Fig 2: A bi-allelic splicing donor site variant in LRRC23 was identified from asthenozoospermia patients.A A consanguineous pedigree with two infertile males (IV-1 and IV-2). IV-1 was subjected for WES (arrow). Genotypes of the variant (blue) in all family members included in this study (III-1, III-2, IV-1, IV-2, IV-3, and IV-4) are confirmed by Sanger sequencing. +, wild-type allele. An infertile female sibling (IV-4) is marked in black circle.B Papanicolaou-stained sperm from the infertile male (IV-2).C Mapping of the LRRC23 variant. Mutation of G to A at the splicing donor site in the 5th intron is predicted to prevent LRRC23 mRNA from splicing.D Sequencing chromatograms presenting the LRRC23 variant in the infertile male (IV-1) and his father (III-2). The variant is underlined and normal splicing donor site (GT) is boxed.E, F Minigene assay for testing altered splicing of LRRC23 by the variant. (E) Minigene constructs expressing LRRC23 ORF containing the 5th intron (sashed) with wild-type (WT) or mutant (Mut, red) splicing donor site were generated. The constructs are tagged with FLAG and HA at N- and C-termini, respectively. (F) RT-PCR of the 293T cells transfected with the minigene constructs reveals the 5th intron is not spliced out and retained by the variant. Intron-spanning primers, F1 and R1, are used. Repeated three times with biological replications.

Fig 3: LRRC23 mutation disrupts the third radial spoke (RS) in sperm flagellum.A Immunostaining of flagellar proteins in different compartments. Shown are midpiece (TOM20), annulus (SEPT4 and SEPT12), fibrous sheath (AKAP4), outer dense fiber (ODF2), and axoneme (acetylated tubulin, AcTub) in Lrrc23+/? (top) and Lrrc23?/? (bottom) sperm. Magnified insets are represented for annulus proteins (scale bars in insets = 2µm). Fluorescence and corresponding DIC images are merged. Sperm heads were counter stained with Hoechst. Lrrc23+/? sperm were used for positive control. Experiments were performed with three biological replications.B Transmission electron microscopy images of Lrrc23+/? (left) and Lrrc23?/? (right) sperm. Shown are longitudinal section of sperm flagella. M, mitochondria; ODF, outer dense fiber; AX, axoneme; CP, central pair; MT, microtubule; FS, fibrous sheath. Lrrc23+/? sperm were used for positive control.C Cryo-electron tomography (cryo-ET) of WT and Lrrc23?/? sperm flagella. Shown are representative tomographic slices from WT (left) and Lrrc23?/? sperm (right). The 9+2 axonemal structure are shown in both WT and Lrrc23?/? in cross-sectional view (left). Axonemal structures are shown with proximal side of the flagellum on the left in longitudinal view (right; see Movie EV3). Magnified insets (bottom) reveal that RS1, 2, and 3 are shown in WT sperm (left, filled arrowheads) but RS3, especially head part, is not clearly visible (right, red arrowheads) in Lrrc23?/? sperm. or WT sperm were used for positive control.

Fig 4: C-terminal truncation of human LRRC23 by the splicing site mutation prevents its interaction with radial spoke (RS) head.A Sub-tomogram averaging images of RSs from Chlamydomonas reinhardtii (left), Trypanosoma brucei (middle), and mouse sperm (right). Original data from Electron Microscopy Data Bank was rendered.B Structure of RS in C. reinhardtii. A schematic cartoon shows the RS1 and 2. The structure of RS2 stalk is shown in inset (PDB Id: 7JRJ).C, D Purification of normal (hLRRC23WT) and the mutant human LRRC23 (hLRRC23Mut) by the splicing site mutation (c.621+1G>A) in this study. (C) Diagrams for the purified recombinant normal and mutant proteins tagged with tagged with GST and HA at N- and C-termini, respectively. (D) Purified proteins by Coomassie blue staining (left) and immunoblotting with a-HA (middle) and a-LRRC23 (right). Proteins matched to the predicted size were marked with asterisks.E A cartoon of the RSPH-trap approach to test LRRC23 interaction with RS proteins. Individual human RS proteins tagged with FLAG (RSPH-FLAG) are expressed in 293T cells and enriched by a-FLAG resin from cell lysates. The recombinant RSPH proteins were incubated with the purified hLRRC23WT or hLRRC23Mut and subjected to immunoblotting.F Interaction of hLRRC23 to a RS head component, RSPH9. The purified hLRRC23 were incubated with the RSPH-Trap (RS head, RSPH6A and RSPH9; stalk, RSPH3 and RSPH22) and subjected to immunoblotting. 5% amount of the hLRRC23s used for the trap assay were loaded as inputs. White lines in individual a-HA blot images indicate marker information (75 kDa, left; 50 kDa, right). Experiments were repeated four times. Purified GST was used for negative control (Fig EV4B). Experiments were repeated three times with biological replications.G A phylogenetic tree constructed by Maximum-likelihood analysis of the protein sequences of the C. reinhardtii RSP15 and the orthologs of LRRC23 and LRRC34. LRR37, the first LRRC23 ortholog identified in Ciona intestinalis is marked in bold.H Comparison of the reported RSP15 from C. reinhardtii and the predicted structure of LRRC23 and LRRC34 from human. Atomic structure of the C. reinhardtii RS2 containing RSP15 are represented by ribbon (RS2) and surface (RSP15) diagram (left, PDB Id: 7JU4). Ribbon diagrams of C. reinhardtii RSP15 and AlphaFold-predicted human LRRC23 (middle) and LRRC34 (right) are shown for structural comparison. Secondary structures are color-coded. Different from C. reinhardtii RSP15 and LRRC34, LRRC23 does not display repeated a-helix (magenta) between ß-sheets (gold).

Fig 5: Lrrc23 mutant mice mimicking human splice variant phenocopy male infertility and reduced sperm motility.A, B Immunoblotting of LRRC23 in testis (A) and epididymal sperm (B) from mutant male mice. Truncated LRRC23 (arrowheads) is detected from testis microsome fraction (filled), but not in mature sperm (empty), of heterozygous (+/?) and homozygous (?/?) males. Acetylated tubulin (AcTub) is a loading control. Experiments were performed with three biological replications.C Confocal images of immunostained LRRC23 in Lrrc23+/? and Lrrc23?/? epididymal sperm Experiments were repeated with three biological replications.D Epididymal sperm counts. n.s., not significant.E Pregnancy rate of Lrrc23+/? and Lrrc23?/? males.F Number of litters from fertile females mated with Lrrc23+/? and Lrrc23?/? males.G Swimming trajectory of Lrrc23+/? and Lrrc23?/? sperm in viscous media (0.3% methylcellulose). Swimming trajectory for 2 seconds is overlaid. Experiments were performed with three biological replications. See Movie EV1.H Flagellar waveforms of Lrrc23+/? and Lrrc23?/? sperm before (0 minute) and after (90 minutes) inducing capacitation. Flagellar movements for two beat cycles are overlaid and color coded in time. Experiments were performed with three biological replications. See Movie EV2.Data information: In (A-C), samples from WT were used for positive or negative control of normal or truncated LRRC23. In (D, F), circles indicate sperm counts from individual males (D) and pup numbers from each litter (F), and data represented as mean ± SEM (D, Mann-whiteny U test; F, Student’s t-test). n.s., non-significant.