Spermatogonial stem cells (SSCs) hold potential to be used as a therapeutic tool to reverse chemotherapy-induced infertility. Unfortunately, in vitro culture techniques that are a precursor to these therapies are not robust, with studies showing a 13-fold reduction in SSC number and a 16-fold reduction in regenerative capacity over 6 months1. To address this deficiency, this project aimed to discover novel growth factors that can promote long-term self-renewal of the SSC population in vitro, through the identification and characterisation of unique membrane receptors.
To identify growth factor receptors that are enriched in self-renewing SSCs, as opposed to downstream progenitor or differentiating spermatogonia, mining of bulk2- and single cell-RNAseq databases (unpublished and 3) was conducted. Platelet-derived growth factor receptor A (PDGFRA) was selected for further investigation due to a 2.77-fold enrichment in transcript levels in SSCs above progenitor spermatogonia (P<0.00005) in the postnatal day 6 testis. Further, transcripts for associated ligands (PDGF-A/B) were found to be expressed in several somatic and germ cell populations, suggesting that this growth factor-receptor interaction occurs in the niche in vivo, but is not necessarily replicated within current in vitro culture conditions. To validate receptor expression at the protein level, a novel transgenic reporter mouse line was used (Id4-eGFP mouse2) that can delineate SSC and progenitor populations by way of GFP intensity. Using immunocytochemistry, we confirmed that PDGFRA was expressed in 50% of the SSC population, a significant enrichment above progenitor spermatogonia (P<0.05). Interestingly, further investigation of ligand expression using immunohistochemistry suggested that PDGF-A production may occur within SSCs themselves, potentially suggesting an autocrine, rather than paracrine, regulatory mechanism. Future experiments will explore the consequences of PDGFRA knockdown on SSC self-renewal capacity. By characterising growth factor-receptor interactions that sustain SSCs, we may be able to adapt in vitro culture conditions to better maintain these cells long-term.