# paper.pdf ## Metadata - PDFFormatVersion=1.4 - Language=en - IsLinearized=false - IsAcroFormPresent=false - IsXFAPresent=false - IsCollectionPresent=false - IsSignaturesPresent=false - Title=TEX43, a Testis-Enriched Microtubule-Associated Gene, Exerts Minimal Impact on Spermatogenesis and Fertility in Mice and Humans - Creator=Chromium - Producer=Skia/PDF m123 - CreationDate=D:20251210152426+00'00' - ModDate=D:20251210152426+00'00' ## Contents ### Page 1 [](https://doi.org/10.21203/rs.3.rs-8181104/v1) # TEX43, a Testis-Enriched Microtubule-Associated Gene, Exerts Minimal Impact on Spermatogenesis and Fertility in Mice and Humans - ###### Qi-qi Chen Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital - ###### Li Lei Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital - ###### Xin Qiu Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital - ###### Yujun Liu Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital - ###### Ping Liu Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital - ###### Rong Li Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital - ###### Xu Zhi Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital - ###### Xinjie Zhuang Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital ###### Research Article ###### Keywords: ###### Posted Date: December 5th, 2025 ###### DOI: [https://doi.org/10.21203/rs.3.rs-8181104/v1](https://doi.org/10.21203/rs.3.rs-8181104/v1) ###### License: This work is licensed under a Creative Commons Attribution 4.0 International License. [Read Full License](https://creativecommons.org/licenses/by/4.0/) ###### Additional Declarations: No competing interests reported. ## ### Page 2 ## Abstract ## Purpose Testis-specic 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) identied 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 agellar microtubules. Tex43-KO mice showed modestly reduced sperm density (28.6 ± 3.2 vs. 41.2 ± 2.9×10 ⁶ sperm/ml in WT; P < 0.01) but normal testicular architecture, sperm motility, and fertility (litter size: KO 6.8 ± 0.7 vs. WT 7.2 ± 0.5 pups/litter; P > 0.05). TEM revealed increased agellar end piece "9 + 2" microtubule disorganization in KO sperm (~ 30% vs. ~5% in WT; P < 0.01). WES identied 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. ## 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- specic 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]. ### Page 3 In male reproductive biology, the testis-specic gene (TEX) family plays a pivotal role in regulating spermatogenesis and sustaining male fertility. The term "TEX gene" was rst coined by Wang et al. in 2001, subsequent to their identication of novel spermatogenic cell-specic transcripts in mice using the technique of cDNA suppression subtractive hybridization (SSH) [7]. Subsequently, orthologs of these TEX genes have been identied 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 usingTex11-decient mice have demonstrated its essentiality in spermatogenesis: loss ofTex11impairs chromosomal synapsis, increases germ cell apoptosis, and ultimately causes male infertility [8–10].Tex12, a conserved vertebrate gene, is one of the meiosis-specic 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].Tex14encodes a testis-specic protein containing ankyrin repeats and protein kinase-C domains, which is critical for forming intercellular bridges during meiosis. Male mice lackingTex14 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 specically 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-specic 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-nger 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, andTex37 appear dispensable for spermatogenesis, as their respective knockout mice remain fertile[28–31]. Tex40 is a novel agellar protein, and its depletion results in rigid sperm agella 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 identied 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 tness. 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). ### Page 4 C57BL/6N mice were housed in a specic 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 eciency 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 conrmed by PCR and western blotting (anti-TEX43 antibody, 1:1000; Bioss Antibodies, bs-15207R). ### Q-PCR for Tex43 Expression Tissue-Specic 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 ⁻ΔΔ Ct method. ### Immunohistochemistry (IHC) and Immunocytochemistry IHC on Tissue Sections Testes and epididymides from WT and Tex43-KO mice (8 weeks old, n = 3) were xed in 4% paraformaldehyde (PFA) for 24h, embedded in paran, and sectioned. Deparanization 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 xed 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. ## ### Page 5 ## Testicular Histology and TUNEL Assay H&E and PAS Staining Testes and epididymides from WT/KO mice (8 weeks old, n = 3) were xed in Bouin’s solution (Sigma-Aldrich) for 24h, embedded in paran, 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 deparanized 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 ltered through 70 μm cell strainers to remove tissue debris. Sperm Density and Morphology Sperm density was quantied using a hemocytometer under a light microscope. For morphology, 5 μl sperm suspension was smeared on slides, air-dried, xed 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 elds 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. ### Page 6 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% CO ₂ ). 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% CO ₂ ). Sperm from WT/KO males were capacitated in HTF for 1h; 15 μl sperm suspension (1×10 ⁶ 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, xed 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 CO ₂ (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-xed in 1% osmium tetroxide for 1.5h at room temperature, washed 3× with distilled water, and dehydrated via ethanol and propylene oxide. Samples were inltrated 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 quantication, ≥ 50 agellar 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 ltered 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; ltering 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 rened 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 quantied via HBPLUS (v3.1); salt bridges were identied using PyMOL’s "nd salt bridges" function. ## ### Page 7 ## 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 signicance was dened as P < 0.05, P < 0.01, P < 0.001, P < 0.0001; "ns" denotes non-signicance (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. 1A). During postnatal testicular development, Tex43 mRNA was rst 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. 1B). 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. 1C). 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. 