Fig 2: QRICH2 is detected in human sperm flagella. a–i 3D STORM images of QRICH2 (green) (a–c), a-Tubulin (red) (d–f) and their co-localization (yellow) (g–i). a, d, g, x-y-z projections (scale bars, 5 µm). b, e, h, x–y projections: sections surrounded by white square (scale bars, 0.5 µm). c, f, i, y-z cross-sections near the annulus (white arrowheads) (scale bars, 0.1 µm). j The domains of QRICH2 and the positions of two mutations in QRICH2. QRICH2 contains three domains: Glutenin hmw super family (amino acid positions 282–864), SMC_N super family (amino acid positions 1036–1402) and DUF4795 (amino acid positions 1252–1432). k Immunofluorescence staining of QRICH2 on human spermatozoa from normal control and the two MMAF individuals. QRICH2 staining (red) was absent in sperm from PA and significantly weak in sperm from PB. (blue, DAPI; scale bars, 5 µm)

Fig 3: The mechanisms were elucidated by proposed models. a The mechanisms of QRICH2 modulating the development of sperm flagella. QRICH2 promoted sperm flagellar formation through upregulating ODF2 and CABYR expression as a transcription factor, inhibiting ubiquitin-mediated degradation of AKAP3, TSSK4, and ROPN1 and finally reinforcing the interaction of AKAP3/CABYR/ROPN1 and TSSK4/ODF2 during the biogenesis of sperm flagella. b–d Effects of QRICH2 mutations on the structural conformation of QRICH2 protein. Superimposed structures of wild type (green) and mutant (purple) of QRICH2. The structures of QRICH2 protein were changed by the mutations of c.3313C>A [p.Q1105K] (b), c.3335G>A [p.G1112E] (c) and c.4039A>G [p.M1347V] (d). e The difference of the abnormal sperm tail number between WT mice and Ht mice was not significant. (Student’s t-test; n = 3 biologically independent WT mice or Ht mice; NS not significant; error bars, s.e.m)

Fig 4: Electron microscopy analysis shows the typical MMAF phenotypes in KO mice. a The MMAF phenotypes in Qrich2-deficient mice by SEM (scale bars, 5 µm). The WT mice showed a normal flagellum, whereas the KO mice had short, coiled, or absent flagella. b numerous defects in the sperm flagellar ultrastructures in the KO mice. The cross-section of flagella from KO mice presents disorganized arrangement of CP and ODF, and incomplete OD in the mid-piece. And the absence of OD and ODF were observed in the principal piece. (scale bars, 150 nm),

Fig 5: The mechanisms of QRICH2 regulating sperm flagellar formation. a The mRNA levels of Akap3, Odf2, Tssk4, March10, Cabyr and Ropn1 in the testes of the KO and WT mice. The reduced mRNA levels of Odf2 and Cabyr in the KO mice were detected by real-time PCR (Student’s t-test; n = 3 independent experiments; *p < 0.05; NS, not significant; error bars, s.e.m). b One QRICH2-occupied site in the ODF2 promoter and two QRICH2-occupied sites in the CABYR promoter were detected by ChIP-PCR assays of human testicular tissues. Input DNA and normal mouse IgG pulldowns were used as positive control and negative control, respectively. c ChIP-qPCR results showed the increased binding of QRICH2 to the ODF2 and CABYR promoter regions in the group with QRICH2 antibody compared to that with IgG (Student’s t-test; n = 3 independent experiments; *p < 0.05; error bars, s.e.m). d QRICH2 could enhance the transcriptional activity of ODF2 and CABYR by luciferase reporter assays (Student’s t-test; n = 3 independent experiments; *p < 0.05; error bars, s.e.m). e The increased ubiquitination levels of Akap3, Tssk4, Ropn1 and March10 in the testes of the KO mice compared with that in the WT mice. f, g QRICH2 decreased the ubiquitylation of exogenous AKAP3 (f) and TSSK4 (g) in HEK 293 cells. The cells were harvested after a 6-h treatment with 10 µM of MG132. h The binding of Ropn1 and Cabyr to Akap3 was weakened in the testes of the KO mice compared with that in the WT mice. i Overexpression of TSSK4 could increase the protein levels of ODF2