{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:34:49Z","timestamp":1760146489837,"version":"build-2065373602"},"reference-count":41,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2024,11,11]],"date-time":"2024-11-11T00:00:00Z","timestamp":1731283200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Grants-in-Aid for Scientific Research on Innovative Areas","award":["21112004","16083203","2370023"],"award-info":[{"award-number":["21112004","16083203","2370023"]}]},{"name":"Scientific Research on Priority Areas","award":["21112004","16083203","2370023"],"award-info":[{"award-number":["21112004","16083203","2370023"]}]},{"name":"Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan","award":["21112004","16083203","2370023"],"award-info":[{"award-number":["21112004","16083203","2370023"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Animals show diverse processes of gametogenesis in the evolutionary pathway. Here, we characterized the spermatogenic cells in the testis of the marine invertebrate Ciona robusta. Ciona sperm differentiate in a non-cystic type of testis, comprising many follicles with various sizes and stages of spermatogenic cells. In the space among follicles, we observed free cells that were recognized by antibody against M\u00fcllerian inhibiting substance, a marker for vertebrate Sertoli cells. We further categorized the spermatogenic cells into four round stages (RI to RIV) and three elongated stages (EI to EIII) by morphological and immunohistochemical criteria. An antibody against a vertebrate Vasa homolog recognized a few large spermatogonium-like cells (RI) near the basal wall of a follicle. Consistent with the period of meiosis, a synaptonemal complex protein SYCP3 was recognized from early spermatocytes (RII) to early spermatids (E1). Acetylated tubulins were detected in spermatids before flagellar elongation at the RIV stage and became distributed along the flagella. Electron microscopy showed that the free cells outside the testicular follicle possessed a characteristic of vertebrate Sertoli cells. These results would provide a basis for basic and comparative studies on the mechanism of spermatogenesis.<\/jats:p>","DOI":"10.3390\/cells13221863","type":"journal-article","created":{"date-parts":[[2024,11,11]],"date-time":"2024-11-11T08:01:27Z","timestamp":1731312087000},"page":"1863","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Immunohistochemical Characterization of Spermatogenesis in the Ascidian Ciona robusta"],"prefix":"10.3390","volume":"13","author":[{"given":"Haruka","family":"Sakurai","sequence":"first","affiliation":[{"name":"Shimoda Marine Research Center, University of Tsukuba, 5-10-1, Shimoda 415-0025, Shizuoka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7742-8158","authenticated-orcid":false,"given":"Kogiku","family":"Shiba","sequence":"additional","affiliation":[{"name":"Shimoda Marine Research Center, University of Tsukuba, 5-10-1, Shimoda 415-0025, Shizuoka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Katsumi","family":"Takamura","sequence":"additional","affiliation":[{"name":"Faculty of Life Science and Biotechnology, Fukuyama University, Fukuyama 729-0292, Hiroshima, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8848-9733","authenticated-orcid":false,"given":"Kazuo","family":"Inaba","sequence":"additional","affiliation":[{"name":"Shimoda Marine Research Center, University of Tsukuba, 5-10-1, Shimoda 415-0025, Shizuoka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,11,11]]},"reference":[{"key":"ref_1","unstructured":"Russell, L.D., Ettlin, R., Sinha Hikim, A.P., and Clegg, E.D. (1990). Mammalian spermatogenesis. Histological and Histopathological Evaluation of the Testis, Cache River."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1152\/physrev.00013.2015","article-title":"Spermatogenesis: The Commitment to meiosis","volume":"96","author":"Griswold","year":"2016","journal-title":"Physiol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1002\/wdev.204","article-title":"From cyst to tubule: Innovations in vertebrate spermatogenesis","volume":"5","author":"Yoshida","year":"2016","journal-title":"Wiley Interdiscip. Rev. Dev. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Satoh, N. (2014). Developmental Genomics of Ascidians, Wiley-Blackwell.","DOI":"10.1002\/9781118656129"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1002\/mrd.10131","article-title":"EST analysis of gene expression in testis of the ascidian Ciona intestinalis","volume":"62","author":"Inaba","year":"2002","journal-title":"Mol. Reprod. Dev."