2A–C), consistent with Q-PCR’s post-meiotic expression pattern. In the epididymis, TEX43 was restricted to the agellar region of maturing spermatids in the lumen, with no staining in epididymal epithelial cells (Fig. 2D–F). Immunocytochemistry conrmed TEX43 co-localization with sperm agella (Fig. 1). ## Successful Generation of Tex43-KO Mice CRISPR/Cas9-mediated deletion of exons 1–3 of Tex43 was conrmed 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. 3A, B). Sanger sequencing veried the deletion breakpoint (Fig. 2). Western blotting and IHC conrmed complete loss of TEX43 protein in KO testes and sperm (Fig. 3C, 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 Deciency 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. 4A, 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, conrming unimpaired spermiation (Fig. 4G). TUNEL assays revealed no signicant 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. 4D). Sperm morphology analysis via H&E staining showed no signicant difference in the proportion of normal sperm between groups (KO: 86.2 ± 3.1% vs. WT: 88.5 ± 2.7%; P > 0.05; Fig. 4B). Abnormalities (head defects, neck/midpiece defects, tail defects) were rare and comparable to WT, indicating Tex43 deciency does not impair sperm morphogenesis. ## Tex43 Deciency Reduces Sperm Density but Preserves Motility Sperm density was signicantly lower in Tex43-KO mice (28.6 ± 3.2×10 ⁶ sperm/ml) compared to WT (41.2 ± 2.9×10 ⁶ sperm/ml; P < 0.01; Fig. 4E). Notably, KO sperm density remained well above the murine fertility threshold (> 10×10 ⁶ sperm/ml), and no difference was observed between heterozygotes and KO mice, suggesting a recessive effect of Tex43 deletion. ### Page 8 CASA revealed no signicant 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. 4F). This indicates Tex43 deciency does not compromise sperm motility, despite its localization to agellar 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. 5A). IVF assays conrmed no signicant 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. 5B). These results demonstrate Tex43 deciency does not impair male fertility or early embryonic development—aligning with the lack of motility defects. ## Tex43 Deciency Causes Subtle Flagellar Ultrastructural Defects SEM showed Tex43-KO sperm had normal head morphology (regular oval shape, intact acrosome) and agellar middle piece structure (tightly packed mitochondrial sheath; Fig. 6A, B). The agellar principal piece retained the canonical "9 + 2" microtubule conguration in most KO sperm, though ~ 15% showed partial loss of outer dense bers (ODFs),a mild defect not observed in WT (Fig. 6C, D). TEM revealed the most prominent defect: increased disorganization of the "9 + 2" microtubule array in the agellar end piece of Tex43-KO sperm (~ 30% of KO sperm vs. ~5% of WT; P < 0.01; Fig. 6E–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 agellum) and did not affect the principal piece or middle piece,explaining the preserved sperm motility. ## TEX43 Variants in Men with Asthenoteratozoospermia WES identied 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 identied 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. 7A, B). This conformational shift (RMSD: 1.8 Å vs. WT) is predicted to weaken TEX43’s binding to microtubules, consistent with the agellar defects observed in KO mice. ### Page 9 Table 1 | - | Semen Characteristics and Clinical Outcomes of Patients with TEX43 Variants | Patient No. | cDNA Change | Semen Volume (ml) | Seminal PH | Concentration (10 ⁶ /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 ndings: (1) TEX43 is a testis-enriched, evolutionarily conserved gene expressed in maturing spermatids; (2) Tex43 deletion in mice causes mild agellar 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 agellar microtubules and stage-specic expression initially suggested a critical role in spermiogenesis (e.g., agellar 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 agellar 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 Modiers The identication 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 modiers rather than major pathogenic factors, consistent with the mild phenotype of KO mice. ### Page 10 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 conrm variant inheritance. Future studies should enroll larger cohorts (stratied 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 agellar 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 agellar 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 ndings advance understanding of testis-specic 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. 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Front Cell Dev Biol. 2024;12:1411162. PubMed PMID: 38835510; PubMed Central PMCID: PMCPMC11148293. ## Figures ### Page 13 ## Figure 1 Expression Pattern of Tex43 and Cross-Species Alignment (A) Tissue-specic 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<0.05, **P<0.01 vs. dpp 0. ### Page 14 ## Figure 2 IHC Localization of TEX43 in Murine Testis and Epididymis (A) Panoramic view of testis: TEX43 (brown) localized to adluminal spermatids. (B) Low-magnication testis: TEX43 restricted to round/elongating spermatids (arrowheads). (C) High-magnication testis: TEX43 in spermatid cytoplasm (arrowheads). (D) Panoramic view of epididymis: TEX43 in lumenal spermatids. (E) Low-magnication epididymis: TEX43 in agellar region (arrowheads). (F) High-magnication epididymis: TEX43 in spermatid agella (arrowheads). Negative controls (insets) show no staining. Scale bars: (A, D) 100 μ m; (B, E) 50 μ m; (C, F) 20 μ m. ### Page 15 ## Figure 3 Generation and Validation of Tex43-KO Mice (A) CRISPR/Cas9 targeting strategy: gRNAs ank 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 μ m. ### Page 16 ## Figure 4 Testicular Phenotype and Sperm Parameters in WT and Tex43-KO Mice (A) Testicular size: Representative images of testes from 8-week-old WT and KO mice (n=3 per group). (B) Sperm morphology: H&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: Signicantly reduced in KO (28.6±3.2×10 ⁶ vs. WT 41.2±2.9×10 ⁶ 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&E ### Page 17 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 μ m; (C–F) Mean ± SEM; *P<0.05, **P<0.01, ns: non-signicant. ## Figure 5 Fertility and Embryonic Development Outcomes (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 signicant 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- signicant. ### Page 18 ## Figure 6 Sperm Ultrastructural Analysis (SEM/TEM) (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) Quantication of end piece disorganization: ~30% of KO sperm affected (n=50 end pieces per group). Scale bars: (A) 2 μ m; (B) 500 nm; (C–F) 100 nm; (G) Mean ± SEM; **P<0.01 vs. WT. ### Page 19 ## Figure 7 Structural Models of WT and Mutant TEX43 (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.