ZMYND12 serves as an IDAd subunit that is essential for sperm motility in mice

ZMYND12 serves as an IDAd subunit that is essential for sperm motility in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ZMYND12 serves as an IDAd subunit that is essential for sperm motility in mice Chang Wang, Qingsong Xie, Xun Xia, Chuanying Zhang, Shan Jiang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4539728/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Jul, 2024 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 3 You are reading this latest preprint version Abstract Inner dynein arms (IDAs) are formed from a protein complex that is essential for appropriate flagellar bending and beating. IDA defects have previously been linked to the incidence of asthenozoospermia (AZS) and male infertility. The testes-enriched ZMYND12 protein is homologous with an IDA component identified in Chlamydomonas . ZMYND12 deficiency has previously been tied to infertility in males, yet the underlying mechanism remains uncertain. Here, a CRISPR/Cas9 approach was employed to generate Zmynd12 knockout ( Zmynd12 −/− ) mice. These Zmynd12 −/− mice exhibited significant male subfertility, reduced sperm motile velocity, and impaired capacitation. Through a combination of co-immunoprecipitation and mass spectrometry, ZMYND12 was found to interact with TTC29 and PRKACA. Decreases in the levels of PRKACA were evident in the sperm of these Zmynd12 −/− mice, suggesting that this change may account for the observed drop in male fertility. Moreover, in a cohort of patients with AZS, one patient carrying a ZMYND12 variant was identified, expanding the known AZS-related variant spectrum. Together, these findings demonstrate that ZMYND12 is essential for flagellar beating, capacitation, and male fertility. Spermatogenesis Knockout mice Male fertility IDA PRKACA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The motility of sperm is vital for male fertility owing to the need for sperm to propel themselves along the length of the female reproductive tract following ejaculation so that they can fertilize the egg [ 1 ]. Impaired sperm motility can directly result in male infertility [ 2 ]. Asthenozoospermia (AZS) is a common type of primary male infertility wherein patients exhibit < 40% motile spermatozoa or < 32% progressive spermatozoa despite the absence of any abnormalities in sperm morphology or sperm counts [ 3 ]. The genetic processes that govern sperm motility, however, remain incompletely understood. As highly specialized cells, sperm consist of a head domain and a unique flagellum required for the oscillatory movement of these cells through the female reproductive tract such that they can fertilize mature oocytes [ 4 ]. The flagellar structure is highly conserved and consists of the cytoskeletal axoneme and a range of specifically organized peri-axonemal elements [ 5 ]. The axoneme is localized within the center of the flagella and is composed of nine peripheral doublet microtubules (DMTs) arranged around a central pair (CP) of microtubules in what has been termed a “9 + 2” arrangement [ 6 ]. Microtubule dynamics are shaped and maintained through the links that are formed among dynein arms, radial spokes, and the nexin-dynein regulatory complex (N-DRC) [ 7 ]. Variations in the many proteins present within sperm flagellum are thought to be closely associated with the pathogenesis of AZS in humans [ 8 , 9 ]. The precise function of these proteins and how they contribute to mammalian infertility, however, has yet to be firmly established. The swinging movement of sperm flagella is strongly dependent on dynein arm function, with axonemal dynein identification having yielded insight into the mechanistic basis for flagellar bending [ 10 ]. Axonemal dyneins are complex molecular motors composed of heavy (DHC), intermediate (IC), light (LC), and light intermediate chain (LIC) polypeptides with various molecular weights and activities [ 11 ]. Dynein motors are located within the axoneme, and are classified into the outer and inner dynein arms (ODAs and IDAs, respectively) [ 12 ]. Any form of axonemal dynein arm defects in Chlamydomonas has been demonstrated to result in the severe impairment of ciliary motility [ 13 , 14 ]. In both mice and humans, biallelic male sterility-related mutations in several genes related to dynein arm component biosynthesis including DNAH1 [ 15 ], DNAH2 [ 16 ], DNAH10 [ 17 ], and DNALI1 [ 1 ], have been identified. While affected patients exhibit reductions in sperm movement and multiple morphological abnormalities of the flagella (MMAF), the specific genetic basis for AZS is incompletely understood. ZMYND12 (Zinc Finger, MYND domain containing 12) is encoded on chromosomes 1 and 4 in humans and mice, respectively. The p38 homolog of ZMYND12 was first identified as an IDA component in Chlamydomonas [ 18 ]. Seven IDA subspecies (a-g) have been identified to date, each of which consists of one or two of eight distinct DHCs [ 19 ]. Prior studies identified p38 in Chlamydomonas as an IDAd-specific accessory subunit [ 20 ]. Coutton et al. recently found that 3 of 167 patients with MMAF harbored variants in the ZMYND12 gene [ 21 ]. In line with its possible functional role in this context, knockdown of the ZMYND12 ortholog TbTAX-1 in Trypanosoma brucei had a pronounced effect on sperm motility [ 21 , 22 ]. These results suggest a potential role for ZMYND12 deficiency in human AZS. Advances in gene editing-based models have enabled the in vivo investigation of the distinct phenotypic effects associated with knockout and knockdown models [ 23 ]. Accordingly, a CRISPR/Cas9-based approach was herein used to generate Zmynd12 -knockout mice as a means of exploring the phenotypic role played by ZMYND12 in vivo . Results Deletion of the testis-enriched ZMYND12 results in male subfertility Initially, murine ZMYND12 expression patterns across tissue types were analyzed via qPCR, revealing that it is expressed a high levels in the spleen and testis, with these levels being highest in the testis (Fig. 1 A). Testis ZMYND12 mRNA levels initially began rising at 3 weeks postpartum and continued to rise with testis development into adulthood (Fig. 1 B). When a STA-PUT (Sedimentation at Unit Gravity) approach was used for spermatocyte and spermatid isolation, the highest levels of Zmynd12 enrichment were detected in the round spermatids (RS) (Fig. 1 C). Subsequent immunofluorescent staining confirmed that ZMYND12 was present in the flagella of elongated spermatids in the testes, in addition to being present within spermatozoa from both humans and mice (Fig. 1 D; Supplementary Fig. S1 ). These results suggest that ZMYND12 may play an important functional role in the flagellum. As a protein with a high degree of evolutionary conservation, human and mouse ZMYND12 exhibit high similarity (Supplementary Fig. S2 ). In an effort to understand its functional role in vivo , a CRISPR/Cas9 strategy was used to generate Zmynd12 -knockout ( Zmynd12 –/– ) mice. For this approach, the zygotes of wild-type ( Zmynd12 +/+ ) mice were microinjected with Cas9 and gRNAs targeting exons 2–7 of Zmynd12 (Fig. 1 E). Subsequent PCR analyses confirmed the deletion of a 17,162 bp segment of the Zmynd12 gene in the resultant mice (Fig. 1 F). Quantitative PCR additionally confirmed that the Zmynd12 was absent from the testes of Zmynd12 –/– mice, indicating that it had been successfully deleted (Fig. 1 G). The Zmynd12 –/– mice exhibited healthy growth with no evidence of any defects (Supplementary Fig. S3 ). When they were subjected to fertility testing, vaginal plus were detected, confirming the ability of adult Zmynd12 –/– males to mate with Zmynd12 +/– and wild-type females. Three males were used in each group, and each male was paired with two females. While the Zmynd12 –/– males were fertile, the average litter size was decreased, suggesting that loss of ZMYND12 can result in male subfertility (Fig. 1 H). Zmynd12 –/– mice exhibit normal spermatogenesis and spermatozoa morphology In an effort to explore drivers of subfertility in male Zmynd12 –/– mice, epididymal and testis samples from adult Zmynd12 –/– and control animals were analyzed. No differences in testicular size or appearance were observed when comparing these two groups of mice (Fig. 2 A), nor was there any significant difference in the testicular/body weight ratio (Fig. 2 B). Hematoxylin and eosin (H&E) staining revealed no evidence of apparent defects in Zmynd12 –/– testis sections (Fig. 2 C), and there were also no differences in average spermatocyte, round spermatid, pachytene, or pre-leptotene counts per tubule between these two groups of mice (Fig. 2 D; Supplementary Fig. S4 A). Furthermore, the flagella of elongated spermatids did not differ between the two genotypes (Supplementary Fig. S4 B). Epididymal morphology was similarly normal in these Zmynd12 –/– mice (Fig. 2 E). In addition, the Zmynd12 –/– mice were capable of producing sufficient morphologically normal spermatozoa (Fig. 2 F-H). ZMYND12 is essential for sperm motility Given that it is an IDA component, ZMYND12 may serve as a regulator of flagellar motility. A computer-assisted sperm analyzer (CASA) was thus used to assess the motility of sperm from prepared knockout mice. While the spermatozoa of Zmynd12 +/+ mice were able to move in a linear manner, those of Zmynd12 –/– mice moved with a circular trajectory (Fig. 3 A). In addition, the data revealed comparable total motility when comparing the samples from Zmynd12 +/+ and Zmynd12 –/– mice (Fig. 3 B). However, these Zmynd12 –/– animals did exhibit significant reductions in sperm average path velocity (VAP), curvilinear velocity (VCL), and straight line velocity (VSL) as compared to controls (Fig. 3 C-E). When these sperm were cultivated in a 37℃, 5% CO 2 incubator, the motility of Zmynd12 −/− sperm declined more dramatically relative to WT sperm (Fig. 3 B-E). These results suggest a role for ZMYND12, with the loss of this protein potentially contributing to reduced sperm velocity. The flagella of ZMYND12-deficient sperm are structurally normal Flagellar structural integrity is vital for effective sperm motility. As such, transmission electron microscopy was used to evaluate the ultrastructural properties of sperm flagella from Zmynd12 +/+ and Zmynd12 –/– mice. The mid-piece of sperm from Zmynd12 –/– animals presented with the expected “9 + 2” axonemal microtubular arrangement, together with IDA, radial spoke, and outer dense fiber structures that were intact and consistent with those of Zmynd12 +/+ sperm (Fig. 4 A-C). This suggests that Zmynd12 –/– spermatozoa do not present with any pronounced abnormalities, as was subsequently confirmed through immunofluorescent staining. In Chlamydomonas flagella, the homolog of ZMYND12 is a structural component of the IDAd. However, analyses of several known DHCs and other IDA components revealed no abnormalities in any of these cases in samples from Zmynd12 –/– mice, even for the known IDAd component DNAH1 (Fig. 4 D-I). These data suggest that the deletion of ZMYND12 does not alter the major structural characteristics of sperm flagella, indicating that ZMYND12 is an accessory IDA subunit the loss of which does not induce significant flagellar abnormalities in murine spermatozoa. ZMYND12 influences the localization of PRKACA and regulates sperm capacitation To further examine the possible mechanisms whereby ZMYND12 can regulate sperm motility, proteins extracted from the testes of adult WT mice were immunoprecipitated in an effort to identify ZMYND12-interacting proteins. Immunoprecipitation was performed using anti-ZMYND12 or anti-IgG as a control (Fig. 5 A; Supplementary Table S1 ). LC-MS/MS analyses of precipitates and western blotting identified PRKACA and TTC29 as candidate ZMYND12 binding partners when assessing only those targets interacting a minimum of three times (Fig. 5 A-B). These interactions are partially consistent with results that have been reported in humans [ 21 ]. Immunofluorescent staining and western blotting for these candidate ZMYND12 binding partners were next performed on Zmynd12 +/+ and Zmynd12 –/– spermatozoa, revealing a significant decrease in PRKACA and TTC29 levels in the absence of functional ZMYND12 (Fig. 5 C-E), indicating that the absence of ZMYND12 may affect the assembly of these two proteins. PRKACA is a catalytic subunit of protein kinase A (PKA) known