Title:
Rad18 is required for long-term maintenance of spermatogenesis in mouse testes

Satoshi Tateishi

Abstract:
Maintaining the integrity of spermatogenic stem cells is essential to transfer genetic information to a descendant. However, knowledge of maintenance of genetic stability in stem cells is still limited. RAD18 is critical for DNA damage tolerance (DDT) through mono- and multiubiquitination of proliferating cell nuclear antigen (PCNA) to maintain genomic stability. Mammalian RAD18 is highly expressed in the spermatocytes and the nuclei of a few spermatogonia in adult mice. To elucidate the physiological function of RAD18, we analyzed a phenotype of Rad18-/- mice. The mice were born and appeared to grow normally. Although the mice were fertile, fertility and testis weight decreased with age. Histological examination revealed normal spermatogenesis in almost all seminiferous tubules in Rad18-/- testes at 2 months old, and abnormal sperm could not be detected in the epididymis. However, 25% of the tubules lost almost all germ cells at 12 months. The seminiferous tubules frequently retained only late differentiated phase germ cells, suggesting that the exhaustion of spermatogonial stem cells leads to the loss of all germ cells in the seminiferous tubules. Wild-type germ cells were successfully transplanted into and colonized in the seminiferous tubules of aged Rad18-/- mice, indicating that Sertoli cells have a normal supportive function even in aged testes. We conclude that RAD18 is intrinsically required for the long-term maintenance of spermatogenesis.
Here we used single-molecule assay on combed DNA fibers to show that mouse embryonic fibroblasts (MEFs) derived from Rad18-/- mice displayed slow fork rates, suggesting replication stress in the MEFs. However it remains to be elucidated whether Rad18 and cell cycle checkpoint contribute to suppress replication stress in concerted manner. To this end, we prepared Chk2 ^-/-Rad18^-/- mice by mating. Unexpectedly MEFs derived from Chk2^-/- Rad18^-/- mice showed very slow growth rate. The MEFs displayed increased amount of RPA foci in nuclei and expressed high levels of p16, p53 and p21 proteins, suggesting that loss of Rad18 and Chk2 genes suppresses cellular growth via induction of the CDK inhibitors. We would like to discuss the role of Rad18 and ATM-Chk2 pathways in regulation of expression of CDK inhibitors.