{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T17:17:47Z","timestamp":1770916667700,"version":"3.50.1"},"reference-count":49,"publisher":"Oxford University Press (OUP)","issue":"5","license":[{"start":{"date-parts":[[2023,3,28]],"date-time":"2023-03-28T00:00:00Z","timestamp":1679961600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/pages\/standard-publication-reuse-rights"}],"content-domain":{"domain":["rep.bioscientifica.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2023,5,1]]},"abstract":"<jats:title>\u2003<\/jats:title>\n                  <jats:sec>\n                    <jats:title>In brief<\/jats:title>\n                    <jats:p>The mechanism by which p32 protein increases during capacitation in boar spermatozoa is unknown. This manuscript shows a new mechanism of induction of p32 in boar spermatozoa: the proteolysis of the phosphorylated and glycosylated form of SPACA1.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Abstract<\/jats:title>\n                    <jats:p>Protein tyrosine phosphorylation (PY) induction is associated with sperm capacitation. We previously showed that calcium-sensing receptor (CASR) inhibition by NPS2143 induces the 32 kDa tyrosine-phosphorylated protein (p32) in boar spermatozoa. We showed that NPS2143 induced an increase in p32 and loss of acrosomal integrity in live and dead spermatozoa in capacitating conditions (Tyrode's complete medium); the p32 rise occurred in dead spermatozoa, as shown by flow cytometry sorting. EGTA addition blunted the increase in p32, the loss of acrosomal integrity, and the increase in dead spermatozoa induced by NPS2143, indicating that the effects of NPS2143 are calcium-dependent. Mass spectrometry was used to identify which tyrosine-phosphorylated proteins were induced by NPS2143, but only serine\/threonine-phosphorylated proteins were found; among these, SPACA1 was identified with different molecular weights (18, 32, and 35\u201345 kDa). We confirmed tyrosine phosphorylation of SPACA1 at 32 and 35\u201345 kDa by immunoprecipitation and co-localization of PY and SPACA1 in the presence of NPS2143 by immunofluorescence. The molecular weight of SPACA1 (35\u201345 kDa) decreased after treatment with peptide-N-glycosidase F, indicating that this protein is N-glycosylated. The soybean trypsin inhibitor (STI), a serine protease inhibitor, suppressed the appearance of p32 and SPACA1 (30 and 32 kDa) induced by NPS2143. Also, 8-Br-cAMP and A23187 treatments induced an increase in p32 and SPACA1 (30\u201332 kDa) and a parallel induction of the acrosome reaction. These findings suggest that CASR inhibition induces loss of acrosomal integrity and proteolysis of the glycosylated and phosphorylated SPACA1 (35\u201345 kDa) resulting in a SPACA1 rise at 32 kDa (p32).<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1530\/rep-22-0299","type":"journal-article","created":{"date-parts":[[2023,2,23]],"date-time":"2023-02-23T12:58:10Z","timestamp":1677157090000},"page":"475-489","update-policy":"https:\/\/doi.org\/10.1530\/crossmarkpolicy-11","source":"Crossref","is-referenced-by-count":6,"title":["The CASR antagonist NPS2143 induces loss of acrosomal integrity and proteolysis of SPACA1 in boar spermatozoa"],"prefix":"10.1093","volume":"165","author":[{"given":"Beatriz","family":"Mac\u00edas-Garc\u00eda","sequence":"first","affiliation":[{"name":"Departamento de Medicina Animal , Facultad de Veterinaria, Universidad de Extremadura, C\u00e1ceres, Spain"},{"name":"Grupo de Investigaci\u00f3n Medicina Interna Veterinaria (MINVET) , Instituto de Investigaci\u00f3n INBIO G\u2009+\u2009C, Universidad de Extremadura, C\u00e1ceres, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5568-548X","authenticated-orcid":false,"given":"Lauro","family":"Gonz\u00e1lez-Fern\u00e1ndez","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica y Biolog\u00eda Molecular y Gen\u00e9tica , Facultad de Veterinaria, Universidad de Extremadura, C\u00e1ceres, Spain"},{"name":"Grupo de Investigaci\u00f3n Se\u00f1alizaci\u00f3n Intracelular y Tecnolog\u00eda de la Reproducci\u00f3n (SINTREP) , Instituto de Investigaci\u00f3n INBIO G\u2009+\u2009C, Universidad de