{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,8]],"date-time":"2026-07-08T05:30:52Z","timestamp":1783488652261,"version":"3.55.0"},"reference-count":43,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2023,4,10]],"date-time":"2023-04-10T00:00:00Z","timestamp":1681084800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science","award":["20H05373"],"award-info":[{"award-number":["20H05373"]}]},{"name":"Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science","award":["21H02384"],"award-info":[{"award-number":["21H02384"]}]},{"name":"Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science","award":["21K05977"],"award-info":[{"award-number":["21K05977"]}]},{"name":"Center for Human and Animal Symbiosis Science, Azabu University","award":["20H05373"],"award-info":[{"award-number":["20H05373"]}]},{"name":"Center for Human and Animal Symbiosis Science, Azabu University","award":["21H02384"],"award-info":[{"award-number":["21H02384"]}]},{"name":"Center for Human and Animal Symbiosis Science, Azabu University","award":["21K05977"],"award-info":[{"award-number":["21K05977"]}]},{"name":"Azabu University Research Services Division","award":["20H05373"],"award-info":[{"award-number":["20H05373"]}]},{"name":"Azabu University Research Services Division","award":["21H02384"],"award-info":[{"award-number":["21H02384"]}]},{"name":"Azabu University Research Services Division","award":["21K05977"],"award-info":[{"award-number":["21K05977"]}]},{"name":"MEXT*-Supported Program for the Private University Research Branding Project","award":["20H05373"],"award-info":[{"award-number":["20H05373"]}]},{"name":"MEXT*-Supported Program for the Private University Research Branding Project","award":["21H02384"],"award-info":[{"award-number":["21H02384"]}]},{"name":"MEXT*-Supported Program for the Private University Research Branding Project","award":["21K05977"],"award-info":[{"award-number":["21K05977"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Life"],"abstract":"<jats:p>During mammalian fertilization, repetitive rises of intracellular calcium called calcium oscillations are required for full activation of oocytes. Therefore, oocytes such as round spermatid injected or somatic cell nuclear transferred require additional artificial activation which mimics the calcium oscillations. It is well recognized that sperm specific phospholipase C (PLC\u03b6) is a strong candidate as the sperm factor which can induce calcium oscillations and, at least in mammals, the genetic mutation of PLC\u03b6 in human causes male infertility due to the lack of calcium oscillations in the oocytes. Recent studies showed that the sperm lacking PLC\u03b6 (Plcz1\u2212\/\u2212) still could induce rise(s) of intracellular calcium in the oocytes after IVF but not intracytoplasmic sperm injection (ICSI). In the ICSI oocytes, no pronuclear formation or development to the two-cell stage was observed. However, it is still unclear whether additional activation treatment can rescue the low developmental ability of Plcz1\u2212\/\u2212-sperm-derived oocytes after ICSI. In this study, we examined whether oocytes injected with a Plcz1\u2212\/\u2212 sperm can develop to term by additional artificial activation. In oocytes injected a Plcz1\u2212\/\u2212 sperm and Plcz1\u2212\/\u2212 and eCS (another candidate of the sperm factor) double knockout sperm (Plcz1\u2212\/\u2212eCS\u2212\/\u2212), the rates of pronuclear formation were very low (2.0 \u00b1 2.3% and 6.1 \u00b1 3.7%, respectively) compared to control (92.1 \u00b1 2.6%). However, these rates were dramatically improved by additional procedures of PLC\u03b6-mRNA injection or SrCl2 treatment (Plcz1\u2212\/\u2212 sperm + PLC\u03b6 mRNA, Plcz1\u2212\/\u2212 sperm + SrCl2 and Plcz1\u2212\/\u2212eCS\u2212\/\u2212 sperm + PLC\u03b6 mRNA; 64.2 \u00b1 10.8%, 89.2 \u00b1 2.4% and 72.6 \u00b1 5.4%, respectively). Most of the oocytes were developed to the two-cell stage. After embryo transfer, healthy pups were obtained in all these groups (Plcz1\u2212\/\u2212 sperm + PLC\u03b6 mRNA:10.0 \u00b1 2.8%, Plcz1\u2212\/\u2212 sperm + SrCl2:4.0 \u00b1 4.3% and Plcz1\u2212\/\u2212eCS\u2212\/\u2212 sperm + PLC\u03b6 mRNA: 10.0 \u00b1 5.7%). The rate in Plcz1\u2212\/\u2212 sperm + SrCl2 group was significantly lower than that in control (26.0 \u00b1 2.4%). Taken together, our present results show that additional activation treatment such as SrCl2 and PLC\u03b6 mRNA can fully support to develop to term even in oocyte injected Plcz1\u2212\/\u2212 sperm. In addition, PLC\u03b6-induced oocyte activation is more suitable for successful development to term compared to that such as phenomenon induced by SrCl2. These findings will contribute to improvement for male-dependent human infertility and reproductive technologies in other mammalian species.