-hydroxyprogesterone occurs in the salmonid ovarian thecal cell layers immediately prior to oocyte maturation, and is a prerequisite for the production of 17α,20β-dihydroxy-4-pregnen-3-one (maturation-inducing hormone of salmonid fishes) by granulosa cells during oocyte maturation. 17α-Hydroxylase/17,20-lyase cytochrome P-450 (P-45017α) and 3β-hydroxysteroid dehydrogenase/Δ(5-4)-isomerase (3β-HSD) are the two major steroidogenic enzymes involved in the production of 17α-hydroxyprogesterone and testosterone. Using mammalian cDNA probes, we isolated and characterized full-length cDNAs encoding these two enzymes from a rainbow trout (Oncorhynchus mykiss) ovarian thecal cell cDNA library. The cloning of 2.4-kilobase cDNA encoding P-45017α and transient expression of this clone in nonsteroidogenic monkey kidney tumor COS-1 cells have recently been reported (Sakai et al. 1992). We have isolated a 1.4-kilobase cDNA which is hybridized to the mammalian 3β-HSD cDNAs. Expression of this cDNA in COS-1 cells led to the production of an enzyme which is capable of converting dehydroepiandrosterone to androstenedione. In this study, enzymatic activities and expression of rainbow trout ovarian P-45017α and 3β-HSD are discussed in relation of the steroidogenic shift occurring in the ovarian follicle layers.

Levels of messenger ribonucleic acid for cholesterol side-chain cleavage cytochrome P-450 and 3 beta-hydroxysteroid dehydrogenase in bovine preovulatory follicles decrease after the luteinizing hormone surge.

During the follicular/luteal phase shift in steroidogenesis, follicular steroid production changes from predominantly estradiol and androgen secretion before the LH surge to decreased androgen and estrogen and increased progesterone after the LH surge. Our objective was to determine whether changes in progesterone production by the preovulatory follicle are effected via changes in mRNA levels for the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD). Bovine preovulatory follicles were obtained in the early follicular phase (n = 9 follicles), the midfollicular phase (n = 4), or the late follicular phase (after the LH surge, but before ovulation; n = 5). Total RNA extracted from granulosa cells and theca interna at the time of cell isolation or after 24 or 72 h of culture in control or LH-containing medium was subjected to Northern analysis, and autoradiographs were scanned densitometrically. P450scc mRNA levels in granulosa cells were high in the early follicular phase and decreased by 96% after the LH surge (P < 0.05). 3 beta HSD mRNA levels in granulosa cells were 4.2-fold higher in early vs. late follicular phase (P < 0.01). In theca interna, 3 beta HSD mRNA levels were 3.6- and 2.6-fold higher in the early vs. the mid- and late follicular phase (P < 0.05), but levels of P450scc mRNA did not differ significantly with stage of follicular development. After granulosa cells had been cultured for 24 h in control or LH-containing medium, P450scc and 3 beta HSD mRNA had declined dramatically compared to mRNA levels at the time of cell isolation during the early follicular phase (P < 0.01). However, after 72 h in control or LH-containing medium, an increase in P450scc and 3 beta HSD mRNA was observed relative to levels at 24 h (P < 0.01). After 72 h of culture, the signal for P450scc and 3 beta HSD mRNA in granulosa cells exposed to LH was higher than the signal detected in cultures without LH (P < 0.01). Similar changes in message for P450scc were observed in cultured thecal cells. Thus, the previously observed increases in production of progesterone by bovine theca interna and granulosa cells obtained after vs. before the LH surge cannot be explained by an increase in message for P450scc and 3 beta HSD.(ABSTRACT TRUNCATED AT 400 WORDS)

Androstenedione and progesterone concentrations in preovulatory follicular fluid correlate with successful fertilization and cleavage of human oocytes in vitro

To determine if androstenedione (A) and progesterone (P) concentrations in preovulatory follicular fluid (FF) correlate with successful fertilization and cleavage of human oocytes in vitro. A retrospective randomized trial. Hospital department of obstetrics and gynecology. Fifty-five patients, ages 24 to 39 years, with normal menstrual cycles undergoing in vitro fertilization (IVF) for tubal infertility. Multiple follicular development was induced with clomiphene citrate and human menopausal gonadotropin. Relationships among FF steroid hormone, morphological maturity of oocyte-corona-cumulus complexes, and fertilization and cleavage of oocytes. Follicles with mature oocyte-corona-cumulus complexes and unfertilized oocytes contained significantly greater amounts of A (P < 0.05) than those with mature oocyte-corona-cumulus complexes and fertilized ova, indicating the occurrence of atretic changes. Follicles yielding successfully fertilized and cleaved ova had significantly greater amounts of P (P < 0.05) and A (P < 0.01) but similar levels of 17β-estradiol compared with follicles yielding fertilized ova that failed to cleave. Follicles yielding oocytes that cleaved as a result of IVF have both a shift in steroidogenesis from estrogen to progestin accumulation and declining aromatase activity, thus reflecting progressive luteinization of the follicles.

