{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,4]],"date-time":"2025-12-04T06:59:14Z","timestamp":1764831554619,"version":"3.46.0"},"reference-count":85,"publisher":"MDPI AG","issue":"23","license":[{"start":{"date-parts":[[2025,12,2]],"date-time":"2025-12-02T00:00:00Z","timestamp":1764633600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Universit\u00e4t St. G\u00e4llen to M\u00f3nica Vazquez-Levin and Lydie Lane"},{"name":"National Research Council of Argentina Grant","award":["CONICET: PIP2015#881"],"award-info":[{"award-number":["CONICET: PIP2015#881"]}]},{"name":"Fundaci\u00f3n R. Bar\u00f3n"},{"DOI":"10.13039\/100020087","name":"Fundaci\u00f3n Williams","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100020087","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Sperm proteases are involved in several gamete interaction events leading to fertilization. This report presents a detailed analysis of the expression and localization of serine protease PRSS38 in human and in mouse spermatozoa and its involvement in fertilization-related events, using bioinformatics, cellular, biochemical, molecular, and functional approaches. Bioinformatics analyses included genomics and data analysis, prediction of protein subcellular localization and post-translational modifications, Self-Organizing Maps (SOMs) unsupervised training with other serine proteases, protein modeling (AlphaFold), and genetic variant analysis. For cellular, biochemical, and functional studies, human semen samples were obtained from healthy normozoospermic volunteers, and cauda epididymal sperm were collected from adult Balb-c\/C57 mice. PRSS38 presence was detected in human and mouse sperm protein extracts by Western immunoblotting. Sperm PRSS38 subcellular localization was determined by fluorescence immunocytochemistry. Human sperm\u2013oocyte interaction events were assessed by means of the mouse Cumulus Penetration Assay (CPA) using mouse COCs, the Human Hemizona Assay (HZA), and the ZP-free hamster egg Sperm Penetration Assay (SPA). Mouse sperm\u2013oocyte interactions were evaluated by means of in vitro fertilization (IVF) with COCs and denuded oocytes. PRSS38 is proposed to be a GPI-anchored serine protease (active site: His-100, Asp-150, and Ser-245) based on bioinformatics analyses. Using commercial antibodies, protein forms of the expected Mr (human: 31 kDa; mouse: 32 and 24 kDa) were specifically immunodetected in protein sperm extracts. Immunocytochemical analysis revealed a specific PRSS38 signal in the human sperm acrosomal region, equatorial segment, and flagellum. Mouse sperm PRSS38 was immunolocalized in the equatorial segment and hook. Human sperm preincubation with specific antibodies resulted in inhibition (p &lt; 0.05) of CPA, HZA, and SPA. Mouse sperm preincubation with PRSS38 antibodies impaired (p &lt; 0.05) homologous IVF using COCs and denuded oocytes. Genetic variants affecting residues involved in the GPI anchor and the catalytic triad were found in individuals from the general population whose PRSS38 protease function could be altered. This study provides, for the first time, an integrated analysis of PRSS38 in human and mouse sperm, contributing to our understanding of mammalian fertilization and male infertility.<\/jats:p>","DOI":"10.3390\/ijms262311680","type":"journal-article","created":{"date-parts":[[2025,12,2]],"date-time":"2025-12-02T15:03:13Z","timestamp":1764687793000},"page":"11680","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["PRSS38 Is a Novel Sperm Serine Protease Involved in Human and Mouse Fertilization"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0546-6964","authenticated-orcid":false,"given":"Ania Antonella","family":"Manjon","sequence":"first","affiliation":[{"name":"Laboratorio de Estudios de Interacci\u00f3n Celular en Reproducci\u00f3n y C\u00e1ncer, Instituto de Biolog\u00eda y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET), Vuelta de Obligado 2490, Buenos Aires C1428ADN, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5929-6828","authenticated-orcid":false,"given":"Gustavo Luis","family":"Ver\u00f3n","sequence":"additional","affiliation":[{"name":"Laboratorio de Estudios de Interacci\u00f3n Celular en Reproducci\u00f3n y C\u00e1ncer, Instituto de Biolog\u00eda y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET), Vuelta de Obligado 2490, Buenos Aires C1428ADN, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-0627-593X","authenticated-orcid":false,"given":"Rosario","family":"Vitale","sequence":"additional","affiliation":[{"name":"Research Institute for Signals, Systems and Computational Intelligence, sinc(i), FICH-UNL, CONICET, Ciudad Universitaria, UNL, Santa Fe S3000, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4459-4560","authenticated-orcid":false,"given":"Georgina","family":"Stegmayer","sequence":"additional","affiliation":[{"name":"Research