Primate-specific SSX1 is required for CPSF6-dependent spermatocyte transcription and human fertility

Primate-specific SSX1 is required for CPSF6-dependent spermatocyte transcription and human fertility | 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 Article Primate-specific SSX1 is required for CPSF6-dependent spermatocyte transcription and human fertility Rong Hua, Qingsong Xie, Xuan Sha, Meizhou Liu, Guotong Li, Xun Xia, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8516172/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Genetic variation is a major cause of male infertility, and defining the underlying mechanisms is essential for developing effective interventions. SSX1 is a primate specific gene whose deficiency has been linked to sperm malformations in humans and cynomolgus monkeys, yet its pathogenic mechanism remains unclear. Here, by whole exome sequencing of a cohort of 536 men with oligoasthenoteratozoospermia(OAT), we identified three previously unreported SSX1 variants, including two deletion alleles and one selective splice site variant. SSX1 deficiency was associated with a characteristic OAT phenotype featuring short, coiled flagella and severe loss of the central pair microtubules. SSX1 is expressed in early spermatocytes, and its loss disrupted the expression of spermiogenesis and sperm function related genes that are transcribed in advance during the spermatocyte stage. Mechanistically, SSX1 acts as a transcriptional regulator by interacting with the transcription and processing factor CPSF6 and promoting nuclear recruitment of the CPSF complex, with functional relevance in both spermatocytes and spermatids. Collectively, our study supports a functional role for SSX1 with CPSF6 in human spermatogenesis, expands the genetic diagnostic spectrum of OAT, and provides a transcription focused framework for precision management of male infertility. Biological sciences/Developmental biology/Germline development/Spermatogenesis Health sciences/Diseases/Reproductive disorders/Infertility Male infertility Oligoasthenoteratozoospermia SSX1 CPSF6 Transcriptional regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Reproductive and genetic disorders affect millions of families worldwide. Infertility is estimated to impact ~ 200 million individuals, with male factors contributing to roughly half of cases. About 1 in 20 men of reproductive age experiences subfertility or infertility. Clinically, this highly heterogeneous condition commonly presents as oligozoospermia, asthenozoospermia, and teratozoospermia (OAT), or, in severe cases, azoospermia. Accumulating evidence indicates that genetic defects account for ~ 30–50% of male infertility, with single-gene mutations representing the most frequent identifiable cause 1 , 2 , 3 . Spermatogenesis is the developmental process through which male germ cells are generated, encompassing spermatogonial proliferation and differentiation, meiosis, and spermiogenesis 4 . As a continuous and tightly coordinated program, disruption at any stage can compromise sperm output and quality, leading to reduced sperm number, impaired motility, and abnormal morphology. Notably, spermatogenesis is characterized by a unique regulatory feature, transcription-translation uncoupling 5 . During spermiogenesis, the nucleus undergoes profound chromatin compaction, driving global transcriptional silencing 6 . Consequently, many mRNAs encoding proteins required for late spermiogenesis are transcribed earlier and stored in the cytoplasm, and some spermiogenesis-related transcripts are even produced during meiosis 7 . This timing suggests that regulatory factors in spermatocytes may preconfigure transcriptional programs that ultimately support post-meiotic sperm morphogenesis. SSX1 (SSX family member 1; also known as cancer/testis antigen family 5, member 1) is normally functional in testicular germ cells 8 but is aberrantly activated in multiple malignancies, including synovial sarcoma and melanoma 9 . As a primate-specific gene, SSX1 deficiency has been linked to OAT phenotypes in humans, nonhuman primates, and tree shrews 8 . However, the physiological role of SSX1 during spermatogenesis remains insufficiently defined, and the mechanisms by which its loss leads to male infertility are still unclear. Because SSX1 contains an N-terminal, conserved Kruppel-associated box, a transcriptional repressor domain previously reported only in Kruppel-type zinc finger proteins 10 , we hypothesized that SSX1 may function as a transcriptional regulator and thereby control key gene-expression programs required for sperm development. In this study, we identified three hemizygous SSX1 variants in unrelated men with primary infertility characterized by OAT, and sperm from these individuals consistently exhibited typical OAT features. By integrating expression profiling, protein-protein interaction analyses, and single-cell transcriptomic datasets, we further uncovered that SSX1 associates with the RNA-processing/transcription-linked factor CPSF6 and functions as a spermatocyte-expressed transcriptional regulatory module that influences the expression of key proteins required for spermiogenesis. Results Identification of hemizygous variants in X-linked SSX1 in human with OAT. In this study, whole-exome sequencing was performed in a cohort of 536 men with OAT. The preliminarily filtered data were further analyzed according to the workflow described in our previous studies 11 , 12 . Three hemizygous variants in SSX1 were identified in three unrelated families with primary male infertility. In consanguineous family (Family AN001), a hemizygous 1- bp- insertion in SSX1(M1: c.189dupC, p.F63fs) were identified in F1- II- 1. In Family AN002, F2- II- 1 harboured hemizygous variants in SSX1 (M2: c.571 + 1G > A). In Family AN003, F3- II- 1 carried SSX1 hemizygous nonsense variant (M3: c.C517T:p.Q173X). Population frequency analysis revealed that all three variants were either absent or present at extremely low frequencies in the 1000 Genomes Project, ExAC (East Asian population), and gnomAD (East Asian population) (Table 1 ). No other pathogenic variants were found in known infertility-associated genes. The variants were verified by Sanger sequencing, and co-segregation analysis demonstrated an X-linked inheritance pattern (Fig. 1 A). Table 1 Genetic analysis of the patients with SSX1 -mutation Genetics analysis Patients AN001 AN0022 AN003 cDNA mutation c.189dupC c.571 + 1G>A c. C517T Protein alteration p.Phe63fs*9 - p.Q173X Exon Exon 4 - Exon 7 Variant type frameshift splice-site nonsense mutation Allele frequency 1KGP 0 0.0026 0 ExAC_EAS 0 0.0005 0 GnomAD_EAS 0 0.0010 0 CADD 27.4 24.4 33 Abbreviations:1KGP, 1000 Genomes Project; ExAC, Exome Aggregation Consortium; gnomAD, Genome Aggregation Database The SSX1 (NM_005635) gene is located on the X chromosome, according to the Human Protein Atlas. The protein contains a Kruppel-associated box domain at the N-terminus and an SSX repression domain at the C-terminus. In silico analysis revealed that the two altered residues (p.F63f and p.Q173X) occupy highly conserved positions among different species (Fig. 1 B). c.571 + 1G > A variant causes aberrant splicing of SSX1 transcripts To assess the effect of the novel c.571 + 1G > A variant on precursor mRNA splicing, we constructed SSX1 wild-type and mutant minigene plasmids for in vitro exon-trapping minigene assays (Figure S1 A). RT-PCR amplification of transcripts from cells transfected with the mutant construct produced a 292 bp band, whereas the wild-type construct yielded a 236 bp fragment corresponding to the correctly spliced product (Figure S1 B). Sanger sequencing further confirmed that the c.571 + 1G > A splice-site mutation led to partial retention of intronic sequence between exons 7 and 8, resulting in aberrant splicing and a frameshift mutation, which generated an unstable transcript (Figures S1 C and S1D). OAT phenotypes in the man harboring SSX1 p.F63f To evaluate the pathogenic effects of the novel variants, H&E staining and Western blot analyses were performed on testicular samples obtained from patient harboring SSX1 p.F63f . The results showed that testicular development and seminiferous tubule architecture were unaffected in the patient, with germ cells present at all stages of spermatogenesis. However, SSX1 protein was completely absent in the testicular tissue of the patients, and no truncated forms were detected (Figs. 2 A and 2 B). Immunofluorescence analysis using peanut agglutinin revealed a marked reduction in the number of round spermatids in the testicular samples from the patients (Figs. 2 C and 2 D). According to the WHO guidelines, semen analyses were performed during routine clinical evaluations. The semen analyses showed that all three patients had markedly reduced sperm concentration, motility, and progressive motility compared with the reference ranges, consistent with a typical OAT phenotype (Table 2 ). To characterize the defects underlying oligoasthenoteratozoospermia, H&E staining was performed to examine sperm morphology, and the proportion of morphologically abnormal spermatozoa was quantified. Compared with the normal control, spermatozoa from the patients displayed predominant tail abnormalities, with most presenting as short-tailed sperm (Fig. 2 E– 2 G). Subsequently, transmission electron microscopy was performed to examine the ultrastructure of spermatozoa. Compared with the typical “9 + 2” axonemal microtubule arrangement observed in the flagella of normal control, sperm from the man carrying the hemizygous SSX1 p.F63f variant exhibited loss of the central pair of microtubules in both the middle and principal pieces of the flagellum, along with disorganized peripheral dense fiber structures in the principal piece (Fig. 3 A and 3 B). To further investigate the impact of SSX1 deficiency on sperm flagellar assembly, we examined representative components of the dynein arms, radial spokes, central microtubules, and mitochondrial sheath. Immunofluorescence analysis revealed that, aside from the previously observed flagellar shortening, the localization of DNAH12, RSPH9, TOMM20, GAPDHS, and SPAG6 remained unaltered in the patient spermatozoa, in addition, peanut agglutinin staining showed no abnormalities in the acrosome structure (Fig. 3 C- 3 H), suggesting that SSX1 deficiency may not directly affect the expression or localization of these flagellar and acrosomal components 13 , 14 , 15 . Table 2 Clinical characteristics of the patients with SSX1 -mutation Clinical characteristics AN001 AN002 AN003 Reference limits Age (years) 32 31 35 Semen parameters Semen volume (mL) 1.5 3.8 5.6 1.5 Sperm concentration (10 6 /ml) 3.5 4 1.1 15 Progressive motility (%) 12.3 17.6 5 32 Motility (%) 25.6 