CD33 as a Leukocyte-Associated Marker Expressed on Human Spermatozoa

CD33 as a Leukocyte-Associated Marker Expressed on Human Spermatozoa | 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 CD33 as a Leukocyte-Associated Marker Expressed on Human Spermatozoa Nasrin Sereshki, Mitra Rafiee, Razieh Alipour, Sasan Navkhasi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-518727/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Sialic acid-binding immunoglobulin-type lectins (Siglecs) are commonly present on immune cells and often mediate cell-to-cell interactions and signaling. Studies have shown the presence of Siglecs 1, 2, 5, 6, 10 and 14 on human spermatozoa. To the best of our knowledge, the expression of CD33 on spermatozoa has not yet been studied. Methods Semen samples were collected from 25 healthy men with normal semen status. CD33 expression on purified spermatozoa was evaluated by flow cytometry methods. Results The results demonstrate the expression of CD33 on the surface of purified spermatozoa. The mean (± SD) of MFI (mean fluorescence intensity) was 12.85 (± 1.33) and the mean percentage of spermatozoa that express CD33 was 73.75 (± 3.75). Conclusion Results were obtained showing that spermatozoa express CD33 (or Siglec-3) on their surface. The physiological role of these molecules on spermatozoa remains to be determined. It is recommended that further research should be undertaken regarding the role of Siglecs (such as CD33) on spermatozoa apoptosis. Molecular Biology Spermatozoa CD33 Siglecs Figures Figure 1 1. Introduction Sialic acid is an essential component of the spermatozoa glycocalyx and is involved in functions of spermatozoa including motility, migration and interaction with cumulus-oocyte complex (COC) ( 1 ). Sialic acids that bind to the end of glycans on the surface of cells and secrete glycoconjugates to form sialoglycans are ligands for sialic acid-binding immunoglobulin-like lectins (Siglecs) ( 2 , 3 ). Siglecs are immunoglobulin-type transmembrane proteins and comprise; a) V-set domain (sialic acid-binding N-terminal), b) C2-set domain (variable numbers of Ig domains), c) a transmembrane region and d) a cytosolic tail ( 1 ). Siglecs are commonly present on immune cells and often mediate cell-to-cell interactions and signaling ( 4 – 6 ). They transmit inhibitory or activating signals based on possessive ITIM or ITAM on the cytosolic tail ( 4 – 6 ). Siglecs bind to their ligands expressed on other cells (in trans) in order to communicate with neighboring cells and also interact with ligands expressed on the same cell (in cis) ( 7 ). There are two primary subsets of Siglecs based on their sequence similarities and evolutionary conservation; a) conserved Siglecs, including Siglecs 1, 2, 4 and 15 and b) rapidly evolved Siglecs, including Siglec-3 (CD33) in humans and Siglecs 5, 6, 7, 8, 9, 10, 11, 14 and 16 ( 3 ). One of the main roles of some of these receptors is the fertilization process ( 1 , 8 , 9 ). Studies have shown the presence of Siglecs 1, 2, 5, 6, 10 and 14 on human spermatozoa ( 1 ). To the best of our knowledge, the expression of CD33 on spermatozoa has not yet been studied. CD33 preferentially binds to α2-6- and α2-3-sialylated glycans on the surface of normal and leukemic cells ( 7 , 10 ). CD33 is mostly expressed on myeloid cells and on some lymphoid cells, such as NK cells ( 7 ). Studies have shown that CD33 is an inhibitory receptor and therefore has a role in immune regulation ( 11 ). We discovered that CD33 expresses on spermatozoa following inadvertently pouring anti-CD33 antibody (instead of the intended antibody) during an experiment on a semen sample. Therefore, this study seeks to address CD33 expression on the surface of human spermatozoa. The results of this study can be beneficial regarding the use of spermatozoa as a model to study the biological function of CD33 molecule. 2. Methods And Materials 2.1 Subjects Twenty-five healthy volunteers aged 25–56 years entered the study. Semen samples were collected by masturbation after 2–3 days of sexual abstinence. After semen analysis according to WHO standard guidelines (WHO, 2010), samples with normal quality (according to WHO reference intervals for values of semen parameters) were selected for the assessment of CD33 expression. Informed consent was obtained from all subjects who participated in this study. The protocol for this study was approved by the Ethics Committee of Isfahan University of Medical Sciences (Isfahan, Iran). The ethics committee approval letter number is IR.MUI.REC.1395.3.480. 