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We prepared IgM and IgG (PFG-5 and PFG-6, respectively) monoclonal antibodies against granulosa cells prepared from healthy antral follicles of porcine ovaries. PFG-5 antibody specifically recognized two cell-membrane proteins (PFG-5 antigen: 55 kD, pl 5.9, and PFG-6 antigen: 42 kD, pl 5.2), and PFG-6 antibody recognized PFG-6 antigen. Immunochemical reactions of these antibodies were only detected in follicular granulosa cells but not any other ovarian tissues for organs. Both antigens were detected in granulose cells of healthy follicles, but PFG-6 antigen disappeard in granulosa cells of atretic follicles. When the isolated granulosa cells prepared from healthy follicles were cultured in medium containing PFG-5 antibody, the cells underwent apoptosis, and co-incubation with PFG-6 antibody inhibited PFG-5 antibody inducible apoptosis. These observations suggested that PFG-5 antigen is a novel cell death receptor, which is different from well-known apoptosis-mediating receptors (Fas or tumor necrosis factor receptor), and that PFG-6 antigen may act as a decoy receptor and inhibit apoptotic signals through PFG-5 antigen.

The process of apoptosis of follicular granulosa cells in porcine ovaries during follicular atresia was investigated by in situ DNA 3'end-labeling at the level of individual cells. Histochemical changes in the follicular basement membrane (BM) were visualized by immunofluorescent staining of BM extracellular matrix (ECM) components, i.e. type IV collagen, laminin and fibronectin. At the first stage, granulosa cells located on the inner surface of the granulosa layer appeared to undergo apoptosis, followed by neighboring granulosa cells. No apoptotic granulosa cells making tight contact with intact BM were observed. Detachment and degeneration of the granulosa cell layer and fragmentation of BM occurred in follicles at the advanced stage of atresia. Finally, intermittent structures of BM and subsequent invasion of macrophages and fibroblasts were observed. Therefore we concluded that granulosa cell apoptosis is an initial symptom of follicular atresia in the porcine ovaries, and the degradation of BM follows granulosa cell apoptosis in the pig. Our results suggest that ECM components of follicular BM act as survival factors on follicular granulosa cells in porcine ovaries.

The present study was designed to investigate the immunohistochemical localization and distribution of basic fibroblast growth factor (bFGF) in the goat pituitary gland. We examined various fixatives for tissue preparation, and confirmed that methyl Carnoy-fixed tissues were associated with a better preservation of bFGF. After fixation, paraffin- embedded tissue sections were prepared, and were stained immunohistochemically with mouse monoclonal antibody against bovine bFGF. Approximately 30% of parenchymal cells in the pars distalis of the goat pituitary gland displayed strong immunoreactivity against the bFGF antibody. Endothelial cells occasionally showed positive immunoreactivity. No immunopositive cells were found in either the pars intermedia of the pituitary gland or posterior lobes. Based on analysis of serial sections stained with antibodies against pituitary hormones (luteinizing hormone and adrenocorticotropic hormone), bFGF-positive cells appear to be a subpopulation of gonadotrophs and or corticotrophs. The present findings indicate that immunohistochemical reactivity for bFGF may be partially attributed to differences in fixatives and that bFGF may play critical roles in the goat pituitary gland.