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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.

Previously, we prepared an IgM monoclonal antibody(PFG-1) which specifically recognized a cell-membraneglycoprotein (PFG-1 antigen; 55 kD, pI 5.9),immunohistochemically reacted with granulosa cells ofhealthy follicles but not of atretic follicles, andinduced granulosa cell apoptosis. In the presentstudy, an IgM monoclonal antibody (PFG-3) capable ofinducing granulosa cell apoptosis and an IgGmonoclonal antibody (PFG-4) not capable of inducingapoptosis were produced against granulosa cellsprepared from healthy antral follicles of porcineovaries. Two-dimensional Western blotting analysisrevealed that PFG-3 specifically recognized twocell-membrane proteins (named PFG-3-1 andPFG-3-2/PFG-1 antigens; 42 kD, pI 5.2 and 55 kD, pI5.9, respectively) of healthy granulosa cells, andthat PFG-4 recognized the same two cell-membraneproteins. In atretic granulosa cells, PFG-3-2/PFG-1antigen disappeared. Immunochemical reactions of theseantibodies were only detected in follicular granulosacells but not any other ovarian tissues or organs.PFG-3 and PFG-4 immunohistochemically reacted withgranulosa cells of healthy and atretic follicles. Whenthe isolated granulosa cells prepared from healthyfollicles were cultured in medium containing PFG-3,the cells underwent apoptosis, and co-incubation withPFG-4 inhibited PFG-3-inducible apoptosis. Theseobservations suggested that PFG-3-2/PFG-1 antigen isa novel cell death receptor which is different fromthe apoptosis-mediating receptors (Fas/Apo-1/CD95 orTNF receptor), and that PFG-3-1 antigen may act as adecoy receptor and inhibit apoptotic signal transmission.

The sugar chains in cellular glycoconjugates have many biological functions. Extensive morphological development and remodeling occur in the ovary of female animals. This caused us to study glycobiological characteristics of ovarian cells, particularly granulosa cells that undergo apoptosis during follicular atresia. The lectin Sambucus sieboldiana agglutinin (SSA) specific for Siaalpha2,6Gal/GalNAc showed positive staining for granulosa cells only in atretic follicles of porcine ovaries by lectin histochemistry. Lectin blot analysis for SSA demonstrated specific glycoproteins only in atretic follicles. Furthermore, we performed analysis of backbone structures of SSA-positive glycans carried by granulosa cell glycoproteins increased during atresia by glycosidase treatment. Most of these structures were Siaalpha2,6Galbeta1,4GlcNAc on complex-type N-glycans, suggesting that only ST6Gal I of four distinct alpha2,6-sialyltransferases catalyzes alpha2,6-sialic acid transfer in most of the increased glycoproteins of granulosa cells during follicular atresia. Reverse transcription-polymerase chain reaction analysis demonstrated that the expression of ST6Gal I mRNA was up-regulated in granulosa cells during atresia. These results suggested that the alteration of glycoconjugates by ST6Gal I in granulosa cells during atresia is involved in some processes of ovarian follicular atresia and granulosa cell apoptosis.

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).