2.2 Purification of Spermatozoa Density-gradient centrifugation technique was used for purification of spermatozoa. The procedure of purification is described in more detail elsewhere ( 12 ). In brief, 1ml of the spermatozoa suspension was carefully layered over a discontinuous gradient made by AllGrad 95% and 45%. After centrifugation at 400g for 18 minutes, the spermatozoa pellet at the bottom of the centrifuge tubes was washed and re-suspended in AllGrad Wash. The purified spermatozoa were assessed by microscopic visualization for lack of non-spermatozoa cell contamination and viability. 2.3 Flow Cytometry The presence of CD33 on the surface of spermatozoa was measured by direct immunofluorescence using a BD FACS Calibur (BD Biosciences, USA) flow cytometer. 1×10 6 spermatozoa were stained with phycoerythrin (PE) mouse anti-human CD33 (clone: MCD5, IQ Products, Groningen, Netherlands) at room temperature for 30 minutes and then run on flow cytometry. Data from 100,000 events were collected using Cell Quest software (Becton Dickinson). Antibody titration was performed and the optimal titer with the minimal background was selected. Unstained control was used as a negative control. Cell viability tests were not performed because abnormal and dead spermatozoa were removed by AllGrad solution before staining. FlowJo software version X was used for the data analysis. 3. Results CD33 expression on spermatozoa was evaluated by flow cytometric assay. A logarithmic mode of the side scatter (SSC) parameter versus a liner mode of the forward scatter (FSC) parameter was used to detect spermatozoa and a fitting gate was set around them. The corresponding histogram was used to determine the expression of CD33. The results demonstrate the expression of CD33 on the surface of purified spermatozoa (Fig. 1 ). The mean (± SD) of MFI (mean fluorescence intensity) was 12.85 (± 1.33) and the mean percentage of spermatozoa that express CD33 was 73.75 (± 3.75). 4. Discussion CD33 (Siglec-3) is an inhibitory sialoadhesin receptor that is expressed by human leukocytes ( 6 , 11 ) and has major roles in the regulation of these cell functions. Interestingly, this study revealed that CD33 (as a leukocyte marker) is expressed on the surface of human spermatozoa. We could not find any study in agreement with, or contrary to, our result and, to the best of our knowledge, this is the first time that the expression of CD33 on spermatozoa has been demonstrated. The role of other Siglecs expressed on spermatozoa has been studied. It has been determined that the interaction of Siglecs and sialic acids on spermatozoa and the cumulus-oocyte complex (COC) plays the main role in regulating the process of fertilization and embryo development ( 1 , 9 , 13 ). An in vitro study showed that removing sialic acid from spermatozoa resulted in decreased motility and mucus penetration, but increased zona pellucida binding and polyspermy ( 1 ). We think Siglecs, including CD33, on spermatozoa bind to cis ligands (bind to sialic acids on spermatozoa) and lead to the transmission of signals that govern and regulate spermatozoa functions such as motility, penetration, capacitation and acrosome reaction. Further work should be done to establish this hypothesis. Studies have shown that CD33 related Siglecs (rapidly evolved Siglecs mentioned in the Introduction) can induce apoptosis ( 2 ). Apoptosis is a process in which the contents of the cell are wrapped into a small package of membrane that these apoptotic bodies are removed by immune cells without induction of inflammation ( 14 ). We believe that CD33, and other CD33 related Siglecs, may have a role in spermatozoa apoptosis. Spermatozoa apoptosis is essential to remove DNA-damaged cells and prevent the fusion of these damaged cells with oocyte ( 15 ). It is recommended that further research should be undertaken regarding the role of Siglecs (such as CD33) on spermatozoa apoptosis. 5. Conclusion We have obtained satisfactory results showing that spermatozoa express CD33 (or Siglec-3) on their surface. The physiological role of this molecule on spermatozoa remains to be determined. It is recommended that further research should be undertaken regarding the role of Siglecs (such as CD33) on spermatozoa apoptosis. Declarations 6.1 Ethics approval and consent to participate Written informed consent was obtained from all subjects who participated in this study. The protocol for this study was approved by the Ethics Committee of Isfahan University of Medical Sciences (Isfahan, Iran). The ethics committee approval letter number is IR.MUI.REC.1395.3.480. 