to serve as a regulator of sperm capacitation [ 24 ]. Spermatozoa capacitation was next assessed. Western blotting using a specific anti-phosphotyrosine antibody indicated increased protein phosphorylation when wild-type spermatozoa underwent 90 min of in vitro capacitation. However, in contrast, the phosphorylation of Zmynd12 –/– spermatozoa showed significantly reduced compare to the controls ( Fig. 5 F). These results indicate that ZMYND12 may interact with TTC29 and PRKACA. As the ortholog of TTC29 in Chlamydomonas , p44, is an IDA component, the interaction between ZMYND12 and TTC29 in mice suggests functional conservation.. Additionally, ZMYND12 loss reduced PRKACA levels in the flagellum. The brain and tracheal ciliary morphology of Zmynd12 –/– mice are normal The “9 + 2” microtubular arrangement is conserved in sperm flagella and in all motile cilia. Furthermore, defects in IDAd have been shown to cause ciliopathies, including primary ciliary dyskinesia (PCD) and MMAF in humans [ 25 – 27 ]. Zmynd12 –/– mice were next evaluated for any potential PCD-related phenotypes. Initial analyses suggested that the brain samples of these knockout mice exhibited weights and external morphological characteristics consistent with those of Zmynd12 +/+ animals (Fig. 6 A-B). Consistently, brain sections from these Zmynd12 –/– mice that had been stained with H&E appeared similar to those of WT mice (Fig. 6 C). Further comparisons of the tracheas of Zmynd12 –/– and WT mice revealed no differences in the length of tracheal cilia, nor were there any differences in the expression or localization of AC-TUBULIN in tracheal sections from these animals (Fig. 6 D). The loss of ZMYND12 function in mice thus fails to give rise to any apparent morphological defects impacting the brain ventricles and tracheal cilia. Additional research, however, will be necessary to characterize the functional performance of Zmynd12 −/− cilia. Identification of biallelic ZMYND12 variants in a patient with AZS Whole exome sequencing (WES) analyses led to the identification of a biallelic ZMYND12 variant in a patient with AZS (A001: II-1, 35 years old) (Fig. 7 A). Through sanger sequencing, this biallelic ZMYND12 variant was confirmed to have originated from two asymptomatic heterozygous parents, suggesting that it is subject to autosomal recessive inheritance (Fig. 7 B). This variant entailed a 1-bp insertion that was predicted to introduce a translational frameshift and a premature stop codon at position 38 of the 53th ZMYND12 amino acid coding sequence (Fig. 7 C). Analyses of semen samples from this proband individual (A001) revealed a total motility of 7.25%, and a progressive motility of 1.5% (Table 1 ). In line with prior reports [ 21 ], this patient presented with a high proportion of morphologically abnormal sperm (Table 1 ). Table 1 Semen data of the patient. Semen parameters A001 Normal values Color gray-white Milk-white, gray-white, yellowwish Semen volume(ml) 3 ≥ 1.5 pH 7.4 7.2–8.5 Sperm concentration (M/ml) 7.25 ≥ 15 Progressive motility (%) 1.5 ≥ 32 Motility 2 ≥ 40 Morphologically normal sperm (%) 0 > 4 Short flagella (%) 31.2 < 1.0 Coiled flagella (%) 17.2 < 17.0 Absent flagella (%) 24.7 < 5.0 Angulation (%) 7.5 < 13.0 Irregular calibre (%) 19.4 < 2.0 Normal Values based on the World Health Organization standards and the distribution ranges of morphologically abnormal spermatozoa observed in fertile individuals [ 46 , 47 ]; M, million. Discussion In this study, ZMYND12 was identified as an IDA component in mice that is expressed at the highest levels in male germ cells and that is required for normal sperm motility and capacitation. Zmynd12 –/– male mice exhibited subfertility phenotypes, with reductions in sperm velocity despite the absence of any overt structural defects. The ability of ZMYND12 and TTC29 to interact supports their association with the IDAd subspecies in murine spermatozoa, and further analyses suggested that ZMYND12 may regulate sperm capacitation by influencing PRKACA assembly. ZMYND12 serves as a specialized IDAd accessory subunit in mice sperm Axonemal dynein consists of the ODA and IDA, with the ODA repeating every 24 nm and 7 IDA subspecies repeating in the 96 nm range [ 28 ]. IDAs are important for bending motions, whereas ODAs provide acceleration [ 29 ]. IDAs have a complex composition, with the major IDA species (a, b, c, d, e, f/I1, and g) exhibiting distinct compositional and location profiles within the axoneme [ 30 ]. These IDAs are composed of multiple subunits that are broadly classified into the DHC, LC, IC, and accessory subunit categories [ 18 ]. Distal DHCs play a vital role in the conversion of ATP-derived chemical energy into mechanical force, thereby driving ciliary motility [ 31 ]. Proximal IC, LC, and LIC light subunits form the foundation of IDA complexes and are thought to regulate dynein activity. Of these, ICs are specific to IDA f/I1, while many other IDA components are believed to perform non-IDA functions. For example, DNALI1 is an IDAd LIC subunit that is also a component of cytoplasmic dynamin and is involved in IMT [ 1 , 32 ]. Here, ZMYND12 was selected as the component of interest, with its Chlamydomonas ortholog, p38, having previously been classified as an IDAd accessory subunit [ 18 ]. While the precise functional role of p38 was not established, it was found to localize to the cilia and to play a role in axonemal IDAd docking [ 33 ]. Here, TTC29 was identified as an interaction partner of ZMYND12. The finding that the ortholog of TTC29 in Chlamydomonas is an IDAd subunit [ 18 ] suggests that ZMYND12 may show IDAd localization in mouse sperm. ZMYND12 serves as a regulator of PRKACA assembly that induces sperm capacitation In contrast to their prior characterization as simple, rigid structures, sperm flagella are now understood to be highly complex organelles that contain an array of specialized enzymes. TSSK4, for example, is a Testis-Specific Serine/Threonine Protein Kinase (TSSK) family protein found in the outer dense fibers where it controls the structural organization and motility of sperm through interactions with ODF2 [ 34 ]. The fibrous sheath scaffold protein AKAP (A kinase anchoring protein) is capable of binding to protein kinase A (PKA) and particular subcellular substrates to protect the biophosphorylation reaction [ 35 ]. The TSSK6 kinase and the DUSP21 phosphatase also exhibit periodic binding activity within the axoneme of murine spermatozoa [ 36 ]. Here, an interaction between ZMYND12 and PRKACA was detected such that ZMYND12 deletion resulted in a pronounced drop in PRKACA levels. This supports a role for ZMYND12 as a regulator of the assembly of PRKACA, potentially by serving as an anchoring site for the binding of this kinase, which is important for sperm capacitation. Consistently, a significant reduction in Zmynd12 −/− sperm capacitation was observed in this study, highlighting a novel function for IDAd. ZMYND12 exhibits species-specific differences in functionality between mice and humans To date, many different dynein-associated genes have been established as candidate factors related to male infertility characterized by impaired sperm motility. The loss of DNAH1 function was the first such variant that was conclusively identified as a cause of MMAF cases of AZS [ 15 ]. More recently, studies have documented links between male fertility and a range of dynein proteins including DNAH2, with many variants in these genes having been linked to MMAF symptoms [ 37 ]. A recent report documented an association between MMAF incidence and ZMYND12 truncating and frameshift variants [ 21 ]. In the present study, a novel variant in ZMYND12 associated with loss of function was identified in an MMAF patient with high sperm malformation rates. These results highlight the potential pathogenic effects of the loss of ZMYND12 as a driver of male infertility, extending the known spectrum of AZS causes and therefore providing potential benefits to the genetic counseling and healthcare management of individuals found to harbor this genetic variant. With progressive advances in the understanding of the genetic basis for male infertility, a growing number of animal models have been developed to confirm and characterize the pathogenicity of certain variants in rodents and primates [ 38 , 39 ]. The knockdown of the ZMYND12 ortholog TbTAX-1 in Trypanosoma brucei , has been reported to markedly alter flagellar motility in a manner akin to the changes evident in the sperm of males bearing homozygous ZMYND12 variants [ 21 ]. While the male knockout mice in this study showed signficantly reduced sperm movement velocity, no corresponding reduction in the total fresh sperm motility was observed. Moreover the sperm from these Zmynd12 −/− mice did not exhibit any overt morphological abnormalities in contrast to the findings from the evaluated human patient. This may suggest that ZMYND12 plays distinct roles in the flagella of sperm from humans and mice. Interestingly, TTC29, as an interaction partner of ZMYND12, has also been found to function differently in humans and mice. Multiple case studies have demonstrated that loss of TTC29 leads to MMAF in humans, while TTC29-deficient mouse sperm showed only subtle morphological defects [ 40 , 41 ]. As in Chlamydomonas , TTC29 and ZMYND12 may function as accessory units in IDAd in mouse sperm, while in human sperm, they play a structural role in the flagella [ 18 ]. These differences reflect the functional differences in IDAd between human and mouse sperm flagella. In conclusion, the present data offer clear evidence that ZMYND12 is required for sperm motility and capacitation in mice. The multiple documented cases of human ZMYND12 variants also support a potential link between variations in this gene and the incidence of MMAF, which is a specific AZS subtype. At the molecular level, ZMYND12 was identified as a binding partner for PRKACA, and TTC29, serving as a key regulator of the functionality of murine flagella. Methods Animal care and ethics Mice were housed under specific pathogen-free conditions in a controlled setting (50–70% humidity, 20–22°C, 12 h light/dark cycle) with free food and water access. Suffering was minimized wherever possible, and mice were sacrificed by cervical dislocation when necessary. Male mice that were 8–10 weeks old were used when harvesting sperm, testes, and brain tissue samples to conduct phenotypic analyses. Sample sizes were not predetermined using any statistical techniques. Mice were assigned to experimental groups at random, and animal studies did not employ any blinding method or exclusion criteria. At the end of the study, all remaining animals were euthanized and used for tissue analyses. Study patients The AZS patients tested in this study were recruited from the Ningbo Women and Children's Hospital. Participants with abnormalities in somatic chromosome karyotypes, genomic azoospermia factor deletions, serum sex hormone levels, and scrotal ultrasonography were excluded from the analysis. This investigation received ethical approval (approval no. EC2020-048) from the above institution and all subject provided written informed consent prior to the initiation of the study. All protocols were conducted in accordance with the Declaration of Helsinki and approved by the institutional ethics review board. Genetic analysis Whole-exome sequencing and bioinformatic analyses were performed as previously described [ 1 ]. Briefly, the extraction of genomic DNA and whole-exome enrichment were performed sequentially, according to a standardized protocol. Subsequently, high-throughput sequencing of the captured DNA was performed on the HiSeq X-TEN or NovaSeq 6000 platforms (Illumina, San Diego, CA, USA). Standard assembly (Burrows–Wheeler Aligner, http://bio-bwa.sourceforge.net/ ), calling (Genome Analysis Toolkit, https://gatk.broadinstitute.org/hc/en-us ), and annotation (ANNOVAR, https://annovar.openbioinformatics.org/en/latest/ ) were then performed. Lastly, Sanger sequencing was conducted to verify the candidate mutations and corresponding origins. CRISPR/Cas9-mediated knockout Zmynd12 −/− mice were produced using a CRISPR/Cas9-based approach. Briefly, guide RNAs (gRNAs) targeting exon 2–7 of Zmynd12 were created (gRNA1: 5′-GTAAGTCCACATACCCACAAAGG-3′ and gRNA2: 5′-GCTGCCACGTCAGCCTACACAGG-3′). Zyotes from C57BL/6 mice were simultaneously injected with these gRNAs and