Investigating a role for PDGF signalling in regulating spermatogonial stem cell function   — ASN Events
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  3. Investigating a role for PDGF signalling in regulating spermatogonial stem cell function  

Investigating a role for PDGF signalling in regulating spermatogonial stem cell function   (#481)

Logan LM McElhone 1 2 , Tessa TL Lord 1 2
  1. Centre for Reproductive Science, College of Engineering, Science and Environment, University of Newcastle, Callaghan, NSW, Australia
  2. Reproductive and Family Health Research Program, Hunter Medical Research Institute, New Lambton Heights, , NSW, New Lambton Heights

Background

Despite survival rates exceeding 80%, approximately half of male childhood cancer survivors experience permanent infertility following chemotherapy. As prepubertal boys cannot bank sperm, development of fertility-preservation strategies depends on understanding the regulation of spermatogonial stem cells (SSCs). We recently identified platelet-derived growth factor receptor alpha (PDGFRA) as highly expressed in SSCs and investigated whether PDGF signalling promotes SSC maintenance and expansion in vitro.

Methods

An ID4-eGFP transgenic mouse line was used to identify SSCs and progenitor spermatogonia, which have ‘Bright’ and ‘Dim’ GFP fluorescence, respectively. Immunofluorescence techniques were used to identify PDGF ligand expression in somatic cells within the testis (n=4). Primary cultures of undifferentiated spermatogonia were treated with either PDGFRA inhibitor (Tyrphostin, n=4) or supplemented with PDGF-BB (n=4). Control and PDGF-BB treated cultures were subjected to spermatogonial transplantation (n=4), and RNAseq analyses.

Results

We confirmed that PDGF ligands are expressed in key somatic cells of the SSC niche, supporting the hypothesis that PDGFRA may be activated in SSCs via paracrine signalling. Inhibition of PDGFRA using Tyrphostin resulted in a significant reduction in GFP-Bright SSCs in culture (p<0.05). Contrastingly, PDGF-BB supplementation increased the percentage of GFP-Bright SSCs (p<0.05), and the number of donor derived colonies following transplantation (p=0.1) however decreased overall cell number over 3 weeks (p<0.05). RNAseq and Ingenuity Pathway Analysis revealed activation of genes and pathways related to SSC self-renewal, however also identified modulation of genes related to cell adhesion, which may have impaired cell attachment during the 3-week culture period.

Conclusion

Although our data suggest that PDGF-BB may stimulate SSC self-renewal in vitro, its superfluous effect on cell adhesion means that this ligand may not be ideal for encouraging expansion in current 2D culture conditions. Regardless, this study represents an important gain-in-knowledge, identifying PDGF signalling as a novel pathway acting on SSCs in the mouse testis.