Aims: Nuclear-cytoplasmic transport is a fundamental process in eukaryotic cells. Importin α proteins play a central role in the nuclear transport processes, and there are 6 subtypes in mouse and 7 in human. Although some subtypes show high expression in specific male germ cells, it is still unclear what function each of these subtypes performs in male reproduction.
Methods: We established a knock-out (KO) mouse of Importin α4 protein, which is encoded by the Kpna4 gene. The Kpna4 KO mouse was subfertile and yielded smaller litter sizes than those of wild-type (WT) males. To understand the molecular function of Importin α4 protein in spermatogenesis, we performed an in vitro fertilization assay, sperm motility assays, transmission electron microscopy and a comprehensive proteomics analysis.
Results: Sperm from the Kpna4 KO mouse had significantly reduced quality and motility, and the acrosome reaction was also impaired. Transmission electron microscopy revealed striking defects in sperm from the KO mice, including abnormal head morphology and multiple axoneme structures. A comprehensive proteomics analysis of testis from the KO mice followed by the ChIP-Atlas enrichment analysis which searches for proteins significantly bound around multiple query genes indicated that genes perturbed by loss of Importin α4 are regulated by Taf7l and Tbpl1 transcription factors, both of which are necessary for spermatogenesis. In addition, genes encoding those proteins identified by the proteomics analysis were characterized by active histone marks such as H3K4me3 and H3K27ac in the testes and sperm-related cells.
Conclusion: Our findings indicate that Importin α4 is critical for establishing normal sperm morphology in mice, and genetic loss of Importin α4 could disrupt the chromatin status in testicular cells, resulting in reduced expression of genes critical for sperm formation.