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.

Fundulus heteroclitus gonadotropins.5: Small scale chromatographic fractionation of pituitary extracts into components with different steroidogenic activities using homologous bioassays.

Fractionation and characterization of gonadotropins (GtH) from Fundulus heteroclitus pituitary extracts were carried out using a biocompatible liquid chromatographic procedure (Pharmacia FPLC system). Chromatographic fractions were monitored for gonadotropic activities (induction of oocyte maturation and steroid production) using homologous follicle bioassays in vitro. Size-exclusion chromatography eluted gonadotropic activity in one major protein peak (Mr approximately 30,000).

Inhibitory action of the gonadopeptide inhibin on amphibian (Rana pipiens) steroidogenesis and oocyte maturation.

In view of recent reports on the production of inhibin- and activin-like proteins in lower vertebrates and their important role during development, we have examined the effects of the gonadopeptide inhibin in the process of oocyte maturation using amphibian (Rana pipiens) fully grown preovulatory ovarian follicles cultured in vitro. In the presence of frog pituitary homogenate (FPH), which stimulates progesterone (P4) levels and the subsequent germinal vesicle breakdown (GVBD), purified porcine inhibin (35-50 IU) inhibited both of these responses in a dose-dependent manner. Inhibin also blocked GVBD initiated by exogenously added P4 in intact as well as denuded oocytes.

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 (17 alpha,20 beta-dihydroxy-4-pregnen-3-one; 17,20 beta P)-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.

Effects of isoquinolinesulfonamide H-8 on Fundulus heteroclitus ovarian follicles: role of cyclic nucleotide-dependent protein kinases on steroidogenesis and oocyte maturation in vitro.

The possible role of cyclic nucleotide-dependent protein kinases in mediating the stimulatory actions of Fundulus heteroclitus pituitary extract (FPE) during ovarian steroidogenesis and oocyte maturation in vitro was investigated. Follicle-enclosed oocytes were cultured in the presence of FPE and/or N-[2-Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), a compound that inhibits protein kinase A (PKA) and cGMP-dependent protein kinase. H-8 alone (0.

Effects of cytoplasmic components upon sperm aster development in Bufo arenarum eggs.

Bufo arenarum oocytes obtained during the winter period, presenting a metabolism similar to that of the differentiated tissues, are not able to form a sperm aster after spermatozoon injection. These oocytes may be considered immature, with respect to the state of their cytoplasm. In the present work, aster formation was induced in winter coelomic oocytes through injection of cytoplasm from summer oocytes, GTP, and EDTA.

Functional heterologous gap junctions in Fundulus ovarian follicles maintain meiotic arrest and permit hydration during oocyte maturation.

The physiological significance of heterologous gap junctions between granulosa cells and the oocyte was investigated in late vitellogenic ovarian follicles of the teleost Fundulus heteroclitus. Lucifer Yellow injected into the oocyte readily passed to the overlying granulosa cells, demonstrating effective dye-coupling. Passage of the fluorescent dye, and hence intercellular communication, was inhibited both by the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and by 1-octanol, known uncouplers of gap junctions in a variety of invertebrate and vertebrate cell types.

Steroidogenesis in Fundulus heteroclitus V.: purification, characterization, and metabolism of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one by intact follicles and its role in oocyte maturation.

In vitro steroidogenesis of ovarian follicles incubated with radioactive precursors or a Fundulus heteroclitus pituitary extract (FPE) was investigated. Steroids were extracted from both the medium and follicular tissue and fractionated by liquid or thin-layer chromatography. A similar pattern of steroid metabolites was obtained with either [14C]pregnenolone or [14C]progesterone as exogenous precursor.

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.

Effect of the isolated germinal vesicle of Bufo arenarum oocytes upon spermatozoa.

Bufo arenarum sperm treated with isolated germinal vesicle stopped motility in a few minutes and lost fertilization capacity. Treated with egg water, it recovered motility and was able to fertilize.

Oogenesis in Fundulus heteroclitus. VI. Establishment and verification of conditions for vitellogenin incorporation by oocytes in vitro.

A procedure was developed for studying vitellogenin (VTG) incorporation by vitellogenic oocytes of Fundulus heteroclitus in vitro. Since homologous VTG can be obtained from this animal only with great difficulty, the use of [32P]VTG from Xenopus laevis was explored as an alternative. Vitellogenic as well as maturational-stage oocytes were found to sequester X.

