1. Genetics and Genomics

DNAH3 deficiency causes flagellar inner dynein arm loss and male infertility in humans and mice

  1. Xiang Wang
  2. Gan Shen
  3. Yihong Yang
  4. Chuan Jiang
  5. Tiechao Ruan
  6. Xue Yang
  7. Liangchai Zhuo
  8. Yingteng Zhang
  9. Yangdi Ou
  10. Xinya Zhao
  11. Shunhua Long
  12. Xiangrong Tang
  13. Tingting Lin  Is a corresponding author
  14. Ying Shen  Is a corresponding author
  1. 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, China
  2. NHC Key Laboratory of Chronobiology, Sichuan University, China
  3. 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, China
  4. Department of Pediatrics, West China Second University Hospital, Sichuan University, China
  5. West China School of Medicine, Sichuan University, China
  6. West China School of Basic Medicine and Forensic Medicine, Sichuan University, China
  7. Chongqing Key Laboratory of Human Embryo Engineering, Center for Reproductive Medicine, Women and Children’s Hospital of Chongqing Medical University, China
  8. Chongqing Clinical Research Center for Reproductive Medicine, Chongqing Health Center for Women and Children, China
https://doi.org/10.7554/eLife.96755.4
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Cite this article
  1. Xiang Wang
  2. Gan Shen
  3. Yihong Yang
  4. Chuan Jiang
  5. Tiechao Ruan
  6. Xue Yang
  7. Liangchai Zhuo
  8. Yingteng Zhang
  9. Yangdi Ou
  10. Xinya Zhao
  11. Shunhua Long
  12. Xiangrong Tang
  13. Tingting Lin
  14. Ying Shen
(2024)
DNAH3 deficiency causes flagellar inner dynein arm loss and male infertility in humans and mice
eLife 13:RP96755.
https://doi.org/10.7554/eLife.96755.4
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7 figures, 5 tables and 1 additional file

Figures

Figure 1
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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.

Figure 1—source data 1

Summary of whole exome sequencing and the candidate variants identified.

https://cdn.elifesciences.org/articles/96755/elife-96755-fig1-data1-v1.xlsx
Figure 1—source data 2

Primers for Sanger sequencing.

https://cdn.elifesciences.org/articles/96755/elife-96755-fig1-data2-v1.docx
Figure 1—source data 3

PDF file containing original western blotting for Figure 1D.

https://cdn.elifesciences.org/articles/96755/elife-96755-fig1-data3-v1.zip
Figure 1—source data 4

Original files for western blotting analysis displayed in Figure 1D.

https://cdn.elifesciences.org/articles/96755/elife-96755-fig1-data4-v1.zip
Figure 2 with 1 supplement
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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.

Figure 2—figure supplement 1
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The histogram of various flagellar morphology in the normal control and patients.
Figure 2—figure supplement 1—source data 1

The percentage distribution of various flagellar morphology in the normal control and patients.

https://cdn.elifesciences.org/articles/96755/elife-96755-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 1 supplement
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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.

Figure 3—figure supplement 1
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The percentage of aberrant ultrastructure in different cross-sections of sperm from the normal control and patients.
Figure 4 with 8 supplements
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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.

Figure 4—figure supplement 1
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The expression of DNAH3 in mouse testis.

(A) qPCR analysis revealed that Dnah3 was highly expressed in the mouse testis. (B) qPCR analysis showed that Dnah3 expression was significantly elevated beginning on postnatal Day 12, peaked at postnatal Day 30, and maintained a stable expression level thereafter.

Figure 4—figure supplement 1—source data 1

PDF file containing original gels for Figure 4—figure supplement 1A, B.

https://cdn.elifesciences.org/articles/96755/elife-96755-fig4-figsupp1-data1-v1.zip
Figure 4—figure supplement 1—source data 2

Original files for gel analysis displayed in Figure 4—figure supplement 1A, B.

https://cdn.elifesciences.org/articles/96755/elife-96755-fig4-figsupp1-data2-v1.zip
Figure 4—figure supplement 2
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DNAH3 is expressed during spermatogenesis in mice and humans.

(A) Immunofluorescence staining of DNAH3 in isolated mouse germ cells. Pink, PNA; green, DNAH3; blue, DAPI; scale bars, 5 μm. (B) Immunofluorescence staining of DNAH3 in isolated human germ cells. Pink, PNA; green, DNAH3; blue, DAPI; scale bars, 5 μm.

