Growth hormone releasing factor and vasoactive intestinal peptide stimulate rat granulosa cell plasminogen activator activity in vitro during follicular development

Abstract

Growth hormone-releasing factor (GRF) and vasoactive intestinal peptide (VIP) are two structurally homologous peptides sharing common target cell receptor and known to enhance FSH-induced steroidogenesis of undifferentiated granulosa cell in vitro. Although VIP, has been reported to stimulate plasminogen activator (PA) activity in rat granulosa cells, our knowledge on the actions and interactions of these two peptides with FSH in the regulation of rat granulosa cell PA system during follicular development remains incomplete. Undifferentiated and differentiated rat granulosa cells from pre-antral (DES-treated rats) and antral (eCG-treated rats) follicles, respectively, were cultured in a chemically defined medium in the absence and presence of FSH (400 ng/ml), GRF (10 −8–10 −5 M) and/or VIP (10 −9–10 −5 M). Net secreted (PAs) and cell-associated (PAc) PA activities was measured by the fibrinolysis assay and characterized by the fibrin overlay method. Granulosa cell differentiative (progestin secretion) and proliferative (DNA synthesis) responses were analyzed by radioimmunoassay and [ 3H]thymidine incorporation, respectively. Both GRF and VIP stimulated PAs and PAc activities in a concentration-dependent manner in 24-h cultures of granulosa cells from the two stages of follicular development. They (10 −5 M) enhanced FSH-stimulated PAs activity in granulosa cell cultures of pre-antral follicles, with GRF being more effective than VIP. On the contrary, only GRF (10 μM) potentiated FSH-induced PAs and PAc activities in cultures of granulosa cell from antral follicles. The stimulation of PA activity by these agonists decreased with the duration of culture irrespective of the stage of follicular development. This decrease in PA activity was not associated with a change in PA inhibitor activity. Whereas two enzyme activities corresponding to 55 and 30 kDa were observed in culture of undifferentiated granulosa cells, only the high molecular weight species was detectable in the differentiated cells and was regulated by the gonadotropin and the peptides. Whereas GRF and VIP (1 μM) enhanced basal and FSH-stimulated progesterone (P) and 20α-dihydroprogesterone (20α-OH-P) secretion by undifferentiated granulosa cells, only basal progestin production were stimulated by the peptides in differentiated cells. GRF, but not VIP, stimulated granulosa cell DNA synthesis, a response independent of the stage of follicular development and markedly suppressed by FSH. The earlier demonstration of GRF and VIP secretion by ovarian cells and the presently observed influence on the proliferative and differentiative responses of granulosa cells to these peptides in vitro suggest that they play an important role in the intra-ovarian regulation of follicular development.