Regulatory Mechanisms of Granulosa Cell Apoptosis in Ovarian Follicle Atresia

Abstract

Porcine ovary samples were prepared for histochemical and ultrastructural analyses. In situ analysis of DNA fragmentation was performed on histological sections of follieles using the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling TUNEL) method. No apoptotic cells were observed in healthy follicles. In atretic follicles, apoptotic TUNEL staining was seen in scattered granulosa cells located on the inner surface of the follicular wall, but not in cumulus cells, internal or external theca cells, or oocytes. Nuclear condensation, a typical feature of apoptosis was seen only in scattered granulosa cells. The neutral Ca²⁺/Mg²⁺-dependent endonuclease is involved in granulosa cell apoptosis. No endonuclease activity was detected in cumulus cells. An IgM monoclonal antibody (PFG-1) capable of inducing granulosa cell apoptosis was then produced against granulosa cells prepared from healthy antral follicles. Two-dimensional (2D) Western blotting analysis revealed that PFG-1 specifically recognized a cell-membrane protein (PFG-1 antigen, 55 kD, pl 5.9). PFG-1 immunohistochemically reacted with granulosa cells of healthy follicles but not those of atretic follicles. When the isolated granulosa cells prepared from healthy follicles were cultured in medium containing 0.1 μg/ml of PFG-1, the cells underwent apoptosis. These observations indicated that apoptosis occurs in granulosa cells but not cumulus cells in the atretic follicles and that the PFG-1 antigen, a novel cell death receptor, is different from the apoptosis-mediating receptors Fas antigen or tumor neerosis factor receptor 1 (TNFR-1).