Steroidogenesis in Fundulus heteroclitus. II. Production of 17 alpha-hydroxy-20 beta-dihydroprogesterone, testosterone, and 17 beta-estradiol by various components of the ovarian follicle.

Fundulus heteroclitus prematurational follicles (1.3-1.4 mm) were dissected into various components and cultured in vitro to examine the type of cells involved in the synthesis of steroids upon F.

Steroidogenesis in Fundulus heteroclitus. I. Production of 17 alpha-hydroxy,20 beta-dihydroprogesterone, testosterone, and 17 beta-estradiol by prematurational follicles in vitro.

In order to understand better the mechanism of gonadotropin action on steroidogenesis in prematurational follicles of Fundulus heteroclitus, follicle synthesis of 17 alpha-hydroxy, 20 beta-dihydroprogesterone (17 alpha-OH,20 beta-DHP), testosterone (T), and 17 beta-estradiol (E2) from a variety of precursors and the maturational response of oocytes were simultaneously followed in vitro. The addition of 25-hydroxycholesterol, pregnenolone, or progesterone to unstimulated follicles increased media 17 alpha-OH,20 beta-DHP, T, and E2, as well as oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner. Inhibition of cholesterol side-chain cleavage by aminoglutethimide blocked 25-hydroxycholesterol-promoted steroid accumulation and GVBD, indicating that 25-hydroxycholesterol does not directly induce GVBD, but rather is metabolized in the follicle to an active steroid (presumably 17 alpha-OH,20 beta-DHP).

Studies on the Mechanism of Action of Estradiol in Regulating Follicular Progesterone Levels: Effects on cAMP Mediated Events and 3β-Hydroxysteroid Dehydrogenase: (ovarian steroidogenesis/cAMP, 3β-hydroxysteroid dehydrogenase/progesterone/estradiol regulation/Rana pipiens).

Previous studies demonstrated that estradiol interferes with pituitary-induced progesterone production and oocyte maturation in cultured amphibian (Rana pipiens) ovarian follicles. To elucidate the mode of action of estradiol in modulating follicular progesterone accumulation we have examined its effects on cAMP-induced progesterone production and enzymatic conversion of pregnenolone to progesterone by 3β-hydroxysteroid dehydrogenase (3β-HSD). Follicular cAMP levels were manipulated with forskolin (an adenylate cyclase activator), isobutyl methyl xanthine (IBMX-phosphodiesterase inhibitor) and exogenously added cAMP.

Cholesterol mediation of progesterone production and oocyte maturation in cultured amphibian (Rana pipiens) ovarian follicles.

Treatment of isolated amphibian ovarian follicles with frog pituitary homogenate (FPH) increases follicular progesterone levels, which, in turn, initiate oocyte maturation. Recent studies have demonstrated that follicular progesterone production requires concomitant protein synthesis at some stage preceding pregnenolone formation. Experiments were carried out to determine whether cholesterol metabolism plays a role in mediating these biochemical and physiological processes.

Sperm Nuclear Transformation and Aster Formation Related to Metabolic Behaviour in Amphibian Eggs.

Bufo arenarum oocyte maturation is related to important modifications in the metabolism of carbohydrates. Such changes involve a different relative participation of the main oxidative routes and are under the influence of seasonal variations of the pituitary activity. Ovulated coelomic oocytes obtained during the winter period, were unable to initiate cleavage after injection of a sperm suspension.

Protein synthesis and steroidogenesis in amphibian (Rana pipiens) ovarian follicles: studies on the conversion of pregnenolone to progesterone.

Previous experiments demonstrated that protein synthesis was involved in frog pituitary homogenate (FPH)-induced follicular progesterone production. In this study the metabolic conversion of pregnenolone to progesterone, and involvement of protein synthesis in this specific step of the progesterone synthetic pathway, was investigated in vitro cultured ovarian follicles of Rana pipiens. Fully grown follicles were incubated with frog pituitary homogenates or exogenous pregnenolone and progesterone content of follicle extracts and medium were measured by radioimmunoassay.

Protein synthesis involvement in regulating pituitary-induced progesterone levels in ovarian follicles of Rana pipiens.

Involvement of protein synthesis in frog pituitary homogenate (FPH)-induced progesterone production and/or accumulation in ovarian follicles was investigated. In amphibians, cycloheximide (C), an inhibitor of protein synthesis, inhibits progesterone and FPH-induced germinal vesicle breakdown (GVBD). However, the site and mechanisms of action of cycloheximide within ovarian follicles have not been elucidated.