TEX43, a Testis-Enriched Microtubule-Associated Gene, Exerts Minimal Impact on Spermatogenesis and Fertility in Mice and Humans

TEX43, a Testis-Enriched Microtubule-Associated Gene, Exerts Minimal Impact on Spermatogenesis and Fertility in Mice and Humans | 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 TEX43, a Testis-Enriched Microtubule-Associated Gene, Exerts Minimal Impact on Spermatogenesis and Fertility in Mice and Humans Qi-qi Chen, Li Lei, Xin Qiu, Yujun Liu, Ping Liu, Rong Li, Xu Zhi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8181104/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Purpose Testis-specific TEX family genes are critical for spermatogenesis, but TEX43’s function remains uncharacterized. This study aimed to delineate TEX43’s role in spermatogenesis and fertility using murine models and clinical data. Methods Tex43 expression was analyzed via quantitative reverse transcription-polymerase chain reaction (Q-PCR), immunohistochemistry (IHC), and western blotting. A Tex43 knockout (KO) mouse model was generated using CRISPR/Cas9 (targeting exons 1–3). Testicular histology (hematoxylin-eosin [H&E] staining), sperm parameters (morphology via H&E smears, density via hemocytometer, motility via computer-assisted sperm analysis [CASA]), and fertility (in vivo breeding assays, in vitro fertilization [IVF]) were evaluated. Sperm ultrastructure was assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Whole-exome sequencing (WES) identified TEX43 variants in 146 infertile men with asthenoteratozoospermia. Structural modeling of WT/mutant TEX43 was performed via SWISS-MODEL. Results Tex43 is testis-enriched: mRNA expression initiated at postnatal day 18 (round spermatid stage) and peaked in elongating spermatids; TEX43 localized to sperm flagellar microtubules. Tex43-KO mice showed modestly reduced sperm density (28.6 ± 3.2 vs. 41.2 ± 2.9×10⁶ sperm/ml in WT; P 0.05). TEM revealed increased flagellar end piece "9 + 2" microtubule disorganization in KO sperm (~ 30% vs. ~5% in WT; P < 0.01). WES identified 9 infertile men with TEX43 variants; 4 with exonic variants (e.g., p.R37Q) achieved live births via intracytoplasmic sperm injection (ICSI). Structural modeling showed p.R37Q disrupted hydrogen bonds critical for microtubule binding. Conclusions TEX43 is minimal impact on murine spermatogenesis and fertility, likely due to genetic redundancy. Human TEX43 variants may exert subtle reproductive effects, requiring validation in larger cohorts with functional studies. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Male infertility represents a prominent challenge in men’s reproductive health globally. According to the World Health Organization (WHO), approximately 15% of reproductive-age couples worldwide face infertility issues, with male factors accounting for nearly 50% of these cases [ 1 , 2 ]. The etiology of male infertility is complex and multifactorial, and genetic perturbations that reduce germ cell quantity or impair sperm quality are well-recognized key contributors [ 3 ].Notably, the incidence of male infertility has continued to rise alongside societal and environmental changes, underscoring the urgency of clarifying its underlying mechanisms. Spermatogenesis is a highly orchestrated process. This complex cascade involves the coordinated regulation of over 2,300 genes [ 4 ].At the molecular level, sperm formation and maturation are driven by the programmed expression of cell- and stage-specific genes. The ordered transcription of testis-enriched genes, alongside the synergistic interactions of transcription factors, metabolic enzymes, cell cycle regulators, forms the molecular backbone of spermatogenesis. Disruption of critical genes or proteins can arrest or perturb spermatogenic cell development, leading to the production of morphologically abnormal, immotile, or non-functional sperm, ultimately resulting in fertilization failure and male infertility [ 5 , 6 ]. In male reproductive biology, the testis-specific gene (TEX) family plays a pivotal role in regulating spermatogenesis and sustaining male fertility. The term "TEX gene" was first coined by Wang et al. in 2001, subsequent to their identification of novel spermatogenic cell-specific transcripts in mice using the technique of cDNA suppression subtractive hybridization (SSH) [ 7 ]. Subsequently, orthologs of these TEX genes have been identified in a wide range of organisms, spanning vertebrates, invertebrates and yeast. Accumulating evidence indicates that many TEX genes are integral to spermatogenesis and closely linked to male fertility. For instance, Tex11 which encodes a protein harboring tetratricopeptide repeat motifs exhibits 74% sequence identity between humans and mice. Studies using Tex11 -deficient mice have demonstrated its essentiality in spermatogenesis: loss of Tex11 impairs chromosomal synapsis, increases germ cell apoptosis, and ultimately causes male infertility [ 8 – 10 ]. Tex12 , a conserved vertebrate gene, is one of the meiosis-specific components of the synaptonemal complex (SC). Its deletion leads to failed chromosomal synapsis in males and ovarian follicle loss in females, underscoring its universal role in meiotic progression [ 11 , 12 ]. Tex13 is exclusively expressed in male germ cells; its nuclear localization and transcriptional repressor activity suggest a regulatory role in spermatogenesis, with expression tightly controlled at the translational level[ 13 ]. Tex14 encodes a testis-specific protein containing ankyrin repeats and protein kinase-C domains, which is critical for forming intercellular bridges during meiosis. Male mice lacking Tex14 are sterile, emphasizing its non-redundant function in spermatogenesis [ 14 – 16 ]. Tex15 , expressed in spermatogonia and early spermatocytes, is required for DNA double-strand break repair. Its interaction with the PIWI protein family further implies roles in epigenetic regulation and transposable element silencing[ 17 – 19 ]. Tex18 , a single-exon gene specifically expressed in male germ cells, is implicated in spermatid differentiation; Tex18-null mice display subfertility, which is attributed to abnormal sperm morphology and impaired sperm motility[ 20 ]. Tex19 , a mammal-specific gene, is essential for meiotic chromosome synapsis and genomic stability; its deletion in mice results in infertility, highlighting its importance in germ cell development [ 21 – 25 ]. Tex27 , which contains a zinc-finger domain, is hypothesized to function as a transcription factor in postmeiotic cells, potentially modulating germ cell maturation and regulatory networks[ 26 , 27 ]. In contrast, Tex33 , Tex36 , and Tex37 appear dispensable for spermatogenesis, as their respective knockout mice remain fertile[ 28 – 31 ]. Tex40 is a novel flagellar protein, and its depletion results in rigid sperm flagella and impaired male fertility[ 32 , 33 ]. Finally, Tex101 is critical for male fertility, and its deletion disrupts sperm physiology and motility[ 34 – 37 ]. Collectively, the TEX gene family comprises a diverse array of proteins that exert indispensable functions throughout spermatogenesis. Elucidating the roles of these genes is fundamental to deciphering the molecular basis of male fertility and infertility. In our clinical investigations, we identified Tex43 mutations in a cohort of infertile individuals, spanning both exonic and intronic regions, with the majority localized to introns. Notably, four patients harboring exonic Tex43 mutations exhibited reduced sperm motility and density upon semen analysis. Given the well-documented roles of other TEX family members in testicular function and our clinical observations of Tex43-mutated individuals, we sought to investigate whether Tex43 contributes to spermatogenesis and male reproductive fitness. To address this question, we generated a Tex43 knockout (KO) mouse model. Using this model, we aim to characterize the spatiotemporal expression pattern, subcellular localization, and functional mechanisms of TEX43 during spermatogenesis. This research will advance our understanding of germ cell developmental mechanisms and potentially deliver novel insights for the diagnosis and treatment of male infertility, paving the way for precision medicine in reproductive health. Materials and Methods Animals and Ethical Approval All experiments were approved by the Institutional Animal Welfare and Ethics Committee of Peking University (Approval No. : A2022156) and complied with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). C57BL/6N mice were housed in a specific pathogen-free (SPF) facility with ad libitum access to food and water. Generation of Tex43-KO Mice CRISPR/Cas9 targeting was designed to delete exons 1–3 of murine Tex43 (Ensembl ID: ENSMUSG00000049101), a region encompassing the microtubule-binding domain. Two guide RNAs (gRNAs) were synthesized: gRNA1 (5′-GACAAAGGAAAGCTACGTGGG-3′) and gRNA2 (5′-CGTAAGCAGCAGAACCGAGGG − 3′). Validation of gRNA efficiency was performed via in vitro cleavage assay. A mixture of gRNAs and Cas9 protein was microinjected into fertilized mice oocytes. Injected oocytes were transferred into the oviducts of pseudo-pregnant ICR females. Founder mice (F0) were genotyped via PCR (primers: forward 5′-TGGCTGAGTTCCTGTTTGTG-3′, reverse 5′-CAGGGCTTCTTTCTGCTTGT-3′) and Sanger sequencing. Homozygous Tex43-KO mice (Tex43⁻/⁻) were generated by intercrossing heterozygotes (Tex43⁺/⁻), with genotyping confirmed by PCR and western blotting (anti-TEX43 antibody, 1:1000; Bioss Antibodies, bs-15207R). Q-PCR for Tex43 Expression Tissue-Specific Expression Tissues (heart, brain, liver, spleen, lung, kidney, thymus, mammary gland, epididymis, skeletal muscle, testis) were collected from adult WT males (8 weeks old, n = 3). Testes were collected from WT mice at postnatal days (dpp) 0, 7, 14, 18, 21, 28, 35, 42, 49, and 56 (n = 3 per time point) to capture spermatogenic stages. RNA Extraction and Q-PCR Total RNA was extracted using TRIzol® reagent (Invitrogen, USA) per manufacturer’s protocol, with DNase I (Thermo Fisher, USA) treatment to eliminate genomic DNA contamination. cDNA synthesis was performed with 1µg total RNA using the PrimeScript™ RT Reagent Kit (Takara, Japan). Q-PCR was run on a StepOnePlus™ Real-Time PCR System (Thermo Fisher) with SYBR® Premix Ex Taq™ (Takara): 95°C for 30s, 40 cycles of 95°C for 5s, 60°C for 30s. Tex43 primers (forward 5′-GTTCGTGCCATTGGTTTGTC-3′, reverse 5′-GAGTGGGATTGAATGCAG AAG-3′) were validated via BLAST and melting curve analysis. Gapdh (forward 5′-AGGTCGGTGTGAACGGATTTG-3′, reverse 