Bi-allelic mutations of DNAH10 cause primary male infertility with asthenoteratozoospermia in humans and mice

Abstract

Multiple morphological abnormalities of the sperm flagella (MMAF)-induced asthenoteratozoospermia is a common cause of male infertility. Previous studies have identified several MMAF-associated genes, highlighting the condition's genetic heterogeneity. To further define the genetic causes underlying MMAF, we performed whole-exome sequencing in a cohort of 643 Chinese MMAF-affected men. Bi-allelic DNAH10 variants were identified in five individuals with MMAF from four unrelated families. These variants were either rare or absent in public population genome databases and were predicted to be deleterious by multiple bioinformatics tools. Morphological and ultrastructural analyses of the spermatozoa obtained from men harboring bi-allelic DNAH10 variants revealed striking flagellar defects with the absence of inner dynein arms (IDAs). DNAH10 encodes an axonemal IDA heavy chain component that is predominantly expressed in the testes. Immunostaining analysis indicated that DNAH10 localized to the entire sperm flagellum of control spermatozoa. In contrast, spermatozoa from the men harboring bi-allelic DNAH10 variants exhibited an absence or markedly reduced staining intensity of DNAH10 and other IDA components, including DNAH2 and DNAH6. Furthermore, the phenotypes were recapitulated in mouse models lacking Dnah10 or expressing a disease-associated variant, confirming the involvement of DNAH10 in human MMAF. Altogether, our findings in humans and mice demonstrate that DNAH10 is essential for sperm flagellar assembly and that deleterious bi-allelic DNAH10 variants can cause male infertility with MMAF. These findings will provide guidance for genetic counseling and insights into the diagnosis of MMAF-associated asthenoteratozoospermia.

Keywords: DNAH10; MMAF; knockout mice; male infertility; sperm flagella.

Figure 1

Identification of bi-allelic DNAH10 variants in men with asthenoteratozoospermia (A) Pedigrees of four families affected by DNAH10 variants (M1–M6) identified via WES. Male individuals with asthenoteratozoospermia in these families are indicated by black-filled squares. The double lines indicate first-degree consanguinity. (B) Sanger sequencing confirmed the presence of bi-allelic DNAH10 variants (M1–M6) in T012 II-2, T089 II-1, H049 II-2, NK067 II-1, and NK067 II-2. The variant positions are indicated by using red arrows. WT, wild-type.

Figure 2

Sperm morphology analyses for men harboring bi-allelic DNAH10 variants (A) H&E staining for the spermatozoa obtained from a fertile control individual (NC) and men harboring bi-allelic DNAH10 variants. Compared to the spermatozoa of NC, which presented long, smooth tails (i), most spermatozoa obtained from men harboring DNAH10 variants displayed typical MMAF phenotypes, such as short (ii), absent (iii), coiled (iv), and irregular flagella (v). The data of H049 II-2 are shown as an example. Scale bars, 5 μm. (B) SEM analysis of the spermatozoa obtained from a fertile control individual (NC) and men harboring bi-allelic DNAH10 variants. (i) Normal morphology of the spermatozoon from a healthy control male. (ii–vii) Most spermatozoa obtained from men harboring bi-allelic DNAH10 variants displayed typical MMAF phenotypes, including short (ii), absent (iii), bent (iv), coiled (v), and irregular flagella (vi and vii). The data of H049 II-2 are shown as an example. Scale bars, 5 μm.

Figure 3

Expression analysis of DNAH10 in the spermatozoa obtained from a male control individual and men harboring bi-allelic DNAH10 variants Representative images of spermatozoa obtained from a fertile control individual (NC) and men harboring bi-allelic DNAH10 variants (T012 II-2, NK067 II-1, and NK067 II-2) stained with the anti-DNAH10 antibody, anti-α-tubulin antibody, and DAPI. Staining results revealed that DNAH10 was localized along the sperm flagella in the sperm obtained from the NC but was almost absent in the sperm flagella in the sperm obtained from men harboring bi-allelic DNAH10 variants. Representative data are provided to illustrate the typical staining observed in DNAH10-associated cases. Scale bars, 5 μm.

