Regulatory mechanisms of granulosa cell apoptosis in porcine ovarian follicle atresia

制备猪卵巢样品进行组织化学和超微结构观察。采用TUNEL法对卵泡组织切片进行DNA片段化原位分析.健康卵泡：颗粒细胞、卵丘细胞、内外卵泡膜细胞、卵母细胞均未见凋亡细胞.闭锁早期：TUNEL染色显示，细胞凋亡的组织化学被认为是分散的颗粒细胞位于卵泡壁的内表面，但不是在卵丘细胞，内部或外部卵泡膜细胞，或卵母细胞。散在的颗粒细胞中可见典型的凋亡特征，即核浓缩，但同一卵泡中也可见超微结构正常的卵丘细胞.闭锁晚期至终末期：散在卵泡壁内表面的颗粒细胞开始凋亡，但卵丘细胞中未检测到TUNEL阳性细胞。双向免疫印迹分析显示PFG-1特异性识别细胞膜蛋白（55 kD，pI 5.9 ; PFG-1抗原）。PFG-1与健康卵泡的颗粒细胞发生化学反应，但不与闭锁卵泡的颗粒细胞发生化学反应。当从健康卵泡制备的分离的颗粒细胞在含有PFG-1的培养基中培养时，通过核形态学、DNA电泳和流式细胞术分析确定细胞发生凋亡。ICE样半胱氨酸蛋白酶的特异性抑制剂可以阻止PFG-1诱导的细胞凋亡过程。PFG-1抗原的组织化学和生物化学性质不同于Fas或TNF受体，PFG-1抗原是一种新的细胞死亡受体。ICE样蛋白酶和Ca^2+依赖性核酸内切酶参与这种新型受体的细胞内信号传递。

Porcine ovary samples were prepared for histochemical and ultrastructural observations. In situ analysis of DNA fragmentation was performed on histological sections of follicles using TUNEL method.1. Healthy follicles : No apoptotic cells were observed among granulosa or cumulus cells, internal or external theca cells, or oocytes.2. Early stage of atresia : Apoptosis demonstrated histochemically by 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. Typical apoptotic features, i.e. condensed nuclei, were seen in the scattered granulosa cells, but cumulus cells with normal ultrastructure were also seen in the same follicles.3. Late to final stages of atresia : Granulosa cells scattered on the inner surface of the follicular wall began to undergo apoptosis, but no TUNEL-positive cells were detected among the cumulus cells.An IgM monoclonal antibody (PFG-1) capable of inducing granulosa cell apoptosis was produced against granulosa cells prepared from healthy antral follicles of pig ovaries. Two-dimensional Western blotting analysis revealed that PFG-l specifically recognized a cell-membrane protein (55 kD, pI 5.9 ; PFG-l antigen). PFG-l immunohistochemically reacted with granulosa cells of healthy follicles but not of atretic follicles. When the isolated granulosa cells prepared from healthy follicles were cultured in medium containing PFG-1, the cells underwent apoptosis as determined by nuclear morphology, DNA electrophoresis and flow cytometric analysis. Specific inhibitors of ICE-like cysteine proteases could stop the apoptotic process induced by PFG-1. Histochemical and biochemical properties of the PFG-l antigen are different from those of Fas or TNF-receptor, and PFG-l antigen is a novel cell death receptor. ICE-like proteases and Ca^<2+>-dependent endonuclease are involved in intracellular signal transmission of this novel receptor.