Using a well-in-drop (WID) oocyte/embryo culture system that allows identification of follicular origin, we have investigated the effects of granulosa cells (GCs) apoptosis, follicle size, cumulus–oocyte complexes (COCs) morphology, and cumulus expansion on the developmental competence of goat oocytes matured and cultured individually following parthenogenetic activation. The WID system supported oocyte maturation and embryo development to a level similar to the conventional group system. The majority of goat oocytes acquired competence for development up to the 8–16 cell stage in follicles larger than 2 mm, but did not gain the ability to form morula/blastocyst (M/Bs) until follicles larger than 3 mm in diameter. The extent of atresia affected M/Bs formation. This effect varied according to the follicle size. Cumulus expansion increased with follicle size and decreased with increasing incidence of GCs apoptosis. Oocyte developmental potential was also correlated with cumulus expansion. Regardless of the degree of follicle atresia, 73–84% of the floating cells in the follicular fluid (FF) underwent apoptosis. Correlation between floating cell density in FF and oocyte developmental potency suggests the possibility to use the floating cell density as a simple and non-invasive marker for oocyte quality. It is concluded that the developmental potential of an oocyte is determined by multifactor interactions, and multiple factors must be considered together to accurately predict the quality of an oocyte.
Zheng-Bin Han, Guo-Cheng Lan, Yan-Guang Wu, Dong Han, Wei-Guo Feng, Jun-Zuo Wang and Jing-He Tan
Jun Yin, Bing Ni, Yi-dong Yang, Zhong-wei Tang, Zhi-qi Gao, Lan Feng, Wei-gong Liao and Yu-qi Gao
Autophagy and apoptosis are interlocked in an extensive crosstalk. Our previous study demonstrated that hypotonic hypoxia-induced marked apoptosis of a spermatocyte-derived cell line (GC-2). However, whether hypoxia-induced apoptosis is mediated by inhibition of autophagy under hypoxic conditions remains unclear. In this study, GC-2 cells were cultured in 1% O2 and harvested at different time points. Autophagy was determined by acridine orange staining, cyto-ID staining, mCherry-GFP-LC3B adenovirus transfection and Western blotting for various autophagy markers. Apoptosis was detected by TUNEL staining, flow cytometry, JC-1 staining and Western blotting of apoptosis-related proteins. We found that hypoxia-induced apoptosis of GC-2 cells through mitochondrial and death receptor pathways and inhibited autophagic flux in GC-2 cells in a time-dependent manner. However, while marked autolysosome formation was observed in GC-2 cells before 24-h culture in hypoxic conditions, apparent apoptosis was observed only after 24-h culture in hypoxic conditions. Caspase-8 siRNA treatment induced cell survival, accompanied by induction of the mature autophagosome, acidic vesicular organelle formation and autophagic flux. Furthermore, Beclin-1 overexpression markedly attenuated the impairment of spermatogenesis in mice by inhibiting apoptosis of spermatocytes. The results of this study demonstrate that hypoxia inhibits autophagy, which further enhances hypoxia-induced apoptosis of mouse spermatocytes by promoting caspase-8 activation in a time-dependent manner, suggesting that combined application of apoptosis inhibition and autophagy activation might be a therapeutic strategy for treating hypoxia-induced male infertility.