Institute for Signals, Systems and Computational Intelligence, sinc(i), FICH-UNL, CONICET, Ciudad Universitaria, UNL, Santa Fe S3000, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-2850-805X","authenticated-orcid":false,"given":"Fernanda","family":"Gonzalez Echeverria-Raffo","sequence":"additional","affiliation":[{"name":"Centro M\u00e9dico Fertilab, Buenos Aires C1116ABI, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9818-3030","authenticated-orcid":false,"given":"Lydie","family":"Lane","sequence":"additional","affiliation":[{"name":"CALIPHO Group, SIB Swiss Institute of Bioinformatics, CH-1211 Geneva, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4463-6532","authenticated-orcid":false,"given":"M\u00f3nica Hebe","family":"Vazquez-Levin","sequence":"additional","affiliation":[{"name":"Laboratorio de Estudios de Interacci\u00f3n Celular en Reproducci\u00f3n y C\u00e1ncer, Instituto de Biolog\u00eda y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET), Vuelta de Obligado 2490, Buenos Aires C1428ADN, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"369","DOI":"10.1038\/s41585-018-0003-3","article-title":"Genetics of male infertility","volume":"15","author":"Krausz","year":"2018","journal-title":"Nat. Rev. Urol."},{"key":"ref_2","unstructured":"World Health Organization (2023). Infertility Prevalence Estimates, 1990\u20132021, World Health Organization. Licence: CC BY-NC-SA 3.0 IGO."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1501","DOI":"10.1016\/j.celrep.2019.01.045","article-title":"The Neonatal and Adult Human Testis Defined at the Single-Cell Level","volume":"26","author":"Sohni","year":"2019","journal-title":"Cell Rep."},{"key":"ref_4","unstructured":"Neill, K. (2006). CHAPTER 22\u2014The Epididymis. Knobil and Neill\u2019s Physiology of Reproduction, Academic Press. [3rd ed.]."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"James, E.R., Carrell, D.T., Aston, K.I., Jenkins, T.G., Yeste, M., and Salas-Huetos, A. (2020). The Role of the Epididymis and the Contribution of Epididymosomes to Mammalian Reproduction. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21155377"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"E59","DOI":"10.1038\/35055178","article-title":"A profile of fertilization in mammals","volume":"3","author":"Wassarman","year":"2001","journal-title":"Nat. Cell Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"dev176966","DOI":"10.1242\/dev.176966","article-title":"The molecular mechanisms mediating mammalian fertilization","volume":"146","author":"Bhakta","year":"2019","journal-title":"Development"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1093\/biolre\/ioy028","article-title":"Sperm-egg interaction and fertilization: Past, present, and future","volume":"99","author":"Okabe","year":"2018","journal-title":"Biol. Reprod."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"644","DOI":"10.1093\/biolre\/ioac037","article-title":"Mysteries and unsolved problems of mammalian fertilization and related topics","volume":"106","author":"Yanagimachi","year":"2022","journal-title":"Biol. Reprod."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1016\/j.bbrc.2013.10.028","article-title":"A testis-specific serine protease, Prss41\/Tessp-1, is necessary for the progression of meiosis during murine in vitro spermatogenesis","volume":"441","author":"Yoneda","year":"2013","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"dev197558","DOI":"10.1242\/dev.197558","article-title":"PRSS50 is a testis protease responsible for proper sperm tail formation and function","volume":"148","author":"Scovell","year":"2021","journal-title":"Development"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1093\/biolre\/ioaf069","article-title":"Ovochymase 2 is a key regulatory factor modulating proteolytic pathways and sperm maturation in the mammalian epididymis","volume":"113","author":"Kent","year":"2025","journal-title":"Biol. Reprod."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1094","DOI":"10.1002\/mrd.23224","article-title":"Proteomic analysis of seminal extracellular vesicle proteins involved in asthenozoospermia by iTRAQ","volume":"86","author":"Lin","year":"2019","journal-title":"Mol. Reprod. Dev."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1093\/biolre\/ioaa041","article-title":"Prss55 but not Prss51 is required for male fertility in mice","volume":"103","author":"Kobayashi","year":"2020","journal-title":"Biol Reprod."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"340","DOI":"10.1095\/biolreprod48.2.340","article-title":"Inhibition of acrosin activity with a trypsin inhibitor blocks human sperm penetration of the zona pellucida","volume":"48","author":"Liu","year":"1993","journal-title":"Biol. Reprod."