<\/jats:p>","DOI":"10.3390\/life13040980","type":"journal-article","created":{"date-parts":[[2023,4,10]],"date-time":"2023-04-10T05:59:33Z","timestamp":1681106373000},"page":"980","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Successful Production of Offspring Derived from Phospholipase C Zeta-Deficient Sperm by Additional Artificial Activation"],"prefix":"10.3390","volume":"13","author":[{"given":"Naoki","family":"Hirose","sequence":"first","affiliation":[{"name":"Faculty of Life and Environmental Science, University of Yamanashi, Yamanashi 400-8510, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yasuyuki","family":"Kikuchi","sequence":"additional","affiliation":[{"name":"Faculty of Life and Environmental Science, University of Yamanashi, Yamanashi 400-8510, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Atsuko","family":"Kageyama","sequence":"additional","affiliation":[{"name":"Laboratory of Animal Reproduction, Graduate School of Veterinary Medicine, Azabu University, Kanagawa 252-0206, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hibiki","family":"Sugita","sequence":"additional","affiliation":[{"name":"School of Veterinary Medicine, Azabu University, Kanagawa 252-0206, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miu","family":"Sakurai","sequence":"additional","affiliation":[{"name":"School of Veterinary Medicine, Azabu University, Kanagawa 252-0206, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yui","family":"Kawata","sequence":"additional","affiliation":[{"name":"Laboratory of Animal Reproduction, Graduate School of Veterinary Medicine, Azabu University, Kanagawa 252-0206, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7643-7976","authenticated-orcid":false,"given":"Jumpei","family":"Terakawa","sequence":"additional","affiliation":[{"name":"School of Veterinary Medicine, Azabu University, Kanagawa 252-0206, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Teruhiko","family":"Wakayama","sequence":"additional","affiliation":[{"name":"Faculty of Life and Environmental Science, University of Yamanashi, Yamanashi 400-8510, Japan"},{"name":"Advanced Biotechnology Center, University of Yamanashi, Yamanashi 400-8510, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9398-7358","authenticated-orcid":false,"given":"Junya","family":"Ito","sequence":"additional","affiliation":[{"name":"Laboratory of Animal Reproduction, Graduate School of Veterinary Medicine, Azabu University, Kanagawa 252-0206, Japan"},{"name":"School of Veterinary Medicine, Azabu University, Kanagawa 252-0206, Japan"},{"name":"Center for Human and Animal Symbiosis Science, Azabu University, Kanagawa 252-0206, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naomi","family":"Kashiwazaki","sequence":"additional","affiliation":[{"name":"Laboratory of Animal Reproduction, Graduate School of Veterinary Medicine, Azabu University, Kanagawa 252-0206, Japan"},{"name":"School of Veterinary Medicine, Azabu University, Kanagawa 252-0206, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,4,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"846","DOI":"10.1002\/mrd.21359","article-title":"PLC\u03b6 and its role as a trigger of development in verte-brates","volume":"78","author":"Ito","year":"2011","journal-title":"Mol. Reprod. Dev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1016\/j.ydbio.2007.12.012","article-title":"The roles of Ca2+, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development","volume":"315","author":"Ducibella","year":"2008","journal-title":"Dev. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/S0070-2153(08)60563-3","article-title":"Molecular basis of mammalian egg activation","volume":"30","author":"Schultz","year":"1995","journal-title":"Curr. Top. Dev. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3533","DOI":"10.1242\/dev.129.15.3533","article-title":"PLC zeta: A sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development","volume":"129","author":"Saunders","year":"2002","journal-title":"Development"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1081","DOI":"10.1095\/biolreprod.108.067801","article-title":"Difference in Ca2+ oscillation-inducing activity and nuclear translocation ability of PLCZ1, an egg-activating sperm factor candidate, between mouse, rat, human, and medaka fish","volume":"78","author":"Ito","year":"2008","journal-title":"Biol. Reprod."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"400","DOI":"10.1016\/j.ceca.2008.02.003","article-title":"Relationship between nuclear sequestration of PLCzeta and termination of PLCzeta-induced Ca2+ oscillations in mouse eggs","volume":"44","author":"Ito","year":"2008","journal-title":"Cell Calcium"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1530\/rep.0.1240611","article-title":"Sperm phospholipase Czeta from humans and cynomolgus monkeys triggers Ca2+ oscillations, activation and development of mouse oocytes","volume":"124","author":"Cox","year":"2002","journal-title":"Reproduction"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1095\/biolreprod.109.079814","article-title":"Specific differences in the activity and nuclear localization of murine and bovine phospholipase C zeta 1","volume":"83","author":"Cooney","year":"2010","journal-title":"Biol. Reprod."