Cas9 mRNA, after which the embryos were transferred into the uterus of pseudopregnant recipient female mice. The genotypes of offspring were then confirmed through PCR amplification with primers detailed in Supplementary Table S2 . qPCR Trizol (Thermo Fisher Scientific, 15596026) was used to extract total RNA from each sample, of which 1 µg per sample was then reverse transcribed with the PrimeScript™ RT reagent Kit (Takara, RR036A) based on provided directions to generate cDNA. SYBR Green Master Mix (Vazyme, Q131) and a LightCycler480II system (Roche) were then used for all qPCR analyses performed with primers shown in supplementary Table S2 , with 18S rRNA serving as a reference control. Western immunoblotting After extracting proteins with RIPA buffer (Beyotime, P0013B) and quantifying their levels with a BCA Kit (Beyotime, P0012), equal protein amounts were separated via 10% SDS-PAGE and transferred to PVDF membranes. Blots were then blocked with 5% BSA (Sigma, v900933) in TBS for 2 h at room temperature, followed by overnight incubation with appropriately diluted primary antibodies (4°C, overnight). Blots were blocked four times using TBST (15 min/wash), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (2 h, room temperature. A chemiluminescence reagent was then used for protein band detection. Antibodies Purchased antibodies included anti-ZMYND12 used for IP, WB and IF (Proteintech, 25587-1-AP), anti-PRKACA used for WB and IF (Proteintech, 24503-1-AP), anti-TTC29 used for WB and IF (Atlas Antibodies, HPA061473), anti-Phosphotyrosine used for WB (Merck Millipore, 05-1050X), anti-AC-TUBULIN used for WB and IF (FineTest, FNab00082), anti-DNAH1 used for IF (Thermo Fisher Scientific, PA5-57826), anti-DNAH2 used for IF (Novus, NBP2-49506), anti-DNAH10 used for IF (Bioss, bs-11022R), anti-DNAH12 used for IF (Thermo Fisher Scientific, PA5-63952), anti-DNALI1 used for IF (Proteintech, 17601-1-AP), Anti-γH 2 AX used for IF (Abcam, ab81299), Normal Rabbit IgG used for IP (Cell Signaling Technology, 2729S), Goat-anti-Mouse IgG (H + L)-HRP used for WB (Beyotime, A0216), Goat-anti-Rabbit IgG (H + L)-HRP used for WB (Beyotime, A0208), IPKine HRP, Mouse Anti-Rabbit IgG LCS used for WB (Abbkine, A25022), Donkey-anti-Mouse IgG, Alexa Fluor488 used for IF (Thermo Fisher Scientific, A-21202), Donkey-anti-Rabbit IgG, Alexa Fluor555 used for IF (Thermo Fisher Scientific, A-31572), Donkey-anti-Rabbit IgG, Alexa Fluor488 used for IF (Thermo Fisher Scientific, A-21206), and the anti-AKAP3 used for IF was a gift from Qi’s lab [ 42 ]. The working concentrations of the antibodies are shown in Supplementary Table S3 . Fertility testing Fertility analyses were performed by mating sexually mature knockout male mice with two wild-type C57BL/6 female mice for a 6-month period during which the female mice were exchanged every other gestation cycle. Knockout male mice and controls were fed under identical conditions, and litter sizes were recorded during fertility testing. All fertility testing was conducted using 8 to 10-week-old mice. Silver staining and LC-MS/MS After separating proteins by 12% SDS-PAGE, they were stained with a Fast Silver Stain Kit (Beyotime, P0017S). Bands of interest were then excised manually, digested using sequencing-grade trypsin (Promega, WI, USA), and the peptides therein were extracted, dried, and analyzed via LC-MS/MS. The IP precipitates were separated on SDS-PAGE and stained with AgNO 3 . The bands were removed from the gels following trypsin digestion. The EASY-nanoLC 1200 system (Thermo Fisher Scientific), equipped with an Orbitrap Q Exactive HFX mass spectrometer (Thermo Fisher Scientific) and a nanospray ion source, was used for LC-MS/MS analysis. Mixtures of tryptic peptides were dissolved in 0.1% formic acid (FA) in LC-grade water and injected into an analytical column (75 µm× 25 cm, C18 column, 1.9 µm, Dr. Maisch). Solution A was 0.1% FA and solution B was 80% ACN and 0.1% FA. A 95-min linear gradient (3–5% B for 5 s, 5–15% B for 40 min, 15–28% B for 34 min and 50 s, 28–38% B for 12 min, 30–100% B for 5 s, and 100% B for 8 min) was applied using a high-resolution MS pre-scan, with a mass range of 350–1500. The normalized collision energy for elevated energy collision-driven dissociation (HCD) was adjusted to 28, and the resulting fragments were identified using a resolution of 15,000. All ions chosen for fragmentation were excluded for 30 s via dynamic exclusion. Data processing was done with Proteome Discoverer software (Thermo Fisher Scientific), and the mouse reference proteome was retrieved from the UniProt database (release 2021.04) using standard variables. Co-immunoprecipitation RIPA buffer (1 mL; Beyotime, P0013C) was used to extract total testicular proteins, followed by centrifugation (40 min, 13,000 rpm). Supernatants were then collected, precleared for 1 h using 30 µL of protein A/G beads (Bimake, B23202) at 4°C, and the lysates were then incubated overnight at 4°C with appropriate antibodies. Protein complexes were then combined with 60 µL of Protein A/G magnetic beads, followed by a further 6 h incubation at 4°C. Supernatants were then removed, and beads were washed with RIPA buffer 5 times, followed by the addition of SDS loading buffer. Samples were then boiled for 10 min at 95°C and dentured proteins were separated by SDS-PAGE and detected with appropriate antibodies. As a negative control, rabbit IgG was also used for co-immunoprecipitation. Histological and immunofluorescent staining Sperm samples were fixed with 4% paraformaldehyde (PFA) for 10 min before spreading on slides. The slides were dried and then rinsed three times with PBS. For the preparation of paraffin-embedded sections, tissues were fixed using 4% paraformaldehyde or modified Davidson's fluid (MDF) for 48 h. Then, these samples were treated with a gradient of 70%, 80%, 90%, and 100% ethanol, a 1:1 mixture of ethanol and xylene, and pure xylene. After embedding these samples in paraffin, 5 µm sections were cut. Before staining, sections were deparaffinized and rehydrated. For H&E staining, these tissues were strained with hematoxylin and eosin staining solution. For IF staining, sections were treated with 10 mM citrate solution (pH 6) while heating for antigen retrieval. Both the sperm samples and sections were blocked using 1% BSA (Sigma, v900933), followed by overnight incubation at 4°C with appropriate primary antibodies, washed, and treated for 2 h with secondary antibodies and Hoechst 33342 at room temperature. Samples were then fixed using glycero, covered using glass coverslips, followed by imaging with an LSM980 confocal microscope (Carl Zeiss). Transmission Electron Microscopy (TEM) For TEM, samples were fixed with 1% osmium tetroxide and dehydrated with an ethanol gradient (50, 70, 90, and 100% ethanol) and 100% acetone. After infiltration with acetone and SPI-Chem resin and embedding with Epon 812, the samples were sectioned using an ultra-microtome and stained with uranyl acetate and lead citrate. A JEM-1400 transmission electron microscope (JEOL) was used for sample evaluation and imaging. Sperm motility analyses When analyzing sperm motility, an approach reported previously was employed [ 43 ]. Briefly, following the resection of the cauda epididymis from an adult mouse, sperm were dislodged by squeezing into modified HTF medium (Irvine Scientific, 90126) containing 10% fetal bovine serum (FBS) and incubated at 37℃ for 10 min. The suspended sperm were then assessed with a computer-assisted sperm analysis (CASA, CEROS v.12, Hamilton Thorne Research), allowing for analyses of motile sperm. Capacitation and tyrosine phosphorylation detection in mice sperm Sperm capacitation and tyrosine phosphorylation detection were conducted based on a published protocol with minor modifications [ 44 , 45 ]. The excised cauda epididymis was placed in HTF medium, consisting of 101.6 mM NaCl, 4.7 mM KCl, 0.37 mM K 2 PO 4 , 0.2 mM MgSO 4 ·7H 2 O, 2 mM CaCl 2 , 25 mM NaHCO 3 , 2.78 mM glucose, 0.33 mM pyruvate, 21.4 mM sodium lactate, 286 mg/L penicillin G, 228 mg/L streptomycin, and 5 mg/ml fatty acid-free BSA (Sangon, A602448), to release sperm. The sperm were divided into non-capacitated and capacitated groups, with the latter incubated at 37°C with 5% CO2 for 90 minutes. After centrifugation at 500g and 4°C, the pellet was collected. The proteins were extracted and subjected to Western blot analysis of tyrosine phosphorylation. Statistical analysis. Student’s two-tailed t-tests were used to compare data in GraphPad Prism. Not significant (ns), P ≥ 0.05; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The exact sample sizes (n) for each experimental group/condition are provided in the figure legends.All analyses were performed at least in triplicate. Declarations Competing Interests The authors declare there are no competing interests. Ethics approval This investigation received ethical approval (approval nos. EC2020-048) from the aforementioned institution and received documented informed consent from all subjects prior to the initiation of the study. All the studies were carried out in accordance with the Declaration of Helsinki and approved by the institutional ethics review board. All animal studies were performed as per the criteria and protocols established by the Institutional Animal Care and Use Committee of Cyagen Biosciences Inc., with all protocols having received institutional ethical approval (Approval No. TACU23-FY025). Data Availability All data relevant to the study are included in the article or uploaded as supplementary information. Author’s Contribution H.Z., R.H. and C.Z. designed the study and reviewed the manuscript. C.W., Q.X. and X.X. performed the most biochemical experiments, analyzed the data, prepared figures and/or tables, and wrote the manuscript. S.W., and S.J. performed some biochemical experiments, analyzed the data, prepared figures and/or tables, reviewed drafts of the paper. H.Z. prepared the mouse models. J.X and X.Z. provided patients’ data and performed clinical assessments. All authors approved the final manuscript. Acknowledgements We would like to thank Huayu Qi from the Chinese Academy of Sciences for the anti-AKAP3 antibody. Consent for publication The author’s consent to publication. Funding This work was supported by National Natural Science Foundation of China (82371622 and 32000584 to R.H.); the Key Project of Natural Science Foundation for Universities of Anhui Province Education Department (2023AH050843 to C.W.); the Natural Science Foundation of Huai’an (HAB202305 to H.Z.); Natural Science Foundation of Anhui Province (2208085Y31 to R.H.); the exceptional support plan of talent introduction of Anhui University of Chinese Medicine (2023rcyb022 to C.W.). the Science and Technology development Fundation of Nanjing Medical Univertisy (NMUB20220214 to X.Z.); Ningbo science and technology project (2023Z178 to J.X.). 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Hum Reprod Update 16(3):231–245 Supplementary Files Supplementary.docx TableS1ZMYND12interactingprotein.xlsx TableS2PrimersusedinPCRandRTqPCR.doc TableS3Detailedinformationontheantibodies.xlsx Cite Share Download PDF Status: Published Journal Publication published 27 Jul, 2024 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Editorial decision: Accept as is 01 Jul, 2024 Reviewers agreed at journal 11 Jun, 2024 First submitted to journal 04 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4539728","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":313188317,"identity":"a62fdc0e-9749-420e-84d2-462576ddf58f","order_by":0,"name":"Chang Wang","email":"","orcid":"","institution":"Anhui University of Traditional Chinese Medicine - East Campus: Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chang","middleName":"","lastName":"Wang","suffix":""},{"id":313188318,"identity":"060b71fd-92ff-4c6f-977c-af3dee40c5fa","order_by":1,"name":"Qingsong Xie","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qingsong","middleName":"","lastName":"Xie","suffix":""},{"id":313188319,"identity":"32f3439d-7833-4d0f-8b0c-67876a8a8aea","order_by":2,"name":"Xun Xia","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xun","middleName":"","lastName":"Xia","suffix":""},{"id":313188320,"identity":"1fcde831-da01-4c15-80d7-bebe3536c652","order_by":3,"name":"Chuanying Zhang","email":"","orcid":"","institution":"Anhui University of Traditional Chinese Medicine - 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Data are means ± SEM, n = 3.