Follicle stimulating hormone-induced DNA synthesis in the granulosa cells of hamster preantral follicles involves activation of cyclin-dependent kinase-4 rather than cyclin d2 synthesis

Although cyclin D2 mRNA synthesis precedes gonadotropin-induced DNA synthesis in quiescent granulosa cells in culture, it is unclear whether a similar mechanism exists for the granulosa cells of growing preantral follicles in cyclic animals. The objective was to evaluate whether the synthesis of cyclin D2 protein was a prerequisite for FSH-induced DNA synthesis in the granulosa cells of intact preantral follicles of cyclic hamsters. Preantral follicles from cyclic hamsters were cultured in the presence or absence of FSH, and cell cycle parameters were examined. FSH stimulated cyclin-dependent kinase (CDK)-4 activity by 2 h and DNA synthesis by 4 h without altering the levels of cyclin D2 in the granulosa cells. The FSH effect was mimicked by epidermal growth factor administered in vivo. Although FSH increased the levels of cyclin D2 mRNA, it also stimulated the degradation of cyclin D2 as well as p27(Kip1) and p19(INK4) proteins. FSH activation of CDK4 was mediated by cAMP and ERK-1/2. In contrast to granulosa cells in intact follicles, FSH or cAMP significantly increased cyclin D2 protein levels in cultured granulosa cells but failed to induce DNA synthesis. Collectively, these data suggest that granulosa cells of preantral follicles, which are destined to enter the S phase during the estrous cycle, contain necessary amounts of cyclin D2 and other G1 phase components. FSH stimulation results in the formation and activation of the cyclin D2/CDK4 complex leading to DNA synthesis. This mechanism may be necessary for rapid movement of follicles from preantral to antral stages during the short duration of the murine estrous cycle.     

Evidence that functional interactions of CREB and SF-1 mediate hormone regulated expression of the aromatase gene in granulosa cells and constitutive expression in R2C cells

The proximal promoter of the rat aromatase CYP19 gene contains two functional domains that can confer hormone/cAMP inducibility in primary cultures of rat granulosa cells and constitutive expression in R2C Leydig cells. Region A contains a hexameric sequence that binds steroidogenic factor-1 (SF-1). Region B contains a CRE-like sequence that binds CREB and two other factors, X and Y. To determine if CRE binding factors X and Y had overlapping functions with CREB, and to determine if the CREB and SF-1 binding sites exhibited functional interactions in the context of the intact promoter, mutations within the CRE and hexameric SF-1 binding site were generated. Mutations within the CRE showed that CREB but not factors X and Y mediated cAMP-dependent activity of chimeric transgenes in primary granulosa cell cultures. Granulosa cells transfected with constructs that bound CREB but not SF-1 (or the converse) resulted in a loss of approximately 50% cAMP-dependent CAT activity. Transgenes that did not bind CREB or SF-1 exhibited no cAMP-dependent CAT activity. When these same constructs where transfected into R2C Leydig cells, mutation of either the CREB or SF-1 binding sites resulted in a greater than 90% loss of CAT activity. Western blot and immunocytochemistry analyses revealed that the amount of phosphorylated CREB increased in response to hormone/cAMP in granulosa cells and was high in R2C Leydig cells, coinciding with expression of the transgenes and endogenous aromatase mRNA in each cell type. Therefore, in both cell types the aromatase promoter is dependent upon a functional CRE and the presence of phosphoCREB. The CREB and SF-1 binding sites interact in an additive manner to mediate cAMP transactivation in granulosa cells, whereas they interact synergistically to confer high basal transactivation in R2C Leydig cells. Taken together, the results indicated that the molecular mechanisms or pathways that activate CREB, SF-1 or their interaction are different in granulosa cells and R2C cells.