6.2 Consent for publication Not applicable. 6.3 Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 6.4 Competing interests None 6.5 Funding This study is financially supported by the authors. 6.6 Authors' contributions N.S.; Designed and performed experiments, analysed data and co-wrote the paper, M.T.; Performed experiments and co-wrote the paper and supervised the research, R.A.; Performed experiments, co-wrote the paper., S.N.; co-wrote the paper, D.W.; co-wrote, reviewed and edited the paper and V.H.; Performed the calculations. All authors contributed to the final manuscript. 6.7 Acknowledgements The authors would like to thank Mohadeseh Thogyani and Faezeh Abbasi Rad for their valuable insight and recommendations and for their contribution in briefing the volunteers and obtaining their informed consent. References Fernandez-Fuertes B, Blanco-Fernandez A, Reid CJ, Meade K, Fair S, Lonergan P. Removal of sialic acid from bull sperm decreases motility and mucus penetration ability but increases zona pellucida binding and polyspermic penetration in vitro. Reproduction. 2018;155(6):481-92. Crocker PR, Redelinghuys P. Siglecs as positive and negative regulators of the immune system. Portland Press Ltd.; 2008. Varki A. Natural ligands for CD33-related Siglecs? : Oxford University Press; 2009. Crocker PR, Paulson JC, Varki A. Siglecs and their roles in the immune system. Nature Reviews Immunology. 2007;7(4):255-66. Crocker PR, Varki A. Siglecs, sialic acids and innate immunity. Trends in immunology. 2001;22(6):337-42. Pillai S, Netravali IA, Cariappa A, Mattoo H. Siglecs and immune regulation. Annual review of immunology. 2012;30:357-92. Hernández‐Caselles T, Martínez‐Esparza M, Pérez‐Oliva AB, Quintanilla‐Cecconi AM, García‐Alonso A, Alvarez‐López DMR, et al. A study of CD33 (SIGLEC‐3) antigen expression and function on activated human T and NK cells: two isoforms of CD33 are generated by alternative splicing. Journal of leukocyte biology. 2006;79(1):46-58. Alkhodair K, Almhanna H, McGetrick J, Gedair S, Gallagher M, Fernandez-Fuertes B, et al. Siglec expression on the surface of human, bull and ram sperm. Reproduction. 2018;155(4):361-71. Ma F, Wu D, Deng L, Secrest P, Zhao J, Varki N, et al. Sialidases on mammalian sperm mediate deciduous sialylation during capacitation. Journal of Biological Chemistry. 2012;287(45):38073-9. Walter RB, Appelbaum FR, Estey EH, Bernstein ID. Acute myeloid leukemia stem cells and CD33-targeted immunotherapy. Blood. 2012;119(26):6198-208. Hernández-Caselles T, Miguel RC-S, Ruiz-Alcaraz AJ, García-Peñarrubia P. CD33 (Siglec-3) inhibitory function: role in the NKG2D/DAP10 activating pathway. Journal of immunology research. 2019;2019. Sereshki N, Andalib A, Ghahiri A, Mehrabian F, Sherkat R, Rezaei A, et al. The expression of human leukocyte antigen by human ejaculated spermatozoa. Molecular genetics & genomic medicine. 2019;7(12):e1005. Tecle E, Reynoso HS, Wang R, Gagneux P. The female reproductive tract contains multiple innate sialic acid-binding immunoglobulin-like lectins (Siglecs) that facilitate sperm survival. Journal of Biological Chemistry. 2019;294(31):11910-9. Taylor RC, Cullen SP, Martin SJ. Apoptosis: controlled demolition at the cellular level. Nature reviews Molecular cell biology. 2008;9(3):231-41. Nakidkina A, Kuzmina T. Apoptosis in Spermatozoa and Its Role in Deteriorating Semen Quality. Russian Journal of Developmental Biology. 2019;50(4):165-72. Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-518727","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":26596321,"identity":"e018f022-7175-4737-be1b-a230cfff11ea","order_by":0,"name":"Nasrin Sereshki","email":"","orcid":"","institution":"Isfahan university of medical sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nasrin","middleName":"","lastName":"Sereshki","suffix":""},{"id":26596322,"identity":"c262c679-05a2-4c1b-b4ed-3dc5a665695e","order_by":1,"name":"Mitra Rafiee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIie3PMUvDQBTA8fd4g1O4tdB+BUEo1KXfxDHQ7XDNEGumZKl7C1K/Ql0y3/ngXAL9ADoUAk4OGeNQ8V1HIYlugveHHFx4Px4HEAr9yfBgAOYE5C+JfETZAKELIYszmZRL5X/gMJHjSYFfg/lpbz+5LBhNk5jxeaFc87G9uVKFkDYpO8mkisGuq9fpjIk2d+WzXjNmuKpeOskIrg1H+VvsCUSl05kQwryHqBr4+Mm3pRA83jv9MEhGMcgMk99CUZbq3TCpwa7cwpMpTZzRj0Js71tUTE2bzmm2tzW+p0u93bM9tEk3+R6fTvPjeWn5m+FQKBT6J30Bsq1cAgrnnw0AAAAASUVORK5CYII=","orcid":"","institution":"Birjand universiry of medical sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mitra","middleName":"","lastName":"Rafiee","suffix":""},{"id":26596323,"identity":"df30e324-2895-4dbc-b949-4b7b8b573aa8","order_by":2,"name":"Razieh Alipour","email":"","orcid":"","institution":"Isfahan university of medical sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Razieh","middleName":"","lastName":"Alipour","suffix":""},{"id":26596324,"identity":"e971d12c-e756-4ed9-840d-f5944c8c66d3","order_by":3,"name":"Sasan Navkhasi","email":"","orcid":"","institution":"Hamadan university of medical sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sasan","middleName":"","lastName":"Navkhasi","suffix":""},{"id":26596325,"identity":"965eae89-d3c6-46d6-a5ae-1268ff2c7a0a","order_by":4,"name":"Vahid Ahmadipanah","email":"","orcid":"","institution":"Hamadan university of medical of sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Vahid","middleName":"","lastName":"Ahmadipanah","suffix":""},{"id":26596326,"identity":"a4124d6e-ac54-4bb4-a091-ac8a7c1b9a32","order_by":5,"name":"david wilkinson","email":"","orcid":"","institution":"university of Aberdeen","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"david","middleName":"","lastName":"wilkinson","suffix":""}],"badges":[],"createdAt":"2021-05-12 13:24:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-518727/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-518727/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":9159144,"identity":"ea023fae-6eb1-4367-a1ac-bc00c4c2a96a","added_by":"auto","created_at":"2021-05-13 21:30:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50582,"visible":true,"origin":"","legend":"Representative flow cytometry gating plots and histogram of unstained (ut) control and sample. Unstained control was used to differentiate the negative and positive population.","description":"","filename":"f1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-518727/v1/67230c08c40944c00f7ef76c.jpg"},{"id":24332981,"identity":"b452b99e-d416-4777-8228-84676d1786ae","added_by":"auto","created_at":"2022-07-26 09:11:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":277074,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-518727/v1/debb5b22-85b9-4e6e-be03-3548a7d16b19.pdf"}],"financialInterests":"","formattedTitle":"CD33 as a Leukocyte-Associated Marker Expressed on Human Spermatozoa","fulltext":[{"header":"1. Introduction","content":" \u003cp\u003eSialic acid is an essential component of the spermatozoa glycocalyx and is involved in functions of spermatozoa including motility, migration and interaction with cumulus-oocyte complex (COC) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Sialic acids that bind to the end of glycans on the surface of cells and secrete glycoconjugates to form sialoglycans are ligands for sialic acid-binding immunoglobulin-like lectins (Siglecs) (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSiglecs are immunoglobulin-type transmembrane proteins and comprise; a) V-set domain (sialic acid-binding N-terminal), b) C2-set domain (variable numbers of Ig domains), c) a transmembrane region and d) a cytosolic tail (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Siglecs are commonly present on immune cells and often mediate cell-to-cell interactions and signaling (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). They transmit inhibitory or activating signals based on possessive ITIM or ITAM on the cytosolic tail (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Siglecs bind to their ligands expressed on other cells (in trans) in order to communicate with neighboring cells and also interact with ligands expressed on the same cell (in cis) (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are two primary subsets of Siglecs based on their sequence similarities and evolutionary conservation; a) conserved Siglecs, including Siglecs 1, 2, 4 and 15 and b) rapidly evolved Siglecs, including Siglec-3 (CD33) in humans and Siglecs 5, 6, 7, 8, 9, 10, 11, 14 and 16 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). One of the main roles of some of these receptors is the fertilization process (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Studies have shown the presence of Siglecs 1, 2, 5, 6, 10 and 14 on human spermatozoa (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, the expression of CD33 on spermatozoa has not yet been studied. CD33 preferentially binds to α2-6- and α2-3-sialylated glycans on the surface of normal and leukemic cells (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). CD33 