Follicle-stimulating hormone enhances somatomedin C binding to cultured rat granulosa cells. Evidence for cAMP dependence

The ovarian granulosa cell has recently been shown to be the site of Somatomedin C (Sm-C) production, reception, and action. To further elucidate the relevance of Sm-C to granulosa cell physiology, we have undertaken to study the regulation of its receptor under in vitro conditions using a primary culture of rat granulosa cells. Granulosa cells cultured without treatment for 72 h displayed limited, albeit measurable, specific Sm-C binding. However, continuous treatment with increasing concentrations of follicle-stimulating hormone (FSH) for the duration of the 72-h incubation period resulted in dose-dependent increments in Sm-C binding (1.7-, 2.9-, 3.9-, and 3.6-fold increases over untreated controls for 50, 100, 180, and 330 ng/ml of FSH, respectively). This apparent up regulatory action of FSH proved time-dependent, with a minimal time requirement of 24-48 h. Granulosa cell Sm-C binding was similarly enhanced following elevation of the intracellular cAMP content by a series of cAMP-generating agonists, inhibition of cAMP-phosphodiesterase activity, or the provision of nondegradable cAMP analogs. Our findings further indicate that high dose forskolin, like FSH, is capable of augmenting Sm-C binding by itself, that a relatively inactive low dose of forskolin synergizes with FSH in this regard, but that combined treatment with maximal stimulatory doses of both agonists does not prove additive. Taken together, these observations indicate that FSH is capable of exerting a stimulatory effect on granulosa cell Sm-C binding and that cAMP, its purported intracellular second messenger, may play an intermediary role in this regard.     

Estrogens augment the stimulation of ovarian aromatase activity by follicle-stimulating hormone in cultured rat granulosa cells

The effects of estrogens on ovarian aromatase activity were investigated in vitro using granulosa cells from immature hypophysectomized estrogen-primed rats. The cells were cultured for 3 days in an androgen-free medium in the presence of follicle-stimulating hormone (FSH), with or without the specified estrogen. After washing, the cells were reincubated for 5 h with 10(-7) M androstenedione, and the formation of estrogens was measured. Estrogen production by control and diethylstilbestrol-treated cells was negligible, while FSH stimulated aromatase activity. Furthermore, concomitant treatment with diethylstilbestrol led to dose-dependent increases in the FSH-induced aromatase activity with an ED50 value of 4 X 10(-9) M and an apparent Vmax value 12- to 16-fold higher than those induced by FSH alone. The direct stimulatory effect of estrogens was time-dependent and was not accounted for by increases in cell protein. Various native and synthetic estrogens also augmented the FSH induction of aromatases (native estrogens: estradiol-17 beta = estrone greater than estradiol-17 alpha greater than estriol; synthetic estrogens: hexestrol greater than moxestrol greater than ethinyl estradiol much greater than chlorotrianisene and mestranol). The effect of estradiol-17 beta was dose-dependent with an ED50 value of 9 X 10(-9) M, which is within the physiological levels of follicular estradiol-17 beta. Although treatment with androgens also enhanced the FSH-induced aromatases, treatment with a progestin (R5020) or a mineralocorticoid (aldosterone) was without effect. Thus, estrogens directly augment the stimulation of granulosa cell aromatase activity by FSH. Follicular estrogens may activate intraovarian autoregulatory positive feedback mechanisms to enhance their own production, resulting in selective follicle maturation and the preovulatory estrogen surge.     

Differential in vivo binding dynamics of somatic and oocyte-specific linker histones in oocytes and during ES cell nuclear transfer

The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional, active processes contribute to the replacement of somatic histones with H1FOO during nuclear transfer. These results provide the first mechanistic insights into the crucial step of linker histone replacement as it occurs during fertilization and somatic cell nuclear transfer.     

