Expression of Y-Chromosome-Encoded Specific Genes in Mouse and Human Brain Neurons and Their Potential Impact on Sex Differences

Expression of Y-Chromosome-Encoded Specific Genes in Mouse and Human Brain Neurons and Their Potential Impact on Sex Differences | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Expression of Y-Chromosome-Encoded Specific Genes in Mouse and Human Brain Neurons and Their Potential Impact on Sex Differences Yuan Junying Yuan, Huang Xiang Huang, Ren Laifeng Ren, Chen Ze Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9434261/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Traditional paradigms hold that Y-chromosome-encoded genes are predominantly expressed in the testes, where they regulate male sexual development and fertility. However, whether these genes are expressed in brain tissue and their functional significance therein have long been overlooked. In this study, we employed C4 single-cell sequencing technology and detected the expression of 11 Y-chromosome-encoded genes in the mouse brain, including DDX3Y, USP9Y, KDM5D, Eif2s3y, Uty, II3ra, II9r, Spry3, Asmt, Zfy1, and Zfy2. The expression of KDM5D, Eif2s3y, and Uty was validated by RNA in situ hybridization (RNA-ISH), and the protein expression of DDX3Y and USP9Y was confirmed by immunofluorescence staining. These genes were found to be predominantly expressed in neurons, with lower expression levels observed in glial cells. Comparative analysis of expression changes between 3-month-old and 14-month-old mouse brains revealed that Uty expression was upregulated in neurons of aged mice and became clearly detectable in glial cells; DDX3Y, KDM5D, and Eif2s3y also showed increased expression in neurons; whereas Usp9y expression remained low but detectable. Finally, DDX3Y expression was examined in human brain tissue and was found to be predominantly distributed in neurons of the cerebral gray matter, with lower expression in the white matter. Collectively, this study provides the first systematic evidence of in situ expression of Y-chromosome-encoded proteins in mammalian brain neurons and glial cells, offering a direct molecular basis for understanding male–female brain differences independent of sex hormones, and provides new insights into sex bias in neurological disorders. Y chromosome brain neuron sex difference single-cell sequencing DDX3Y Uty Figures Figure 1 Figure 2 Figure 3 Introduction Sex differences are widely observed in brain structure and function, encompassing cognitive abilities, emotional regulation, and the incidence and severity of various neurological disorders, including autism spectrum disorder, Parkinson's disease, and multiple sclerosis (de Vries & Forger, 2015 ; Berry et al., 2024 ). Traditional explanations have focused primarily on the regulatory effects of sex hormones, such as testosterone and estrogen, on brain development. However, the sex hormone theory cannot fully account for certain sex-specific neural phenotypes, particularly those that emerge early in development or in the absence of pronounced hormonal fluctuations (Johansson et al., 2016 ). The Y chromosome has long been regarded as the "male sex-determining chromosome," with its primary function thought to be confined to the reproductive system. In recent years, a few studies have suggested that Y-chromosome genes may be expressed in certain brain regions; however, systematic evidence at single-cell resolution remains lacking (Vakilian et al., 2015 ; Venkataramanan et al., 2021 ). In the present study, we employed single-cell sequencing, RNA in situ hybridization, immunofluorescence, and cross-age comparisons to systematically investigate the expression patterns of Y-chromosome-encoded genes in mouse and human brains, aiming to provide a new molecular perspective for sex-related neuroscience. 2. Materials and Methods Experimental Animals and Tissue Samples Mice: Male C57BL/6J mice were divided into two cohorts: young (3 months old) and aged (14 months old). Wild-type male mice from both age groups were euthanized by cervical dislocation. Brain tissues were collected and either fixed in 4% paraformaldehyde for coronal paraffin sectioning, or snap-frozen in liquid nitrogen and stored at − 80°C for subsequent experiments. Human brain tissue: Peritumoral tissue resected during glioma surgery was obtained. The study was approved by the Ethics Committee of Shanghai Outdo Biotech Co., Ltd. (Ethics approval number: SHYJS-CP-240101). Tissue samples were collected from the gray matter and white matter of the frontal lobe. Single-Cell Sequencing Single-cell sequencing was performed as described previously (Zhong et al., 2021 ). The detailed procedures were as follows. Sample preparation: The target region of the mouse brain (excluding the cerebellum and brainstem) was dissected and placed in pre-chilled 1× phosphate-buffered saline (PBS). Fresh tissues were snap-frozen in liquid nitrogen and stored at − 80°C. A single-nucleus suspension was subsequently prepared for single-nucleus sequencing. Droplet generation and cell capture: Using the DNBelab C-TaiM 4 single-cell droplet generator, the nuclei suspension, reverse transcription reagents, and dual beads (capture beads carrying cell barcodes and unique molecular identifiers [UMIs]; labeling beads carrying droplet barcodes) were loaded onto a microfluidic chip. Negative pressure was applied to generate water-in-oil droplets, each encapsulating a single nucleus and a single bead. Cell lysis and reverse transcription: Nuclei were lysed within the droplets to release mRNA. PolyT primers on the bead surface captured the mRNA, and reverse transcription was performed to generate cDNA with cell-specific barcodes. Library construction: Droplets were broken to recover the cDNA products, and PCR amplification was carried out to construct the sequencing library. Sequencing: Paired-end sequencing was performed on the MGISEQ-2000 platform (MGI), targeting 20,000–50,000 reads per cell. Data analysis: DNBC4 tools software was used for data quality control, alignment, and