Single-cell RNA sequencing and PANDORA-seq reveal the pivotal role of piRNA in heat stress-induced testicular injury.

Global climate change and the greenhouse effect have accelerated the decline in semen quality of male animals. PIWI-interacting RNAs (piRNAs), a newly discovered class of non-coding RNAs, play a crucial role in spermatogenesis. However, the molecular mechanisms of piRNAs in testicular heat stress remain poorly understood. In this study, we analyzed the impact of environmental temperature on semen quality in mammals, using 23,385 data points from pigs as a representative species. We constructed a mouse testicular heat stress model and performed in vitro functional validation of selected piRNAs in spermatogonia. Our results revealed a significant negative correlation between environmental temperature and semen quality, with rapid declines occurring when temperatures exceeded 27 °C. Heat stress caused testicular atrophy and spermatogenic arrest, while decreasing piRNA-related gene expression in spermatogenic cells. PANDORA-seq identified piRNAs as the most responsive small RNAs to heat stress. We further identified piR_021767 as a key regulator of spermatogenic disorders, with its expression negatively correlated with testicular weight and semen density. Inhibition of piR_021767 expression in C18-4 cells suppressed proliferation and promoted apoptosis. Dual luciferase assays showed that piR_021767 inhibits BAX expression, regulating spermatogonial apoptosis. This study provides new insights into the role of piRNAs in heat stress-induced spermatogenic disorders.