Modulation of granulosa cell deoxyribonucleic acid synthesis and differentiation by activin

Abstract

FSH stimulates follicular growth through inducing granulosa cell proliferation. Our working hypothesis is that mitogenesis is facilitated by a locally produced growth/differentiation factor(s) that modulates FSH action in developing granulosa cells. The present study was undertaken to examine the effect of the related gonadal peptides activin and inhibin on granulosa cell proliferation. Monolayer cultures of granulosa cells isolated from preantral/early antral follicles in immature rat ovaries were established by incubation overnight in serum-free medium 199. Treatment was initiated with serum-free medium containing recombinant human (rh) activin and/or rh-inhibin in the presence or absence of FSH. After incubation for 18 h, medium was collected for progesterone determination (as a marker of cell differentiation), and the cell monolayers were incubated for 2 h in the presence of [3H]thymidine to measure DNA synthesis. Activin dose dependently (1-100 ng/ml) stimulated DNA synthesis (minimal effective dose, 1 ng/ml), whereas inhibin or FSH alone was without effect. When activin (1 ng/ml), but not inhibin, was present with FSH, the gonadotropin caused dose-dependent increases in [3H]DNA synthesis over a wide range of FSH concentrations (1-100 ng/ml). This property of activin was unaltered by the additional presence of inhibin (1-100 ng/ml). To analyze the role of cAMP in mediating the mitogenic action of FSH in the presence of activin, experiments were repeated substituting a membrane-permeable cAMP agonist, 8-bromo-cAMP (8br-cAMP; 0.1-3 mM). Similar to FSH, 8br-cAMP had no effect on granulosa cell DNA synthesis in the absence of activin. However, in the presence of activin (1 ng/ml) 8br-cAMP was stimulatory. The dose response to 8br-cAMP revealed a biphasic effect on DNA synthesis and differentiation: DNA synthesis rose to a maximum in the presence of 0.5 mM 8br-cAMP and declined thereafter. Progesterone synthesis only started to increase in the presence of 0.1 mM 8br-cAMP, rising to a maximum at 3 mM 8br-cAMP, at which concentration DNA synthesis was fully suppressed. We conclude that activin induces DNA replication in rat granulosa cells. In the presence of activin, FSH and 8br-cAMP are mitogens. These actions of FSH and 8br-cAMP occur at doses too low to stimulate steroidogenesis, emphasizing the role of intracellular cAMP tone in granulosa cell proliferation and differentiation.

Follicle-Stimulating Hormone (FSH)-dependent Regulation of Extracellular Regulated Kinase (ERK) Phosphorylation by the Mitogen-activated Protein (MAP) Kinase Phosphatase MKP3

“…Although we were unable to identify a positive control for this inhibitor, as rat GCs do not divide in primary culture (45)(46)(47) and, hence, do not expressthecyclinsnecessaryforcyclin-dependentkinase2phos-phorylation (48), this result suggests that CDC25s are not functioning as ERK phosphatases in GCs. Additional indirect evidence that the CDC25 DUSPs are not functioning as ERK phosphatases in GCs is a report that Na 2 VO 3 inhibits CDC25 DUSPs (49), whereas this phosphatase inhibitor does not inhibit the ERK phosphatase in GCs (Fig.…”

FSH signaling pathways in immature granulosa cells that regulate target gene expression: Branching out from protein kinase A

“…Indeed, it is recognized that FSH is not sufficient to stimulate activation of the cyclin D 2 gene and consequent proliferation of rat granulosa cells (reviewed in [23]); rather, activin or another member of the transforming growth factor (TGF) β family plus FSH is required [78][79][80][81] (see Fig. 1).…”

The Parkes lecture: controlled ovarian stimulation in women

“…FSH activates paracrine mechanisms in mammalian granulosa cells through the occupation of cell-surface FSH receptors and the stimulation of cyclic AMPmediated post-receptor signalling (Richards et al, 1998), which leads to a pattern of gene expression that promotes cellular proliferation and steroidogenesis (Yong et al, 1992(Yong et al, , 1994. Genes affected by FSH include those encoding growth-differentiation factors such as members of the activin-TGF-β superfamily, which activate serine-threonine kinase-mediated post-receptor signalling (Dorrington et al, 1988;Gaddy-Kurten et al, 1995;Miró and Hillier, 1995) in adjacent thecal cells, as well as within the granulosa cells themselves. Conversely, LH acts on thecal cells to induce the formation of paracrine factors such as androgens, which are also granulosa cell aromatase substrates, and which exert receptor mediated action on adjacent granulosa cells.…”