IQUB mutation induces radial spoke 1 deficiency causing asthenozoospermia with normal sperm morphology in humans and mice

Abstract

Background: Asthenozoospermia (ASZ) accounts for about 20-40% of male infertility, and genetic factors, contributing to 30-40% of the causes of ASZ, still need further exploration. Radial spokes (RSs), a T-shaped macromolecular complex, connect the peripheral doublet microtubules (DMTs) to a central pair (CP), forming a CP-RS-DMT structure to regulate the beat frequency and amplitude of sperm flagella. To date, many components of RSs and their functions in human sperm flagella remain unclear.

Methods: We recruited a cohort of 323 infertile males with ASZ between August 2019 and June 2024. Genetic mutations were identified by whole-exome sequencing. Computer-aided sperm analysis, Papanicolaou staining, and electron microscopy were applied to evaluate the motility, morphology, and ultrastructure of spermatozoa, respectively. Protein mass spectrometry, western blotting, and bioinformatic analyses were performed to identify critical components of mammalian RS1 to model its structure and explore the pathological mechanism of IQUB deficiency. Intracytoplasmic sperm injection (ICSI) was applied for the patient and Iqub^-/- mice.

Results: We identified a novel homozygous IQUB mutation [c.842del (p.L281Pfs*28)] in an ASZ male with normal sperm morphology (ANM), which resulted in the complete loss of IQUB in sperm flagella. Deficiency of RS1, but not RS2 or RS3, was observed in both IQUB^842del patient and Iqub^-/- mice, and resulted in the reduction of sperm kinetic parameters, indicating the critical role of IQUB in regulating mammalian RS1 assembly and sperm flagellar beat. More importantly, we identified twelve critical components of RS1 in humans and mice, among which RSPH3, RSPH6A, RSPH9 and DYDC1 constituting the head, DYDC1, NME5, DNAJB13 and PPIL6 assembling into the head-neck complex, AK8, ROPN1L, RSPH14, DYNLL1, and IQUB forming the stalk of RS1. Along with the RS1 defect, the IQUB deficiency caused significant down-regulation of the inner dynein arms of DNAH7 and DNAH12, highlighting their nearby location with RS1. Finally, ICSI can effectively resolve the male infertility caused by IQUB genetic defects.

Conclusions: We demonstrate that IQUB may serve as an adapter for sperm flagellar RS1 in both humans and mice and consolidated the causal relationship between IQUB genetic mutations and ANM, further enriching the genetic spectrum of male infertility.

Keywords: Assisted reproductive technology; Asthenozoospermia; IQUB; Male infertility; Radial spoke.

Fig. 1

Identification of a IQUB^842del mutation in an infertile patient with asthenozoospermia. A Pedigree of the consanguineous family with the infertile patient. The black arrow points to the proband, the “〧” sign indicates infertility. B Sanger sequencing chromatograms of the family. The red arrow indicates mutation loci. C The position of the identified IQUB^842del mutation at the chromosome, transcript (ENST00000324698.6) and protein (Q8NA54) levels. D Immunofluorescence staining of spermatozoa from the normal control and IQUB^842del patient against β-TUBULIN (green) and IQUB (red), DNA (blue) was counterstained with DAPI. Scale bars, 5 μm. E Western blotting of spermatozoa revealed that IQUB was absent in IQUB^842del patient. Three times independently collected sperm samples from normal control and the IQUB^842del patient

Fig. 2

Morphological and ultrastructural evaluations of the spermatozoa from IQUB^842del patient. A and B Papanicolaou staining and scanning electron microscopy both showed normal sperm morphology of IQUB^842del patient. Scale bars, 5 μm. C Spermatozoa cross-sections of transmission electron microscopy (TEM) showed nine radial spokes (RSs) arranged in normal control and partial loss of RSs (red arrow) in IQUB^842del patient. D Percentage of cross-sections with RSs loss in normal control and IQUB^842del patient. E Spermatozoa longitudinal-sections of TEM showed that RS1-3 were arranged in 96-nm repeats in normal control, whereas in IQUB^842del patient there were only two intact RSs per 96-nm repeat without RS1. Red arrow indicates RS1, green arrow indicates RS2, and cyan arrow indicates RS3. Yellow rectangles represent the region where the gray values are measured. F Sperm movement paths and flagellar swing amplitudes in normal control and IQUB^842del patient

