{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T11:42:42Z","timestamp":1791459762075,"version":"4.3.4"},"reference-count":43,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2024,11,22]],"date-time":"2024-11-22T00:00:00Z","timestamp":1732233600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"JSPS KAKENHI","award":["20K06621"],"award-info":[{"award-number":["20K06621"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>We previously reported that five astacin-like metalloproteases with thrombospondin type-1 repeats (Tasts) located on the sperm surface are a promising candidate as the protease involved in sperm penetration of the vitelline coat (VC) during fertilization of the ascidian Ciona intestinalis type A (Phlebobranchia). However, whether such a protease is involved in the fertilization of other ascidians is unknown. Here, we investigated the effects of four metalloprotease inhibitors on the fertilization of the ascidian Halocynthia roretzi (Stolidobranchia). Three metalloprotease inhibitors, GM6001, TAPI-0, and TAPI-1, strongly inhibited fertilization at 33 and 11 \u03bcM, whereas TAPI-2 weakly inhibited fertilization at 33 \u03bcM. In contrast, GM6001NC (negative control) had no effect on fertilization at 100 \u03bcM. Furthermore, GM6001 had no inhibitory effect on the fertilization of VC-deprived eggs. The metalloprotease appears to function at the middle or late stage of fertilization. Ten Tast genes were identified in the H. roretzi genome database, among which four genes (HrTast1, HrTast2b, HrTast2c, and HrTast3c) possessed a single transmembrane domain in the N-terminal region. These four genes are transcribed in the testis and ovary, as revealed by RT-PCR. Anti-HrTast2c IgG raised against a peptide corresponding to the Zn-binding consensus sequence weakly inhibited fertilization at 0.5 mg\/mL. These results led us to propose that sperm astacin-like metalloproteases may be involved in sperm penetration of the VC during H. roretzi fertilization.<\/jats:p>","DOI":"10.3390\/biom14121487","type":"journal-article","created":{"date-parts":[[2024,11,22]],"date-time":"2024-11-22T04:21:36Z","timestamp":1732249296000},"page":"1487","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Involvement of Metalloproteases in the Fertilization of the Ascidian Halocynthia roretzi"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4363-7696","authenticated-orcid":false,"given":"Hitoshi","family":"Sawada","sequence":"first","affiliation":[{"name":"Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, 429-63 Sugashima, Toba 517-0004, Japan"},{"name":"Department of Food and Nutritional Environment, College of Human Life and Environment, Kinjo Gakuin University, Omori, Moriyama-ku, Nagoya 463-8521, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ikuya","family":"Hattori","sequence":"additional","affiliation":[{"name":"Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, 429-63 Sugashima, Toba 517-0004, Japan"},{"name":"Department of Food and Nutritional Environment, College of Human Life and Environment, Kinjo Gakuin University, Omori, Moriyama-ku, Nagoya 463-8521, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noritaka","family":"Hashii","sequence":"additional","affiliation":[{"name":"Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki 210-9501, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3969-9192","authenticated-orcid":false,"given":"Takako","family":"Saito","sequence":"additional","affiliation":[{"name":"Department of Applied Life Sciences, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,11,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"37R","DOI":"10.1093\/glycob\/11.4.37R","article-title":"Glycobiology of sperm-egg interactions in deuterostomes","volume":"11","author":"Mengerink","year":"2001","journal-title":"Glycobiology"},{"key":"ref_2","unstructured":"Litscher, E.S., and Wassarman, P.M. (2018). Extracellular Matrix and Egg Coats, Academic Press."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/B978-012515400-0\/50007-5","article-title":"Fertilization in mammals","volume":"Volume 1","author":"Neil","year":"2006","journal-title":"Knobil and Neill\u2019s Physiology of Reproduction"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1093\/molehr\/gat004","article-title":"The molecular basis of gamete recognition in mice and humans","volume":"19","author":"Avella","year":"2013","journal-title":"Mol. Human. Reprod."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"984","DOI":"10.1172\/JCI41585","article-title":"Fertilization: A sperm\u2019s journey to and interaction with the oocyte","volume":"120","author":"Ikawa","year":"2010","journal-title":"J. Clin. Investig."