Male infertility is an issue of growing global concern, with various environmental, genetic and physiological factors contributing to declining sperm quality and function. Among these, testicular heat stress has emerged as a critical disruptor of spermatogenesis, particularly affecting the thermosensitive precursor germ cells, pachytene spermatocytes and round spermatids, manifesting as low amounts of poor-quality sperm being produced. The aim of this work is to understand the consequence of testicular heat stress and elucidate the initial molecular basis by which hyperthermia leads to poor quality sperm formation.
Herein we show that testicular hyperthermia causes a rapid (<6 hours) loss of structural integrity in the liquid-liquid phase separated (LLPS) chromatoid body (CB), a large ribonucleoprotein granule essential for spermatogenesis. Using transmission electron microscopy, we observed that heat exposure (42OC water bath 30 min) resulted in a significantly less dense chromatoid body compared to anaesthetic control (33OC water bath 30 min). Further visualisation of the CB through poly-dt staining resulted in a significant loss of signal, suggesting a loss of mRNA retention in the chromatoid body.
As a LLPS complex, formation of the CB is influenced by both temperature and post-translational modifications. To examine this further, we have employed a phospho-proteomic approach using two models. Firstly, pachytene spermatocytes and round spermatids were enriched, and subject to in vitro heat stress (33OC – 38OC) for 30 min. Secondly, individual mice were subject to water bath heating (33OC – 42OC) for 30 min to achieve testicular hyperthermia. Following treatment, phospho-peptides were enriched (TiO2) and quantitative total and phospho-proteomic analyses (TMT, 16-plex coupled LC-MS/MS) was performed to investigate temperature-induced changes in global protein expression and phosphorylation patterns.