{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T04:19:18Z","timestamp":1789532358616,"version":"build-2803163510"},"reference-count":44,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2020,1,24]],"date-time":"2020-01-24T00:00:00Z","timestamp":1579824000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000780","name":"European Commission","doi-asserted-by":"publisher","award":["H2020-MSCA-IF-79212"],"award-info":[{"award-number":["H2020-MSCA-IF-79212"]}],"id":[{"id":"10.13039\/501100000780","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100014440","name":"Ministerio de Ciencia, Innovaci\u00f3n y Universidades","doi-asserted-by":"publisher","award":["RYC-2014-15581"],"award-info":[{"award-number":["RYC-2014-15581"]}],"id":[{"id":"10.13039\/100014440","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100014440","name":"Ministerio de Ciencia, Innovaci\u00f3n y Universidades","doi-asserted-by":"publisher","award":["AGL2017-88329-R"],"award-info":[{"award-number":["AGL2017-88329-R"]}],"id":[{"id":"10.13039\/100014440","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100014440","name":"Ministerio de Ciencia, Innovaci\u00f3n y Universidades","doi-asserted-by":"publisher","award":["FJCI-2017-31689"],"award-info":[{"award-number":["FJCI-2017-31689"]}],"id":[{"id":"10.13039\/100014440","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100014440","name":"Ministerio de Ciencia, Innovaci\u00f3n y Universidades","doi-asserted-by":"publisher","award":["FPU18\/00666"],"award-info":[{"award-number":["FPU18\/00666"]}],"id":[{"id":"10.13039\/100014440","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Ag\u00e8ncia de Gesti\u00f3 d\u2019Ajuts Universitaris i de Recerca","award":["2017-SGR-1229"],"award-info":[{"award-number":["2017-SGR-1229"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antioxidants"],"abstract":"<jats:p>Glutathione S-transferases (GSTs) are essential sperm antioxidant enzymes involved in cell protection against oxidative stress and toxic chemicals, preserving sperm function and fertilising ability. Artificial insemination (AI) in pigs is commonly carried out through the use of liquid-stored semen at 17 \u00b0C, which not only reduces sperm metabolic activity but also sperm quality and AI-farrowing rates within the 72 h of storage. While one may reasonably suggest that such enzymes are implicated in the physiology and maintenance of mammalian sperm function during liquid-storage, no previous studies conducted on any species have addressed this hypothesis. Therefore, the objective of the present work was to characterise the presence and function of sperm GSTs in mammalian sperm, using the pig as a model. In this regard, inhibition of such enzymes by ethacrynic acid (EA) during semen storage at 17 \u00b0C was performed to evaluate the effects of GSTs in liquid-preserved boar sperm by flow cytometry, immunofluorescence, and immunoblotting analysis. The results of this study have shown, for the first time in mammalian species, that the inhibition of GSTs reduces sperm quality and functionality parameters during their storage at 17 \u00b0C. These findings highlight the key role of such enzymes, especially preserving mitochondrial function and maintaining plasma membrane stability. In addition, this study has identified and localised GSTM3 in the tail and equatorial subdomain of the head of boar sperm. Finally, this study has set grounds for future investigations testing supplementation of semen extenders with GSTs, as this may improve fertility outcomes of swine AIs.<\/jats:p>","DOI":"10.3390\/antiox9020100","type":"journal-article","created":{"date-parts":[[2020,1,24]],"date-time":"2020-01-24T11:01:00Z","timestamp":1579863660000},"page":"100","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":37,"title":["Glutathione S-Transferases Play a Crucial Role in Mitochondrial Function, Plasma Membrane Stability and Oxidative Regulation of Mammalian Sperm"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0685-7534","authenticated-orcid":false,"given":"Marc","family":"Llavanera","sequence":"first","affiliation":[{"name":"Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, E-17003 Girona Spain"},{"name":"Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, E-17003 Girona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5655-5003","authenticated-orcid":false,"given":"Ariadna","family":"Delgado-Berm\u00fadez","sequence":"additional","affiliation":[{"name":"Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, E-17003 Girona Spain"},{"name":"Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, E-17003 