Apoptosis in ovarian granulosa cells of cattle: Morphological features and clearance by homologous phagocytosis

Apoptosis is involved in many physiological processes of the ovary, such as recruitment of prenatal germ cells, follicular atresia, ovulation, and luteolysis. Based on the need for the involvement of phagocytic cells to achieve apoptosis clearance and that follicular atresia is triggered by weak apoptotic stimuli, we postulate that granulosa cells engullng apoptotic corpses (ACs) must carry out this macrophagic process. Since apoptosis was early defined in terms of morphological aspects, here we describe apoptosis induced by a GnRH analog (leuprolide acetate, LA) at histological level on bovine granulosa cells (primary culture, CPGB, and an established cell line, BGC-1). We observed two main types of apoptosis. In type A, the whole cell or most of it is compacted into a single large AC that is then engulfed by neighboring cells or simply detached. In type B, small portions of cells, either with or without nuclear material, become ACs that are also phagocytosed. Apoptosis and homologous phagocytosis were confirmed by TUNEL and immunocytochemistry for Bax and active caspase 3. Induction of apoptosis was significant in BGC-1 cells treated for 24 h with 100 nM LA. CPGB cells showed two types of response with different doses of LA. Fetal calf serum was necessary to find apoptosis induced by LA.