is mostly expressed on myeloid cells and on some lymphoid cells, such as NK cells (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Studies have shown that CD33 is an inhibitory receptor and therefore has a role in immune regulation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). We discovered that CD33 expresses on spermatozoa following inadvertently pouring anti-CD33 antibody (instead of the intended antibody) during an experiment on a semen sample. Therefore, this study seeks to address CD33 expression on the surface of human spermatozoa. The results of this study can be beneficial regarding the use of spermatozoa as a model to study the biological function of CD33 molecule.\u003c/p\u003e "},{"header":"2. Methods And Materials","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Subjects\u003c/h2\u003e \u003cp\u003eTwenty-five healthy volunteers aged 25\u0026ndash;56 years entered the study. Semen samples were collected by masturbation after 2\u0026ndash;3 days of sexual abstinence. After semen analysis according to WHO standard guidelines (WHO, 2010), samples with normal quality (according to WHO reference intervals for values of semen parameters) were selected for the assessment of CD33 expression. Informed consent was obtained from all subjects who participated in this study. The protocol for this study was approved by the Ethics Committee of Isfahan University of Medical Sciences (Isfahan, Iran). The ethics committee approval letter number is IR.MUI.REC.1395.3.480.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Purification of Spermatozoa\u003c/h2\u003e \u003cp\u003eDensity-gradient centrifugation technique was used for purification of spermatozoa. The procedure of purification is described in more detail elsewhere (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In brief, 1ml of the spermatozoa suspension was carefully layered over a discontinuous gradient made by AllGrad 95% and 45%. After centrifugation at 400g for 18 minutes, the spermatozoa pellet at the bottom of the centrifuge tubes was washed and re-suspended in AllGrad Wash. The purified spermatozoa were assessed by microscopic visualization for lack of non-spermatozoa cell contamination and viability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Flow Cytometry\u003c/h2\u003e \u003cp\u003eThe presence of CD33 on the surface of spermatozoa was measured by direct immunofluorescence using a BD FACS Calibur (BD Biosciences, USA) flow cytometer. 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e spermatozoa were stained with phycoerythrin (PE) mouse anti-human CD33 (clone: MCD5, IQ Products, Groningen, Netherlands) at room temperature for 30 minutes and then run on flow cytometry. Data from 100,000 events were collected using Cell Quest software (Becton Dickinson). Antibody titration was performed and the optimal titer with the minimal background was selected. Unstained control was used as a negative control. Cell viability tests were not performed because abnormal and dead spermatozoa were removed by AllGrad solution before staining. FlowJo software version X was used for the data analysis.\u003c/p\u003e \u003c/div\u003e "},{"header":"3. Results","content":" \u003cp\u003eCD33 expression on spermatozoa was evaluated by flow cytometric assay. A logarithmic mode of the side scatter (SSC) parameter versus a liner mode of the forward scatter (FSC) parameter was used to detect spermatozoa and a fitting gate was set around them. The corresponding histogram was used to determine the expression of CD33. The results demonstrate the expression of CD33 on the surface of purified spermatozoa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The mean (\u0026plusmn;\u0026thinsp;SD) of MFI (mean fluorescence intensity) was 12.85 (\u0026plusmn;\u0026thinsp;1.33) and the mean percentage of spermatozoa that express CD33 was 73.75 (\u0026plusmn;\u0026thinsp;3.75).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"4. Discussion","content":" \u003cp\u003eCD33 (Siglec-3) is an inhibitory sialoadhesin receptor that is expressed by human leukocytes (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) and has major roles in the regulation of these cell functions. Interestingly, this study revealed that CD33 (as a leukocyte marker) is expressed on the surface of human spermatozoa. We could not find any study in agreement with, or contrary to, our result and, to the best of our knowledge, this is the first time that the expression of CD33 on spermatozoa has been demonstrated.