Mutations in DNAH1, which encodes an inner arm heavy chain dynein, lead to male infertility from multiple morphological abnormalities of the sperm flagella

Abstract

Ten to fifteen percent of couples are confronted with infertility and a male factor is involved in approximately half the cases. A genetic etiology is likely in most cases yet only few genes have been formally correlated with male infertility. Homozygosity mapping was carried out on a cohort of 20 North African individuals, including 18 index cases, presenting with primary infertility resulting from impaired sperm motility caused by a mosaic of multiple morphological abnormalities of the flagella (MMAF) including absent, short, coiled, bent, and irregular flagella. Five unrelated subjects out of 18 (28%) carried a homozygous variant in DNAH1, which encodes an inner dynein heavy chain and is expressed in testis. RT-PCR, immunostaining, and electronic microscopy were carried out on samples from one of the subjects with a mutation located on a donor splice site. Neither the transcript nor the protein was observed in this individual, confirming the pathogenicity of this variant. A general axonemal disorganization including mislocalization of the microtubule doublets and loss of the inner dynein arms was observed. Although DNAH1 is also expressed in other ciliated cells, infertility was the only symptom of primary ciliary dyskinesia observed in affected subjects, suggesting that DNAH1 function in cilium is not as critical as in sperm flagellum.

Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Location of DNAH1 Mutations in the Intron-Exon Structure and in the Protein Representation of DNAH1 (A) DNAH1 genomic structure. (B) DNAH1 domain map showing the location of the four identified mutations. The red boxes indicate the six known AAA-ATPase domains (AAA 1 to 6) as detected by homology (Uniprot server). The microtubule-binding domain (MBT) lies between AAA4 and AAA5. The N-terminal part of the protein binds to the intermediate, light-intermediate dynein chains. The position of the stalk and the microtubule-binding domain (MTB) are indicated.

Figure 2

Analysis of P3 Carrying the c.11788−1G>A Variant Evidencing DNAH1 mRNA Decay by RT-PCR and the Absence of DNAH1 in Sperm by Immunolocalization (A) RT-PCR analyses of subject P3 (c.11788−1G>A homozygote) and control individuals from the general population (C1–C3). Electrophoresis showing the RT-PCR amplification of DNAH1 exons 30–32, 64–66, 73–75. C1, C2, and C3 yield a normal fragment of 228, 293, and 241 bp, whereas subject P3 shows no amplification. There is no amplification from the RT-negative blank control (column B). (B) Electrophoresis showing the amplification of the same cDNAs with GAPDH and RPL0 primers. Bands of equivalent intensity are obtained from all samples including P3. Reverse transcription was carried out with 500 ng of extracted RNA and oligo dT priming. Two microliters of the obtained cDNA mix was used for the subsequent PCR. PCR amplification was carried out with three couples of primers located in exons 30–32, 64–66, and 73–75 of DNAH1 at an elongation temperature of 57°C (40 cycles), in parallel to amplification of the same samples with the control housekeeping GAPDH and RPL0, respectively, at an elongation temperature of 60°C (35 cycles). RT-PCR primers are listed in Table S4. (C and D) Immunofluorescence staining of human spermatozoa with DNAH1 antibodies (green) and DNAI2 (red). DNAH1 is observed throughout the flagellum in control sperm, whereas it is absent from P3’s sperm. In both control and P3 sperm, ODA is present as witnessed by the immunostaining of DNAI2. (E and F) Immunofluorescence staining of human spermatozoa with DNALI1 antibodies (green) and DNAI2 (red). DNALI1, a marker of IDA, is localized throughout flagella in control sperm, whereas it is strongly reduced in sperm from P3. No difference is noticed in the coimmunostaining of DNAI2. Sperm were counterstained with Hoechst 33342 (blue) as nuclei marker. White scale bars represent 5 μm. Sperm cells were washed in phosphate-buffered saline (PBS), fixed in 4% PFA for 2 min at room temperature (RT), and washed twice in PBS. Fixed spermatozoa were allowed to air-dry on poly-L-lysine coated slides followed by permeabilization with 0.5% Triton X-100. Samples were then blocked with (PBS)/1% bovine serum albumin (BSA)/2% normal goat serum (NGS) for 30 min at RT. Slides were incubated with the primary antibodies 2 hr followed by an incubation with the secondary antibodies for 45 min at RT and mounting in Dako mounting medium (Dako). Appropriate controls were performed, omitting the primary antibodies. Polyclonal mouse DNALI1 and monoclonal mouse DNAI2 were purchased from Abcam (UK) and Abnova Corporation (Taiwan), respectively. Polyclonal DNAH1 antibodies were purchased from Prestige Antibodies (Sigma-Aldrich). Monoclonal mouse anti-acetylated-α-tubulin were purchased from Sigma-Aldrich. Highly cross-adsorbed secondary antibodies (Alexa Fluor 488 and Alexa Fluor 546) were obtained from Molecular Probes (Invitrogen).

