Interactions of Apoptosis and Extracellular Matrices in Granulosa Cells of Atretic Follicles in Porcine Ovaries.

Abstract

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...

Changes in Cell Adhesion Molecules during Follicular Atresia in Porcine Ovaries.

“…Strong expression of VE-cadherin was observed in a single cell layer directly attached to the basement membrane. As previously reported, ECM components of the follicular basement membrane may act as survival factors for follicular granulosa cells and theca interna cells directly a t ta ch ed to t he ba se men t m e mb ra ne [ 25 ] . Therefore, it is presumed that contact between the basement membrane and granulosa cells or theca interna cells has an essential role in maintaining growth and development of healthy follicles, and loss of contact induces apoptotic degeneration in follicular cells.…”

“…Many studies of follicular atresia have revealed the morphological and biochemical characteristics of the atretic follicles, which include detachment and degeneration of the granulosa cell layer [2][3][4], fragmentation of the basal membrane of the follicles [25], reduced DNA synthesis [26] and decreased estrogen production [27]. Recent studies h a v e s u g g es te d t h a t a po pt o s i s i s t h e k e y mechanism underlying ovarian follicular atresia [28], and have confirmed that granulosa cells first undergo apoptosis and that no apoptotic cell death occurs in cumulus cells in porcine atretic follicles [6].…”

Species-specific Differences in Apoptotic Cell Localization in Granulosa and Theca Interna Cells during Follicular Atresia in Porcine and Bovine Ovaries.

“…A substantial number of studies of follicular atresia have been reported, and they have revealed the morphological and biochemical characteristics of the atretic follicles, which include detachment and degeneration of the granulosa cell layer [2,3], fragmentation of the basal membrane of the follicles [15], reduced DNA synthesis [16] and decreased estrogen production [17]. Recent findings have suggested that apoptosis is the key mechanism underlying ovarian follicular atresia [18], and have confirmed that granulosa cells first undergo apoptosis and that no apoptotic cell death occurs in cumulus cells in porcine atretic follicles [11].…”

“…On the other hand, larger numbers of proliferating cells were mainly seen in the outer region of the granulosa layer in healthy porcine follicles, and the granulosa cells located in the outer surface were in direct contact with the basement membrane. One likely candidate as a survival factor(s) is an extracellular matrix (ECM) component(s) in the basement membrane of follicles [15]. A previous in vitro study reported by Aharoni et al [29] revealed that ECM components of the basement membrane protected granulosa cells from apoptosis.…”

TRAIL-Decoy Receptor-1 Disappears in Granulosa Cells of Atretic Follicles in Porcine Ovaries.

“…Frozen sections of follicular sections of pig ovaries were prepared as described previously [38][39][40][41][42]. Briefly, individual preovulatory antral follicles, 3-5 mm in diameter, were dissected from ovaries obtained from mature sows at a slaughterhouse.…”