Previous studies on the molecular basis of hormone action during granulosa cell differentiation in vitro have shown the importance of growth factors and peptide regulators, as well as gonadotropic hormones in the control of ovarian follicular maturation. Several hypothalamic peptides, including GnRH, vasopressin, and CRF have been found to act as local regulators of gonadal function. The 43 amino acid hypothalamic polypeptide, growth hormone releasing factor (GRF) is found in several peripheral sites as well as in the brain, and acts on a common VIP/GRF receptor in several tissues as well as on the specific GRF receptor of somatotrophs in the anterior pituitary gland. Treatment with GRF has been shown to stimulate follicular development when administered with FSH to infertile women who are resistant to conventional gonadotropin therapy. The synergistic action of GRF on ovarian function could reflect the known actions of growth hormone and IGF-I in the granulosa cell, but the presence of GRF in the gonads, and in rat and human ovarian follicular fluid, suggests that local actions of the hypothalamic peptide could also be involved in this process. In cultured granulosa cells, GRF and its potent analogs and derivatives were found to bind to high-affinity receptors that also recognized VIP. Both GRF and VIP stimulated cAMP production, and markedly potentiated FSH-induced cAMP production, as well as progesterone production, aromatase activity, and the expression of LH receptors. These findings indicate that GRF acts on a common VIP/GRF receptor to promote gonadotropin-induced maturation of ovarian granulosa cells, and suggest that the peptide's action in gonadotropin-resistant infertile women is at least partly mediated by this mechanism. In other studies, procedures for the measurement of renin and angiotensinogen gene expression in the granulosa cell were evaluated for use in studies on hormonal regulation of these components of the ovarian renin-angiotensin system in vitro. While solution hybridization procedures were applicable to the detection of mRNAs for renin and angiotensinogen, the low levels of these mRNAs necessitated the use of more sensitive methods for their quantitation in cultured granulosa cells. This is being performed by the polymerase chain reaction, using conditions optimized for amplification of granulosa-cell mRNA, employing an appropriate internal standard to permit quantitation of the amplified sequences and their changes during stimulation of granulosa cell maturation in vitro by gonadotropins and other regulatory factors.
