Acute testicular hyperthermia leads to a rapid loss of global piRNA levels and a consequent increase in transcript abundance, including LINE1 activity within heat-sensitive male germ cells

Spermatogenesis is a highly temperature sensitive process, occurring 2-8°C lower than core body temperature. Testicular hyperthermia rapidly affects male precursor germ cells including spermatocytes and spermatids. To understand the immediate transcriptional response, we gave mice testicular heat stress and isolated round spermatids. Using next-generation RNA sequencing we found 134 differentially expressed transcripts, 93% of which were upregulated. In addition, testicular hyperthermia induced 395 differential splicing events and altered the usage of 61 polyadenylation sites. To explain these observations and understand why transcript abundance appears to favour upregulation following testicular hyperthermia, we assessed global piRNA levels and found an overall, rapid reduction. Concomitantly, we observed an increase in transposable element RNA (LINE1) and protein (ORF1p) abundance. Furthermore, increased LINE1 expression appeared to be correlated with DNA damage in the male germline. At 24 hours post heat stress, piRNA levels recovered close to control levels, coincident with a significant reduction in LINE1 transcript expression within spermatocytes. These findings suggest that piRNA-mediated transposon suppression is a critical mechanism underlying the temperature sensitivity of spermatogenesis and may be one of the reasons why the testis require lower temperatures.