Regulatory Mechanisms of Granulosa Cell Apoptosis in Ovarian Follicle Atresia

Abstract

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).

Molecular Characteristics of Porcine Fas-associated Death Domain (FADD) and Procaspase-8

“…Overexpression of pFADD and pprocaspase-8 induced cell death in the HeLa-K cells. To date, we have investigated the follicular atretic mechanisms of follicular atresia in porcine ovaries [18][19][20][21][22][26][27][28][29][30][31] and have shown that g r a n u l o s a c e l l a p o p t o s i s i s a d o m i n a n t phenomenon in follicular atresia. Therefore, we examined whether overexpressed pFADD and pprocaspase-8 can induce apoptosis in the granulosa cells of human, murine, and porcine origin (KGN, KK1 and JC410, respectively).…”

“…Cell death ligand and receptor systems [such as the Fas ligand (FasL)/Fas system and TNF relating apoptosis inducing ligand (TRAIL)/TRAIL-receptor system] mediate apoptosis during follicular aresia in porcine ovaries [18][19][20][21][22][23][24][25][26][27]. In porcine granulosa cells, apoptosis is regulated by a cell death ligand and receptor system, TRAIL/TRAIL-R, as follows [22].…”

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

“…Ovarian sections were stained by the TUNEL method using a commercial kit (Apop Tag; Intergen, Purchase, NY, USA) according to a modification of the method reported previously [6][7][8][9][10] to allow visualization of the apoptotic cells. Briefly, the sections were fixed in 10% neutralbuffered formalin (pH.…”

“…Recent findings have suggested that apoptosis, originally described by Kerr et al [5], is the mechanism underlying ovarian follicular atresia. Ovarian follicular atresia is affected by various factors such as steroids, gonadotrophins and cytokines, but the mechanism that regulates follicle selection during follicular development is not well understood [6][7][8]. Our previous studies of follicular atresia in porcine ovaries demonstrated that degeneration of the atretic follicles can be explained partly by apoptosis of granulosa cells [9,10].…”

“…In the present study, the expression of classic cadherins and β-catenin, which is associated with the membrane-anchored cytoplasmic domain of classic cadherins [13], in developing and/or atretic follicles of porcine ovaries was investigated by immunocytochemical analysis. The principal aims were: first, to determine what types of cadherins are expressed in porcine follicles; second, to investigate the pattern of expression in follicles during follicular atresia, especially in granulosa cells, apoptotic cell death of which is considered to be a key phenomenon in follicular atresia and which contributes to determination of the follicle population in each estrous cycle [6][7][8][9][10]; and third, to determine whether there are any differences in expression of cadherins in differentiating follicular cells.…”