Figure 4—figure supplement 3
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Generation of Dnah3 KO mice.

(A) Schematic illustration of the strategy for the generation of Dnah3 KO mice. (B, C) PCR sequencing (B) and qPCR (C) were used to confirm the genotype and KO efficiency (n=three biologically independent WT mice or KO mice; Student’s t test; *, p<0.05; error bars, s.e.m.). (D) Immunofluorescence staining of DNAH3 in testis of Dnah3 KO mice and WT mice. Green, DNAH3; blue, DAPI; scale bars, 75 μm. (E) Immunofluorescence staining of DNAH3 in spermatozoa isolated from the cauda epididymis of Dnah3 KO mice and WT mice. Red, DNAH3; green, α-Tubulin; blue, DAPI; scale bars, 5 μm.

Figure 4—figure supplement 3—source data 1

Primers for Sanger sequencing and qPCR.

https://cdn.elifesciences.org/articles/96755/elife-96755-fig4-figsupp3-data1-v1.docx
Figure 4—figure supplement 4
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Ciliary development of Dnah3 KO mice.

(A) H&E staining of lung, brain, eye, and oviduct from Dnah3 KO mice and WT mice. Scale bars, 100 μm. (B) Analysis of ciliary development in the lung, brain, eye, and oviduct from Dnah3 KO mice and WT mice by using immunofluorescence staining. Green, Ac-Tubulin; blue, DAPI; scale bars, 20 μm.

Figure 4—figure supplement 5
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Fertility of Dnah3 KO mice.

(A) H&E staining of ovary tissue sections from 8-week-old Dnah3 KO female mice and WT female mice. Scale bars, 75 μm (n=three biologically independent WT mice or KO mice). (B) Sizes of the testis and epididymis of the 8-week-old Dnah3 KO and WT mice (n=three biologically independent WT mice or KO mice; Student’s t test; NS, no significance; error bars, s.e.m.).

Figure 4—figure supplement 6
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Morphology and ultrastructure of sperm isolated from Dnah3 KO mice.

(A, B) Papanicolaou staining (A), and SEM analysis (B) revealed morphological defects in partial spermatozoa from Dnah3 KO mice compared to WT mice. Scale bars in (A), 5 μm; scale bars in (B), 2.5 μm. (n=three biologically independent WT mice or KO mice; Student’s t test; error bars, s.e.m.). (C) The percentage of aberrant axonemal arrangement in different cross-sections of sperm from WT mice and Dnah3 KO mice. (n=three biologically independent WT mice or KO mice; error bars, s.e.m.). (D) The percentage of microtubule doublets that presented IDAs in WT mice and Dnah3 KO mice. (n=three biologically independent WT mice or KO mice; Student’s t test; error bars, s.e.m.). (E) Statistics of malformed mitochondria in the midpiece of sperm from WT mice and Dnah3 KO mice. (n=three biologically independent WT mice or KO mice; Student’s t test; error bars, s.e.m.).

Figure 4—video 1
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CASA of sperm from WT mice.

Sperm from the epididymis of WT mice were collected, incubated, and recorded under a phase-contrast microscope. A normal quantity and motility of sperm were observed in the WT mice (n=three biologically independent WT mice).

Figure 4—video 2
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CASA of sperm from Dnah3 KO mice.

Epididymal sperm of Dnah3 KO mice were collected, incubated in HTF medium at 37 °C for 10 min, and recorded under a phase-contrast microscope. The movie showed a significantly reduced motility of sperm from Dnah3 KO (n=three biologically independent Dnah3 mice).

Figure 5 with 1 supplement
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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.

Figure 5—source data 1

PDF file containing original western blotting for Figure 5D–F and Figure 6D–F.

https://cdn.elifesciences.org/articles/96755/elife-96755-fig5-data1-v1.zip
Figure 5—source data 2

Original files for western blotting analysis displayed in Figure 5D–F and Figure 6D–F.

https://cdn.elifesciences.org/articles/96755/elife-96755-fig5-data2-v1.zip
Figure 5—figure supplement 1
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Immunofluorescence staining of ODA-associated proteins in spermatozoa obtained from variants within DNAH3 patients.