\u003c/p\u003e\n\u003cp\u003e(B) \u003cem\u003eZmynd12\u003c/em\u003e mRNA levels in the testes of mice at different ages, with 18S as a normalization control. W, weeks. Data are means ± SEM, n = 3.\u003c/p\u003e\n\u003cp\u003e(C) qPCR was used to measure \u003cem\u003eZmynd12\u003c/em\u003e expression in male germ cells isolated from the testes of mice, with 18S as a normalization control. Data are means ± SEM, n = 3.\u003c/p\u003e\n\u003cp\u003e(D) IF staining for ZMYND12 (red) and PNA (green) in the testes of WT mice，n = 3.\u003c/p\u003e\n\u003cp\u003e(E) Schematic overview of the approach to generating \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice using a CRISPR/Cas9 approach.\u003c/p\u003e\n\u003cp\u003e(F) PCR was used to identify murine genotypes with the F1, R1, and R2 primers. Wildtype and knockout mice were respectively identified using the F1/R1 and F1/R2 primer pairs.\u003c/p\u003e\n\u003cp\u003e(G) qPCR was used to measure \u003cem\u003eZmynd12\u003c/em\u003e expression in the testes of \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice, with 18S as a normalization control. Data are means ± SEM, n = 3.\u003c/p\u003e\n\u003cp\u003e(H) Average numbers of pups per litter for male \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice. Data are means ± SEM, n = 3.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figures01.png","url":"https://assets-eu.researchsquare.com/files/rs-4539728/v1/d3a209e26f2cc75ebd155a61.png"},{"id":59606185,"identity":"26f29fba-700a-4de4-82d6-5cfaa04c65f7","added_by":"auto","created_at":"2024-07-03 18:51:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3442142,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNormal spermatogenic phases are evident in ZMYND12-deficient mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The testes of male \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e(B) Average testis weights normalized to body weight. Data are means ± SEM, n =6.\u003c/p\u003e\n\u003cp\u003e(C) H\u0026amp;E-stained testicular sections from male \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice, n = 3.\u003c/p\u003e\n\u003cp\u003e(D) Relative composition ratios for different cell types at spermatogenic stage VIII in the testes of \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice. P-L, pre-leptotene; P, pachytene; RS, round spermatids. Data are means ± SEM, n\u003cem\u003e \u003c/em\u003e= 3.\u003c/p\u003e\n\u003cp\u003e(E) H\u0026amp;E-stained cauda epididymal sections and caput epididymal sections from male \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice, n = 3.\u003c/p\u003e\n\u003cp\u003e(F) H\u0026amp;E-stained spermatozoa from male \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e cauda epididymidis, n = 3.\u003c/p\u003e\n\u003cp\u003e(G) Percentages of spermatozoa from \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice exhibiting morphological abnormalities. Data are means ± SEM, n\u003cem\u003e \u003c/em\u003e= 3.\u003c/p\u003e\n\u003cp\u003e(H) AC-TUBULIN (green) staining of the spermatozoa from \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice, n = 3.\u003c/p\u003e","description":"","filename":"Figures02.png","url":"https://assets-eu.researchsquare.com/files/rs-4539728/v1/1a458e3c40e148f6500c33c4.png"},{"id":59606192,"identity":"de6d2866-caee-4634-a3ca-a9109bdd587d","added_by":"auto","created_at":"2024-07-03 18:51:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1700594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eZmynd12\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e\u003cstrong\u003emice exhibit impaired swimming parameters.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Sperm motility tracing performed using a computer-assisted sperm analysis system after incubation for 10, 90, and 180 minutes, n = 3.\u003c/p\u003e\n\u003cp\u003e(B) Total cauda epididymal sperm motility for samples from male \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice were assessed following an incubation period for 10, 90, and 180 min. Data are means ± SEM, n\u003cem\u003e \u003c/em\u003e=3.\u003c/p\u003e\n\u003cp\u003e(C-E) VAP (average path velocity) (C), VCL (curvilinear velocity) (D), and VSL (straight line velocity) (E) were assessed following an incubation period for 10, 90, and 180 min. Data are means ± SEM, n\u003cem\u003e \u003c/em\u003e= 3.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figures03.png","url":"https://assets-eu.researchsquare.com/files/rs-4539728/v1/050e72818c31d5861154a77e.png"},{"id":59606187,"identity":"64c962e6-794d-4bc8-91cf-718d137a5b14","added_by":"auto","created_at":"2024-07-03 18:51:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2608049,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZMYND12-deficient sperm flagella do not exhibit any substantial abnormalities.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) TEM-based ultrastructural analyses of flagellar cross-sections from the spermatozoa of \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice. M, mid-piece; P, principal piece.\u003c/p\u003e\n\u003cp\u003e(B) Percentages of sperm from \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice exhibiting ultrastructural abnormalities detected via TEM. Data are means ± SEM, n\u003cem\u003e \u003c/em\u003e= 3.\u003c/p\u003e\n\u003cp\u003e(C) Abnormal ODF quantification for cross-sections of the mid-piece and principal piece of spermatozoa from \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003emice. Data are means ± SEM, n\u003cem\u003e \u003c/em\u003e= 3.\u003c/p\u003e\n\u003cp\u003e(D-I) Immunofluorescent staining was used to detect DNAH1 (D), DNAH2 (E), DNAH10 (F), DNAH12 (G), DNALI1 (H), and AKAP3 (I) in \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003espermatozoa,\u003cem\u003e \u003c/em\u003en\u003cem\u003e \u003c/em\u003e= 4.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figures04.png","url":"https://assets-eu.researchsquare.com/files/rs-4539728/v1/d49543bfe5c07cc3766d2f09.png"},{"id":59606191,"identity":"21b881fd-8691-4eab-8e0f-4a0998989bbd","added_by":"auto","created_at":"2024-07-03 18:51:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1220400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZMYND12 regulates sperm capacitation and engages in interactions with PRKACA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) IP was performed using wild-type mouse testis samples, after which silver staining and mass spectrometry were performed, revealing that PRKACA and TTC29 were possible interacting proteins. Exp, experimental group, in which interacting proteins were precipitated with an anti-ZMYND12 antibody; Ctl, control group, where an anti-IgG was used as a negative control for immunoprecipitation; Coverage, the coverage of the identified peptide relative to the protein; #Peptides, the types of peptides identified, n\u003cem\u003e \u003c/em\u003e= 3.\u003c/p\u003e\n\u003cp\u003e(B) Co-immunoprecipitation analysis of the interaction partners of ZMYND12 in testicular protein extracts.\u003c/p\u003e\n\u003cp\u003e(C) TTC29 IF staining (red) of \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003espermatozoa,\u003cem\u003e \u003c/em\u003en\u003cem\u003e \u003c/em\u003e= 3.\u003c/p\u003e\n\u003cp\u003e(D) PRKACA IF staining (red) of \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003espermatozoa,\u003cem\u003e \u003c/em\u003en\u003cem\u003e \u003c/em\u003e= 3.\u003c/p\u003e\n\u003cp\u003e(E) Western blotting of PRKACA from \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003espermatozoa,\u003cem\u003e \u003c/em\u003ewith AC-TUBULIN as the internal control, n\u003cem\u003e \u003c/em\u003e= 3.\u003c/p\u003e\n\u003cp\u003e(F) Protein tyrosine phosphorylation associated with capacitation in spermatozoa from both \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice, with AC-TUBULIN as the internal control, n\u003cem\u003e \u003c/em\u003e= 3.\u003c/p\u003e","description":"","filename":"Figures05.png","url":"https://assets-eu.researchsquare.com/files/rs-4539728/v1/dd94f64dc0c190a94aaf61ba.png"},{"id":59606991,"identity":"54bdc4fc-c786-4a9b-a472-da1213e88a83","added_by":"auto","created_at":"2024-07-03 18:59:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2288989,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZMYND12 is not required for normal brain or tracheal cilia morphology.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Brain images for male\u003cem\u003e Zmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e(B) Brain and body weight ratios for male \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice\u003cem\u003e. \u003c/em\u003eData are means ± SEM, n\u003cem\u003e \u003c/em\u003e=3.\u003c/p\u003e\n\u003cp\u003e(C) H\u0026amp;E-stained brain sections from male \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice,\u003cem\u003e \u003c/em\u003en\u003cem\u003e \u003c/em\u003e= 3.\u003c/p\u003e\n\u003cp\u003e(D) AC-TUBULIN IF staining (green) in \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003etracheal ciliated columnar epithelial cells,\u003cem\u003e \u003c/em\u003en\u003cem\u003e \u003c/em\u003e= 3.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figures06.png","url":"https://assets-eu.researchsquare.com/files/rs-4539728/v1/8efbb787da8c6e3762b5ef19.png"},{"id":59606992,"identity":"c2ca49e9-5d87-4da1-8b27-bc2574feec49","added_by":"auto","created_at":"2024-07-03 18:59:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":891906,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of a ZMYND12 mutation in a male with AZS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Pedigree analysis of the family affected by biallelic \u003cem\u003eZMYND12\u003c/em\u003e variations. Males suffering from infertility are marked with filled black squares.\u003c/p\u003e\n\u003cp\u003e(B) Sanger sequencing was used to verify \u003cem\u003eZMYND12\u003c/em\u003e variants identified using whole-exome sequencing in a male with AZS (A001). Inserted bases are marked with a black dashed box.\u003c/p\u003e\n\u003cp\u003e(C) The locations of variations in the \u003cem\u003eZMYND12\u003c/em\u003egene.\u003c/p\u003e","description":"","filename":"Figures07.png","url":"https://assets-eu.researchsquare.com/files/rs-4539728/v1/efc47cf5ad1df0c4b8c1b0da.png"},{"id":61597244,"identity":"f43fa800-c3df-4501-91fd-2c776b862b13","added_by":"auto","created_at":"2024-08-01 17:32:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20873523,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4539728/v1/ec807f0e-f103-4270-92e4-057a9893ee8c.pdf"},{"id":59606189,"identity":"ee84f003-1aa1-4e7b-beb8-48508c6c97a8","added_by":"auto","created_at":"2024-07-03 18:51:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":991064,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-4539728/v1/eab21106befe5543f04fad0e.docx"},{"id":59606184,"identity":"57cb497a-5193-41ff-ae6c-d08c6208d88c","added_by":"auto","created_at":"2024-07-03 18:51:58","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":765774,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1ZMYND12interactingprotein.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4539728/v1/b5f3d3d92d268d45cd235341.xlsx"},{"id":59606990,"identity":"b6e6850a-dbe0-4e71-ab54-43efe4412472","added_by":"auto","created_at":"2024-07-03 18:59:58","extension":"doc","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17408,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2PrimersusedinPCRandRTqPCR.doc","url":"https://assets-eu.researchsquare.com/files/rs-4539728/v1/b7ff9f8574f7c5f404314950.doc"},{"id":59606188,"identity":"f6235848-b704-4c93-9601-d17b195c1501","added_by":"auto","created_at":"2024-07-03 18:51:59","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11519,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3Detailedinformationontheantibodies.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4539728/v1/22a3c7540f57e01accd44a49.xlsx"}],"financialInterests":"","formattedTitle":"ZMYND12 serves as an IDAd subunit that is essential for sperm motility in mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe motility of sperm is vital for male fertility owing to the need for sperm to propel themselves along the length of the female reproductive tract following ejaculation so that they can fertilize the egg [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Impaired sperm motility can directly result in male infertility [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Asthenozoospermia (AZS) is a common type of primary male infertility wherein patients exhibit\u0026thinsp;\u0026lt;\u0026thinsp;40% motile spermatozoa or \u0026lt;\u0026thinsp;32% progressive spermatozoa despite the absence of any abnormalities in sperm morphology or sperm counts [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The genetic processes that govern sperm motility, however, remain incompletely understood.