Acute testicular hyperthermia leads to rapid loss of global piRNA and a concurrent increase in LINE1 activity within heat sensitive male germ cells

Spermatogenesis is a highly temperature sensitive process, which occurs 2–6 °C below core body temperature. Testicular hyperthermia rapidly affects male precursor cells, including spermatocytes and round spermatids, leading to elevated DNA damage. To understand the immediate transcriptional response of these cell types, we subjected mice to testicular hyperthermia and performed whole transcriptome sequencing on round spermatids following testicular hyperthermia. Analysis of sequencing data revealed that 93% of differentially expressed transcripts were upregulated following heat stress, with a notable trend to upregulation of transposable elements. Further investigation revealed a > 50% global reduction in piRNA levels, which coincided with increased LINE1 transcript abundance, elevated expression of ORF1p and increased DNA damage in heat stressed spermatocytes. The loss of piRNA appears to be driven by the rapid deformation of liquid–liquid phase separated ribonucleoprotein biocondensates, as demonstrated by the chromatoid body, which serves as an epicentre for piRNA processing. This loss of piRNA can cause widespread RNA dysregulation, transposon activation, DNA damage and impair spermatogenesis. These findings suggest that testicular hyperthermia disrupts piRNA biogenesis by destabilisation of liquid–liquid phase separated ribonucleoprotein biocondensates, including the chromatoid body. As a result, many transcripts, including transposable elements, become dysregulated which leads to DNA damage and impaired sperm quality.