Extremadura, C\u00e1ceres, Spain"}]}],"member":"286","published-online":{"date-parts":[[2023,3,28]]},"reference":[{"key":"2026021211241505300_bib1","doi-asserted-by":"publisher","first-page":"581","DOI":"10.1071\/bi9510581","article-title":"Observations on the penetration of the sperm in the mammalian egg","volume":"4","author":"Austin","year":"1951","journal-title":"Australian Journal of Scientific Research. Ser. B"},{"key":"2026021211241505300_bib2","doi-asserted-by":"publisher","first-page":"599","DOI":"10.1016\/j.theriogenology.2004.09.034","article-title":"Use of phosphoproteomics to study tyrosine kinase activity in capacitating boar sperm. Kinase activity and capacitation","volume":"63","author":"Bailey","year":"2005","journal-title":"Theriogenology"},{"key":"2026021211241505300_bib3","doi-asserted-by":"publisher","DOI":"10.1210\/clinem\/dgaa937","article-title":"The calcium-sensing receptor is essential for calcium and bicarbonate sensitivity in human spermatozoa","volume":"106","author":"Boisen","year":"2021","journal-title":"Journal of Clinical Endocrinology and Metabolism"},{"key":"2026021211241505300_bib4","doi-asserted-by":"publisher","first-page":"88","DOI":"10.1002\/mrd.20286","article-title":"Changes in tyrosine phosphorylation associated with true capacitation and capacitation-like state in boar spermatozoa","volume":"71","author":"Bravo","year":"2005","journal-title":"Molecular Reproduction and Development"},{"key":"2026021211241505300_bib5","doi-asserted-by":"publisher","first-page":"245","DOI":"10.1089\/omi.2016.0173","article-title":"Posttranslational modifications in spermatozoa and effects on male fertility and sperm viability","volume":"21","author":"Brohi","year":"2017","journal-title":"Omics"},{"key":"2026021211241505300_bib6","doi-asserted-by":"publisher","first-page":"333","DOI":"10.1016\/j.beem.2013.02.006","article-title":"Role of the calcium-sensing receptor in extracellular calcium homeostasis","volume":"27","author":"Brown","year":"2013","journal-title":"Best Practice and Research. Clinical Endocrinology and Metabolism"},{"key":"2026021211241505300_bib7","doi-asserted-by":"publisher","first-page":"575","DOI":"10.1038\/366575a0","article-title":"Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid","volume":"366","author":"Brown","year":"1993","journal-title":"Nature"},{"key":"2026021211241505300_bib8","doi-asserted-by":"publisher","first-page":"697","DOI":"10.1038\/168697b0","article-title":"Fertilizing capacity of spermatozoa deposited into the fallopian tubes","volume":"168","author":"Chang","year":"1951","journal-title":"Nature"},{"key":"2026021211241505300_bib9","doi-asserted-by":"publisher","first-page":"129","DOI":"10.1146\/annurev-physiol-021113-170259","article-title":"Control of life-or-death decisions by RIP1 kinase","volume":"76","author":"Christofferson","year":"2014","journal-title":"Annual Review of Physiology"},{"key":"2026021211241505300_bib10","doi-asserted-by":"publisher","first-page":"425","DOI":"10.1530\/rep-09-0134","article-title":"Ca2+ stores in sperm: their identities and functions","volume":"138","author":"Costello","year":"2009","journal-title":"Reproduction"},{"key":"2026021211241505300_bib11","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1093\/molehr\/gag017","article-title":"Role of protein tyrosine phosphorylation in the thapsigargin-induced intracellular Ca(2+) store depletion during human sperm acrosome reaction","volume":"9","author":"Dorval","year":"2003","journal-title":"Molecular Human Reproduction"},{"key":"2026021211241505300_bib13","doi-asserted-by":"publisher","first-page":"727","DOI":"10.1002\/j.1939-4640.2003.tb02734.x","article-title":"The importance of calcium in the appearance of p32, a boar sperm tyrosine phosphoprotein, during in vitro capacitation","volume":"24","author":"Dube","year":"2003","journal-title":"Journal of Andrology"},{"key":"2026021211241505300_bib12","doi-asserted-by":"publisher","first-page":"519","DOI":"10.2164\/jandrol.04163","article-title":"The proacrosin binding protein, sp32, is tyrosine phosphorylated during capacitation of pig sperm","volume":"26","author":"Dube","year":"2005","journal-title":"Journal of