5′-TGTAGACCATGTAGTTGAGGTCA-3′) served as the internal control. Relative expression was calculated via the 2⁻ΔΔCt method. Immunohistochemistry (IHC) and Immunocytochemistry IHC on Tissue Sections Testes and epididymides from WT and Tex43-KO mice (8 weeks old, n = 3) were fixed in 4% paraformaldehyde (PFA) for 24h, embedded in paraffin, and sectioned. Deparaffinization was performed via xylene and graded ethanol. Antigen retrieval was done in 10 mM citrate buffer (pH 6.0) at 95°C for 20 min. Sections were blocked with 5% bovine serum albumin (BSA) for 1h at 37°C, then incubated with anti-TEX43 antibody (1:400, Bioss bs-15207R) overnight at 4°C. Secondary antibody (goat anti-rabbit IgG-HRP, 1:500; ZSGB-BIO, China) was applied for 1h at 37°C, with visualization via 3,3′-diaminobenzidine (DAB; ZSGB-BIO). Negative controls omitted primary antibody. Images were acquired via Nikon Eclipse 70i microscope. Immunocytochemistry on Sperm Cauda epididymides from WT/KO mice (n = 3) were dissected, minced in Human Tubal Fluid (HTF) medium, and incubated for 15 min to release sperm. Sperm were fixed in 4% PFA for 10 min, smeared on glass slides, and blocked with 5% BSA for 1h. Slides were incubated with anti-TEX43 (1:400) overnight at 4°C, followed by Alexa Fluor 555-conjugated secondary antibody (1:1000; Invitrogen) for 1h. PNA-FITC (1:200; Sigma-Aldrich) stained the acrosome, and DAPI (1:1000; Invitrogen) stained nuclei. Images were captured via Zeiss LSM 880 confocal microscope. Testicular Histology and TUNEL Assay H&E and PAS Staining Testes and epididymides from WT/KO mice (8 weeks old, n = 3) were fixed in Bouin’s solution (Sigma-Aldrich) for 24h, embedded in paraffin, and sectioned. H&E staining: Sections were stained with hematoxylin (Beyotime, China) for 2 min, differentiated in 1% HCl-ethanol (6 dips), and counterstained with eosin for 3s. Periodic acid-Schiff (PAS) staining: Sections were treated with 0.5% periodic acid for 5 min, incubated with Schiff’s reagent for 10 min (dark), and counterstained with hematoxylin. Images were acquired via Nikon Eclipse 70i microscope; ≥10 seminiferous tubules per mouse were analyzed for spermatogenic stage progression. TUNEL Assay Testicular sections were deparaffinized and treated with proteinase K for 20 min at 37°C. TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche) per protocol: incubation with TUNEL reaction mixture, DAPI counterstaining, and visualization via confocal microscopy. Apoptotic cells per seminiferous tubule were counted (≥ 10 tubules per mouse, n = 5 per group). Sperm Parameter Analysis Sperm Isolation Cauda epididymides from WT/KO mice were dissected, minced in pre-warmed HTF medium and incubated for 15 min to allow sperm release. Sperm suspensions were filtered through 70 µm cell strainers to remove tissue debris. Sperm Density and Morphology Sperm density was quantified using a hemocytometer under a light microscope. For morphology, 5 µl sperm suspension was smeared on slides, air-dried, fixed in 4% PFA, and stained with H&E. Two blinded observers counted ≥ 1000 sperm per mouse, categorizing morphology as normal or abnormal (head/neck/tail defects) per WHO guidelines. CASA for Sperm Motility A 5 µl sperm suspension was loaded onto a pre-warmed CASA counting slide. Motility parameters were analyzed via Sperm Class Analyzer v4.0.0 with a phase-contrast microscope (Olympus BX53) and high-speed camera (25 frames/s). Parameters measured: total motility (%), progressive motility (%), average path velocity (VAP, µm/s), straight-line velocity (VSL, µm/s), curvilinear velocity (VCL, µm/s), and beat-cross frequency (BCF, Hz). ≥5 fields and 200 sperm per sample were evaluated; each sample was tested in triplicate. Fertility Assays In Vivo Fertility WT (Tex43⁺/⁺) and KO (Tex43⁻/⁻) males were individually housed with two WT females for 3 months. Females were checked daily for vaginal plugs (mating marker). Upon plug detection, females were separated and monitored for pregnancy. Litter size (live pups per litter) and pregnancy rate were recorded. IVF Assay Superovulation: 6–8-week-old females were injected intraperitoneally with 5 IU pregnant mare serum gonadotropin (PMSG; Ningbo Second Hormone Factory, China), followed by 5 IU human chorionic gonadotropin (hCG) 48h later. In Vivo Fertilization Validation: Superovulated females were mated with WT/KO males (1:1). Zygotes were collected from oviducts 12–16h post-mating, washed in G1-plus medium (Vitrolife, Sweden), and cultured (37°C, 5% CO₂). Fertilization rate = (2-cell embryos / collected zygotes) × 100%. In Vitro Fertilization: Females were euthanized 12h post-hCG; oocyte-corona cumulus complexes (COCs) were collected from oviducts and placed in 200 µl HTF drops (pre-equilibrated 37°C, 5% CO₂). Sperm from WT/KO males were capacitated in HTF for 1h; 15 µl sperm suspension (1×10⁶ sperm/ml) was added to COCs. After 4h co-incubation, oocytes were washed in G1-plus and cultured. IVF rate = (2-cell embryos / inseminated oocytes) × 100%. Embryonic development (4-cell, morula, blastocyst) was monitored daily for 3 days. SEM and TEM for Sperm Ultrastructure SEM Preparation Sperm from WT/KO mice (n = 3) were washed in PBS, fixed in 2.5% glutaraldehyde (0.1 M cacodylate buffer, pH 7.4) for 2h at 4°C, and washed 3× with cacodylate buffer. Samples were dehydrated via graded ethanol (30%–100%, 15 min each), critical-point dried with liquid CO₂ (Leica EM CPD300), mounted on aluminum stubs, and sputter-coated with gold-palladium (Leica EM ACE600). Images were acquired via JEOL JSM-7800F SEM. TEM Preparation Fixed sperm were post-fixed in 1% osmium tetroxide for 1.5h at room temperature, washed 3× with distilled water, and dehydrated via ethanol and propylene oxide. Samples were infiltrated with epoxy resin and polymerized. Ultrathin sections were cut via Leica EM UC7 ultramicrotome, collected on copper grids, and stained with 2% uranyl acetate and lead citrate. Images were acquired via JEOL JEM-1400 TEM. For microtubule disorganization quantification, ≥ 50 flagellar end pieces per group were analyzed. Human Subjects and WES Study Cohort 146 Chinese men with asthenoteratozoospermia were recruited from the Center for Reproductive Medicine, Peking University Third Hospital (2019–2024). Inclusion criteria: no history of endocrine disorders, genital tract infection, or chemotherapy/radiotherapy. Exclusion criteria: Y-chromosome microdeletions or AZF gene mutations. All participants provided written informed consent; the study was approved by the Ethics Committee of Peking University Third Hospital (Approval No.: 2021SZ-003) and complied with the Declaration of Helsinki. WES and Variant Analysis Genomic DNA was extracted from peripheral blood leukocytes using the QIAamp DNA Blood Mini Kit. WES libraries were prepared with the Agilent SureSelect Human All Exon V6 capture system, targeting ~ 60 Mb of coding regions. Sequencing was performed on the Illumina NovaSeq 6000 platform, achieving ≥ 100× average coverage of target regions. Raw reads were filtered via Trimmomatic (v0.39), aligned to the human reference genome (GRCh38) via BWA-MEM (v0.7.17), and variant calling was done via GATK (v4.2.6). Variants were annotated using ANNOVAR; filtering criteria: minor allele frequency (MAF) < 0.01 in gnomAD, ExAC, and 1000 Genomes Project databases; missense variants with CADD score ≥ 20; splice-site variants within ± 20 bp of exons. Sanger sequencing validated TEX43 variants in patients and their family members. Structural Modeling of TEX43 3D models of WT and mutant (p.R37Q) TEX43 (human: NP_997291.1; mouse: NP_080375.2) were generated via SWISS-MODEL ( https://swissmodel.expasy.org/ ) using PDB 7ZXY (68% sequence identity to human TEX43) as the template. Models were refined via molecular dynamics simulations (GROMACS v2021.4) for 10 ns. Structural analysis (residue interactions, root-mean-square deviation [RMSD]) was performed using PyMOL (v2.5; Schrödinger, USA). Hydrogen bonds were quantified via HBPLUS (v3.1); salt bridges were identified using PyMOL’s "find salt bridges" function. Statistical Analysis Data are presented as mean ± standard error of the mean (SEM). Comparisons between two groups (WT vs. KO) were made using unpaired two-tailed Student’s t-test (continuous variables: litter size, sperm density, motility) or chi-square test (categorical variables: pregnancy rate, fertilization rate). One-way ANOVA was used for multi-group comparisons (WT vs. HET vs. KO). Statistical significance was defined as P < 0.05, P < 0.01, P < 0.001, P 0.05). All analyses were performed using GraphPad Prism (v9.0; GraphPad Software, USA). Results Tex43 Is a Testis-Enriched Gene Expressed in Maturing Spermatids Q-PCR showed Tex43 mRNA was exclusively expressed in murine testes,no detectable expression was observed in 10 other somatic tissues (Fig. 1 A). During postnatal testicular development, Tex43 mRNA was first detected at 18 dpp (coinciding with the emergence of round spermatids), with expression increasing progressively and peaking at 56 dpp (sexual maturity in male mice; Fig. 1 B). Amino acid sequence alignment of TEX43 orthologs (mouse, human, rhesus macaque) revealed high conservation: 93% identity between human and rhesus macaque, 72% between mouse and human (Fig. 1 C). IHC localized TEX43 protein to the cytoplasm of round and elongating spermatids in WT testes (adluminal compartment of seminiferous tubules); no staining was observed in spermatogonia, spermatocytes, or somatic cells (Fig. 2 A–C), consistent with Q-PCR’s post-meiotic expression pattern. In the epididymis, TEX43 was restricted to the flagellar region of maturing spermatids in the lumen, with no staining in epididymal epithelial cells (Fig. 2 D–F). Immunocytochemistry confirmed TEX43 co-localization with sperm flagella (Fig. 1 ). Successful Generation of Tex43-KO Mice CRISPR/Cas9-mediated deletion of exons 1–3 of Tex43 was confirmed by genotyping PCR: WT mice showed a single ~ 400 bp band, heterozygotes (Tex43⁺/⁻) showed two bands (~ 400 bp and ~ 200 bp), and homozygotes (Tex43⁻/⁻) showed a single ~ 200 bp band (Fig. 3 A, B). Sanger sequencing verified the deletion breakpoint (Fig. 2 ). Western blotting and IHC confirmed complete loss of TEX43 protein in KO testes and sperm (Fig. 3 C, D); WT testes showed robust TEX43 expression in spermatids. Tex43-KO mice were viable and fertile, with no overt developmental abnormalities (body weight, general health) up to 6 months of age, distinguishing them from KO models of other TEX genes (e.g., Tex11, Tex14) that cause sterility. Tex43 Deficiency Does Not