Varicocele (VC) is a common cause of male infertility. An increasing number of studies have confirmed that chronic hypoxia plays an important role in male infertility caused by VC. Our previous study suggested that autophagy may have a crucial function in VC. Whether autophagy plays an important role in hypoxic spermatogonia remains unknown. We established a new method to study VC by culturing spermatogonia under hypoxic conditions to simulate the chronic hypoxic environment of VC. Moreover, the effects of autophagy on hypoxic spermatogonia were observed by regulating autophagy. Spermatogonia from GC-1 spg mice were cultured in vitro in chronic hypoxic conditions (2% O2, 93% N2, 5% CO2) with a tri-gas incubator. Cells were separately cultured under normal oxygen and hypoxic conditions, and then, the autophagy blocker drug LY294002 was subsequently administered. Cytoactivity and proliferation were tested by CCK8 assays. Cells cultured in hypoxic conditions were divided into the hypoxia for 24 h, 48 h, and 72 h groups and the LY294002+hypoxia for 24 h, 48 h, and 72 h groups. Hoechst staining and flow cytometry/Annexin-V-FITC/PI staining were used to detect cell apoptosis, while transmission electron microscopy was used to observe the structure of the spermatogonia. Western blot analysis was used to observe the expression of Hif-1α, Beclin-1, LC3, SOD2, GPX4, and Cyt C. As the hypoxia time increased, CCK8 detection showed that the cytoactivity of the spermatogonia was suppressed. After administration of the inhibitor LY294002, the inhibition ratio of the spermatogonia increased. As the hypoxia time was prolonged, the apoptosis index of spermatogonia increased. After administration of LY294002, the apoptosis index increased significantly. Transmission electron microscopy showed that there were abundant structurally abnormal mitochondria and autophagosomes and autolysosomes in hypoxic spermatogonia. In addition, LY294002 reduced the expression of autophagosomes and autophagosomes, but the number of abnormal mitochondria and vacuoles in hypoxic cells increased significantly. As the hypoxia time was prolonged, the expression levels of Hif-1α, Beclin-1, LC3 II/LC3-I, and Cyt C increased gradually, but there were no significant changes in SOD2 and GPX4. After administration of LY294002, the expression levels of Beclin-1, LC3 II/LC3-I, and GPX4 significantly decreased, but Cyt C significantly increased. Thus, chronic hypoxia decreases cytoactivity and proliferation of spermatogonia. Moreover, hypoxia damages the mitochondria and other organelles. At the same time, hypoxia activates the autophagic system to clear these damaged organelles. The inhibition of autophagy in spermatogonia under chronic hypoxic conditions prevents the removal of damaged mitochondria and eventually results in a significant decrease in the antioxidant system molecule GPX4, rather than SOD2, and a significant increase in the apoptosis rate.
Keywords: GPX4; Hif-1α; LY294002; autophagy; hypoxia; spermatogonia; varicocele.