quantification to generate a gene expression matrix. Subsequent cell clustering and differential expression analysis were conducted using the Seurat tool kit. RNA In Situ Hybridization (RNA-ISH) Specific probes targeting KDM5D, Eif2s3y, and Uty were designed. Mouse brain paraffin sections were deparaffinized, digested with proteinase K, and dehydrated through a graded ethanol series. Pre-warmed (78°C) denaturation solution was applied to the slides and incubated for 8 minutes, followed by another round of graded ethanol dehydration. The probe mixture was then added and incubated at 37°C for 12–16 hours. Sections were washed once with hybridization solution and twice with Burrer E (10 minutes each wash). Finally, DAPI was applied and incubated for 20 minutes at room temperature in the dark. After two washes with PBS, glycerol was applied for mounting. Sections were observed under a fluorescence microscope to analyze cellular expression. Immunofluorescence Staining Paraffin-embedded mouse brain sections were first deparaffinized with xylene and then dehydrated through a graded series of alcohol solutions (100%, 95%, 85%, 75%, and 50%). Permeabilization was performed with 0.3% Triton X-100 for 10 minutes at 37°C. Sections were then immersed in antigen retrieval solution (sodium citrate, cat. no. C1032, Solarbio) and heated in a pressure cooker for antigen retrieval. After three washes with PBS, sections were blocked with bovine serum albumin (BSA) and subsequently incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-DDX3Y (A23794, Abconal) and anti-USP9Y (ab181842, Abcam). Following PBS washing, sections were incubated with fluorescence-labeled secondary antibodies for 40 minutes at 37°C, washed again with PBS, counterstained with DAPI (ZH0922, Merck) for nuclei, and observed under a fluorescence microscope. Reverse Transcription–Quantitative Polymerase Chain Reaction (RT-qPCR) Mouse brain tissues were collected, and total RNA was extracted using the Tiangen RNA Simple Total RNA Extraction Kit (lot no. B1201B). Extracted RNA was reverse-transcribed into cDNA using the Promega Reverse Transcription Kit. Real-time PCR was performed using the Promega GoTaq qPCR Master Mix (lot no. 0000577234) with the cDNA as template. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. All experiments were performed in at least triplicate. Relative mRNA levels were normalized to GAPDH mRNA values, and fold changes were quantified using the comparative CT (2 − ΔΔCT) method. Primer sequences are listed in Supplementary Table 1. Statistical Analysis Comparisons between two groups were performed using t-tests, and comparisons among multiple groups were performed using one-way analysis of variance. P < 0.05 was considered statistically significant. 3. Results Single-Cell Sequencing Identified 11 Y-Chromosome Genes Expressed in the Mouse Brain The markers used for cell cluster identification in single-cell sequencing are presented in Supplementary Table 2. Transcripts of the following Y-chromosome genes were detected in the male mouse brain: DDX3Y, USP9Y, KDM5D, Eif2s3y, Uty, II3ra, II9r, Spry3, Asmt, Zfy1, and Zfy2. Expression of these genes exhibited cell-type specificity, being predominantly enriched in neuronal subpopulations, with low expression levels observed in a subset of glial cells (Fig. 1 /2). RNA-ISH and Immunofluorescence Validated Y-Chromosome Gene Expression in Neurons RNA in situ hybridization (RNA-ISH) revealed that positive signals for KDM5D, Eif2s3y, and Uty were predominantly detected in neurons, with only minimal signals observed in glial cells. Immunofluorescence staining further confirmed that DDX3Y and USP9Y proteins were localized to the cytoplasm and perinuclear regions of neurons, but were not detected in glial cells. In human brain tissue, immunofluorescence staining showed that DDX3Y protein was widely distributed in neurons of the cerebral gray matter, whereas it was almost undetectable in the white matter, which is predominantly composed of axons and glial cells. This distribution pattern was highly consistent with that observed in mice. Age-Dependent Expression Changes: Uty Showed Clear Expression in Glial Cells In 14-month-old mice, Uty expression in neurons was significantly upregulated compared with that in 12-week-old mice (P < 0.01). Moreover, clear Uty signals were detected in glial cells for the first time. Expression levels of DDX3Y, KDM5D, and Eif2s3y were also increased in neurons of aged mice (P < 0.05). In contrast, Usp9y expression remained low but was still detectable by RT-PCR, with no significant difference observed between the two age groups. 4. Discussion In this study, we provide the first systematic evidence at single-cell resolution that multiple Y-chromosome-encoded genes are expressed in situ in neurons of male mouse and human brains. Notably, some of these genes exhibit age-dependent upregulation and ectopic expression in glial cells. These findings challenge the conventional view that Y-chromosome genes are confined to the reproductive system and open new avenues for sex-related neuroscience research. Challenging Traditional Views: Direct Effects of Y-Chromosome Genes on the Brain It has long been assumed that Y-chromosome genes are expressed exclusively in the testes and exert their effects on the brain indirectly, solely through sex hormones such as testosterone. Using single-cell sequencing, RNA-ISH, and immunofluorescence, our study provides direct evidence that Y-chromosome genes are expressed in situ in brain neurons and glial cells. This finding overturns the single-model paradigm of sex hormone mediation and suggests that Y-chromosome gene products may act as direct molecular regulators of brain function (Tanida et al., 2025 ; Xu et al., 2002 ). New Explanations for Sex Bias in Neurological Disorders Many neurological disorders—including autism spectrum disorder, Parkinson's disease, and schizophrenia—exhibit significantly higher incidence or greater severity in males. Traditional explanations have largely attributed this bias to sex