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% O 2 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.Reproduction (2018) 156 545-558 546 Reproduction (2018) 156 545-558 https://rep.bioscientifica.com The activation of apoptosis can also inhibit autophagy under hypoxia. Caspases that are activated during apoptosis, mainly caspase-8 and caspase-3, can cleave autophagy-related proteins, such as ATG3, ATG4D and Beclin1, suppressing autophagy and resulting in cell damage (Li et al. 2011, Oral et al. 2012, Lamy et al. 2013). In addition, autophagy can block the induction of apoptosis and attenuate cellular injury under hypoxia. The activation of autophagy inhibits apoptosis by clearing damaged mitochondria, inhibiting caspase-3 activation and clearing reactive oxygen species (ROS)-damaged proteins. Thus, autophagy plays a protective role under hypoxia (Zhang et al. 2013, Li et al. 2014). However, in specific cases, autophagy or autophagy-relevant proteins may help in inducing apoptosis, which can aggravate cellular injury under hypoxia. The underlying mechanisms include the activation of caspases and clearance of the Bruce protein (an anti-apoptotic protein, a member of the inhibitors of apoptosis proteins family) (Nezis et al. , Young et al. 2012. Taking all the above into consideration, the interaction between autophagy and apoptosis in spermatocytes under hypoxia treatment remains unclear.In our previous study, we...