Background:

As one of the factors of male infertility, high temperature induces apoptosis of differentiated spermatogenic cells, sperm DNA oxidative damage, and changes in morphology and function of Sertoli cells. Spermatogonial stem cells (SSCs) is a kind of germline stem cells which maintain the spermatogenesis through self-renewal and differentiation. At present, however, the effect of high temperature on SSC differentiation in vitro has not been reported. Methods In the present study, we used in vitro differentiation model of SSCs to research the effect of heat stress treatment on SSC differentiation. Firstly, real-time PCR was used to detect the expression levels of self-renewal and differentiation marker genes in differentiation-cultured SSCs after heat stress treatment. Then, the effect of heat stress on the transcriptome of differentiation cultured SSCs was analyzed by RNA-seq. Enrichment of functions and signaling pathways analysis were performed based on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Results We found that 2 days of 37℃ or 43℃ (30min/d) heat stress treatment significantly inhibited SSC differentiation. The differentiation related genes c-kit , stra8 , Rec8 , Sycp3 and Ovol1 were down-regulated after 2 days and 4 days of heat stress at 37℃. The transcriptome of SSCs was significantly differentially expressed on the second day and fourth day after heat stress treatment at 37℃. In total, 1660 and 7252 differentially expressed genes (DEGs) were identified by RNA-Seq in SSCs treated with heat stress at 37℃ for 2 days and 4 days respectively, compared with those cultured at 34℃. KEGG pathway analysis showed that P53, ribosome and carbon metabolism signaling pathways promoting stem cell differentiation were significantly enriched after heat stress treatment at 37℃. Conclusion These results indicate that high temperature at 37℃ significantly inhibits SSC differentiation and promotes enrichment of P53, ribosome and carbon metabolism signaling pathways in stem cell differentiation, providing a reference for the pathogenesis of heat-induced azoospermia.