丝裂原活化蛋白激酶(MAPK)参与调节斑马鱼卵母细胞第一次减数分裂的恢复

旨在阐明MAPK的激活在斑马鱼(Danio rerio)卵母细胞第一次减数分裂恢复中的作用。用17α,20β双羟孕酮(17α,20β-DHP)单独作用及17α,20β-DHP与U0126(MAPK上游信号MEK阻断剂)共同作用两种方法,分别培养斑马鱼卵母细胞。采集不同培养时间的卵母细胞,经裂解变性后利用SDS-PAGE电泳和Western Blot蛋白质免疫印迹技术,检测MAPK磷酸化变化,并且观察生发泡破裂(GVBD)发生情况。结果表明,斑马鱼卵母细胞在17α,20β-DHP诱发下6h基本发生GVBD,MAPK活性在2h明显升高,一直到6h都保持在较高水平。而加入U0126后,斑马鱼卵母细胞MAPK活性受到明显抑制,同一时间GVBD发生率均低于对照组斑马鱼卵母细胞。以上结果表明,MAPK参与调节斑马鱼卵母细胞第一次减数分裂的恢复,是斑马鱼卵母细胞GVBD正常发生所必需的。     

Androgen regulation of progestin biosynthetic enzymes in FSH-treated rat granulosa cells in vitro

The influence of androgens on the FSH modulation of progestin biosynthetic enzymes was studied $\text{in vitro}$. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 3 days in a serum-free medium containing FSH (20 ng/ml) with or without increasing concentrations (10^?9?10^?6 M) of 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone; DHT), 5α-androstane-3α, 17β-diol (3α-diol), or the synthetic androgen 17β-hydroxy-17-methyl-4,9,11-estratrien-3-one (methyltrienolone; R1881). FSH treatment increased progesterone and 20α-hydroxy-4-pregnen-3-one(20α-OH-P) production by 10.2- and 11-fold, respectively. Concurrent androgen treatment augmented FSH-stimulated progesterone and 20α-OH-P production in a dose-related manner (R1881 > 3α-diol > DHT). In the presence of an inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), the FSH-stimulated pregnenolone (3β-hydroxy-5-pregnen-20-one) production (a 20-fold increase) was further enhanced by co-treatment with R1881, 3α-diol or DHT. Furthermore, FSH treatment increased 4.4-fold the activity of 3β-HSD, which converts pregnenolone to progesterone. This stimulatory action of FSH was further augmented by concurrent androgen treatment. In contrast, androgen treatment did not affect FSH-stimulated activity of a progesterone breakdown enzyme, 20α-hydroxysteroid dehydrogenase(20α-HSD). These results demonstrate that the augmenting effect of androgens upon FSH-stimulated progesterone biosynthesis is not due to changes in the conversion of progesterone to 20α-OH-P, but involves an enhancing action upon 3β-HSDΔ⁵, Δ⁴-isomerase complexes and additional enzymes prior to pregnenolone biosynthesis.     

Serotonin inhibition of steroid-induced meiotic maturation in the teleost Fundulus heteroclitus: Role of cyclic AMP and protein kinases