hormones or dosage effects of X-linked genes. The present discovery of Y-chromosome gene expression in the brain, along with its age-dependent changes, raises the possibility that these genes directly modulate disease susceptibility (Dewing et al., 2006 ). For instance, the ectopic expression of Uty observed in glial cells of aged mice may contribute to the heightened susceptibility of males to certain neurodegenerative diseases, such as Parkinson's disease. Dynamic Changes of Y-Chromosome Genes During Aging: Beyond Y-Chromosome Loss X-chromosome inactivation and Y-chromosome loss (LOY) have emerged as major foci in aging research (Fritz García et al., 2024 ). Our study offers a complementary perspective: rather than focusing on global Y-chromosome loss, we examined how retained Y genes dynamically change with age. We found that Uty expression increases in neurons of aged mice and becomes clearly detectable in glial cells, while DDX3Y, KDM5D, and Eif2s3y also show increasing trends in neurons. These results indicate that the Y chromosome in the aging brain is not merely passively lost but may actively influence cellular function through transcriptional regulatory changes, including up regulation or down regulation (Ocañas et al., 2022 ). This dynamic view opens a new dimension in aging research, suggesting that Y-chromosome genes may function as active regulators of the aging process rather than passive markers of genomic instability. Potential Biomarkers This study identified 11 Y-chromosome genes expressed in the brain, some of which exhibit age-dependent expression changes. These observations point to potential clinical applications. If expression changes of a particular Y gene (e.g., Uty or DDX3Y) are significantly correlated with cognitive decline, such a gene could serve as a blood or cerebrospinal fluid biomarker for monitoring male brain aging or assessing the risk of neurodegenerative diseases (Graham et al., 2019 ; Rastad et al., 2023 ). Limitations Several limitations should be acknowledged. First, single-cell resolution was essential because traditional bulk RNA sequencing would have masked the low-abundance but cell-type-specific signals of Y-chromosome genes. In this study, only 11 of the 28 Y genes were detected (SRY was not detected), and these were largely confined to neuronal subpopulations, with some (e.g., Usp9y) expressed at very low levels. Second, functional experiments such as gene knockout or over expression were not performed; therefore, the specific functions of Y-chromosome genes in the brain require further validation. Third, only DDX3Y was examined in human samples; the expression profiles of other Y-chromosome genes in the human brain remain to be systematically characterized. Fourth, this study did not distinguish expression differences across distinct brain regions (e.g., cortex, hippocampus, hypothalamus); future studies should investigate the functional significance of Y genes in specific nuclei. Finally, the age comparison included only two time points (3 months and 14 months); finer age gradients will be necessary to delineate the temporal patterns of expression changes. Conclusion In summary, this study provides systematic evidence for the direct expression of Y-chromosome genes in the mammalian brain, challenges the traditional sex hormone mediation model, and offers new molecular perspectives on sex differences, neurological disease susceptibility, and brain aging mechanisms. Future research should integrate gene editing, behavioral studies, and human genetics to further elucidate the functions of Y-chromosome genes in the brain and their clinical implications in disease. Declarations This article has no funding support. Author Contribution H and Y wrote the main manuscript text.Y prepared Figures 2, 3, Supplemental Figure 1, and Table 1. C)prepared Figure 1 and Supplemental Table 2. All authors reviewed and approved the manuscript. References Berry ASF, Finucane BM, Myers SM, et al. A genome-first study of sex chromosome aneuploidies provides evidence of Y chromosome dosage effects on autism risk. Nat Commun. 2024;15(1):8897. Published 2024 Oct 15. doi:10.1038/s41467-024-53211-7 de Vries GJ, Forger NG. Sex differences in the brain: a whole body perspective. Biol Sex Differ. 2015;6:15. Published 2015 Aug 15. doi:10.1186/s13293-015-0032-z. Dewing P, Chiang CW, Sinchak K, et al. Direct regulation of adult brain function by the male-specific factor SRY. Curr Biol. 2006;16(4):415-420. doi:10.1016/j.cub.2006.01.017. Fritz García JHG, Keller Valsecchi CI, Basilicata MF. Sex as a biological variable in ageing: insights and perspectives on the molecular and cellular hallmarks. Open Biol. 2024 Oct;14(10):240177. doi: 10.1098/rsob.240177. Epub 2024 Oct 30. PMID: 39471841; PMCID: PMC11521605. Graham EJ, Vermeulen M, Vardarajan B, Bennett D, De Jager P, Pearse RV 2nd, Young-Pearse TL, Mostafavi S. Somatic mosaicism of sex chromosomes in the blood and brain. Brain Res. 2019 Oct 15;1721:146345. doi: 10.1016/j.brainres.2019.146345. Epub 2019 Jul 23. PMID: 31348909; PMCID: PMC6717667. Johansson MM, Lundin E, Qian X, et al. Spatial sexual dimorphism of X and Y homolog gene expression in the human central nervous system during early male development. Biol Sex Differ. 2016;7:5. Published 2016 Jan 12. doi:10.1186/s13293-015-0056-4. Ocañas SR, Ansere VA, Tooley KB, et al. Differential Regulation of Mouse Hippocampal Gene Expression Sex Differences by Chromosomal Content and Gonadal Sex. Mol Neurobiol. 2022;59(8):4669-4702. doi:10.1007/s12035-022-02860-0. Rastad S, Barjaste N, Lanjanian H, Moeini A, Kiani F, Masoudi-Nejad A. Parallel molecular alteration between Alzheimer's disease and major depressive disorder in the human brain dorsolateral prefrontal cortex: an insight from gene expression and methylation profile analyses. Genes Genet Syst. 2023;97(6):311-324. doi:10.1266/ggs.22-00022. Tanida T, Yokoyama T, Nakajima T, et al. Morphological Sex Reversal in the Sexually Dimorphic Nucleus of the Preoptic Area in the Hypothalamus Delineated by Calbindin D28k-Immunoreactive Cell Clusters in Y POS mice. Acta Histochem Cytochem. 