},{"key":"ref_6","unstructured":"Barret, A.J. (1977). The occurrence and function of proteolytic enzymes in the reproductive tract of mammals. Proteinases in Mammalian Cells and Tissues, North\u2013Holland."},{"key":"ref_7","unstructured":"Metz, C.B., and Monroy, A. (1985). Lysins. Biology of Fertilization, Academic Press."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"139","DOI":"10.2108\/zsj.19.139","article-title":"Ascidian sperm lysin system","volume":"19","author":"Sawada","year":"2002","journal-title":"Zool. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1530\/REP-11-0041","article-title":"Sperm proteasome and fertilization","volume":"142","author":"Sutovsky","year":"2011","journal-title":"Reproduction"},{"key":"ref_10","first-page":"385","article-title":"Mechanism of sperm-zona pellucida penetration during mammalian fertilization: 26S proteasome as a candidate egg coat lysin","volume":"63","author":"Yi","year":"2007","journal-title":"Soc. Reprod. Fertil."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1315","DOI":"10.1016\/j.cell.2017.05.033","article-title":"Structural basis of egg-coat sperm recognition at fertilization","volume":"169","author":"Raj","year":"2017","journal-title":"Cell"},{"key":"ref_12","first-page":"113","article-title":"The action of acrosin on the zona pellucida","volume":"207","author":"Urch","year":"1986","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_13","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_14","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_15","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1095\/biolreprod.109.083089","article-title":"Mice lacking two sperm serine proteases, ACR and PRSS21, are subfertile, but the mutant sperm are infertile in vitro","volume":"83","author":"Kawano","year":"2010","journal-title":"Biol. Reprod."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1093\/humupd\/8.5.405","article-title":"Role of acrosomal matrix proteases in sperm-zona pellucida interactions","volume":"8","author":"Honda","year":"2002","journal-title":"Hum. Reprod. Update"},{"key":"ref_17","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_18","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1016\/0012-1606(81)90322-5","article-title":"Evidence for participation of sperm proteinases in fertilization of the solitary ascidian, Halocynthia roretzi: Effects of protease inhibitors","volume":"86","author":"Hoshi","year":"1981","journal-title":"Dev. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1002\/mrd.1120050309","article-title":"Evidence for acrosin-like enzyme in sperm extract and its involvement in fertilization of the ascidian, Halocynthia roretzi","volume":"5","author":"Sawada","year":"1982","journal-title":"Gamete Res."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Saito, T., and Sawada, H. (2022). Fertilization of ascidians: Gamete interaction, self\/nonself recognition, and sperm penetration of egg-coat. Front. Cell Dev. Biol., 9.","DOI":"10.3389\/fcell.2021.827214"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Sawada, H., and Saito, T. (2022). Mechanisms of sperm-egg interactions: What ascidian fertilization research has taught us. Cells, 11.","DOI":"10.3390\/cells11132096"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1016\/j.bbrc.2010.01.006","article-title":"Vitellongenin C-terminal fragments participate in fertilization as egg-coat binding partners of sperm trypsin-like proteases in the ascidian Halocynthia roretzi","volume":"392","author":"Akasaka","year":"2010","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1002\/jez.b.22488","article-title":"Identification of novel isoforms of vitellogenin expressed in ascidian eggs","volume":"320","author":"Akasaka","year":"2013","journal-title":"J. Exp. Zool. Part B Mol. Dev. Evol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"238","DOI":"10.1006\/dbio.1993.1182","article-title":"High molecular weight protease complexes (proteasomes) of sperm of the ascidian, Halocynthia roretzi: Isolation, characterization, and physiological roles in fertilization","volume":"158","author":"Saitoh","year":"1993","journal-title":"Dev. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"312","DOI":"10.1016\/0012-1606(81)90174-3","article-title":"The ascidian sperm reaction: Ca2+ uptake in relation to H+ efflux","volume":"88","author":"Lambert","year":"1981","journal-title":"Dev. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1223","DOI":"10.1073\/pnas.032389499","article-title":"Extracellular ubiquitination and proteasome-mediated degradation of the ascidian sperm receptor","volume":"99","author":"Sawada","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1111\/jzs.12101","article-title":"Morphological evidence that the molecularly determined Ciona intestinalis type A and type B are different species: Ciona robusta and Ciona intestinalis","volume":"53","author":"Brunetti","year":"2015","journal-title":"J. Zool. Syst. Evol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1002\/(SICI)1098-2795(199808)50:4<493::AID-MRD13>3.0.CO;2-3","article-title":"Participation of sperm proteasome in fertilization of the Phlebobranch ascidian, Ciona intestinalis","volume":"50","author":"Sawada","year":"1998","journal-title":"Mol. Reprod. Develop."