Hypoxia exposure impairs male fertility via inhibiting Septin2-mediated spermatogonial proliferation

What are the molecular mechanisms underlying hypoxia-induced male reproductive impairment?Hypoxia compromises Septin2 (Sept2) transcription in spermatogonia, which impedes spermatogonial proliferation through protein phosphatase 2A (PP2A)-dependent AKT dephosphorylation.Hypoxia is associated with impaired spermatogenesis and poor sperm parameters in men. Spermatogonia proliferation, a crucial early step in spermatogenesis, is essential for maintaining the spermatogenic cell population and ensuring sperm quality. However, the connection between hypoxia and spermatogonial proliferation remains poorly understood, and treatment options for hypoxia-related reproductive disorders are limited.A cross-sectional study analyzed semen samples from 24 high-altitude (HA) residents, 6 pathological hypoxia (PH) patients, and 19 healthy controls to evaluate hypoxia-associated sperm parameter alterations. Complementary animal studies employing a hypobaric chamber-induced hypoxic mouse model (n = 5) confirmed reproductive impairments through assessment of birth rates, sperm quality, and testicular histopathology. Transcriptomic profiling of hypoxic versus normoxic mouse testes (n = 3/group) identified spermatogonial proliferation defects as a predominant pathological feature and pinpointed Sept2 as a candidate mediator. Subsequent mechanistic investigations employed in vitro hypoxic culture of spermatogonial cell lines under hypoxic conditions coupled with pharmacological modulation of PP2A activity in mice (n = 3-5 per intervention group) to delineate the underlying molecular pathways.Semen parameters were evaluated using computer-assisted sperm analysis (CASA; for sperm concentration, count, and motility), morphological staining (Pap staining for sperm deformity), and eosin-nigrosin staining (for sperm viability). In hypoxic mouse model, fertility outcomes were assessed through fertility assessment (mating experiments), sperm parameters (CASA), testicular histology (H&E staining), and spermatogonia proliferation (immunohistochemistry and qPCR). In hypoxic spermatogonial cell models, cell proliferation was detected using CCK-8, EdU incorporation, flow cytometry, and western blotting. Sept2 manipulation (knockdown/overexpression), followed by mechanistic analyses (dual-luciferase reporter assay, DNA pulldown/mass spectrometry, TMT-based quantitative proteomics, co-immunoprecipitation, etc), was performed to investigate the mechanism underlying hypoxia-regulated spermatogonia proliferation. The SEPT2 inhibitor forchlorfenuron (FCF), the PP2A agonists celastrol, erlotinib, and FTY720, as well as PP2A inhibitor okadaic acid (OA) were used to investigate the role of the SEPT2-PP2A-AKT axis in male fertility regulation.Both human populations (HA residents and PH patients) and mouse model consistently demonstrated hypoxia-related reproductive dysfunction. Mechanistic analyses revealed that hypoxia significantly downregulated Sept2 expression in spermatogonia, concomitant with impaired proliferative capacity. Sept2 knockdown in normoxic mice the hypoxia-induced defects in spermatogenesis. Complementary in vitro studies confirmed that Sept2 depletion impaired spermatogonial proliferation by inducing G1-S phase arrest, while its overexpression mitigated hypoxia-related proliferative defects. Further investigation revealed that hypoxia disrupts Sept2 transcription by interfering with the binding of RNA polymerase II subunit A (POLR2A) to the Sept2 promoter. The consequent reduction in Sept2 expression led to stabilization of the B56γ regulatory subunit of PP2A, resulting in enhanced AKT dephosphorylation and subsequent suppressed proliferation. Pharmacological intervention with the PP2A inhibitor OA restored reproductive competence and sperm quality in hypoxic mice, whereas PP2A agonists exacerbated these deficits.RNA-seq data are deposited in China National Center for Bioinformation (CNCB) under accession number PRJCA035733.This study focused on the effects of hypoxia on sperm parameters. Additional factors such as alterations in reproductive hormones and sexual function may contribute to hypoxia-induced infertility and warrant further research.This study identifies the SEPT2-PP2A/B56γ-AKT axis as a key regulator in hypoxia-related spermatogonia proliferation impairment. PP2A inhibitors such as OA may offer a therapeutic strategy to protect male fertility under hypoxic conditions.This work was supported by the National Natural Science Foundation of China (No. 82101688) and Natural Science Foundation of Chongqing (No. CSTB2022NSCQ-MSX0943). The authors have no conflicts of interest to declare.