The transduction of the serotonin (5-HT) signal in Fundulus heteroclitusovarian follicles leading to the inhibition of oocyte meiosis reinitiation (oocyte maturation) in vitro induced by the naturally occurring maturation-inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) was investigated. Steroid-induced oocyte maturation was inhibited by 5-HT in a dose-dependent manner; maximum inhibition (90%) was observed with 10⁻⁴ M 5-HT. Groups of follicle-enclosed oocytes were cultured in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and treated with increasing doses of 5-HT. Serotonin was found to slightly increase the levels of follicular 3′,5′-cyclic adenosine monophosphate (cAMP) in a dose-dependent manner; 10⁻⁴ M 5-HT induced approximately a 3-fold increase in cAMP with respect to the controls. The changes in cAMP were then evaluated in follicles treated with 17,20βP in IBMX-free culture media in the presence or absence of 10⁻⁴ M 5-HT. The exposure of follicles to 17,20βP alone produced a small and transient reduction in cAMP (40%) within 1–3 hr of steroid stimulation, and these early changes in cAMP appeared associated with a high incidence of germinal vesicle breakdown (80% GVBD) by 24 hr of incubation. Under these conditions, treatment of follicles with 5-HT also increased significantly the production of cAMP, and when 5-HT was combined with 17,20βP, the steroid-mediated reduction in cAMP was prevented and the levels of GVBD inhibited by 95%. Meiosis also was reinitiated with either the protein kinase A (PKA) inhibitor H8 or the protein kinase C (PKC) activator PMA, and the 5-HT inhibitory action on GVBD was found to be 100-fold reduced or completely ineffective, respectively. Preincubation of follicles with the PKC inhibitor GF109203x abolished PMA-induced GVBD in a dose-dependent manner, whereas this inhibitor had no effect on 17,20βP-triggered meiotic maturation, indicating that activation of PKC is apparently sufficient but not necessary to reinitiate meiosis. Taken together, these findings suggest that 5-HT may inhibit 17,20βP-induced meiotic reinitiation through the activation of a cAMP-PKA transduction pathway and that PKC possibly induces oocyte maturation by a different pathway than the steroid and thus is not affected by 5-HT. Mol. Reprod. Dev. 49:333–341, 1998. © 1998 Wiley-Liss, Inc.     

Cyclic AMP level and phosphodiesterase activity during 17alpha,20beta-dihydroxy-4-pregnen-3-one induction and theophylline inhibition of oocyte maturation in the catfish,Clarias batrachus

This study directly tested the hypothesis that the induction of oocyte maturation in the catfish Clarias batrachus is followed by a transient decrease in oocyte cyclic AMP (cAMP) level that is due to an increase in phosphodiesterase (PDE) activity. Further, the PDE inhibitor theophylline was used to investigate the possible role of PDE in the maturation-inducing action of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), the physiological maturation-inducing steroid of this catfish species. The results obtained from batches of oocytes taken from the same donor at the same time clearly show a close relationship between dose-dependent induction of germinal vesicle breakdown (GVBD) and PDE activity with a concomitant decrease in cAMP in the oocytes treated with different concentrations of 17alpha,20beta-DP. In contrast, theophylline prevents GVBD and inhibits PDE activity by promoting cAMP accumulation in oocytes. A time-dependent decrease in PDE activity and an increase in cAMP content with a marked inhibition of GVBD were recorded even in oocytes pre-stimulated with 1 microgram/ml 17alpha,20beta-DP for 6 h and then treated with 1 mM theophylline for various times. These results suggest that cAMP plays a key role in the regulation of oocyte maturation in C. batrachus which may be mediated by PDE activity.     

Cyclic AMP-mediated control of oocyte maturation in the catfish, Clarias batrachus (Bloch): effects of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one and phosphodiesterase inhibitors

An increase in the percentage of germinal vesicle breakdown (GVBD) with a corresponding decrease in cAMP was found in the oocytes which were incubated for 36 hr with different concentrations of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP). At its highest concentration (1 microgram/ml), 17 alpha,20 beta-DP induced 91.9 +/- 2.3% GVBD and decreased cAMP level to 0.8 +/- 0.1 pmol/oocyte from 2.9 +/- 0.2 pmol/oocyte (control). The two different known inhibitors of phosphodiesterase viz. 3-isobutyl-1-methyl-xanthine (IBMX) and theophylline inhibited GVBD in vitro and promoted the accumulation of cAMP in a dose-dependent manner irrespective of whether the oocytes were treated for a short duration (2 hr) or for a long duration (36 hr). Evaluation of time course response to 1 mM IBMX or 1 mM theophylline revealed that cAMP levels increased at all the time points when compared with their respective controls and blocked maturation. In contrast, 1 microgram/ml 17 alpha,20 beta-DP not only induced oocyte maturation but also caused an immediate decrease in cAMP within the first 2 hr (from 3.2 +/- 1.3 to 1.3 +/- 0.1 pmol/oocyte) of incubation which was maintained till the end of experiment (36 hr). Likewise, a significant inhibition of GVBD and accumulation of cAMP was recorded even in oocytes pre-stimulated with 1 microgram/ml 17 alpha,20 beta-DP for 6 hr and then treated with different concentrations of IBMX or theophylline. Taken together, these data strongly suggest that in C. batrachus a decrease of oocyte cAMP concentration is a prerequisite for the induction of oocyte maturation, and its increase is associated with the maintenance of meiotic arrest.     

