{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T15:19:01Z","timestamp":1790435941858,"version":"4.1.0"},"reference-count":46,"publisher":"Public Library of Science (PLoS)","issue":"2","license":[{"start":{"date-parts":[[2026,2,5]],"date-time":"2026-02-05T00:00:00Z","timestamp":1770249600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"kakenhi","doi-asserted-by":"crossref","award":["25K02455"],"award-info":[{"award-number":["25K02455"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001691","name":"kakenhi","doi-asserted-by":"crossref","award":["24K21284"],"award-info":[{"award-number":["24K21284"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001691","name":"kakenhi","doi-asserted-by":"crossref","award":["22K19588"],"award-info":[{"award-number":["22K19588"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001691","name":"kakenhi","doi-asserted-by":"crossref","award":["21H03068"],"award-info":[{"award-number":["21H03068"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Grant for Strategic Research Promotion of Yokohama City University","award":["SK202403"],"award-info":[{"award-number":["SK202403"]}]},{"name":"JST CREST grant","award":["JPMJCR21N1"],"award-info":[{"award-number":["JPMJCR21N1"]}]},{"name":"Japan Agency for Medical Research and Development grant","award":["JP23jf0126001"],"award-info":[{"award-number":["JP23jf0126001"]}]},{"name":"Japan Agency for Medical Research and Development grant","award":["24gn0110086h0001"],"award-info":[{"award-number":["24gn0110086h0001"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    <jats:italic>In vitro<\/jats:italic>\n                    spermatogenesis (IVS) remains a major challenge due to its complexity and extended duration. Although organ culture of neonatal mouse testes can support complete spermatogenesis, its three-dimensional structure limits precise observation and control of culture conditions, resulting in heterogeneous oxygen and nutrient gradients. Two-dimensional (2-D) systems provide greater accessibility but often induce accelerated or abnormal meiosis. In this study, we present a simplified, serum free 2-D culture system that enables meiotic progression of mouse germ cells to the mid pachytene stage. Testicular cells from\n                    <jats:italic>Acrosin<\/jats:italic>\n                    (\n                    <jats:italic>Acr<\/jats:italic>\n                    )-GFP transgenic mice (2.5 to 10.5 days\n                    <jats:italic>postpartum<\/jats:italic>\n                    : dpp) were enzymatically dissociated and cultured in APEL medium, a chemically defined and serum free basal formulation. The medium was supplemented with bovine pituitary extract (BPE), follicle stimulating hormone (FSH), and testosterone (BFT medium). Meiotic progression was monitored in real time by GFP fluorescence and further assessed by immunocytochemistry and nuclear spread analysis. Testicular cells consistently formed Sertoli cell monolayers and supported germ cell differentiation to mid pachytene stage. GFP-positive cells appeared around days 17.5 to 21.5 of culture, reflecting a modest delay compared to organ culture. GFP-positive cells were observed not only in 7.5 to 10.5 dpp cultures, but also in 2.5 to 6.5 dpp testes lacking preexisting spermatocytes, indicating\n                    <jats:italic>de novo<\/jats:italic>\n                    induction of meiosis. BFT medium, compared to APEL medium, increased the frequency of GFP-positive wells and the per-well duration of GFP expression, reaching statistical significance in the 9.5\u201310.5 dpp group (p\u2009=\u20090.021). Nuclear spread analysis confirmed synapsis through pachytene, although cells did not progress to diplotene. This simplified culture system offers a robust and accessible platform for studying early meiotic events and optimizing IVS.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0342007","type":"journal-article","created":{"date-parts":[[2026,2,5]],"date-time":"2026-02-05T18:34:05Z","timestamp":1770316445000},"page":"e0342007","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["A simplified two-dimensional culture system supports meiotic progression during mouse spermatogenesis in vitro"],"prefix":"10.1371","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-9966-6407","authenticated-orcid":true,"given":"Yuki","family":"Yokoi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuki","family":"Yamashita","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Koichi","family":"Uemura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hisakazu","family":"Odaka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7449-2015","authenticated-orcid":true,"given":"Kazuhide","family":"Makiyama","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1422-0317","authenticated-orcid":true,"given":"Takehiko","family":"Ogawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6198-2234","authenticated-orcid":true,"given":"Takuya","family":"Sato","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0744-7492","authenticated-orcid":true,"given":"Mitsuru","family":"Komeya","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,2,5]]},"reference":[{"issue":"1","key":"pone.0342007.ref001","doi-asserted-by":"crossref","first-page":"3458","DOI":"10.1038\/s41598-021-82792-2","article-title":"Rat