\u003c/p\u003e \u003cp\u003eAs highly specialized cells, sperm consist of a head domain and a unique flagellum required for the oscillatory movement of these cells through the female reproductive tract such that they can fertilize mature oocytes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The flagellar structure is highly conserved and consists of the cytoskeletal axoneme and a range of specifically organized peri-axonemal elements [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The axoneme is localized within the center of the flagella and is composed of nine peripheral doublet microtubules (DMTs) arranged around a central pair (CP) of microtubules in what has been termed a \u0026ldquo;9\u0026thinsp;+\u0026thinsp;2\u0026rdquo; arrangement [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Microtubule dynamics are shaped and maintained through the links that are formed among dynein arms, radial spokes, and the nexin-dynein regulatory complex (N-DRC) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Variations in the many proteins present within sperm flagellum are thought to be closely associated with the pathogenesis of AZS in humans [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The precise function of these proteins and how they contribute to mammalian infertility, however, has yet to be firmly established.\u003c/p\u003e \u003cp\u003eThe swinging movement of sperm flagella is strongly dependent on dynein arm function, with axonemal dynein identification having yielded insight into the mechanistic basis for flagellar bending [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Axonemal dyneins are complex molecular motors composed of heavy (DHC), intermediate (IC), light (LC), and light intermediate chain (LIC) polypeptides with various molecular weights and activities [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Dynein motors are located within the axoneme, and are classified into the outer and inner dynein arms (ODAs and IDAs, respectively) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Any form of axonemal dynein arm defects in \u003cem\u003eChlamydomonas\u003c/em\u003e has been demonstrated to result in the severe impairment of ciliary motility [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In both mice and humans, biallelic male sterility-related mutations in several genes related to dynein arm component biosynthesis including \u003cem\u003eDNAH1\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], \u003cem\u003eDNAH2\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], \u003cem\u003eDNAH10\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and \u003cem\u003eDNALI1\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], have been identified. While affected patients exhibit reductions in sperm movement and multiple morphological abnormalities of the flagella (MMAF), the specific genetic basis for AZS is incompletely understood.\u003c/p\u003e \u003cp\u003eZMYND12 (Zinc Finger, MYND domain containing 12) is encoded on chromosomes 1 and 4 in humans and mice, respectively. The p38 homolog of ZMYND12 was first identified as an IDA component in \u003cem\u003eChlamydomonas\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Seven IDA subspecies (a-g) have been identified to date, each of which consists of one or two of eight distinct DHCs [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Prior studies identified p38 in \u003cem\u003eChlamydomonas\u003c/em\u003e as an IDAd-specific accessory subunit [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Coutton et al. recently found that 3 of 167 patients with MMAF harbored variants in the \u003cem\u003eZMYND12\u003c/em\u003e gene [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In line with its possible functional role in this context, knockdown of the ZMYND12 ortholog TbTAX-1 in \u003cem\u003eTrypanosoma brucei\u003c/em\u003e had a pronounced effect on sperm motility [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These results suggest a potential role for ZMYND12 deficiency in human AZS.\u003c/p\u003e \u003cp\u003eAdvances in gene editing-based models have enabled the \u003cem\u003ein vivo\u003c/em\u003e investigation of the distinct phenotypic effects associated with knockout and knockdown models [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Accordingly, a CRISPR/Cas9-based approach was herein used to generate \u003cem\u003eZmynd12\u003c/em\u003e-knockout mice as a means of exploring the phenotypic role played by ZMYND12 \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDeletion of the testis-enriched ZMYND12 results in male subfertility\u003c/h2\u003e \u003cp\u003eInitially, murine ZMYND12 expression patterns across tissue types were analyzed via qPCR, revealing that it is expressed a high levels in the spleen and testis, with these levels being highest in the testis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Testis ZMYND12 mRNA levels initially began rising at 3 weeks postpartum and continued to rise with testis development into adulthood (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). When a STA-PUT (Sedimentation at Unit Gravity) approach was used for spermatocyte and spermatid isolation, the highest levels of \u003cem\u003eZmynd12\u003c/em\u003e enrichment were detected in the round spermatids (RS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Subsequent immunofluorescent staining confirmed that ZMYND12 was present in the flagella of elongated spermatids in the testes, in addition to being present within spermatozoa from both humans and mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD; Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These results suggest that ZMYND12 may play an important functional role in the flagellum.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs a protein with a high degree of evolutionary conservation, human and mouse ZMYND12 exhibit high similarity (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In an effort to understand its functional role \u003cem\u003ein vivo\u003c/em\u003e, a CRISPR/Cas9 strategy was used to generate \u003cem\u003eZmynd12\u003c/em\u003e-knockout (\u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e) mice. For this approach, the zygotes of wild-type (\u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e) mice were microinjected with Cas9 and gRNAs targeting exons 2\u0026ndash;7 of \u003cem\u003eZmynd12\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Subsequent PCR analyses confirmed the deletion of a 17,162 bp segment of the \u003cem\u003eZmynd12\u003c/em\u003e gene in the resultant mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Quantitative PCR additionally confirmed that the \u003cem\u003eZmynd12\u003c/em\u003e was absent from the testes of \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e mice, indicating that it had been successfully deleted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e mice exhibited healthy growth with no evidence of any defects (Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). When they were subjected to fertility testing, vaginal plus were detected, confirming the ability of adult \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e males to mate with \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e and wild-type females. Three males were used in each group, and each male was paired with two females. While the \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e males were fertile, the average litter size was decreased, suggesting that loss of ZMYND12 can result in male subfertility (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003cb\u003eZmynd12\u003c/b\u003e \u003csup\u003e \u003cb\u003e\u0026ndash;/\u0026ndash;\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emice exhibit normal spermatogenesis and spermatozoa morphology\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn an effort to explore drivers of subfertility in male \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e mice, epididymal and testis samples from adult \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e and control animals were analyzed. No differences in testicular size or appearance were observed when comparing these two groups of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), nor was there any significant difference in the testicular/body weight ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Hematoxylin and eosin (H\u0026amp;E) staining revealed no evidence of apparent defects in \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e testis sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and there were also no differences in average spermatocyte, round spermatid, pachytene, or pre-leptotene counts per tubule between these two groups of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD; Supplementary Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA). Furthermore, the flagella of elongated spermatids did not differ between the two genotypes (Supplementary Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB). Epididymal morphology was similarly normal in these \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In addition, the \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e mice were capable of producing sufficient morphologically normal spermatozoa (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eZMYND12 is essential for sperm motility\u003c/h2\u003e \u003cp\u003eGiven that it is an IDA component, ZMYND12 may serve as a regulator of flagellar motility. A computer-assisted sperm analyzer (CASA) was thus used to assess the motility of sperm from prepared knockout mice. While the spermatozoa of \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice were able to move in a linear manner, those of \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e mice moved with a circular trajectory (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In addition, the data revealed comparable total motility when comparing the samples from \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). However, these \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e animals did exhibit significant reductions in sperm average path velocity (VAP), curvilinear velocity (VCL), and straight line velocity (VSL) as compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-E). When these sperm were cultivated in a 37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator, the motility of \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e sperm declined more dramatically relative to WT sperm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-E). These results suggest a role for ZMYND12, with the loss of this protein potentially contributing to reduced sperm velocity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eThe flagella of ZMYND12-deficient sperm are structurally normal\u003c/h2\u003e \u003cp\u003eFlagellar structural integrity is vital for effective sperm motility. As such, transmission electron microscopy was used to evaluate the ultrastructural properties of sperm flagella from \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e mice. The mid-piece of sperm from \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e animals presented with the expected \u0026ldquo;9\u0026thinsp;+\u0026thinsp;2\u0026rdquo; axonemal microtubular arrangement, together with IDA, radial spoke, and outer dense fiber structures that were intact and consistent with those of \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e sperm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). This suggests that \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e spermatozoa do not present with any pronounced abnormalities, as was subsequently confirmed through immunofluorescent staining.