Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism

Abstract

Recent biochemical studies have suggested that apoptotic cell death is the molecular mechanism underlying the degeneration of ovarian follicles during atresia. Using a sensitive autoradiographic method for the detection of DNA fragmentation, we studied apoptosis in ovarian granulosa cells or intact follicles placed in serum-free culture as model systems to elucidate the hormonal regulation of atresia. Immature rats (25 days old) were primed for 2 days with 10 IU equine CG to induce a homogeneous population of mature preovulatory follicles. Granulosa cells isolated from these follicles contained predominantly intact high mol wt DNA. However, a time-dependent, spontaneous onset of internucleosomal DNA fragmentation characteristic of apoptotic cell death occurred in granulosa cells during culture. Treatment of granulosa cells with epidermal growth factor (EGF), transforming growth factor-α (TGFα), or basic fibroblast growth factor (bFGF) inhibited the spontaneous onset of apoptotic DNA cleavage found during culture by 40-60%. In contrast, insulin-like growth factor I, insulin, TGFβ and tumor necrosis factor-α were ineffective. Likewise, activation of the protein kinase A or C pathways with forskolin or phorbol 12-myristate 13-acetate, respectively, did not prevent the onset of DNA fragmentation, although inclusion of a tyrosine kinase inhibitor (genistein) completely blocked the ability of EGF, TGFα, and bFGF to suppress apoptosis in granulosa cells. Similar to cultured granulosa cells, a spontaneous onset of apoptosis was also observed to occur in isolated preovulatory follicles during culture. Furthermore, treatment of follicles with EGF or bFGF inhibited the spontaneous initiation of apoptosis, and the suppressive effects of these growth factors were also attenuated by co-treatment with genistein. Our findings indicate that: 1) both granulosa cells and follicles cultured under serum-free conditions undergo a spontaneous onset of apoptosis; and 2) EGF, TGFα, and bFGF, acting through the tyrosine kinase pathway, are potential physiological inhibitors of apoptotic cell death in the ovary. Coupled with the sensitive autoradiographic method for analysis of apoptotic cell death, serum-free cultures of granulosa cells and follicles provide useful models to elucidate the role of growth factors and other hormones in modulating follicle atresia, as well as to study the basic mechanisms that regulate apoptosis. Copyright © 1992 by The Endocrine Society.