35.1 20 40 Spermmorphology Normal sperm morphology (%) 2.46 0.99 1.21 ≥ 4 Abnormal sperm morphology (%) 97.54 99.01 98.79 ≤ 96 Karyotype 46, XY 46, XY 46, XY AZF deletion Undetectable Undetectable Undetectable SSX1 deficiency disrupts spermatogenic transcriptional programs and reduces spermatocyte number Given the testis-specific expression pattern of SSX1 , we further analyzed its expression dynamics across different stages of spermatogenesis. With ethical approval, 10× Genomics single-cell RNA sequencing was performed on testicular samples from patient harboring SSX1 p.F63fs and a normal control to construct a high-resolution transcriptional atlas of human spermatogenesis. We successfully identified 12 distinct cell clusters, including undifferentiated spermatogonia, differentiated spermatogonia (Diff SG), early spermatocyte and late spermatocyte, round spermatid and elongating spermatid, as well as various somatic cell compartments (Figure S4 A and S4B). Figure 4 A shows that SSX1 expression was initiated in differentiated spermatogonia and gradually increased, reaching a peak in spermatocytes, whereas little or no expression was detected in undifferentiated spermatogonia or elongating spermatids. To investigate the potential impact of SSX1 deficiency on the testicular transcriptome, we compared the single-cell transcriptomic profiles between patient harboring SSX1 p.F63f and a normal control. As shown in Fig. 4 B, extensive transcriptional alterations were observed from spermatocytes to elongating spermatids. Notably, the elongating spermatid stage exhibited a predominance of down-regulated genes, suggesting impaired germ-cell differentiation (Figure S4 D). Therefore, Gene Ontology (GO) analysis of the down-regulated genes in elongating spermatids revealed significant enrichment in pathways related to microtubule-based movement, cilium or flagellum-dependent cell motility, sperm motility, and fertilization (Fig. 4 C). Given that the proportion of round spermatids was reduced in the patient, possibly due to transcriptional dysregulation in spermatocytes caused by SSX1 deficiency, we further performed GO enrichment analysis on the down-regulated genes from early and late spermatocytes. As shown in Fig. 4 D, the significantly enriched biological processes in both cell types included cytoplasmic translation, ribonucleoprotein complex biogenesis. To quantify the relative number of spermatocytes, γH2AX (a marker of spermatocytes)–positive cells were counted and normalized to the number of SOX9-positive Sertoli cells, which served as an internal reference for seminiferous tubule cross-sections. The results showed that the number of spermatocytes in the testis of patient harboring SSX1 p.F63f was significantly reduced compared with that of the normal control (Fig. 4 E and 4 F). Collectively, these results suggest that SSX1 loss impairs global transcriptional programs essential for ribosomal function, protein synthesis, and cytoskeletal organization, which are required for meiotic progression and subsequent spermatid differentiation. SSX1 interacts with CPSF6 to regulate spermatogenic transcription To further elucidate the molecular mechanism by which SSX1 regulates spermatogenic transcription, we sought to identify its potential interacting proteins. Therefore, immunoprecipitation followed by mass spectrometry was performed using testicular lysates from a normal control to screen for SSX1-interacting proteins (Figure S3 A and S3B). Among the interacting proteins identified by mass spectrometry, CPSF6 (cleavage and polyadenylation specificity factor 6) attracted our attention (Fig. 5 A). CPSF6 is a component of the cleavage and polyadenylation complex that plays a crucial role in pre-mRNA 3’-end processing, transcription termination, and RNA transport, and has been implicated in the regulation of germ cell gene expression 16 . Further Western blot analysis revealed that the level of CPSF6 protein was markedly reduced in the testis of patient harboring SSX1 p.F63 compared with the control (Fig. 5 B). Immunofluorescence staining showed that in the control testis, CPSF6 was predominantly localized in the nuclei of spermatocytes, whereas its fluorescence signal was notably decreased in patient harboring SSX1 p.F63f (Fig. 5 C). These findings suggest that SSX1 may interact with CPSF6, and that loss of SSX1 could lead to decreased expression or stability of CPSF6, thereby perturbing transcriptional regulation essential for spermatogenesis. Intracytoplasmic sperm injection could rescue infertility in SSX1 -deficient men rather than in vitro fertilization In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are widely used assisted reproductive technologies (ART) for infertile couples with reduced sperm motility or abnormal morphology. Patient harboring SSX1 p.F63f initially underwent IVF treatment; however, no pregnancy was achieved. Following further evaluation and clinical counseling, the couple proceeded with ICSI. In the first ICSI cycle, fertilization was attempted but did not result in pregnancy. In the second ICSI cycle, 13 oocytes were retrieved from the patient’s partner, 3 of which were successfully fertilized, yielding a cleavage rate of 100%. Two embryos were subsequently transferred, leading to one successful implantation and the birth of a healthy full-term female infant. The partner of patient AN002 had 36 oocytes retrieved, 27 of which were successfully fertilized, with a cleavage rate of 96%; two embryos were transferred, leading to the birth of healthy twins (Table 3 ). Overall, our clinical data indicate that male infertility caused by SSX1 variants can be rescued through ICSI treatment. Table 3 ICSI outcomes of the SSX1- mutant patients AN001 AN002 ICSI treatment No. of ICSI cycles 2 1 No. of oocytes infected 13 36 Fertilization rate (%) 23 (3/13) 75 (27/36) Cleavage rate (%) 100 (3/3) 96 (26/27) Number of embryos transferred 2 2 Implantation rate (%) 50 (1/2) 100 (2/2 ) Clinical pregnancy Y Y Delivery Y Y Discussion In a cohort of 536 men with idiopathic OAT, we identified three individuals carrying homozygous SSX1 variants, including two loss-of-function deletions and one splice-site mutation. Analyses of SSX1 -deficient sperm revealed central pair microtubule defects in the flagellum and abnormal localization of key flagellar components. We further showed that SSX1 acts in spermatocytes, and its loss disrupts spermatocyte transcriptional programs, reduces spermatocyte abundance, and impairs the expression of proteins required for sperm formation, likely through association with CPSF6. These findings provide mechanistic insight into SSX1-related OAT and highlight SSX1 as a candidate gene for genetic diagnosis. Expanding the Genetic Basis of OAT, Informing Treatment Choices Genetic defects are an important cause of OAT. According to the Online Mendelian Inheritance in Man (OMIM) database, pathogenic variants in more than 100 genes have been implicated in male infertility 17 , yet a substantial fraction of OAT cases remain unexplained. By whole-exome sequencing of a cohort of 536 men with OAT, we identified three previously unreported SSX1 variants, thereby expanding the genetic landscape of OAT. In families AN001 and AN003, the variants c.189dupC (p.F63fs) and c.C517T (p.Q173X) were predicted loss-of-function alleles, and the AN002 proband carried a variant that was experimentally shown to cause aberrant splicing. Protein-level assessment and sperm phenotyping supported pathogenicity for the AN001 proband, consistent with prior reports. However, because sufficient sperm samples from AN002 and AN003 were not available, the pathogenicity of these two variants requires further validation. Identifying the causal genotype can also inform clinical decision-making. Intracytoplasmic sperm injection (ICSI) is an effective option for many men with OAT, but it is not universally successful. For example, embryos from patients with DZIP1-associated flagellar defects may arrest at the cleavage stage after ICSI 18 . In our study, two SSX1-mutant probands underwent ICSI at our center and achieved successful pregnancies, consistent with published findings 8 . These observations suggest that SSX1 deficiency does not cause a catastrophic defect of the sperm head and support ICSI as a reasonable treatment option for OAT patients carrying SSX1 variants. SSX1 is a spermatocyte-stage transcriptional regulator SSX1 is a primate-conserved gene, making it time-consuming to establish a stably breeding gene-edited animal model 8 . In addition, the limited performance of available antibodies has hampered the characterization of SSX1 expression and its interaction partners, including both proteins and RNAs. To address these challenges, we used transcriptomic analyses to show that SSX1 expression begins in early spermatocytes. We further identified CPSF6 as an SSX1-interacting protein by immunoprecipitation. Notably, SSX1 deficiency markedly altered CPSF6 protein abundance and subcellular localization, supporting the notion that SSX1 protein is already functional in early spermatocytes. Given the conserved transcriptional repressor-related domain within SSX1, we hypothesize that SSX1 contributes to transcriptional regulation during spermatogenesis. The concurrent disruption of CPSF6, a factor implicated in mRNA processing and reported to function in spermatogenesis 16 , 19 , further supports this model. Although direct genetic models for CPSF6 in germ cells remain limited, our data indicate that the SSX1-CPSF6 complex is tightly linked to spermatocyte transcriptome regulation. SSX1-CPSF6 loss reshapes spermatocyte transcriptomes to impair spermiogenesis Single-cell transcriptomic profiling showed minimal differences between the probands and controls before SSX1 expression, whereas pronounced transcriptomic perturbations emerged after meiotic initiation. This temporal pattern is consistent with SSX1-CPSF6 acting at the spermatocyte stage to shape downstream gene-expression programs. The precise mechanisms, including direct target RNAs, warrant further molecular studies. Because transcription and translation are uncoupled during spermatogenesis, many transcripts required for spermiogenesis are produced in late spermatocytes and stored for later use 6 , 20 . Functional annotation of differentially expressed genes in spermatocytes revealed that downregulated genes were enriched for sperm motility and fertilization functions rather than meiosis-related pathways. This finding suggests that SSX1 loss more directly compromises transcriptional programs supporting spermiogenesis. Moreover, many downregulated genes encoded post-transcriptional regulators, raising the possibility of a cascade effect that amplifies dysregulation during subsequent spermiogenesis. This