| In ordor to clarify and reveal (1) the origination of ovarian follicular atresia and follicular degeneration in different ages of buffalo cows, (2) the morphological and biochemical characteristic of granulosa cells (GCs) of healthy dominant follicle(hDF) and atretic dominant follicle (aDF) in buffalo,(3)the death characteristic of GCs and preantrum follicles cultured in vitro,(4) the effects of several antioxidants (including vitamin C(VC), N-acetyl-L-cysteine(NAC), superoxide dismutase(SOD), catalase(CAT), )and oxide(hydrogen peroxide.H2O2) on the viability of GCs cultured in vitro, several experiments were conducted in this study and corresponding results were gained as follows: (1) the follicular cell apoptosis was observed on ovarian slides from 5-month-old fetal buffalo, prepuberty and adult buffalo cows by paraffin wax embedded, HE stained and DNA in situ end labeling(TUNEL). Apoptosis of a large number of follicular cells and some oocytes is widely and evenly existed among preantra in ovarian cortex area, single cell apoptosis occurs dominantly among living cells. Buffalo cows of Prepuberty have the all kids of follicles as adult except for graafian follicle. There are 2 patterns of apoptosis in the antrum follicle of prepuberty and adult buffalo cows, the first pattern is single cell apoptosis among living cells in the membrane GC layer, the characteristic morphological features of apoptosis include the cell with a single smallnucleus with distinctly dense homogeneously staining chromatin and the cell containing multiple smaller, densely staining nuclear fragment; the second pattern is many apoptotic bodies with different shapes and sizes appearing in the antrum of follicle, some apoptotic bodies are much bigger than normal GC.(2) apoptotic GCs were existed in both hDF and aDF, but aDF has much more apoptotic GCs than hDF. GC apoptosis is characterizied by cell with smaller size and dense nuclear under HE staining or fluorescence staining examination. DNA electrophoresis of GCs from hDF and aDF reveals DNA integrity and DNA cleavage ("smear" but not "ladder") feature, respectively. Very few apoptotic GCs are found by flow cytemetry(FCM) examination of GCs from aDF, a DNA peak appears far before G0/G1 peak in diagram. The DNA cleavage of GCs in aDF occurs randomly from the results of DNA electrophoresis and FCM examination. (3) GCs apoptosis induced by serum withdrew in culture in vitro was demonstrated by HE staining and TUNEL examination; Under electron microscope, the apoptotic oocyte from preantrum cultured and induced in vitro by serum withdrew has several big nuclear frgments with chromatin condensation, apoptotic GCs contain some distinct endoplasmic reticulum structure with whorl shape. (4) the low concentrations of VC, NAC, SOD, CAT, and H2O2 have an inhibitory action to apoptosis of GCs induced by serum withdrew in culture in vitro, and the high concentration of them decrease the viability of GCs. Take together, these results suggest that apoptosis of follicular GCs and oocyte are involved in follicular atresia in buffalo cows, this phenomenon is widely existence on the ovaries from 5-month-old fetal, prepuberty to adult buffalo cows, and that the apoptosis occurs for short time at first and then secondary necrosis develops for long time during the ovarian follicular atresia in buffalo cows. The apoptosis easy occurs during the culture of GCs andpreantum follicles in vitro, antioxidants with proper concentration can increase the GCs viability.
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