Effect of extracellular matrix on bovine spermatogonial stem cells and gene expression of niche factors regulating their development in vitro

• Extracellular matrix (ECM) potentiated bovine SSCs differentiation.
• Possible role of KITLG in differentiation of bovine SSCs in response to ECM.
• Conventional culture enhances bovine SSCs self-renewal.
• Potential contribution of GDNF to SSCs self-renewal during conventional culture.

Extracellular matrix (ECM) could influence cells function through providing structural and functional networks facilitating the cellular interactions. The present study was conducted to evaluate the effect of culture on ECM versus plastic on bovine spermatogonial stem cells (SSCs) and growth factors regulating their development. Following isolation, bovine testicular cells were cultured on ECM-coated or uncoated (control) plates for 12 days. The colonization of SSCs was assessed by inverted microscope and the gene expression was evaluated using quantitative real-time PCR. The colonization rate was greater in ECM than the control group (P < 0.05). The expression of markers of undifferentiated spermatogonia increased in response to conventional culture (P < 0.05). Conversely, the expression of ckit as a marker for differentiated spermatogonia was reduced following culture in the control and ECM groups (P < 0.05), but this decrease was less in ECM group (P < 0.05). Accordingly, while cells cultured on uncoated plates had greater expression of markers of undifferentiated spermatogonia (P < 0.05), cells cultured on ECM-coated plates showed higher expression of ckit (P < 0.05). Moreover, culture on ECM resulted in higher expression of kit ligand (Kitlg; P < 0.05), whereas culture on plastic led to greater expression of glial cell line-derived neurotrophic factor (Gdnf; P < 0.05). In conclusion, the present study revealed that the permissive effect of ECM on bovine SSCs differentiation in vitro, which was probably mediated through upregulation of KITLG expression. Moreover, the results imply that GDNF might contribute to germ cells self-renewal during conventional culture.