Human spermatogonial stem cells (SSCs) are crucial for spermatogenesis and male reproduction. Although abnormal pyroptosis caused by inflammation impacts male fertility, the molecular mechanisms underlying the pyroptosis of human SSCs are elusive. To induce pyroptosis, lipopolysaccharide (LPS) was introduced into the vas deferens of mice. RNA sequencing (RNA-Seq) of human SSCs was employed to identify NOD-like receptor thermal protein domain associated protein 3 (NLRP3), and RNA interference (RNAi) was used to determine the function and mechanism of NLRP3 in controlling pyroptosis of human SSCs. Guanylate-binding protein 4 (GBP4) was analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein–protein interaction (PPI) analyses, and the impact of GBP4 on NLRP3 in human SSCs was subsequently assessed using GBP4 short hairpin RNA (shRNA). LPS reduced the testicular weight of mice, disrupted spermatogenesis, and increased the levels of inflammatory factors (interleukin-18 [IL-18] and interleukin-1β [IL-1β]). NLRP3 siRNAs antagonized the LPS-induced increases in IL-18 and IL-1β, proliferative inhibition, and pyroptosis (Caspase 1 and Gasdermin D) in human SSCs. An association between GBP4 and NLRP3 in human SSCs was identified by co-immunoprecipitation (Co-IP). Human SSCs with stable GBP4 shRNA decreased the expression level of NLRP3 in human SSCs under inflammatory conditions. Collectively, these results imply that LPS-induced NLRP3 activation enhances the pyroptosis of human SSCs via the regulation of GBP4. This study offers novel insights into molecular mechanisms underlying the fate determinations of human SSCs.

Keywords: GBP4; human spermatogonial stem cells; male infertility; NLRP3; pyroptosis