Background High temperature has a very adverse effect on mammalian spermatogenesis and eventually leads to sub- or infertility through either apoptosis or DNA damage. However, the direct effects of heat stress on the development of spermatogonial stem cells (SSCs) are still unknown because SSCs are rare in the testes. Methods In the present study, we first used in vitro-cultured SSCs to study the effect of heat shock treatment on SSC development. Then, we used RNA-Seq analysis to identify new genes or signalling pathways implicated in the heat stress response. Results We found that 45 min of 43 °C heat shock treatment significantly inhibited the proliferation of SSCs 2 h after treatment but did not lead to apoptosis. In total, 17,822 genes were identified by RNA-Seq after SSC heat shock treatment. Among these genes, we found that 200 of them had significantly changed expression, with 173 upregulated and 27 downregulated genes. The number of differentially expressed genes in environmental information processing pathways was 37, which was the largest number. We screened the candidate JAK-STAT signalling pathway on the basis of inhibition of cell cycle progression and found that the JAK-STAT pathway was inhibited after heat shock treatment. The flow cytometry results further confirmed that heat stress caused S phase cycle arrest of SSCs. Conclusion Our results showed that heat shock treatment at 43 °C for 45 min significantly inhibited SSC self-renewal through S phase cell cycle arrest but not apoptosis.

Safety is the chief consideration in recipient preparation of spermatogonial stem cell (SSC) transplantation in mammals, especially humans. In this study, we compared the safety of the SSC transplantation recipients that were prepared both by testes heat shock plus testes busulfan injection (heat shock+busulfan(t)) and by busulfan intraperitoneal injection (busulfan i.p.) only. Our results showed that heat shock+busulfan(t) treatment significantly (p < 0.05) reduced mortality in mice and did not produce bone marrow cell toxicity. Furthermore, heat shock+busulfan(t) treatment directly damaged SSCs and exhausted almost all of the germ cells in the testis; the exhaustion of these cells is considered a key factor in the successful preparation of the recipients. Therefore, we used heat shock+busulfan(t) treatment to prepare recipients of SSC transplantation. Two months after SSC transplantation, the number and length of donor SSC-derived colonies in the testis of recipient in heat shock+busulfan(t) group was closed to that in busulfan i.p. group. Therefore, compared with busulfan i.p. treatment, heat shock+busulfan(t) treatment improved the safety of recipient preparation without reducing the efficiency of SSC transplantation. Two GFP-positive offspring were produced from 1 of the 20 recipients that had mated with female mice 72 days after SSC transplantation. In conclusion, heat shock with busulfan treatment is a safe method to prepare the recipient of SSC transplantation in mice.

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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.