2025;58(4):153-160. doi:10.1267/ahc.25-00020. Vakilian H, Mirzaei M, Sharifi Tabar M, et al. DDX3Y, a Male-Specific Region of Y Chromosome Gene, May Modulate Neuronal Differentiation. J Proteome Res. 2015;14(9):3474-3483. doi:10.1021/acs.jproteome.5b00512. Venkataramanan S, Gadek M, Calviello L, Wilkins K, Floor SN. DDX3X and DDX3Y are redundant in protein synthesis. RNA. 2021;27(12):1577-1588. doi:10.1261/rna.078926.121. Xu J, Burgoyne PS, Arnold AP. Sex differences in sex chromosome gene expression in mouse brain. Hum Mol Genet. 2002;11(12):1409-1419. doi:10.1093/hmg/11.12.1409. Zhong J, Tang G, Zhu J, et al. Single-cell brain atlas of Parkinson's disease mouse model. J Genet Genomics. 2021;48(4):277-288. doi:10.1016/j.jgg.2021.01.003. Additional Declarations No competing interests reported. Supplementary Files file.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 19 May, 2026 Reviews received at journal 14 May, 2026 Reviews received at journal 11 May, 2026 Reviewers agreed at journal 29 Apr, 2026 Reviewers agreed at journal 29 Apr, 2026 Reviewers agreed at journal 29 Apr, 2026 Reviewers agreed at journal 28 Apr, 2026 Reviewers invited by journal 27 Apr, 2026 Editor assigned by journal 22 Apr, 2026 Submission checks completed at journal 22 Apr, 2026 First submitted to journal 16 Apr, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9434261","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633091012,"identity":"9714faf8-fd8e-462c-9584-0087553a3de6","order_by":0,"name":"Yuan Junying Yuan","email":"","orcid":"","institution":"Central Laboratory of Shanxi Provincial Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"Junying","lastName":"Yuan","suffix":""},{"id":633091013,"identity":"bde889cb-d02c-4624-be0d-41f8d8ef7859","order_by":1,"name":"Huang Xiang Huang","email":"","orcid":"","institution":"Central Laboratory of Shanxi Provincial Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huang","middleName":"Xiang","lastName":"Huang","suffix":""},{"id":633091014,"identity":"702b1409-b3f9-4c96-ad0e-177d9183b389","order_by":2,"name":"Ren Laifeng Ren","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIie3QsQqCUBTG8RMXdLnleoOGHuFAUIvog7QYglvQ6OAgBDn2MELzkQNOQi/QYEuzvUB0DZq9bkH3v5zl+w33Athsv5gLQKm+nhDcmhGhSaPvvHASNCbQE7zKpTISm6O4E6W3oGQJCJm/HSQLdpCoecQXnlILdbLPh4gSgPw8cbzmWYSTnE2I21H14nh1lKgMiUSqcg5QjCAHopojxfqTI6O3uEXZUcahd2Zuu8wfJt92n2VkOu8Lx4xtNpvtz3oDaxBCOYcY5cAAAAAASUVORK5CYII=","orcid":"","institution":"Central Laboratory of Shanxi Provincial Cancer Hospital","correspondingAuthor":true,"prefix":"","firstName":"Ren","middleName":"Laifeng","lastName":"Ren","suffix":""},{"id":633091016,"identity":"a5b880e5-1813-429d-b06c-9379ceefb1b6","order_by":3,"name":"Chen Ze Chen","email":"","orcid":"","institution":"Central Laboratory of Shanxi Provincial Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"Ze","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2026-04-16 06:56:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9434261/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9434261/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108591393,"identity":"6bf6756e-6b82-4277-97a9-41ad0e912024","added_by":"auto","created_at":"2026-05-06 09:50:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":333072,"visible":true,"origin":"","legend":"\u003cp\u003eSingle-Cell Sequencing Identified 11 Y-Chromosome Genes Expressed in the Mouse Brain. (A) UMAP plot of single-cell subcluster classification. Left: 3M brain tissue; Right: 14M brain tissue. Purple: Astrocytes; Red: Excitatory neurons; Blue: Inhibitory neurons; Green: Microglia; Light blue: Oligodendrocytes. (B–K) Violin plots. Expression levels of Y-chromosome genes Ddx3y, Eif2s3y, Kdm5d, Uty, and Usp9y in astrocytes, excitatory neurons, inhibitory neurons, microglia, and oligodendrocytes from 3M and 14M brain tissues. Gray: Astrocytes; Blue: Excitatory neurons; Dark blue: Inhibitory neurons; Purple: Microglia; Red: Oligodendrocytes.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9434261/v1/561e69fdbebbdcfa810e6063.png"},{"id":108591394,"identity":"af5f72f7-4e6a-4526-8365-0a51c6aeff42","added_by":"auto","created_at":"2026-05-06 09:50:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":247346,"visible":true,"origin":"","legend":"\u003cp\u003e(A–D) RNA-ISH and Immunofluorescence Validated Y-Chromosome Gene Expression in Neurons.Coronal section FISH staining of brain tissue from 3M wild-type male mice. Detection of Y-chromosome genes Eif2s3y, Kdm5d, and Uty expression in brain tissue. Blue: DAPI; Green: Negative control, Eif2s3y, Kdm5d, Uty. (E–F) Immunofluorescence staining detecting DDX3Y and USP9Y expression in brain tissue. Blue: DAPI; Red: DDX3Y, USP9Y. (G) Immunofluorescence staining detecting DDX3Y expression in human brain gray matter and white matter. Blue: DAPI; Red: DDX3Y.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9434261/v1/6d683f2d80a8b0a9ae8283db.png"},{"id":108591395,"identity":"db019ec8-38d7-4412-b00b-c4701ea7d6b3","added_by":"auto","created_at":"2026-05-06 09:50:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":273424,"visible":true,"origin":"","legend":"\u003cp\u003eAge-Dependent Expression Changes: Uty Showed Clear Expression in Glial Cells. (A–E) Expression of Y-chromosome genes Ddx3y, Eif2s3y, Kdm5d, Uty, and Usp9y in brain astrocytes, excitatory neurons, inhibitory neurons, microglia, and oligodendrocytes. Red: 3M brain tissue; Blue: 14M brain tissue. (F–I) RT-qPCR detection of Ddx3y, Eif2s3y, Kdm5d, and Uty expression levels in brain tissues of wild-type male mice at 1M, 3M, 6M, and 10M (data are presented as mean of three independent experiments, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9434261/v1/53a557a9b19364b6ee138a22.png"},{"id":108805616,"identity":"d36c39c5-690c-40d0-82d5-2971f518b0b2","added_by":"auto","created_at":"2026-05-08 15:26:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1444341,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9434261/v1/14cd58ef-a63b-483f-bfe6-d1b5766c07eb.pdf"},{"id":108591392,"identity":"4c29b534-c9d1-472a-8368-f54d6894a212","added_by":"auto","created_at":"2026-05-06 