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Their testis-specific expression, together with their putative role in phosphorylation pathways, suggests that TSSKs have relevant roles in spermiogenesis, sperm function, or both. Independent Tssk3 and Tssk6 knockout mice, as well as the double Tssk1\/Tssk2 KO males, are sterile. However, the double KO results are silent regarding the individual roles of TSSK1 and TSSK2. The aim of this study was to develop independent mutant mouse models of Tssk1 and Tssk2, using CRISPR\/Cas9, to evaluate their independent roles in reproduction. Male heterozygous pups were used to establish one Tssk1 and two independent Tssk2 mutant lines. Natural mating mutant Tssk1 and Tssk2 homozygous males but not females were found to be sterile. Additionally, homozygous males have lower sperm numbers and decreased motility, and were infertile in vitro. Anti-TSSK2 antibodies were validated against Tssk2 mutants and used in Western blot and immunofluorescence experiments. TSSK2 is localized to the sperm head; importantly, it is present in the testes and sperm from Tssk1 mutant mice, confirming individual mutation. Our results indicate that both TSSK1 and TSSK2 are individually essential for male reproduction and support both kinases as suitable nonhormonal male contraceptive targets.<\/jats:p>","DOI":"10.3390\/biom15040601","type":"journal-article","created":{"date-parts":[[2025,4,18]],"date-time":"2025-04-18T06:23:56Z","timestamp":1744957436000},"page":"601","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Identification of TSSK1 and TSSK2 as Novel Targets for Male Contraception"],"prefix":"10.3390","volume":"15","author":[{"given":"Saman","family":"Nayyab","sequence":"first","affiliation":[{"name":"Department of Veterinary and Animal Sciences, University of Massachusetts-Amherst, Amherst, MA 01003, USA"},{"name":"Molecular and Cellular Biology Program, University of Massachusetts, Amherst, MA 01003, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u0131\u00e1 Gracia","family":"Gervasi","sequence":"additional","affiliation":[{"name":"Department of Animal Science, University of Connecticut, Storrs, CT 06269, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Darya A.","family":"Tourzani","sequence":"additional","affiliation":[{"name":"Department of Veterinary and Animal Sciences, University of Massachusetts-Amherst, Amherst, MA 01003, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yeva","family":"Shamailova","sequence":"additional","affiliation":[{"name":"Department of Animal Science, Rutgers University, New Brunswick, NJ 08901, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1091-5629","authenticated-orcid":false,"given":"Hiroki","family":"Akizawa","sequence":"additional","affiliation":[{"name":"Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mahboubeh","family":"Taghavi","sequence":"additional","affiliation":[{"name":"Department of Veterinary and Animal Sciences, University of Massachusetts-Amherst, Amherst, MA 01003, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7281-3471","authenticated-orcid":false,"given":"Wei","family":"Cui","sequence":"additional","affiliation":[{"name":"Animal Models Core Facility, Institute for Applied Life Sciences (IALS), University of Massachusetts-Amherst, Amherst, MA 01003, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rafael","family":"Fissore","sequence":"additional","affiliation":[{"name":"Department of Veterinary and Animal Sciences, University of Massachusetts-Amherst, Amherst, MA 01003, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana Maria","family":"Salicioni","sequence":"additional","affiliation":[{"name":"Department of Veterinary and Animal Sciences, University of Massachusetts-Amherst, Amherst, MA 01003, USA"},{"name":"Molecular and Cellular Biology Program, University of Massachusetts, Amherst, MA 01003, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8900-9460","authenticated-orcid":false,"given":"Gunda I.","family":"Georg","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry, Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3786-3753","authenticated-orcid":false,"given":"Elizabeth","family":"Snyder","sequence":"additional","affiliation":[{"name":"Department of Animal Science, Rutgers University, New Brunswick, NJ 08901, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9320-7518","authenticated-orcid":false,"given":"Pablo E.","family":"Visconti","sequence":"additional","affiliation":[{"name":"Department of Veterinary and Animal Sciences, University of Massachusetts-Amherst, Amherst, MA 01003, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,4,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1093\/molehr\/gaq071","article-title":"Expression and Localization of Five Members of the Testis-Specific Serine Kinase (Tssk) Family in Mouse and Human Sperm and Testis","volume":"17","author":"Li","year":"2010","journal-title":"Mol. Hum. Reprod."