Fig. 2

Homozygous knock-out mutation in Actl7a leads to male infertility and abnormal sperm head morphology in the mouse model. (A) Schematic illustration of strategies for the generation of Actl7a knock-out (KO) mice using CRISPR/Cas9 technology. 2 bp of Actl7a were deleted from Exon. (B) Genotyping of the constructed Actl7a knockout mutation in mice. WT, wild type; +/KO, heterozygous mutation; KO/KO, homozygous mutation. (C) The ACTL7A protein was completely absent in the testes of Actl7a-KO mice. α-tubulin was used as a loading control. (D) The Actl7a-KO mice were completely infertile. The fertility assessment experiments were performed among the male WT (WT; n = 5) mice, the male heterozygous (+/KO; n = 5) mutation mice and the male homozygous (KO/KO; n = 5) mutation mice. One-way ANOVA, ***P < 0.001; NS, not significant. Data are means ± SEM. (E-G) Sperm head morphology analysis of sperm cells in WT and Actl7a-KO mice. (E) Sperm nuclei stained with DAPI in WT and Actl7a-KO mice. 75% of the mutant sperm lacked the hook-like structure and showed smaller head compared with normal sperm. (F) Head morphology of sperm cells in WT and Actl7a-KO mice. (G) Area, bounding width, circularity, length of hook, perimeter, and regularity of sperm head in WT and Actl7a-KO mice