Girona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Samuel","family":"Olives","sequence":"additional","affiliation":[{"name":"Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, E-17003 Girona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yentel","family":"Mateo-Otero","sequence":"additional","affiliation":[{"name":"Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, E-17003 Girona Spain"},{"name":"Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, E-17003 Girona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandra","family":"Recuero","sequence":"additional","affiliation":[{"name":"Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, E-17003 Girona Spain"},{"name":"Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, E-17003 Girona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5125-7274","authenticated-orcid":false,"given":"Sergi","family":"Bonet","sequence":"additional","affiliation":[{"name":"Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, E-17003 Girona Spain"},{"name":"Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, E-17003 Girona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Beatriz","family":"Fern\u00e1ndez-Fuertes","sequence":"additional","affiliation":[{"name":"Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, E-17003 Girona Spain"},{"name":"Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, E-17003 Girona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2209-340X","authenticated-orcid":false,"given":"Marc","family":"Yeste","sequence":"additional","affiliation":[{"name":"Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, E-17003 Girona Spain"},{"name":"Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, E-17003 Girona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9873-814X","authenticated-orcid":false,"given":"Isabel","family":"Barranco","sequence":"additional","affiliation":[{"name":"Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, E-17003 Girona Spain"},{"name":"Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, E-17003 Girona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,1,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1016\/S0378-4320(00)00157-3","article-title":"Storage of boar semen","volume":"62","author":"Johnson","year":"2000","journal-title":"Anim. Reprod. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"69","DOI":"10.21451\/1984-3143-AR895","article-title":"State-of-the-art of boar sperm preservation in liquid and frozen state","volume":"14","author":"Yeste","year":"2017","journal-title":"Anim. Reprod."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1111\/j.1439-0531.2011.01836.x","article-title":"Assessment of storage effects in liquid preserved boar semen","volume":"46","author":"Waberski","year":"2011","journal-title":"Reprod. Domest. Anim."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1016\/j.anireprosci.2008.01.006","article-title":"Preservation of boar semen at 18 \u00b0C induces lipid peroxidation and apoptosis like changes in spermatozoa","volume":"110","author":"Kumaresan","year":"2008","journal-title":"Anim. Reprod. Sci."},{"key":"ref_5","first-page":"1","article-title":"Harmful and beneficial role of ROS","volume":"2018","author":"Reed","year":"2018","journal-title":"Oxid. Med. Cell. Longev."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/S0026-0495(00)80077-3","article-title":"What is oxidative stress?","volume":"49","author":"Betteridge","year":"2000","journal-title":"Metabolism"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/j.anireprosci.2018.08.047","article-title":"Effects of oligomeric proanthocyanidins on quality of boar semen during liquid preservation at 17 \u00b0C","volume":"198","author":"Li","year":"2018","journal-title":"Anim. Reprod. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1007\/BF02790102","article-title":"Antioxidant effect of vitamin E and glutathione on lipid peroxidation in boar semen plasma","volume":"47","author":"Pietras","year":"1995","journal-title":"Biol. Trace Elem. Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"502","DOI":"10.1016\/j.freeradbiomed.2008.11.004","article-title":"Reactive oxygen-induced reactive oxygen formation during human sperm capacitation","volume":"46","author":"Lamothe","year":"2009","journal-title":"Free Radic. Biol. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"33048","DOI":"10.1074\/jbc.M112.366690","article-title":"Electrophilic aldehydes generated by sperm metabolism activate mitochondrial reactive oxygen species generation and apoptosis by targeting succinate dehydrogenase","volume":"287","author":"Aitken","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1155\/2014\/626374","article-title":"The enzymatic antioxidant system of human spermatozoa","volume":"2014","year":"2014","journal-title":"Adv. Androl."