C9orf72 Overexpression in Asthenozoospermia: Implications for Spermatogonia Proliferation through mitophagy and glucose metabolism

Abstract

Background The role of the C9orf72 gene in sperm function and asthenozoospermia remains unclear. This study aimed to investigate whether the expression of C9orf72 was abnormal in patients with asthenozoospermia and its effects on spermatogonia proliferation. Methods Semen samples were collected from 27 men with asthenozoospermia and 31 normal men. Immunofluorescence and Western blotting were employed to detect the protein expression of C9orf72 in semen samples. Mouse spermatogonia (GC-1 spg) transfected with C9orf72-overexpressing lentivirus were assessed for cell viability, apoptosis, ROS levels, mitochondrial membrane potential changes, colocalization between autophagosomes and mitochondria, glucose content and glucose energy metabolism-related protein expression. The effect of the mitophagy inhibitor Mdivi-1 on C9orf72-induced apoptosis in GC-1 spg cells was investigated. Results The results showed that the C9orf72 protein was significantly upregulated in asthenozoospermic semen samples and negatively correlated with sperm progressive motility. Overexpression of C9orf72 inhibited proliferation, increased apoptosis, and elevated ROS levels in GC-1 spg cells. Enhanced mitophagy was observed in C9orf72-overexpressing cells, as indicated by mitochondrial membrane potential reduction, decreased MitoTracker Red CMXRos-labeled mitochondria, and increased LC-3-labeled autophagy. Moreover, glucose energy metabolism was impaired in C9orf72-overexpressing cells, with decreased glucose uptake and reduced protein expression of GLUT1, GLUT3, HK2 and LDHA. The mitophagy inhibitor Mdivi-1 partially reversed C9orf72-induced apoptosis in GC-1 spg cells. Conclusions This study demonstrates that C9orf72 is significantly overexpressed in asthenozoospermic semen samples and negatively correlated with sperm progressive motility. C9orf72 overexpression inhibits spermatogonia proliferation, and the mechanism by which it inhibits the proliferation of spermatogonia may be through promoting mitophagy and inhibiting glucose metabolism, representing a potential regulatory mechanism underlying asthenozoospermia.