09:50:31","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1541218,"visible":true,"origin":"","legend":"","description":"","filename":"file.docx","url":"https://assets-eu.researchsquare.com/files/rs-9434261/v1/f3da766312b061ca7840d9b7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Expression of Y-Chromosome-Encoded Specific Genes in Mouse and Human Brain Neurons and Their Potential Impact on Sex Differences","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSex differences are widely observed in brain structure and function, encompassing cognitive abilities, emotional regulation, and the incidence and severity of various neurological disorders, including autism spectrum disorder, Parkinson's disease, and multiple sclerosis (de Vries \u0026amp; Forger, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Berry et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Traditional explanations have focused primarily on the regulatory effects of sex hormones, such as testosterone and estrogen, on brain development. However, the sex hormone theory cannot fully account for certain sex-specific neural phenotypes, particularly those that emerge early in development or in the absence of pronounced hormonal fluctuations (Johansson et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The Y chromosome has long been regarded as the \"male sex-determining chromosome,\" with its primary function thought to be confined to the reproductive system. In recent years, a few studies have suggested that Y-chromosome genes may be expressed in certain brain regions; however, systematic evidence at single-cell resolution remains lacking (Vakilian et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Venkataramanan et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the present study, we employed single-cell sequencing, RNA in situ hybridization, immunofluorescence, and cross-age comparisons to systematically investigate the expression patterns of Y-chromosome-encoded genes in mouse and human brains, aiming to provide a new molecular perspective for sex-related neuroscience.\u003c/p\u003e "},{"header":"2. Materials and Methods","content":"\u003ch2\u003eExperimental Animals and Tissue Samples\u003c/h2\u003e\u003cp\u003eMice: Male C57BL/6J mice were divided into two cohorts: young (3 months old) and aged (14 months old). Wild-type male mice from both age groups were euthanized by cervical dislocation. Brain tissues were collected and either fixed in 4% paraformaldehyde for coronal paraffin sectioning, or snap-frozen in liquid nitrogen and stored at − 80°C for subsequent experiments. Human brain tissue: Peritumoral tissue resected during glioma surgery was obtained. The study was approved by the Ethics Committee of Shanghai Outdo Biotech Co., Ltd. (Ethics approval number: SHYJS-CP-240101). Tissue samples were collected from the gray matter and white matter of the frontal lobe.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSingle-Cell Sequencing\u003c/h2\u003e \u003cp\u003eSingle-cell sequencing was performed as described previously (Zhong et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). The detailed procedures were as follows. Sample preparation: The target region of the mouse brain (excluding the cerebellum and brainstem) was dissected and placed in pre-chilled 1× phosphate-buffered saline (PBS). Fresh tissues were snap-frozen in liquid nitrogen and stored at − 80°C. A single-nucleus suspension was subsequently prepared for single-nucleus sequencing. Droplet generation and cell capture: Using the DNBelab C-TaiM 4 single-cell droplet generator, the nuclei suspension, reverse transcription reagents, and dual beads (capture beads carrying cell barcodes and unique molecular identifiers [UMIs]; labeling beads carrying droplet barcodes) were loaded onto a microfluidic chip. Negative pressure was applied to generate water-in-oil droplets, each encapsulating a single nucleus and a single bead. Cell lysis and reverse transcription: Nuclei were lysed within the droplets to release mRNA. PolyT primers on the bead surface captured the mRNA, and reverse transcription was performed to generate cDNA with cell-specific barcodes. Library construction: Droplets were broken to recover the cDNA products, and PCR amplification was carried out to construct the sequencing library. Sequencing: Paired-end sequencing was performed on the MGISEQ-2000 platform (MGI), targeting 20,000–50,000 reads per cell. Data analysis: DNBC4 tools software was used for data quality control, alignment, and quantification to generate a gene expression matrix. Subsequent cell clustering and differential expression analysis were conducted using the Seurat tool kit.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA In Situ Hybridization (RNA-ISH)\u003c/h3\u003e\n\u003cp\u003eSpecific probes targeting KDM5D, Eif2s3y, and Uty were designed. Mouse brain paraffin sections were deparaffinized, digested with proteinase K, and dehydrated through a graded ethanol series. Pre-warmed (78°C) denaturation solution was applied to the slides and incubated for 8 minutes, followed by another round of graded ethanol dehydration. The probe mixture was then added and incubated at 37°C for 12–16 hours. Sections were washed once with hybridization solution and twice with Burrer E (10 minutes each wash). Finally, DAPI was applied and incubated for 20 minutes at room temperature in the dark. After two washes with PBS, glycerol was applied for mounting. Sections were observed under a fluorescence microscope to analyze cellular expression.