β-Adrenoceptor antagonists reinitiate meiotic maturation in Clarias batrachus oocytes

Effects of β-adrenoceptor antagonists propranolol and alprenolol in the oocyte maturation of the catfish (Clarias batrachus) were investigated under in vitro. Cyclic AMP (cAMP) levels were also measured in the control, propranolol and phosphodiesterase (PDE) inhibitor treated oocytes. When full-grown folliculated oocytes were cultured in vitro in the presence of different concentrations of propranolol or alprenolol, both the substances induced germinal vesicle breakdown (GVBD) in a dose-dependent manner. The maturational effect of alprenolol at the concentration of 1.0 mM was similar to that of the 1.5 mM dose of propranolol inducing more than 88% GVBD. In the time course study, when the oocytes were treated with 1.5 mM propranolol or with 1.0 mM alprenolol for various times, both the antagonists induced more than 80% GVBD after 4 h of incubations and this induction gradually increased with the increased duration of treatments. On the other hand, 1.5 mM propranolol treatment caused a significant decrease in oocyte cAMP which was maintained upto the duration of the study (36 h). When the oocytes were first stimulated by 1.5 mM propranolol or 1.0 mM alprenolol for 4 h and then treated with various doses of cAMP or PDE inhibitors (IBMX and theophylline), all these substances effectively blocked β-adrenoceptor antagonist-induced GVBD. Both these PDE inhibitors promoted the accumulation of cAMP in the oocytes. These results provide the first example of an existence of a cAMP-mediated mechanism of action of β-adrenoceptor antagonists in the induction of oocyte maturation in fish.     

Effects of cAMP forskolin and cyanoketone on invitro oocyte maturation in the catfish,Clarias batrachus

This study investigated the interactive effects of cyanoketone (CK), an inhibitor of 3β-hydroxysteroid dehydrogenase on the effects of cAMP and forskolin (FK) on oocyte maturation inClarias batrachus using an in vitro incubation technique. When the oocytes were incubated in the presence of 1 Μg/ml 17α, 20β-dihydroxy-4-pregnen-3-one[l7α, 20Β-DP, the maturation-inducing steroid (MIS) of this species] for 6h, they matured [85.3 + 1.36% germinal vesicle breakdown (GVBD)] normally after additional incubation for 20–30 h in plain medium. On the other hand, exposure to 1.0 and 8 0 mM of cAMP after MIS stimulation caused significant inhibition of GVBD but lower concentrations (0.1 and 0.5 mM) of cAMP were noninhibitory. However, when the oocytes were preincubated for 1 h with 1 μg/mI CK, a significant inhibition in the percentage of GVBD was recorded including the lower concentrations of cAMP. FK, an activator of adenylate cyclase, could significantly induce GVBD at all of its concentrations (0.1, 0.5, 1.0 and 10.0 μM) in a dose- and time-dependent manner. However, when the oocytes were exposed to 1 μg/ml CK for 1 h, prior to FK stimulation, a complete inhibition of GVBD occurred but when CK treatment was given after the FK stimulation, only a partial inhibition of maturation was observed. Taken together, these data indirectly suggest that FK induces catfish oocyte maturation probably by stimulating follicular production of Δ⁴ steroid ( 17α,20 β-DP)through an adenylate cyclase-c AMP-mediated pathway, a mechanism identical to the gonadotropin-induced oocyte maturation.     

