Down-Regulation of Neogenin Decreases Proliferation and Differentiation of Spermatogonia during the Early Phase of Spermatogenesis

Non-obstructive azoospermia is a major clinical issue associated with male infertility that remains to be addressed. Although neogenin is reportedly abundantly expressed in the testis, its role in mammalian spermatogenesis is unknown. We systematically investigated the role of neogenin during spermatogenesis by performing loss-of-function studies. Testis-specific neogenin conditional knock-out (cKO) mice were generated using CRISPR/Cas9 and neogenin-targeting guide RNAs. We analyzed the expression profiles of germ cell factors by RT-PCR and Western blotting. Neogenin localized mainly to spermatogonia in seminiferous tubules of mouse testes. RT-PCR and Western blot analyses further demonstrated that neogenin expression varied during spermatogenesis and was dramatically increased at postnatal day 12–25 during the pubertal stage. In neogenin-cKO mouse testes, the ratio of primary and secondary spermatocytes was significantly decreased compared with the control, while the number of apoptotic testicular cells was significantly increased. Taken together, these results suggest that neogenin plays a pivotal role in the maintenance and proliferation of spermatogonia during the early stage of spermatogenesis in mice.