LRRC23 is a conserved component of the radial spoke that is necessary for sperm motility and male fertility in mice

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction of structures including dynein arms, radial spokes (RSs), microtubules, and the dynein regulatory complex (DRC). RSs facilitate the beating motion of these organelles by mediating signal transduction between dyneins and a central pair (CP) of singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled the detection of 23 different proteins (RSP1-23), with the roles of RSP13, RSP15, RSP18, RSP19, and RSP21 remained poorly understood. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. Through immunoelectron microscopy, we demonstrate that LRRC23 localizes to the RS complex within murine sperm flagella. We further found that LRRC23 was able to interact with RSHP9 and RSPH3A/B. The knockout of Lrrc23 resulted in RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was unaffected by the loss of this protein. Spermatozoa lacking LRRC23 were unable to efficiently pass through the uterotubal junction and exhibited defective zona penetration. Together these data indicate that LRRC23 is a key regulator underpinning the integrity of RS complex within the flagella of mammalian spermatozoa, whereas it is dispensable in cilia. Author summary

precursor complex, with the remaining RSPs being assembled following transportation 87 to the axoneme [21]. Nevertheless, the specific roles of RSP13, 15, 18, 19, and 21 in 88 the maturely assembled RS complex are less understood. Chlamydomonas RSP15 is 89 known to be a leucine-rich repeat (LRR) protein that is thought to be homologous to 90 LRR37 protein found within the RSs in the spermatozoa of Ciona intestinalis [14,22].  Herein, we show that Lrrc23 is an evolutionarily conserved gene preferentially 97 5 enriched in testis and required for male fertility in mice. LRRC23 localized to the RS 98 and interacted with RSHP9 and RSPH3A/B, and the knockout of Lrrc23 resulted in RS 99 complex disorganization and impaired murine sperm motility, whereas RS distribution 100 and motility in cilia from these mice was unaffected. Together, these data suggest that 101 LRRC23 plays an essential role in stabilizing the RS complex in sperm flagella, but is 102 dispensable in respiratory cilia.

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Lrrc23 is an evolutionarily conserved gene enriched in the testis 105 Lrrc23 is encoded by the genomes of known basal eukaryotic species that utilize  Lrrc23 is essential for male fertility and sperm motility 118 To test the functional importance of Lrrc23 in mice, we employed the CRISPR/Cas9 119 genome editing system to generate two Lrrc23 mutant mouse lines. We first prepared 120 a stable Lrrc23 mutant mouse line harboring a deletion of the exons 3-7 of this gene 121 (Lrrc23 Δ1/Δ1 ; Fig 1C). Male Lrrc23 Δ1/Δ1 mice did not exhibit any overt developmental 122 or behavioral abnormalities. We assessed their fertility by housing individual Lrrc23 +/Δ1 123 and Lrrc23 Δ1/Δ1 males with wildtype (WT) females and counting the numbers of 124 6 offspring. Lrrc23 Δ1/Δ1 males were unable to sire any offspring despite successful 125 copulation with WT females confirmed by vaginal plugs (Fig 1D), suggesting the loss 126 of Lrrc23 expression results in male infertility in mice. 127 In order to determine whether the male infertility was due to arrested 128 spermatogenesis, we next investigated the sperm formation and production in 129 Lrrc23 Δ1/Δ1 male animals. However, no differences in testis appearance and weight were 130 detected when comparing Lrrc23 +/Δ1 and Lrrc23 Δ1/Δ1 littermates (Fig 1E and 1F). Mayer hematoxylin solution of testicular sections failed to reveal any differences in the 133 spermatogenesis between Lrrc23 +/Δ1 and Lrrc23 Δ1/Δ1 males (Fig 1G and 1H).

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Epididymal ducts filled with spermatozoa were observed in both the cauda and caput 135 regions (Fig 1I-1L).