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"606","DOI":"10.1095\/biolreprod62.3.606","article-title":"Expression of human proacrosin in Escherichia coli and binding to zona pellucida","volume":"62","author":"Furlong","year":"2000","journal-title":"Biol. Reprod."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1780","DOI":"10.1016\/j.fertnstert.2004.12.042","article-title":"Binding of recombinant human proacrosin\/acrosin to zona pellucida (ZP) glycoproteins. I. Studies with recombinant human ZPA, ZPB, and ZPC","volume":"83","author":"Furlong","year":"2005","journal-title":"Fertil. Steril."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"10470","DOI":"10.1074\/jbc.273.17.10470","article-title":"Acrosin accelerates the dispersal of sperm acrosomal proteins during acrosome reaction","volume":"273","author":"Yamagata","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1245","DOI":"10.1016\/j.fertnstert.2007.12.072","article-title":"Acrosin antibodies and infertility. I. Detection of antibodies towards proacrosin\/acrosin in women consulting for infertility and evaluation of their effects upon the sperm protease activities","volume":"91","author":"Veaute","year":"2009","journal-title":"Fertil. Steril."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2456","DOI":"10.1016\/j.fertnstert.2009.08.040","article-title":"Anti-human proacrosin antibody inhibits the zona pellucida (ZP)-induced acrosome reaction of ZP-bound spermatozoa","volume":"93","author":"Veaute","year":"2010","journal-title":"Fertil. Steril."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1111\/j.1600-0897.2012.01127.x","article-title":"DNA immunization against proacrosin impairs fertility in male mice","volume":"68","author":"Veiga","year":"2012","journal-title":"Am. J. Reprod. Immunol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1676","DOI":"10.1016\/S0015-0282(03)00372-8","article-title":"Biochemical and molecular studies of the proacrosin\/acrosin system in patients with unexplained infertility","volume":"79","author":"Rawe","year":"2003","journal-title":"Fertil. Steril."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Zhao, S., Zhang, R., Wang, L., Zhao, D., Tang, L., and Rao, M. (2024). Acrosin activity negatively influences the cumulative live birth rate in patients undergoing IVF treatment. Reprod. Biomed. Online, 49.","DOI":"10.1016\/j.rbmo.2024.103993"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1213","DOI":"10.1093\/humrep\/dead059","article-title":"ACROSIN deficiency causes total fertilization failure in humans by preventing the sperm from penetrating the zona pellucida","volume":"38","author":"Hua","year":"2023","journal-title":"Hum. Reprod."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"31845","DOI":"10.1016\/S0021-9258(18)31772-1","article-title":"Sperm from mice carrying a targeted mutation of the acrosin gene can penetrate the oocyte zona pellucida and effect fertilization","volume":"269","author":"Baba","year":"1994","journal-title":"J. Biol. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"370","DOI":"10.1002\/(SICI)1098-2795(199703)46:3<370::AID-MRD16>3.0.CO;2-2","article-title":"Spermatozoa lacking acrosin protein show delayed fertilization","volume":"46","author":"Adham","year":"1997","journal-title":"Mol. Reprod. Dev."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"16976","DOI":"10.1074\/jbc.M112470200","article-title":"A mouse serine protease TESP5 is selectively included into lipid rafts of sperm membrane presumably as a glycosylphosphatidylinositol-anchored protein","volume":"277","author":"Honda","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1001","DOI":"10.1111\/j.1365-2443.2008.01222.x","article-title":"Reduced fertility of mouse epididymal sperm lacking Prss21\/Tesp5 is rescued by sperm exposure to uterine microenvironment","volume":"13","author":"Yamashita","year":"2008","journal-title":"Genes Cells."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"3998","DOI":"10.1021\/acs.jproteome.6b00400","article-title":"Looking for Missing Proteins in the Proteome of Human Spermatozoa: An Update","volume":"15","author":"Vandenbrouck","year":"2016","journal-title":"J. Proteome. Res."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Pierleoni, A., Martelli, P.L., and Casadio, R. (2008). PredGPI: A GPI-anchor predictor. BMC Bioinform., 9.","DOI":"10.1186\/1471-2105-9-392"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3529","DOI":"10.1074\/jbc.M114.628560","article-title":"Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin","volume":"290","author":"Driesbaugh","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"21434","DOI":"10.1074\/jbc.M011423200","article-title":"Prostasin is a glycosylphosphatidylinositol-anchored active serine protease","volume":"276","author":"Chen","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"32941","DOI":"10.1074\/jbc.C600208200","article-title":"Evidence for a matriptase-prostasin proteolytic cascade regulating terminal epidermal differentiation","volume":"281","author":"Currie","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"507","DOI":"10.1093\/molehr\/5.6.507","article-title":"Progesterone promotes the acrosome reaction in capacitated human spermatozoa as judged by flow cytometry and CD46 staining","volume":"5","author":"Bronson","year":"1999","journal-title":"Mol. Hum. Reprod."