Disrupt Spermatogenesis or Sperm Morphology Testicular weight and testis-to-body weight ratio were slightly reduced in Tex43-KO mice (KO: 0.45 ± 0.02% vs. WT: 0.52 ± 0.03%; P < 0.05) but remained within the physiological range (Fig. 4 A, C). H&E and PAS staining showed Tex43-KO testes had intact seminiferous tubule architecture** and all spermatogenic stages (spermatogonia to mature spermatids); epididymal lumens contained abundant mature sperm, confirming unimpaired spermiation (Fig. 4 G). TUNEL assays revealed no significant difference in germ cell apoptosis between KO and WT mice (KO: 0.91 ± 0.18 vs. WT: 0.82 ± 0.15 apoptotic cells per tubule; P > 0.05; Fig. 4 D). Sperm morphology analysis via H&E staining showed no significant difference in the proportion of normal sperm between groups (KO: 86.2 ± 3.1% vs. WT: 88.5 ± 2.7%; P > 0.05; Fig. 4 B). Abnormalities (head defects, neck/midpiece defects, tail defects) were rare and comparable to WT, indicating Tex43 deficiency does not impair sperm morphogenesis. Tex43 Deficiency Reduces Sperm Density but Preserves Motility Sperm density was significantly lower in Tex43-KO mice (28.6 ± 3.2×10⁶ sperm/ml) compared to WT (41.2 ± 2.9×10⁶ sperm/ml; P 10×10⁶ sperm/ml), and no difference was observed between heterozygotes and KO mice, suggesting a recessive effect of Tex43 deletion. CASA revealed no significant differences in any motility parameter between WT and KO sperm: total motility (KO: 62.3 ± 4.2% vs. WT: 65.7 ± 3.8%), progressive motility (KO: 41.5 ± 3.5% vs. WT: 43.2 ± 3.1%), VAP (KO: 68.2 ± 4.1 µm/s vs. WT: 70.5 ± 3.9 µm/s), VSL (KO: 45.3 ± 3.2 µm/s vs. WT: 47.1 ± 2.8 µm/s), VCL (KO: 98.6 ± 5.3 µm/s vs. WT: 101.2 ± 4.9 µm/s), and BCF (KO: 14.2 ± 1.1 Hz vs. WT: 14.8 ± 0.9 Hz; all P > 0.05; Fig. 4 F). This indicates Tex43 deficiency does not compromise sperm motility, despite its localization to flagellar microtubules. Tex43-KO Mice Exhibit Normal Fertility and Embryonic Development In vivo breeding assays showed Tex43-KO males sired litters with sizes (6.8 ± 0.7 pups/litter) comparable to WT (7.2 ± 0.5 pups/litter; P > 0.05); pregnancy rates were also similar (KO: 82.3% vs. WT: 85.7%; P > 0.05; Fig. 5 A). IVF assays confirmed no significant difference in fertilization rate (KO: 61.9 ± 5.2% vs. WT: 66.7 ± 4.8%; P > 0.05) or embryonic development to the blastocyst stage (KO: 45.1 ± 3.9% vs. WT: 47.9 ± 3.8%; P > 0.05; Fig. 5 B). These results demonstrate Tex43 deficiency does not impair male fertility or early embryonic development—aligning with the lack of motility defects. Tex43 Deficiency Causes Subtle Flagellar Ultrastructural Defects SEM showed Tex43-KO sperm had normal head morphology (regular oval shape, intact acrosome) and flagellar middle piece structure (tightly packed mitochondrial sheath; Fig. 6 A, B). The flagellar principal piece retained the canonical "9 + 2" microtubule configuration in most KO sperm, though ~ 15% showed partial loss of outer dense fibers (ODFs),a mild defect not observed in WT (Fig. 6 C, D). TEM revealed the most prominent defect: increased disorganization of the "9 + 2" microtubule array in the flagellar end piece of Tex43-KO sperm (~ 30% of KO sperm vs. ~5% of WT; P < 0.01; Fig. 6 E–G). Disorganization included splayed microtubule doublets and loss of the outer junction (OJ) between A- and B-tubules,critical for axonemal stability. Notably, this defect was restricted to the end piece (a short, terminal segment of the flagellum) and did not affect the principal piece or middle piece,explaining the preserved sperm motility. TEX43 Variants in Men with Asthenoteratozoospermia WES identified 9 infertile men with TEX43 variants (Table 1 ), including 2 intronic variants (intron1:c.86 + 141G > A, intron2:c.196-25C > T) and 2 exonic variants (exon1:c.1-23T > A, exon2:c.110G > A:p.R37Q). The intronic variant pair (c.86 + 141G > A/c.196-25C > T) was co-carried by 8 patients, suggesting a potential founder effect in the Chinese population. Five patients with exonic variants underwent ICSI: 4 achieved live births, and 1 had a biochemical pregnancy (Table 1 ). No TEX43 variants were identified in 50 fertile male controls. Structural modeling of the p.R37Q variant (exon2:c.110G > A) showed the arginine-to-glutamine substitution disrupted two key interactions: (1) loss of a salt bridge between R37 and E62 (a negatively charged residue in the microtubule-binding domain), and (2) reduction in hydrogen bonds from 2 (WT) to 1 (mutant; Fig. 7 A, B). This conformational shift (RMSD: 1.8 Å vs. WT) is predicted to weaken TEX43’s binding to microtubules, consistent with the flagellar defects observed in KO mice. Table 1 Semen Characteristics and Clinical Outcomes of Patients with TEX43 Variants Patient No. cDNA Change Semen Volume (ml) Seminal PH Concentration (10⁶/ml) Total Motility (%) Progressive Motility (%) Insemination Clinical Outcome 1 intron1:c.86 + 141G > A, intron2:c.196-25C > T, exon1:c.1-23T > A 2.2 7.5 17.36 23.78 7.86 ICSI Live Birth 2 intron1:c.86 + 141G > A, intron2:c.196-25C > T, exon1:c.1-23T > A 1.5 7.5 5.13 34.62 28.85 ICSI Live Birth 3 exon1:c.1-23T > A 3.2 7.5 2.25 11.76 5.88 ICSI Live Birth 4 intron1:c.86 + 141G > A, exon1:c.1-23T > A 2.6 7.5 8.79 30.34 15.73 ICSI Biochemical Pregnancy 5 exon2:c.110G > A:p.R37Q 3.8 7.2 36.36 40.94 35.63 ICSI Live Birth Discussion This study delineates TEX43’s role in mammalian reproduction, with three key findings: (1) TEX43 is a testis-enriched, evolutionarily conserved gene expressed in maturing spermatids; (2) Tex43 deletion in mice causes mild flagellar ultrastructural defects and reduced sperm density but preserves fertility; (3) human TEX43 variants are associated with asthenoteratozoospermia but do not prevent successful pregnancy via ICSI. These results align with the Journal of Assisted Reproduction and Genetics’ focus on bridging basic reproductive biology with clinical practice. TEX43’s Role in Spermatogenesis: Redundancy Explains Lack of Overt Phenotype TEX43’s localization to sperm flagellar microtubules and stage-specific expression initially suggested a critical role in spermiogenesis (e.g., flagellar assembly). However, Tex43-KO mice showed only subtle defects, consistent with genetic redundancy, a hallmark of male reproductive biology[ 4 , 29 , 38 ]. Redundancy may involve: TEX family paralogs: TEX40 and TEX44 share 35–40% sequence identity with TEX43 and are expressed in spermatids; their upregulation in KO testes could compensate for TEX43 loss. Microtubule-associated proteins: CFAP77 and CCDC105 form a ternary complex with TEX43 at the flagellar OJ [ 39 ]; CFAP77-KO mice have severe axonemal defects and sterility, but CCDC105-KO mice are fertile, suggesting TEX43 and CCDC105 are redundant [ 40 ]. Our TEM data support this: Tex43-KO sperm had OJ defects restricted to the end piece, while CFAP77-KO sperm have widespread OJ disruption [ 39 ]. Clinical Relevance: TEX43 Variants as Subtle Fertility Modifiers The identification of TEX43 variants in men with asthenoteratozoospermia, including the functionally relevant p.R37Q variant, links TEX43 to human infertility. Notably, all patients with exonic variants achieved pregnancy via ICSI, mirroring the murine data (preserved fertilization capacity). This suggests TEX43 variants are subtle fertility modifiers rather than major pathogenic factors, consistent with the mild phenotype of KO mice. Limitations of the clinical analysis include: (1) small cohort size (9 variant carriers); (2) lack of functional validation for intronic variants (their impact on splicing is unknown); (3) absence of family segregation data to confirm variant inheritance. Future studies should enroll larger cohorts (stratified by infertility subtype) and perform minigene assays to test intronic variant effects on TEX43 expression. For clinical practice, TEX43 variants do not preclude successful ICSI, reassuring for couples with these variants. For basic research, Tex43-KO mice provide a model to study genetic redundancy in flagellar biology; future work could use RNA-seq/proteomics to identify compensatory genes in KO testes. Conclusions TEX43 is a testis-enriched, microtubule-localized gene with conserved expression across mammals. Its deletion in mice causes mild sperm density reduction and flagellar end piece defects but preserves fertility, likely due to genetic redundancy. Human TEX43 variants are associated with asthenoteratozoospermia but do not prevent ICSI success. These findings advance understanding of testis-specific gene function and highlight the importance of functional validation in interpreting genetic variants in infertility. Declarations Author Contribution Q. C performed experiments. L.L wrote the manuscript. Y. L designed the experiments and generated the CRISPR mice.X.Q performed on the sample collection and clinical case analysis. P.L and R.L provided valuable advice for the manuscript. X. Z and X.Z supervised the study and reviewed the manuscript. X.Z and X.Z are the correspondence authors of this research.All authors reviewed the manuscript. References Jiao SY, Yang YH, Chen SR. Molecular genetics of infertility: loss-of-function mutations in humans and corresponding knockout/mutated mice. Hum Reprod Update. 2021;27(1):154–89. 10.1093/humupd/dmaa034 . PubMed PMID: 33118031. Fainberg J, Kashanian JA, F1000Res. Recent advances in understanding and managing male infertility. 2019;8. Epub 20190516. 10.12688/f1000research.17076.1 . PubMed PMID: 31143441; PubMed Central PMCID: PMCPMC6524745. O'Flynn O'Brien KL, Varghese AC, Agarwal A. The genetic causes of male factor infertility: a review. 