Gene expression changes in male germ cells differ following in vivo versus in vitro exposure to hyperthermia

Testicular heat stress is a well-established disruptor of spermatogenesis, yet the immediate transcriptional responses in heat-sensitive germ cells remain poorly understood.

To investigate the transcriptional responses of isolated spermatocytes and round spermatids to heat stress, comparing in vivo and in vitro models.

We employed two methods of testicular hyperthermia, an in vivo (water bath) and an in vitro (isolated cell) model, and analysed transcriptional changes in spermatocytes and round spermatids. These were compared with in vitro heat responses in somatic cell lines (NIH3T3, B16F10 and TM4 cells). Transcriptional responses were assessed by real-time quantitative polymerase chain reaction (PCR).

In vivo heat stress resulted in significant upregulation of heat-responsive genes (Hspa1a and Hsph1), the long non-coding RNA 4930555K19Rik, and transposable element LINE1. However, in vitro heat stress showed only minimal induction of Hspa1a and failed to replicate the broader transcriptional responses observed in vivo. Somatic cell lines demonstrated greater heat shock gene activation than isolated primary germ cells following in vitro heat exposure.

The results suggest that the testicular microenvironment plays a critical role in the transcriptional response to heat stress. Isolated germ cells may not adequately represent the molecular response to testicular hyperthermia.

These results have provided new insight into the molecular basis of male germ cell heat sensitivity, with implications for fertility preservation under environmental or occupational heat exposure. Understanding the dependence of transcriptional responses on the testicular microenvironment could guide future interventions to protect spermatogenesis and inform assisted conception in humans and livestock.