Steroid production by amago salmon ( Oncorhynchus rhodurus) testes at different developmental stages

Developmental changes in the steroidogenic capacity of amago salmon ( Oncorhynchus rhodurus) testicular fragments at six different stages during spermatogenesis and spermiation were examined using 18-hr incubations. Although both basal and chum salmon gonadotropin (SGA)-induced production of 11-ketotestosterone (11-ketoT) and 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOHprog) remained relatively low in testicular fragments isolated in August (GSI 1.06; primarily spermatocytes and spermatids in the testes) and September (GSI 2.33–4.85; many spermatids and spermatozoa in the testes), 11-ketoT production was two to three times higher than 17α,20β-diOHprog production. In mid October (GSI 4.31; primarily spermatozoa in the testes, with a few spermatids remaining), SGA greatly stimulated 11-ketoT production with no further stimulation in late October (GSI 3.54; advanced spermiation). In contrast, SGA dramatically increased 17α,20β-diOHprog production in late October and November (GSI 3.02), coincident with the period of active spermiation. A time course study showed that SGA caused a marked increase in production of 17α,20β-diOHprog within the first 3 hr. Testes collected in August and September produced a considerable amount of 17α,20β-diOHprog in the presence of an exogenous precursor, 17α-hydroxyprogesterone. These results indicate that a shift in steroidogenesis from 11-ketoT to 17α,20β-diOHprog occurs in the amago salmon testis immediately prior to or during the spermiation period and further suggest that mechanisms of gonadotropin action on 17α,20β-diOHprog production in testes differ from those of ovaries ( Y. Nagahama, 1987, Dev. Growth Differ. 29, 1–12).

Quantitative studies of steroid bioconversions in the seminal vesicles of spawning male African catfish, Clarias gariepinus (Burchell), under natural conditions, and of non-spawning catfish under natural and fish farm conditions

1. 1. Steroid bioconversions in the seminal vesicles of Clarias gariepinus were studied quantitatively in vitro by tissue incubations with [ 3H]pregnenolone, [ 3H]androstenedione and [ 14C]11β-hydroxyandrostenedione, respectively. 2. 2. Spawning and non-spawning catfish, collected in the Hula nature reserve in northern Israel during the spawning period, and non-spawning animals, collected from a fish pond in the same region during the same period, were studied. 3. 3. Spawning animals showed a significantly higher production of 17α-hydroxyprogesterone, 5β-pregnan-17α-ol-3,20-dione and 5β-reduced androgens than non-spawning feral and pond catfish, as a result of a significantly increased contribution of the enzymes 5β-reductase and 3α-hydroxysteroid dehydrogenase (HSD). 4. 4. In spawning catfish the concentrations of gonadotropin in blood plasma were also significantly higher than in the plasma of non-spawning feral and pond catfish. This increase in gonadotropin level might have induced the rise in enzyme activity of 5β-reductase and 3α-HSD. 5. 5. It is concluded that the absence of a shift in steroidogenesis towards the production of 5β-reduced steroids may be among the factors preventing spontaneous spawning in male African catfish under husbandry conditions.

Shift in Steroidogenesis in the Ovarian Follicles of the Goldfish (Carassius auratus) during Gonadotropin-Induced Oocyte Maturation: (oocyte maturation/gonadotropin/steroidogenesis/ovarian follicle/goldfish).

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.

Relationship between the preovulatory luteinizing hormone (LH) surge and androstenedione synthesis of preantral follicles in the cyclic hamster: detection by in vitro responses to LH.

Preantral follicles of cyclic hamsters were isolated on proestrus, estrus and diestrus I, incubated for 3 h in 1 ml TC-199 containing 1 microgram ovine luteinizing hormone (LH) (NIH-S22), and the concentrations of progesterone (P), androstenedione (A) and estradiol (E2) determined by radioimmunoassay. At 0900-1000 h on proestrus (pre-LH surge) preantral follicles produced 2.4 +/- 0.3 ng A/follicle per 3 h, less than 100 pg E2/follicle and less than 250 pg P/follicle. At the peak of the LH surge (1500-1600 h) preantral follicles produced 1.8 +/- 0.2 ng P and 1.9 +/- 0.1 A and less than 100 pg E2/follicle. After the LH surge (1900-2000 h proestrus and 0900-1000 h estrus) preantral follicles were unable to produce A and E2 but produced 4.0 +/- 1.0 and 5.0 +/- 1.1 ng P/follicle, respectively. By 1500-1600 h estrus, the follicles produced 8.1 +/- 3.1 ng P/follicle but synthesized A (1.6 +/- 0.2 ng/follicle) and E2 (362 +/- 98 pg/follicle). On diestrus 1 (0900-1000 h), the large preantral-early antral follicles produced 1.9 +/- 0.3 ng A, 2.4 +/- 0.4 ng E2 and 0.7 +/- 0.2 ng P/follicle. Thus, there was a shift in steroidogenesis by preantral follicles from A to P coincident with the LH surge; then, a shift from P to A to E2 after the LH surge. The LH/follicle-stimulating hormone (FSH) surges were blocked by administration of 6.5 mg phenobarbital (PB)/100 g BW at 1300 h proestrus. On Day 1 of delay (0900-1000 h) these follicles produced large quantities of A (2.2 +/- 0.2 ng/follicle) and small amounts of E2 (273 +/- 27 pg/follicle) but not P (less than 250 pg/follicle).(ABSTRACT TRUNCATED AT 250 WORDS)