aligns with the observation that spermiogenic defects in the probands’ testes were more prominent than meiotic abnormalities. Together, our single-cell transcriptomic and protein-interaction data support a model in which the SSX1-CPSF6 complex regulates the expression of RNAs required for sperm morphogenesis. Disruption of this module leads to severe OAT. Overall, in a cohort of 536 men with OAT, we identified three probands carrying pathogenic SSX1 variants. Their sperm exhibited severe flagellar malformations with profound disruption of the central pair microtubules. Using patient-derived samples, we characterized the expression pattern of SSX1, profiled the single-cell transcriptome of SSX1-deficient testes, and mapped SSX1-interacting proteins. These data support a model in which the SSX1-CPSF6 complex regulates spermatocyte-stage transcriptional programs associated with spermiogenesis, thereby shaping sperm morphogenesis. Collectively, our study expands the genetic etiology of OAT and offers a transcriptional regulatory perspective on the mechanisms underlying male infertility. Materials and methods Human subjects A total of 536 Chinese men diagnosed with OAT were recruited from the Reproductive Medical Center of the First Affiliated Hospital of Anhui Medical University (Hefei, China). Individuals with potential causes of infertility, such as abnormal chromosomal karyotypes or hormone levels, androgenic or endocrine disorders, testicular trauma or tumors, pathogenic Y-chromosome microdeletions, or seminal duct obstruction, were excluded. Semen analyses were performed according to the World Health Organization guidelines (5th edition, Kruger/Strict morphology criteria) 21 . Ethical approval for the study, granted under approval numbers of PJ2017-11-10 and PJ2020-13-10, was attained from the institution’s ethics committee. Patients provided explicit written consent, and the study strictly adhered to the principles set forth in the Declaration of Helsinki. Genetic analysis Whole-exome sequencing and subsequent bioinformatic analyses were performed as previously reported 2 . Genomic DNA was isolated from peripheral blood using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Exonic regions were captured with the Agilent SureSelectXT Human All Exon Kit (Agilent, San Jose, CA, USA) and sequenced on the Illumina HiSeq X-TEN platform (Illumina, San Diego, CA, USA). Sequence reads were aligned to the human reference genome (GRCh37/hg19) using Burrows–Wheeler Aligner, followed by variant calling with Genome Analysis Toolkit and functional annotation via ANNOVAR. Candidate variants and their parental inheritance were validated by Sanger sequencing. Histopathological analysis Testicular tissues were fixed in modified Davidson’s fluid for 24 hours, followed by dehydration through a graded ethanol series (70%, 80%, 90%, and 100%) and clearing with xylene. The tissues were then embedded in paraffin, sectioned continuously at a thickness of 5 μm, mounted on glass slides, stained with hematoxylin and eosin (H&E), and examined under a light microscope. Cell culture and transfection HEK293T cells were cultured in DMEM (Bio-Channel) with 10% fetal bovine serum (Bio-Channel) and penicillin/streptomycin (Biosharp). Plasmid transfection was carried out using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. For transfections in 6-well plates, 3 μg plasmid, 3.75 μl Lipofectamine 3000, and 6 μl P3000 were added to each well. Cells were harvested 24-36 hours post-transfection for subsequent analysis. Minigene splicing assay The SSX1-WT and SSX1-Mut plasmids were separately transfected into HEK293T cells. Cells were harvested 48 h after transfection, and total RNA was first extracted using TRIzol reagent. The isolated RNA was then reverse-transcribed into cDNA using a Takara reverse transcription kit. Subsequently, PCR amplification was performed using a Taq PCR Master Mix.The PCR products were resolved on a 3% agarose gel containing a nucleic acid staining dye. Two specific DNA bands were excised from the gel, purified, and the corresponding cDNA fragments were subjected to Sanger sequencing. Semen parameter analysis Semen analyses of oligoasthenoteratozoospermic subjects were performed in accredited clinical laboratories according to the World Health Organization (WHO) guidelines. Semen samples from the man carrying SSX1 variants were obtained by masturbation after 2–7 days of sexual abstinence and evaluated following 30 min of liquefaction at 37 ℃. The morphology and proportion of morphologically abnormal spermatozoa were assessed using H&E staining and transmission electron microscopy. Western Blot Analysis Human testicular tissues were lysed in RIPA buffer (P0013B, Beyotime) supplemented with protease and phosphatase inhibitors (P1049, Beyotime). The extracted proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (ISEQ00010, Merck Millipore). The membranes were blocked with 5% non-fat milk in TBST buffer at room temperature for 2 hours and incubated overnight at 4 ℃ with primary antibodies. After washing three times with TBST the next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 hours. Protein bands were visualized using the High-signal ECL chemiluminescent detection kit (Tanon, Shanghai, China). Immunofluorescence Immunofluorescence was carried out as described previously 22 . Paraffin-embedded tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval by heating in a microwave oven with Improved Citrate Antigen Retrieval Solution for 10 minutes. For sperm samples, semen smears were prepared on glass slides and fixed with 4% paraformaldehyde for 10 minutes, followed by permeabilization with 0.3% Triton X-100 at room temperature for 40 minutes. Both tissue sections and sperm samples were blocked with 10% donkey serum in PBS at room temperature for 2 hours, and then incubated with primary antibodies overnight at 4 ℃. The following day, samples were washed three times with PBST and incubated with Alexa Fluor 555-conjugated anti-rabbit or Alexa Fluor 488-conjugated anti-mouse secondary antibodies at room temperature for 2 hours. Before imaging, nuclei were stained with Hoechst 33342 for 5 minutes, washed with PBS, and mounted with glycerol.Images were acquired using an LSM980 confocal laser scanning microscope (Carl Zeiss AG). Immunoprecipitation IP–MS analysis was conducted as previously described 23 . Human testicular proteins were extracted, digested, and immunoprecipitated with anti-SSX1 (Proteintech) or anti-IgG (Proteintech) antibodies cross-linked to protein G magnetic beads. The resulting peptides were separated on a NanoLC Ultimate 3000 system equipped with an EasySpray column and analyzed on an Orbitrap Fusion Lumos mass spectrometer operating in data-dependent acquisition (DDA) mode. Raw spectra were processed using MaxQuant (v1.6.1.0) for label-free quantification, and peptide identification was performed against the human UniProt database. Common contaminants were excluded during data processing. The immunoprecipitation results were further validated by Western blotting using anti-CPSF6 (Abcam) antibodies. Single-cell RNA sequencing data analysis The matrix data were merged and re-analyzed by using the Seurat package (version: 4.4.0) under the R environment (version: 4.2) 24 . First, cell doublets were removed by using the Scrublet workflow 25 . The Harmony package (version: 1.0.1) was used to remove the batch effects of different samples. Then, cells were retained with at least 800 expressed genes and less than 25% of the reads mapped to the mitochondrial genome. The PCA reduction analysis was performed by using the top 5000 highly variable genes. Cell classification and annotation were based on the UMAP clusters, according to the canonical markers of different somatic and germ cell types 26, 27 . The FindMarker function embedded in Seurat was used to identify DEGs between different groups, based on the normalized expression values. Only genes with an average log2-transformed difference greater than 0.25, and an adjusted p value (FDR) less than 0.05 were defined as DEGs. The WebGestaltR package was used to perform functional enrichments analysis based on the embeded datasets of Gene Ontology (GO; including biological processes, cellular components, and molecular functions) and pathways (including Reactome and KEGG 28 . Statistical analysis Statistical analyses were performed using GraphPad Prism version 10.0. All data are presented as mean ± standard deviation. Differences between two genotypes were analyzed using a paired, two-tailed Student’s t -test. A P value < 0.05 was considered statistically significant. Declarations Data availability The supplementary materials contain all additional data used in this study. Further inquiries can be directed to the corresponding authors. Conflict of interest The authors declare that they have no conflict of interest. Author contribution Q.X. and X.S. conducted the primary experiments, analyzed the data, organized the datasets, and prepared the figures and tables for the manuscript. G.L., X.X., Y.L., M.L., Y.G. and M.X. participated in some experiments or provided technical support. C.X., H.G., X.H., Y.C., and H.W. collected clinical cases, established the disease specific cohort, and identified the probands included in this study. H.W., R.H. and Y.C. initiated the project, designed the experiments, and supervised the study. Q.X. and R.H. drafted the manuscript. H.W. and Y.C. revised the manuscript. All authors approved the final version of the manuscript. Acknowledgments We thank the patients and their families for their crucial support in our research. This study was funded by National Natural Science Foundation of China (82471648 to H.W.; 82430051 to Y.C. and 82571862 to R.H.). The National Key R&D Program of China (2023YFC2705504 to Y.C.); Anhui Provincial Natural Science Foundation (2308085QH253 to C.X.); the Key Research Program of Anhui Science and Technology Innovation Platform (202305a12020016 to Y.C.); the Research Funds of Center for Big Data and Population Health of IHM (JKS2023004 to H.W.); Scientific Research Platform Base Construction Foundation of Anhui Medical University (2023xkjT053 to Y.C.). and the Natural Foundation of Anhui Educational Committee (2022AH010072 to Y.C.). References Asero P, Calogero AE, Condorelli RA, Mongioi L, Vicari E, Lanzafame F , et al. Relevance of genetic investigation in male infertility. J Endocrinol Invest 2014, 37 (5) : 415-427. Wu H, Liu Y, Li Y, Li K, Xu C, Gao Y , et al. DNALI1 deficiency causes male infertility with severe asthenozoospermia in humans and mice by disrupting the assembly of the flagellar inner dynein arms and fibrous sheath. Cell Death Dis 2023, 14 (2) : 127. 