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1095\/biolreprod.104.037333","article-title":"Fertilization and inositol 1,4,5-trisphosphate (IP3)-induced calcium release in type-1 inositol 1,4,5-trisphosphate receptor down-regulated bovine eggs","volume":"73","author":"Malcuit","year":"2005","journal-title":"Biol. Reprod."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/j.biolcel.2003.11.003","article-title":"Mammalian fertilization: From sperm factor to phospholipase Czeta","volume":"96","author":"Kurokawa","year":"2004","journal-title":"Biol. Cell"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"393","DOI":"10.1530\/rep.1.01018","article-title":"Molecular cloning, testicular postnatal expression, and oocyte-activating potential of porcine phospholipase Czeta","volume":"132","author":"Yoneda","year":"2006","journal-title":"Reproduction"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1111\/asj.12044","article-title":"Molecular characteristics of horse phospholipase C zeta (PLC\u03b6)","volume":"84","author":"Sato","year":"2013","journal-title":"Anim. Sci. J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e13345","DOI":"10.1111\/asj.13345","article-title":"Phospholipase Czeta (PLCzeta) versus postacrosomal sheath WW domain-binding protein (PAWP): Which molecule will survive as a sperm factor?","volume":"91","author":"Nakai","year":"2020","journal-title":"Anim. Sci. J."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"10408","DOI":"10.1074\/jbc.M313801200","article-title":"Recombinant phospholipase Czeta has high Ca2+ sensitivity and induces Ca2+ oscillations in mouse eggs","volume":"279","author":"Kouchi","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2417","DOI":"10.1093\/humrep\/dep207","article-title":"Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men","volume":"24","author":"Heytens","year":"2009","journal-title":"Hum. Reprod."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"3671","DOI":"10.1172\/JCI36942","article-title":"Human sperm devoid of PLC, zeta 1 fail to induce Ca(2+) release and are unable to initiate the first step of embryo development","volume":"118","author":"Yoon","year":"2008","journal-title":"J. Clin. Invest."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1016\/j.fertnstert.2012.05.002","article-title":"Characterization of two heterozygous mutations of the oocyte activation factor phospholipase C zeta (PLCzeta) from an infertile man by use of minisequencing of individual sperm and expression in somatic cells","volume":"98","author":"Kashir","year":"2012","journal-title":"Fertil. Steril."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"878","DOI":"10.1093\/hmg\/ddv617","article-title":"Homozygous mutation of PLCZ1 leads to defective human oocyte activation and infertility that is not rescued by the WW-binding protein PAWP","volume":"25","author":"Escoffier","year":"2016","journal-title":"Hum. Mol. Genet."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2914","DOI":"10.1242\/dev.150227","article-title":"PLC\u03b6 is the physiological trigger of the Ca2+ oscillations that induce embryogenesis in mammals but conception can occur in its absence","volume":"144","author":"Hachem","year":"2017","journal-title":"Development"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1315","DOI":"10.1038\/s41598-018-19497-6","article-title":"Sperm-borne phospholipase C zeta-1 ensures monospermic fertilization in mice","volume":"8","author":"Nozawa","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1089\/clo.2006.0002","article-title":"Viable offspring derived from fetal and adult mammalian cells","volume":"9","author":"Wilmut","year":"2007","journal-title":"Cloning Stem. Cells"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2095","DOI":"10.1126\/science.282.5396.2095","article-title":"Eight calves cloned from somatic cells of a single adult","volume":"282","author":"Kato","year":"1998","journal-title":"Science"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"369","DOI":"10.1038\/28615","article-title":"Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei","volume":"394","author":"Wakayama","year":"1998","journal-title":"Nature"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1188","DOI":"10.1126\/science.289.5482.1188","article-title":"Pig cloning by microinjection of fetal fibroblast nuclei","volume":"289","author":"Onishi","year":"2000","journal-title":"Science"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1863","DOI":"10.1095\/biolreprod.103.025171","article-title":"Similar time restriction for intracytoplasmic sperm injection and round spermatid injection into activated oocytes for efficient offspring production","volume":"70","author":"Kishigami","year":"2004","journal-title":"Biol. Reprod."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"pdb.prot094425","DOI":"10.1101\/pdb.prot094425","article-title":"Cloning Mice","volume":"2017","author":"Ogura","year":"2017","journal-title":"Cold Spring Harb. Protoc."