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Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 20 Dec, 2025 Reviews received at journal 12 Dec, 2025 Reviews received at journal 11 Dec, 2025 Reviewers agreed at journal 04 Dec, 2025 Reviewers agreed at journal 03 Dec, 2025 Reviewers invited by journal 03 Dec, 2025 Editor assigned by journal 27 Nov, 2025 Submission checks completed at journal 25 Nov, 2025 First submitted to journal 22 Nov, 2025 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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09:33:13","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":119543,"visible":true,"origin":"","legend":"","description":"","filename":"37b033b19c004edfafe0d68462d21fd01structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8181104/v1/354bf18220b827afc577e012.xml"},{"id":97669769,"identity":"c298ef87-cf12-4b21-aab9-0c0946ccc2a0","added_by":"auto","created_at":"2025-12-08 09:28:51","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":129210,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8181104/v1/245c421d8721181b701fcc49.html"},{"id":97512746,"identity":"c3752888-6a1c-4aa4-a36e-5565731cd291","added_by":"auto","created_at":"2025-12-05 09:30:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":221131,"visible":true,"origin":"","legend":"\u003cp\u003eExpression Pattern of Tex43 and Cross-Species Alignment\u003c/p\u003e\n\u003cp\u003e(A) Tissue-specific Tex43 mRNA expression (Q-PCR): Tex43 is exclusively expressed in testes (n=3 per tissue). (B) Temporal Tex43 expression during postnatal testicular development: Expression initiates at 18 dpp (round spermatids) and peaks at 56 dpp (sexual maturity; n=3 per time point). (C) Amino acid sequence alignment of TEX43 orthologs (mouse, human, rhesus macaque): Residues color-coded by biochemical properties (green: polar; red: acidic; blue: basic; pink: hydrophobic); asterisks denote fully conserved residues. Scale bars: (A, B) Mean ± SEM; *P\u0026lt;0.05, **P\u0026lt;0.01 vs. dpp 0.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8181104/v1/bbfc30f729a1f7c52b030129.png"},{"id":97672015,"identity":"b6189533-61f9-4c13-b79b-d2329c854c26","added_by":"auto","created_at":"2025-12-08 09:33:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":828407,"visible":true,"origin":"","legend":"\u003cp\u003eIHC Localization of TEX43 in Murine Testis and Epididymis\u003c/p\u003e\n\u003cp\u003e(A) Panoramic view of testis: TEX43 (brown) localized to adluminal spermatids. (B) Low-magnification testis: TEX43 restricted to round/elongating spermatids (arrowheads). (C) High-magnification testis: TEX43 in spermatid cytoplasm (arrowheads). (D) Panoramic view of epididymis: TEX43 in lumenal spermatids. (E) Low-magnification epididymis: TEX43 in flagellar region (arrowheads). (F) High-magnification epididymis: TEX43 in spermatid flagella (arrowheads). Negative controls (insets) show no staining. Scale bars: (A, D) 100 μm; (B, E) 50 μm; (C, F) 20 μm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8181104/v1/56be2a33ba597a53cb5650b6.png"},{"id":97512747,"identity":"b4a777aa-1040-4b7c-b1e2-b17394b6cf2b","added_by":"auto","created_at":"2025-12-05 09:30:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":576948,"visible":true,"origin":"","legend":"\u003cp\u003eGeneration and Validation of Tex43-KO Mice\u003c/p\u003e\n\u003cp\u003e(A) CRISPR/Cas9 targeting strategy: gRNAs flank exons 1–3 of Tex43; deletion removes the microtubule-binding domain. (B) Genotyping PCR: WT (Tex43⁺/⁺) = ~400 bp, heterozygote (Tex43⁺/⁻) = ~400/+200 bp, KO (Tex43⁻/⁻) = ~200 bp. (C) IHC of WT testis: Robust TEX43 expression in spermatids (arrowheads). (D) IHC of KO testis: No detectable TEX43 staining. Scale bars: (C, D) 50 μm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8181104/v1/6c3726fffa3857f13c711950.png"},{"id":97512754,"identity":"d364f51b-67c0-45a0-b8b1-93083ce431cd","added_by":"auto","created_at":"2025-12-05 09:30:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1122109,"visible":true,"origin":"","legend":"\u003cp\u003eTesticular Phenotype and Sperm Parameters in WT and Tex43-KO Mice\u003c/p\u003e\n\u003cp\u003e(A) Testicular size: Representative images of testes from 8-week-old WT and KO mice (n=3 per group). (B) Sperm morphology: H\u0026amp;E staining shows no difference in normal sperm proportion (n=3 per group). (C) Testis-to-body weight ratio: Slightly reduced in KO (0.45±0.02% vs. WT 0.52±0.03%; n=5 per group). (D) TUNEL assay: No difference in apoptotic germ cells per tubule (n=5 per group). (E) Sperm density: Significantly reduced in KO (28.6±3.2×10⁶ vs. WT 41.2±2.9×10⁶ sperm/ml; n=3 per group). (F) CASA motility parameters: No differences in total motility, progressive motility, or velocity metrics (n=3 per group). (G) H\u0026amp;E staining: WT and KO testes have intact seminiferous tubules (top) and epididymal lumens with mature sperm (bottom). Scale bars: (A) 5 mm; (B, G) 50 μm; (C–F) Mean ± SEM; *P\u0026lt;0.05, **P\u0026lt;0.01, ns: non-significant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8181104/v1/e8afc4ec7a1f811d2a67de00.png"},{"id":97512753,"identity":"00a9b3f1-4000-4a8f-ac77-ba85447061ca","added_by":"auto","created_at":"2025-12-05 09:30:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":652640,"visible":true,"origin":"","legend":"\u003cp\u003eFertility and Embryonic Development Outcomes\u003c/p\u003e\n\u003cp\u003e(A) In vivo fertility: Litter size (KO 6.8±0.7 vs. WT 7.2±0.5 pups/litter) and pregnancy rate (KO 82.3% vs. WT 85.7%) are comparable (n=5 males per group, 2 females per male). (B) IVF embryonic development: No significant differences in 2-cell, 4–8-cell, morula, or blastocyst rates (n=3 replicates, ≥50 oocytes per replicate). Scale bars: (A, B) Mean ± SEM; ns: non-significant.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8181104/v1/91aab933cee3c8a45b103621.png"},{"id":97670683,"identity":"9273b33b-368d-4945-a5ee-956f659ada9a","added_by":"auto","created_at":"2025-12-08 09:31:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":657571,"visible":true,"origin":"","legend":"\u003cp\u003eSperm Ultrastructural Analysis (SEM/TEM)\u003c/p\u003e\n\u003cp\u003e(A) SEM of sperm head: KO sperm have normal oval shape (arrow) and acrosome (arrowhead). (B) SEM of middle piece: KO sperm have intact mitochondrial sheath (arrow). (C) TEM of principal piece: WT sperm show canonical \"9+2\" microtubules (arrow) and ODFs (arrowhead). (D) TEM of principal piece (KO): ~15% of KO sperm show partial ODF loss (arrowhead). (E) TEM of end piece (WT): Orderly \"9+2\" array (arrow). (F) TEM of end piece (KO): Disorganized microtubules (arrow) and OJ loss (arrowhead). (G) Quantification of end piece disorganization: ~30% of KO sperm affected (n=50 end pieces per group). Scale bars: (A) 2 μm; (B) 500 nm; (C–F) 100 nm; (G) Mean ± SEM; **P\u0026lt;0.01 vs. WT.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8181104/v1/c70247bf978e84ea2a01d71d.png"},{"id":97671206,"identity":"6cb35c62-be6c-48f4-9aaf-baf31cef9178","added_by":"auto","created_at":"2025-12-08 09:32:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":181234,"visible":true,"origin":"","legend":"\u003cp\u003eStructural Models of WT and Mutant TEX43\u003c/p\u003e\n\u003cp\u003e(A) WT TEX43: 3D model shows R37 (blue) forms a salt bridge with E62 (red) and hydrogen bonds with L35/L89 (green)—critical for microtubule binding. (B) p.R37Q mutant: Q37 (orange) loses the salt bridge with E62 and one hydrogen bond, causing a conformational shift (RMSD = 1.8 Å vs. WT). Dashed lines denote interactions; insets show close-up of binding interface.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8181104/v1/4b10eed74da0acb46324a7d6.png"},{"id":97892949,"identity":"ea5a2f9d-1073-45ce-b095-65e045e6a960","added_by":"auto","created_at":"2025-12-10 15:24:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5200742,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8181104/v1/a06b301f-52fa-4526-a30b-bb7d8bf369d1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"TEX43, a Testis-Enriched Microtubule-Associated Gene, Exerts Minimal Impact on Spermatogenesis and Fertility in Mice and Humans","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMale infertility represents a prominent challenge in men\u0026rsquo;s reproductive health globally. According to the World Health Organization (WHO), approximately 15% of reproductive-age couples worldwide face infertility issues, with male factors accounting for nearly 50% of these cases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The etiology of male infertility is complex and multifactorial, and genetic perturbations that reduce germ cell quantity or impair sperm quality are well-recognized key contributors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].Notably, the incidence of male infertility has continued to rise alongside societal and environmental changes, underscoring the urgency of clarifying its underlying mechanisms.\u003c/p\u003e\u003cp\u003eSpermatogenesis is a highly orchestrated process. This complex cascade involves the coordinated regulation of over 2,300 genes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].At the molecular level, sperm formation and maturation are driven by the programmed expression of cell- and stage-specific genes. The ordered transcription of testis-enriched genes, alongside the synergistic interactions of transcription factors, metabolic enzymes, cell cycle regulators, forms the molecular backbone of spermatogenesis. Disruption of critical genes or proteins can arrest or perturb spermatogenic cell development, leading to the production of morphologically abnormal, immotile, or non-functional sperm, ultimately resulting in fertilization failure and male infertility [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn male reproductive biology, the testis-specific gene (TEX) family plays a pivotal role in regulating spermatogenesis and sustaining male fertility. The term \"TEX gene\" was first coined by Wang et al. in 2001, subsequent to their identification of novel spermatogenic cell-specific transcripts in mice using the technique of cDNA suppression subtractive hybridization (SSH) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Subsequently, orthologs of these TEX genes have been identified in a wide range of organisms, spanning vertebrates, invertebrates and yeast. Accumulating evidence indicates that many TEX genes are integral to spermatogenesis and closely linked to male fertility.