The Effects And Mechanism Of Heat Stress On Granulosa Cell Apoptosis And Follicular Atresia In Mouse Ovary

Heat stress is known to alter follicular dynamics and granulosa cell function, and may contribute to the diminished reproductive efficiency commonly observed in mammals during the summer. Although several investigators have studied heat-induced ovarian injury, few reports have focused on the effects of chronic heat stress on ovarian function and the molecular mechanisms through which it induces ovarian injury.In Exp.1, we investigated the effects of acute heat stress on the development of small and medium follicles in the mice ovaries. A total of 20 female CD-1 mice of 7 weeks old were assigned to a control or heat-stressed treatment. After superovulation, the mice were housed at a constant 25℃ with ambient humidity for 6 d (n=10) or at a constant 37℃ with 50%-60% relative humidity for 10 h per d for 6 d (n=10). Everyday, before and after exposure to heat treatment the body weight and rectal temperature were recorded. After a 6-day heat treatment, the mice were superovulated, and their eggs were count and analyzed for the percent of abnormalty, and their ovaries were analyzed for follicular atresia. The results showed that the body weight of heat-stressed mice decreased compared with control mice, while the rectal temperature and the number of superovulation were increased in heat-stressed mice than in control mice.In Exp.2, the mechanisms of chronic heat stress on follicular atresia in mice ovaries were investigated. A total of 48 female CD-1 mice were assigned to a control or heat-stressed treatment. After exposure to a constant temperature of 25℃ for 7,14,21 or 28 d (n=6) or to 42℃ for 3 h per d for 7,14, 21 or 28 d (n=6), the mice were euthanized and their ovaries were analyzed for follicular atresia, granulosa cell apoptosis, changes in the abundance of HSP70, aromatase and Bim protein and serum concentrations of estradiol. The results showed that the body weight and food intake of heat-stressed mice decreased compared with control mice while the concentration of estradiol in serum was lower in heat-stressed mice than in control mice. Compared with control mice, the percentage of atretic follicles and the number of antral follicles with severe apoptotic signals were increased after 21 d of heat-stressed treatment. HSP70 protein was more abundant in heat-stressed mice than control mice.In Exp.3, the mechanism of heat stress on follicular atresia and granulosa cell apoptosis was investigated. Firstly, the expression of HSP70 and aromatase was quantified in antral follicles cultured in vitro at 37 or 42℃ for 24 h. Sencondly, granulosa cells maintained at 37 or 42℃ for 12 h or 24 h were analyzed for E2 concentration, aromatse, HSP70 and Bim. Thirdly, granulosa cells from ovaries maintained at 37 or 41℃ for 2 h were divided into 3 parts, one part were not cultured, one part were cultured at 37℃ for 6h without serum, and one part were cultured at 37℃ for 24h with serum. All of the granulosa cells were analyzed for their expression of HSP70, Bim, caspase-3 and cleaved caspase, furthermore, those granulosa cells cultured with serum were analyzed for apoptosis too. Lastly, primordial follicles and growing follicle from ovaries maintained at 37 or 41℃ for 2 h were cultured at 37℃ for 6h and analyzed for the expression of HSP70. The results showed that heat stress increased HSP70 and decreased aromatase proteins in antral follicles. Heat stress increased HSP70, but decreased E2 and aromatase in granulosa cells maintained at 42℃ for 12 h or 24 h. The TUNEL-positive granulosa cells from heat-stressed ovaries maintained at 41℃ for 2 h were observed concomitant with a significant increase in HSP70, Bim and cleaved caspase-3 protein. The HSP70 level of primordial follicles from ovaries maintained at 41℃ for 2 h was significantly higher than that of growing follicles.Conclusions:(1) acute heat stress increase rectal temperature and the number of eggs after superovulation, but decrease the body weight. (2) chronic heat-stress in mice decreased estradiol in serum and aromatase in antral follicles but increased number of atretic follicles and granulosa cell undergoing apoptosis which might explain the decreased fertility commonly observed in heat-stressed animals. (3) Bim and caspase-3 were involved in granulosa cell apoptosis induced by heat stress.