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.     

Ovarian development and serum sex steroid and vitellogenin profiles in the female cultured sturgeon hybrid, the bester

Changes in serum levels of estradiol-17/J (E₂), testosterone (T), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and vitellogenin (VTG), in cultured adult female bester were examined in relation to ovarian development during a 1-year sampling period. Considerable variations in oocyte development were found among fish. Oocytes at previtellogenic stage (≥0–6mm in diameter) generally started to develop concomitantly with the degeneration of the first batch of oocytes. In vitellogenic individuals, ovaries were comprised of more advanced oocytes with diameter ranging from 0–6 to 2–6 mm and in the post-vitellogenic class, oocytes attained their largest size (>2–6mm) while the germinal vesicle was migrating towards the animal pole. Oocytes with a migrating nucleus were maintained during the winter period and massive degeneration started in April–May without germinal vesicle breakdown (GVBD) or ovulation occurring. Seasonal changes in E₂, T and VTG levels were well correlated with the advancement of oogenesis. Their levels increased during vitellogenesis, whereas in the post-vitellogenic (migratory nucleus) stage the levels of E₂ declined from 2–4 ng ml ^?1± to 1–2 ng ml ^?1 and VTG from 4–10 mg ml ^?1 to 0.–0.5 mg ml ^?1 while T levels remained high (50–60 ng ml ^?1). In contrast, serum levels of 17,20?-P were constantly low (less than 0.2 ng ml^?L) throughout the reproductive cycle. These results indicate that the time appropriate for induction of artificial reproduction would be from October–November to April–May when the oocytes are in the late Stages of the development.     

Reproductive biology and endocrine regulation of final oocyte maturation of captive white bass

The ovarian development, and plasma levels of gonadotropin II (GtH II) and sex-steroid hormones at the end of vitellogenesis were examined in captive white bass Morone chrysops. The changes in plasma hormone levels and oocyte morphology associated with gonadotropinreleasing hormone agonist (GnRHa)-induced final oocyte maturation (FOM) were studied. Although plasma 17β-oestradiol (E₂) and oocyte diameter increased, there were no changes in GtH II, testosterone (T), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) or 17,20β,21-dihydroxy-4-pregnen-3-one (17,20β,21-P) in non-hormone-treated females, and no FOM was observed. Treatment with a sustained-release GnRHa delivery system (GnRHa implant) induced two FOM cycles separated by about 24 h, with the release of approximately equal numbers of eggs in each spawn. Plasma GtH II levels were elevated significantly throughout FOM, reaching a maximum of 9·07 ± 1·55 ng ml^?1 in ovulated fish. Both plasma E₂ and T increased soon after the GnRHa treatment, but E₂ declined in fish undergoing germinal vesicle (GV) migration. Plasma T increased further during FOM (7·55 ± 2·87 ng ml^?1), but declined precipitously at ovulation. A surge in plasma 17,20β-P and 17,20β,21-P (4·11 ± 0·97 ng ml^?1 and 3·10 ± 0·77 ng ml^?1, respectively) was observed in females undergoing GV breakdown (GVBD). Based on the involvement of different sex-steroid hormones, FOM was separated into two stages. Early FOM included lipid-droplet coalescence and GV migration, and was associated with elevations in plasma GtH II and T. Late FOM included GVBD and yolk-globule coalescence, and was associated with elevations in plasma GtH II, 17,20β-P and 17,20β,21-P. The results of this study point to the absence of a surge in plasma GtH II as the missing link in the reproductive axis responsible for the failure of captive white bass to undergo FOM at the end of vitellogenesis. Sustained elevation of plasma GtH II via treatment with a GnRHa implant induced two consecutive spawns with an overall egg production two- to eightfold higher than previously obtained from captive broodstocks, and similar to annual egg production Values reported for wild fish.     