\u003c/p\u003e\u003cp\u003eFor instance, \u003cem\u003eTex11\u003c/em\u003e which encodes a protein harboring tetratricopeptide repeat motifs exhibits 74% sequence identity between humans and mice. Studies using \u003cem\u003eTex11\u003c/em\u003e-deficient mice have demonstrated its essentiality in spermatogenesis: loss of \u003cem\u003eTex11\u003c/em\u003e impairs chromosomal synapsis, increases germ cell apoptosis, and ultimately causes male infertility [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. \u003cem\u003eTex12\u003c/em\u003e, a conserved vertebrate gene, is one of the meiosis-specific components of the synaptonemal complex (SC). Its deletion leads to failed chromosomal synapsis in males and ovarian follicle loss in females, underscoring its universal role in meiotic progression [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. \u003cem\u003eTex13\u003c/em\u003e is exclusively expressed in male germ cells; its nuclear localization and transcriptional repressor activity suggest a regulatory role in spermatogenesis, with expression tightly controlled at the translational level[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eTex14\u003c/em\u003e encodes a testis-specific protein containing ankyrin repeats and protein kinase-C domains, which is critical for forming intercellular bridges during meiosis. Male mice lacking \u003cem\u003eTex14\u003c/em\u003e are sterile, emphasizing its non-redundant function in spermatogenesis [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. \u003cem\u003eTex15\u003c/em\u003e, expressed in spermatogonia and early spermatocytes, is required for DNA double-strand break repair. Its interaction with the PIWI protein family further implies roles in epigenetic regulation and transposable element silencing[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. \u003cem\u003eTex18\u003c/em\u003e, a single-exon gene specifically expressed in male germ cells, is implicated in spermatid differentiation; Tex18-null mice display subfertility, which is attributed to abnormal sperm morphology and impaired sperm motility[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eTex19\u003c/em\u003e, a mammal-specific gene, is essential for meiotic chromosome synapsis and genomic stability; its deletion in mice results in infertility, highlighting its importance in germ cell development [\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. \u003cem\u003eTex27\u003c/em\u003e, which contains a zinc-finger domain, is hypothesized to function as a transcription factor in postmeiotic cells, potentially modulating germ cell maturation and regulatory networks[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In contrast, \u003cem\u003eTex33\u003c/em\u003e, \u003cem\u003eTex36\u003c/em\u003e, and \u003cem\u003eTex37\u003c/em\u003e appear dispensable for spermatogenesis, as their respective knockout mice remain fertile[\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Tex40 is a novel flagellar protein, and its depletion results in rigid sperm flagella and impaired male fertility[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Finally, \u003cem\u003eTex101\u003c/em\u003e is critical for male fertility, and its deletion disrupts sperm physiology and motility[\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCollectively, the TEX gene family comprises a diverse array of proteins that exert indispensable functions throughout spermatogenesis. Elucidating the roles of these genes is fundamental to deciphering the molecular basis of male fertility and infertility.\u003c/p\u003e\u003cp\u003eIn our clinical investigations, we identified Tex43 mutations in a cohort of infertile individuals, spanning both exonic and intronic regions, with the majority localized to introns. Notably, four patients harboring exonic Tex43 mutations exhibited reduced sperm motility and density upon semen analysis. Given the well-documented roles of other TEX family members in testicular function and our clinical observations of Tex43-mutated individuals, we sought to investigate whether Tex43 contributes to spermatogenesis and male reproductive fitness. To address this question, we generated a Tex43 knockout (KO) mouse model. Using this model, we aim to characterize the spatiotemporal expression pattern, subcellular localization, and functional mechanisms of TEX43 during spermatogenesis. This research will advance our understanding of germ cell developmental mechanisms and potentially deliver novel insights for the diagnosis and treatment of male infertility, paving the way for precision medicine in reproductive health.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals and Ethical Approval\u003c/h2\u003e\u003cp\u003e All experiments were approved by the Institutional Animal Welfare and Ethics Committee of Peking University (Approval No. : A2022156) and complied with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). C57BL/6N mice were housed in a specific pathogen-free (SPF) facility with ad libitum access to food and water.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGeneration of Tex43-KO Mice\u003c/h3\u003e\n\u003cp\u003eCRISPR/Cas9 targeting was designed to delete exons 1\u0026ndash;3 of murine Tex43 (Ensembl ID: ENSMUSG00000049101), a region encompassing the microtubule-binding domain. Two guide RNAs (gRNAs) were synthesized: gRNA1 (5\u0026prime;-GACAAAGGAAAGCTACGTGGG-3\u0026prime;) and gRNA2 (5\u0026prime;-CGTAAGCAGCAGAACCGAGGG \u0026minus;\u0026thinsp;3\u0026prime;). Validation of gRNA efficiency was performed via in vitro cleavage assay. A mixture of gRNAs and Cas9 protein was microinjected into fertilized mice oocytes. Injected oocytes were transferred into the oviducts of pseudo-pregnant ICR females. Founder mice (F0) were genotyped via PCR (primers: forward 5\u0026prime;-TGGCTGAGTTCCTGTTTGTG-3\u0026prime;, reverse 5\u0026prime;-CAGGGCTTCTTTCTGCTTGT-3\u0026prime;) and Sanger sequencing. Homozygous Tex43-KO mice (Tex43⁻/⁻) were generated by intercrossing heterozygotes (Tex43⁺/⁻), with genotyping confirmed by PCR and western blotting (anti-TEX43 antibody, 1:1000; Bioss Antibodies, bs-15207R).\u003c/p\u003e\n\u003ch3\u003eQ-PCR for Tex43 Expression\u003c/h3\u003e\n\u003cp\u003eTissue-Specific Expression\u003c/p\u003e\u003cp\u003eTissues (heart, brain, liver, spleen, lung, kidney, thymus, mammary gland, epididymis, skeletal muscle, testis) were collected from adult WT males (8 weeks old, n\u0026thinsp;=\u0026thinsp;3). Testes were collected from WT mice at postnatal days (dpp) 0, 7, 14, 18, 21, 28, 35, 42, 49, and 56 (n\u0026thinsp;=\u0026thinsp;3 per time point) to capture spermatogenic stages.\u003c/p\u003e\u003cp\u003eRNA Extraction and Q-PCR\u003c/p\u003e\u003cp\u003eTotal RNA was extracted using TRIzol\u0026reg; reagent (Invitrogen, USA) per manufacturer\u0026rsquo;s protocol, with DNase I (Thermo Fisher, USA) treatment to eliminate genomic DNA contamination. cDNA synthesis was performed with 1\u0026micro;g total RNA using the PrimeScript\u0026trade; RT Reagent Kit (Takara, Japan). Q-PCR was run on a StepOnePlus\u0026trade; Real-Time PCR System (Thermo Fisher) with SYBR\u0026reg; Premix Ex Taq\u0026trade; (Takara): 95\u0026deg;C for 30s, 40 cycles of 95\u0026deg;C for 5s, 60\u0026deg;C for 30s. Tex43 primers (forward 5\u0026prime;-GTTCGTGCCATTGGTTTGTC-3\u0026prime;, reverse 5\u0026prime;-GAGTGGGATTGAATGCAG AAG-3\u0026prime;) were validated via BLAST and melting curve analysis. Gapdh (forward 5\u0026prime;-AGGTCGGTGTGAACGGATTTG-3\u0026prime;, reverse 5\u0026prime;-TGTAGACCATGTAGTTGAGGTCA-3\u0026prime;) served as the internal control. Relative expression was calculated via the 2⁻ΔΔCt method.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry (IHC) and Immunocytochemistry\u003c/h3\u003e\n\u003cp\u003eIHC on Tissue Sections\u003c/p\u003e\u003cp\u003eTestes and epididymides from WT and Tex43-KO mice (8 weeks old, n\u0026thinsp;=\u0026thinsp;3) were fixed in 4% paraformaldehyde (PFA) for 24h, embedded in paraffin, and sectioned. Deparaffinization was performed via xylene and graded ethanol. Antigen retrieval was done in 10 mM citrate buffer (pH 6.0) at 95\u0026deg;C for 20 min. Sections were blocked with 5% bovine serum albumin (BSA) for 1h at 37\u0026deg;C, then incubated with anti-TEX43 antibody (1:400, Bioss bs-15207R) overnight at 4\u0026deg;C. Secondary antibody (goat anti-rabbit IgG-HRP, 1:500; ZSGB-BIO, China) was applied for 1h at 37\u0026deg;C, with visualization via 3,3\u0026prime;-diaminobenzidine (DAB; ZSGB-BIO). Negative controls omitted primary antibody. Images were acquired via Nikon Eclipse 70i microscope.\u003c/p\u003e\n\u003ch3\u003eImmunocytochemistry on Sperm\u003c/h3\u003e\n\u003cp\u003eCauda epididymides from WT/KO mice (n\u0026thinsp;=\u0026thinsp;3) were dissected, minced in Human Tubal Fluid (HTF) medium, and incubated for 15 min to release sperm. Sperm were fixed in 4% PFA for 10 min, smeared on glass slides, and blocked with 5% BSA for 1h. Slides were incubated with anti-TEX43 (1:400) overnight at 4\u0026deg;C, followed by Alexa Fluor 555-conjugated secondary antibody (1:1000; Invitrogen) for 1h. PNA-FITC (1:200; Sigma-Aldrich) stained the acrosome, and DAPI (1:1000; Invitrogen) stained nuclei. Images were captured via Zeiss LSM 880 confocal microscope.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eTesticular Histology and TUNEL Assay\u003c/h2\u003e\u003cp\u003eH\u0026amp;E and PAS Staining\u003c/p\u003e\u003cp\u003eTestes and epididymides from WT/KO mice (8 weeks old, n\u0026thinsp;=\u0026thinsp;3) were fixed in Bouin\u0026rsquo;s solution (Sigma-Aldrich) for 24h, embedded in paraffin, and sectioned. H\u0026amp;E staining: Sections were stained with hematoxylin (Beyotime, China) for 2 min, differentiated in 1% HCl-ethanol (6 dips), and counterstained with eosin for 3s. Periodic acid-Schiff (PAS) staining: Sections were treated with 0.5% periodic acid for 5 min, incubated with Schiff\u0026rsquo;s reagent for 10 min (dark), and counterstained with hematoxylin. Images were acquired via Nikon Eclipse 70i microscope; \u0026ge;10 seminiferous tubules per mouse were analyzed for spermatogenic stage progression.\u003c/p\u003e\u003cp\u003eTUNEL Assay\u003c/p\u003e\u003cp\u003eTesticular sections were deparaffinized and treated with proteinase K for 20 min at 37\u0026deg;C. TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche) per protocol: incubation with TUNEL reaction mixture, DAPI counterstaining, and visualization via confocal microscopy. Apoptotic cells per seminiferous tubule were counted (\u0026ge;\u0026thinsp;10 tubules per mouse, n\u0026thinsp;=\u0026thinsp;5 per group).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSperm Parameter Analysis\u003c/h3\u003e\n\u003cp\u003eSperm Isolation\u003c/p\u003e\u003cp\u003eCauda epididymides from WT/KO mice were dissected, minced in pre-warmed HTF medium and incubated for 15 min to allow sperm release. Sperm suspensions were filtered through 70 \u0026micro;m cell strainers to remove tissue debris.\u003c/p\u003e\u003cp\u003eSperm Density and Morphology\u003c/p\u003e\u003cp\u003eSperm density was quantified using a hemocytometer under a light microscope. For morphology, 5 \u0026micro;l sperm suspension was smeared on slides, air-dried, fixed in 4% PFA, and stained with H\u0026amp;E. Two blinded observers counted\u0026thinsp;\u0026ge;\u0026thinsp;1000 sperm per mouse, categorizing morphology as normal or abnormal (head/neck/tail defects) per WHO guidelines.