Unique metabolic phenotype and its transition during maturation of juvenile male germ cells.

Human male reproductive development has a prolonged prepubertal period characterized by juvenile quiescence of germ cells with immature spermatogonial stem cell (SSC) precursors (gonocytes) present in the testis for an extended period of time. The metabolism of gonocytes is not defined. We demonstrate with mitochondrial ultrastructure studies via TEM and IHC and metabolic flux studies with UHPLC-MS that a distinct metabolic transition occurs during the maturation to SSCs. The mitochondrial ultrastructure of prepubertal human spermatogonia is shared with prepubertal pig spermatogonia. The metabolism of early prepubertal porcine spermatogonia (gonocytes) is characterized by the reliance on OXPHOS fuelled by oxidative decarboxylation of pyruvate. Interestingly, at the same time, a high amount of the consumed pyruvate is also reduced and excreted as lactate. With maturation, prepubertal spermatogonia show a metabolic shift with decreased OXHPOS and upregulation of the anaerobic metabolism-associated uncoupling protein 2 (UCP2). This shift is accompanied with stem cell specific promyelocytic leukemia zinc finger protein (PLZF) protein expression and glial cell-derived neurotropic factor (GDNF) pathway activation. Our results demonstrate that gonocytes differently from mature spermatogonia exhibit unique metabolic demands that must be attained to enable their maintenance and growth in vitro.

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.

Comparing the adult and pre-pubertal testis: Metabolic transitions and the change in the spermatogonial stem cell metabolic microenvironment.

BACKGROUND: Survivors of childhood cancer often suffer from infertility. While sperm cryopreservation is not feasible before puberty, the patient's own spermatogonial stem cells could serve as a germ cell reservoir, enabling these patients to father their own children in adulthood through the isolation, in vitro expansion, and subsequent transplantation of spermatogonial stem cells. However, this approach requires large numbers of stem cells, and methods for successfully propagating spermatogonial stem cells in the laboratory are yet to be established for higher mammals and humans. The improvement of spermatogonial stem cell culture requires deeper understanding of their metabolic requirements and the mechanisms that regulate metabolic homeostasis. AIM: This review gives a summary on our knowledge of spermatogonial stem cell metabolism during maintenance and differentiation and highlights the potential influence of Sertoli cell and stem cell niche maturation on spermatogonial stem cell metabolic requirements during development. RESULTS AND CONCLUSIONS: Fetal human spermatogonial stem cell precursors, or gonocytes, migrate into the seminiferous cords and supposedly mature to adult stem cells within the first year of human development. However, the spermatogonial stem cell niche does not fully differentiate until puberty, when Sertoli cells dramatically rearrange the architecture and microenvironment within the seminiferous epithelium. Consequently, pre-pubertal and adult spermatogonial stem cells experience two distinct niche environments potentially affecting spermatogonial stem cell metabolism and maturation. Indeed, the metabolic requirements of mouse primordial germ cells and pig gonocytes are distinct from their adult counterparts, and novel single-cell RNA sequencing analysis of human and porcine spermatogonial stem cells during development confirms this metabolic transition. Knowledge of the metabolic requirements and their changes and regulation during spermatogonial stem cell maturation is necessary to implement laboratory-based techniques and enable clinical use of spermatogonial stem cells. Based on the advancement in our understanding of germline metabolism circuits and maturation events of niche cells within the testis, we propose a new definition of spermatogonial stem cell maturation and its amendment in the light of metabolic change.

Targeted Gene Editing in Porcine Germ Cells.

As the genetic mutations driving human disease are identified, there is an increasing need for a biomedical model that can accurately represent the disease of interest and provide a platform for potential therapeutic testing. Pigs are a better model for human disease than rodents because of their genetic and physiological similarities to humans. However, current methods to generate porcine models are both technically challenging and expensive. Germline genetic modification through gene edited spermatogonia provides an effective alternative to how these models are developed. Here, we report an improved technique of gene editing in spermatogonia of pigs using CRISPR-Cas9 to generate different edits that reflect the genotypes of human diseases.