Inhibition of oocyte maturation in the mouse: Participation of cAMP,steroid hormones,and a putative maturation-inhibitory factor

The hypothesis that cumulus cells inhibit oocyte maturation by a cAMP-dependent process was tested (R. M. Schultz, R. Montgomery, P. F. Ward-Bailey, and J. J. Eppig (1983). Dev. Biol.95, 294–304.). Treatment of isolated cumulus cell-oocyte complexes with follicle-stimulating hormone (FSH) resulted in a dose-dependent increase in both cumulus cell cAMP levels and in the extent of inhibition of germinal vesicle breakdown (GVBD), the first morphological manifestation of oocyte maturation. Furthermore, it was found that concentrations of a membrane-permeable analog of cAMP, dibutyryl cAMP (dbcAMP), that were below those required for complete meiotic inhibition had a greater inhibitory effect on cumulus cell-enclosed oocytes than on denuded oocytes. Cumulus cell-enclosed and denuded oocytes matured at the same time in the absence of dbcAMP. Ablation of the gap junctions that couple cumulus cells to the oocyte abolished the maturation-inhibitory action of cumulus cells that was promoted either by FSH or low concentrations of dbcAMP. These results are consistent with the hypothesis that inhibition of oocyte maturation is mediated by a factor of granulosa/cumulus cell origin, other than cAMP, which requires cAMP for its activity and/or generation, and an intact intercellular coupling pathway between cumulus cells and the oocyte. A variety of steroid hormones potentiated the FSH-induced inhibition of maturation in cumulus cell-enclosed oocytes. In addition, steroid hormones inhibited maturation in denuded oocytes, but only when oocyte cAMP levels were elevated by cAMP analogs or forskolin. Steroids alone did not inhibit maturation of either cumulus cell-enclosed or denuded oocytes. Moreover, the steroids alone or in combination with FSH did not affect metabolic coupling between the cumulus cells and oocytes, nor did testosterone affect the forskolin-induced level of cAMP in denuded oocytes. Therefore, it is proposed that the oocyte is a site for the synergistic activity of steroid hormones with a cAMP-dependent process in inhibiting maturation. Results of these studies are discussed in terms of the roles of intercellular communication, cAMP, a putative maturation-inhibiting factor, and steroid hormones in the inhibition of maturation of mouse oocytes.     

Characterization of prostaglandin F2α receptor of mouse 3T3 fibroblasts and its functional expression in Xenopus laevis oocytes

Prostaglandin (PG) F_2α increased [³H]thymidine incorporation into quiescent NIH 3T3 cells, stimulated phosphoinositide breakdown, and raised intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner with ED₅₀ values of 2.0 × 10^?8 M, 4.6 × 10^?8 M, and 7.5 × 10^?8 M, respectively. The increase in [³H]thymidine incorporation with PGF_2α was additive with that seen with epidermal growth factor (EGF) or insulin. The peak [Ca²⁺]_i increase with PGF_2α was still obvious in the absence of extracellular Ca²⁺ and was insensitive to islet activating protein (IAP) pretreatment. Membranes prepared from NIH 3T3 cells exhibited a specific binding for PGF_2α, which was sensitive to GTP_γS but not sensitive to IAP pretreatment. Xenopus laevis oocytes injected with NIH 3T3 cell mRNA between 18S and 28S rRNA fractionated by sucrose gradient, expressed a PGF_2α-specific Cl^? current when examined by voltage clamp. This Cl^? current was also insensitive to IAP pretreatment and not affected by extracellular Ca²⁺ concentration ([Ca²⁺]_o). These results indicate 1) that the NIH 3T3 cells expressed a specific PGF_2α receptor which is linked to phosphoinositide-specific phospholipase C (PLC) activation and to mobilization of Ca²⁺ via an IAP-insensitive G-proteins(s), 2) that this PGF_2α receptor may play an active role in the proliferation of NIH 3T3 cells, and 3) that this PGF_2α receptor can be expressed in the oocyte system. © 1993 Wiley-Liss, Inc.     