Figure 4

Sperm ultrastructure analyses for men harboring bi-allelic DNAH10 variants TEM analysis of spermatozoa obtained from a fertile control individual (NC) and men harboring bi-allelic DNAH10 variants. Cross-sections of the midpiece and principal piece of the sperm flagella in the sperm obtained from NC displayed typical “9 + 2” microtubule structure and peri-axoneme structure. The axoneme microtubule structure, including nine pairs of peripheral doublet microtubules (DMT; indicated with white arrows) and the central pair of microtubules (CP; indicated with blue arrows), is visible. The outer dynein arms (ODA; indicated with yellow arrows) and inner dynein arms (IDA; indicated with red arrows) are also visible. The peri-axoneme structure includes a helical mitochondrial sheath (MS; indicated with green arrows), nine outer dense fibers (ODFs; indicated with orange arrows), and the fiber sheath (FS; indicated with pink arrows). Cross-sections of the midpiece, principal piece, and endpiece of the spermatozoa obtained from men harboring bi-allelic DNAH10 variants revealed typical axonemal anomalies with the absence of inner dynein arms, while other axoneme microtubule structures seemed to be unaffected. The data of NK067 II-1 and NK067 II-2 are shown as an example. Scale bars, 200 nm.

Figure 5

DNAH2 and DNAH6 immunostaining is altered in spermatozoa obtained from men harboring bi-allelic DNAH10 variants (A and B) The spermatozoa obtained from a fertile control individual (NC) and men harboring bi-allelic DNAH10 variants were stained with anti-DNAH2 (indicated as red in [A]), anti-DNAH6 (indicated as red in [B]), anti-a-tubulin (indicated as green) antibodies, and DAPI (indicated as blue). DNAH2 and DNAH6 normally localized along the sperm flagella in the control sperm. However, expression of both DNAH2 and DNAH6 was almost absent in the sperm obtained from men harboring bi-allelic DNAH10 variants. Scale bars, 10 μm.

Figure 6

Dnah10 deficiency results in typical MMAF phenotypes and infertility in male mice (A) Fertility test of male mice at 2–5 months of age after mating with Dnah10^+/− females (^∗∗∗p < 0.001). (B) The size (left) and weight (right) of testes were comparable between Dnah10^−/− and Dnah10^+/− male mice at 2 months of age. n.s., not significant. (C) The sperm concentration of Dnah10^−/− male mice was significantly lower than that of Dnah10^+/− male mice (^∗∗∗p < 0.001). (D) Percentages of motile sperms in Dnah10^−/− male mice and Dnah10^+/− male mice at 2 months of age (^∗∗∗p < 0.001). (E) H&E staining of the spermatozoa obtained from mouse cauda epididymis. When compared with the normal morphology of spermatozoa obtained from Dnah10^+/− male mice, Dnah10^−/−male mouse spermatozoa exhibited aberrant flagellar morphologies, which were consistent with the clinical phenotypes observed in men harboring bi-allelic DNAH10 variants. (F) Cross-sectional ultrastructure of cauda epididymal spermatozoa obtained from Dnah10^−/− male mice and Dnah10^+/− male mice at 2 months of age via TEM. Compared with the normal ultrastructure in Dnah10^+/− male mice, cross-sections of the midpiece and principal piece of the sperm flagella in Dnah10^−/− male mice revealed disorganization of the axoneme, mitochondrial sheaths, and outer dense fibers. Scale bars, 100 nm.

Figure 7

Dnah10^M/M male mice exhibit typical MMAF phenotypes and infertility (A) Fertility test of male mice at 2–5 months of age after mating with Dnah10^+/M females (^∗∗∗p < 0.001). (B) The size (left) and weight (right) of testes were comparable between Dnah10^M/M and Dnah10^+/M male mice at 2 months of age. n.s., not significant. (C) The sperm concentration of Dnah10^M/M male mice was significantly lower than that of Dnah10^+/M male mice (^∗∗∗p < 0.001). (D) Percentages of motile sperms (e) in Dnah10^M/M male mice and Dnah10^+/M male mice at 2 months of age (^∗∗∗p < 0.001). (E) H&E staining of the spermatozoa obtained from mouse cauda epididymis. When compared with the normal morphology of spermatozoa obtained from Dnah10^+/M male mice, Dnah10^M/M male mouse spermatozoa exhibited aberrant flagellar morphologies, which were consistent with the clinical phenotypes in men harboring bi-allelic DNAH10 variants. (F) Cross-sectional ultrastructure of cauda epididymal spermatozoa obtained from Dnah10^M/M male mice and Dnah10^+/M male mice at 2 months of age via TEM. Compared with the normal ultrastructure in Dnah10^+/M male mice, cross-sections of the midpiece and principal piece of the sperm flagella in Dnah10^M/M male mice revealed disorganization of the axoneme, mitochondrial sheaths, and outer dense fibers. Scale bars, 100 nm.