Proper timing of a quiescence period in precursor prospermatogonia is required for stem cell pool establishment in the male germline.

The stem cell-containing undifferentiated spermatogonial population in mammals, which ensures continual sperm production, arises during development from prospermatogonial precursors. Although a period of quiescence is known to occur in prospermatogonia prior to postnatal spermatogonial transition, the importance of this has not been defined. Here, using mouse models with conditional knockout of the master cell cycle regulator Rb1 to disrupt normal timing of the quiescence period, we found that failure to initiate mitotic arrest during fetal development leads to prospermatogonial apoptosis and germline ablation. Outcomes of single-cell RNA-sequencing analysis indicate that oxidative phosphorylation activity and inhibition of meiotic initiation are disrupted in prospermatogonia that fail to enter quiescence on a normal timeline. Taken together, these findings suggest that key layers of programming are laid down during the quiescent period in prospermatogonia to ensure proper fate specification and fitness in postnatal life.

Donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout males.

Spermatogonial stem cell transplantation (SSCT) is an experimental technique for transfer of germline between donor and recipient males that could be used as a tool for biomedical research, preservation of endangered species, and dissemination of desirable genetics in food animal populations. To fully realize these potentials, recipient males must be devoid of endogenous germline but possess normal testicular architecture and somatic cell function capable of supporting allogeneic donor stem cell engraftment and regeneration of spermatogenesis. Here we show that male mice, pigs, goats, and cattle harboring knockout alleles of the NANOS2 gene generated by CRISPR-Cas9 editing have testes that are germline ablated but otherwise structurally normal. In adult pigs and goats, SSCT with allogeneic donor stem cells led to sustained donor-derived spermatogenesis. With prepubertal mice, allogeneic SSCT resulted in attainment of natural fertility. Collectively, these advancements represent a major step toward realizing the enormous potential of surrogate sires as a tool for dissemination and regeneration of germplasm in all mammalian species.