in vitro spermatogenesis promoted by chemical supplementations and oxygen-tension control","volume":"11","author":"T Matsumura","year":"2021","journal-title":"Sci Rep"},{"issue":"5","key":"pone.0342007.ref002","doi-asserted-by":"crossref","first-page":"469","DOI":"10.2164\/jandrol.107.004655","article-title":"The cycle of the seminiferous epithelium in humans: a need to revisit?","volume":"29","author":"RP Amann","year":"2008","journal-title":"J Androl"},{"issue":"7339","key":"pone.0342007.ref003","doi-asserted-by":"crossref","first-page":"504","DOI":"10.1038\/nature09850","article-title":"In vitro production of functional sperm in cultured neonatal mouse testes","volume":"471","author":"T Sato","year":"2011","journal-title":"Nature"},{"issue":"2","key":"pone.0342007.ref004","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0116660","article-title":"Retinol improves in vitro differentiation of pre-pubertal mouse spermatogonial stem cells into sperm during the first wave of spermatogenesis","volume":"10","author":"B Arkoun","year":"2015","journal-title":"PLoS One"},{"issue":"7","key":"pone.0342007.ref005","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1002\/bdr2.1002","article-title":"Evaluation of Culture Time and Media in an In Vitro Testis Organ Culture System","volume":"109","author":"N Nakamura","year":"2017","journal-title":"Birth Defects Res"},{"issue":"1","key":"pone.0342007.ref006","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/j.ydbio.2019.08.007","article-title":"Strains matter: Success of murine in vitro spermatogenesis is dependent on genetic background","volume":"456","author":"JMD Portela","year":"2019","journal-title":"Dev Biol"},{"issue":"12","key":"pone.0342007.ref007","first-page":"819","article-title":"Vitamin A prevents round spermatid nuclear damage and promotes the production of motile sperm during in vitro maturation of vitrified pre-pubertal mouse testicular tissue","volume":"22","author":"L Dumont","year":"2016","journal-title":"Mol Hum Reprod"},{"issue":"1","key":"pone.0342007.ref008","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1186\/s12958-017-0305-y","article-title":"Cellular and molecular characterization of gametogenic progression in ex vivo cultured prepuberal mouse testes","volume":"15","author":"J Isoler-Alcaraz","year":"2017","journal-title":"Reprod Biol Endocrinol"},{"issue":"6","key":"pone.0342007.ref009","doi-asserted-by":"crossref","first-page":"911","DOI":"10.1002\/j.1939-4640.2001.tb03430.x","article-title":"A century of research on mammalian male germ cell meiotic differentiation in vitro","volume":"22","author":"C Staub","year":"2001","journal-title":"J Androl"},{"issue":"3","key":"pone.0342007.ref010","doi-asserted-by":"crossref","first-page":"330","DOI":"10.1016\/j.stem.2016.01.017","article-title":"Complete Meiosis from Embryonic Stem Cell-Derived Germ Cells In Vitro","volume":"18","author":"Q Zhou","year":"2016","journal-title":"Cell Stem Cell"},{"key":"pone.0342007.ref011","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1016\/S0014-5793(99)00433-0","article-title":"Real-time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein","volume":"449","author":"T Nakanishi","year":"1999","journal-title":"FEBS Lett"},{"key":"pone.0342007.ref012","doi-asserted-by":"crossref","first-page":"30","DOI":"10.3389\/fcell.2014.00030","article-title":"Generation of a dual-color reporter mouse line to monitor spermatogenesis in vivo","volume":"2","author":"Y Makino","year":"2014","journal-title":"Front Cell Dev Biol"},{"issue":"5","key":"pone.0342007.ref013","doi-asserted-by":"crossref","first-page":"768","DOI":"10.1038\/nprot.2008.42","article-title":"A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies","volume":"3","author":"ES Ng","year":"2008","journal-title":"Nat Protoc"},{"issue":"6","key":"pone.0342007.ref014","doi-asserted-by":"crossref","first-page":"1514","DOI":"10.1038\/nprot.2014.102","article-title":"Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells","volume":"9","author":"VV Orlova","year":"2014","journal-title":"Nat Protoc"},{"issue":"9","key":"pone.0342007.ref015","doi-asserted-by":"crossref","first-page":"1681","DOI":"10.1038\/nprot.2016.098","article-title":"Generation of kidney organoids from human pluripotent stem cells","volume":"11","author":"M Takasato","year":"2016","journal-title":"Nat Protoc"},{"issue":"12","key":"pone.0342007.ref016","doi-asserted-by":"crossref","first-page":"1037","DOI":"10.1038\/nmeth.1740","article-title":"NKX2-5(eGFP\/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes","volume":"8","author":"DA