Dysregulation of the miR-29c-3p/collagen axis induces spermatogenic failure in heat stress and non-obstructive azoospermia

Rising global temperatures have intensified male infertility linked to testicular heat stress. While microRNAs play pivotal roles in spermatogenesis, the reason for aberrant microRNA expression following heat stress-induced spermatogenic disorders remains largely unclear. We established a murine heat stress model and performed small RNA sequencing. Candidate microRNAs were obtained via intersection with additional spermatogenic arrest and Sertoli cell-only syndrome datasets. The proliferation of germ cells was assessed by cholecystokinin-8 and 5-ethynyl-2’-deoxyuridine assays. Bioinformatic analysis was applied to predict downstream miRNA targets, and enrichment analysis was performed. Fluorescence in situ hybridization and immunofluorescence assays were utilized to determine the expression of miRNAs and their targets in Sertoli cell-only syndrome models and clinical testicular samples. In total, testicular heat stress resulted in 102 upregulated and 114 downregulated miRNAs. Four candidate microRNAs (miR-378a-3p, miR-29c-3p, let-7a-5p, and miR-9-5p) were screened for further investigation. Functional assays demonstrated that elevated expression of miR-378a-3p, miR-29c-3p, or silencing of miR-9-5p, notably inhibited the proliferation ability of spermatogonia (GC-1 cells). Common targets of these microRNAs were highly enriched in collagen-related pathways, predominantly through the inhibition of miR-29c-3p-targeted collagen type I alpha 1 and type III alpha 1 chain 1. Compared with normal tissue samples, tissue samples from Sertoli cell-only syndrome model mice and from those with spermatogenic disorders also exhibited high miR-29c-3p expression levels. In summary, our study identified microRNA expression patterns during testicular heat stress and preliminarily investigated the roles of specific microRNAs. miR-29c-3p and the collagen synthesis pathway could be utilized as biomarkers and therapeutic targets for heat-induced spermatogenic disorders.