Oral Presentation ESA-SRB-NZSE-CaSR 2026 in conjunction with ENSA

Thermal and proteotoxic stressors drive dynamic remodelling of protein solubility in the male germline (143922)

Shannon P Smyth 1 2 3 , David A Skerrett-Byrne 2 4 5 6 7 , Ching-Seng Ang 8 , Heather C Murray 9 10 , Nathan D Burke 1 2 3 , Amanda L Anderson 2 3 , John E Schjenken 2 3 , Brett Nixon 2 3 , Elizabeth G Bromfield 1 2 3
  1. School of BioSciences, Faculty of Science, Bio21 Institute, University of Melbourne, Parkville, VIC, Australia
  2. Reproductive and Family Health Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia 2305
  3. Centre for Reproductive Science, School of Science, University of Newcastle, Callaghan, NSW, Australia 2308
  4. Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany
  5. German Center for Diabetes Research (DZD), Neuherberg, Germany
  6. Mercy Perinatal, Mercy Hospital for Women, Heidelburg, VIC, Australia 3084
  7. Therapeutics Discovery and Vascular Function in Pregnancy Group, Department of Obstetrics, Gynaecology and Newborn Health, Melbourne Medical School, The University of Melbourne, Heidelburg, VIC, Australia 3084
  8. Bio21 Mass Spectrometry and Proteomics Facility, The University of Melbourne, Parkville, VIC, Australia
  9. School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, Australia
  10. Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia

Background: Stringent regulation of the proteome during periods of stress is essential for the maintenance of cellular homeostasis, function and survival. While proteome regulation has been extensively explored in somatic cells, knowledge of how germ cells respond to proteotoxic stress is lacking.

Methods: This study aimed to investigate how the proteome of mouse spermatocytes and spermatids (n=5 per treatment) are remodelled in response to three proteotoxic stressors; 4-hydroxynonenal (a lipid aldehyde), MG132 (a proteasome inhibitor) and whole-body heat stress (8h at 35°C/16h at 25°C for 14-days). To achieve this, we employed protein solubility fractionation coupled to a high-depth proteomic workflow. This approach permitted the quantification of stress-dependent changes in total protein abundance as well as changes in protein solubility from the same population of cells.

Results: Interrogation of total protein abundance revealed that each proteotoxic stressor induces a unique proteomic signature, with heat stress eliciting the largest response. Further assessment of protein solubility revealed distinct germ cell stage-specific responses to heat stress, with spermatocyte proteins preferentially adopting a ‘less soluble’ state suggesting a shift towards misfolded and/or oligomeric proteoforms while spermatid proteins adopted a ‘more insoluble’ state suggesting accumulation of aggregated proteins. As such, protein aggregation in the germline appears to be an adaptive mechanism, driving the activation of key pathways associated with regulation of the cell cycle, metabolism, transcription and protein homeostasis. Among those proteins identified as potentially aggregating in response to heat stress were several with established roles in spermatogenesis (e.g., heat shock factor protein 5 (HSF5)) and sperm structure/function (e.g., testis-expressed protein 38 (TEX38) and izumo sperm-egg fusion protein 3 (IZUMO3)).

Conclusion: Ultimately, this study provides an increased understanding of stress response pathways in the male germline; a crucial step to inform future strategies to fortify germ cells against stress with implications for fertility and offspring health.