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn \u003cem\u003eChlamydomonas\u003c/em\u003e flagella, the homolog of ZMYND12 is a structural component of the IDAd. However, analyses of several known DHCs and other IDA components revealed no abnormalities in any of these cases in samples from \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e mice, even for the known IDAd component DNAH1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-I). These data suggest that the deletion of ZMYND12 does not alter the major structural characteristics of sperm flagella, indicating that ZMYND12 is an accessory IDA subunit the loss of which does not induce significant flagellar abnormalities in murine spermatozoa.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eZMYND12 influences the localization of PRKACA and regulates sperm capacitation\u003c/h2\u003e \u003cp\u003eTo further examine the possible mechanisms whereby ZMYND12 can regulate sperm motility, proteins extracted from the testes of adult WT mice were immunoprecipitated in an effort to identify ZMYND12-interacting proteins. Immunoprecipitation was performed using anti-ZMYND12 or anti-IgG as a control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). LC-MS/MS analyses of precipitates and western blotting identified PRKACA and TTC29 as candidate ZMYND12 binding partners when assessing only those targets interacting a minimum of three times (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). These interactions are partially consistent with results that have been reported in humans [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescent staining and western blotting for these candidate ZMYND12 binding partners were next performed on \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e spermatozoa, revealing a significant decrease in PRKACA and TTC29 levels in the absence of functional ZMYND12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-E), indicating that the absence of ZMYND12 may affect the assembly of these two proteins. PRKACA is a catalytic subunit of protein kinase A (PKA) known to serve as a regulator of sperm capacitation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Spermatozoa capacitation was next assessed. Western blotting using a specific anti-phosphotyrosine antibody indicated increased protein phosphorylation when wild-type spermatozoa underwent 90 min of in vitro capacitation. However, in contrast, the phosphorylation of \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e spermatozoa showed significantly reduced compare to the controls \u003cem\u003e(\u003c/em\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). These results indicate that ZMYND12 may interact with TTC29 and PRKACA. As the ortholog of TTC29 in \u003cem\u003eChlamydomonas\u003c/em\u003e, p44, is an IDA component, the interaction between ZMYND12 and TTC29 in mice suggests functional conservation.. Additionally, ZMYND12 loss reduced PRKACA levels in the flagellum.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe brain and tracheal ciliary morphology of\u003c/b\u003e \u003cb\u003eZmynd12\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026ndash;/\u0026ndash;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice are normal\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u0026ldquo;9\u0026thinsp;+\u0026thinsp;2\u0026rdquo; microtubular arrangement is conserved in sperm flagella and in all motile cilia. Furthermore, defects in IDAd have been shown to cause ciliopathies, including primary ciliary dyskinesia (PCD) and MMAF in humans [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e mice were next evaluated for any potential PCD-related phenotypes. Initial analyses suggested that the brain samples of these knockout mice exhibited weights and external morphological characteristics consistent with those of \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). Consistently, brain sections from these \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e mice that had been stained with H\u0026amp;E appeared similar to those of WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther comparisons of the tracheas of \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e and WT mice revealed no differences in the length of tracheal cilia, nor were there any differences in the expression or localization of AC-TUBULIN in tracheal sections from these animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). The loss of ZMYND12 function in mice thus fails to give rise to any apparent morphological defects impacting the brain ventricles and tracheal cilia. Additional research, however, will be necessary to characterize the functional performance of \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cilia.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of biallelic\u003c/b\u003e \u003cb\u003eZMYND12\u003c/b\u003e \u003cb\u003evariants in a patient with AZS\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhole exome sequencing (WES) analyses led to the identification of a biallelic \u003cem\u003eZMYND12\u003c/em\u003e variant in a patient with AZS (A001: II-1, 35 years old) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Through sanger sequencing, this biallelic \u003cem\u003eZMYND12\u003c/em\u003e variant was confirmed to have originated from two asymptomatic heterozygous parents, suggesting that it is subject to autosomal recessive inheritance (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). This variant entailed a 1-bp insertion that was predicted to introduce a translational frameshift and a premature stop codon at position 38 of the 53th ZMYND12 amino acid coding sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Analyses of semen samples from this proband individual (A001) revealed a total motility of 7.25%, and a progressive motility of 1.5% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In line with prior reports [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], this patient presented with a high proportion of morphologically abnormal sperm (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSemen data of the patient.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSemen parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA001\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal values\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003egray-white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMilk-white, gray-white, yellowwish\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSemen volume(ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.2\u0026ndash;8.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSperm concentration (M/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProgressive motility (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMotility\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMorphologically normal sperm (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShort flagella (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoiled flagella (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;17.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsent flagella (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;5.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAngulation (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;13.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIrregular calibre (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNormal Values based on the World Health Organization standards and the distribution ranges of morphologically abnormal spermatozoa observed in fertile individuals [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e];\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eM, million.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, ZMYND12 was identified as an IDA component in mice that is expressed at the highest levels in male germ cells and that is required for normal sperm motility and capacitation. \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026ndash;/\u0026ndash;\u003c/em\u003e\u003c/sup\u003e male mice exhibited subfertility phenotypes, with reductions in sperm velocity despite the absence of any overt structural defects. The ability of ZMYND12 and TTC29 to interact supports their association with the IDAd subspecies in murine spermatozoa, and further analyses suggested that ZMYND12 may regulate sperm capacitation by influencing PRKACA assembly.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eZMYND12 serves as a specialized IDAd accessory subunit in mice sperm\u003c/h2\u003e \u003cp\u003eAxonemal dynein consists of the ODA and IDA, with the ODA repeating every 24 nm and 7 IDA subspecies repeating in the 96 nm range [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. IDAs are important for bending motions, whereas ODAs provide acceleration [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. IDAs have a complex composition, with the major IDA species (a, b, c, d, e, f/I1, and g) exhibiting distinct compositional and location profiles within the axoneme [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These IDAs are composed of multiple subunits that are broadly classified into the DHC, LC, IC, and accessory subunit categories [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Distal DHCs play a vital role in the conversion of ATP-derived chemical energy into mechanical force, thereby driving ciliary motility [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Proximal IC, LC, and LIC light subunits form the foundation of IDA complexes and are thought to regulate dynein activity. Of these, ICs are specific to IDA f/I1, while many other IDA components are believed to perform non-IDA functions. For example, DNALI1 is an IDAd LIC subunit that is also a component of cytoplasmic dynamin and is involved in IMT [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Here, ZMYND12 was selected as the component of interest, with its \u003cem\u003eChlamydomonas\u003c/em\u003e ortholog, p38, having previously been classified as an IDAd accessory subunit [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. While the precise functional role of p38 was not established, it was found to localize to the cilia and to play a role in axonemal IDAd docking [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Here, TTC29 was identified as an interaction partner of ZMYND12. The finding that the ortholog of TTC29 in \u003cem\u003eChlamydomonas\u003c/em\u003e is an IDAd subunit [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] suggests that ZMYND12 may show IDAd localization in mouse sperm.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eZMYND12 serves as a regulator of PRKACA assembly that induces sperm capacitation\u003c/h3\u003e\n\u003cp\u003eIn contrast to their prior characterization as simple, rigid structures, sperm flagella are now understood to be highly complex organelles that contain an array of specialized enzymes. TSSK4, for example, is a Testis-Specific Serine/Threonine Protein Kinase (TSSK) family protein found in the outer dense fibers where it controls the structural organization and motility of sperm through interactions with ODF2 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The fibrous sheath scaffold protein AKAP (A kinase anchoring protein) is capable of binding to protein kinase A (PKA) and particular subcellular substrates to protect the biophosphorylation reaction [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The TSSK6 kinase and the DUSP21 phosphatase also exhibit periodic binding activity within the axoneme of murine spermatozoa [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Here, an interaction between ZMYND12 and PRKACA was detected such that ZMYND12 deletion resulted in a pronounced drop in PRKACA levels. This supports a role for ZMYND12 as a regulator of the assembly of PRKACA, potentially by serving as an anchoring site for the binding of this kinase, which is important for sperm capacitation. Consistently, a significant reduction in \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e sperm capacitation was observed in this study, highlighting a novel function for IDAd.