Identification of a homozygous splicing mutation in 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 exhibited a homozygous variant in IQCH. Sequence chromatograms are shown below the pedigree. (B) (i) The electrophoresis results of the minigene assay show a decrease in the molecular weight of the RT-PCR products generated from Mut-IQCH (263 bp) compared with those from WT-IQCH (381 bp). (ii) Sanger sequencing of the complementary DNA of the splicing mutation showing the deletion of exon 4 in IQCH. (iii) The pattern diagram demonstrating the splicing effects caused by the IQCH mutation. (iv) Western blotting results showed that the Mut–cIQCH plasmids did not express IQCH. NC, negative control. Three independent experiments were performed. (C) Immunofluorescence staining showed that the expression of IQCH was barely detected in the proband’s sperm compared with that in the control (blue, DAPI; green, α-Tubulin; red, IQCH; scale bars, 5 μm).

Results of the semen analysis and sperm morphology examination of the patient

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 showing flagellar morphological abnormalities (scale bars in A, 5 μm; scale bars in B, 2.5 μm). The dotted boxes and red arrowheads denote axoneme cracking and exposure. (C) Transmission electron microscopy (TEM) results showing deformed acrosomes and abnormal arrangement and diameter of mitochondria (scale bars, 500 nm). The red arrowheads denote abnormal mitochondria. N, nucleus; M, mitochondria. (D and E) Defects in the acrosome and mitochondria were observed in the proband’s sperm by PNA (D) and TFAM (E) staining (blue, DAPI; green, α-Tubulin; red, PNA or TFAM; scale bars, 5 μm). PNA, peanut agglutinin; TFAM, transcription factor A, mitochondrial.

The absence of Iqch impaired spermatogenesis in mice.

(A and B) Papanicolaou staining (B) and SEM (C) results showing unmasking, bending, or cracking of the axoneme in spermatozoa from Iqch KO mice (n = 3 biologically independent WT mice and KO mice; scale bars in B, 5 μm; scale bars in C, 2.5 μm). The red arrowheads point to axoneme abnormalities. (C) TEM revealing dilated intermembrane spaces of mitochondria and a normal annulus in the testicular and epididymal spermatozoa of Iqch KO mice (n = 3 biologically independent WT mice and KO mice; scale bars, 500 nm). The red arrowheads point to the dilated intermembrane spaces of mitochondria. The green arrowheads point to the normal annulus. M, mitochondria.

Semen analysis using CASA in the mouse model of Iqch KO

Poor IVF treatment outcomes resulting from the use of sperm from Iqch KO mice.

(A) Representative pronucleus embryos, two-cell embryos, and blastocysts obtained from WT mice and Iqch KO mice. Iqch KO mice exhibited significantly lower fertilization rates, cleavage rates, and blastocyst formation rates than WT mice (n = 3 biologically independent WT mice and KO mice; scale bars, 100 μm; Student’s t test; *p < 0.05; error bars, s.e.m.). (B) The acrosome reaction rates in the capacitated spermatozoa from the WT mice and Iqch KO mice were determined by Coomassie brilliant blue staining. The acrosome reaction rates were reduced in the spermatozoa from the Iqch KO mice (n = 3 biologically independent WT mice and KO mice; scale bars, 5 μm; Student’s t test; *p < 0.05; error bars, s.e.m.). The green arrowheads indicate the reacted acrosomes. The red arrowheads indicate intact acrosomes. (C and D) PNA (C) and PLCζ (D) staining showing abnormal acrosome morphology and aberrant PLCζ localization and expression in spermatozoa from Iqch KO mice (n = 3 biologically independent WT mice and KO mice; scale bars, scale bars, 5 μm). The dotted box indicates the typical pattern of PLCζ localization and expression in spermatozoa from WT mice. PNA, peanut agglutinin; PLCζ, phospholipase C zeta 1.

IQCH bound and upregulated male reproduction-related proteins in mouse sperm

(A) Bubble plots of the GO analysis showing that the IQCH-interacting proteins are significantly enriched in spermatogenesis and RNA processing in three categories: biological process, cellular component, and molecular function. GO, Gene Ontology. (B) Thirty-three ribosomal proteins interacted with IQCH in mouse sperm. (C) A heatmap showing the differential protein results from the proteomic analysis of the sperm from the WT and Iqch KO mice. (D) Bubble plots showing the decreased enrichment of proteins related to the spermatogenetic process and RNA processing according to the GO analysis of the Iqch KO mice compared to the WT mice. (E) Venn diagram depicting the 76 overlapping proteins among the 1186 downregulated proteins in sperm from Iqch KO mice and the 288 proteins that bind to IQCH. (F) Chord diagram showing 21 proteins involved in RNA binding, 8 proteins involved in mitochondrial function, and 4 proteins involved in calcium channel activity among the 76 overlapping proteins. HNRPAB showed the greatest reduction in expression in the Iqch KO mice.

IQCH interacted with CaM to regulate HNRPAB expression and spermatogenesis.

(A) co-IP of mouse sperm lysates revealed that the seven proteins most relevant to the phenotype of the Iqch KO mice were associated with IQCH. (B) Western blotting showing the reduced expression of the seven proteins in the sperm from the Iqch KO mice compared to the WT mice. (C) qPCR analysis of RNA immunoprecipitation (RIP) using HNRPAB antibodies and IgG antibodies on mouse sperm lysates showed that HNRPAB interacted with several RNAs associated with fertilization and axoneme assembly. qPCR analysis of RIP in the sperm lysates from the Iqch KO mice revealed a decrease in the interaction between HNRPAB and the RNA targets compared to that in the WT mice (Student’s t test; *p < 0.05; error bars, s.e.m.). (D) co-IP assays showing the binding of IQCH and CaM in WT sperm. (E) co-IP assays showed that the decreased expression of HNRPAB was due to the reduced binding of IQCH to CaM resulting from the knockdown of IQCH or CaM. (F) The overexpression of IQCH and the simultaneous knockdown of CaM or the overexpression of CaM and the simultaneous knockdown of IQCH in K562 cells further confirmed that the downregulation of HNRPAB was due to the diminished interaction between IQCH and CaM, as determined by western blotting analysis. (G) Analysis of the binding affinity between GFP-IQCH from the cell lysates (target) and recombinant CaM (ligand) by a microscale thermophoresis (MST) assay showing the interaction between IQCH and CaM. Their interaction was disrupted after deletion of the IQ motif within IQCH. (H) co-IP of HEK293 cells cotransfected with the WT-IQCH and WT-CaM plasmids, cotransfected with the IQCH (△IQ) and WT-CaM plasmids, or cotransfected with the control and WT-CaM plasmids showed that CaM interacted with the IQ motif. The downregulation of HNRPAB was due to the disrupted interaction between IQCH and CaM. Three independent experiments were performed.

Proposed model for the mechanisms underlying the involvement of IQCH in spermatogenesis.

IQCH interacts with 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 development of the acrosome, mitochondria, and axoneme, thereby playing a critical role in spermatogenesis.