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2157","DOI":"10.1126\/science.1080049","article-title":"The draft genome of Ciona intestinalis: Insights into chordate and vertebrate origins","volume":"298","author":"Dehal","year":"2002","journal-title":"Science"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"D808","DOI":"10.1093\/nar\/gkv966","article-title":"ANISEED 2015: A digital framework for the comparative developmental biology of ascidians","volume":"44","author":"Brozovic","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4064","DOI":"10.1002\/pmic.201500162","article-title":"Proteomics of ionomycin-induced ascidian sperm reaction: Released and exposed sperm proteins in the ascidian Ciona intestinalis","volume":"15","author":"Nakazawa","year":"2015","journal-title":"Proteomics"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Nakazawa, S., Shirae-Kurabayashi, M., and Sawada, H. (2019). The role of metalloproteases in fertilisation in the ascidian Ciona robusta. Sci. Rep., 9.","DOI":"10.1038\/s41598-018-37721-1"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"4158","DOI":"10.1073\/pnas.1115086109","article-title":"Self-incompatibility response induced by calcium increase in sperm of the ascidian Ciona intestinalis","volume":"109","author":"Saito","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1002\/mrd.10089","article-title":"Localization and roles in fertilization of sperm proteasomes in the ascidian Halocynthia roretzi","volume":"62","author":"Sawada","year":"2002","journal-title":"Mol. Reprod. Develop."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1007\/BF01975902","article-title":"Timing of action of sperm proteases in ascidian fertilization","volume":"42","author":"Sawada","year":"1986","journal-title":"Experientia"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"164","DOI":"10.1038\/358164a0","article-title":"Structure of astacin and implications for activation of astacins and zinc-ligation of collagenases","volume":"358","author":"Bode","year":"1992","journal-title":"Nature"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1209","DOI":"10.1021\/jm970404a","article-title":"Inhibition of membrane-type 1 matrix metalloproteinase by hydroxamate inhibitors: An examination of the subsite pocket","volume":"41","author":"Yamamoto","year":"1998","journal-title":"J. Med. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"765","DOI":"10.1016\/S0968-0896(97)00028-X","article-title":"Synthesis and biological evaluation of orally active matrix metalloproteinase inhibitors","volume":"5","author":"Hirayama","year":"1997","journal-title":"Bioorganic Med. Chem."},{"key":"ref_39","unstructured":"Rawlings, N.D., and Salvesen, G. (2013). Handbook of Proteolytic Enzymes, Elsevier."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Giebeler, N., and Zigrino, P. (2016). A disintegrin and metalloprotease (ADAM): Historical overview of their functions. Toxins, 8.","DOI":"10.3390\/toxins8040122"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1247","DOI":"10.1002\/pro.5560040701","article-title":"The astacin family of metallopeptidases","volume":"4","author":"Bond","year":"1995","journal-title":"Protein Sci."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1007\/s00441-012-1429-1","article-title":"MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization","volume":"349","author":"Ferrer","year":"2012","journal-title":"Cell Tissue Res."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Sald\u00edvar-Hern\u00e1ndez, A., Gonz\u00e1lez-Gonz\u00e1lez, M.E., S\u00e1nchez-Tusi\u00e9, A., Maldonado-Rosas, I., L\u00f3pez, P., Trevi\u00f1o, C.L., Larrea, F., and Chirinos, M. (2015). Human sperm degradation of zona pellucida proteins contributes to fertilization. Reprod. Biol. Endocrinol., 13.","DOI":"10.1186\/s12958-015-0094-0"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/14\/12\/1487\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:37:18Z","timestamp":1760114238000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/14\/12\/1487"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,11,22]]},"references-count":43,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2024,12]]}},"alternative-id":["biom14121487"],"URL":"https:\/\/doi.org\/10.3390\/biom14121487","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,11,22]]}}}