Identification of IQCH as a calmodulin-associated protein required for sperm motility in humans

Abstract

Sperm fertilization ability mainly relies on proper sperm progression through the female genital tract and capacitation, which involves phosphorylation signaling pathways triggered by calcium and bicarbonate. We performed exome sequencing of an infertile asthenozoospermic patient and identified truncating variants in MAP7D3, encoding a microtubule-associated protein, and IQCH, encoding a protein of unknown function with enzymatic and signaling features. We demonstrate the deleterious impact of both variants on sperm transcripts and proteins from the patient. We show that, in vitro, patient spermatozoa could not induce the phosphorylation cascades associated with capacitation. We also provide evidence for IQCH association with calmodulin, a well-established calcium-binding protein that regulates the calmodulin kinase. Notably, we describe IQCH spatial distribution around the sperm axoneme, supporting its function within flagella. Overall, our work highlights the cumulative pathological impact of gene mutations and identifies IQCH as a key protein required for sperm motility and capacitation.

Keywords: Cell biology; Clinical genetics; Health sciences.

Graphical abstract

Figure 1

Characterization of the MAP7D3 hemizygous c.850C>T variant identified in the patient (A) Representation of the MAP7D3 gene with exons (bars) and introns (connections) adapted from Ensembl genome browser. The red asterisk indicates the identified c.850C>T mutation, which was also confirmed by Sanger sequencing on genomic DNA from the patient (see electropherogram). (B) RT-PCR analysis of the MAP7D3-201 transcript isoform (exons 7–8) and MAP7D3-202 transcript isoform (exon 7-8 junction) in a panel of human tissues and mature spermatozoa. Tissues: 1: heart, 2: brain, 3: placenta, 4: lung, 5: liver, 6: muscle, 7: kidney, 8: pancreas, 9: colon, 10: ovary, 11: leukocytes, 12: prostate, 13: intestine, 14: spleen, 15: testis (highlighted by the red box), and 16: thymus. (C) MAP7D3 expression profile throughout human spermatogenesis. Transcript analyses of single-cell RNA quantitative sequencing datasets from human adult testis are available in the literature using the ReproGenomics viewer. Transcript levels are indicated in UMI (unique molecular identifier) normalized for all stages of germ cell differentiation (spermatogonia to differentiated spermatids) and somatic cell types (Leydig cells, Sertoli cells, macrophages). (D) Left panel: semi-quantitative RT-PCR amplifying MAP7D3-201 from spermatozoa from the control individual and the patient and normalization against HPRT. Middle panel: electropherogram of amplicon sequencing. Right panel: RT-qPCR of patient spermatozoa. (E) MAP7D3 structure showing the coiled-coil domains (CC), mutation site (red asterisk), and antibody epitope. Immunoblot assay of MAP7D3 from the patient spermatozoa (red asterisk, expected size of MAP7D3). Immunofluorescence assay of MAP7D3 from the patient spermatozoa with tubulin co-staining. Scale bar 10 μm.

Figure 2

Characterization of the IQCH homozygous c.1456+1G>C variant identified in the patient (A) Linear structure of the IQCH gene showing exons (bars) and introns (junctions). The red asterisk indicates the mutation site (source: Ensembl genome browser). Chromatograms of IQCH Sanger sequencing for the patient (right) and control individual (left). (B) Analysis of IQCH isoforms in human adult tissues and spermatozoa samples. Samples lacking reverse transcriptase for cDNA amplification (-RT) were used as a negative control. Tissues: 1: heart, 2: brain, 3: placenta, 4: lung, 5: liver, 6: muscle, 7: kidney, 8: pancreas, 9: colon, 10: ovary, 11: leukocytes, 12: prostate, 13: intestine, 14: spleen, 15: testis (highlighted by the red box), and 16: thymus. (C) IQCH expression profile throughout human spermatogenesis according to single-cell RNA-seq quantitative sequencing (data from the ReproGenomics viewer). Transcript levels are indicated in UMI (unique molecular identifier) normalized for all stages of germ cell differentiation (spermatogonia to differentiated spermatids) and somatic cell type (Leydig cells, Sertoli cells, macrophages). (D) Left panel: semi-quantitative RT-PCR analysis of IQCH isoforms in spermatozoa from the control individual and the patient. The RT-PCR signal was normalized against that of HPRT (352 bp amplicon). Middle panel: chromatograms showing the amplicon sequencing, with the red lines marking the exon boundaries. Right panel: RT-qPCR analysis of IQCH transcripts in sperm from the control individual and the patient after normalization against GAPDH. (E) Linear structure of the IQCH protein showing multiple enzymatic, metabolic, and signaling domains, together with the mutation site identified in the patient of the present study and the localization of antibody epitopes used in the experimental analyses. CK-l, carbamate kinase-like domain; IQ, IQ motif; Zn-exo, Zn-dependent exopeptidase domain; LDH-l, lactate dehydrogenase C-terminal domain-like (figure created using DOG software²²). Western blot and immunodetection analyses of IQCH from spermatozoa from the patient using antibody Ab-B. α-tubulin is stained in green and DNA with DAPI, in blue. Scale bar 10 μm.

Figure 3

Association of IQCH with calmodulin in flagella and its requirement for sperm capacitation (A) Western blot analysis of protein tyrosine phosphorylation following in vitro capacitation of spermatozoa from the patient and a control individual, at 37°C for 3 h. The data presented for the control individual are representative of the profiles obtained with semen samples from five control individuals displaying normal semen parameters following WHO criteria (Figure S3A). (B) Assessment of the IQCH/calmodulin interaction by proximity ligation assay (PLA). Analysis of the physical proximity of IQCH and calmodulin (CaM) relative to the biological negative control (DNAI2-SPAG6, two proteins from different axonemal complexes) and positive control (DNAI2-DNAH17, two proteins from the same axonemal complex). (C) 3D structures of IQ motifs bound to calmodulin in the calcium free status. Side chains of the residues involved in IQ-CaM interactions are shown as sticks and colored blue or orange for polar and hydrophobic contacts, respectively. Structural and sequence alignments of the IQ motifs from IQCH, IQCG, and Myosin V are shown.

Figure 4

Analysis of the spatial distribution of IQCH within sperm flagella by super-resolution microscopy (A) STORM imaging of sperm flagella stained for IQCH (red) and α-tubulin (green) or calmodulin (CaM, green). Scale bars 1 μm. (B) STED images of co-staining IQCH (violet) and α-tubulin (green), highlighting the helicoidal spatial distribution of IQCH along the sperm flagellum (dashed frames) and its co-localization with calmodulin. Distribution profiles of the signal in the selected regions highlight the longitudinal and transversal periodicity of IQCH. Scale bars 2 μm and 2.5 μm. (C) Schematic representation of a transversal section of a sperm flagellum with the localization of proteins identifying the various complexes and subcellular regions of sperm flagella localization [Graphical elements from Servier Medical Art, licensed under a Creative Commons Attribution 3.0 Unported License]. Distribution data were obtained by analyzing the STORM and STED images shown in Figure S4.