Identification of a 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. The affected males exhibited the homozygous variant in IQCH by Sanger sequencing. 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 the Mut-IQCH (263 bp) compared with the 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 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) The Papanicolaou staining (A) and scanning electron microscopy (SEM) (B) results showed flagellar morphological abnormalities (scale bars in A, 5 μm; scale bars in B, 2.5 μm). The dotted boxes and red arrowheads denote the axoneme cracking and exposure. (C) Transmission electron microscopy (TEM) results showing the deformed acrosomes and abnormal arrangement and diameter mitochondria (scale bars, 500 nm). The red arrowheads denote the abnormal mitochondria. N, nucleus; M, mitochondria. (D and E) Defects of 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) The Papanicolaou staining (B) and SEM (C) results showing unmasking, bending, or cracking axoneme in the spermatozoa from the 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 the axoneme abnormalities. (C) TEM revealing dilated intermembrane spaces of mitochondria and a normal annulus in the testicular and epididymal spermatozoa from the 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 from using sperm from the Iqch KO mice.

(A) Representative pronucleus embryos, two-cell embryos, and blastocysts obtained from the WT mice and the Iqch KO mice. Significantly reduced fertilization rates, cleavage rates, and blastocyst formation rates were observed in the Iqch KO mice compared to the WT mice (n = 3 biologically independent WT mice and KO mice; (B) 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 the abnormal acrosome morphology and aberrant PLCζ location and expression in the spermatozoa from the 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 the PLCζ location and expression in the spermatozoa from the WT mice. PNA, peanut agglutinin; PLCζ, phospholipase C zeta 1.

IQCH bound and activated male reproduction-related proteins in the sperm of the mice.

(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 the sperm from the mice. (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 between the 1186 downregulated proteins in the sperm from the Iqch KO mice and the 288 proteins binding with 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 of expression in the Iqch KO mice.

IQCH interacted with CaM to regulate the expression of HNRPAB and spermatogenesis.

(A) A Co-IP assay of the mouse sperm lysates revealed that the seven proteins most relevant to the phenotype of the Iqch KO mice bound with IQCH. (C) 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 the mouse sperm lysates showed that HNRPAB interacted with several RNAs associated with fertilization and axoneme assembly. The qPCR analysis of RIP performed on the sperm lysates from the Iqch KO mice revealed a decrease in the interaction between HNRPAB and the RNA targets compared to the WT mice (Student’s t-test; *p < 0.05; error bars, s.e.m). (D) The Co-IP assay showed the binding of IQCH and CaM in the WT sperm but not in the Iqch KO sperm. (E) The Co-IP assay showed that the decreased expression of HNRPAB was due to the reduced binding of IQCH and CaM by the knockout of IQCH or CaM. (F) The overexpression of IQCH and the simultaneous knockout of CaM or the overexpression of CaM and the simultaneous (D) knockout of IQCH in K562 cells further confirmed that the downregulation of HNRPAB was due to the diminished interaction between IQCH and CaM 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 destroyed after the deletion of the IQ motif within IQCH. (H) The Co-IP assay of the HEK 293 cells co-transfected with WT-IQCH and WT-CaM plasmids or co-transfected with IQCH (△IQ) 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 of action underlying the involvement of IQCH in spermatogenesis.

IQCH interacts with CaM by 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 acrosome, mitochondria, and axoneme, thereby playing a critical role in spermatogenesis.