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).

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.

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Abstract. Changes of glycoconjugates in granulosa cells during follicular atresia were examined by lectin histochemical and biochemical techniques. Twenty-four lectins were used to visualize the different glycoconjugates of granulosa cells in frozen sections of the pig ovary. Four lectins showed stronger staining of the granulosa cells in atretic than in healthy follicles. Very strong and/or strong staining with Sambucus sieboldiana agglutinin (SSA) was seen in scattered granulosa cells of atretic follicles, but those of healthy follicles were not stained with this lectin. In atretic follicles, very strong-moderate diffuse staining was seen with Aleuria aurantia lectin (AAL), Pisum sativum agglutinin (PSA) and Lens culinaris agglutinin (LCA) in granulosa cells, in contrast with very weak staining in those of healthy follicles. Furthermore, lectin blot analysis with three lectins (SSA, AAL, and PSA) indicated several glycoproteins with increased staining in atretic relative to healthy follicles for these three lectins. Especially, two SSA-positive glycoproteins (62 and 64 kD) of granulosa cells in atretic follicles were specific and characteristic which were not detected in those in healthy follicles. These results suggest that changes of some glycoconjugates in granulosa cells may be involved in granulosa cell apoptosis and follicular atresia.

Abstract. We prepared an IgM monoclonal (PAG-1) antibody against follicular granulosa cells of porcine ovaries. Immunohistochemical reaction of the antibody was detected only in follicular granulosa cells, not in any other ovarian tissues or organs. Western blotting analysis demonstrated that 38-and 42-kD bands were present in the granulosa cell membrane samples prepared from healthy follicles. In the samples from early atretic and progressed atretic follicles, 38-and 64-kD bands were shown. During follicular atresia, the 64-kD band increased. After glycosidase treatment, the 64-kD band shifted to 42-kD, but no change was seen in the 38-and 42-kD bands. The present results indicate that the 42-kD band membrane protein is glucoconjugated during atresia, resulting in a glycoconjugated 64-kD protein appearing in the granulosa cells of the atretic follicles. We hypothesize that the glycoconjugated cell membrane protein (64-kD glycoprotein) may act as a trigger for phagocytosis in neighboring granulosa cells in atretic follicles.