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence Staining\u003c/h3\u003e\n\u003cp\u003eParaffin-embedded mouse brain sections were first deparaffinized with xylene and then dehydrated through a graded series of alcohol solutions (100%, 95%, 85%, 75%, and 50%). Permeabilization was performed with 0.3% Triton X-100 for 10 minutes at 37°C. Sections were then immersed in antigen retrieval solution (sodium citrate, cat. no. C1032, Solarbio) and heated in a pressure cooker for antigen retrieval. After three washes with PBS, sections were blocked with bovine serum albumin (BSA) and subsequently incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-DDX3Y (A23794, Abconal) and anti-USP9Y (ab181842, Abcam). Following PBS washing, sections were incubated with fluorescence-labeled secondary antibodies for 40 minutes at 37°C, washed again with PBS, counterstained with DAPI (ZH0922, Merck) for nuclei, and observed under a fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eReverse Transcription–Quantitative Polymerase Chain Reaction (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003eMouse brain tissues were collected, and total RNA was extracted using the Tiangen RNA Simple Total RNA Extraction Kit (lot no. B1201B). Extracted RNA was reverse-transcribed into cDNA using the Promega Reverse Transcription Kit. Real-time PCR was performed using the Promega GoTaq qPCR Master Mix (lot no. 0000577234) with the cDNA as template. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. All experiments were performed in at least triplicate. Relative mRNA levels were normalized to GAPDH mRNA values, and fold changes were quantified using the comparative CT (2 − ΔΔCT) method. Primer sequences are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eComparisons between two groups were performed using t-tests, and comparisons among multiple groups were performed using one-way analysis of variance. P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e "},{"header":"3. Results","content":"\u003ch2\u003eSingle-Cell Sequencing Identified 11 Y-Chromosome Genes Expressed in the Mouse Brain\u003c/h2\u003e\u003cp\u003eThe markers used for cell cluster identification in single-cell sequencing are presented in Supplementary Table\u0026nbsp;2. Transcripts of the following Y-chromosome genes were detected in the male mouse brain: DDX3Y, USP9Y, KDM5D, Eif2s3y, Uty, II3ra, II9r, Spry3, Asmt, Zfy1, and Zfy2. Expression of these genes exhibited cell-type specificity, being predominantly enriched in neuronal subpopulations, with low expression levels observed in a subset of glial cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e/2).\u003c/p\u003e\u003ch3\u003eRNA-ISH and Immunofluorescence Validated Y-Chromosome Gene Expression in Neurons\u003c/h3\u003e\u003cp\u003eRNA in situ hybridization (RNA-ISH) revealed that positive signals for KDM5D, Eif2s3y, and Uty were predominantly detected in neurons, with only minimal signals observed in glial cells. Immunofluorescence staining further confirmed that DDX3Y and USP9Y proteins were localized to the cytoplasm and perinuclear regions of neurons, but were not detected in glial cells. In human brain tissue, immunofluorescence staining showed that DDX3Y protein was widely distributed in neurons of the cerebral gray matter, whereas it was almost undetectable in the white matter, which is predominantly composed of axons and glial cells. This distribution pattern was highly consistent with that observed in mice.\u003c/p\u003e\u003ch3\u003eAge-Dependent Expression Changes: Uty Showed Clear Expression in Glial Cells\u003c/h3\u003e\u003cp\u003eIn 14-month-old mice, Uty expression in neurons was significantly upregulated compared with that in 12-week-old mice (P \u0026lt; 0.01). Moreover, clear Uty signals were detected in glial cells for the first time. Expression levels of DDX3Y, KDM5D, and Eif2s3y were also increased in neurons of aged mice (P \u0026lt; 0.05). In contrast, Usp9y expression remained low but was still detectable by RT-PCR, with no significant difference observed between the two age groups.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we provide the first systematic evidence at single-cell resolution that multiple Y-chromosome-encoded genes are expressed in situ in neurons of male mouse and human brains. Notably, some of these genes exhibit age-dependent upregulation and ectopic expression in glial cells. These findings challenge the conventional view that Y-chromosome genes are confined to the reproductive system and open new avenues for sex-related neuroscience research.\u003c/p\u003e\u003ch2\u003eChallenging Traditional Views: Direct Effects of Y-Chromosome Genes on the Brain\u003c/h2\u003e\u003cp\u003eIt has long been assumed that Y-chromosome genes are expressed exclusively in the testes and exert their effects on the brain indirectly, solely through sex hormones such as testosterone. Using single-cell sequencing, RNA-ISH, and immunofluorescence, our study provides direct evidence that Y-chromosome genes are expressed in situ in brain neurons and glial cells. This finding overturns the single-model paradigm of sex hormone mediation and suggests that Y-chromosome gene products may act as direct molecular regulators of brain function (Tanida et al., \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e; Xu et al., \u003cspan class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eNew Explanations for Sex Bias in Neurological Disorders\u003c/h2\u003e\u003cp\u003eMany neurological disorders—including autism spectrum disorder, Parkinson's disease, and schizophrenia—exhibit significantly higher incidence or greater severity in males. Traditional explanations have largely attributed this bias to sex hormones or dosage effects of X-linked genes. The present discovery of Y-chromosome gene expression in the brain, along with its age-dependent changes, raises the possibility that these genes directly modulate disease susceptibility (Dewing et al., \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e). For instance, the ectopic expression of Uty observed in glial cells of aged mice may contribute to the heightened susceptibility of males to certain neurodegenerative diseases, such as Parkinson's disease.