Andrology"},{"key":"2026021211241505300_bib14","doi-asserted-by":"publisher","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 and Tissue Research"},{"key":"2026021211241505300_bib15","doi-asserted-by":"publisher","first-page":"733","DOI":"10.1007\/s00441-012-1433-5","article-title":"The composition, protein genesis and significance of the inner acrosomal membrane of eutherian sperm","volume":"349","author":"Ferrer","year":"2012","journal-title":"Cell and Tissue Research"},{"key":"2026021211241505300_bib16","doi-asserted-by":"publisher","first-page":"3583","DOI":"10.1242\/dev.081778","article-title":"SPACA1-deficient male mice are infertile with abnormally shaped sperm heads reminiscent of globozoospermia","volume":"139","author":"Fujihara","year":"2012","journal-title":"Development"},{"key":"2026021211241505300_bib17","doi-asserted-by":"publisher","DOI":"10.1095\/biolreprod.112.107078","article-title":"Focal adhesion kinases and calcium\/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm","volume":"88","author":"Gonz\u00e1lez-Fern\u00e1ndez","year":"2013","journal-title":"Biology of Reproduction"},{"key":"2026021211241505300_bib18","doi-asserted-by":"publisher","first-page":"735","DOI":"10.1095\/biolreprod66.3.735","article-title":"SAMP32, a testis-specific, isoantigenic sperm acrosomal membrane-associated protein","volume":"66","author":"Hao","year":"2002","journal-title":"Biology of Reproduction"},{"key":"2026021211241505300_bib19","doi-asserted-by":"publisher","first-page":"194","DOI":"10.1002\/mrd.20099","article-title":"A unique mechanism for cyclic adenosine 3',5'-monophosphate-induced increase of 32-kDa tyrosine-phosphorylated protein in boar spermatozoa","volume":"69","author":"Harayama","year":"2004","journal-title":"Molecular Reproduction and Development"},{"key":"2026021211241505300_bib20","doi-asserted-by":"publisher","first-page":"1083","DOI":"10.1042\/BST20180276","article-title":"2018 cell cycle-dependent phosphorylation and regulation of cellular differentiation","volume":"46","author":"Hardwick","year":"2018","journal-title":"Biochemical Society Transactions"},{"key":"2026021211241505300_bib21","doi-asserted-by":"publisher","first-page":"389","DOI":"10.1038\/ng1295-389","article-title":"A mouse model of human familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism","volume":"11","author":"Ho","year":"1995","journal-title":"Nature Genetics"},{"key":"2026021211241505300_bib22","doi-asserted-by":"publisher","first-page":"2143","DOI":"10.1016\/j.bbamem.2013.05.026","article-title":"AMP-activated kinase, AMPK, is involved in the maintenance of plasma membrane organization in boar spermatozoa","volume":"1828","author":"Hurtado de Llera","year":"2013","journal-title":"Biochimica et Biophysica Acta"},{"key":"2026021211241505300_bib23","doi-asserted-by":"publisher","first-page":"910","DOI":"10.1016\/j.bbrc.2008.10.073","article-title":"Putative sperm fusion protein IZUMO and the role of N-glycosylation","volume":"377","author":"Inoue","year":"2008","journal-title":"Biochemical and Biophysical Research Communications"},{"key":"2026021211241505300_bib24","doi-asserted-by":"publisher","first-page":"574","DOI":"10.1016\/0003-2697(7890784-4","article-title":"Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers","volume":"86","author":"James","year":"1978","journal-title":"Analytical Biochemistry"},{"key":"2026021211241505300_bib25","doi-asserted-by":"publisher","first-page":"3600","DOI":"10.18632\/oncotarget.12274","article-title":"Factors and pathways involved in capacitation: how are they regulated?","volume":"8","author":"Jin","year":"2017","journal-title":"Oncotarget"},{"key":"2026021211241505300_bib26","doi-asserted-by":"publisher","first-page":"421","DOI":"10.1095\/biolreprod.107.067314","article-title":"The equatorial subsegment in mammalian spermatozoa is enriched in tyrosine phosphorylated proteins","volume":"79","author":"Jones","year":"2008","journal-title":"Biology of Reproduction"},{"key":"2026021211241505300_bib27","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0251973","article-title":"ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model","volume":"16","author":"Kato","year":"2021","journal-title":"PLoS