Oral Presentation ESA-SRB-ANZBMS 2024 in conjunction with ENSA

Phosphoproteomic analyses identifying novel regulators of spermatogonial stem cell function (#34)

David A Skerrett-Byrne 1 2 3 4 , Brett Nixon 1 4 , Jon M Oatley 5 , Connor Cason 1 , Ilana R Berntstein 1 , Tessa Lord 1 4
  1. The University of Newcastle, CALLAGHAN, NSW, Australia
  2. Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health Neuherberg, Munich, Germany
  3. German Center for Diabetes Research (DZD) , Neuherberg, Germany
  4. Hunter Medical Research Institute, New Lambton Heights, NSW, Australia
  5. Washington State University, Pullman, WA, USA

Spermatogonial stem cells (SSCs) hold the potential to be used for ground-breaking technologies, spanning from infertility treatments for childhood cancer survivors to biobanking for endangered species conservation. While a wealth of transcriptomic data have been produced, proteomic data is scarce, and no phosphoproteomic databases exist for this rare cell population. Here we have used an Id4-eGfp transgenic mouse line to capture populations of mouse SSCs and progenitor spermatogonia, producing the first phosphoproteomic database using the EasyPhos platform1. In overlaying these analyses with existing RNAseq data, we have created a comprehensive roadmap representing the transition from self-renewing to differentiation-poised germ cell.

Proteomic analysis identified 8,461 and 8,446 proteins in SSCs and progenitor spermatogonia, respectively (average 15.2 unique peptides/protein, 33.6% coverage). Of these, 258 were significantly upregulated in SSCs (foldchange ±1.5, p ≤ 0.05; LHX1, GFRA1), and 274 in progenitors (CRABP, cKIT). In overlaying the proteome with equivalent RNAseq data2, a discordance between RNA and protein expression was clear (Pearson R2=0.236) highlighting the need for caution when interpreting transcriptomic data as a proxy for protein activity/function. Ingenuity pathway analysis (IPA) of differentially expressed proteins identified canonical pathways such as RET, HIF1 and Sirtuin signalling, and predicted upstream regulators that included well defined self-renewal factors (GDNF), and novel growth factors (PDGF-BB). Analysis of the phosphoproteome identified 1,760 and 1,815 phosphoproteins, respectively: 60 significantly increased in SSCs, and 257 in progenitors. IPA analysis predicted activation of canonical pathways such as Mitosis and DNA repair (Z-score ≥2) upon the progenitor transition. A shortlist of predicted master kinases was produced, and pilot studies using targeted inhibitors have revealed significant modulation of spermatogonia behaviour in vitro. Cumulatively, data produced here have identified novel growth factors and kinase inhibitor compounds that dictate SSC activity in vitro, potentially providing a gateway to development of SSC technologies for real-world applications.

  1. Skerrett-Byrne, D. A. et al. Phosphoproteomic analysis of the adaption of epididymal epithelial cells to corticosterone challenge. Andrology 12, 1038-1057, doi:https://doi.org/10.1111/andr.13636 (2024).
  2. Helsel, A. R. et al. ID4 levels dictate the stem cell state in mouse spermatogonia. Development 144, 624-634, doi:10.1242/dev.146928 (2017).