\u003c/p\u003e\u003ch2\u003eDynamic Changes of Y-Chromosome Genes During Aging: Beyond Y-Chromosome Loss\u003c/h2\u003e\u003cp\u003eX-chromosome inactivation and Y-chromosome loss (LOY) have emerged as major foci in aging research (Fritz García et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). Our study offers a complementary perspective: rather than focusing on global Y-chromosome loss, we examined how retained Y genes dynamically change with age. We found that Uty expression increases in neurons of aged mice and becomes clearly detectable in glial cells, while DDX3Y, KDM5D, and Eif2s3y also show increasing trends in neurons. These results indicate that the Y chromosome in the aging brain is not merely passively lost but may actively influence cellular function through transcriptional regulatory changes, including up regulation or down regulation (Ocañas et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). This dynamic view opens a new dimension in aging research, suggesting that Y-chromosome genes may function as active regulators of the aging process rather than passive markers of genomic instability.\u003c/p\u003e\u003ch2\u003ePotential Biomarkers\u003c/h2\u003e\u003cp\u003eThis study identified 11 Y-chromosome genes expressed in the brain, some of which exhibit age-dependent expression changes. These observations point to potential clinical applications. If expression changes of a particular Y gene (e.g., Uty or DDX3Y) are significantly correlated with cognitive decline, such a gene could serve as a blood or cerebrospinal fluid biomarker for monitoring male brain aging or assessing the risk of neurodegenerative diseases (Graham et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Rastad et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eLimitations\u003c/h2\u003e\u003cp\u003eSeveral limitations should be acknowledged. First, single-cell resolution was essential because traditional bulk RNA sequencing would have masked the low-abundance but cell-type-specific signals of Y-chromosome genes. In this study, only 11 of the 28 Y genes were detected (SRY was not detected), and these were largely confined to neuronal subpopulations, with some (e.g., Usp9y) expressed at very low levels. Second, functional experiments such as gene knockout or over expression were not performed; therefore, the specific functions of Y-chromosome genes in the brain require further validation. Third, only DDX3Y was examined in human samples; the expression profiles of other Y-chromosome genes in the human brain remain to be systematically characterized. Fourth, this study did not distinguish expression differences across distinct brain regions (e.g., cortex, hippocampus, hypothalamus); future studies should investigate the functional significance of Y genes in specific nuclei. Finally, the age comparison included only two time points (3 months and 14 months); finer age gradients will be necessary to delineate the temporal patterns of expression changes.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study provides systematic evidence for the direct expression of Y-chromosome genes in the mammalian brain, challenges the traditional sex hormone mediation model, and offers new molecular perspectives on sex differences, neurological disease susceptibility, and brain aging mechanisms. Future research should integrate gene editing, behavioral studies, and human genetics to further elucidate the functions of Y-chromosome genes in the brain and their clinical implications in disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThis article has no funding support.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH and Y wrote the main manuscript text.Y prepared Figures 2, 3, Supplemental Figure 1, and Table 1. C)prepared Figure 1 and Supplemental Table 2. All authors reviewed and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBerry ASF, Finucane BM, Myers SM, et al. A genome-first study of sex chromosome aneuploidies provides evidence of Y chromosome dosage effects on autism risk. Nat Commun. 2024;15(1):8897. Published 2024 Oct 15. doi:10.1038/s41467-024-53211-7\u003c/li\u003e\n\u003cli\u003ede Vries GJ, Forger NG. Sex differences in the brain: a whole body perspective. Biol Sex Differ. 2015;6:15. Published 2015 Aug 15. doi:10.1186/s13293-015-0032-z.\u003c/li\u003e\n\u003cli\u003eDewing P, Chiang CW, Sinchak K, et al. Direct regulation of adult brain function by the male-specific factor SRY. Curr Biol. 2006;16(4):415-420. doi:10.1016/j.cub.2006.01.017.\u003c/li\u003e\n\u003cli\u003eFritz Garc\u0026iacute;a JHG, Keller Valsecchi CI, Basilicata MF. Sex as a biological variable in ageing: insights and perspectives on the molecular and cellular hallmarks. Open Biol. 2024 Oct;14(10):240177. doi: 10.1098/rsob.240177. Epub 2024 Oct 30. PMID: 39471841; PMCID: PMC11521605.\u003c/li\u003e\n\u003cli\u003eGraham EJ, Vermeulen M, Vardarajan B, Bennett D, De Jager P, Pearse RV 2nd, Young-Pearse TL, Mostafavi S. Somatic mosaicism of sex chromosomes in the blood and brain. Brain Res. 2019 Oct 15;1721:146345. doi: 10.1016/j.brainres.2019.146345. Epub 2019 Jul 23. PMID: 31348909; PMCID: PMC6717667.\u003c/li\u003e\n\u003cli\u003eJohansson MM, Lundin E, Qian X, et al. Spatial sexual dimorphism of X and Y homolog gene expression in the human central nervous system during early male development. Biol Sex Differ. 2016;7:5. Published 2016 Jan 12. doi:10.1186/s13293-015-0056-4.\u003c/li\u003e\n\u003cli\u003eOca\u0026ntilde;as SR, Ansere VA, Tooley KB, et al. Differential Regulation of Mouse Hippocampal Gene Expression Sex Differences by Chromosomal Content and Gonadal Sex. Mol Neurobiol. 2022;59(8):4669-4702. doi:10.1007/s12035-022-02860-0.\u003c/li\u003e\n\u003cli\u003eRastad S, Barjaste N, Lanjanian H, Moeini A, Kiani F, Masoudi-Nejad A. Parallel molecular alteration between Alzheimer\u0026apos;s disease and major depressive disorder in the human brain dorsolateral prefrontal cortex: an insight from gene expression and methylation profile analyses. Genes Genet Syst. 2023;97(6):311-324. doi:10.1266/ggs.22-00022.\u003c/li\u003e\n\u003cli\u003eTanida T, Yokoyama T, Nakajima T, et al. Morphological Sex Reversal in the Sexually Dimorphic Nucleus of the Preoptic Area in the Hypothalamus Delineated by Calbindin D28k-Immunoreactive Cell Clusters in Y POS mice. Acta Histochem Cytochem. 