Department of Pathophysiology/Key Laboratory of High Altitude Environment Medicine, Key Laboratory of High Altitude Medicine, College of High Altitude Military Medicine, Army Medical University, Third Military Medical University, Chongqing, People's Republic of China.
Institute of Medicine and Hygienic Equipment for High Altitude Region/Key Laboratory of High Altitude Environment Medicine, Key Laboratory of High Altitude Medicine, College of High Altitude Military Medicine, Army Medical University, Third Military Medical University, Chongqing, People's Republic of China.
Reproduction. 2018 Dec;156(6):545-558. doi: 10.1530/REP-18-0243.
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.
自噬和细胞凋亡在广泛的相互作用中相互关联。我们之前的研究表明，低渗缺氧诱导精母细胞衍生细胞系（GC-2）发生明显的细胞凋亡。然而，在低氧条件下，缺氧诱导的细胞凋亡是否通过自噬的抑制来介导尚不清楚。在这项研究中，将 GC-2 细胞在 1%O2 中培养，并在不同时间点收获。通过吖啶橙染色、Cyto-ID 染色、mCherry-GFP-LC3B 腺病毒转染和各种自噬标志物的 Western blot 检测自噬。通过 TUNEL 染色、流式细胞术、JC-1 染色和凋亡相关蛋白的 Western blot 检测细胞凋亡。我们发现，低氧诱导 GC-2 细胞通过线粒体和死亡受体途径凋亡，并在时间依赖性方式抑制 GC-2 细胞中的自噬流。然而，在低氧条件下培养 24 小时之前，GC-2 细胞中观察到明显的自噬溶酶体形成，但仅在低氧条件下培养 24 小时后观察到明显的细胞凋亡。Caspase-8 siRNA 处理诱导细胞存活，同时诱导成熟自噬体、酸性囊泡细胞器形成和自噬流。此外，Beclin-1 的过表达通过抑制精母细胞凋亡显著减轻了对小鼠生精功能的损害。这项研究的结果表明，低氧抑制自噬，进一步通过促进 caspase-8 的激活，从而增强低氧诱导的小鼠精母细胞凋亡，这提示联合应用凋亡抑制和自噬激活可能是治疗低氧诱导男性不育的一种治疗策略。
