Deficiency of IQCH causes male infertility in humans and mice
- Key Laboratory of Obstetrics, Gynecologic and Pediatric Diseases and Birth Defects of the Ministry of Education, West China Second University Hospital, Sichuan University, China
- Department of Pediatrics, West China Second University Hospital, Sichuan University, 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, China
- Sichuan University-The Chinese University of Hong Kong (SCU-CUHK) Joint Laboratory for Reproductive Medicine, Key Laboratory of Obstetric, Gynaecologic and Paediatric Diseases and Birth Defects of Ministry of Education, West China Second University Hospital, Sichuan University, China
- Reproductive Endocrinology and Regulation Laboratory, Department of Obstetric and Gynaecologic, West China Second University Hospital, Sichuan University, China
- Education Key Laboratory of Cell Proliferation & Regulation Biology, College of Life Sciences, Beijing Normal University, China
- NHC Key Laboratory of Chronobiology, Sichuan University, China
Figures
Figure 1
Identification of a homozygous splicing mutation in IQ motif-containing H (IQCH) in a consanguineous family with male infertility.
(A) Pedigree analysis of the consanguineous family with two infertile males (II-1 and IV-1), with the black arrow pointing to the proband. Sanger sequencing revealed that the affected males …
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Figure 1—source data 1
Primers for Sanger sequencing and Minigene.
- https://cdn.elifesciences.org/articles/88905/elife-88905-fig1-data1-v1.docx
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Figure 1—source data 2
Original blots and gels of Figure 1.
- https://cdn.elifesciences.org/articles/88905/elife-88905-fig1-data2-v1.zip
Figure 2 with 1 supplement
Abnormal flagellar morphology and defective acrosomes and mitochondria in the infertile patient.
(A and B) Papanicolaou staining (A) and scanning electron microscopy (SEM) (B) results show flagellar morphological abnormalities (scale bars in A, 5 μm; scale bars in B, 2.5 μm). The dotted boxes …
Figure 2—figure supplement 1
Transmission electron microscopy (TEM) and immunofluorescence analysis of the abnormal phenotype of spermatozoa in the patient.
(A) TEM results show the deranged or incomplete ‘9+2’ microtubule structure of the flagella. The percentage of abnormal ‘9+2’ microtubule structures in the control and proband (scale bars, 500 nm; …
Figure 3 with 7 supplements
The absence of IQ motif-containing H (Iqch) impaired spermatogenesis in mice.
(A and B) Papanicolaou staining (B) and scanning electron microscopy (SEM) (C) results showing unmasking, bending, or cracking of the axoneme in spermatozoa from Iqch knockout (KO) mice (n=3 …
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Figure 3—source data 1
Primers for RT-PCR and sgRNA sequences.
- https://cdn.elifesciences.org/articles/88905/elife-88905-fig3-data1-v1.docx
Figure 3—figure supplement 1
The expression pattern of IQ motif-containing H (IQCH) in testes.
(A) qPCR analysis showed that Iqch was predominantly expressed in the testes, and Iqch began to be significantly expressed on P21, reached its peak on P35, and then showed a stable expression …
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Figure 3—figure supplement 1—source data 1
Original blots and gels of Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/88905/elife-88905-fig3-figsupp1-data1-v1.zip
Figure 3—figure supplement 2
Generation of IQ motif-containing H (Iqch) knockout (KO) mice.
(A) A schematic illustration of the strategy used to generate the Iqch KO mice. Exons 2 through 3 of the Iqch gene were targeted by the CRISPR-Cas9 system. (B) PCR, RT‒PCR, and western blotting were …
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Figure 3—figure supplement 2—source data 1
Original blots and gels of Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/88905/elife-88905-fig3-figsupp2-data1-v1.zip
Figure 3—figure supplement 3
No obvious abnormalities in the histology of the testes or epididymis were observed in the IQ motif-containing H (Iqch) knockout (KO) mice.
