The Threshold of Spermatogonial Stem Cell Numbers That Allows Male Fertility Restoration in Mice after a Treatment with an Alkylating Agent, Busulfan.

Abstract

As the cancer survival rate has been increasing steadily in recent years, the risk of infertility induced by anti-cancer regimen has become a significant concern for the quality of post-cancer life. Spermatogonial stem cells (SSCs) constitute the foundation of spermatogenesis and represent a crucial resource for the male fertility restoration. Using the mouse as a model, we investigated in this study the cytotoxic effect on SSCs of an alkylating agent, busulfan, and correlated the kinetics of SSC loss and proliferation with those of male fertility loss and restoration. We treated male transgenic mice, which carry LacZ in all male germ cells, with busulfan at three doses (15, 30 and 45 mg/kg) and examined their fertility by mating with intact females at different times post treatment. We measured SSC numbers by a transplantation assay using immunocompatible recipient mice. Colonies of donor-derived spermatogenesis were visualized by incubating recipient testes with 5-bromo-4chloro-3-indolyl β-galactosidase (X-gal) to determine the number of functional donor SSCs at each time point. We also measured testis weights and sperm counts and assessed spermatogenic recovery in histology. We found that fertility was lost within 4 weeks after treatment, regardless of busulfan doses. However, most SSCs were lost (>95%) in a dose-dependent manner within 3 days after treatment. Male fertility was restored in 12 weeks following treatment with 15 mg/kg busulfan, while 26 and 30 weeks were required when the males received 30 and 45 mg/kg busulfan, respectively, demonstrating a dose-dependent effect on SSC recovery. Interestingly, however, SSC numbers reached 30% of the before-treatment level by 4 weeks prior to the time of fertility restoration, regardless of busulfan doses (i.e., 8, 22, and 26 weeks with 15, 30, and 45 mg/kg busulfan, respectively). In agreement with these results, testis weights and spermatogenic recovery showed a dose-dependent effect of busulfan and its preferential killing of SSCs. The results of sperm counts showed that 17.5% of the before-treatment sperm number is required to grant fertility in mice, below which mice remained infertile. These results demonstrate that the loss and restoration of fertility after busulfan treatment are a direct consequence of SSC loss and proliferation. Importantly, the fact that the SSC numbers at 4 weeks before fertility recovery are nearly identical across the busulfan doses used indicate that there is a threshold in SSC numbers that allows the restoration of male fertility, which is 30% of the initial SSC numbers. In addition, these results set 17.5% of sperm count as a critical size of sperm population that allows male fertility in mice. (poster)