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.

Abstract. Species-specific differences in the process of apoptosis in granulosa and theca layers during follicular atresia in porcine and bovine ovaries were investigated by in situ DNA 3'endlabeling (TUNEL) at the level of individual cells. In porcine ovaries, granulosa cells located on the inner surface of the granulosa layer appeared to undergo apoptosis at the first stage, followed by neighboring granulosa cells. In the contrast, granulosa cells located on the middle region appeared to undergo apoptosis at the first stage in bovine ovaries. In both porcine and bovine ovaries, detachment and degeneration of the granulosa cell layer and fragmentation of basement membrane occurred in the follicles at advanced stage of atresia. In the ovaries of both species, theca interna cells were still intact at the stage of early atresia and then apoptotic cells appeared in the late to final stages of atresia, but no apoptotic cells were observed in theca externa layers during follicular atresia. The present findings indicated that apoptosis occurring in granulosa cells is an initial symptom of follicular atresia in the ovaries of both species, and that the apoptosis inducing factor(s) and/or survival factor(s) for the granulosa cells may be different between porcine and bovine ovaries.

Apoptosis in granulosa cells plays a crucial role in ovarian follicular atresia, but the intracellular regulating mechanism, especially the mitochondrion-dependent apoptosis signalling pathway, is still largely unknown. This study examined whether the mitochondrial pathway is associated with granulosa cell apoptosis during atresia in pig ovaries. Both mRNAs of caspase-9 and apoptotic protease-activating factor 1 (Apaf1), which are major signal transducing components in the mitochondrial pathway, were detected in granulosa cells in healthy, early atretic and progressed atretic follicles by RT-PCR. No changes in the expression of Apaf1 mRNA were seen during follicular atresia, but the expression of caspase-9 mRNA increased during atresia. Apaf1 protein was steadily detected in granulosa cells prepared from healthy, early atretic and progressed atretic follicles by western blot analysis, but high expression of the precursor of caspase-9 (procaspase-9) was detected only in granulosa cells of healthy follicles. Decreased procaspase-9 protein was demonstrated during follicular atresia. Proteolytic activity of caspase-9 increased during atresia, in agreement with the diminution of procaspase-9 protein. Intensive expression of caspase-9 mRNA was demonstrated in the granulosa cells of early atretic and progressed atretic follicles but not in those of healthy follicles. These results indicate that the mitochondrial signalling pathway, which is mediated by Apaf1 and caspase-9, plays a crucial role in determining the fate of granulosa cells during atresia in pig ovaries.

Abstract: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. Key words: Apoptosis, Extracellular Matrix, Granulosa cell, Atretic follicle, Porcine ovary.More than 99% of ovarian follicles undergo atresia at various stages of development in mammalian ovaries [1,2]. Follicular atresia is a key reproductive phenomenon which decided the follicles that will not ovulate in the ovary. Despite its critical role during the recruitment of follicles for ovulation, the mechanisms of the cellular and molecular events are not well understood yet. Despite species-specific differences, it is generally accepted that the earlier stages of ovarian follicular atresia are correlated with disorganization and degeneration of follicular granulosa cells [2,3]. Early studies [4][5][6][7] showed that both enhanced DNA degradation and ladder formation, hallmarks of apoptotic cell death, occur in granulosa cells of atretic follicles, and that apoptosis, originally reported by Kerr et al. [8], plays a key role in mammalian follicular atresia. Our previous detailed studies of the mechanism of follicular atresia in porcine ovaries demonstrated that degeneration of the atretic follicles can be explained in part by apoptosis of granulosa cells [9][10][11][12][13][14][15][16][17][18][19][20][21], and that the alteration of glycoconjugates of cellsurface and intercellular components in granulosa cells during follicular atresia are involved in some processes of ovarian follicular atresia and granulosa cell apoptosis [22][23][24][25].The extracellular matrix (ECM) components play important roles in the maintenance of homeostasis of organs and cells [26]. The relationship between ECM and cells that adhere to it can play important regulatory roles in many basic cellular processes by influencing enzyme activity [27,28] and phospholipid metabolism [29], and by modifying transcriptional and transitional activities of the cell [30]. These...

Abstract. Determination of the expression level and localization of cell adhesion molecules is crucial for understanding the mechanism of maintenance and remodeling of the ovarian follicle structure. We immunocytochemically investigated expression of the cell adhesion molecules, "classic" cadherins and β-catenin, in developing and/or atretic follicles of porcine ovaries. Healthy follicles showed strong staining for cadherin-8 and β-catenin in granulosa cells tightly attached to the basement membrane, and moderate/weak staining was seen on the inner surface of the granulosa cell layer. Strong VE-cadherin expression was seen in a single cell layer attached to the basement membrane in the theca interna layer of healthy follicles. The expression of cadherin-8, β-catenin and VE-cadherin decreased during follicular atresia. No positive staining was observed for R-cadherin, E-cadherin, Tcadherin, BR-cadherin or P-cadherin, and a weak positive reaction for N-cadherin was seen only in the granulosa cells of healthy and early atretic follicles. These findings indicate that cadherin-8, Ncadherin and VE-cadherin have important roles in follicular development and/or degeneration, and that decreases in the expression of these cadherins are involved in follicular atresia in porcine follicles.