Elliott","year":"2011","journal-title":"Nat Methods"},{"issue":"1","key":"pone.0342007.ref017","doi-asserted-by":"crossref","DOI":"10.1016\/j.cell.2022.11.026","article-title":"A transcription factor atlas of directed differentiation","volume":"186","author":"J Joung","year":"2023","journal-title":"Cell"},{"issue":"10","key":"pone.0342007.ref018","doi-asserted-by":"crossref","first-page":"939","DOI":"10.1016\/j.jcyt.2021.05.001","article-title":"Differentiation of natural killer cells from induced pluripotent stem cells under defined, serum- and feeder-free conditions","volume":"23","author":"KB Lupo","year":"2021","journal-title":"Cytotherapy"},{"key":"pone.0342007.ref019","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1242\/jcs.65.1.249","article-title":"Morphological and temporal sequence of meiotic prophase development at puberty in the male mouse","volume":"65","author":"P Goetz","year":"1984","journal-title":"J Cell Sci"},{"issue":"12","key":"pone.0342007.ref020","doi-asserted-by":"crossref","first-page":"3030","DOI":"10.1002\/bit.26822","article-title":"Neonatal testis growth recreated in vitro by two-dimensional organ spreading","volume":"115","author":"K Kojima","year":"2018","journal-title":"Biotechnol Bioeng"},{"issue":"4","key":"pone.0342007.ref021","doi-asserted-by":"crossref","first-page":"362","DOI":"10.1002\/rmb2.12291","article-title":"In vitro spermatogenesis in two-dimensionally spread mouse testis tissues","volume":"18","author":"M Komeya","year":"2019","journal-title":"Reprod Med Biol"},{"issue":"6","key":"pone.0342007.ref022","doi-asserted-by":"crossref","first-page":"594","DOI":"10.1101\/gad.237313.113","article-title":"Meiosis-specific cohesin mediates homolog recognition in mouse spermatocytes","volume":"28","author":"K-I Ishiguro","year":"2014","journal-title":"Genes Dev"},{"issue":"1","key":"pone.0342007.ref023","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1023\/A:1018445520117","article-title":"A drying-down technique for the spreading of mammalian meiocytes from the male and female germline","volume":"5","author":"AH Peters","year":"1997","journal-title":"Chromosome Res"},{"issue":"1","key":"pone.0342007.ref024","doi-asserted-by":"crossref","first-page":"504","DOI":"10.1038\/s42003-022-03449-y","article-title":"Temperature sensitivity of DNA double-strand break repair underpins heat-induced meiotic failure in mouse spermatogenesis","volume":"5","author":"K Hirano","year":"2022","journal-title":"Commun Biol"},{"issue":"13","key":"pone.0342007.ref025","doi-asserted-by":"crossref","first-page":"5091","DOI":"10.1002\/j.1460-2075.1992.tb05616.x","article-title":"A coiled-coil related protein specific for synapsed regions of meiotic prophase chromosomes","volume":"11","author":"RL Meuwissen","year":"1992","journal-title":"EMBO J"},{"key":"pone.0342007.ref026","doi-asserted-by":"crossref","first-page":"525","DOI":"10.1146\/annurev.cellbio.19.111301.155141","article-title":"The genetics and molecular biology of the synaptonemal complex","volume":"20","author":"SL Page","year":"2004","journal-title":"Annu Rev Cell Dev Biol"},{"issue":"3","key":"pone.0342007.ref027","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1038\/85830","article-title":"Recombinational DNA double-strand breaks in mice precede synapsis","volume":"27","author":"SK Mahadevaiah","year":"2001","journal-title":"Nat Genet"},{"issue":"3","key":"pone.0342007.ref028","doi-asserted-by":"crossref","first-page":"452","DOI":"10.1038\/bmt.2012.244","article-title":"Investigation of the freely available easy-to-use software \u201cEZR\u201d for medical statistics","volume":"48","author":"Y Kanda","year":"2013","journal-title":"Bone Marrow Transplant"},{"issue":"6","key":"pone.0342007.ref029","doi-asserted-by":"crossref","first-page":"501","DOI":"10.1016\/S0040-8166(00)80006-3","article-title":"Regulation of acrosome formation in mice expressing green fluorescent protein as a marker","volume":"32","author":"S Ventel\u00e4","year":"2000","journal-title":"Tissue Cell"},{"key":"pone.0342007.ref030","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1290\/1543-706X(2003)039<0388:BPEPRP>2.0.CO;2","article-title":"Bovine pituitary extract provides remarkable protection against oxidative stress in human prostate epithelial cells","volume":"39","author":"KD Kent","year":"2003","journal-title":"In Vitro Cell Dev Biol Anim"},{"key":"pone.0342007.ref031","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/j.semcdb.2014.02.010","article-title":"Hormonal control of germ cell development and spermatogenesis","volume":"29","author":"PJ O\u2019Shaughnessy","year":"2014","journal-title":"Semin Cell Dev Biol"},{"issue":"1","key":"pone.0342007.ref032","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1038\/ng0996-62","article-title":"Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds","volume":"14","author":"S Morais da