(A) Hematoxylin-eosin (H&E) staining of testicular sections from adult Iqch KO mice shows no obvious abnormalities at different stages of spermatogenesis compared to those from wild-type (WT) mice …
Figure 3—figure supplement 4
Cracking of the axoneme occurred during spermatogenesis in the IQ motif-containing H (Iqch) knockout (KO) mice.
(A) Papanicolaou staining of the isolated germ cells from the testes of the Iqch KO mice revealed cracking of the axoneme at steps 13-14 and 15-16 (n=3 biologically independent wild-type (WT) mice …
Figure 3—figure supplement 5
The spermatozoa from the IQ motif-containing H (Iqch) knockout (KO) mice exhibited defective mitochondria.
(A) Immunofluorescence staining of SEPTIN4 revealed no significant differences at the annulus between the spermatozoa from the wild-type (WT) mice and those from the Iqch KO mice (n=3 biologically …
Figure 3—video 1
The movie showed the in vitro motility of epididymal sperm from wild-type (WT) mice.
Sperm were collected, incubated, and recorded by video-recording under a phase-contrast microscope. The movie demonstrates normal sperm quantity and motility in the WT mice (n=3 biologically …
Figure 3—video 2
Sperm with reduced motility were collected from the cauda epididymis and vas deferens of IQ motif-containing H (Iqch) knockout (KO) male mice and analyzed under a phase-contrast microscope(n=3 biologically independent KO mice).
Figure 4 with 1 supplement
Poor in vitro fertilization (IVF) treatment outcomes resulting from the use of sperm from IQ motif-containing H (Iqch) knockout (KO) mice.
(A) Representative pronucleus embryos, two-cell embryos, and blastocysts were obtained from wild-type (WT) mice and Iqch KO mice. Iqch KO mice exhibited significantly lower fertilization rates, …
Figure 4—figure supplement 1
The loss of function of IQ motif-containing H (IQCH) resulted in deficiencies in oocyte activation and acrosomes.
(A) Representative images of the in vitro fertilization (IVF) treatments show that the spermatozoa from the Iqch knockout (KO) mice did not penetrate the zona pellucida (n=3 biologically independent …
Figure 5
IQ motif-containing H (IQCH) bound and upregulated male reproduction-related proteins in mouse sperm.
(A) Bubble plots of the GO analysis show that the IQCH-interacting proteins are significantly enriched in spermatogenesis and RNA processing in three categories: biological process, cellular …
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Figure 5—source data 1
List of 288 interactors of IQ motif-containing H (IQCH) about LC-MS/MS analysis of mouse sperm lysates.
- https://cdn.elifesciences.org/articles/88905/elife-88905-fig5-data1-v1.xlsx
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Figure 5—source data 2
List of differentially down-regulated proteins about proteomic analysis of sperm from IQ motif-containing H (Iqch) knockout (KO) and wild-type (WT) mice.
- https://cdn.elifesciences.org/articles/88905/elife-88905-fig5-data2-v1.xlsx
Figure 6 with 4 supplements
IQ motif-containing H (IQCH) interacted with calmodulin (CaM) to regulate HNRPAB expression and spermatogenesis.
(A) Co-immunoprecipitation (co-IP) of mouse sperm lysates revealed that the seven proteins most relevant to the phenotype of the Iqch knockout (KO) mice were associated with IQCH. (B) Western …
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Figure 6—source data 1
Primers for qPCR and siRNA sequences.
- https://cdn.elifesciences.org/articles/88905/elife-88905-fig6-data1-v1.docx
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Figure 6—source data 2
Overview of the antibodies or dyes used in this study.
- https://cdn.elifesciences.org/articles/88905/elife-88905-fig6-data2-v1.docx
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Figure 6—source data 3
Original blots of Figure 6.