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1146\/annurev.pharmtox.45.120403.095857","article-title":"Glutathione transferases","volume":"45","author":"Hayes","year":"2005","journal-title":"Annu. Rev. Pharmacol. Toxicol."},{"key":"ref_13","first-page":"1","article-title":"The triple role of glutathione S-transferases in mammalian male fertility","volume":"77","author":"Llavanera","year":"2019","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2053","DOI":"10.1242\/jcs.115.10.2053","article-title":"Sperm plasma-membrane-associated glutathione S-transferases as gamete recognition molecules","volume":"115","author":"Hemachand","year":"2002","journal-title":"J. Cell Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1002\/j.1939-4640.1996.tb01764.x","article-title":"Effect of sperm lipid peroxidation on fertilization","volume":"17","author":"Kodama","year":"1996","journal-title":"J. Androl."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/srep13821","article-title":"Proteomic approaches for profiling negative fertility markers in inferior boar spermatozoa","volume":"5","author":"Kwon","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s40104-019-0370-5","article-title":"GSTM3, but not IZUMO1, is a cryotolerance marker of boar sperm","volume":"10","author":"Llavanera","year":"2019","journal-title":"J. Anim. Sci. Biotechnol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1517\/13543776.16.4.431","article-title":"Glutathione S-transferase (GST) inhibitors","volume":"16","author":"Mathew","year":"2006","journal-title":"Expert Opin. Ther. Pat."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"913","DOI":"10.3109\/00498259309059418","article-title":"Ethacrynic acid and its glutathione conjugate as inhibitors of glutathione S-transferases","volume":"23","author":"Ploement","year":"1993","journal-title":"Xenobiotica"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1205","DOI":"10.1016\/0006-2952(80)90420-7","article-title":"Binding of ethacrynic acid to hepatic glutathione-transferases in the rat","volume":"29","author":"Yamada","year":"1980","journal-title":"Biochem. Pharmacol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/0167-4838(93)90245-M","article-title":"Interactions of glutathione S-transferase-\u03c0 with ethacrynic acid and its glutathione conjugate","volume":"1164","author":"Awasthi","year":"1993","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1038\/jhg.2010.59","article-title":"The association of glutathione-S-transferase gene polymorphisms (GSTM1, GSTT1, GSTP1) with idiopathic male infertility","volume":"55","author":"Safarinejad","year":"2010","journal-title":"J. Hum. Genet."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"296","DOI":"10.1016\/S0014-5793(98)00032-5","article-title":"Inhibition of sperm glutathione S-transferase leads to functional impairment due to membrane damage","volume":"422","author":"Gopalakrishnan","year":"1998","journal-title":"FEBS Lett."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1095\/biolreprod53.2.276","article-title":"Viability assessment of mammalian sperm using SYBR-14 and propidium iodide","volume":"53","author":"Garner","year":"1995","journal-title":"Biol. Reprod."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Yeste, M., Estrada, E., Rivera del \u00c1lamo, M.-M., Bonet, S., Rigau, T., and Rodr\u00edguez-Gil, J.-E. (2014). The increase in phosphorylation levels of serine residues of protein HSP70 during holding time at 17 \u00b0C is concomitant with a higher cryotolerance of boar spermatozoa. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0090887"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1828","DOI":"10.1095\/biolreprod.102.011445","article-title":"A triple-stain flow cytometric method to assess plasma- and acrosome-membrane integrity of cryopreserved bovine sperm immediately after thawing in presence of egg-yolk particles","volume":"68","author":"Nagy","year":"2003","journal-title":"Biol. Reprod."},{"key":"ref_27","first-page":"213","article-title":"Detection of \u201capoptosis-like\u201d changes during the cryopreservation process in equine sperm","volume":"29","author":"Muriel","year":"2007","journal-title":"J. Androl."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1002\/mrd.1080350214","article-title":"Flow cytometric studies of bicarbonate-mediated Ca2+ influx in boar sperm populations","volume":"35","author":"Harrison","year":"1993","journal-title":"Mol. Reprod. Dev."