One"},{"key":"2026021211241505300_bib28","doi-asserted-by":"publisher","first-page":"4505","DOI":"10.1021\/pr500524p","article-title":"Diagnosis and prognosis of male infertility in mammal: the focusing of tyrosine phosphorylation and phosphotyrosine proteins","volume":"13","author":"Kwon","year":"2014","journal-title":"Journal of Proteome Research"},{"key":"2026021211241505300_bib29","doi-asserted-by":"publisher","first-page":"93","DOI":"10.1007\/s12275-019-9021-y","article-title":"Determination of protein phosphorylation by polyacrylamide gel electrophoresis","volume":"57","author":"Lee","year":"2019","journal-title":"Journal of Microbiology"},{"key":"2026021211241505300_bib30","doi-asserted-by":"publisher","first-page":"391","DOI":"10.1071\/RD16338","article-title":"Calcium-sensing receptor (CASR) is involved in porcine in vitro fertilisation and early embryo development","volume":"30","author":"Liu","year":"2018","journal-title":"Reproduction, Fertility, and Development"},{"key":"2026021211241505300_bib31","doi-asserted-by":"publisher","first-page":"499","DOI":"10.1007\/s10719-020-09924-0","article-title":"Identification of lipid raft glycoproteins obtained from boar spermatozoa","volume":"37","author":"Lopez-Salguero","year":"2020","journal-title":"Glycoconjugate Journal"},{"key":"2026021211241505300_bib34","doi-asserted-by":"publisher","first-page":"236","DOI":"10.1002\/mrd.22615","article-title":"Extracellular calcium regulates protein tyrosine phosphorylation through calcium-sensing receptor (CaSR) in stallion sperm","volume":"83","author":"Macias-Garcia","year":"2016","journal-title":"Molecular Reproduction and Development"},{"key":"2026021211241505300_bib33","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1016\/j.theriogenology.2017.03.002","article-title":"Role of the calcium-sensing receptor (CaSR) in bovine gametes and during in vitro fertilization","volume":"95","author":"Macias-Garcia","year":"2017","journal-title":"Theriogenology"},{"key":"2026021211241505300_bib32","doi-asserted-by":"publisher","first-page":"751","DOI":"10.1002\/mrd.23160","article-title":"The calcium-sensing receptor regulates protein tyrosine phosphorylation through PDK1 in boar spermatozoa","volume":"86","author":"Macias-Garcia","year":"2019","journal-title":"Molecular Reproduction and Development"},{"key":"2026021211241505300_bib35","doi-asserted-by":"publisher","first-page":"1396","DOI":"10.1016\/s0015-0282(0300267-x","article-title":"Calcium requirements for human sperm function in vitro","volume":"79","author":"Marin-Briggiler","year":"2003","journal-title":"Fertility and Sterility"},{"key":"2026021211241505300_bib36","doi-asserted-by":"publisher","first-page":"2013","DOI":"10.1242\/jcs.02326","article-title":"Evidence of the presence of calcium\/calmodulin-dependent protein kinase IV in human sperm and its involvement in motility regulation","volume":"118","author":"Marin-Briggiler","year":"2005","journal-title":"Journal of Cell Science"},{"key":"2026021211241505300_bib37","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1262\/jrd.2021-075","article-title":"Influence of different cellular concentrations of boar sperm suspensions on the induction of capacitation and acrosome reaction","volume":"68","author":"Martin-Hidalgo","year":"2022","journal-title":"Journal of Reproduction and Development"},{"key":"2026021211241505300_bib38","doi-asserted-by":"publisher","first-page":"96","DOI":"10.2164\/jandrol.110.011254","article-title":"Localization, distribution, and function of the calcium-sensing receptor in sperm","volume":"33","author":"Mendoza","year":"2012","journal-title":"Journal of Andrology"},{"key":"2026021211241505300_bib39","doi-asserted-by":"publisher","first-page":"337","DOI":"10.1007\/978-1-60761-913-0_18","article-title":"Immobilized metal affinity chromatography\/reversed-phase enrichment of phosphopeptides and analysis by CID\/ETD tandem mass spectrometry","volume":"681","author":"Navajas","year":"2011","journal-title":"Methods in Molecular Biology"},{"key":"2026021211241505300_bib40","doi-asserted-by":"publisher","first-page":"94","DOI":"10.1016\/j.anireprosci.2016.07.006","article-title":"Changes