Set7-Mediated Repression of the HIF-1α Adaptive Response Triggers Apoptosis in Hypoxic Spermatogonia

Male infertility, often caused by oligospermia, is a major global health concern. Hypoxia is a known inducer of germ cell death, a process governed by the hypoxia-inducible factor 1α (HIF-1α). The methyltransferase Set7 represses HIF-1α activity, but its specific role in the hypoxic male germline remains unknown.

The mouse spermatogonia-derived GC-2 cell line was used. The effect of hypoxia on SET domain-containing lysine methyltransferase 7 (Setd7) mRNA expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Cells were transfected with a Set7 overexpression plasmid, and its impact was measured via luciferase reporter assays using an erythropoietin (EPO) promoter and qRT-PCR for HIF-1α targets (glucose transporter 1 (Glut1), phosphoglycerate kinase 1 (Pgk1), pyruvate kinase, muscle (Pkm)). Stable cell lines expressing wild-type Set7 or a catalytically dead mutant (Set7-H297A) were generated. Apoptosis under hypoxia was analyzed by flow cytometry and fluorescence microscopy.

Hypoxia (1% O₂) significantly suppressed Setd7 mRNA expression while inducing HIF-1α target gene expression (Glut1, Pgk1, vascular endothelial growth factor (Vegf), Pkm). Set7 overexpression inhibited hypoxia-induced EPO promoter activity and blunted the hypoxic induction of Glut1, Pgk1, and Pkm. Although Set7 overexpression did not alter HIF-1α protein levels, it markedly increased hypoxia-induced apoptosis. This pro-apoptotic effect was significantly attenuated in cells expressing the enzymatically inactive Set7-H297A mutant.

Set7 represses the essential HIF-1α-mediated adaptive response to hypoxia in spermatogonial cells. Paradoxically, this repression potentiates hypoxia-induced apoptosis in a methyltransferase-dependent manner. These findings identify Set7 as a critical molecular switch that shifts cells from cellular adaptation to death under hypoxic stress, suggesting a regulatory pathway potentially relevant to hypoxia-associated male infertility.