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Saito, T., and Sawada, H. (2021). Fertilization of ascidians: Gamete interaction, self\/nonself recognition and sperm penetration of egg coat. Front. Cell Dev. Biol., 9.","DOI":"10.3389\/fcell.2021.827214"},{"key":"ref_7","unstructured":"Metz, C.B., and Monroy, A. (1985). Sperm chemo-orientation in metazoan. Biology of Fertilization, Academic Press."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"14831","DOI":"10.1073\/pnas.242470599","article-title":"A chemoattractant for ascidian spermatozoa is a sulfated steroid","volume":"99","author":"Yoshida","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1043","DOI":"10.2108\/zsj.20.1043","article-title":"Molecular architecture of the sperm flagella: Molecules for motility and signaling","volume":"20","author":"Inaba","year":"2003","journal-title":"Zool. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"20497","DOI":"10.1073\/pnas.1217018109","article-title":"Calaxin drives sperm chemotaxis by Ca2+-mediated direct modulation of a dynein motor","volume":"109","author":"Mizuno","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Yoshida, K., Shiba, K., Sakamoto, A., Ikenaga, J., Matsunaga, S., Inaba, K., and Yoshida, M. (2018). Ca2+ efflux via plasma membrane Ca2+-ATPase mediates chemotaxis in ascidian sperm. Sci. Rep., 8.","DOI":"10.1038\/s41598-018-35013-2"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1093\/icb\/22.4.841","article-title":"The ascidian sperm reaction","volume":"22","author":"Lambert","year":"1982","journal-title":"Am. Zool."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1111\/j.1440-169X.1988.00325.x","article-title":"Sperm binding and penetration during ascidian fertilization","volume":"30","author":"Lambert","year":"1988","journal-title":"Dev. Growth Differ."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1073\/pnas.9.5.170","article-title":"Removal of the block to self-fertilization in the ascidian Ciona","volume":"9","author":"Morgan","year":"1923","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"548","DOI":"10.1126\/science.1152488","article-title":"Mechanism of self-sterility in a hermaphroditic chordate","volume":"320","author":"Harada","year":"2008","journal-title":"Science"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"4158","DOI":"10.1073\/pnas.1115086109","article-title":"Self-incompatiblity response induced by calcium increase in sperm of the ascidian Ciona intestinalis","volume":"109","author":"Saito","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","first-page":"391","article-title":"Spermatogenese et spermiogenese de Ciona intestinalis L. Observees au electronique","volume":"8","author":"Georges","year":"1969","journal-title":"J. Microscopie."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1111\/j.1440-169X.1993.00495.x","article-title":"Identification of early oogenetic cells in the solitary ascidians, Ciona savignyi and Ciona intestinalis: An immunoelectron microscopic study","volume":"35","author":"Okada","year":"1993","journal-title":"Dev. Growth Differ."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"759","DOI":"10.1046\/j.1440-169x.1999.00471.x","article-title":"Differentiation of the gonad rudiment into ovary and testis in the solitary ascidian, Ciona intestinalis","volume":"41","author":"Okada","year":"1999","journal-title":"Dev. Growth Differ."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1007\/s00427-001-0204-1","article-title":"Primordial germ cells originate from the endodermal strand cells in the ascidian Ciona intestinalis","volume":"212","author":"Takamura","year":"2002","journal-title":"Dev. Genes Evol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1111\/j.1440-169X.1980.00561.x","article-title":"Acrosome differentiation in the spermatogenesis of Ciona intestinalis","volume":"22","author":"Cotelli","year":"1980","journal-title":"Dev. Growth Differ."},{"key":"ref_22","unstructured":"Wilson, W.H., Stricker, S.A., and Shinn, G.L. (1994). Spermatogenesis in the ovotestes of the solitary ascidian Boltenia villosa. Reproduction and Development of Marine Invertebrates, Hopkins University Press."},{"key":"ref_23","first-page":"221","article-title":"Urochordata: Ascidiacea","volume":"Volume 15","author":"Harrison","year":"1997","journal-title":"Microscopic Anatomy of Invertebrates"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1002\/dvdy.22542","article-title":"Germline cell formation and gonad regeneration in solitary and colonial ascidians","volume":"240","author":"Kawamura","year":"2011","journal-title":"Dev. Dyn."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"204","DOI":"10.2108\/zsj.27.204","article-title":"Multidimensional analysis of uncharacterized sperm proteins in Ciona intestinalis: EST-based analysis and functional immunoscreening of testis-expressed genes","volume":"27","author":"Konno","year":"2010","journal-title":"Zool. Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1016\/j.ydbio.2009.10.012","article-title":"Distribution and structural diversity of cilia in tadpole larvae of the ascidian Ciona intestinalis","volume":"337","author":"Konno","year":"2010","journal-title":"Dev. Biol."},{"key":"ref_27","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":"Yuan","year":"2000","journal-title":"Mol. Cell"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"773","DOI":"10.1038\/nrm3227","article-title":"Post-translational regulation of the microtubule cytoskeleton: Mechanisms and functions","volume":"12","author":"Janke","year":"2011","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2085","DOI":"10.1083\/jcb.101.6.2085","article-title":"Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms","volume":"101","author":"Piperno","year":"1985","journal-title":"J. Cell Biol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Konno, A., Shiba, K., Cai, C., and Inaba, K. (2015). Branchial cilia and sperm flagella recruit distinct axonemal components. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0126005"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"eabf3621","DOI":"10.1126\/sciadv.abf3621","article-title":"A dynein-associated photoreceptor protein prevents ciliary acclimation to blue light","volume":"7","author":"Kutomi","year":"2021","journal-title":"Sci. Adv."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e983400","DOI":"10.4161\/21565562.2014.983400","article-title":"Comparative testicular structure and spermatogenesis in bony fishes","volume":"4","author":"Uribe","year":"2015","journal-title":"Spermatogenesis"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"4755514","DOI":"10.1155\/2022\/4755514","article-title":"Models and molecular markers of spermatogonial stem cells in vertebrates: To find models in nonmammals","volume":"2022","author":"Song","year":"2022","journal-title":"Stem Cells Int."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1007\/s00441-019-03004-y","article-title":"Spermatogenesis and lobular cyst type of testes organization in marine gastropod Littorina saxatilis (Olivi 1792)","volume":"376","author":"Demin","year":"2019","journal-title":"Cell Tissue Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"31","DOI":"10.2307\/3593140","article-title":"Nutritive phagocyte incubation chambers provide a structural and nutritive microenvironment for germ cells of Strongylocentrotus droebachiensis, the green sea urchin","volume":"209","author":"Walker","year":"2005","journal-title":"Biol. Bull."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/B978-0-12-396491-5.00003-4","article-title":"Chapter 3. Sea urchin gametogenesis\u2014Structural, functional and molecular\/genomic biology","volume":"Volume 38","author":"Walker","year":"2013","journal-title":"Sea Urchins: Biology and Ecology"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1083\/jcb.103.1.13","article-title":"Cytoplasmic microtubules containing acetylated alpha-tubulin in Chlamydomonas reinhardtii: Spatial arrangement and properties","volume":"103","author":"LeDizet","year":"1986","journal-title":"J. Cell Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"4237","DOI":"10.1007\/s00018-015-2000-5","article-title":"Tubulin acetylation: Responsible enzymes, biological functions and human diseases","volume":"72","author":"Li","year":"2015","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"R1143","DOI":"10.1016\/j.cub.2015.11.001","article-title":"Shared and distinct mechanisms of compartmentalized and cytosolic ciliogenesis","volume":"25","author":"Leroux","year":"2015","journal-title":"Curr. Biol."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Fingerhut, J.M., and Yamashita, Y.M. (2020). mRNA localization mediates maturation of cytoplasmic cilia in Drosophila spermatogenesis. J. Cell Biol., 219.","DOI":"10.1083\/jcb.202003084"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Riparbelli, M.G., Persico, V., and Callaini, G. (2020). The microtubule cytoskeleton during the early Drosophila spermiogenesis. Cells, 9.","DOI":"10.3390\/cells9122684"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/13\/22\/1863\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:29:53Z","timestamp":1760113793000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/13\/22\/1863"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,11,11]]},"references-count":41,"journal-issue":{"issue":"22","published-online":{"date-parts":[[2024,11]]}},"alternative-id":["cells13221863"],"URL":"https:\/\/doi.org\/10.3390\/cells13221863","relation":{},"ISSN":["2073-4409"],"issn-type":[{"type":"electronic","value":"2073-4409"}],"subject":[],"published":{"date-parts":[[2024,11,11]]}}}