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Additional Declarations There is no conflict of interest Supplementary Files SupplementaryTables.docx Supplemental Table. 1 Originalwesternblots.tif Original western blots SupplementaryFigures.docx Supplemental Figures Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 25 Mar, 2026 Review # 3 received at journal 17 Mar, 2026 Review # 2 received at journal 08 Mar, 2026 Review # 1 received at journal 04 Mar, 2026 Reviewer # 3 agreed at journal 23 Feb, 2026 Reviewer # 2 agreed at journal 23 Feb, 2026 Reviewer # 1 agreed at journal 23 Feb, 2026 Reviewers invited by journal 10 Feb, 2026 Submission checks completed at journal 07 Jan, 2026 Editor assigned by journal 04 Jan, 2026 First submitted to journal 04 Jan, 2026 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8516172","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":589255238,"identity":"dc380287-41f7-43c0-a39f-fbcc418ea3ed","order_by":0,"name":"Rong 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University","correspondingAuthor":false,"prefix":"","firstName":"Huan","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2026-01-05 02:26:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8516172/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8516172/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102962991,"identity":"b88f62a9-b925-4247-a1ad-3e1e3b9cb21d","added_by":"auto","created_at":"2026-02-19 04:12:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6367391,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of hemizygous \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSSX1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e variants in men with X-linked asthenoteratozoospermia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Pedigrees and Sanger sequencing chromatograms of the three affected families. The red boxes indicate the mutated nucleotide positions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e Evolutionary conservation and exon locations of the \u003cem\u003eSSX1\u003c/em\u003e\u003csup\u003e189dupC\u003c/sup\u003e and \u003cem\u003eSSX1\u003c/em\u003e\u003csup\u003eC517T \u003c/sup\u003evariants. KRAB, Kruppel-associated box; SSXRD, SSX repression domain.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8516172/v1/f688fd9654ac178075b4cbce.png"},{"id":102770966,"identity":"32ed8903-38bd-4f3c-8557-a73e42a251bd","added_by":"auto","created_at":"2026-02-16 12:33:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27835169,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDefective spermatogenesis and abnormal sperm morphology in a man carrying the SSX1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eF63fs\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e variant.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e H\u0026amp;E staining of testicular sections from a normal control and the man harboring the hemizygous SSX1 variant c.189dupC (p.F63fs). P, pachytene spermatocyte; rS, round spermatid; Sp, spermatid.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB \u003c/strong\u003eWestern blot analysis of SSX1 expression in testicular tissue from a normal control and the patient carrying the SSX1\u003csup\u003eF63fs\u003c/sup\u003e spermatozoa.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e Immunofluorescence staining of testicular sections from a normal control and the SSX1\u003csup\u003eF63fs\u003c/sup\u003e patient using PNA (red) and Hoechst (blue).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e Relative number of round spermatids per lumen in a normal control and the SSX1\u003csup\u003eF63fs\u003c/sup\u003e\u003cem\u003e \u003c/em\u003epatient. Data are presented as mean ± SD from three technical replicates (n = 3). Two-tailed unpaired Student’s t test, ****, P \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE \u003c/strong\u003eH\u0026amp;E staining of ejaculated spermatozoa from a normal control and the SSX1\u003csup\u003eF63fs\u003c/sup\u003e\u003cem\u003e \u003c/em\u003epatient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF \u003c/strong\u003ePercentage of morphologically abnormal spermatozoa in a normal control and the SSX1\u003csup\u003eF63fs\u003c/sup\u003e\u003cem\u003e \u003c/em\u003epatient based on routine semen analysis. Data are presented as mean ± SD from three technical replicates (n = 3). Two-tailed Student’s t test, ****, P \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG \u003c/strong\u003ePercentage of sperm with different tail morphology patterns in a normal control and the SSX1\u003csup\u003eF63fs\u003c/sup\u003e\u003cem\u003e \u003c/em\u003epatient based on routine semen analysis. n = 307 (Control), n = 224\u003c/p\u003e\n\u003cp\u003e(SSX1\u003csup\u003eF63fs\u003c/sup\u003e).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8516172/v1/1650741e9c96f967f77fd1e7.png"},{"id":102962424,"identity":"799f5e14-a202-465e-81e6-ba27816d398a","added_by":"auto","created_at":"2026-02-19 04:08:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11024175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUltrastructural and molecular defects in spermatozoa carrying the SSX1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eF63fs\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e variant.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Transmission electron microscopy (TEM) analysis of spermatozoa from a normal control and the man harboring the hemizygous SSX1\u003csup\u003eF63fs\u003c/sup\u003e\u003cem\u003e \u003c/em\u003evariant. CP, central pair of microtubules; ODF, outer dense fiber; DMT, double microtubule.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e Percentage of spermatozoa with central pair damage in the normal control and the SSX1\u003csup\u003eF63fs\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eindividual.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC \u003c/strong\u003eImmunofluorescence detection of the dynein arm–associated protein DNAH12 in spermatozoa of the normal control and the SSX1\u003csup\u003eF63fs\u003c/sup\u003e\u003cem\u003e \u003c/em\u003epatient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD \u003c/strong\u003eLocalization pattern of the radial spoke protein RSPH9 in control and SSX1\u003csup\u003eF63fs\u003c/sup\u003e spermatozoa as revealed by immunofluorescence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE \u003c/strong\u003eMitochondrial sheath integrity assessed by TOMM20 staining in spermatozoa from the normal control and the SSX1\u003csup\u003eF63fs\u003c/sup\u003e patient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF \u003c/strong\u003eCentral pair–associated protein SPAG6 visualized by immunofluorescence in control and SSX1\u003csup\u003eF63fs\u003c/sup\u003e spermatozoa.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG \u003c/strong\u003eDistribution of the fibrous sheath protein GAPDHS in spermatozoa of the normal control and the SSX1\u003csup\u003eF63fs\u003c/sup\u003e patient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH \u003c/strong\u003eAcrosomal status evaluated by PNA staining in control and SSX1\u003csup\u003eF63fs\u003c/sup\u003e spermatozoa.\u003c/p\u003e","description":"","filename":"Figure32.png","url":"https://assets-eu.researchsquare.com/files/rs-8516172/v1/4e6c8a3be16bec3006203df8.png"},{"id":102770969,"identity":"dc23e21c-de5e-4f18-92d6-2b02392ee4bd","added_by":"auto","created_at":"2026-02-16 12:33:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8402915,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSSX1 expression patterns and the impact of the SSX1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eF63fs\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e variant on spermatocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Violin plots showing gene expression in spermatogenic cells from a normal control and the patient carrying the SSX1\u003csup\u003eF63fs\u003c/sup\u003e loss-of-function variant, based on scRNA-seq data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB \u003c/strong\u003eVolcano plots depicting differentially expressed genes across testicular cell types between the control and the patient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e Gene Ontology (GO) enrichment analysis of differentially expressed genes in elongating spermatids from the control and the patient. ST, spermatids.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e GO enrichment analysis of differentially expressed genes in early spermatocytes (left) and late spermatocytes (right) from the control and the patient. SC, spermatocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE\u003c/strong\u003e Immunofluorescence staining for γH2AX and SOX9 in testicular sections from the control and the SSX1\u003csup\u003eF63fs\u003c/sup\u003e patient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e Quantification of the γH2AX/SOX9 signal ratio in testicular sections from the control and the patient. Data are presented as mean ± SD from three technical replicates (n = 3). Two-tailed unpaired Student’s t-test, ***, P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8516172/v1/bf704b572495311796237149.png"},{"id":102770965,"identity":"2ba1c5a7-ab79-4263-8562-f94c29b1108e","added_by":"auto","created_at":"2026-02-16 12:33:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10948508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification and validation of the SSX1-interacting protein CPSF6 in human testis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Scatter plot of SSX1-interacting proteins identified by mass spectrometry in testicular tissue from a man with obstructive azoospermia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e Western blot analysis of CPSF6, identified as an SSX1-interacting protein, in testicular tissue from a normal control and the SSX1\u003csup\u003eF63fs\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eindividual.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e Immunofluorescence staining for CPSF6 in testicular sections from a normal control and the patient.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8516172/v1/c609a71f4c06fdc7c2db040d.png"},{"id":109296196,"identity":"c10ddbeb-df3b-4870-b5b3-8bc1cd16f3be","added_by":"auto","created_at":"2026-05-15 08:46:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":91345558,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8516172/v1/bf2d1b67-1c02-4bce-8a9a-313fa23df537.pdf"},{"id":102770961,"identity":"d91add6c-00bb-4e9a-8540-a02fc16c1d59","added_by":"auto","created_at":"2026-02-16 12:33:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11849,"visible":true,"origin":"","legend":"Supplemental Table. 