Figure 3

Electron Microscopy Analysis of Spermatozoa from P3, Carrying the c.11788−1G>A Variant, Reveals Numerous Ultrastructural Defects (A) EM cross-section of a flagellum from a control individual sperm sample. (B) Cross-section from the individual P3 sample showing numerous defects: lack of IDA (red arrow) and axonemal disorganization with mislocalized peripheral doublets (green arrows) associated with a displacement of the central pair (blue asterisk). (C) Cross-section from individual P3 sperm flagellum showing a complete absence of the central pair. (D) Cross-section from individual P3 sperm flagellum showing supernumerary dense fibers with absence of mitochondrion on the right side of the mid-piece. (E and F) Drawings describing the normal sperm axoneme ultrastructure with their different components (E) and different defects observed in DNAH1 mutated subjects (F). Sperm cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) during 2 hr at room temperature. Details of transmission electron microscopy technique were detailed previously.

Figure 4

Relative mRNA Expression of DNAH1, DNAH3, DNAH7, and DNAH12 in Testis and in Tracheal Cells Expression of DNAH1 mRNA (in green) in testis is significantly higher (8-fold) than in tracheal cells (in blue). Tracheal cell expression is set to 1. Expression of DNAH3 and DNAH7 mRNA are significantly lower in testis than in tracheal cells, whereas DNAH12 expression is not significantly different. Data are presented as mean ± standard deviation of three independent quantitative real-time PCR experiments. Statistical tests (paired t test) with a two-tailed p value ≤ 0.05 were considered as significant (∗). Human testis and trachea cDNA were obtained from Amsbio (Abingdon). mRNA expression were assessed by qPCR with a Biorad CFX9 (Biorad). PCR primers used to amplify DNAH1 and three other inner dynein arm heavy chain genes (DNAH3, DNAH7, and DNAH12) and the reference gene ACTB are listed in Table S5. The PCR cycle was as follows: 10 min 95°C, 1 cycle; 10 s 95°C, 30 s 58°C + fluorescence acquisition, 55 cycles. Analysis was performed with Biorad software CFX Manager v.3.0, with advanced relative quantification mode. Values for each gene were normalized to expression level of beta-actin gene (ACTB) via the 2-ΔΔCT method. The 2-ΔΔCT value was set at 0 in tracheal cells, resulting in an arbitrary expression of 1.

Figure 5

Proposed Schematic Model for the Location and the Function of the Inner Arm Heavy Chain DNAH1 in the Axoneme of Human Sperm Flagellum (A) Simplified representation showing a cross-sectional view of one microtubule doublet of an axoneme surrounding the central pair complex; the viewing is from the flagellar base. Light gray, central pair complex; light blue, outer doublets; dark blue, outer arm dyneins (OAD); dark pink, inner arm dynein heavy chain DNAH1; dark gray, radial spoke 3. (B) Longitudinal view illustrating the approximate localization on the outer doublet A-tubule of the various dyneins and regulatory structures within a single 96 nm axonemal repeat. RS3 stalk is directly connected to the DNAH1 tail through an arc-like structure. DNAH1 may therefore stabilize the RS3. Light pink, inner arm dyneins (IAD); yellow, IC/LC, intermediate chain/light chain; orange, nexin-dynein regulatory complex (N-DRC); green, modifier of inner arms (MIA) complex; blue, calmodulin- and spoke-associated complex (CSC); purple, distal protrusion (DP).