Gonadal development and sex steroids in a female Arctic charr broodstock

Gonadal development and plasma levels of sex steroids were investigated in female Arctic charr at 3-week intervals over a 12-month period. Circulating levels of oestradiol-17β (E₂), testosterone (T) and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) were measured by radioimmunoassay, and gonadal status assessed through histological examination and measurement of gonadosomatic index (GSI) and frequency distribution of oocyte size-classes. Gonadal recrudescence during March-July was characterized by modest but insignificant increases in plasma levels of E₂ (2–4 ng ml^?1) and T (2–5 ng ml^?1) and recruitment of oocytes into yolk accumulation. Only a small and insignificant rise in GSI and no apparent increase in oocyte diameter occurred during this period, indicating that the rate of yolk formation and oocyte growth was low. Following transformation from stage V (peripheral yolk granule stage) to stage VI (yolk granule migration stage) in late July, the vitellogenic oocytes entered a phase of rapid growth which resulted in a marked rise in GSI until ovulation commenced in late September. Gonadal growth during this period was accompanied by increases in plasma levels of E₂ and T which peaked at 11 ± 1 (mid-August) and 71 ± 5ng ml^?1 (late September), respectively. The levels of both steroids dropped rapidly during final maturation and ovulation, followed by a surge in plasma levels of 17,20β-P which peaked at an average of 74 ± 17 ng ml^?1 in early October. All three steroids returned to basal levels within a month after ovulation, and all steroids, except E₂, remained low until March of the following year. A slight increase in E₂ detected in February and March during the second season may have been associated with recruitment into vitellogenesis of a new generation of oocytes. It is suggested that the abrupt increase in vitellogenesis in late July may reflect a condition-dependent decision to proceed with maturation, once the energy reserves have been repleted during spring-early summer.     

Out‐of‐season production of 17,20β‐dihydroxypregn‐4‐en‐3‐one in the roach Rutilus rutilus

In this study, although the highest production of two physiologically significant progestins in teleosts [17,20β‐dihydroxypregn‐4‐en‐3‐one (17,20β‐P) and 17,20β,21‐trihydroxypregn‐4‐en‐3‐one (17,20β,21‐P)] was observed in the period just prior to spawning in both male and female roach Rutilus rutilus, there was also a substantial production (mean levels of 5–10 ng ml^?1 in blood; and a rate of release of 5–20 ng fish^?1 h^?1 into the water) in males and females in the late summer and early autumn (at least 7 months prior to spawning). During this period, the ovaries were increasing rapidly in size and histological sections were dominated by oocytes in the secondary growth phase [i.e. incorporation of vitellogenin (VTG)]. At the same time, the testes were also increasing rapidly in size and histological sections were dominated by cysts containing mainly spermatogonia type B. Measurements were also made of 11‐ketotestosterone (11‐KT) in males and 17β‐oestradiol and VTG in females. The 3 months with the highest production of 11‐KT coincided with the period that spermatozoa were present in the testes. In females, the first sign of a rise in 17β‐oestradiol concentrations coincided with the time of the first appearance of yolk globules in the oocytes (in August). The role of the progestins during the late summer and autumn has not been established.     