(A – C) The expression of DNAH8 (A), DNAH17 (B) and DNAI1 (C) in spermatozoa of the patients was comparable to that in normal controls. Red, DNAH8 in (A), DNAH17 in (B), DNAI1 in (C); green, α-Tubulin; blue, DAPI; scale bars, 5 μm.

Figure 6 with 1 supplement
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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.

Figure 6—figure supplement 1
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Immunofluorescence staining of ODA-associated proteins in spermatozoa of Dnah3 KO and WT mice.

(A – C) The expression of DNAH8 (A), DNAH17 (B) and DNAI1 (C) in spermatozoa from Dnah3 KO mice was comparable to that in spermatozoa from WT mice. Red, DNAH8 in (A), DNAH17 in (B), DNAI1 in (C); green, α-Tubulin; blue, DAPI; scale bars, 5 μm.

Figure 7
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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.).

Tables

Table 1
Semen analysis of the patients in the present study.
Patient 1Patient 2Patient 3Patient 4Reference
Semen parametersSemen volume (ml)4.33.30.84.6≥1.5
Semen concentration (106 /ml)2.00.511.021.0≥15.0
Motility (%)6.03.000≥40.0
Progressive motility (%)02.300≥32.0
Sperm morphologyNormal (%)1.31.11.81.2≥4.0
Tail defects (%)91.387.596.597.5-

  1. -,not applicable.

Table 2
Variants analysis of the patients in the present study.
Patient 1Patient 2Patient 3Patient 4
VariantcDNA mutation *c.3590C>Tc.3590C>Gc.4837G>Tc.5587delc.10355C>Tc.2314C>Tc.4045G>A
Protein alterationp.Pro1197Leup.Pro1197Argp.Ala1613Serp.Leu1863*p.Ser3452Leup.Arg772Trpp.Asp1349Asn
Mutation typeMissenseMissenseMissenseNonsenseMissenseMissenseMissense
Allele frequencyExAC_EAS0.000100.00416500.00060.00190.0065
GnomAD_EAS0.0000501600.0027741500.00080.0020.007
1000 Genomes Project_EAS000.0050000.00400.0069
Function predictionSIFTDeleteriousDeleteriousDeleterious/ToleratedDeleteriousDeleterious
Polyphen-2Probably damagingProbably damagingProbably damaging/Probably damagingProbably damagingProbably damaging
Mutation TasterDisease causingDisease causingDisease causing/Disease causingDisease causingDisease causing
CADD †3329.527.5/25.427.934

  1. /,not applicable.

  2. *

    NM_017539.2.

  3. †

    score >4.0 is predicted to be damaging.

Table 3
Semen analysis using CASA in the Dnah3 KO mice.
WTKOp* value
Semen parameters
Sperm concentration (106/ml) †112.32±18.26105.17±11.150.059
Motility (%) *71.56±3.974.37±1.15<0.01
Progressive motility (%) *60.36±4.324.37±1.15<0.01
Sperm locomotion parameters
Curvilinear velocity (VCL) (μm/s)*67.54±6.799.07±1.22<0.01
Straight-line velocity (VSL) (μm/s)*28.91±4.862.68±0.52<0.01
Average path velocity (VAP) (μm/s)*39.02±5.313.85±0.82<0.01
Amplitude of lateral head displacement (ALH) (μm)*0.71±0.030.13±0.04<0.01
Linearity (LIN)*0.43±0.070.30±0.020.037
Wobble (WOB,=VAP/VCL)*0.58±0.060.42±0.050.024
Straightness (STR,=VSL/VAP)0.74±0.220.70±0.130.80
Beat-cross frequency (BCF) (Hz)*4.86±0.120.73±0.08<0.01
  1. *

    A significant difference, two-sided student’s t-test. n=3 biologically independent WT mice or KO mice.

  2. †

    Epididymides and vas deferens.