Fig. 3

Iqub knockout causes asthenozoospermia with RS1 loss in mice. A Pup birth quantification per vaginal plug of Iqub^+/+, Iqub^+/−, and Iqub^−/− mice. No pregnancy occurred in females mated with Iqub^−/− male mice. Data are presented as means ± SEM (n = 6, *** P < 0.001). B Testes and epididymis from 9-week-old Iqub^+/+, Iqub^+/−, and Iqub^−/− mice. C The ratios of testis/body in Iqub^+/+, Iqub^+/−, and Iqub^−/− mice were not significantly different. n = 4, P > 0.05. D Sperm number obtained from cauda epididymal of Iqub^+/+ and Iqub^−/− mice. n = 4. E and F Percentage of motile spermatozoa and progressively motile spermatozoa from Iqub^+/+ and Iqub^−/− mice. n = 4, *** P < 0.001. G and H Both Papanicolaou staining and scanning electron microscopy showed normal sperm morphology of Iqub^−/− mice. Scale bars, 5 μm. I Spermatozoa cross-sections of transmission electron microscopy (TEM) showed nine radial spokes (RSs) regularly arranged in Iqub^+/+ mice and partial loss of RSs (red arrow) in Iqub^−/− mice. J Percentage of cross-sections with RSs loss in Iqub^+/+ and Iqub^−/− mice. K Spermatozoa longitudinal-sections of TEM showed that RS1-3 were arranged in 96-nm repeats in Iqub^+/+ mice, while in Iqub^−/− mice there were only two intact RSs per 96-nm repeat without RS1. Red arrow indicates RS1, green arrow indicates RS2, and cyan arrow indicates RS3. Blue rectangles represent the region where the gray values are measured. L Sperm movement paths and flagellar swing amplitudes in Iqub^+/+ and Iqub^−/− mice

Fig. 4

The identification of sperm RS1 components in human and mice. A Schematic showing the procedures of comparative proteomics performed in the spermatozoa of human and mice. n = 3. B Venn diagram showing the significantly down regulated proteins involving in sperm RS1 components (yellow and white font) and inner dynein arms (IDAs, blue font) in human and mice. C and D Western blotting showed a significant decrease of RS1 proteins in the spermatozoa of IQUB^842del patient and Iqub^−/− mice. For humans, three times independently collected sperm samples from normal control and the IQUB^842del patient. For mice, three individual animals were analyzed as biological replicates. NC, normal control. E-H Co-IP revealed that IQUB interacted with CALM, RSPH14 and AK8, but not MORN3. I and J The IDAs of DNAH7 and DNAH12 were significantly decreased in the spermatozoa of IQUB^842del patient and Iqub^−/− mice. NC, normal control. Data are presented as means ± SEM (* P < 0.05, ** P < 0.01)

Fig. 5

The RS1 models and ICSI results of human and mice. A and B The predicted RS1 complexes of human and mice, which were based on the RS1 structure of Chlamydomonas reinhardtii. C The development of two embryos from the IQUB^842del patient underwent ICSI. D The ICSI results of Iqub^+/+ and Iqub^−/− males, with no significant differences in the percentage of 2 PN (pronucleus), 2 cell, and blastocyst. Data are presented as means ± SEM, n = 3

Fig. 6

The proposed model of IQUB mutation in male infertility. IQUB mutation induced the loss of RS1 in sperm flagella, further reduced flagella swing amplitude and led to asthenozoospermia with normal sperm morphology (ANM) in human and mice