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Zhang, Z.D., Frankish, A., Hunt, T., Harrow, J., and Gerstein, M. (2010). Identification and Analysis of Unitary Pseudogenes: Historic and Contemporary Gene Losses in Humans and Other Primates. Genome Biol., 11.","DOI":"10.1186\/gb-2010-11-3-r26"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/0378-1119(94)90762-5","article-title":"Characterization of a Novel Murine Testis-Specific Serine\/Threonine Kinase","volume":"139","author":"Bielke","year":"1994","journal-title":"Gene"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1851","DOI":"10.1083\/jcb.139.7.1851","article-title":"A Novel Family of Serine\/Threonine Kinases Participating in Spermiogenesis","volume":"139","author":"Kueng","year":"1997","journal-title":"J. Cell Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1006\/geno.2001.6628","article-title":"Cloning and Chromosomal Localization of a Gene Encoding a Novel Serine\/Threonine Kinase Belonging to the Subfamily of Testis-Specific Kinases","volume":"77","author":"Visconti","year":"2001","journal-title":"Genomics"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1016\/S0925-4773(00)00255-0","article-title":"A Novel Member of the Testis Specific Serine Kinase Family, Tssk-3, Expressed in the Leydig Cells of Sexually Mature Mice","volume":"93","author":"Zuercher","year":"2000","journal-title":"Mech. Dev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1093\/biolre\/ioaa064","article-title":"Testis-Specific Serine Kinase Protein Family in Male Fertility and as Targets for Non-Hormonal Male Contraception","volume":"103","author":"Salicioni","year":"2020","journal-title":"Biol. Reprod."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1007\/s11596-015-1417-2","article-title":"Testis Specific Serine\/Threonine Protein Kinase 4 (TSSK4) Leads to Cell Apoptosis Relying on Its Kinase Activity","volume":"35","author":"Wang","year":"2015","journal-title":"J. Huazhong Univ. Sci. Technol. [Med. Sci.]"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2741","DOI":"10.1242\/jcs.047225","article-title":"Tssk6 Is Required for Izumo Relocalization and Gamete Fusion in the Mouse","volume":"122","author":"Sosnik","year":"2009","journal-title":"J. Cell Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"4250","DOI":"10.1128\/MCB.25.10.4250-4261.2005","article-title":"Identification and Characterization of SSTK, a Serine\/Threonine Protein Kinase Essential for Male Fertility","volume":"25","author":"Spiridonov","year":"2005","journal-title":"Mol. Cell. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"718","DOI":"10.1002\/mrd.23539","article-title":"TSSK3, a Novel Target for Male Contraception, Is Required for Spermiogenesis","volume":"88","author":"Nayyab","year":"2021","journal-title":"Mol. Reprod. Dev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"826","DOI":"10.1111\/andr.13314","article-title":"Testis-Specific Serine Kinase 3 Is Required for Sperm Morphogenesis and Male Fertility","volume":"11","author":"Nozawa","year":"2022","journal-title":"Andrology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1242\/jcs.059949","article-title":"Functional Transformation of the Chromatoid Body in Mouse Spermatids Requires Testis-Specific Serine\/Threonine Kinases","volume":"123","author":"Shang","year":"2010","journal-title":"J. Cell Sci."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/j.ydbio.2008.03.047","article-title":"Targeted Deletion of Tssk1 and 2 Causes Male Infertility due to Haploinsufficiency","volume":"319","author":"Xu","year":"2008","journal-title":"Dev. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of structural proteins during the assembly of the head of bacteriophage T4","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"808","DOI":"10.1016\/j.cell.2014.02.056","article-title":"Structurally distinct Ca2+ signaling domains of sperm flagella orchestrate tyrosine phosphorylation and motility","volume":"157","author":"Chung","year":"2014","journal-title":"Cell"},{"key":"ref_17","first-page":"523","article-title":"ICSI in the Mouse","volume":"476","author":"Stein","year":"2010","journal-title":"Methods Enzymol."},{"key":"ref_18","first-page":"e59913","article-title":"A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry","volume":"125","author":"Lima","year":"2017","journal-title":"J. Vis. Exp."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"340","DOI":"10.1093\/biolre\/ioad069","article-title":"ADAD1 Is Required for Normal Translation of Nuclear Pore and Transport Protein Transcripts in Spermatids of Mus musculus","volume":"109","author":"Potgieter","year":"2023","journal-title":"Biol. Reprod."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1111\/j.1365-2605.1993.tb01156.x","article-title":"Histological and Histopathological Evaluation of the Testis","volume":"16","author":"Russell","year":"1993","journal-title":"Int. J. Androl."