Comparative analysis of the effects of gonadotropin-releasing hormone agonist on the proliferative activity, apoptosis, and steroidogenesis in cultured porcine granulosa cells at varying stages of follicular growth

This study was conducted to analyze comparative effects of gonadropin-releasing hormone (GnRH) agonist on the proliferation, apoptosis, and differentiated function of cultured porcine granulosa cells from varying follicle stages. Comparative analyses of porcine granulosa cells from varying follicle stages to respond to GnRH agonist were performed in terms of proliferating cell nuclear antigen (PCNA) expression, occurrence of apoptosis, and 17β-estradiol (E2) and progesterone (P) secretion. PCNA expression was examined by the avidin/biotin immunoperoxidase method with a monoclonal antibody to PCNA, and apoptosis was assessed by in situ DNA 3′-end labeling method and DNA fragmentation analysis. E2 and P were measured by radioimmunoassays. The PCNA positive rate of granulosa cells cultured in the presence of GnRH agonist (10−9M) was lower compared with that of cells cultured in the absence of GnRH agonist. However, the apoptosis positive rate was higher, and E2 and P secretion by cultured granulosa cells was lower in the presence of GnRH agonist (10−9M) compared with that in the absence of GnRH agonist. The inhibitory effect of GnRH agonist on PCNA positive rate of cultured cells was prominent in granulosa cells from small and medium but not from large follicles. By contrast, the inhibitory effect of GnRH agonist on E2 and P secretion by cultured cells was prominent in granulosa cells from large but not small and medium follicles. The stimulatory effect of GnRH agonist on apoptosis positive rate of cultured cells was, however, uniform regardless of the stages of follicular growth. These results demonstrate that GnRH agonist exerts diverse actions on granulosa cells over the course of follicular growth. One downregulates granulosa proliferation in immature follicles as well as steroidogenesis in mature follicles, and the other upregulates apoptosis of granulosa cells regardless of the stages of follicular growth.