Table 4
Outcomes of ICSI treatment in the patients with DNAH3 mutations.
SubjectsPatientPatient 1Patient 2Patient 3Patient 4
Female age (y)24303036
Length of primary infertility history (y)6138
Basal hormonesFSH (IU/L)7.18.53.494.3
LH (IU/L)4.442.54.22.6
E2 (pg/mL)835043.4968
Prog (ng/mL)0.20.60.30.3
ICSI CyclesCycle 1Cycle 1Cycle 2Cycle 1Cycle 1Cycle 2Cycle 3Cycle 4
E2 level on the trigger day (pg/ml)33661519.61582.4>5,0004440500031522206
No. of follicles ≥14 mm on the trigger day15652013121411
No. of follicles ≥18 mm on the trigger day1034811975
No. of oocytes retrieved24652516202114
ICSI progressNo. of MII oocytes216520121387
Fertilization rate (%)80.95 (17/21)50 (3/6)100 (5/5)95 (19/20)41.67 (5/12)46.15 (6/13)50 (4/8)42.86 (3/7)
Cleavage Rate (%)100 (17/17)100 (2/2)100 (5/5)100 (19/19)100 (5/5)100 (6/6)100 (4/4)66.7 (2/3)
Available D3 embryos1325152200
Blastocyst formation rate (%)61.5 (8/13)066.7 (2/3)66.7 (10/15)----
Clinical outcomesNo. of embryos transferred2 blastocysts02 D31 blastocyst2 D32 D3--
Implantation rate (%)50 (1/2)00100 (1/1)00--
Clinical pregnancyYesNoNoYes----
No. of live birthngoing--ngoing----

  1. -,not applicable.

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-DNAH1
(Rabbit polyclonal)		CusabioCSB-PA878961LA01HUIF (1:100)
AntibodyAnti-DNAH3
(Rabbit polyclonal)		CusabioCSB-PA823461LA01HUIF (1:100)
AntibodyAnti-DNAH3
(Rabbit polyclonal)		Gift from Prof. Yueqiu Tan, Central South University, China.WB (1:200)
AntibodyAnti-DNAH6
(Rabbit polyclonal)		Proteintech18080–1-AP,
RRID: AB_2878493		IF (1:50),
WB (1:150)
AntibodyAnti-DNAH8
(Rabbit polyclonal)		AtlasHPA028447,
RRID: AB_10599600		IF (1:200)
AntibodyAnti-DNAH17
(Rabbit polyclonal)		Proteintech24488–1-AP,
RRID: AB_2879568		IF (1:50)
AntibodyAnti-DNAI1
(Rabbit polyclonal)		Proteintech12756–1-AP,
RRID: AB_10643244		IF (1:50)
AntibodyAnti-DNALI1
(Rabbit polyclonal)		Proteintech17601–1-AP,
RRID: AB_2095372		IF (1:50),
WB (1:150)
AntibodyAnti-TOM20
(Rabbit polyclonal)		Proteintech11802–1-AP,
RRID: AB_2207530		IF (1:50)
AntibodyAnti-SLC25A4
(Rabbit polyclonal)		Signalway32484,
RRID: AB_2941094		IF (1:100)
AntibodyAnti-alpha tubulin
(Mouse monoclonal)		Abcamab7291,
RRID: AB_2241126		IF (1:500)
AntibodyAnti-alpha tubulin
(Rabbit polyclonal)		Proteintech11224–1-AP,
RRID: AB_2210206		WB (1:1000)
AntibodyAnti-Rabbit IgG,
Alexa Fluor 488
(Goat polyclonal)		InvitrogenA11008,
RRID: AB_143165		IF (1:1000)
AntibodyAnti-Mouse IgG,
Alexa Fluor 594
(Goat polyclonal)		InvitrogenA11005,
RRID: AB_141372		IF (1:1000)
AntibodyAnti-acetylated alpha tubulin
(Mouse monoclonal)		Abcamab24610,
RRID: AB_448182		IF (1:500)
AntibodyAnti-Mouse IgG,
HRP-conjugated
(Goat polyclonal)		ProteintechSA00001-1,
RRID: AB_2722565		WB (1:5000)
AntibodyAnti-Rabbit IgG,
HRP-conjugated
(Goat polyclonal)		ProteintechSA00001-2,
RRID: AB_2722564		WB (1:5000)
OtherLectin PNAInvitrogenL-32460IF (1:50)

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  1. Xiang Wang
  2. Gan Shen
  3. Yihong Yang
  4. Chuan Jiang
  5. Tiechao Ruan
  6. Xue Yang
  7. Liangchai Zhuo
  8. Yingteng Zhang
  9. Yangdi Ou
  10. Xinya Zhao
  11. Shunhua Long
  12. Xiangrong Tang
  13. Tingting Lin
  14. Ying Shen
(2024)
DNAH3 deficiency causes flagellar inner dynein arm loss and male infertility in humans and mice
eLife 13:RP96755.
https://doi.org/10.7554/eLife.96755.4