Effects of chronic heat stress on granulosa cell apoptosis and follicular atresia in mouse ovary

Background： 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.Methods： In Exp. 1, 48 female 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 protein and serum concentrations of estradiol. In Exp. 2, the expression of HSP70 and aromatase was quantified in antral follicles cultured in vitro at 37 or 42 ℃ for 24 h. In Exp. 3, granulosa cells from ovaries maintained at 37 or 41 ℃ for 2 h were analyzed for their expression of HSP70, Bim, caspase-3 and cleaved caspase-3.Results： In Exp. 1, body weight and food intake of heat-stressed mice decreased（P 〈 0.05） compared with control mice while the concentration of estradiol in serum was lower（P 〈 0.05） 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（P 〈 0.05） after 21 d of heat-stressed treatment. HSP70 protein was more abundant（P 〈 0.05） in heat-stressed mice than control mice. In Exp. 2, heat stress increased HSP70 and decreased aromatase proteins（P 〈 0.05） in antral follicles. In Exp. 3, TUNEL-positive granulosa cells from heat-stressed ovaries were observed concomitant with a significant increase in HSP70, Bim and cleaved caspase-3 protein.Conclusion： Heat-stress in mice decrease estradiol in serum and aromatase in antral follicles but increased number of atretic follicles and granulosa cell undergoing apoptosis which may explain the decreased fertility commonly observed in heat-stressed animals.

Effect of calcium on porcine ICSI embryos expressing EGFP is related to activation of ooplasmic DNase I

Several reliable methods to produce transgenic animals use the male genome. After penetration into oocytes, sperm DNA undergoes dramatic conformational changes that might represent an opportunity for exogenous DNA to integrate into the zygote genome. A nuclease,DNase I, with Ca2+/Mg2+dependent activity and Zn2+inhibition, is one of the enzymes responsible for sperm DNA remodeling. To date, the effect of different calcium concentrations in manipulation media on porcine intracytoplasmic sperm injection has not been fully investigated.The present study was conducted to examine the effect of calcium in the surrounding media, and we found that the number of embryos expressing green fluorescent protein(EGFP) was increased in media containing Ca2+. However, the number did not change over Ca2+concentrations from 2 to 8 mmol$L–1(P > 0.05). Moreover, free Ca2+concentrations in the media were found to affect the efficiency which is porcine intracytoplasmic sperm injection(ICSI) embryos expressing EGFP protein, which was related to the activation of ooplasmic DNase I. These findings reveal a mechanism and pathway involving EGFP expression in ICSI embryos.