- https://cdn.elifesciences.org/articles/88905/elife-88905-fig6-data3-v1.zip
Figure 6—figure supplement 1
The reduced expression levels of the seven proteins were most relevant to the phenotype of the spermatozoa of the IQ motif-containing H (Iqch) knockout (KO) mice.
Immunofluorescence staining shows that the expression levels of the seven proteins most relevant to the phenotype of the Iqch KO mice, including HNRNPK, HNRPAB, EWSR1, SYNCRIP, FUS, and ANXA7, were …
Figure 6—figure supplement 2
Statistics results about protein expression levels by western blotting analysis.
(A) Densitometry analysis was performed on corresponding Western blotting to assess the expression levels of seven proteins most relevant to the IQ motif-containing H (Iqch) knockout (KO) mouse …
Figure 6—figure supplement 3
Gene Ontology (GO) analysis of the downregulated genes of the IQ motif-containing H (Iqch) knockout (KO) mice through RNA sequencing.
Bubble plots of the GO analysis revealed the effects of IQCH deficiency on male fertility in three categories: BP, biological process; CC, cellular component; and MF, molecular function.
Figure 6—figure supplement 4
IQ motif-containing H (IQCH) interacted with calmodulin (CaM) to regulate spermatogenesis.
(A) A schematic representation of IQCH showing an IQ motif conserved between humans and mice. The domains were predicted via SMART (http://smart.embl-heidelberg.de/). (B) The protein-protein docking …
Figure 7
Proposed model for the mechanisms underlying the involvement of IQ motif-containing H (IQCH) in spermatogenesis.
IQCH interacts with calmodulin (CaM) via the IQ motif to regulate the expression of RNA-binding proteins. RNA binding proteins, particularly HNRPAB, bind and regulate several RNAs that influence the …
Tables
Table 1
Results of the semen analysis and sperm morphology examination of the patient.
| Parameter | Patient | Reference* |
|---|---|---|
| Sperm volume, ml | 2.8 | ≥1.5 |
| Sperm concentration, million/ml | 10.0 | ≥15 |
| Vitality, % | 30 | ≥58 |
| Motility, % | 4 | ≥32 |
| Abnormal morphology (%) | 99.5 | - |
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*
The reference values were based on the guidelines provided in the WHO manual fifth edition (2010).
Table 2
Semen analysis using computer-assisted sperm analysis (CASA) in the mouse model of IQ motif-containing H (Iqch) knockout (KO).
| Adult Male Mice | |||
|---|---|---|---|
| WT | KO | p† value | |
| Semen parameters | |||
| Sperm concentration (106 /ml) * | 94.51±10.68 | 67.75±3.70 | 0.038 |
| Motility (%) | 57.08±2.18 | 5.49±3.25 | <0.001 |
| Progressive motility (%) | 57.00±2.05 | 5.49±3.25 | <0.001 |
| Sperm locomotion parameters | |||
| Curvilinear velocity (VCL) (μm/s) | 78.29±6.23 | 9.94±4.03 | <0.001 |
| Straight-line velocity (VSL) (μm/s) | 36.18±2.09 | 2.90±1.98 | <0.001 |
| Average path velocity (VAP) (μm/s) | 45.10±0. 19 | 4.17±2.49 | <0.001 |
| Amplitude of lateral head displacement (ALH) (μm) | 0.77±0. 02 | 0.13±0.03 | <0.001 |
| Linearity (LIN) | 0.46±0.01 | 0.27±0.08 | 0.047 |
| Wobble (WOB,=VAP/VCL) | 0.58±0.04 | 0.40±0.08 | 0.033 |
| Straightness (STR,=VSL/VAP) | 0.80±0.05 | 0.67±0.07 | 0.069 |
| Beat-cross frequency (BCF) (Hz) | 4.33±0.19 | 0.65±0.26 | <0.001 |
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n = 3 biologically independent wild-type (WT) mice or knockout (KO) mice.
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*
Epididymides and vas deferens.
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†
two-tailed Student’s t-test.
