A Role for Exchange of Extracellular Vesicles in Porcine Spermatogonial Co-Culture

Spermatogonial stem cells (SSCs) provide the basis for lifelong male fertility through self-renewal and differentiation. Prepubertal male cancer patients may be rendered infertile by gonadotoxic chemotherapy and, unlike sexually mature men, cannot store sperm. Testicular biopsies taken prior to treatment may be used to restore fertility in adulthood. Testicular SSC populations are limited, necessitating in vitro culture systems to increase the numbers of SSCs available for downstream applications. Using the pig as a non-rodent model, we developed spermatogonial culture systems to expand spermatogonia from 1- and 8-week-old porcine testes, comparing feeder layers consisting of populations enriched for Sertoli cells, peritubular myoid cells (PMCs), pig fetal fibroblasts (PFFs), and testicular endothelial cells (TECs). As previously developed porcine spermatogonial culture systems relied exclusively on Sertoli cell feeder layers, we explored whether constituent cells of the SSC niche, such as PMCs and TECs, or fibroblastic cells like PFFs, may also support SSC expansion. Spermatogonia co-cultured with PMCs and PFFs had comparable rates of proliferation and apoptosis to spermatogonia co-cultured with Sertoli cells. To elucidate the mechanism behind the beneficial nature of feeder layers, we investigated the role of extracellular vesicles in the dynamic crosstalk between spermatogonia and feeder cells. Sertoli cell-released exosomes were found to be taken up by spermatogonia, and the inhibition of exosomal release reduced spermatogonial proliferation

A Role for Exchange of Extracellular Vesicles in Porcine Spermatogonial Co-Culture

Spermatogonial stem cells (SSCs) provide the basis for lifelong male fertility through self-renewal and differentiation. Prepubertal male cancer patients may be rendered infertile by gonadotoxic chemotherapy and, unlike sexually mature men, cannot store sperm. Alternatively, testicular biopsies taken prior to treatment may be used to restore fertility in adulthood. Testicular SSC populations are limited, and in vitro culture systems are required to increase numbers of SSCs for treatment, demanding culture systems for SSC propagation. Using the pig as a non-rodent model, we developed culture systems to expand spermatogonia from immature testis tissue, comparing different feeders (Sertoli cells, peritubular myoid cells (PMCs) and pig fetal fibroblasts (PFFs)). Spermatogonia co-cultured with Sertoli cells, PMCs and PFFs had comparable rates of proliferation and apoptosis. To elucidate the mechanism behind the beneficial nature of feeder layers, we investigated the role of extracellular vesicles in crosstalk between spermatogonia and feeder cells. Sertoli cell-released exosomes are incorporated by spermatogonia, and inhibition of exosomal release reduces spermatogonial proliferation. Together, these results show that PMCs, PFFs and Sertoli cells promote spermatogonial proliferation in co-culture, with exosomal exchange representing one possible mechanism. Further characterization of exosomal cargo may ultimately allow the development of feeder-free culture systems for clinical use

Metabolic Requirements for Spermatogonial Stem Cell Establishment and Maintenance In Vivo and In Vitro

The spermatogonial stem cell (SSC) is a unique adult stem cell that requires tight physiological regulation during development and adulthood. As the foundation of spermatogenesis, SSCs are a potential tool for the treatment of infertility. Understanding the factors that are necessary for lifelong maintenance of a SSC pool in vivo is essential for successful in vitro expansion and safe downstream clinical usage. This review focused on the current knowledge of prepubertal testicular development and germ cell metabolism in different species, and implications for translational medicine. The significance of metabolism for cell biology, stem cell integrity, and fate decisions is discussed in general and in the context of SSC in vivo maintenance, differentiation, and in vitro expansion