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/j.mce.2009.02.006","article-title":"Nongenomic activation of spermatozoa by steroid hormones: Facts and fictions","volume":"308","author":"Baldi","year":"2009","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2513","DOI":"10.1073\/pnas.1917595117","article-title":"Acrosin is essential for sperm penetration through the zona pellucida in hamsters","volume":"117","author":"Hirose","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"4501","DOI":"10.1021\/cr000033x","article-title":"Serine protease mechanism and specificity","volume":"102","author":"Hedstrom","year":"2002","journal-title":"Chem. Rev."},{"key":"ref_38","first-page":"1","article-title":"An overview of the proacrosin\/acrosin system in human spermatozoa","volume":"56","author":"Furlong","year":"2005","journal-title":"Treballs de la SCB"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"921","DOI":"10.1095\/biolreprod.109.076273","article-title":"The glycosylphosphatidylinositol-anchored serine protease PRSS21 (testisin) imparts murine epididymal sperm cell maturation and fertilizing ability","volume":"81","author":"Bugge","year":"2009","journal-title":"Biol. Reprod."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1186\/s13048-016-0228-9","article-title":"The serine protease prostasin (PRSS8) is a potential biomarker for early detection of ovarian cancer","volume":"9","author":"Tamir","year":"2016","journal-title":"J. Ovarian Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2040","DOI":"10.1111\/jcmm.16116","article-title":"PRSS55 plays an important role in the structural differentiation and energy metabolism of sperm and is required for male fertility in mice","volume":"25","author":"Zhu","year":"2021","journal-title":"J. Cell. Mol. Med."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Prochetto, S., Reinheimer, R., and Stegmayer, G. (2024). evolSOM: An R package for analyzing conservation and displacement of biological variables with self-organizing maps. Bioinform. Adv., 4.","DOI":"10.1093\/bioadv\/vbae124"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1870","DOI":"10.1093\/jxb\/erz546","article-title":"Transcriptional and biochemical analyses of gibberellin expression and content in germinated barley grain","volume":"71","author":"Betts","year":"2020","journal-title":"J. Exp. Bot."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Milone, D.H., Stegmayer, G.S., Kamenetzky, L., L\u00f3pez, M., Lee, J.M., Giovannoni, J.J., and Carrari, F. (2010). *omeSOM: A software for clustering and visualization of transcriptional and metabolite data mined from interspecific crosses of crop plants. BMC Bioinform., 11.","DOI":"10.1186\/1471-2105-11-438"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Milone, D.H., Stegmayer, G., L\u00f3pez, M., Kamenetzky, L., and Carrari, F. (2014). Improving clustering with metabolic pathway data. BMC Bioinform., 15.","DOI":"10.1186\/1471-2105-15-101"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"18843","DOI":"10.1016\/S0021-9258(17)32244-5","article-title":"Prostasin is a novel human serine proteinase from seminal fluid. Purification, tissue distribution, and localization in prostate gland","volume":"269","author":"Yu","year":"1994","journal-title":"J. Biol. Chem."},{"key":"ref_47","first-page":"3199","article-title":"Testisin, a new human serine proteinase expressed by premeiotic testicular germ cells and lost in testicular germ cell tumors","volume":"59","author":"Hooper","year":"1999","journal-title":"Cancer Res."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1250","DOI":"10.1046\/j.1432-1327.2001.01986.x","article-title":"Organization and chromosomal localization of the murine Testisin gene encoding a serine protease temporally expressed during spermatogenesis","volume":"268","author":"Scarman","year":"2001","journal-title":"Eur. J. Biochem."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1002\/jcp.21575","article-title":"Composition and Significance of Detergent Resistant Membranes in Mouse Spermatozoa","volume":"218","author":"Nixon","year":"2009","journal-title":"J. Cell. Physiol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1111\/andr.12569","article-title":"The serine protease testisin is present on the surface of capacitated stallion spermatozoa and interacts with key zona pellucida binding proteins","volume":"7","author":"Swegen","year":"2019","journal-title":"Andrology"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"66","DOI":"10.4103\/aja202275","article-title":"Identification of risk genes in Chinese nonobstructive azoospermia patients based on whole-exome sequencing","volume":"25","author":"Liu","year":"2023","journal-title":"Asian J. Androl."