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.

To study intracellular pathways by which the human papillomavirus 16 oncogene E7 participates in carcinogenesis, we expressed an inducible chimera of E7 by fusion to the hormone-binding domain of the estrogen receptor. The chimeric protein (E7ER) transformed rodent fibroblast cell lines and induced DNA synthesis on addition of estradiol. In coimmunoprecipitation experiments, E7ER preferentially bound p130 when compared to p107 and pRb. After estradiol addition, E7ER localization changed to a more intense intranuclear staining. Induction of E7 function was not correlated with binding to p130 or pRb but rather with intranuclear localization and modest induction of binding to p107.Human papillomavirus (HPV) infection of genital tract epithelium is associated with dysplastic changes in epithelial growth and is an important prerequisite for the development of cervical cancer. Two viral genes, E6 and E7, mediate transformation by HPV (1-4). The majority of cervical carcinomas contain truncated forms of HPV, which retains E6 and E7, integrated into the cellular genome (5-9). HPV16 E7 induces growth of cells in soft agar (1), stimulates DNA synthesis (10, 11), cooperates with ras to transform primary cells (12, 13), and overcomes G 1 arrest induced by serum deprivation, actinomycin D, or overexpression of p21 (14-17). The protein encoded by E7 has a region of shared sequence homology with both adenovirus E1A and SV40 large T antigen, which mediates binding to the retinoblastoma gene product pRb (13,18). By binding to pRb, E7 alters the interaction of pRb with the transcription factor E2F-1, resulting in activation of E2F-responsive genes (19). E7 protein from high-risk HPV types, HPV16 and HPV18, binds with a higher affinity to pRb than E7 protein from low-risk HPV types such as HPV6 and HPV11 (20). Therefore, the prevailing model is that E7 protein, by binding to pRb, promotes progression through the cell cycle. The role of E7 binding to other Rb-family members such as p107 and p130 has not been entirely elucidated. However, it is known that induction of B-myb transcription by E2F-1 is the result of E7 binding to p107 rather than to pRb (21).To assess the relationship between transformation and E7 binding to Rb-family members, we have constructed an inducible chimeric molecule consisting of HPV16 E7 fused in-frame to the hormone-binding domain of the estrogen receptor (ER), an approach used to render other oncogenes steroiddependent (22)(23)(24)(25)(26)(27). By using this inducible chimera of E7, we demonstrate that HPV16 E7 activity is correlated with intranuclear localization of the chimera. In addition, E7 binds to greater proportions of intracellular p130 and p107 than pRb in quiescent cells.

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.