1","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8516172/v1/932dc7da14e5aa7c53a93362.docx"},{"id":102770963,"identity":"dd307dcb-e940-431c-b81c-2776b2e79177","added_by":"auto","created_at":"2026-02-16 12:33:34","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":875100,"visible":true,"origin":"","legend":"\u003cp\u003eOriginal western blots\u003c/p\u003e","description":"","filename":"Originalwesternblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-8516172/v1/cac54e14185c44c7ec5bd7f9.tif"},{"id":102770964,"identity":"64200255-2463-4017-95e7-01b50b587243","added_by":"auto","created_at":"2026-02-16 12:33:34","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5870846,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figures\u003c/p\u003e","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8516172/v1/34ce392db77466b597b2391f.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Primate-specific SSX1 is required for CPSF6-dependent spermatocyte transcription and human fertility","fulltext":[{"header":"Introduction","content":"\u003cp\u003eReproductive and genetic disorders affect millions of families worldwide. Infertility is estimated to impact\u0026thinsp;~\u0026thinsp;200\u0026nbsp;million individuals, with male factors contributing to roughly half of cases. About 1 in 20 men of reproductive age experiences subfertility or infertility. Clinically, this highly heterogeneous condition commonly presents as oligozoospermia, asthenozoospermia, and teratozoospermia (OAT), or, in severe cases, azoospermia. Accumulating evidence indicates that genetic defects account for ~\u0026thinsp;30\u0026ndash;50% of male infertility, with single-gene mutations representing the most frequent identifiable cause \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSpermatogenesis is the developmental process through which male germ cells are generated, encompassing spermatogonial proliferation and differentiation, meiosis, and spermiogenesis \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. As a continuous and tightly coordinated program, disruption at any stage can compromise sperm output and quality, leading to reduced sperm number, impaired motility, and abnormal morphology. Notably, spermatogenesis is characterized by a unique regulatory feature, transcription-translation uncoupling \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. During spermiogenesis, the nucleus undergoes profound chromatin compaction, driving global transcriptional silencing \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Consequently, many mRNAs encoding proteins required for late spermiogenesis are transcribed earlier and stored in the cytoplasm, and some spermiogenesis-related transcripts are even produced during meiosis \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. This timing suggests that regulatory factors in spermatocytes may preconfigure transcriptional programs that ultimately support post-meiotic sperm morphogenesis.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSSX1\u003c/em\u003e (SSX family member 1; also known as cancer/testis antigen family 5, member 1) is normally functional in testicular germ cells \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e but is aberrantly activated in multiple malignancies, including synovial sarcoma and melanoma \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. As a primate-specific gene, SSX1 deficiency has been linked to OAT phenotypes in humans, nonhuman primates, and tree shrews \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, the physiological role of SSX1 during spermatogenesis remains insufficiently defined, and the mechanisms by which its loss leads to male infertility are still unclear. Because SSX1 contains an N-terminal, conserved Kruppel-associated box, a transcriptional repressor domain previously reported only in Kruppel-type zinc finger proteins \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, we hypothesized that SSX1 may function as a transcriptional regulator and thereby control key gene-expression programs required for sperm development.\u003c/p\u003e \u003cp\u003eIn this study, we identified three hemizygous SSX1 variants in unrelated men with primary infertility characterized by OAT, and sperm from these individuals consistently exhibited typical OAT features. By integrating expression profiling, protein-protein interaction analyses, and single-cell transcriptomic datasets, we further uncovered that SSX1 associates with the RNA-processing/transcription-linked factor CPSF6 and functions as a spermatocyte-expressed transcriptional regulatory module that influences the expression of key proteins required for spermiogenesis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification of hemizygous variants in X-linked\u003c/b\u003e \u003cb\u003eSSX1\u003c/b\u003e \u003cb\u003ein human with OAT.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study, whole-exome sequencing was performed in a cohort of 536 men with OAT. The preliminarily filtered data were further analyzed according to the workflow described in our previous studies \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Three hemizygous variants in SSX1 were identified in three unrelated families with primary male infertility. In consanguineous family (Family AN001), a hemizygous 1- bp- insertion in SSX1(M1: c.189dupC, p.F63fs) were identified in F1- II- 1. In Family AN002, F2- II- 1 harboured hemizygous variants in \u003cem\u003eSSX1\u003c/em\u003e (M2: c.571\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A). In Family AN003, F3- II- 1 carried \u003cem\u003eSSX1\u003c/em\u003e hemizygous nonsense variant (M3: c.C517T:p.Q173X). Population frequency analysis revealed that all three variants were either absent or present at extremely low frequencies in the 1000 Genomes Project, ExAC (East Asian population), and gnomAD (East Asian population) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). No other pathogenic variants were found in known infertility-associated genes. The variants were verified by Sanger sequencing, and co-segregation analysis demonstrated an X-linked inheritance pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenetic analysis of the patients with \u003cem\u003eSSX1\u003c/em\u003e-mutation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenetics analysis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003ePatients\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAN001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAN0022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAN003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecDNA mutation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.189dupC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.571\u0026thinsp;+\u0026thinsp;1G\u0026gt;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ec. C517T\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein alteration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep.Phe63fs*9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep.Q173X\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExon 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExon 7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariant type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eframeshift\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003esplice-site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enonsense mutation\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAllele frequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1KGP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExAC_EAS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGnomAD_EAS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCADD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eAbbreviations:1KGP, 1000 Genomes Project; ExAC, Exome Aggregation Consortium; gnomAD, Genome Aggregation Database\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe \u003cem\u003eSSX1\u003c/em\u003e (NM_005635) gene is located on the X chromosome, according to the Human Protein Atlas. The protein contains a Kruppel-associated box domain at the N-terminus and an SSX repression domain at the C-terminus. In silico analysis revealed that the two altered residues (p.F63f and p.Q173X) occupy highly conserved positions among different species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003ec.571\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A variant causes aberrant splicing of\u003c/b\u003e \u003cb\u003eSSX1\u003c/b\u003e \u003cb\u003etranscripts\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess the effect of the novel c.571\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A variant on precursor mRNA splicing, we constructed \u003cem\u003eSSX1\u003c/em\u003e wild-type and mutant minigene plasmids for in vitro exon-trapping minigene assays (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). RT-PCR amplification of transcripts from cells transfected with the mutant construct produced a 292 bp band, whereas the wild-type construct yielded a 236 bp fragment corresponding to the correctly spliced product (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Sanger sequencing further confirmed that the c.571\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A splice-site mutation led to partial retention of intronic sequence between exons 7 and 8, resulting in aberrant splicing and a frameshift mutation, which generated an unstable transcript (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC and S1D).