Synergistic interactions of somatomedin-C with adenosine 3',5'-cyclic monophosphate-dependent granulosa cell agonists

Recent studies have demonstrated the ability of somatomedin-C (Sm-C) to synergize with follicle-stimulating hormone (FSH) in the activation of cultured rat granulosa cell progesterone biosynthesis as well as the induction of luteinizing hormone (LH) receptors. Neither effect could be attributed to Sm-C-enhanced granulosa cell survival or replication, but could be accounted for, in part, by increased adenosine 3',5'-cyclic monophosphate (cAMP) generation. The present study was undertaken to determine if the synergistic property of Sm-C is FSH-selective and hence limited in relevance to follicular maturation, as well as to clarify further the role of cAMP in Sm-C-amplified agonist action. To this end, the ability of Sm-C to modulate the hormonal action of a series of physiologic as well as pharmacologic granulosa cell agonists was examined in vitro using cultured granulosa cells from immature, hypophysectomized, diethylstilbestrol-treated rats. Concurrent treatment with highly purified Sm-C (50 ng/ml) resulted in marked increases over controls in the LH-stimulated [1 ng human chorionic gonadotropin (hCG)]-and beta 2-adrenergic-stimulated (10(-6) M terbutaline) accumulation of cAMP (3.8- and 2.6-fold, respectively and progesterone (3.2- and 7.4-fold, respectively). Similarly, concurrent treatment with Sm-C also augmented the vasoactive intestinal peptidergic stimulation of granulosa cell cAMP generation (4.1-fold) and progesterone biosynthesis (2.1-fold). In contrast, Sm-C was incapable of enhancing progesterone accumulation in response to stimulation with rat prolactin, a cAMP-independent granulosa cell agonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

The steroidogenic characteristics of primary testicular cell cultures from adult hypophysectomized rats: enhanced formation of C21 steroids

To characterize and clarify the time-related pattern of steroidogenesis in primary testicular cultures from adult hypophysectomized rats, we have determined the pattern of C19 and C21 steroids using novel enzymatic assay techniques that rely on highly specific bacterial hydroxysteroid dehydrogenases. Steroids contained in culture media were separated in a standardized high performance liquid chromatography system and the 17 beta-hydroxy- and 17-oxosteroids were quantified by a transydrogenase assay. The individual 3 alpha-, 3 beta-, 17 beta-, and 20 alpha-hydroxysteroids were in turn measured by enzymatic oxidation. Presumptive steroid identities were confirmed by enzymatic oxidation or reduction to products that were rechromatographed and identified by co-elution with standards. Although human chorionic gonadotropin stimulated an increase in the "adult" hormones, testosterone and 4-androstene-3, 17-dione, on both Days 1 and 11 of culture, the majority of the steroids found, even on Day 1, were 3 alpha-hydroxy-5 alpha-dihydrosteroids rather than delta 4-3-oxosteroids. A specific 5 alpha-reduced, C21 steroid: 5 alpha-pregnane-3 alpha, 20 alpha-diol, increased over time and became the most abundant gonodotropin-stimulated steroid (about 5-fold in excess of testosterone) by Day 11. In contrast, testosterone was the identifying steroid of nondispersed testes from both intact and hypophysectomized rats. Studies with tracer quantities of [3H]pregnenolone in culture confirmed the initial (Day 1) preponderance of 3 alpha-hydroxy-5 alpha-dihydrosteroids, as well as the accumulation with time of 20 alpha-hydroxysteroids. These findings suggest that contrary to expectation, cultured testicular cells from young adult hypophysectomized rats display a relatively atypical steroidogenic pattern. Although the cellular mechanisms underlying the time-dependent accumulation of C21 steroids remain uncertain, these patterns suggest either regressive changes in the original parent cells or the emergence of a population of latent cells. Although of limited utility as a model for examining adult testicular physiology, primary cultures of dispersed whole testes should prove useful in studies of culture-induced phenotypic regression and the attendant alteration at the level of gene expression.