Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility

Abstract

Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF. To further define genetic factors underlying MMAF, we performed whole-exome sequencing in a cohort of 90 Chinese MMAF-affected men. Two cases (2.2%) were identified as carrying bi-allelic missense DNAH8 variants, variants which were either absent or rare in the control human population and were predicted to be deleterious by multiple bioinformatic tools. Re-analysis of exome data from a second cohort of 167 MMAF-affected men from France, Iran, and North Africa permitted the identification of an additional male carrying a DNAH8 homozygous frameshift variant. DNAH8 encodes a dynein axonemal heavy-chain component that is expressed preferentially in the testis. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring bi-allelic DNAH8 variants showed a highly aberrant morphology and ultrastructure of the sperm flagella. Immunofluorescence assays performed on the spermatozoa from men harboring bi-allelic DNAH8 variants revealed the absent or markedly reduced staining of DNAH8 and its associated protein DNAH17. Dnah8-knockout male mice also presented typical MMAF phenotypes and sterility. Interestingly, intracytoplasmic sperm injections using the spermatozoa from Dnah8-knockout male mice resulted in good pregnancy outcomes. Collectively, our experimental observations from humans and mice demonstrate that DNAH8 is essential for sperm flagellar formation and that bi-allelic deleterious DNAH8 variants lead to male infertility with MMAF.

Keywords: CRISPR; DNAH17; DNAH8; ICSI; dynein; exome; flagella; infertility; knockout; sperm.

Figure 1

Identification of Bi-allelic DNAH8 Variants in Men with MMAF (A–C) The pedigrees of three families affected by DNAH8 variants. The NCBI reference sequence number for DNAH8 transcript is NM_001206927.2. Sanger sequencing results are shown below the pedigrees. The variant positions are indicated by red arrows or a dashed box. WT, wild type. (D) Variant locations and phylogenetic conservation of the mutated residues in DNAH8 protein. The NCBI reference sequence number for DNAH8 protein is NP_001193856.1. Colored squares denote different domains according to the NCBI browser. DHC_N1—dynein heavy chain, N-terminal region 1; CKAP2_C—cytoskeleton-associated protein 2 C terminus; DHC_N2—dynein heavy chain, N-terminal region 2; P-loop_NTPase—P-loop containing nucleoside triphosphate hydrolases; MT—microtubule-binding stalk of dynein motor; AAA_9—ATP-binding dynein motor region D5; Dynein_heavy—dynein heavy chain and region D6 of dynein motor.

Figure 2

Morphology of the Spermatozoa from Men Harboring Bi-allelic DNAH8 Variants (A) Normal morphology of the spermatozoon from a healthy control male evident under light microscopy. Scale bars: 5 μm. (B–E) Most spermatozoa from men harboring bi-allelic DNAH8 variants presented MMAF phenotypes, including absent (B), short (C), coiled (D), and irregular-caliberflagella (E). The data of subject A051 as an example. (F) Normal morphology of the spermatozoon from a healthy control male as revealed by scanning electronic microscopy. (G–J) The MMAF phenotypes, including absent (G), short (H), coiled (I), irregular-caliber flagella (J), were clearly revealed in the spermatozoa from subject A051 harboring bi-allelic DNAH8 variants.

Figure 3

TEM Analyses of Sperm Cells from Men Harboring Bi-allelic DNAH8 Variants (A and B) Cross-sections of the mid-piece (A) and principal piece (B) of the sperm flagella in a control individual show the typical ‘‘9 + 2’’ microtubule structure, including nine pairs of peripheral microtubule doublets (DMT; blue arrows), nine outer dense fibers (ODF; yellow arrows), and the central pair of microtubules (CP; red arrows). The outer dynein arms (ODA; green arrows) and inner dynein arms (IDA; pink arrows) are also visible. Scale bars: 200 μm. (C–F) In the spermatozoa from men harboring bi-allelic DNAH8 variants, various axonemal anomalies can be observed, including the lack of CP (C) or DMT (D). Misarranged (D) or supernumerary ODFs (E) were also observed. ODAs were disassembled or absent in the samples from men carrying bi-allelic DNAH8 variants (C–F).

