Homozygous ACTL9 mutations cause irregular mitochondrial sheath arrangement and abnormal flagellum assembly in spermatozoa and male infertility

Purpose To identify novel variants in ACTL9 and new phenotypes responsible for male infertility. Methods Genomic DNA was extracted from peripheral blood samples for whole-exome sequencing (WES). Computer-assisted sperm analysis (CASA) was used to test the motility of spermatozoa. The ultrastructure of flagella and the mitochondrial sheath were assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Immunostaining was used to validate the localization and expression of ACTL9 and ACTL7A. An Actl9 -mutated mouse model was used to validate the phenotypes by CASA and TEM. Results We identified novel homozygous variants in ACTL9 in two independent Chinese families. Spermatozoa with ACTL9 mutations showed decreased CASA parameters and a higher proportion of spermatozoa with abnormal morphology, exhibiting coiled flagella and a thickened midpiece. The spermatozoa were characterized by chaotic or irregular ‘9+2’ structures and irregular mitochondrial sheath arrangements in the flagellum. Actl9 knock-in mice also showed abnormal CASA parameters and irregular ‘9+2’ structures in flagella. Conclusions Our study expands the mutation spectrum and phenotypic spectrum of ACTL9 .