{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T21:10:12Z","timestamp":1765228212119,"version":"3.46.0"},"reference-count":27,"publisher":"Public Library of Science (PLoS)","issue":"12","license":[{"start":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T00:00:00Z","timestamp":1765152000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["CRU326"],"award-info":[{"award-number":["CRU326"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    Classical histology struggles to preserve three-dimensional spatial context, prompting the emergence of optical tissue clearing techniques that enable imaging of intact specimens at cellular or subcellular resolution. These techniques have revolutionised fields like cell biology, developmental biology, and neuroscience. However, their application in reproductive biology remains unexplored \u2013 particularly in studying the complexities of testicular development. We developed a novel, efficient and affordable toolbox for studying intact testicular tissues, PT-CLEAR3D, that stands for primate testis \u2013 whole mount staining, tissue clearing and three-dimensional imaging. Intact testicular tissues from humans (transgender model), common marmosets and macaques underwent antibody labelling, clearing with organic solvents, and three-dimensional imaging using light sheet fluorescence microscopy. Marker specificity was confirmed by immunofluorescence staining of 3 and 25 \u00b5m testicular sections, followed by imaging with confocal microscopy. The testicular structure was evaluated using several markers: spermatogonia (melanoma-associated antigen 4), least differentiated spermatogonia (Piwi-like protein 4), Sertoli cells (vimentin and SRY-Box transcription Factor 9), peritubular myoid cells and vasculature (alpha-smooth muscle actin), and NucSpot as a nuclear dye. PT-CLEAR3D efficiently achieved optical transparency while a commercial kit that was run in parallel was inefficient. This study presents a pioneering three-dimensional visualization of intact testicular samples of up to 50\u2009mm\n                    <jats:sup>3<\/jats:sup>\n                    in size and imaging depth of up to 4.5\u2009mm across three primate species. Remarkably, PT-CLEAR3D revealed critical details at both tissue and cellular levels such as the spatial distribution of germ and somatic cells, cellular bridges, and vasculature. Furthermore, PT-CLEAR3D enabled three-dimensional reconstructions that effectively reduces confirmation bias enhancing our observation of spermatogonial clones organized as single cells, pairs, and quartets. Importantly, it adeptly identified testicular pathology and the persistence of germ cell clones in select tubules within the transgender testis following hormonal suppression of spermatogenesis. This technological development offers a versatile toolbox with benefits such as applicability across multiple species, fluorophore multiplexing, compatibility with different fixatives and deep tissue volumetric imaging with cellular resolution. Overall, PT-CLEAR3D establishes a foundation for spatial evaluation of testicular development, presenting substantial potential for advancing our understanding of the intricate kinetics of spermatogenesis in health and disease.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0327287","type":"journal-article","created":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T18:23:57Z","timestamp":1765218237000},"page":"e0327287","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Optical tissue clearing and 3D imaging of intact primate testicular tissue: a novel technology development"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0964-7604","authenticated-orcid":true,"given":"Pauline Wanjiku","family":"Kibui","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7292-8308","authenticated-orcid":true,"given":"Sarah","family":"Weischer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicola","family":"von Ostau","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9902-5368","authenticated-orcid":true,"given":"Jochen","family":"Hess","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nils","family":"Kirschnick","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thomas","family":"Zobel","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7571-5313","authenticated-orcid":true,"given":"Stefan","family":"Schlatt","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2025,12,8]]},"reference":[{"issue":"3","key":"pone.0327287.ref001","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1159\/000526024","article-title":"Evolutionary changes in pathology and our understanding of disease","volume":"90","author":"EJC Chew","year":"2023","journal-title":"Pathobiology"},{"key":"pone.0327287.ref002","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1007\/978-3-030-47483-6_5","article-title":"From scope to screen: the evolution of histology education","volume":"1260","author":"JA Chapman","year":"2020","journal-title":"Adv Exp Med Biol"},{"issue":"7","key":"pone.0327287.ref003","doi-asserted-by":"crossref","first-page":"1153","DOI":"10.1038\/s41592-024-02327-1","article-title":"Deep 3D histology powered by tissue clearing, omics and AI","volume":"21","author":"A Ert\u00fcrk","year":"2024","journal-title":"Nat Methods"},{"issue":"2","key":"pone.0327287.ref004","doi-asserted-by":"crossref","first-page":"246","DOI":"10.1016\/j.cell.2015.06.067","article-title":"Clarifying tissue clearing","volume":"162","author":"DS Richardson","year":"2015","journal-title":"Cell"},{"issue":"6","key":"pone.0327287.ref005","doi-asserted-by":"crossref","first-page":"102650","DOI":"10.1016\/j.isci.2021.102650","article-title":"Rapid methods for the evaluation of fluorescent reporters in tissue clearing and the segmentation of large vascular structures","volume":"24","author":"N Kirschnick","year":"2021","journal-title":"iScience"},{"issue":"18","key":"pone.0327287.ref006","doi-asserted-by":"crossref","DOI":"10.1242\/dev.199369","article-title":"Tissue clearing and 3D imaging in developmental biology","volume":"148","author":"A Vieites-Prado","year":"2021","journal-title":"Development"},{"issue":"11","key":"pone.0327287.ref007","doi-asserted-by":"crossref","DOI":"10.1083\/jcb.202307143","article-title":"Successful 3D imaging of cleared biological samples with light sheet fluorescence microscopy","volume":"222","author":"E Delage","year":"2023","journal-title":"J Cell Biol"},{"key":"pone.0327287.ref008","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1016\/j.semcdb.2014.03.007","article-title":"Regulation of spermatogenesis: an evolutionary biologist\u2019s perspective","volume":"29","author":"S Schlatt","year":"2014","journal-title":"Semin Cell Dev Biol"},{"key":"pone.0327287.ref009","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1007\/978-1-4615-9008-8_6","article-title":"Dynamics of human spermatogenesis.","author":"Y Clermont","year":"1970","journal-title":"Advances in experimental medicine and biology. 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