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOAT phenotypes in the man harboring SSX1\u003csup\u003ep.F63f\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eTo evaluate the pathogenic effects of the novel variants, H\u0026amp;E staining and Western blot analyses were performed on testicular samples obtained from patient harboring SSX1\u003csup\u003ep.F63f\u003c/sup\u003e. The results showed that testicular development and seminiferous tubule architecture were unaffected in the patient, with germ cells present at all stages of spermatogenesis. However, SSX1 protein was completely absent in the testicular tissue of the patients, and no truncated forms were detected (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Immunofluorescence analysis using peanut agglutinin revealed a marked reduction in the number of round spermatids in the testicular samples from the patients (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e According to the WHO guidelines, semen analyses were performed during routine clinical evaluations. The semen analyses showed that all three patients had markedly reduced sperm concentration, motility, and progressive motility compared with the reference ranges, consistent with a typical OAT phenotype (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To characterize the defects underlying oligoasthenoteratozoospermia, H\u0026amp;E staining was performed to examine sperm morphology, and the proportion of morphologically abnormal spermatozoa was quantified. Compared with the normal control, spermatozoa from the patients displayed predominant tail abnormalities, with most presenting as short-tailed sperm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Subsequently, transmission electron microscopy was performed to examine the ultrastructure of spermatozoa. Compared with the typical \u0026ldquo;9\u0026thinsp;+\u0026thinsp;2\u0026rdquo; axonemal microtubule arrangement observed in the flagella of normal control, sperm from the man carrying the hemizygous SSX1\u003csup\u003ep.F63f\u003c/sup\u003e variant exhibited loss of the central pair of microtubules in both the middle and principal pieces of the flagellum, along with disorganized peripheral dense fiber structures in the principal piece (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To further investigate the impact of \u003cem\u003eSSX1\u003c/em\u003e deficiency on sperm flagellar assembly, we examined representative components of the dynein arms, radial spokes, central microtubules, and mitochondrial sheath. Immunofluorescence analysis revealed that, aside from the previously observed flagellar shortening, the localization of DNAH12, RSPH9, TOMM20, GAPDHS, and SPAG6 remained unaltered in the patient spermatozoa, in addition, peanut agglutinin staining showed no abnormalities in the acrosome structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), suggesting that \u003cem\u003eSSX1\u003c/em\u003e deficiency may not directly affect the expression or localization of these flagellar and acrosomal components \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical characteristics of the patients with \u003cem\u003eSSX1\u003c/em\u003e-mutation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical characteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAN001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAN002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAN003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReference limits\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSemen parameters\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSemen volume (mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSperm concentration (10\u003csup\u003e6\u003c/sup\u003e/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProgressive motility (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMotility (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpermmorphology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal sperm morphology (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbnormal sperm morphology (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKaryotype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46, XY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46, XY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e46, XY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAZF deletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUndetectable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUndetectable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUndetectable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSSX1 deficiency disrupts spermatogenic transcriptional programs and reduces spermatocyte number\u003c/h3\u003e\n\u003cp\u003eGiven the testis-specific expression pattern of \u003cem\u003eSSX1\u003c/em\u003e, we further analyzed its expression dynamics across different stages of spermatogenesis. With ethical approval, 10\u0026times; Genomics single-cell RNA sequencing was performed on testicular samples from patient harboring SSX1\u003csup\u003ep.F63fs\u003c/sup\u003e and a normal control to construct a high-resolution transcriptional atlas of human spermatogenesis. We successfully identified 12 distinct cell clusters, including undifferentiated spermatogonia, differentiated spermatogonia (Diff SG), early spermatocyte and late spermatocyte, round spermatid and elongating spermatid, as well as various somatic cell compartments (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA and S4B). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA shows that SSX1 expression was initiated in differentiated spermatogonia and gradually increased, reaching a peak in spermatocytes, whereas little or no expression was detected in undifferentiated spermatogonia or elongating spermatids. To investigate the potential impact of SSX1 deficiency on the testicular transcriptome, we compared the single-cell transcriptomic profiles between patient harboring SSX1\u003csup\u003ep.F63f\u003c/sup\u003e and a normal control. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, extensive transcriptional alterations were observed from spermatocytes to elongating spermatids. Notably, the elongating spermatid stage exhibited a predominance of down-regulated genes, suggesting impaired germ-cell differentiation (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eD). Therefore, Gene Ontology (GO) analysis of the down-regulated genes in elongating spermatids revealed significant enrichment in pathways related to microtubule-based movement, cilium or flagellum-dependent cell motility, sperm motility, and fertilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Given that the proportion of round spermatids was reduced in the patient, possibly due to transcriptional dysregulation in spermatocytes caused by SSX1 deficiency, we further performed GO enrichment analysis on the down-regulated genes from early and late spermatocytes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, the significantly enriched biological processes in both cell types included cytoplasmic translation, ribonucleoprotein complex biogenesis. To quantify the relative number of spermatocytes, γH2AX (a marker of spermatocytes)\u0026ndash;positive cells were counted and normalized to the number of SOX9-positive Sertoli cells, which served as an internal reference for seminiferous tubule cross-sections. The results showed that the number of spermatocytes in the testis of patient harboring SSX1\u003csup\u003ep.F63f\u003c/sup\u003e was significantly reduced compared with that of the normal control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Collectively, these results suggest that SSX1 loss impairs global transcriptional programs essential for ribosomal function, protein synthesis, and cytoskeletal organization, which are required for meiotic progression and subsequent spermatid differentiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSSX1 interacts with CPSF6 to regulate spermatogenic transcription\u003c/h3\u003e\n\u003cp\u003eTo further elucidate the molecular mechanism by which SSX1 regulates spermatogenic transcription, we sought to identify its potential interacting proteins. Therefore, immunoprecipitation followed by mass spectrometry was performed using testicular lysates from a normal control to screen for SSX1-interacting proteins (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA and S3B). Among the interacting proteins identified by mass spectrometry, CPSF6 (cleavage and polyadenylation specificity factor 6) attracted our attention (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). CPSF6 is a component of the cleavage and polyadenylation complex that plays a crucial role in pre-mRNA 3\u0026rsquo;-end processing, transcription termination, and RNA transport, and has been implicated in the regulation of germ cell gene expression \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther Western blot analysis revealed that the level of CPSF6 protein was markedly reduced in the testis of patient harboring SSX1\u003csup\u003ep.F63\u003c/sup\u003e compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Immunofluorescence staining showed that in the control testis, CPSF6 was predominantly localized in the nuclei of spermatocytes, whereas its fluorescence signal was notably decreased in patient harboring SSX1\u003csup\u003ep.F63f\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These findings suggest that SSX1 may interact with CPSF6, and that loss of SSX1 could lead to decreased expression or stability of CPSF6, thereby perturbing transcriptional regulation essential for spermatogenesis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIntracytoplasmic sperm injection could rescue infertility in\u003c/b\u003e \u003cb\u003eSSX1\u003c/b\u003e\u003cb\u003e-deficient men rather than in vitro fertilization\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are widely used assisted reproductive technologies (ART) for infertile couples with reduced sperm motility or abnormal morphology. Patient harboring SSX1\u003csup\u003ep.F63f\u003c/sup\u003e initially underwent IVF treatment; however, no pregnancy was achieved. Following further evaluation and clinical counseling, the couple proceeded with ICSI. In the first ICSI cycle, fertilization was attempted but did not result in pregnancy. In the second ICSI cycle, 13 oocytes were retrieved from the patient\u0026rsquo;s partner, 3 of which were successfully fertilized, yielding a cleavage rate of 100%. Two embryos were subsequently transferred, leading to one successful implantation and the birth of a healthy full-term female infant. The partner of patient AN002 had 36 oocytes retrieved, 27 of which were successfully fertilized, with a cleavage rate of 96%; two embryos were transferred, leading to the birth of healthy twins (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Overall, our clinical data indicate that male infertility caused by SSX1 variants can be rescued through ICSI treatment.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eICSI outcomes of the \u003cem\u003eSSX1-\u003c/em\u003emutant patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAN001\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAN002\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eICSI treatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of ICSI cycles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of oocytes infected\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFertilization rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23 (3/13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75 (27/36)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCleavage rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 (3/3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96 (26/27)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of embryos transferred\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImplantation rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 (1/2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100 (2/2 )\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical pregnancy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eY\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDelivery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eY\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn a cohort of 536 men with idiopathic OAT, we identified three individuals carrying homozygous \u003cem\u003eSSX1\u003c/em\u003e variants, including two loss-of-function deletions and one splice-site mutation. Analyses of \u003cem\u003eSSX1\u003c/em\u003e-deficient sperm revealed central pair microtubule defects in the flagellum and abnormal localization of key flagellar components. We further showed that SSX1 acts in spermatocytes, and its loss disrupts spermatocyte transcriptional programs, reduces spermatocyte abundance, and impairs the expression of proteins required for sperm formation, likely through association with CPSF6. These findings provide mechanistic insight into SSX1-related OAT and highlight SSX1 as a candidate gene for genetic diagnosis.