Abstract
Acupuncture is an integral component of complementary and alternative medicine that has been reported to enhance sperm motility, improve semen quality, and consequently augment male fertility. However, the precise mechanisms of action and the underlying molecular pathways remain unclear. In the present study, we aimed to elucidate the potential mechanisms through which acupuncture improves reproductive function in a mouse model of cyclophosphamide-induced asthenozoospermia. We collected sperm from the epididymis for semen analysis, collected serum to determine gonadotropin and oxidative stress marker levels, conducted histological examination of testicular tissue using hematoxylin and eosin (HE) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and observed mitochondrial morphology using transmission electron microscopy (TEM). We also assessed oxidative stress levels and total iron content in testicular tissue and validated the proteomic and transcriptomic analysis results of testicular tissue using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), protein imprinting analysis, and immunohistochemistry (IHC). Our results indicate that acupuncture enhances sperm quality in asthenozoospermic mice; increases serum testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels; and attenuates oxidative damage, iron accumulation, and mitochondrial injury in mouse testicular tissues. Through protein and transcriptomic analyses, we identified 21 key genes, of which cytochrome b-245 heavy chain (CYBB), glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 1 (ACSL1), and ferritin mitochondria (FTMT) were closely associated with ferroptosis. RT-qPCR, protein imprinting, and immunofluorescence (IF) analyses collectively indicated that acupuncture reduced ACSL1 and CYBB expression, and increased GPX4 and FTMT expression. Overall, the ferroptosis pathway associated with ACSL1/CYBB/FTMT/GPX4 represents a potential strategy through which acupuncture can improve the reproductive function in asthenozoospermic mice.