\u003c/p\u003e\n\u003ch3\u003eZMYND12 exhibits species-specific differences in functionality between mice and humans\u003c/h3\u003e\n\u003cp\u003eTo date, many different dynein-associated genes have been established as candidate factors related to male infertility characterized by impaired sperm motility. The loss of DNAH1 function was the first such variant that was conclusively identified as a cause of MMAF cases of AZS [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. More recently, studies have documented links between male fertility and a range of dynein proteins including DNAH2, with many variants in these genes having been linked to MMAF symptoms [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. A recent report documented an association between MMAF incidence and \u003cem\u003eZMYND12\u003c/em\u003e truncating and frameshift variants [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In the present study, a novel variant in \u003cem\u003eZMYND12\u003c/em\u003e associated with loss of function was identified in an MMAF patient with high sperm malformation rates. These results highlight the potential pathogenic effects of the loss of ZMYND12 as a driver of male infertility, extending the known spectrum of AZS causes and therefore providing potential benefits to the genetic counseling and healthcare management of individuals found to harbor this genetic variant.\u003c/p\u003e \u003cp\u003eWith progressive advances in the understanding of the genetic basis for male infertility, a growing number of animal models have been developed to confirm and characterize the pathogenicity of certain variants in rodents and primates [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The knockdown of the \u003cem\u003eZMYND12\u003c/em\u003e ortholog TbTAX-1 in \u003cem\u003eTrypanosoma brucei\u003c/em\u003e, has been reported to markedly alter flagellar motility in a manner akin to the changes evident in the sperm of males bearing homozygous \u003cem\u003eZMYND12\u003c/em\u003e variants [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. While the male knockout mice in this study showed signficantly reduced sperm movement velocity, no corresponding reduction in the total fresh sperm motility was observed. Moreover the sperm from these \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice did not exhibit any overt morphological abnormalities in contrast to the findings from the evaluated human patient. This may suggest that ZMYND12 plays distinct roles in the flagella of sperm from humans and mice. Interestingly, TTC29, as an interaction partner of ZMYND12, has also been found to function differently in humans and mice. Multiple case studies have demonstrated that loss of TTC29 leads to MMAF in humans, while TTC29-deficient mouse sperm showed only subtle morphological defects [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. As in \u003cem\u003eChlamydomonas\u003c/em\u003e, TTC29 and ZMYND12 may function as accessory units in IDAd in mouse sperm, while in human sperm, they play a structural role in the flagella [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These differences reflect the functional differences in IDAd between human and mouse sperm flagella. In conclusion, the present data offer clear evidence that ZMYND12 is required for sperm motility and capacitation in mice. The multiple documented cases of human \u003cem\u003eZMYND12\u003c/em\u003e variants also support a potential link between variations in this gene and the incidence of MMAF, which is a specific AZS subtype. At the molecular level, ZMYND12 was identified as a binding partner for PRKACA, and TTC29, serving as a key regulator of the functionality of murine flagella.\u003c/p\u003e"},{"header":"Methods","content":" \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eAnimal care and ethics\u003c/h2\u003e \u003cp\u003eMice were housed under specific pathogen-free conditions in a controlled setting (50\u0026ndash;70% humidity, 20\u0026ndash;22\u0026deg;C, 12 h light/dark cycle) with free food and water access. Suffering was minimized wherever possible, and mice were sacrificed by cervical dislocation when necessary. Male mice that were 8\u0026ndash;10 weeks old were used when harvesting sperm, testes, and brain tissue samples to conduct phenotypic analyses. Sample sizes were not predetermined using any statistical techniques. Mice were assigned to experimental groups at random, and animal studies did not employ any blinding method or exclusion criteria. At the end of the study, all remaining animals were euthanized and used for tissue analyses.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStudy patients\u003c/h2\u003e \u003cp\u003eThe AZS patients tested in this study were recruited from the Ningbo Women and Children's Hospital. Participants with abnormalities in somatic chromosome karyotypes, genomic azoospermia factor deletions, serum sex hormone levels, and scrotal ultrasonography were excluded from the analysis. This investigation received ethical approval (approval no. EC2020-048) from the above institution and all subject provided written informed consent prior to the initiation of the study. All protocols were conducted in accordance with the Declaration of Helsinki and approved by the institutional ethics review board.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGenetic analysis\u003c/h2\u003e \u003cp\u003eWhole-exome sequencing and bioinformatic analyses were performed as previously described [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Briefly, the extraction of genomic DNA and whole-exome enrichment were performed sequentially, according to a standardized protocol. Subsequently, high-throughput sequencing of the captured DNA was performed on the HiSeq X-TEN or NovaSeq 6000 platforms (Illumina, San Diego, CA, USA). Standard assembly (Burrows\u0026ndash;Wheeler Aligner, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bio-bwa.sourceforge.net/\u003c/span\u003e\u003cspan address=\"http://bio-bwa.sourceforge.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), calling (Genome Analysis Toolkit, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gatk.broadinstitute.org/hc/en-us\u003c/span\u003e\u003cspan address=\"https://gatk.broadinstitute.org/hc/en-us\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and annotation (ANNOVAR, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://annovar.openbioinformatics.org/en/latest/\u003c/span\u003e\u003cspan address=\"https://annovar.openbioinformatics.org/en/latest/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were then performed. Lastly, Sanger sequencing was conducted to verify the candidate mutations and corresponding origins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCRISPR/Cas9-mediated knockout\u003c/h2\u003e \u003cp\u003e\u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were produced using a CRISPR/Cas9-based approach. Briefly, guide RNAs (gRNAs) targeting exon 2\u0026ndash;7 of \u003cem\u003eZmynd12\u003c/em\u003e were created (gRNA1: 5\u0026prime;-GTAAGTCCACATACCCACAAAGG-3\u0026prime; and gRNA2: 5\u0026prime;-GCTGCCACGTCAGCCTACACAGG-3\u0026prime;). Zyotes from C57BL/6 mice were simultaneously injected with these gRNAs and Cas9 mRNA, after which the embryos were transferred into the uterus of pseudopregnant recipient female mice. The genotypes of offspring were then confirmed through PCR amplification with primers detailed in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eqPCR\u003c/h2\u003e \u003cp\u003eTrizol (Thermo Fisher Scientific, 15596026) was used to extract total RNA from each sample, of which 1 \u0026micro;g per sample was then reverse transcribed with the PrimeScript\u0026trade; RT reagent Kit (Takara, RR036A) based on provided directions to generate cDNA. SYBR Green Master Mix (Vazyme, Q131) and a LightCycler480II system (Roche) were then used for all qPCR analyses performed with primers shown in supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, with 18S rRNA serving as a reference control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eWestern immunoblotting\u003c/h2\u003e \u003cp\u003eAfter extracting proteins with RIPA buffer (Beyotime, P0013B) and quantifying their levels with a BCA Kit (Beyotime, P0012), equal protein amounts were separated via 10% SDS-PAGE and transferred to PVDF membranes. Blots were then blocked with 5% BSA (Sigma, v900933) in TBS for 2 h at room temperature, followed by overnight incubation with appropriately diluted primary antibodies (4\u0026deg;C, overnight). Blots were blocked four times using TBST (15 min/wash), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (2 h, room temperature. A chemiluminescence reagent was then used for protein band detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003ePurchased antibodies included anti-ZMYND12 used for IP, WB and IF (Proteintech, 25587-1-AP), anti-PRKACA used for WB and IF (Proteintech, 24503-1-AP), anti-TTC29 used for WB and IF (Atlas Antibodies, HPA061473), anti-Phosphotyrosine used for WB (Merck Millipore, 05-1050X), anti-AC-TUBULIN used for WB and IF (FineTest, FNab00082), anti-DNAH1 used for IF (Thermo Fisher Scientific, PA5-57826), anti-DNAH2 used for IF (Novus, NBP2-49506), anti-DNAH10 used for IF (Bioss, bs-11022R), anti-DNAH12 used for IF (Thermo Fisher Scientific, PA5-63952), anti-DNALI1 used for IF (Proteintech, 17601-1-AP), Anti-γH\u003csub\u003e2\u003c/sub\u003eAX used for IF (Abcam, ab81299), Normal Rabbit IgG used for IP (Cell Signaling Technology, 2729S), Goat-anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L)-HRP used for WB (Beyotime, A0216), Goat-anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L)-HRP used for WB (Beyotime, A0208), IPKine HRP, Mouse Anti-Rabbit IgG LCS used for WB (Abbkine, A25022), Donkey-anti-Mouse IgG, Alexa Fluor488 used for IF (Thermo Fisher Scientific, A-21202), Donkey-anti-Rabbit IgG, Alexa Fluor555 used for IF (Thermo Fisher Scientific, A-31572), Donkey-anti-Rabbit IgG, Alexa Fluor488 used for IF (Thermo Fisher Scientific, A-21206), and the anti-AKAP3 used for IF was a gift from Qi\u0026rsquo;s lab [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The working concentrations of the antibodies are shown in Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFertility testing\u003c/h2\u003e \u003cp\u003eFertility analyses were performed by mating sexually mature knockout male mice with two wild-type C57BL/6 female mice for a 6-month period during which the female mice were exchanged every other gestation cycle. Knockout male mice and controls were fed under identical conditions, and litter sizes were recorded during fertility testing. All fertility testing was conducted using 8 to 10-week-old mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eSilver staining and LC-MS/MS\u003c/h2\u003e \u003cp\u003eAfter separating proteins by 12% SDS-PAGE, they were stained with a Fast Silver Stain Kit (Beyotime, P0017S). Bands of interest were then excised manually, digested using sequencing-grade trypsin (Promega, WI, USA), and the peptides therein were extracted, dried, and analyzed via LC-MS/MS.\u003c/p\u003e \u003cp\u003eThe IP precipitates were separated on SDS-PAGE and stained with AgNO\u003csub\u003e3\u003c/sub\u003e. The bands were removed from the gels following trypsin digestion. The EASY-nanoLC 1200 system (Thermo Fisher Scientific), equipped with an Orbitrap Q Exactive HFX mass spectrometer (Thermo Fisher Scientific) and a nanospray ion source, was used for LC-MS/MS analysis. Mixtures of tryptic peptides were dissolved in 0.1% formic acid (FA) in LC-grade water and injected into an analytical column (75 \u0026micro;m\u0026times; 25 cm, C18 column, 1.9 \u0026micro;m, Dr. Maisch). Solution A was 0.1% FA and solution B was 80% ACN and 0.1% FA. A 95-min linear gradient (3\u0026ndash;5% B for 5 s, 5\u0026ndash;15% B for 40 min, 15\u0026ndash;28% B for 34 min and 50 s, 28\u0026ndash;38% B for 12 min, 30\u0026ndash;100% B for 5 s, and 100% B for 8 min) was applied using a high-resolution MS pre-scan, with a mass range of 350\u0026ndash;1500. The normalized collision energy for elevated energy collision-driven dissociation (HCD) was adjusted to 28, and the resulting fragments were identified using a resolution of 15,000. All ions chosen for fragmentation were excluded for 30 s via dynamic exclusion. Data processing was done with Proteome Discoverer software (Thermo Fisher Scientific), and the mouse reference proteome was retrieved from the UniProt database (release 2021.04) using standard variables.