Biallelic pathogenic variants in DNAH3 cause male infertility in humans and mice

Xiang Wang
Gan Shen
Yihong Yang
Chuan Jiang
Tiechao Ruan
Xue Yang
Liangchai Zhuo
Yingteng Zhang
Yangdi Ou
Xinya Zhao
Shunhua Long
Xiangrong Tang
Tingting Lin author has email address
Ying Shen author has email address

Department of Obstetrics/Gynecology, Key Laboratory of Obstetric, Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu 610041, China
NHC Key Laboratory of Chronobiology, Sichuan University, Chengdu 610041, China
Reproduction Medical Center of West China Second University Hospital, Key Laboratory of Obstetric, Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, Sichuan University, Chengdu 610041, China
Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, China
West China School of Medicine, Sichuan University, Chengdu 610041, China
West China School of Basic Medicine and Forensic Medicine, Sichuan University, Chengdu 610041, China
Chongqing Key Laboratory of Human Embryo Engineering, Center for Reproductive Medicine, Women and Children’s Hospital of Chongqing Medical University, Chongqing 400010, China
Chongqing Clinical Research Center for Reproductive Medicine, Chongqing Health Center for Women and Children, Chongqing 400010, China

https://doi.org/10.7554/eLife.96755.1

Open access
Copyright information

Figures and data

Identification of biallelic pathogenic variants in DNAH3 from four unrelated infertile families.
(A) Pedigrees of four families affected by DNAH3 variants (M1–M7). Black arrows indicate the probands in these families. (B) Location of the variants and conservation of affected amino acids in DNAH3. Black arrows indicate the position of the variants. (C) Immunofluorescence staining of DNAH3 in sperm from the patients and normal control. Red, DNAH3; green, α-Tubulin; blue, DAPI; scale bars, 5 μm. (D) Western blotting analysis of DNAH3 expressed in spermatozoa from the patients and normal control.

Semen analysis of the patients in the present study.

Variants analysis of the patients in the present study.

Defects in sperm morphology of the patients harboring DNAH3 variants.
(A, B) Abnormal sperm morphology was observed through Papanicolaou staining (A), and SEM analysis (B) compared to normal control. Scale bars, 5 μm.

Ultrastructural and mitochondrial defects in sperm from infertile men with DNAH3 variants.
(A) TEM analysis of sperm obtained from a normal control and patients harboring DNAH3 variants. Cross-sections of the midpiece, principal piece and endpiece of sperm from normal control showed the typical ‘‘9 + 2’’ microtubule structure, and an IDA and an ODA were displayed on the A-tube of each microtubule doublet. Cross-sections of the midpiece, principal piece and endpiece of sperm from the patients displayed absent or disordered CPs, MTDs and ODFs, as well as an evident missing of the IDAs in different pieces of the flagella. M, mitochondria sheath; ODF, outer dense fiber; MTD, microtubule doublets; CP, central pair; IDA, inner dynein arms; ODA, outer dynein arms. Scale bars, 200 nm. (B) Immunofluorescence staining of TOM20 in sperm from the patients and normal control. Red, TOM20; green, α-Tubulin; blue, DAPI; scale bars, 5 μm.

Dnah3 KO male mice are infertile.
(A) Fertility of Dnah3 KO mice. The KO male mice were infertile (n = five biologically independent WT mice or KO mice; Student’s t test; *, P < 0.05; NS, no significance; error bars, s.e.m.). (B) TEM analysis of the cross-sections of spermatozoa from Dnah3 KO mice revealed an obvious absence of IDAs in different pieces of the flagella compared to WT mice. M, mitochondrion sheath; ODF, outer dense fiber; MTD, microtubule doublet; CP, central pair; IDA, inner dynein arm; ODA, outer dynein arm. Scale bars, 200 nm. (C) Disrupted mitochondria were observed in spermatozoa tail from Dnah3 KO mice by TEM analysis. The yellow arrows indicate the normal mitochondria. The red arrowheads indicate the dilated intermembrane spaces and dissolved mitochondrial material. M, mitochondrion sheath. Scale bars, 200 nm. (D) Immunofluorescence staining of SLC25A4 indicated impaired mitochondrial formation in Dnah3 KO mice compared to WT mice. Red, SLC25A4; green, α-Tubulin; blue, DAPI; scale bars, 5 µm.

Semen analysis using CASA in the Dnah3 KO mice.

Immunofluorescence staining and western blotting analysis of IDA-associated proteins in spermatozoa obtained from normal control and patients with DNAH3 variants.
(A – C) Immunofluorescence staining of DNAH1 (A), DNAH6 (B) and DNALI1 (C) in spermatozoa from patients and normal controls. Red, DNAH1 in (A), DNAH6 in (B), DNALI1 in (C); green, α-Tubulin; blue, DAPI; scale bars, 5 μm. (D – F) Western blotting analysis of DNAH1(D), DNAH6 (E), DNALI1 (F) in sperm lysates from the patients and normal control.

Immunofluorescence staining and western blotting analysis of IDA-associated proteins in spermatozoa from WT and Dnah3 KO mice.
(A – C) Immunofluorescence staining of DNAH1 (A), DNAH6 (B) and DNALI1 (C) in spermatozoa from Dnah3 KO and WT mice. Red, DNAH1 in (A), DNAH6 in (B), DNALI1 in (C); green, α-Tubulin; blue, DAPI; scale bars, 5 μm. (D – F) Western blotting analysis of DNAH1(D), DNAH6 (E) and DNALI1 (F) in spermatozoa lysates from Dnah3 KO and WT mice.

ICSI outcomes of DNAH3-deficient patients and Dnah3 KO mice.
(A) The embryonic development of Patient 1 and Patient 3 after ICSI treatment. MII, metaphase II; PN, pronucleus; scale bars, 40 μm. (B) There was no difference in the fertilization rate or 2-cell and blastocyst embryo formation rates between the Dnah3 KO and WT groups (n = three biologically independent WT mice or KO mice; Student’s t test; NS, no significance; error bars, s.e.m.).

Outcomes of ICSI treatment in the patients with DNAH3 mutations.

Sign up for email alerts