\u003c/p\u003e\u003cp\u003eCASA for Sperm Motility\u003c/p\u003e\u003cp\u003eA 5 \u0026micro;l sperm suspension was loaded onto a pre-warmed CASA counting slide. Motility parameters were analyzed via Sperm Class Analyzer v4.0.0 with a phase-contrast microscope (Olympus BX53) and high-speed camera (25 frames/s). Parameters measured: total motility (%), progressive motility (%), average path velocity (VAP, \u0026micro;m/s), straight-line velocity (VSL, \u0026micro;m/s), curvilinear velocity (VCL, \u0026micro;m/s), and beat-cross frequency (BCF, Hz). \u0026ge;5 fields and 200 sperm per sample were evaluated; each sample was tested in triplicate.\u003c/p\u003e\n\u003ch3\u003eFertility Assays\u003c/h3\u003e\n\u003cp\u003eIn Vivo Fertility\u003c/p\u003e\u003cp\u003eWT (Tex43⁺/⁺) and KO (Tex43⁻/⁻) males were individually housed with two WT females for 3 months. Females were checked daily for vaginal plugs (mating marker). Upon plug detection, females were separated and monitored for pregnancy. Litter size (live pups per litter) and pregnancy rate were recorded.\u003c/p\u003e\u003cp\u003eIVF Assay\u003c/p\u003e\u003cp\u003eSuperovulation: 6\u0026ndash;8-week-old females were injected intraperitoneally with 5 IU pregnant mare serum gonadotropin (PMSG; Ningbo Second Hormone Factory, China), followed by 5 IU human chorionic gonadotropin (hCG) 48h later.\u003c/p\u003e\u003cp\u003eIn Vivo Fertilization Validation: Superovulated females were mated with WT/KO males (1:1). Zygotes were collected from oviducts 12\u0026ndash;16h post-mating, washed in G1-plus medium (Vitrolife, Sweden), and cultured (37\u0026deg;C, 5% CO₂). Fertilization rate = (2-cell embryos / collected zygotes) \u0026times; 100%.\u003c/p\u003e\u003cp\u003eIn Vitro Fertilization: Females were euthanized 12h post-hCG; oocyte-corona cumulus complexes (COCs) were collected from oviducts and placed in 200 \u0026micro;l HTF drops (pre-equilibrated 37\u0026deg;C, 5% CO₂). Sperm from WT/KO males were capacitated in HTF for 1h; 15 \u0026micro;l sperm suspension (1\u0026times;10⁶ sperm/ml) was added to COCs. After 4h co-incubation, oocytes were washed in G1-plus and cultured. IVF rate = (2-cell embryos / inseminated oocytes) \u0026times; 100%. Embryonic development (4-cell, morula, blastocyst) was monitored daily for 3 days.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSEM and TEM for Sperm Ultrastructure\u003c/h2\u003e\u003cp\u003eSEM Preparation\u003c/p\u003e\u003cp\u003eSperm from WT/KO mice (n\u0026thinsp;=\u0026thinsp;3) were washed in PBS, fixed in 2.5% glutaraldehyde (0.1 M cacodylate buffer, pH 7.4) for 2h at 4\u0026deg;C, and washed 3\u0026times; with cacodylate buffer. Samples were dehydrated via graded ethanol (30%\u0026ndash;100%, 15 min each), critical-point dried with liquid CO₂ (Leica EM CPD300), mounted on aluminum stubs, and sputter-coated with gold-palladium (Leica EM ACE600). Images were acquired via JEOL JSM-7800F SEM.\u003c/p\u003e\u003cp\u003eTEM Preparation\u003c/p\u003e\u003cp\u003eFixed sperm were post-fixed in 1% osmium tetroxide for 1.5h at room temperature, washed 3\u0026times; with distilled water, and dehydrated via ethanol and propylene oxide. Samples were infiltrated with epoxy resin and polymerized. Ultrathin sections were cut via Leica EM UC7 ultramicrotome, collected on copper grids, and stained with 2% uranyl acetate and lead citrate. Images were acquired via JEOL JEM-1400 TEM. For microtubule disorganization quantification, \u0026ge;\u0026thinsp;50 flagellar end pieces per group were analyzed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eHuman Subjects and WES\u003c/h2\u003e\u003cp\u003eStudy Cohort\u003c/p\u003e\u003cp\u003e146 Chinese men with asthenoteratozoospermia were recruited from the Center for Reproductive Medicine, Peking University Third Hospital (2019\u0026ndash;2024). Inclusion criteria: no history of endocrine disorders, genital tract infection, or chemotherapy/radiotherapy. Exclusion criteria: Y-chromosome microdeletions or AZF gene mutations. All participants provided written informed consent; the study was approved by the Ethics Committee of Peking University Third Hospital (Approval No.: 2021SZ-003) and complied with the Declaration of Helsinki.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eWES and Variant Analysis\u003c/h2\u003e\u003cp\u003eGenomic DNA was extracted from peripheral blood leukocytes using the QIAamp DNA Blood Mini Kit. WES libraries were prepared with the Agilent SureSelect Human All Exon V6 capture system, targeting\u0026thinsp;~\u0026thinsp;60 Mb of coding regions. Sequencing was performed on the Illumina NovaSeq 6000 platform, achieving\u0026thinsp;\u0026ge;\u0026thinsp;100\u0026times; average coverage of target regions. Raw reads were filtered via Trimmomatic (v0.39), aligned to the human reference genome (GRCh38) via BWA-MEM (v0.7.17), and variant calling was done via GATK (v4.2.6). Variants were annotated using ANNOVAR; filtering criteria: minor allele frequency (MAF)\u0026thinsp;\u0026lt;\u0026thinsp;0.01 in gnomAD, ExAC, and 1000 Genomes Project databases; missense variants with CADD score\u0026thinsp;\u0026ge;\u0026thinsp;20; splice-site variants within \u0026plusmn;\u0026thinsp;20 bp of exons. Sanger sequencing validated TEX43 variants in patients and their family members.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStructural Modeling of TEX43\u003c/h2\u003e\u003cp\u003e3D models of WT and mutant (p.R37Q) TEX43 (human: NP_997291.1; mouse: NP_080375.2) were generated via SWISS-MODEL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using PDB 7ZXY (68% sequence identity to human TEX43) as the template. Models were refined via molecular dynamics simulations (GROMACS v2021.4) for 10 ns. Structural analysis (residue interactions, root-mean-square deviation [RMSD]) was performed using PyMOL (v2.5; Schr\u0026ouml;dinger, USA). Hydrogen bonds were quantified via HBPLUS (v3.1); salt bridges were identified using PyMOL\u0026rsquo;s \"find salt bridges\" function.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Comparisons between two groups (WT vs. KO) were made using unpaired two-tailed Student\u0026rsquo;s t-test (continuous variables: litter size, sperm density, motility) or chi-square test (categorical variables: pregnancy rate, fertilization rate). One-way ANOVA was used for multi-group comparisons (WT vs. HET vs. KO). Statistical significance was defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; \"ns\" denotes non-significance (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). All analyses were performed using GraphPad Prism (v9.0; GraphPad Software, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eTex43 Is a Testis-Enriched Gene Expressed in Maturing Spermatids\u003c/h2\u003e\n \u003cp\u003eQ-PCR showed Tex43 mRNA was exclusively expressed in murine testes,no detectable expression was observed in 10 other somatic tissues (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). During postnatal testicular development, Tex43 mRNA was first detected at 18 dpp (coinciding with the emergence of round spermatids), with expression increasing progressively and peaking at 56 dpp (sexual maturity in male mice; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Amino acid sequence alignment of TEX43 orthologs (mouse, human, rhesus macaque) revealed high conservation: 93% identity between human and rhesus macaque, 72% between mouse and human (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003eIHC localized TEX43 protein to the cytoplasm of round and elongating spermatids in WT testes (adluminal compartment of seminiferous tubules); no staining was observed in spermatogonia, spermatocytes, or somatic cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;C), consistent with Q-PCR\u0026rsquo;s post-meiotic expression pattern. In the epididymis, TEX43 was restricted to the flagellar region of maturing spermatids in the lumen, with no staining in epididymal epithelial cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;F). Immunocytochemistry confirmed TEX43 co-localization with sperm flagella (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eSuccessful Generation of Tex43-KO Mice\u003c/h2\u003e\n \u003cp\u003eCRISPR/Cas9-mediated deletion of exons 1\u0026ndash;3 of Tex43 was confirmed by genotyping PCR: WT mice showed a single\u0026thinsp;~\u0026thinsp;400 bp band, heterozygotes (Tex43⁺/⁻) showed two bands (~\u0026thinsp;400 bp and ~\u0026thinsp;200 bp), and homozygotes (Tex43⁻/⁻) showed a single\u0026thinsp;~\u0026thinsp;200 bp band (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Sanger sequencing verified the deletion breakpoint (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Western blotting and IHC confirmed complete loss of TEX43 protein in KO testes and sperm (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, D); WT testes showed robust TEX43 expression in spermatids. Tex43-KO mice were viable and fertile, with no overt developmental abnormalities (body weight, general health) up to 6 months of age, distinguishing them from KO models of other TEX genes (e.g., Tex11, Tex14) that cause sterility.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eTex43 Deficiency Does Not Disrupt Spermatogenesis or Sperm Morphology\u003c/h2\u003e\n \u003cp\u003eTesticular weight and testis-to-body weight ratio were slightly reduced in Tex43-KO mice (KO: 0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02% vs. WT: 0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03%; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but remained within the physiological range (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, C). H\u0026amp;E and PAS staining showed Tex43-KO testes had intact seminiferous tubule architecture** and all spermatogenic stages (spermatogonia to mature spermatids); epididymal lumens contained abundant mature sperm, confirming unimpaired spermiation (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG). TUNEL assays revealed no significant difference in germ cell apoptosis between KO and WT mice (KO: 0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 vs. WT: 0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 apoptotic cells per tubule; P\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\n \u003cp\u003eSperm morphology analysis via H\u0026amp;E staining showed no significant difference in the proportion of normal sperm between groups (KO: 86.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1% vs. WT: 88.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7%; P\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Abnormalities (head defects, neck/midpiece defects, tail defects) were rare and comparable to WT, indicating Tex43 deficiency does not impair sperm morphogenesis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eTex43 Deficiency Reduces Sperm Density but Preserves Motility\u003c/h2\u003e\n \u003cp\u003eSperm density was significantly lower in Tex43-KO mice (28.