Acrosin is essential for sperm penetration through the zona pellucida in hamsters.

During natural fertilization, mammalian spermatozoa must pass through the zona pellucida before reaching the plasma membrane of the oocyte. It is assumed that this step involves partial lysis of the zona by sperm acrosomal enzymes, but there has been no unequivocal evidence to support this view. Here we present evidence that acrosin, an acrosomal serine protease, plays an essential role in sperm penetration of the zona. We generated acrosin-knockout (KO) hamsters, using an in vivo transfection CRISPR/Cas9 system. Homozygous mutant males were completely sterile. Acrosin-KO spermatozoa ascended the female genital tract and reached ovulated oocytes in the oviduct ampulla, but never fertilized them. In vitro fertilization (IVF) experiments revealed that mutant spermatozoa attached to the zona, but failed to penetrate it. When the zona pellucida was removed before IVF, all oocytes were fertilized. This indicates that in hamsters, acrosin plays an indispensable role in allowing fertilizing spermatozoa to penetrate the zona. This study also suggests that the KO hamster system would be a useful model for identifying new gene functions or analyzing human and animal disorders because of its technical facility and reproducibility.

Behavior of ACRBP-deficient mouse sperm in the female reproductive tract.

Gene-knockout mice lacking ACRBP, a proacrosin-binding protein localized in the acrosome of sperm, have been shown to exhibit male subfertility, owing to abnormal formation of the acrosome. In this study, to elucidate the mechanism contributing to the subfertility phenotype, we examined the behavior of ACRBP-deficient mouse sperm in the female reproductive tract. When sperm that had migrated into the uterus and oviduct after mating were counted, the number of ACRBP-deficient sperm was noticeably smaller in the oviduct of mice post mating. However, ACRBP-deficient sperm recovered from the oviduct possessed morphologically normal head shape and retained normal motility. Importantly, ACRBP-deficient sperm displayed a marked reduction in the ability to successfully gain access to unfertilized oocytes. These data suggest that male subfertility of ACRBP-deficient mice may be attributed to incompleteness of the acrosome reaction rather than impairment in sperm migration from the uterus to the oviduct.

Biogenesis of sperm acrosome is regulated by pre-mRNA alternative splicing of Acrbp in the mouse.

Proper biogenesis of a sperm-specific organelle, the acrosome, is essential for gamete interaction. An acrosomal matrix protein, ACRBP, is known as a proacrosin-binding protein. In mice, two forms of ACRBP, wild-type ACRBP-W and variant ACRBP-V5, are generated by pre-mRNA alternative splicing of Acrbp Here, we demonstrate the functional roles of these two ACRBP proteins. ACRBP-null male mice lacking both proteins showed a severely reduced fertility, because of malformation of the acrosome. Notably, ACRBP-null spermatids failed to form a large acrosomal granule, leading to the fragmented structure of the acrosome. The acrosome malformation was rescued by transgenic expression of ACRBP-V5 in ACRBP-null spermatids. Moreover, exogenously expressed ACRBP-W blocked autoactivation of proacrosin in the acrosome. Thus, ACRBP-V5 functions in the formation and configuration of the acrosomal granule during early spermiogenesis. The major function of ACRBP-W is to retain the inactive status of proacrosin in the acrosome until acrosomal exocytosis.