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation\u003c/h2\u003e \u003cp\u003eRIPA buffer (1 mL; Beyotime, P0013C) was used to extract total testicular proteins, followed by centrifugation (40 min, 13,000 rpm). Supernatants were then collected, precleared for 1 h using 30 \u0026micro;L of protein A/G beads (Bimake, B23202) at 4\u0026deg;C, and the lysates were then incubated overnight at 4\u0026deg;C with appropriate antibodies. Protein complexes were then combined with 60 \u0026micro;L of Protein A/G magnetic beads, followed by a further 6 h incubation at 4\u0026deg;C. Supernatants were then removed, and beads were washed with RIPA buffer 5 times, followed by the addition of SDS loading buffer. Samples were then boiled for 10 min at 95\u0026deg;C and dentured proteins were separated by SDS-PAGE and detected with appropriate antibodies. As a negative control, rabbit IgG was also used for co-immunoprecipitation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eHistological and immunofluorescent staining\u003c/h2\u003e \u003cp\u003eSperm samples were fixed with 4% paraformaldehyde (PFA) for 10 min before spreading on slides. The slides were dried and then rinsed three times with PBS. For the preparation of paraffin-embedded sections, tissues were fixed using 4% paraformaldehyde or modified Davidson's fluid (MDF) for 48 h. Then, these samples were treated with a gradient of 70%, 80%, 90%, and 100% ethanol, a 1:1 mixture of ethanol and xylene, and pure xylene. After embedding these samples in paraffin, 5 \u0026micro;m sections were cut. Before staining, sections were deparaffinized and rehydrated. For H\u0026amp;E staining, these tissues were strained with hematoxylin and eosin staining solution. For IF staining, sections were treated with 10 mM citrate solution (pH 6) while heating for antigen retrieval. Both the sperm samples and sections were blocked using 1% BSA (Sigma, v900933), followed by overnight incubation at 4\u0026deg;C with appropriate primary antibodies, washed, and treated for 2 h with secondary antibodies and Hoechst 33342 at room temperature. Samples were then fixed using glycero, covered using glass coverslips, followed by imaging with an LSM980 confocal microscope (Carl Zeiss).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eTransmission Electron Microscopy (TEM)\u003c/h2\u003e \u003cp\u003eFor TEM, samples were fixed with 1% osmium tetroxide and dehydrated with an ethanol gradient (50, 70, 90, and 100% ethanol) and 100% acetone. After infiltration with acetone and SPI-Chem resin and embedding with Epon 812, the samples were sectioned using an ultra-microtome and stained with uranyl acetate and lead citrate. A JEM-1400 transmission electron microscope (JEOL) was used for sample evaluation and imaging.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eSperm motility analyses\u003c/h2\u003e \u003cp\u003eWhen analyzing sperm motility, an approach reported previously was employed [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Briefly, following the resection of the cauda epididymis from an adult mouse, sperm were dislodged by squeezing into modified HTF medium (Irvine Scientific, 90126) containing 10% fetal bovine serum (FBS) and incubated at 37℃ for 10 min. The suspended sperm were then assessed with a computer-assisted sperm analysis (CASA, CEROS v.12, Hamilton Thorne Research), allowing for analyses of motile sperm.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eCapacitation and tyrosine phosphorylation detection in mice sperm\u003c/h2\u003e \u003cp\u003eSperm capacitation and tyrosine phosphorylation detection were conducted based on a published protocol with minor modifications [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The excised cauda epididymis was placed in HTF medium, consisting of 101.6 mM NaCl, 4.7 mM KCl, 0.37 mM K\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 25 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, 2.78 mM glucose, 0.33 mM pyruvate, 21.4 mM sodium lactate, 286 mg/L penicillin G, 228 mg/L streptomycin, and 5 mg/ml fatty acid-free BSA (Sangon, A602448), to release sperm. The sperm were divided into non-capacitated and capacitated groups, with the latter incubated at 37\u0026deg;C with 5% CO2 for 90 minutes. After centrifugation at 500g and 4\u0026deg;C, the pellet was collected. The proteins were extracted and subjected to Western blot analysis of tyrosine phosphorylation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis.\u003c/h2\u003e \u003cp\u003eStudent\u0026rsquo;s two-tailed t-tests were used to compare data in GraphPad Prism. Not significant (ns), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.05; *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. The exact sample sizes (n) for each experimental group/condition are provided in the figure legends.All analyses were performed at least in triplicate.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare there are no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis investigation received ethical approval (approval nos. EC2020-048) from the aforementioned institution and received documented informed consent from all subjects prior to the initiation of the study. All the studies were carried out in accordance with the Declaration of Helsinki and approved by the institutional ethics review board.\u003c/p\u003e\n\u003cp\u003eAll animal studies were performed as per the criteria and protocols established by the Institutional Animal Care and Use Committee of Cyagen Biosciences Inc., with all protocols having received institutional ethical approval (Approval No. TACU23-FY025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data relevant to the study are included in the article or uploaded as supplementary information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.Z., R.H. and C.Z. designed the study and reviewed the manuscript. C.W., Q.X. and X.X. performed the most biochemical experiments, analyzed the data, prepared figures and/or tables, and wrote the manuscript. S.W., and S.J. performed some biochemical experiments, analyzed the data, prepared figures and/or tables, reviewed drafts of the paper. H.Z. prepared the mouse models. J.X and X.Z. provided patients\u0026rsquo; data and performed clinical assessments. All authors approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Huayu Qi from the Chinese Academy of Sciences for the anti-AKAP3 antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author\u0026rsquo;s consent to publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (82371622 and 32000584 to R.H.); the\u0026nbsp;Key Project of Natural Science Foundation for Universities of Anhui Province Education Department (2023AH050843 to C.W.); the Natural Science Foundation of Huai\u0026rsquo;an (HAB202305 to H.Z.); Natural Science Foundation of Anhui Province (2208085Y31 to R.H.); the exceptional support plan of talent introduction of\u0026nbsp;Anhui University of Chinese Medicine\u0026nbsp;(2023rcyb022 to C.W.). the Science and Technology development Fundation of Nanjing Medical Univertisy (NMUB20220214 to X.Z.);\u0026nbsp;Ningbo science and technology project\u0026nbsp;(2023Z178 to J.X.).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWu H et al (2023) DNALI1 deficiency causes male infertility with severe asthenozoospermia in humans and mice by disrupting the assembly of the flagellar inner dynein arms and fibrous sheath. 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Cell 186(13):2897\u0026ndash;2910e19\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLevkova M, Radanova M, Angelova L (2022) Potential role of dynein-related genes in the etiology of male infertility: A systematic review and a meta-analysis. Andrology 10(8):1484\u0026ndash;1499\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu C et al (2023) Deficiency of primate-specific SSX1 induced asthenoteratozoospermia in infertile men and cynomolgus monkey and tree shrew models. Am J Hum Genet 110(3):516\u0026ndash;530\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J et al (2021) Loss of DRC1 function leads to multiple morphological abnormalities of the sperm flagella and male infertility in human and mouse. Hum Mol Genet 30(21):1996\u0026ndash;2011\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLor\u0026egrave;s P et al (2019) Mutations in TTC29, Encoding an Evolutionarily Conserved Axonemal Protein, Result in Asthenozoospermia and Male Infertility. Am J Hum Genet 105(6):1148\u0026ndash;1167\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu C et al (2019) Bi-allelic Mutations in TTC29 Cause Male Subfertility with Asthenoteratospermia in Humans and Mice. Am J Hum Genet 105(6):1168\u0026ndash;1181\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu K et al (2020) \u003cem\u003eLack of AKAP3 disrupts integrity of the subcellular structure and proteome of mouse sperm and causes male sterility.\u003c/em\u003e 147(2)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCastaneda JM et al (2017) TCTE1 is a conserved component of the dynein regulatory complex and is required for motility and metabolism in mouse spermatozoa. Proc Natl Acad Sci U S A 114(27):E5370\u0026ndash;E5378\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP E, V., et al., Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development, (1995) 121(4)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP Q (1985) K. J F, and W. G M, Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril, 44(4)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAuger J, Jouannet P, Eustache F (2016) Another look at human sperm morphology. Hum Reprod 31(1):10\u0026ndash;23\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCooper TG et al (2010) World Health Organization reference values for human semen characteristics. Hum Reprod Update 16(3):231\u0026ndash;245\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Spermatogenesis, Knockout mice, Male fertility, IDA, PRKACA","lastPublishedDoi":"10.21203/rs.3.rs-4539728/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4539728/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInner dynein arms (IDAs) are formed from a protein complex that is essential for appropriate flagellar bending and beating. IDA defects have previously been linked to the incidence of asthenozoospermia (AZS) and male infertility. The testes-enriched ZMYND12 protein is homologous with an IDA component identified in \u003cem\u003eChlamydomonas\u003c/em\u003e. ZMYND12 deficiency has previously been tied to infertility in males, yet the underlying mechanism remains uncertain. Here, a CRISPR/Cas9 approach was employed to generate \u003cem\u003eZmynd12\u003c/em\u003e knockout (\u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice. These \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited significant male subfertility, reduced sperm motile velocity, and impaired capacitation. Through a combination of co-immunoprecipitation and mass spectrometry, ZMYND12 was found to interact with TTC29 and PRKACA. Decreases in the levels of PRKACA were evident in the sperm of these \u003cem\u003eZmynd12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, suggesting that this change may account for the observed drop in male fertility. Moreover, in a cohort of patients with AZS, one patient carrying a \u003cem\u003eZMYND12\u003c/em\u003e variant was identified, expanding the known AZS-related variant spectrum. Together, these findings demonstrate that ZMYND12 is essential for flagellar beating, capacitation, and male fertility.\u003c/p\u003e","manuscriptTitle":"ZMYND12 serves as an IDAd subunit that is essential for sperm motility in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 18:51:53","doi":"10.21203/rs.3.rs-4539728/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept as is","date":"2024-07-01T22:49:09+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-06-11T14:14:11+00:00","index":0,"fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2024-06-04T11:03:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1dc163aa-79b8-4e74-aa2c-73da8bc817b3","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T17:19:40+00:00","versionOfRecord":{"articleIdentity":"rs-4539728","link":"https://doi.org/10.1007/s00018-024-05344-7","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2024-07-27 16:16:02","publishedOnDateReadable":"July 27th, 2024"},"versionCreatedAt":"2024-07-03 18:51:53","video":"","vorDoi":"10.1007/s00018-024-05344-7","vorDoiUrl":"https://doi.org/10.1007/s00018-024-05344-7","workflowStages":[]},"version":"v1","identity":"rs-4539728","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4539728","identity":"rs-4539728","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}