Silva","year":"1996","journal-title":"Nat Genet"},{"issue":"2","key":"pone.0342007.ref033","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1006\/dbio.1994.1152","article-title":"Developmentally regulated expression of a mouse germ cell nuclear antigen examined from embryonic day 11 to adult in male and female mice","volume":"163","author":"GC Enders","year":"1994","journal-title":"Dev Biol"},{"issue":"6","key":"pone.0342007.ref034","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1046\/j.1365-2605.1998.00080.x","article-title":"A germ cell-specific nuclear antigen recognized by a monoclonal antibody raised against mouse testicular germ cells","volume":"20","author":"H Tanaka","year":"1997","journal-title":"Int J Androl"},{"issue":"5","key":"pone.0342007.ref035","doi-asserted-by":"crossref","first-page":"658","DOI":"10.1016\/j.stem.2014.01.019","article-title":"Mouse spermatogenic stem cells continually interconvert between equipotent singly isolated and syncytial states","volume":"14","author":"K Hara","year":"2014","journal-title":"Cell Stem Cell"},{"issue":"3","key":"pone.0342007.ref036","doi-asserted-by":"crossref","first-page":"109875","DOI":"10.1016\/j.celrep.2021.109875","article-title":"A multistate stem cell dynamics maintains homeostasis in mouse spermatogenesis","volume":"37","author":"T Nakagawa","year":"2021","journal-title":"Cell Rep"},{"issue":"1","key":"pone.0342007.ref037","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/S1097-2765(00)80404-9","article-title":"The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility","volume":"5","author":"L Yuan","year":"2000","journal-title":"Mol Cell"},{"issue":"7","key":"pone.0342007.ref038","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pgen.1006904","article-title":"Tex19.1 promotes Spo11-dependent meiotic recombination in mouse spermatocytes","volume":"13","author":"JH Crichton","year":"2017","journal-title":"PLoS Genet"},{"issue":"9","key":"pone.0342007.ref039","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pgen.1000199","article-title":"Deletion of the pluripotency-associated Tex19.1 gene causes activation of endogenous retroviruses and defective spermatogenesis in mice","volume":"4","author":"R Ollinger","year":"2008","journal-title":"PLoS Genet"},{"issue":"1","key":"pone.0342007.ref040","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1530\/jrf.0.0930233","article-title":"Proliferative activity of gonocytes, Sertoli cells and interstitial cells during testicular development in mice","volume":"93","author":"RP Vergouwen","year":"1991","journal-title":"J Reprod Fertil"},{"issue":"2","key":"pone.0342007.ref041","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1095\/biolreprod.110.083899","article-title":"In vitro murine spermatogenesis in an organ culture system","volume":"83","author":"A Gohbara","year":"2010","journal-title":"Biol Reprod"},{"issue":"1","key":"pone.0342007.ref042","doi-asserted-by":"crossref","first-page":"1488","DOI":"10.1038\/s41467-025-56742-9","article-title":"Intercellular bridges are essential for transposon repression and meiosis in the male germline","volume":"16","author":"J Sorkin","year":"2025","journal-title":"Nat Commun"},{"issue":"1","key":"pone.0342007.ref043","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1002\/j.1939-4640.1986.tb00873.x","article-title":"Polarized Sertoli cell functions in a new two-compartment culture system","volume":"7","author":"A Janecki","year":"1986","journal-title":"J Androl"},{"key":"pone.0342007.ref044","doi-asserted-by":"crossref","first-page":"21472","DOI":"10.1038\/srep21472","article-title":"Long-term ex vivo maintenance of testis tissues producing fertile sperm in a microfluidic device","volume":"6","author":"M Komeya","year":"2016","journal-title":"Sci Rep"},{"issue":"4","key":"pone.0342007.ref045","doi-asserted-by":"crossref","first-page":"885","DOI":"10.1016\/j.bbrc.2018.04.180","article-title":"A monolayer microfluidic device supporting mouse spermatogenesis with improved visibility","volume":"500","author":"H Yamanaka","year":"2018","journal-title":"Biochem Biophys Res Commun"},{"issue":"5","key":"pone.0342007.ref046","doi-asserted-by":"crossref","first-page":"1140","DOI":"10.1016\/j.stemcr.2020.10.006","article-title":"In Vitro Meiosis of Male Germline Stem Cells","volume":"15","author":"Q Lei","year":"2020","journal-title":"Stem Cell Reports"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0342007","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,5]],"date-time":"2026-02-05T18:34:13Z","timestamp":1770316453000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0342007"}},"subtitle":[],"editor":[{"given":"Suresh","family":"Yenugu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,2,5]]},"references-count":46,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2026,2,5]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0342007","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,5]]}}}