\u003c/p\u003e \u003cp\u003eThe role of other Siglecs expressed on spermatozoa has been studied. It has been determined that the interaction of Siglecs and sialic acids on spermatozoa and the cumulus-oocyte complex (COC) plays the main role in regulating the process of fertilization and embryo development (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAn in vitro study showed that removing sialic acid from spermatozoa resulted in decreased motility and mucus penetration, but increased zona pellucida binding and polyspermy (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). We think Siglecs, including CD33, on spermatozoa bind to cis ligands (bind to sialic acids on spermatozoa) and lead to the transmission of signals that govern and regulate spermatozoa functions such as motility, penetration, capacitation and acrosome reaction. Further work should be done to establish this hypothesis.\u003c/p\u003e \u003cp\u003eStudies have shown that CD33 related Siglecs (rapidly evolved Siglecs mentioned in the Introduction) can induce apoptosis (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Apoptosis is a process in which the contents of the cell are wrapped into a small package of membrane that these apoptotic bodies are removed by immune cells without induction of inflammation (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). We believe that CD33, and other CD33 related Siglecs, may have a role in spermatozoa apoptosis. Spermatozoa apoptosis is essential to remove DNA-damaged cells and prevent the fusion of these damaged cells with oocyte (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). It is recommended that further research should be undertaken regarding the role of Siglecs (such as CD33) on spermatozoa apoptosis.\u003c/p\u003e "},{"header":"5. Conclusion","content":"\u003cp\u003eWe have obtained satisfactory results showing that spermatozoa express CD33 (or Siglec-3) on their surface. The physiological role of this molecule on spermatozoa remains to be determined. It is recommended that further research should be undertaken regarding the role of Siglecs (such as CD33) on spermatozoa apoptosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e6.1 Ethics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all subjects who participated in this study. The protocol for this study was approved by the Ethics Committee of Isfahan University of Medical Sciences (Isfahan, Iran). The ethics committee approval letter number is IR.MUI.REC.1395.3.480.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.2 Consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.3 Availability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ecorresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.4 Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.5 \u003c/strong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is financially supported by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.6 \u003c/strong\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.S.; Designed and performed experiments, analysed data and co-wrote the paper, M.T.; Performed experiments and co-wrote the paper and supervised the research, R.A.; Performed experiments, co-wrote the paper., S.N.; co-wrote the paper, D.W.; co-wrote, reviewed and edited the paper and V.H.; Performed the calculations. All authors contributed to the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.7 Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Mohadeseh Thogyani and Faezeh Abbasi Rad for their valuable insight and recommendations and for their contribution in briefing the volunteers and obtaining their informed consent.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFernandez-Fuertes B, Blanco-Fernandez A, Reid CJ, Meade K, Fair S, Lonergan P. Removal of sialic acid from bull sperm decreases motility and mucus penetration ability but increases zona pellucida binding and polyspermic penetration in vitro. Reproduction. 2018;155(6):481-92.\u003c/li\u003e\n\u003cli\u003eCrocker PR, Redelinghuys P. Siglecs as positive and negative regulators of the immune system. Portland Press Ltd.; 2008.\u003c/li\u003e\n\u003cli\u003eVarki A. Natural ligands for CD33-related Siglecs? : Oxford University Press; 2009.\u003c/li\u003e\n\u003cli\u003eCrocker PR, Paulson JC, Varki A. Siglecs and their roles in the immune system. Nature Reviews Immunology. 2007;7(4):255-66.\u003c/li\u003e\n\u003cli\u003eCrocker PR, Varki A. Siglecs, sialic acids and innate immunity. Trends in immunology. 2001;22(6):337-42.