Genetic Loss Of Importin Alpha 4 Causes Abnormal Sperm Morphology And Impacts On Male Fertility In Mouse

Importin alpha proteins play a central role in the transport of cargo from the cytoplasm to the nucleus. In this study, we observed that male knock-out mice for importin alpha 4, which is encoded by theKpna4gene (Kpna4(-/-)), were subfertile and yielded smaller litter sizes than those of wild-type (WT) males. In contrast, mice lacking the closely related importin alpha 3 (Kpna3(-/-)) were fertile. In vitro fertilization and sperm motility assays demonstrated that sperm fromKpna4(-/-)mice had significantly reduced quality and motility. In addition, acrosome reaction was also impaired inKpna4(-/-)mice. Transmission electron microscopy revealed striking defects, including abnormal head morphology and multiple axoneme structures in the flagella ofKpna4(-/-)mice. A five-fold increase in the frequency of abnormalities inKpna4(-/-)mice compared toWTmice indicates the functional importance of importin alpha 4 in normal sperm development. Moreover, Nesprin-2, which is a component of the linker of nucleus and cytoskeleton complex, was expressed at lower levels in sperm fromKpna4(-/-)mice and was localized with abnormal axonemes, suggesting incorrect formation of the nuclear membrane-cytoskeleton structure during spermiogenesis. Proteomics analysis ofKpna4(-/-)testis showed significantly altered expression of proteins related to sperm formation, which provided evidence that genetic loss of importin alpha 4 perturbed chromatin status. Collectively, these findings indicate that importin alpha 4 is critical for establishing normal sperm morphology in mice, providing new insights into male germ cell development by highlighting the requirement of importin alpha 4 for normal fertility.