},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Sharma, N., Liu, S., Tang, L., Irwin, J., Meng, G., and Rancourt, D.E. (2006). Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation. BMC Dev. Biol., 6.","DOI":"10.1186\/1471-213X-6-61"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1071\/RD03102","article-title":"Uterine expression of implantation serine proteinase 2 during the implantation period and in vivo inhibitory effect of its antibody on embryo implantation in mice","volume":"16","author":"Huang","year":"2004","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1530\/rep.0.1220235","article-title":"Regulation of the strypsin-related proteinase ISP2 by progesterone in endometrial gland epithelium during implantation in mice","volume":"122","author":"Liu","year":"2001","journal-title":"Reproduction"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"254","DOI":"10.1093\/biolre\/ioaa060","article-title":"Tmprss12 is required for sperm motility and uterotubal junction migration in mice","volume":"103","author":"Larasati","year":"2020","journal-title":"Biol Reprod."},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"Zhang, J., Zhou, X., Wan, D., Yu, L., Chen, X., Yan, T., Wu, Z., Zheng, M., Zhu, F., and Zhu, H. (2022). TMPRSS12 Functions in Meiosis and Spermiogenesis and Is Required for Male Fertility in Mice. Front. Cell Dev. Biol., 10.","DOI":"10.3389\/fcell.2022.757042"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"971","DOI":"10.1016\/j.fertnstert.2008.07.1760","article-title":"Cumulus cells and their extracellular matrix affect the quality of the spermatozoa penetrating the cumulus mass","volume":"92","author":"Hong","year":"2009","journal-title":"Fertil. Steril."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"4892","DOI":"10.1073\/pnas.1018202108","article-title":"Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization","volume":"108","author":"Jin","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1007\/s13238-011-1022-5","article-title":"Acrosome reaction in the cumulus oophorus revisited: Involvement of a novel sperm-released factor NYD-SP8","volume":"2","author":"Sun","year":"2011","journal-title":"Protein Cell."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1016\/j.ceca.2012.11.007","article-title":"Ca2+ mobilization in cumulus cells: Role in oocyte maturation and acrosome reaction","volume":"53","author":"Chen","year":"2013","journal-title":"Cell Calcium."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"823","DOI":"10.1016\/j.fertnstert.2017.12.026","article-title":"Cumulus oophorus complexes favor physiologic selection of spermatozoa for intracytoplasmic sperm injection","volume":"109","author":"Wang","year":"2018","journal-title":"Fertil. Steril."},{"key":"ref_62","first-page":"1250","article-title":"Human sperm degradation of zona pellucida proteins contributes to fertilization","volume":"99","author":"Larrea","year":"2015","journal-title":"Reprod. Biol. Endocrinol."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"gaab059","DOI":"10.1093\/molehr\/gaab059","article-title":"A mouse testis serine protease, TESP1, as the potential SPINK3 receptor protein on mouse sperm acrosome","volume":"27","author":"Ramachandran","year":"2021","journal-title":"Mol. Hum. Reprod."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"RP93131","DOI":"10.7554\/eLife.93131","article-title":"Deep learning insights into the architecture of the mammalian egg-sperm fusion synapse","volume":"13","author":"Elofsson","year":"2024","journal-title":"eLife"},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"e70040","DOI":"10.1002\/mrd.70040","article-title":"Uncovering the Role of IZUMO Family Members in Human Fertilization","volume":"92","author":"Manjon","year":"2025","journal-title":"Mol. Reprod. Dev."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"R43","DOI":"10.1530\/REP-15-0310","article-title":"Formation and function of the manchette and flagellum during spermatogenesis","volume":"151","author":"Lehti","year":"2016","journal-title":"Reproduction"},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/S0303-7207(01)00692-X","article-title":"Bypassing natural sperm selection during fertilization: The azh mutant offspring experience and the alternative of spermiogenesis in vitro","volume":"187","author":"Kierszenbaum","year":"2002","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_68","doi-asserted-by":"crossref","unstructured":"Hook, K.A., Wilke, L.M., and Fisher, H.S. (2021). Apical Sperm Hook Morphology Is Linked to Sperm Swimming Performance and Sperm Aggregation in Peromyscus Mice. Cells, 10.","DOI":"10.3390\/cells10092279"},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1002\/prot.21018","article-title":"Prediction of protein subcellular localization","volume":"64","author":"Yu","year":"2006","journal-title":"Proteins"},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"W585","DOI":"10.1093\/nar\/gkm259","article-title":"WoLF PSORT: Protein localization predictor","volume":"35","author":"Horton","year":"2007","journal-title":"Nucleic Acids Res."