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u0026times;10⁶ sperm/ml) compared to WT (41.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u0026times;10⁶ sperm/ml; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). Notably, KO sperm density remained well above the murine fertility threshold (\u0026gt;\u0026thinsp;10\u0026times;10⁶ sperm/ml), and no difference was observed between heterozygotes and KO mice, suggesting a recessive effect of Tex43 deletion.\u003c/p\u003e\n \u003cp\u003eCASA revealed no significant differences in any motility parameter between WT and KO sperm: total motility (KO: 62.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2% vs. WT: 65.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8%), progressive motility (KO: 41.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5% vs. WT: 43.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1%), VAP (KO: 68.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1 \u0026micro;m/s vs. WT: 70.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 \u0026micro;m/s), VSL (KO: 45.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 \u0026micro;m/s vs. WT: 47.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 \u0026micro;m/s), VCL (KO: 98.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3 \u0026micro;m/s vs. WT: 101.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9 \u0026micro;m/s), and BCF (KO: 14.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 Hz vs. WT: 14.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 Hz; all P\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF). This indicates Tex43 deficiency does not compromise sperm motility, despite its localization to flagellar microtubules.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eTex43-KO Mice Exhibit Normal Fertility and Embryonic Development\u003c/h2\u003e\n \u003cp\u003eIn vivo breeding assays showed Tex43-KO males sired litters with sizes (6.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 pups/litter) comparable to WT (7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 pups/litter; P\u0026thinsp;\u0026gt;\u0026thinsp;0.05); pregnancy rates were also similar (KO: 82.3% vs. WT: 85.7%; P\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). IVF assays confirmed no significant difference in fertilization rate (KO: 61.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2% vs. WT: 66.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8%; P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) or embryonic development to the blastocyst stage (KO: 45.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9% vs. WT: 47.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8%; P\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). These results demonstrate Tex43 deficiency does not impair male fertility or early embryonic development\u0026mdash;aligning with the lack of motility defects.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eTex43 Deficiency Causes Subtle Flagellar Ultrastructural Defects\u003c/h2\u003e\n \u003cp\u003eSEM showed Tex43-KO sperm had normal head morphology (regular oval shape, intact acrosome) and flagellar middle piece structure (tightly packed mitochondrial sheath; Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). The flagellar principal piece retained the canonical \u0026quot;9\u0026thinsp;+\u0026thinsp;2\u0026quot; microtubule configuration in most KO sperm, though ~\u0026thinsp;15% showed partial loss of outer dense fibers (ODFs),a mild defect not observed in WT (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, D).\u003c/p\u003e\n \u003cp\u003eTEM revealed the most prominent defect: increased disorganization of the \u0026quot;9\u0026thinsp;+\u0026thinsp;2\u0026quot; microtubule array in the flagellar end piece of Tex43-KO sperm (~\u0026thinsp;30% of KO sperm vs. ~5% of WT; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE\u0026ndash;G). Disorganization included splayed microtubule doublets and loss of the outer junction (OJ) between A- and B-tubules,critical for axonemal stability. Notably, this defect was restricted to the end piece (a short, terminal segment of the flagellum) and did not affect the principal piece or middle piece,explaining the preserved sperm motility.\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eTEX43 Variants in Men with Asthenoteratozoospermia\u003c/h2\u003e\n \u003cp\u003eWES identified 9 infertile men with TEX43 variants (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), including 2 intronic variants (intron1:c.86\u0026thinsp;+\u0026thinsp;141G\u0026thinsp;\u0026gt;\u0026thinsp;A, intron2:c.196-25C\u0026thinsp;\u0026gt;\u0026thinsp;T) and 2 exonic variants (exon1:c.1-23T\u0026thinsp;\u0026gt;\u0026thinsp;A, exon2:c.110G\u0026thinsp;\u0026gt;\u0026thinsp;A:p.R37Q). The intronic variant pair (c.86\u0026thinsp;+\u0026thinsp;141G\u0026thinsp;\u0026gt;\u0026thinsp;A/c.196-25C\u0026thinsp;\u0026gt;\u0026thinsp;T) was co-carried by 8 patients, suggesting a potential founder effect in the Chinese population. Five patients with exonic variants underwent ICSI: 4 achieved live births, and 1 had a biochemical pregnancy (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). No TEX43 variants were identified in 50 fertile male controls.\u003c/p\u003e\n \u003cp\u003eStructural modeling of the p.R37Q variant (exon2:c.110G\u0026thinsp;\u0026gt;\u0026thinsp;A) showed the arginine-to-glutamine substitution disrupted two key interactions: (1) loss of a salt bridge between R37 and E62 (a negatively charged residue in the microtubule-binding domain), and (2) reduction in hydrogen bonds from 2 (WT) to 1 (mutant; Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). This conformational shift (RMSD: 1.8 \u0026Aring; vs. WT) is predicted to weaken TEX43\u0026rsquo;s binding to microtubules, consistent with the flagellar defects observed in KO mice.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSemen Characteristics and Clinical Outcomes of Patients with TEX43 Variants\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePatient No.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ecDNA Change\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSemen Volume (ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSeminal PH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConcentration (10⁶/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal Motility (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProgressive Motility (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInsemination\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClinical Outcome\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eintron1:c.86\u0026thinsp;+\u0026thinsp;141G\u0026thinsp;\u0026gt;\u0026thinsp;A, intron2:c.196-25C\u0026thinsp;\u0026gt;\u0026thinsp;T, exon1:c.1-23T\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eICSI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLive Birth\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eintron1:c.86\u0026thinsp;+\u0026thinsp;141G\u0026thinsp;\u0026gt;\u0026thinsp;A, intron2:c.196-25C\u0026thinsp;\u0026gt;\u0026thinsp;T, exon1:c.1-23T\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eICSI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLive Birth\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eexon1:c.1-23T\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eICSI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLive Birth\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eintron1:c.86\u0026thinsp;+\u0026thinsp;141G\u0026thinsp;\u0026gt;\u0026thinsp;A, exon1:c.1-23T\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eICSI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiochemical Pregnancy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eexon2:c.110G\u0026thinsp;\u0026gt;\u0026thinsp;A:p.R37Q\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eICSI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLive Birth\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study delineates TEX43\u0026rsquo;s role in mammalian reproduction, with three key findings: (1) TEX43 is a testis-enriched, evolutionarily conserved gene expressed in maturing spermatids; (2) Tex43 deletion in mice causes mild flagellar ultrastructural defects and reduced sperm density but preserves fertility; (3) human TEX43 variants are associated with asthenoteratozoospermia but do not prevent successful pregnancy via ICSI. These results align with the Journal of Assisted Reproduction and Genetics\u0026rsquo; focus on bridging basic reproductive biology with clinical practice.\u003c/p\u003e\u003cp\u003eTEX43\u0026rsquo;s Role in Spermatogenesis: Redundancy Explains Lack of Overt Phenotype\u003c/p\u003e\u003cp\u003e TEX43\u0026rsquo;s localization to sperm flagellar microtubules and stage-specific expression initially suggested a critical role in spermiogenesis (e.g., flagellar assembly). However, Tex43-KO mice showed only subtle defects, consistent with genetic redundancy, a hallmark of male reproductive biology[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Redundancy may involve: TEX family paralogs: TEX40 and TEX44 share 35\u0026ndash;40% sequence identity with TEX43 and are expressed in spermatids; their upregulation in KO testes could compensate for TEX43 loss.\u003c/p\u003e\u003cp\u003eMicrotubule-associated proteins: CFAP77 and CCDC105 form a ternary complex with TEX43 at the flagellar OJ [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]; CFAP77-KO mice have severe axonemal defects and sterility, but CCDC105-KO mice are fertile, suggesting TEX43 and CCDC105 are redundant [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Our TEM data support this: Tex43-KO sperm had OJ defects restricted to the end piece, while CFAP77-KO sperm have widespread OJ disruption [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eClinical Relevance: TEX43 Variants as Subtle Fertility Modifiers\u003c/p\u003e\u003cp\u003eThe identification of TEX43 variants in men with asthenoteratozoospermia, including the functionally relevant p.R37Q variant, links TEX43 to human infertility. Notably, all patients with exonic variants achieved pregnancy via ICSI, mirroring the murine data (preserved fertilization capacity). This suggests TEX43 variants are subtle fertility modifiers rather than major pathogenic factors, consistent with the mild phenotype of KO mice.