Steroid-induced oocyte maturation in Atlantic croaker (Micropogonias undulatus) is dependent on activation of the phosphatidylinositol 3-kinase/Akt signal transduction pathway

Exposure of fully grown fish and amphibian oocytes to a maturation-inducing steroid (MIS) activates numerous signal transduction pathways to initiate the final stage of oocyte maturation. These events culminate in the activation of maturation-promoting factor and germinal vesicle breakdown (GVBD). In most species, exposure to MIS causes a transient decrease in oocyte cAMP levels. Whether this reduction in oocyte cAMP concentration is sufficient to induce GVBD is unclear. The current study tested the hypothesis that activation of cAMP-independent signal transduction pathways by the naturally occurring MIS, 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), is necessary for GVBD in Atlantic croaker (Micropogonias undulatus) oocytes. Results indicate that although 20beta-S treatment of oocyte membranes significantly reduced cAMP production, incubation of follicles with the cell-permeable cAMP-dependent protein kinase (Prka) inhibitors Rp-cAMP or KT5720 did not promote GVBD in the absence of 20beta-S. Additionally, treatment of follicles with the phosphodiesterase (Pde) inhibitors Cilostamide (Pde3) or Rolipram (Pde4) significantly reduced GVBD, but they were not able to completely block it. In contrast, pharmacologic inhibition of the cAMP-independent phosphatidylinositol 3-kinase (Pik3)/Akt signal transduction pathway using the Pik3 inhibitors Wortmannin or LY294002, or the Akt inhibitor ML-9, blocked 20beta-S-induced GVBD. Finally, mitogen-activated protein kinase (Mapk1/3) activity increased after treatment with 20beta-S; however, inhibition of Mapk1/3 activity using PD98059 or U0126 had no effect on GVBD. These results demonstrate that activation of cAMP-independent signaling pathways, especially the Pik3/Akt pathway, is necessary for 20beta-S-induced GVBD in Atlantic croaker oocytes.     

Cyclic nucleotide content and protein phosphorylation during maturation of Spisula oocytes

Cyclic nucleotide levels in the oocytes of the surf clam Spisula solidissima were measured during germinal vesicle breakdown (GVBD) induced by fertilization. The level of cAMP and cGMP in untreated oocytes was 8.23 ± 0.95 and 4.89 ± 0.39 pmol/10⁶ oocytes. The ratio of cAMP to cGMP ranged from 1.5 to 2.0. The cAMP level in Spisula oocytes fluctuated after fertilization and before GVBD. The cGMP level showed minimal fluctuation, with a tendency to decrease initially followed by a subsequent rise to the basal level in a nonsynchronous manner. These changes were not statistically significant. There was a general increase in protein phosphorylation during the period after fertilization and before GVBD. The greatest increase occurred with proteins of estimated molecular weights of 52, 18, and 12 kD, analyzed by gel electrophoresis and autoradiography.     

Bifunctional enzyme activity at the same active site: Competitive inhibition kinetics with 3α/20β-hydroxysteroid dehydrogenase

20β-Hydroxy-5α-pregnan-3-one (HPO) is a competitive inhibitor of reduction by 3a/20β-hydroxysteroid dehydrogenase (3α/20β-HSD; E.C.1.1.1.53) of 17β-hydroxy-5α-androstan-3-one (DHT; 3α-activity; K_i = 4.6 × 10^?5M) and of 6β-acetoxyprogesterone (6β-AP; 20β-activity; K_i = 4.34 × 10^?5M). HPO and DHT inhibit affinity alkylation of 3α/20β-HSD by 6β-bromoacetoxyprogesterone (6β-BAP). The facts that 1) enzyme 3α-activity and 20β-activity are both competitively inhibited by HPO with practically identical K_i-values, 2) 6β-BAP is solely a 20β-activity substrate for 3α/20β-HSD, 3) one mole of 6β-BAP reacts with one mole of 30/20β-HSD to simultaneously inactivate 3α- and 20β-activity and 4) inactivation of 3α/20β-HSD by 6β-BAP is inhibited by DHT (a Cig-steroid) or HPO (a C₂₁-steroid), support the view that the same active site of 3α/20β-HSD possesses both 3α- and 20β-activity. Bifunctional activity at the same active site is considered for other steroid-specific enzymes in female mammalian reproductive systems.     

