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

Abstract

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.

Keywords: ACTL9; Flagella assembly; Male infertility; Mitochondrial arrangement.

Fig. 1

Identification of mutations in ACTL9 in the male infertility cohort. (A) Pedigree analysis of two families with ACTL9 mutations in our cohort. Two individuals (black squares) exhibited homozygous mutations in ACTL9. Arrows (red) show the positions of the ACTL9 mutations. (B) H&E staining of spermatozoa from two males carrying mutations in ACTL9. The red arrow represents the thickened midpiece of spermatozoa. Scale bars: 5 μm. (C) The proportion of spermatozoa with coiled flagella and a thickened midpiece of spermatozoa in normal controls and males carrying mutations in ACTL9. (D) Representative SEM micrographs of deficiency in flagella and mid-piece of spermatozoa from the patient 1. The red arrow represents the thickened mid-piece of spermatozoa. Scale bars: 2 μm. ** represents P < 0.01

Fig. 2

ACTL9 mutations caused abnormal flagellar assembly and an irregular mitochondrial sheath. (A) Representative TEM micrographs of flagellar assembly in the mid-piece, principal piece and end piece from the normal control and patient 1. DMT represents peripheral doublet microtubule. CP represents central pair microtubules. ODF represents the outer dense fiber. MS represents the mitochondrial sheath. Scale bars: 100 nm. (B) The proportion of sperm with defects in flagellar assembly from the normal control and patient 1. (C) Representative TEM micrographs of the mitochondrial sheath in the midpiece of spermatozoa from normal controls and Patient 1. MS represents the mitochondrial sheath. Scale bars: 500 nm. (D) Immunostaining of TOMM20 (red)/a-tubulin (green) in spermatozoa from normal controls and two males carrying mutations in ACTL9. Scale bars, 5 μm. (E) The relative width of mitochondrial sheaths in spermatozoa from normal controls and two males carrying mutations in ACTL9. ** represents P < 0.01

Fig. 3

The expression and localization of ACTL9 and its binding protein ACTL7A. (A) Immunostaining of ACTL9 (red)/lectin staining (green) in spermatozoa from normal controls and males carrying mutations in ACTL9. Scale bars: 5 μm. (B) Immunostaining of ACTL7A/lectin (green) in spermatozoa from normal controls and males carrying mutations in ACTL9. Scale bars: 5 μm

Fig. 4

Characterization in Actl9^Mut/Mut male mice. (A) Immunoblotting analysis of ACTL9 protein expression in testes from WT and Actl9^Mut/Mut male mice. (B) Indensity ratio of ACTL9/GAPDH in Immunoblotting analysis. (C) Fertility assessment experiments in WT and Actl9^Mut/Mut male mice after mating with WT females. (D) VCL, VSL and VAP of sperm from WT and Actl9^Mut/Mut male mice. (E) Representative TEM micrographs of flagellar assembly in the midpiece, principal piece and end piece from WT and Actl9^Mut/Mut male mice. Scale bars: 200 nm. (F) The proportion of sperm with defects in the ‘9+2’ structure from WT and Actl9.^Mut/Mut male mice. ** represents P < 0.01, * represents P < 0.05