2025;58(4):153-160. doi:10.1267/ahc.25-00020.\u003c/li\u003e\n\u003cli\u003eVakilian H, Mirzaei M, Sharifi Tabar M, et al. DDX3Y, a Male-Specific Region of Y Chromosome Gene, May Modulate Neuronal Differentiation. J Proteome Res. 2015;14(9):3474-3483. doi:10.1021/acs.jproteome.5b00512.\u003c/li\u003e\n\u003cli\u003eVenkataramanan S, Gadek M, Calviello L, Wilkins K, Floor SN. DDX3X and DDX3Y are redundant in protein synthesis. RNA. 2021;27(12):1577-1588. doi:10.1261/rna.078926.121.\u003c/li\u003e\n\u003cli\u003eXu J, Burgoyne PS, Arnold AP. Sex differences in sex chromosome gene expression in mouse brain. Hum Mol Genet. 2002;11(12):1409-1419. doi:10.1093/hmg/11.12.1409.\u003c/li\u003e\n\u003cli\u003eZhong J, Tang G, Zhu J, et al. Single-cell brain atlas of Parkinson\u0026apos;s disease mouse model. J Genet Genomics. 2021;48(4):277-288. doi:10.1016/j.jgg.2021.01.003.\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":"behavioral-and-brain-functions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"babf","sideBox":"Learn more about [Behavioral and Brain Functions](http://behavioralandbrainfunctions.biomedcentral.com)","snPcode":"12993","submissionUrl":"https://submission.nature.com/new-submission/12993/3","title":"Behavioral and Brain Functions","twitterHandle":"@BBF_Journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Y chromosome, brain, neuron, sex difference, single-cell sequencing, DDX3Y, Uty","lastPublishedDoi":"10.21203/rs.3.rs-9434261/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9434261/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTraditional paradigms hold that Y-chromosome-encoded genes are predominantly expressed in the testes, where they regulate male sexual development and fertility. However, whether these genes are expressed in brain tissue and their functional significance therein have long been overlooked. In this study, we employed C4 single-cell sequencing technology and detected the expression of 11 Y-chromosome-encoded genes in the mouse brain, including DDX3Y, USP9Y, KDM5D, Eif2s3y, Uty, II3ra, II9r, Spry3, Asmt, Zfy1, and Zfy2. The expression of KDM5D, Eif2s3y, and Uty was validated by RNA in situ hybridization (RNA-ISH), and the protein expression of DDX3Y and USP9Y was confirmed by immunofluorescence staining. These genes were found to be predominantly expressed in neurons, with lower expression levels observed in glial cells. Comparative analysis of expression changes between 3-month-old and 14-month-old mouse brains revealed that Uty expression was upregulated in neurons of aged mice and became clearly detectable in glial cells; DDX3Y, KDM5D, and Eif2s3y also showed increased expression in neurons; whereas Usp9y expression remained low but detectable. Finally, DDX3Y expression was examined in human brain tissue and was found to be predominantly distributed in neurons of the cerebral gray matter, with lower expression in the white matter. Collectively, this study provides the first systematic evidence of in situ expression of Y-chromosome-encoded proteins in mammalian brain neurons and glial cells, offering a direct molecular basis for understanding male\u0026ndash;female brain differences independent of sex hormones, and provides new insights into sex bias in neurological disorders.\u003c/p\u003e","manuscriptTitle":"Expression of Y-Chromosome-Encoded Specific Genes in Mouse and Human Brain Neurons and Their Potential Impact on Sex Differences","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-06 09:50:27","doi":"10.21203/rs.3.rs-9434261/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-19T04:31:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-14T10:05:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T19:07:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"29526237036550987415383371118904420082","date":"2026-04-29T22:49:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16286153081118109092556271302703038917","date":"2026-04-29T19:16:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"131158340708620291194256515870260253870","date":"2026-04-29T16:30:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"126401292565743653927321219880116063975","date":"2026-04-28T13:41:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-27T15:17:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-22T05:11:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-22T05:11:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Behavioral and Brain Functions","date":"2026-04-16T06:43:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"behavioral-and-brain-functions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"babf","sideBox":"Learn more about [Behavioral and Brain Functions](http://behavioralandbrainfunctions.biomedcentral.com)","snPcode":"12993","submissionUrl":"https://submission.nature.com/new-submission/12993/3","title":"Behavioral and Brain Functions","twitterHandle":"@BBF_Journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c042caba-7175-46de-9682-81a7fb8df785","owner":[],"postedDate":"May 6th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-19T04:31:45+00:00","index":67,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-14T10:05:19+00:00","index":66,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T19:07:16+00:00","index":65,"fulltext":""},{"type":"reviewerAgreed","content":"29526237036550987415383371118904420082","date":"2026-04-29T22:49:31+00:00","index":61,"fulltext":""},{"type":"reviewerAgreed","content":"16286153081118109092556271302703038917","date":"2026-04-29T19:16:45+00:00","index":60,"fulltext":""},{"type":"reviewerAgreed","content":"131158340708620291194256515870260253870","date":"2026-04-29T16:30:26+00:00","index":59,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T09:50:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-06 09:50:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9434261","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9434261","identity":"rs-9434261","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}