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"W228","DOI":"10.1093\/nar\/gkac278","article-title":"DeepLoc 2.0: Multi-label subcellular localization prediction using protein language models","volume":"50","author":"Thumuluri","year":"2022","journal-title":"Nucleic Acids. Res."},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"1700262","DOI":"10.1002\/pmic.201700262","article-title":"HPSLPred: An Ensemble Multi-Label Classifier for Human Protein Subcellular Location Prediction with Imbalanced Source","volume":"17","author":"Wan","year":"2017","journal-title":"Proteomics"},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"3524","DOI":"10.1093\/bioinformatics\/btx476","article-title":"pLoc-mAnimal: Predict subcellular localization of animal proteins with both single and multiple sites","volume":"33","author":"Cheng","year":"2017","journal-title":"Bioinformatics"},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"458","DOI":"10.1093\/jxb\/erac422","article-title":"N-Hydroxy pipecolic acid methyl ester is involved in Arabidopsis immunity","volume":"74","author":"Mohnike","year":"2023","journal-title":"J. Exp. Bot."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"eadg7492","DOI":"10.1126\/science.adg7492","article-title":"Accurate proteome-wide missense variant effect prediction with AlphaMissense","volume":"381","author":"Cheng","year":"2023","journal-title":"Science"},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1038\/s41597-024-03327-8","article-title":"Analysis of AlphaMissense data in different protein groups and structural context","volume":"11","author":"Tordai","year":"2024","journal-title":"Sci. Data."},{"key":"ref_77","unstructured":"World Health Organization (2010). WHO Laboratory Manual for the Examination and Processing of Human Semen, World Health Organization. [5th ed.]."},{"key":"ref_78","first-page":"227","article-title":"\u2018How to count sperm properly\u2019: Checklist for acceptability of studies based on human semen analysis","volume":"31","author":"Barratt","year":"2016","journal-title":"Hum. Reprod."},{"key":"ref_79","unstructured":"Biggers, J.D., Whitten, W.K., and Whittingham, D.G. (1971). The culture of mouse embryos in vitro. Methods in Mammalian Embryology, W. H. Freeman & Co."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/S0165-0378(01)00080-8","article-title":"Evaluation of the proacrosin\/acrosin system and its mechanism of activation in human sperm extracts","volume":"54","author":"Zahn","year":"2002","journal-title":"J. Reprod. Immunol."},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1095\/biolreprod13.5.513","article-title":"The relationship between sperm concentration and fertilization in vitro of mouse eggs","volume":"13","author":"Fraser","year":"1975","journal-title":"Biol. Reprod."},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An open-source platform for biological-image analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"1054","DOI":"10.1093\/molehr\/gau073","article-title":"Evidence for the involvement of proline-rich tyrosine kinase 2 in tyrosine phosphorylation downstream of protein kinase A activation during human sperm capacitation","volume":"20","author":"Battistone","year":"2014","journal-title":"Mol. Hum. Reprod."},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"688","DOI":"10.1016\/S0015-0282(16)59841-0","article-title":"The hemizona assay (HZA): Development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential","volume":"49","author":"Burkman","year":"1988","journal-title":"Fertil. Steril."},{"key":"ref_85","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1095\/biolreprod15.4.471","article-title":"The use of zona-free animal o va as a test-system for the assessment of the fertilizing capacity of human spermatozoa","volume":"15","author":"Yanagimachi","year":"1976","journal-title":"Biol. Reprod."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/23\/11680\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,4]],"date-time":"2025-12-04T05:25:44Z","timestamp":1764825944000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/23\/11680"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,2]]},"references-count":85,"journal-issue":{"issue":"23","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["ijms262311680"],"URL":"https:\/\/doi.org\/10.3390\/ijms262311680","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2025,12,2]]}}}