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3810","DOI":"10.1016\/S0021-9258(17)41932-6","article-title":"p95, the major phosphotyrosine-containing protein in mouse spermatozoa, is a hexokinase with unique properties","volume":"269","author":"Kalab","year":"1994","journal-title":"J. Biol. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e979113","DOI":"10.4161\/21565562.2014.979113","article-title":"Kinases as Targets for Chemical Modulators: Structural Aspects and Their Role in Spermatogenesis","volume":"4","author":"Jenardhanan","year":"2014","journal-title":"Spermatogenesis"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"73","DOI":"10.14715\/cmb\/2024.70.12.10","article-title":"Investigating intrauterine exposure to methamphetamine on serine-threonine kinase pathway in male rat testis: METH effects serine-threonine kinase in spermatogenesis","volume":"70","author":"Zare","year":"2025","journal-title":"Cell. Mol. Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e50209","DOI":"10.7554\/eLife.50209","article-title":"HIPK4 Is Essential for Murine Spermiogenesis","volume":"9","author":"Crapster","year":"2020","journal-title":"eLife"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"100564","DOI":"10.1016\/j.mcpro.2023.100564","article-title":"STK33 Phosphorylates Fibrous Sheath Protein AKAP3\/4 to Regulate Sperm Flagella Assembly in Spermiogenesis","volume":"22","author":"Yu","year":"2023","journal-title":"Mol. Cell. Proteom."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2629","DOI":"10.1038\/s41467-023-38357-0","article-title":"Broad Phosphorylation Mediated by Testis-Specific Serine\/Threonine Kinases Contributes to Spermiogenesis and Male Fertility","volume":"14","author":"Zhang","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1159\/000492516","article-title":"Interstitial Deletion of 5q22.2q23.1 Including APC and TSSK1B in a Patient with Adenomatous Polyposis and Asthenoteratozoospermia","volume":"9","author":"Kadiyska","year":"2019","journal-title":"Mol. Syndromol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"204","DOI":"10.3892\/mmr.2022.12720","article-title":"Role of Testis-Specific Serine Kinase 1B in Undiagnosed Male Infertility","volume":"25","author":"Kadiyska","year":"2022","journal-title":"Mol. Med. Rep."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"374","DOI":"10.2164\/jandrol.107.004598","article-title":"Mutation Screening and Association Study of the TSSK4 Gene in Chinese Infertile Men with Impaired Spermatogenesis","volume":"29","author":"Su","year":"2008","journal-title":"J. Androl."},{"key":"ref_30","first-page":"12","article-title":"Unintended Pregnancy and Its Adverse Social and Economic Consequences on Health System: A Narrative Review Article","volume":"44","author":"Yazdkhasti","year":"2015","journal-title":"Iran. J. Public Health"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1007\/s13669-016-0140-8","article-title":"Male Hormonal Contraception: Where Are We Now?","volume":"5","author":"Wang","year":"2016","journal-title":"Curr. Obstet. Gynecol. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/j.pharmthera.2016.03.012","article-title":"The Current State of Male Hormonal Contraception","volume":"163","author":"Chao","year":"2016","journal-title":"Pharmacol. Ther."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1111\/andr.12110","article-title":"Male Hormonal Contraception: Looking Back and Moving Forward","volume":"4","author":"Roth","year":"2015","journal-title":"Andrology"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1016\/j.pep.2016.01.009","article-title":"Recombinant Production of Enzymatically Active Male Contraceptive Drug Target hTSSK2-Localization of the TSKS Domain Phosphorylated by TSSK2","volume":"121","author":"Shetty","year":"2016","journal-title":"Protein Express. Purif."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1857","DOI":"10.1002\/cmdc.201700503","article-title":"Potent Pyrimidine and Pyrrolopyrimidine Inhibitors of Testis-Specific Serine\/Threonine Kinase 2 (TSSK2)","volume":"12","author":"Hawkinson","year":"2017","journal-title":"ChemMedChem"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Delgado, M., Gallegos, Z., Stippec, S., McGlynn, K., Cobb, M.H., and Whitehurst, A.W. (2024). Testis-specific serine kinase 6 (TSSK6) is abnormally expressed in colorectal cancer and promotes oncogenic behaviors. J. Biol. Chem., 300.","DOI":"10.1101\/2024.01.08.574658"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/15\/4\/601\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:17:18Z","timestamp":1760030238000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/15\/4\/601"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,18]]},"references-count":36,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2025,4]]}},"alternative-id":["biom15040601"],"URL":"https:\/\/doi.org\/10.3390\/biom15040601","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,18]]}}}