\u003c/p\u003e\n\u003ch3\u003eExpanding the Genetic Basis of OAT, Informing Treatment Choices\u003c/h3\u003e\n\u003cp\u003eGenetic defects are an important cause of OAT. According to the Online Mendelian Inheritance in Man (OMIM) database, pathogenic variants in more than 100 genes have been implicated in male infertility \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, yet a substantial fraction of OAT cases remain unexplained. By whole-exome sequencing of a cohort of 536 men with OAT, we identified three previously unreported \u003cem\u003eSSX1\u003c/em\u003e variants, thereby expanding the genetic landscape of OAT. In families AN001 and AN003, the variants c.189dupC (p.F63fs) and c.C517T (p.Q173X) were predicted loss-of-function alleles, and the AN002 proband carried a variant that was experimentally shown to cause aberrant splicing. Protein-level assessment and sperm phenotyping supported pathogenicity for the AN001 proband, consistent with prior reports. However, because sufficient sperm samples from AN002 and AN003 were not available, the pathogenicity of these two variants requires further validation.\u003c/p\u003e \u003cp\u003eIdentifying the causal genotype can also inform clinical decision-making. Intracytoplasmic sperm injection (ICSI) is an effective option for many men with OAT, but it is not universally successful. For example, embryos from patients with DZIP1-associated flagellar defects may arrest at the cleavage stage after ICSI \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In our study, two SSX1-mutant probands underwent ICSI at our center and achieved successful pregnancies, consistent with published findings \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These observations suggest that SSX1 deficiency does not cause a catastrophic defect of the sperm head and support ICSI as a reasonable treatment option for OAT patients carrying SSX1 variants.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSSX1 is a spermatocyte-stage transcriptional regulator\u003c/h2\u003e \u003cp\u003eSSX1 is a primate-conserved gene, making it time-consuming to establish a stably breeding gene-edited animal model \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In addition, the limited performance of available antibodies has hampered the characterization of SSX1 expression and its interaction partners, including both proteins and RNAs. To address these challenges, we used transcriptomic analyses to show that SSX1 expression begins in early spermatocytes. We further identified CPSF6 as an SSX1-interacting protein by immunoprecipitation. Notably, SSX1 deficiency markedly altered CPSF6 protein abundance and subcellular localization, supporting the notion that SSX1 protein is already functional in early spermatocytes.\u003c/p\u003e \u003cp\u003eGiven the conserved transcriptional repressor-related domain within SSX1, we hypothesize that SSX1 contributes to transcriptional regulation during spermatogenesis. The concurrent disruption of CPSF6, a factor implicated in mRNA processing and reported to function in spermatogenesis \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, further supports this model. Although direct genetic models for CPSF6 in germ cells remain limited, our data indicate that the SSX1-CPSF6 complex is tightly linked to spermatocyte transcriptome regulation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSSX1-CPSF6 loss reshapes spermatocyte transcriptomes to impair spermiogenesis\u003c/h3\u003e\n\u003cp\u003eSingle-cell transcriptomic profiling showed minimal differences between the probands and controls before SSX1 expression, whereas pronounced transcriptomic perturbations emerged after meiotic initiation. This temporal pattern is consistent with SSX1-CPSF6 acting at the spermatocyte stage to shape downstream gene-expression programs. The precise mechanisms, including direct target RNAs, warrant further molecular studies.\u003c/p\u003e \u003cp\u003eBecause transcription and translation are uncoupled during spermatogenesis, many transcripts required for spermiogenesis are produced in late spermatocytes and stored for later use \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Functional annotation of differentially expressed genes in spermatocytes revealed that downregulated genes were enriched for sperm motility and fertilization functions rather than meiosis-related pathways. This finding suggests that SSX1 loss more directly compromises transcriptional programs supporting spermiogenesis. Moreover, many downregulated genes encoded post-transcriptional regulators, raising the possibility of a cascade effect that amplifies dysregulation during subsequent spermiogenesis. This aligns with the observation that spermiogenic defects in the probands\u0026rsquo; testes were more prominent than meiotic abnormalities.\u003c/p\u003e \u003cp\u003eTogether, our single-cell transcriptomic and protein-interaction data support a model in which the SSX1-CPSF6 complex regulates the expression of RNAs required for sperm morphogenesis. Disruption of this module leads to severe OAT.\u003c/p\u003e \u003cp\u003eOverall, in a cohort of 536 men with OAT, we identified three probands carrying pathogenic SSX1 variants. Their sperm exhibited severe flagellar malformations with profound disruption of the central pair microtubules. Using patient-derived samples, we characterized the expression pattern of SSX1, profiled the single-cell transcriptome of SSX1-deficient testes, and mapped SSX1-interacting proteins. These data support a model in which the SSX1-CPSF6 complex regulates spermatocyte-stage transcriptional programs associated with spermiogenesis, thereby shaping sperm morphogenesis. Collectively, our study expands the genetic etiology of OAT and offers a transcriptional regulatory perspective on the mechanisms underlying male infertility.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eHuman subjects\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 536 Chinese men diagnosed with OAT were recruited from the Reproductive Medical Center of the First Affiliated Hospital of Anhui Medical University (Hefei, China). Individuals with potential causes of infertility, such as abnormal chromosomal karyotypes or hormone levels, androgenic or endocrine disorders, testicular trauma or tumors, pathogenic Y-chromosome microdeletions, or seminal duct obstruction, were excluded. Semen analyses were performed according to the World Health Organization guidelines (5th edition, Kruger/Strict morphology criteria) \u003csup\u003e21\u003c/sup\u003e. Ethical approval for the study, granted under approval numbers of PJ2017-11-10 and PJ2020-13-10, was attained from the institution’s ethics committee. Patients provided explicit written consent, and the study strictly adhered to the principles set forth in the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole-exome sequencing and subsequent bioinformatic analyses were performed as previously reported \u003csup\u003e2\u003c/sup\u003e. Genomic DNA was isolated from peripheral blood using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Exonic regions were captured with the Agilent SureSelectXT Human All Exon Kit (Agilent, San Jose, CA, USA) and sequenced on the Illumina HiSeq X-TEN platform (Illumina, San Diego, CA, USA). Sequence reads were aligned to the human reference genome (GRCh37/hg19) using Burrows–Wheeler Aligner, followed by variant calling with Genome Analysis Toolkit and functional annotation via ANNOVAR. Candidate variants and their parental inheritance were validated by Sanger sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTesticular tissues were fixed in modified Davidson’s fluid for 24 hours, followed by dehydration through a graded ethanol series (70%, 80%, 90%, and 100%) and clearing with xylene. The tissues were then embedded in paraffin, sectioned continuously at a thickness of 5 μm, mounted on glass slides, stained with hematoxylin and eosin (H\u0026amp;E), and examined under a light microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and transfection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293T cells were cultured in DMEM (Bio-Channel) with 10% fetal bovine serum (Bio-Channel) and penicillin/streptomycin (Biosharp). Plasmid transfection was carried out using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. For transfections in 6-well plates, 3 μg plasmid, 3.75 μl Lipofectamine 3000, and 6 μl P3000 were added to each well. Cells were harvested 24-36 hours post-transfection for subsequent analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMinigene splicing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SSX1-WT and SSX1-Mut plasmids were separately transfected into HEK293T cells. Cells were harvested 48 h after transfection, and total RNA was first extracted using TRIzol reagent. The isolated RNA was then reverse-transcribed into cDNA using a Takara reverse transcription kit. Subsequently, PCR amplification was performed using a Taq PCR Master Mix.The PCR products were resolved on a 3% agarose gel containing a nucleic acid staining dye. Two specific DNA bands were excised from the gel, purified, and the corresponding cDNA fragments were subjected to Sanger sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSemen parameter analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSemen analyses of oligoasthenoteratozoospermic subjects were performed in accredited clinical laboratories according to the World Health Organization (WHO) guidelines. Semen samples from the man carrying \u003cem\u003eSSX1\u003c/em\u003e variants were obtained by masturbation after 2–7 days of sexual abstinence and evaluated following 30 min of liquefaction at 37 ℃. The morphology and proportion of morphologically abnormal