Lightning Talk + Poster ESA-SRB-ANZOS 2025 in conjunction with ENSA

The germline proteome undergoes dynamic remodelling in response to in vitro and in vivo proteotoxic stress (128654)

Shannon P Smyth 1 2 3 , David A Skerrett-Byrne 1 2 4 5 , Ching-Seng Ang 6 , Heather C Murray 7 8 , Nathan D Burke 1 2 3 , Amanda L Anderson 1 2 , Brett Nixon 1 2 , Elizabeth G Bromfield 1 2 3
  1. Infertility and Reproduction Research Program, Hunter Medical Research Institute, Newcastle, NSW, Australia
  2. Centre for Reproductive Biology, School of Environmental and Life Sciences, College of Engineering, Science and Environment, University of Newcastle, Callaghan, NSW, Australia
  3. School of BioSciences, Faculty of Science, Bio21 Institute, University of Melbourne, Parkville, VIC, Australia
  4. School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, Australia
  5. German Center for Diabetes Research (DZD), Neuherberg, Germany
  6. Bio21 Mass Spectrometry and Proteomics Facility, The University of Melbourne, Parkville, VIC, Australia
  7. School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Newcastle, NSW, Australia
  8. Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia

Stringent regulation of the proteome during periods of stress is necessary 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. This study aimed to investigate how the proteome of mouse spermatocytes and spermatids are remodelled in response to three proteotoxic stressors; 4HNE (lipid aldehyde; in vitro), MG132 (proteosome inhibitor; in vitro) and heat stress (8h 35°C/16h 25°C for 14-days; in vivo). Here, we adapted a somatic cell protein solubility fractionation protocol1,2 for germ cells and coupled it to a label-free proteomic workflow3. This approach permits the quantification of stress-dependent changes in total protein abundance as well as changes in protein solubility from the same population of cells. Interrogation of total protein abundance revealed that in vivo heat exposure elicited the largest response in spermatocytes with 409 proteins downregulated and 437 proteins upregulated in response to heat stress. Comparatively, later stage spermatids had a dampened response with 145 proteins upregulated and 204 proteins downregulated following heat exposure. Functional assessment of these heat-dysregulated proteins revealed an enrichment in membrane remodelling proteins (e.g., MMGT1 and ZDHHC21) and a reduction in proteins involved in lipid metabolism (e.g., CYP2E1 and ALDH1B1). Despite different protein subsets being modulated between spermatocyte and spermatid populations, the modulated proteins appeared to contribute to analogous functions. Finally, dysregulated proteomic signatures across the stress conditions had substantial conservation (e.g. 47% conservation in 4HNE and MG132 in spermatocytes) whilst distinct impacts from the individual stressors could still be observed. Ultimately, this study will provide an increased understanding of proteostasis and stress response pathways in the male germline; a crucial step to inform future strategies to fortify germ cells against environmental stressors with potential implications for fertility and offspring health.

 

  1. 1. Wallace EW, Kear-Scott JL, Pilipenko EV, et al. Reversible, Specific, Active Aggregates of Endogenous Proteins Assemble upon Heat Stress. Cell 2015;162(6):1286-1298; doi: 10.1016/j.cell.2015.08.041.
  2. 2. Sui X, Pires DEV, Ormsby AR, et al. Widespread remodeling of proteome solubility in response to different protein homeostasis stresses. Proceedings of the National Academy of Sciences 2020;117(5):2422-2431; doi: doi:10.1073/pnas.1912897117.
  3. 3. Humphrey SJ, Karayel O, James DE, et al. High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform. Nat Protoc 2018;13(9):1897-1916; doi: 10.1038/s41596-018-0014-9.