\u003c/p\u003e\u003cp\u003eLimitations of the clinical analysis include: (1) small cohort size (9 variant carriers); (2) lack of functional validation for intronic variants (their impact on splicing is unknown); (3) absence of family segregation data to confirm variant inheritance. Future studies should enroll larger cohorts (stratified by infertility subtype) and perform minigene assays to test intronic variant effects on TEX43 expression. For clinical practice, TEX43 variants do not preclude successful ICSI, reassuring for couples with these variants. For basic research, Tex43-KO mice provide a model to study genetic redundancy in flagellar biology; future work could use RNA-seq/proteomics to identify compensatory genes in KO testes.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTEX43 is a testis-enriched, microtubule-localized gene with conserved expression across mammals. Its deletion in mice causes mild sperm density reduction and flagellar end piece defects but preserves fertility, likely due to genetic redundancy. Human TEX43 variants are associated with asthenoteratozoospermia but do not prevent ICSI success. These findings advance understanding of testis-specific gene function and highlight the importance of functional validation in interpreting genetic variants in infertility.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eQ. C performed experiments. L.L wrote the manuscript. Y. L designed the experiments and generated the CRISPR mice.X.Q performed on the sample collection and clinical case analysis. P.L and R.L provided valuable advice for the manuscript. X. Z and X.Z supervised the study and reviewed the manuscript. X.Z and X.Z are the correspondence authors of this research.All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJiao SY, Yang YH, Chen SR. Molecular genetics of infertility: loss-of-function mutations in humans and corresponding knockout/mutated mice. Hum Reprod Update. 2021;27(1):154\u0026ndash;89. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/humupd/dmaa034\u003c/span\u003e\u003cspan address=\"10.1093/humupd/dmaa034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PubMed PMID: 33118031.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFainberg J, Kashanian JA, F1000Res. 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PubMed PMID: 32561905; PubMed Central PMCID: PMCPMC7401030.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhan M, Jabeen N, Khan T, Hussain HMJ, Ali A, Khan R, et al. The evolutionarily conserved genes: Tex37, Ccdc73, Prss55 and Nxt2 are dispensable for fertility in mice. Sci Rep. 2018;8(1):4975. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-018-23176-x\u003c/span\u003e\u003cspan address=\"10.1038/s41598-018-23176-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Epub 20180321.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChung JJ, Miki K, Kim D, Shim SH, Shi HF, Hwang JY et al. CatSperzeta regulates the structural continuity of sperm Ca(2+) signaling domains and is required for normal fertility. Elife. 2017;6. Epub 20170223. doi: 10.7554/eLife.23082. PubMed PMID: 28226241; PubMed Central PMCID: PMCPMC5362262.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSinha A, Singh V, Singh S, Yadav S. Proteomic analyses reveal lower expression of TEX40 and ATP6V0A2 proteins related to calcium ion entry and acrosomal acidification in asthenozoospermic males. Life Sci. 2019;218:81\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.lfs.2018.12.016\u003c/span\u003e\u003cspan address=\"10.1016/j.lfs.2018.12.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Epub 20181211.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchiza C, Korbakis D, Panteleli E, Jarvi K, Drabovich AP, Diamandis EP. Discovery of a Human Testis-specific Protein Complex TEX101-DPEP3 and Selection of Its Disrupting Antibodies. Mol Cell Proteom. 2018;17(12):2480\u0026ndash;95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1074/mcp.RA118.000749\u003c/span\u003e\u003cspan address=\"10.1074/mcp.RA118.000749\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Epub 20180810.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFujihara Y, Tokuhiro K, Muro Y, Kondoh G, Araki Y, Ikawa M, et al. Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa. Proc Natl Acad Sci U S A. 2013;110(20):8111\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1222166110\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1222166110\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Epub 20130430.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEndo S, Yoshitake H, Tsukamoto H, Matsuura H, Kato K, Sakuraba M, et al. 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Epub 20181114.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGirault MS, Dupuis S, Ialy-Radio C, Stouvenel L, Viollet C, Pierre R et al. Deletion of the Spata3 Gene Induces Sperm Alterations and In Vitro Hypofertility in Mice. Int J Mol Sci. 2021;22(4). Epub 20210216. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms22041959\u003c/span\u003e\u003cspan address=\"10.3390/ijms22041959\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PubMed PMID: 33669425; PubMed Central PMCID: PMCPMC7920483.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXia L, Yin GL, Long Y, Sun F, Wang BB, Zhu Y, et al. The core outer junction protein CFAP77 connects A- and B-tubules within doublet microtubules of cilia and flagella. PLoS Biol. 2025;23(10):e3003442. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pbio.3003442\u003c/span\u003e\u003cspan address=\"10.1371/journal.pbio.3003442\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Epub 20251021.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNguyen TTT, Tokuhiro K, Shimada K, Wang H, Mashiko D, Tonai S, et al. Gene-deficient mouse model established by CRISPR/Cas9 system reveals 15 reproductive organ-enriched genes dispensable for male fertility. Front Cell Dev Biol. 2024;12:1411162. PubMed PMID: 38835510; PubMed Central PMCID: PMCPMC11148293.\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":false,"email":"","identity":"journal-of-assisted-reproduction-and-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Journal of Assisted Reproduction and Genetics","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8181104/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8181104/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eTestis-specific TEX family genes are critical for spermatogenesis, but TEX43\u0026rsquo;s function remains uncharacterized. This study aimed to delineate TEX43\u0026rsquo;s role in spermatogenesis and fertility using murine models and clinical data.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eTex43 expression was analyzed via quantitative reverse transcription-polymerase chain reaction (Q-PCR), immunohistochemistry (IHC), and western blotting. A Tex43 knockout (KO) mouse model was generated using CRISPR/Cas9 (targeting exons 1\u0026ndash;3). Testicular histology (hematoxylin-eosin [H\u0026amp;E] staining), sperm parameters (morphology via H\u0026amp;E smears, density via hemocytometer, motility via computer-assisted sperm analysis [CASA]), and fertility (in vivo breeding assays, in vitro fertilization [IVF]) were evaluated. Sperm ultrastructure was assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Whole-exome sequencing (WES) identified TEX43 variants in 146 infertile men with asthenoteratozoospermia. Structural modeling of WT/mutant TEX43 was performed via SWISS-MODEL.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eTex43 is testis-enriched: mRNA expression initiated at postnatal day 18 (round spermatid stage) and peaked in elongating spermatids; TEX43 localized to sperm flagellar microtubules. Tex43-KO mice showed modestly reduced sperm density (28.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 vs. 41.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u0026times;10⁶ sperm/ml in WT; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) but normal testicular architecture, sperm motility, and fertility (litter size: KO 6.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 vs. WT 7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 pups/litter; P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). TEM revealed increased flagellar end piece \"9\u0026thinsp;+\u0026thinsp;2\" microtubule disorganization in KO sperm (~\u0026thinsp;30% vs. ~5% in WT; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). WES identified 9 infertile men with TEX43 variants; 4 with exonic variants (e.g., p.R37Q) achieved live births via intracytoplasmic sperm injection (ICSI). Structural modeling showed p.R37Q disrupted hydrogen bonds critical for microtubule binding.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eTEX43 is minimal impact on murine spermatogenesis and fertility, likely due to genetic redundancy. Human TEX43 variants may exert subtle reproductive effects, requiring validation in larger cohorts with functional studies.\u003c/p\u003e","manuscriptTitle":"TEX43, a Testis-Enriched Microtubule-Associated Gene, Exerts Minimal Impact on Spermatogenesis and Fertility in Mice and Humans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-05 09:30:22","doi":"10.21203/rs.3.rs-8181104/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-20T23:16:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-12T10:21:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-11T12:43:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163146768687442091352080205857987537396","date":"2025-12-04T13:28:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"318612226787827674886786676714107342058","date":"2025-12-03T11:22:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-03T05:27:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-27T15:34:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-25T06:06:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Assisted Reproduction and Genetics","date":"2025-11-22T14:28:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"journal-of-assisted-reproduction-and-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Journal of Assisted Reproduction and Genetics","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5b12e6b0-72ac-4dec-bbb2-3b79b634d6bb","owner":[],"postedDate":"December 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T11:11:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-05 09:30:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8181104","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8181104","identity":"rs-8181104","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}