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1207","DOI":"10.1262\/jrd.19067","article-title":"Efficient strontium-induced activation of mouse oocytes in standard culture media by chelating calcium","volume":"53","author":"Kishigami","year":"2007","journal-title":"J. Reprod. Dev."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1038\/s41374-019-0353-3","article-title":"Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction","volume":"100","author":"Kang","year":"2020","journal-title":"Lab. Invest."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1262\/jrd.2019-043","article-title":"Production of mouse offspring from inactivated spermatozoa using horse PLC\u03b6 mRNA","volume":"66","author":"Yamamoto","year":"2020","journal-title":"J. Reprod. Dev."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"709","DOI":"10.1095\/biolreprod52.4.709","article-title":"Intracytoplasmic sperm injection in the mouse","volume":"52","author":"Kimura","year":"1995","journal-title":"Biol. Reprod."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1530\/jrf.0.0860679","article-title":"An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro","volume":"86","author":"Chatot","year":"1989","journal-title":"J. Reprod. Fertil."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1530\/REP-20-0054","article-title":"Birth of offspring from spermatid or somatic cell by co-injection of PLC\u03b6-cRNA","volume":"160","author":"Hirose","year":"2020","journal-title":"Reproduction"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"E1","DOI":"10.1530\/REP-22-0148","article-title":"Sperm Factors and Egg Activation: PLCzeta as the sperm factor that activates eggs: 20 years on","volume":"164","author":"Swann","year":"2022","journal-title":"Reproduction"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"797","DOI":"10.1016\/j.ydbio.2007.04.019","article-title":"Characterization of a sperm factor for egg activation at fertilization of the newt Cynops pyrrhogaster","volume":"306","author":"Harada","year":"2007","journal-title":"Dev. Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1038\/nbt0402-366","article-title":"Cloned rabbits produced by nuclear transfer from adult somatic cells","volume":"20","author":"Adenot","year":"2002","journal-title":"Nat. Biotechnol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.1126\/science.1088313","article-title":"Generation of fertile cloned rats by regulating oocyte activation","volume":"302","author":"Zhou","year":"2003","journal-title":"Science"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1273","DOI":"10.1093\/humrep\/deg212","article-title":"Pregnancy by the tubal transfer of embryos developed after injection of round spermatids into oocyte cytoplasm of the cynomolgus monkey (Macaca fascicularis)","volume":"18","author":"Ogonuki","year":"2003","journal-title":"Hum. Reprod."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1002\/mrd.20984","article-title":"Activation regimens for full-term development of rabbit oocytes injected with round spermatids","volume":"76","author":"Hirabayashi","year":"2009","journal-title":"Mol. Reprod. Dev."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"534","DOI":"10.1016\/j.ydbio.2006.08.041","article-title":"Ca2+ oscillatory pattern in fertilized mouse eggs affects gene expression and development to term","volume":"300","author":"Ozil","year":"2006","journal-title":"Dev. Biol."},{"key":"ref_40","first-page":"5714","article-title":"Regulation of endoplasmic reticulum Ca(2+) oscillations in mammalian eggs","volume":"126","author":"Wakai","year":"2013","journal-title":"J. Cell Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1080\/19396368.2018.1516000","article-title":"Artificial oocyte activation: Physiological, pathophysiological and ethical aspects","volume":"65","author":"Anifandis","year":"2019","journal-title":"Syst. Biol. Reprod. Med."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"28","DOI":"10.3389\/fcell.2020.00028","article-title":"Essential Role of Sperm-Specific PLC-Zeta in Egg Activation and Male Factor Infertility: An Update","volume":"8","author":"Saleh","year":"2020","journal-title":"Front. Cell Dev. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"F1","DOI":"10.1530\/REP-21-0477","article-title":"Sperm Factors and Egg Activation: The structure and function relationship of sperm PLCZ1","volume":"164","author":"Thanassoulas","year":"2022","journal-title":"Reproduction"}],"container-title":["Life"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-1729\/13\/4\/980\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:13:07Z","timestamp":1760123587000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-1729\/13\/4\/980"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,4,10]]},"references-count":43,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2023,4]]}},"alternative-id":["life13040980"],"URL":"https:\/\/doi.org\/10.3390\/life13040980","relation":{},"ISSN":["2075-1729"],"issn-type":[{"value":"2075-1729","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,4,10]]}}}