in the distribution and molecular mass of boar sperm acrosome-associated 1 proteins during the acrosome reaction; their validity as indicators for occurrence of the true acrosome reaction","volume":"172","author":"Ogura","year":"2016","journal-title":"Animal Reproduction Science"},{"key":"2026021211241505300_bib41","doi-asserted-by":"publisher","first-page":"850","DOI":"10.1021\/ac950914h","article-title":"Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels","volume":"68","author":"Shevchenko","year":"1996","journal-title":"Analytical Chemistry"},{"key":"2026021211241505300_bib42","doi-asserted-by":"publisher","first-page":"765","DOI":"10.1007\/s00441-012-1370-3","article-title":"Kinases, phosphatases and proteases during sperm capacitation","volume":"349","author":"Signorelli","year":"2012","journal-title":"Cell and Tissue Research"},{"key":"2026021211241505300_bib43","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1007\/s10930-017-9696-z","article-title":"Phosphorylation: implications in cancer","volume":"36","author":"Singh","year":"2017","journal-title":"Protein Journal"},{"key":"2026021211241505300_bib44","doi-asserted-by":"publisher","first-page":"502","DOI":"10.1262\/jrd.20021","article-title":"A 32-kDa tyrosine-phosphorylated protein shows a protease-dependent increase in dead boar spermatozoa","volume":"54","author":"Tabuchi","year":"2008","journal-title":"Journal of Reproduction and Development"},{"key":"2026021211241505300_bib45","doi-asserted-by":"publisher","first-page":"784","DOI":"10.1095\/biolreprod65.3.784","article-title":"Capacitation is associated with tyrosine phosphorylation and tyrosine kinase-like activity of pig sperm proteins","volume":"65","author":"Tardif","year":"2001","journal-title":"Biology of Reproduction"},{"key":"2026021211241505300_bib46","doi-asserted-by":"publisher","first-page":"1139","DOI":"10.1242\/dev.121.4.1139","article-title":"Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway","volume":"121","author":"Visconti","year":"1995","journal-title":"Development"},{"key":"2026021211241505300_bib47","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.theriogenology.2022.02.017","article-title":"Cysteine is highly enriched in the canonical N-linked glycosylation motif of bovine spermatozoa N-Glycoproteome","volume":"184","author":"Wang","year":"2022","journal-title":"Theriogenology"},{"key":"2026021211241505300_bib48","doi-asserted-by":"publisher","DOI":"10.1016\/j.mcpro.2022.100214","article-title":"Precision glycoproteomics reveals distinctive N-glycosylation in human spermatozoa","volume":"21","author":"Xin","year":"2022","journal-title":"Molecular and Cellular Proteomics"},{"key":"2026021211241505300_bib49","doi-asserted-by":"publisher","first-page":"750","DOI":"10.1093\/biolre\/ioz223","article-title":"Cleavage of SPACA1 regulates assembly of sperm-egg membrane fusion machinery in mature spermatozoadagger","volume":"102","author":"Yamatoya","year":"2020","journal-title":"Biology of Reproduction"}],"container-title":["Reproduction"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/rep.bioscientifica.com\/view\/journals\/rep\/165\/5\/REP-22-0299.xml","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/rep.bioscientifica.com\/view\/journals\/rep\/165\/5\/REP-22-0299.xml","content-type":"text\/html","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/reproduction\/article-pdf\/165\/5\/475\/63880322\/rep-22-0299.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/reproduction\/article-pdf\/165\/5\/475\/63880322\/rep-22-0299.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T16:24:23Z","timestamp":1770913463000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/reproduction\/article\/165\/5\/475\/8217199"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,3,28]]},"references-count":49,"journal-issue":{"issue":"5","published-print":{"date-parts":[[2023,5,1]]}},"URL":"https:\/\/doi.org\/10.1530\/rep-22-0299","relation":{},"ISSN":["1470-1626","1741-7899"],"issn-type":[{"value":"1470-1626","type":"print"},{"value":"1741-7899","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2023,5,1]]},"published":{"date-parts":[[2023,3,28]]}}}