Garin3 regulates sperm-oocyte fusion and male fertility.

The interaction between sperm and the zona pellucida (ZP), which involves the binding of sperm surface ligands to complementary carbohydrates on the ZP, is the initial direct contact between gametes and is crucial for subsequent gamete fusion and successful mammalian fertilization. In this study, we show that Garin3 is a critical gene for sperm-oocyte ZP binding. Garin3 expression begins during the acrosomal stage of spermatogenesis, and the gene is highly expressed in the male reproductive system. We created a Garin3 gene-knockout mouse model, and spermatozoa lacking Garin3 exhibited only mild morphological changes and showed no deficiency in motility; however, their ability to bind to the oocyte ZP was severely impaired and this ultimately resulted in failure of in vitro fertilization. These findings suggest that Garin3 is not directly involved in sperm-ZP binding and that it instead indirectly affects such binding by regulating the expression and localization of acrosome-related proteins, including RAB2, SPACA1, ADAM2, and ACR. Notably, fertilization failure was effectively rescued by bypassing the ZP-binding step by removing the ZP altogether or by performing intracytoplasmic sperm injection (ICSI). In human sperm samples, the expression levels of GARIN3 were significantly reduced in sperm with poor fertilization potential compared to sperm with normal fertilization potential, and this was consistent with the phenotypic observations in animal models. Our findings highlight the crucial role of Garin3 in fertilization and the potential molecular mechanism behind its effects.