Abstract

Spermatogonial stem cells (SSCs) are able to self-renew and differentiate into mature sperm therefore affording life-long male fertility. SSCs are rare in testes, and we previously established a culture system that enabled in vitro propagation of porcine spermatogonia (including SSCs) for up to 2 months. Testicular endothelial cells (TECs), as a key component of the germline niche, have been reported to support long-term in vitro expansion of SSCs from rodents and humans. To further optimize the porcine SSC culture, here we systematically assessed the ability of porcine TECs and Sertoli cells (SCs) as feeder layers to sustain porcine spermatogonial proliferation in vitro. We found that under the optimized condition, SCs significantly promoted the formation of typical grape-like colonies derived from SSEA4+ spermatogonia, with stable expansion for > 100 days and expression of stem and germ cell markers. The cultured cells also retained normal karyotype and successfully colonized the busulfan-treated mouse testes after xenotransplantation. In contrast, TECs only supported short-term spermatogonial proliferation (< 60 days), and the co-cultured spermatogonia exhibited loose aggregation and salient apoptosis. The secretomic analysis further revealed that SCs were enriched with adhesion molecules, anti-stress proteins, and self-renewal-associated factors, whereas TECs preferentially secreted differentiation, extracellular matrix (ECM) remodeling-, and metabolism-related proteins. Collectively, this study demonstrates that SCs, rather than TECs, contribute to optimized culture of spermatogonia in pigs, and our SC-based optimized culture system for porcine spermatogonia provides a scalable platform for genetic manipulation of porcine SSCs therefore facilitating their application to breeding and germplasm innovation.