Pharmacology of the serotonergic inhibition of steroid-induced reinitiation of oocyte meiosis in the teleost Fundulus heteroclitus

Serotonin (5-HT) was found to inhibit steroid (17α,20β-dihydroxy-4-pregnen-3-one; 17,20βP)-induced resumption of oocyte meiosis (oocyte maturation) in vitro in the teleost Fundulus heteroclitus. Serotonin inhibited both follicle-enclosed and denuded oocytes, which indicates the presence of oocyte-associated 5-HT sensitive sites. The response of oocytes to 5-HT was characterized pharmacologically, i.e., the capacity of serotonergic agonists and antagonists to mimic or block the 5-HT inhibition of the steroid-induced oocyte maturation was assessed by the changes in the percentage of oocyte germinal vesicle breakdown (GVBD). Dose-response curves for each compound were drawn and compared. The rank order of potency among the agonists was: 5-HT > 5-methoxytryptamine > tryptamine = 5,6-diHT = 5-carboxidotryptamine > 5,7-diHT = 5-methoxy-dimethyltryptamine > α-methyl-5-HT > 2-methyl-5-HT. Incubation of ovarian follicles with high doses of some antagonists (mianserin and metergoline) induced oocyte GVBD, although this effect was associated with high levels of oocyte atresia during GVBD or shortly after maturation. Consequently, doses of the antagonist too low to induce GVBD were tested for their ability to block the 5-HT inhibitory action; the rank order of potency was: MDL-72222 = metoclopramide > metergoline > propanolol > ketanserin. Dopamine, acetylcholine, epinephrine, and norepinephrine could also inhibit 17,20βP-induced GVBD, although at doses much higher than those of 5-HT; melatonin and histamine had no effect on oocyte maturation. These results suggest that specific receptors mediate the inhibitory action of 5-HT on the steroid-triggered meiosis resumption. The pharmacological profile of these 5-HT receptors is different from those of any known mammalian 5-HT receptor, although they showed some similarities to the 5-HT_1A, 5-HT₂, and 5-HT₃ receptors, as well as to 5-HT receptors on oocytes of some bivalve molluscs. Mol. Reprod. Dev. 48:282–291, 1997. © 1997 Wiley-Liss, Inc.     

Signaling pathways in ascidian oocyte maturation: the roles of cAMP/Epac, intracellular calcium levels, and calmodulin kinase in regulating GVBD

Most mature ascidian oocytes undergo germinal vesicle breakdown (GVBD) when released by the ovary into sea water (SW). Acidic SW blocks this but they can be stimulated by raising the pH, increasing intracellular cAMP levels by cell permeant forms, inhibiting its breakdown or causing synthesis. Boltenia villosa oocytes undergo GVBD in response to these drugs. However, the cAMP receptor protein kinase A (PKA) does not appear to be involved, as oocytes are not affected by the kinase inhibitor H-89. Also, the PKA independent Epac agonist 8CPT-2Me-cAMP stimulates GVBD in acidic SW. GVBD is inhibited in calcium free sea water (CaFSW). The intracellular calcium chelator BAPTA-AM blocks GVBD at 10?μM. GVBD is also inhibited when the ryanodine receptors (RYR) are blocked by tetracaine or ruthenium red but not by the IP(3) inhibitor D-609. However, dimethylbenzanthracene (DMBA), a protein kinase activator, stimulates GVBD in BAPTA, tetracaine or ruthenium red blocked oocytes. The calmodulin kinase inhibitor KN-93 blocks GVBD at 10?μM. This and preceding papers support the hypothesis that the maturation inducing substance (MIS) produced by the follicle cells in response to increased pH causes activation of a G protein which triggers cAMP synthesis. The cAMP then activates an Epac molecule, which causes an increase in intracellular calcium from the endoplasmic reticulum ryanodine receptor. The increased intracellular calcium subsequently activates calmodulin kinase, which causes an increase in cdc25 phosphatase activity, activating MPF and the progression of the oocyte into meiosis.