\u003c/li\u003e\n\u003cli\u003ePillai S, Netravali IA, Cariappa A, Mattoo H. Siglecs and immune regulation. Annual review of immunology. 2012;30:357-92.\u003c/li\u003e\n\u003cli\u003eHern\u0026aacute;ndez‐Caselles T, Mart\u0026iacute;nez‐Esparza M, P\u0026eacute;rez‐Oliva AB, Quintanilla‐Cecconi AM, Garc\u0026iacute;a‐Alonso A, Alvarez‐L\u0026oacute;pez DMR, et al. A study of CD33 (SIGLEC‐3) antigen expression and function on activated human T and NK cells: two isoforms of CD33 are generated by alternative splicing. Journal of leukocyte biology. 2006;79(1):46-58.\u003c/li\u003e\n\u003cli\u003eAlkhodair K, Almhanna H, McGetrick J, Gedair S, Gallagher M, Fernandez-Fuertes B, et al. Siglec expression on the surface of human, bull and ram sperm. Reproduction. 2018;155(4):361-71.\u003c/li\u003e\n\u003cli\u003eMa F, Wu D, Deng L, Secrest P, Zhao J, Varki N, et al. Sialidases on mammalian sperm mediate deciduous sialylation during capacitation. Journal of Biological Chemistry. 2012;287(45):38073-9.\u003c/li\u003e\n\u003cli\u003eWalter RB, Appelbaum FR, Estey EH, Bernstein ID. Acute myeloid leukemia stem cells and CD33-targeted immunotherapy. Blood. 2012;119(26):6198-208.\u003c/li\u003e\n\u003cli\u003eHern\u0026aacute;ndez-Caselles T, Miguel RC-S, Ruiz-Alcaraz AJ, Garc\u0026iacute;a-Pe\u0026ntilde;arrubia P. CD33 (Siglec-3) inhibitory function: role in the NKG2D/DAP10 activating pathway. Journal of immunology research. 2019;2019.\u003c/li\u003e\n\u003cli\u003eSereshki N, Andalib A, Ghahiri A, Mehrabian F, Sherkat R, Rezaei A, et al. The expression of human leukocyte antigen by human ejaculated spermatozoa. Molecular genetics \u0026amp; genomic medicine. 2019;7(12):e1005.\u003c/li\u003e\n\u003cli\u003eTecle E, Reynoso HS, Wang R, Gagneux P. The female reproductive tract contains multiple innate sialic acid-binding immunoglobulin-like lectins (Siglecs) that facilitate sperm survival. Journal of Biological Chemistry. 2019;294(31):11910-9.\u003c/li\u003e\n\u003cli\u003eTaylor RC, Cullen SP, Martin SJ. Apoptosis: controlled demolition at the cellular level. Nature reviews Molecular cell biology. 2008;9(3):231-41.\u003c/li\u003e\n\u003cli\u003eNakidkina A, Kuzmina T. Apoptosis in Spermatozoa and Its Role in Deteriorating Semen Quality. Russian Journal of Developmental Biology. 2019;50(4):165-72.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Spermatozoa, CD33, Siglecs","lastPublishedDoi":"10.21203/rs.3.rs-518727/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-518727/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSialic acid-binding immunoglobulin-type lectins (Siglecs) are commonly present on immune cells and often mediate cell-to-cell interactions and signaling. Studies have shown the presence of Siglecs 1, 2, 5, 6, 10 and 14 on human spermatozoa. To the best of our knowledge, the expression of CD33 on spermatozoa has not yet been studied.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSemen samples were collected from 25 healthy men with normal semen status. CD33 expression on purified spermatozoa was evaluated by flow cytometry methods.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results demonstrate the expression of CD33 on the surface of purified spermatozoa. The mean (\u0026plusmn;\u0026thinsp;SD) of MFI (mean fluorescence intensity) was 12.85 (\u0026plusmn;\u0026thinsp;1.33) and the mean percentage of spermatozoa that express CD33 was 73.75 (\u0026plusmn;\u0026thinsp;3.75).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eResults were obtained showing that spermatozoa express CD33 (or Siglec-3) on their surface. The physiological role of these molecules on spermatozoa remains to be determined. It is recommended that further research should be undertaken regarding the role of Siglecs (such as CD33) on spermatozoa apoptosis.\u003c/p\u003e","manuscriptTitle":"CD33 as a Leukocyte-Associated Marker Expressed on Human Spermatozoa","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-05-13 21:30:26","doi":"10.21203/rs.3.rs-518727/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"08501dc7-63e1-4f07-a032-4d0c8e49c2c6","owner":[],"postedDate":"May 13th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":4309453,"name":"Molecular Biology"}],"tags":[],"updatedAt":"2022-07-26T09:11:17+00:00","versionOfRecord":[],"versionCreatedAt":"2021-05-13 21:30:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-518727","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-518727","identity":"rs-518727","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}