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"729","DOI":"10.1111\/andr.12054","article-title":"Intracellular calcium movements of boar spermatozoa during \u201cin vitro\u201d capacitation and subsequent acrosome exocytosis follow a multiple-storage place, extracellular calcium-dependent model","volume":"3","author":"Yeste","year":"2015","journal-title":"Andrology"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"400","DOI":"10.1016\/j.ceca.2011.02.011","article-title":"\u03b23-Adrenergic activation of sequential Ca2+ release from mitochondria and the endoplasmic reticulum and the subsequent Ca2+ entry in rodent brown adipocytes","volume":"49","author":"Hayato","year":"2011","journal-title":"Cell Calcium"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/j.ceca.2011.10.007","article-title":"Dynamics of mitochondrial [Ca2+] measured with the low-Ca2+-affinity dye rhod-5N","volume":"51","author":"Fonteriz","year":"2012","journal-title":"Cell Calcium"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2089","DOI":"10.2527\/jas.2005-766","article-title":"Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry","volume":"84","author":"Guthrie","year":"2006","journal-title":"J. Anim. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1195","DOI":"10.1111\/asj.12545","article-title":"Effects of glutathione on sperm quality during liquid storage in boars","volume":"87","author":"Zhang","year":"2016","journal-title":"Anim. Sci. J."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1016\/S0014-5793(03)00103-0","article-title":"Functional role of sperm surface glutathione S-transferases and extracellular glutathione in the haploid spermatozoa under oxidative stress","volume":"538","author":"Hemachand","year":"2003","journal-title":"FEBS Lett."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1093\/humupd\/dmi053","article-title":"Glycolysis and sperm motility: Does a spoonful of sugar help the flagellum go round?","volume":"12","author":"Ford","year":"2006","journal-title":"Hum. Reprod. Update"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1530\/REP-13-0178","article-title":"Mitochondria functionality and sperm quality","volume":"146","author":"Amaral","year":"2013","journal-title":"Reproduction"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"427","DOI":"10.1387\/ijdb.072522bs","article-title":"Mammalian sperm metabolism: Oxygen and sugar, friend and foe","volume":"52","author":"Storey","year":"2008","journal-title":"Int. J. Dev. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1042\/bj3530411","article-title":"Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space","volume":"353","author":"Han","year":"2001","journal-title":"Biochem. J."},{"key":"ref_39","first-page":"238","article-title":"Hydrogen peroxide modifies human sperm peroxiredoxins in a dose-dependent manner","volume":"84","year":"2010","journal-title":"Biol. Reprod."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1450","DOI":"10.1016\/j.bbagen.2016.04.001","article-title":"Post-translational modifications in sperm proteome: The chemistry of proteome diversifications in the pathophysiology of male factor infertility","volume":"1860","author":"Samanta","year":"2016","journal-title":"Biochim. Biophys. Acta Gen. Subj."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1016\/j.theriogenology.2013.11.012","article-title":"Glutathione-S-transferase: Role in buffalo (Bubalus bubalis) sperm capacitation and cryopreservation","volume":"81","author":"Kumar","year":"2014","journal-title":"Theriogenology"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1016\/j.fertnstert.2015.11.005","article-title":"Association between the seminal plasma proteome and sperm functional traits","volume":"105","author":"Intasqui","year":"2015","journal-title":"Fertil. Steril."},{"key":"ref_43","first-page":"14","article-title":"Identificaci\u00f3n de diferencias prote\u00f3micas en muestras oligozoosp\u00e9rmicas","volume":"7","author":"Botta","year":"2009","journal-title":"Rev. Int. Androl."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s12958-015-0007-2","article-title":"Major protein alterations in spermatozoa from infertile men with unilateral varicocele","volume":"13","author":"Agarwal","year":"2015","journal-title":"Reprod. Biol. Endocrinol."}],"container-title":["Antioxidants"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-3921\/9\/2\/100\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T13:44:36Z","timestamp":1760363076000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-3921\/9\/2\/100"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,1,24]]},"references-count":44,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2020,2]]}},"alternative-id":["antiox9020100"],"URL":"https:\/\/doi.org\/10.3390\/antiox9020100","relation":{},"ISSN":["2076-3921"],"issn-type":[{"value":"2076-3921","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,1,24]]}}}