Figure 4

Localization of DNAH8 and Associated Protein DNAH17 in the Spermatozoa from Men Harboring Bi-allelic DNAH8 Variants (A) Immunofluorescence staining of the spermatozoa from a normal control male and subjects carrying bi-allelic DNAH8 variants. Anti-DNAH8 (red) and anti-α-tubulin (green) antibodies were used. Spermatozoa were counterstained with 4′,6-diamidino-2-phenylindole as a marker of the cell nucleus. In the fertile control male, DNAH8 immunostaining (red) concentrated along the sperm flagella, but this signal was almost absent from the sperm flagella from subjects harboring bi-allelic DNAH8 variants. The data of subject A051 are provided to illustrate the typical staining observed in the DNAH8-associated cases. Scale bars: 5 μm. (B) DNAH17 immunostaining is affected in the spermatozoa from men harboring bi-allelic DNAH8 variants. The spermatozoa were stained with anti-DNAH17 (red) and anti-α-tubulin (green) antibodies. DNAH17 staining mainly localized along the spermflagella from a control male, while being evidently reduced in the spermatozoa from subject A051. Scale bars: 5 μm.

Figure 5

Sperm Morphology and Ultrastructure Analyses for Dnah8-KO Male Mice (A) H&E staining of the spermatozoa obtained from mouse cauda epididymis. The spermatozoa from heterozygous mutated (Dnah8^wt/em1) male mice showed normal morphology. In contrast, Dnah8-KO (Dnah8^em1/em1) male mice manifested aberrantflagellar morphologies, which were consistent with the clinical phenotypes in the MMAF-affected men. Scale bar: 5 μm. (B) TEM of cross-sections of the spermatozoa from Dnah8-KO male mice. Cross-sections of the mid-piece (i) and principal piece (iii) of the sperm flagella in heterozygous mutated male mice are shown as controls. (ii) Cross-sections of the mid-piece of the sperm flagella in Dnah8-KO male mice revealed disorganization of mitochondrial sheaths, outer dense fibers, and microtubules. (iv) Cross-sections of the principal piece of the sperm flagella in Dnah8-KO male mice showed disorganization of fibrous sheaths, outer dense fibers, and microtubules. White arrows indicate mitochondrial sheath, orange arrows indicate fibrous sheaths, yellow arrows indicate outer dense fibers, blue arrows indicate peripheral microtubule doublets, and red arrows indicate the central pair of microtubules. Scale bars: 200 nm.

Figure 6

Dnah8 is Essential for Normal Spermatogenesis in Mice (A) The development of sperm flagella was investigated in mouse testis through the use of H&E staining. In stage VII–VIII seminiferous tubules, normal round spermatids (arrowheads) were observed, but elongated tails (arrows) were not observed in the testes from Dnah8-KO (Dnah8^em1/em1) male mice. Scale bar: 20 μm. (B) PAS staining of the cauda epididymis from male mice. Decreased sperm quantity was observed in the epididymis from Dnah8-KO male mice, when compared with that in heterozygous mutated (Dnah8^wt/em1) male mice. Scale bar: 20 μm.

Figure 7

Pups Obtained upon ICSI using the Spermatozoa from Dnah8-KO Male Mice (A) Development of ICSI embryos. Fourteen (17.3%) of the 81 oocytes injected with the spermatozoa from Dnah8-KO (Dnah8^em1/em1) male mice developed to the two-cell stage, and three pups were obtained after embryo transfer. ICSI data from the use of sperm from wild-type male mice are shown as controls. (B) The pups obtained from ICSI using the spermatozoa from Dnah8-KO male mice. (C) Genotyping of these pups. All of the pups carried the heterozygous Dnah8-mutated allele. KO denotes the mutated allele and WT denotes the wild-type allele.