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To further determine the cause of male infertility in Lrrc23 knockout mice, we 137 conducted an in vivo fertilization assay. No fertilized eggs were found in Lrrc23 +/+ 138 females after mating with Lrrc23 Δ1/Δ1 males, suggesting the male sterility originated 139 from impaired fertilization instead of defective embryogenesis (Fig 2A and 2B). To 140 investigate if the impaired fertilization was derived from problems in sperm migration 141 or downstream zona pellucida (ZP) penetration or gamete fusion, we then carried out a 142 uterotubal junction (UTJ) penetration assay. Lrrc23 knockout spermatozoa exhibited 143 inability to pass through the UTJ (Fig 2C). Computer-Assisted Sperm Analysis (CASA) 144 was further carried out to assess the sperm motility in Lrrc23 Δ1/Δ1 males, revealing a 145 significant reduction in motile spermatozoa and spermatozoa exhibiting progressive 146 motility, a condition known as asthenozoospermia (Fig 2D and 2E). While progressive 147 motility was impaired, the flagella of spermatozoa from Lrrc23 Δ1/Δ1 mice did beat, albeit 148 across a more limited range (Fig 2F, S1 Video, S2 Video). We additionally conducted  In summary, these findings together confirm that Lrrc23 regulates 164 sperm motility and is required for male fertility in mice.

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LRRC23 is a radial spoke component that localizes to sperm flagella 166 We assessed the subcellular localization of LRRC23 using internally generated 167 antibodies. Western blotting revealed LRRC23 was approximately 40 kDa in size and 168 was absent in Lrrc23 Δ2/Δ2 mice ( Fig 3A). Immunoblot analyses of sperm protein extracts 169 indicated that LRRC23 was present within the Triton X-100-resistant, SDS-soluble 170 fraction (Fig 3B), which is the fraction associated with the axoneme [25]. We next  Table 1). Among these putative LRRC23-interacting proteins, the RS 175 head component RSPH9 was detected, which was further confirmed by co-176 immunoprecipitation and Western blot analysis ( Fig 3C).

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Confocal microscopic observation revealed LRRC23 presented within the flagella 178 and cytoplasm in murine testicular spermatids (Fig 3D). High-resolution microscopy 179 8 confirmed LRRC23 localized to the flagella of murine spermatozoa, and was closer to 180 the CP than the acetylated tubulin ( Fig 4A). Immunoelectron microscopy uncovered 181 that LRRC23 localized to the RS within spermatozoa (Fig 4B-4K

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To determine whether the absence of Lrrc23 was responsible for the male infertility, we [28] (Fig 8A). Western blot analysis confirmed that FLAG or 1D4-tagged LRRC23 was 232 detected in both testis and sperm lysates, whereas no signal was detected in the testis 233 or spermatozoa of WT males that did not carry the transgene (Fig 8B). Lrrc23 knockout-

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Tg males were housed with WT females and resulted in normal litter sizes (8.9 ± 2.9, 235 number of litters n = 152; Fig 8C), indicating that the knockout phenotype was rescued 236 by the transgene. These results confirm that LRRC23 is required for normal sperm 237 motility and male reproduction. have been no reports to date linked LRRC23 or TCTE1 mutations to asthenospermia in 250 humans, we nonetheless believe that these findings would offer important insights into 251 the tissue-specific differences in disease-related phenotypes that manifest in PCD 252 patients.

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Through high resolution fluorescence and immunoelectron microscopic analyses, 254 we determined that LRRC23 localized near the center of the axonemal structure. Co-IP 255 analyses further indicated that LRRC23 was able to interact with RSPH9, suggesting 256 that the latter protein may also play a role in regulating the RS-related sperm flagellar 257 functionality [35]. No significant abnormality in a 12S precursor RS component,  Through IP-MS analysis, we found that LRRC23 was able to interact with 273 additional proteins beyond RSPH9, including SDF4, SSSCA1, PASK, and TDRD1.

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Since PASK, PAS domain-containing serine/threonine-protein kinase, was identified as 275 a potential LRRC23-interacting protein, it is tempting to speculate that LRRC23 may     Table S1.       Table) for 15 min in air at 37 ℃. After 15 min at 37 ℃, these 392 samples were analyzed via CASA system and western blotting.