spermatozoa were assessed using H\u0026amp;E staining and transmission electron microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman testicular tissues were lysed in RIPA buffer (P0013B, Beyotime) supplemented with protease and phosphatase inhibitors (P1049, Beyotime). The extracted proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (ISEQ00010, Merck Millipore). The membranes were blocked with 5% non-fat milk in TBST buffer at room temperature for 2 hours and incubated overnight at 4 ℃ with primary antibodies. After washing three times with TBST the next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 hours. Protein bands were visualized using the High-signal ECL chemiluminescent detection kit (Tanon, Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence was carried out as described previously\u003csup\u003e22\u003c/sup\u003e. Paraffin-embedded tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval by heating in a microwave oven with Improved Citrate Antigen Retrieval Solution for 10 minutes. For sperm samples, semen smears were prepared on glass slides and fixed with 4% paraformaldehyde for 10 minutes, followed by permeabilization with 0.3% Triton X-100 at room temperature for 40 minutes. Both tissue sections and sperm samples were blocked with 10% donkey serum in PBS at room temperature for 2 hours, and then incubated with primary antibodies overnight at 4 ℃. The following day, samples were washed three times with PBST and incubated with Alexa Fluor 555-conjugated anti-rabbit or Alexa Fluor 488-conjugated anti-mouse secondary antibodies at room temperature for 2 hours. Before imaging, nuclei were stained with Hoechst 33342 for 5 minutes, washed with PBS, and mounted with glycerol.Images were acquired using an LSM980 confocal laser scanning microscope (Carl Zeiss AG).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIP–MS analysis was conducted as previously described \u003csup\u003e23\u003c/sup\u003e. Human testicular proteins were extracted, digested, and immunoprecipitated with anti-SSX1 (Proteintech) or anti-IgG (Proteintech) antibodies cross-linked to protein G magnetic beads. The resulting peptides were separated on a NanoLC Ultimate 3000 system equipped with an EasySpray column and analyzed on an Orbitrap Fusion Lumos mass spectrometer operating in data-dependent acquisition (DDA) mode. Raw spectra were processed using MaxQuant (v1.6.1.0) for label-free quantification, and peptide identification was performed against the human UniProt database. Common contaminants were excluded during data processing. The immunoprecipitation results were further validated by Western blotting using anti-CPSF6 (Abcam) antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-cell RNA sequencing data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe matrix data were merged and re-analyzed by using the Seurat package (version: 4.4.0) under the R environment (version: 4.2)\u003csup\u003e24\u003c/sup\u003e. First, cell doublets were removed by using the Scrublet workflow\u003csup\u003e25\u003c/sup\u003e. The Harmony package (version: 1.0.1) was used to remove the batch effects of different samples. Then, cells were retained with at least 800 expressed genes and less than 25% of the reads mapped to the mitochondrial genome. The PCA reduction analysis was performed by using the top 5000 highly variable genes. Cell classification and annotation were based on the UMAP clusters, according to the canonical markers of different somatic and germ cell types\u003csup\u003e26, 27\u003c/sup\u003e. The FindMarker function embedded in Seurat was used to identify DEGs between different groups, based on the normalized expression values. Only genes with an average log2-transformed difference greater than 0.25, and an adjusted p value (FDR) less than 0.05 were defined as DEGs. The WebGestaltR package was used to perform functional enrichments analysis based on the embeded datasets of Gene Ontology (GO; including biological processes, cellular components, and molecular functions) and pathways (including Reactome and KEGG\u003csup\u003e28\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism version 10.0. All data are presented as mean ± standard deviation. Differences between two genotypes were analyzed using a paired, two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. A \u003cem\u003eP\u003c/em\u003e value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supplementary materials contain all additional data used in this study. Further inquiries can be directed to the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQ.X. and X.S. conducted the primary experiments, analyzed the data, organized the datasets, and prepared the figures and tables for the manuscript. G.L., X.X., Y.L., M.L., Y.G. and M.X. participated in some experiments or provided technical support. C.X., H.G., X.H., Y.C., and H.W. collected clinical cases, established the disease specific cohort, and identified the probands included in this study. H.W., R.H. and Y.C. initiated the project, designed the experiments, and supervised the study. Q.X. and R.H. drafted the manuscript. H.W. and Y.C. revised the manuscript. All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the patients and their families for their crucial support in our research. This study was funded by National Natural Science Foundation of China (82471648 to H.W.; 82430051 to Y.C. and 82571862 to R.H.). The National Key R\u0026amp;D Program of China (2023YFC2705504 to Y.C.); Anhui Provincial Natural Science Foundation (2308085QH253 to C.X.); the Key Research Program of Anhui Science and Technology Innovation Platform (202305a12020016 to Y.C.); the Research Funds of Center for Big Data and Population Health of IHM (JKS2023004 to H.W.); Scientific Research Platform Base Construction Foundation of Anhui Medical University (2023xkjT053 to Y.C.). and the Natural Foundation of Anhui Educational Committee (2022AH010072 to Y.C.).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAsero P, Calogero AE, Condorelli RA, Mongioi L, Vicari E, Lanzafame F\u003cem\u003e, et al.\u003c/em\u003e Relevance of genetic investigation in male infertility. \u003cem\u003eJ Endocrinol Invest\u003c/em\u003e 2014, \u003cstrong\u003e37\u003c/strong\u003e(5)\u003cstrong\u003e:\u003c/strong\u003e 415-427.\u003c/li\u003e\n\u003cli\u003eWu H, Liu Y, Li Y, Li K, Xu C, Gao Y\u003cem\u003e, et al.\u003c/em\u003e DNALI1 deficiency causes male infertility with severe asthenozoospermia in humans and mice by disrupting the assembly of the flagellar inner dynein arms and fibrous sheath. \u003cem\u003eCell Death Dis\u003c/em\u003e 2023, \u003cstrong\u003e14\u003c/strong\u003e(2)\u003cstrong\u003e:\u003c/strong\u003e 127.\u003c/li\u003e\n\u003cli\u003eZhou S, Wu H, Zhang J, He X, Liu S, Zhou P\u003cem\u003e, et al.\u003c/em\u003e Bi-allelic variants in human TCTE1/DRC5 cause asthenospermia and male infertility. \u003cem\u003eEur J Hum Genet\u003c/em\u003e 2022, \u003cstrong\u003e30\u003c/strong\u003e(6)\u003cstrong\u003e:\u003c/strong\u003e 721-729.\u003c/li\u003e\n\u003cli\u003eD M dK, K L L, A M, D S, N W. 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WebGestalt 2024: faster gene set analysis and new support for metabolomics and multi-omics. \u003cem\u003eNucleic Acids Research\u003c/em\u003e 2024, \u003cstrong\u003e52\u003c/strong\u003e(W1)\u003cstrong\u003e:\u003c/strong\u003e W415-W421.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Male infertility, Oligoasthenoteratozoospermia, SSX1, CPSF6, Transcriptional regulation","lastPublishedDoi":"10.21203/rs.3.rs-8516172/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8516172/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGenetic variation is a major cause of male infertility, and defining the underlying mechanisms is essential for developing effective interventions. \u003cem\u003eSSX1\u003c/em\u003e is a primate specific gene whose deficiency has been linked to sperm malformations in humans and cynomolgus monkeys, yet its pathogenic mechanism remains unclear. Here, by whole exome sequencing of a cohort of 536 men with oligoasthenoteratozoospermia(OAT), we identified three previously unreported \u003cem\u003eSSX1\u003c/em\u003e variants, including two deletion alleles and one selective splice site variant. \u003cem\u003eSSX1\u003c/em\u003edeficiency was associated with a characteristic OAT phenotype featuring short, coiled flagella and severe loss of the central pair microtubules. SSX1 is expressed in early spermatocytes, and its loss disrupted the expression of spermiogenesis and sperm function related genes that are transcribed in advance during the spermatocyte stage. Mechanistically, SSX1 acts as a transcriptional regulator by interacting with the transcription and processing factor CPSF6 and promoting nuclear recruitment of the CPSF complex, with functional relevance in both spermatocytes and spermatids. Collectively, our study supports a functional role for SSX1 with CPSF6 in human spermatogenesis, expands the genetic diagnostic spectrum of OAT, and provides a transcription focused framework for precision management of male infertility.\u003c/p\u003e","manuscriptTitle":"Primate-specific SSX1 is required for CPSF6-dependent spermatocyte transcription and human fertility","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-16 12:33:25","doi":"10.21203/rs.3.rs-8516172/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-03-25T09:58:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-18T02:34:48+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-09T01:47:06+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-05T01:45:29+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-24T01:50:30+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-24T00:47:00+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-24T00:10:05+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-02-10T23:39:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-07T11:21:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-05T02:24:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death Discovery","date":"2026-01-05T02:24:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1dc163aa-79b8-4e74-aa2c-73da8bc817b3","owner":[],"postedDate":"February 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":62697208,"name":"Biological sciences/Developmental biology/Germline development/Spermatogenesis"},{"id":62697209,"name":"Health sciences/Diseases/Reproductive disorders/Infertility"}],"tags":[],"updatedAt":"2026-03-25T10:01:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-16 12:33:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8516172","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8516172","identity":"rs-8516172","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}