{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T00:35:58Z","timestamp":1759970158852,"version":"build-2065373602"},"reference-count":56,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2025,1,7]],"date-time":"2025-01-07T00:00:00Z","timestamp":1736208000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Taishan Scholars Program","award":["2023BBB172","2023TD36","YFI20240501"],"award-info":[{"award-number":["2023BBB172","2023TD36","YFI20240501"]}]},{"name":"Key R&amp;D Program of Hubei Province","award":["2023BBB172","2023TD36","YFI20240501"],"award-info":[{"award-number":["2023BBB172","2023TD36","YFI20240501"]}]},{"name":"Central Public-Interest Scientific Institution Basal Research Fund, Chinese Academy of Fishery Sciences (CASF)","award":["2023BBB172","2023TD36","YFI20240501"],"award-info":[{"award-number":["2023BBB172","2023TD36","YFI20240501"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Monopterus albus is a protogynous hermaphroditic fish that changes from female to male, but the underlying sex change mechanism remains as-yet unknown. In this study, we firstly cloned and characterized the sequence and protein structure of unc-13d of M. albus. We found that the genomic structure of unc-13d was different from other species. Expression was detected in the developing gonad by applying qRT-PCR and in situ hybridization. We found that the expression of unc-13d in the ovotestis was higher than in the ovary and testes. A strong signal of unc-13d was detected in oocytes and granulosa cells in the ovary and spermatogonia and primary spermatocytes in the testes. We found that the promoter methylation of unc-13d was negatively correlated with gene expression in developing gonads, especially at site 114. A dual-luciferase assay was designed and revealed that dmrt1 regulates promoter activity opposite to foxl2. In summary, during sex reversal, DNA methylation affects the binding of the transcription factor dmrt1 and foxl2 in the promoter region through methylation and demethylation interactions to regulate the expression of unc-13d during gonadal development.<\/jats:p>","DOI":"10.3390\/ani15020122","type":"journal-article","created":{"date-parts":[[2025,1,7]],"date-time":"2025-01-07T07:42:19Z","timestamp":1736235739000},"page":"122","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Molecular Characterization and Expression of unc-13d in the Sex Reversal of Monopterus albus"],"prefix":"10.3390","volume":"15","author":[{"given":"Zitong","family":"Lian","sequence":"first","affiliation":[{"name":"Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fang","family":"Meng","sequence":"additional","affiliation":[{"name":"Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xueping","family":"Xia","sequence":"additional","affiliation":[{"name":"Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Junchao","family":"Fang","sequence":"additional","affiliation":[{"name":"Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8264-5579","authenticated-orcid":false,"given":"Haifeng","family":"Tian","sequence":"additional","affiliation":[{"name":"Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qiaomu","family":"Hu","sequence":"additional","affiliation":[{"name":"Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China"},{"name":"State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"R481","DOI":"10.1016\/S0960-9822(01)00287-1","article-title":"Gonad development: Signals for sex","volume":"11","author":"Koopman","year":"2001","journal-title":"Curr. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Manolakou, P., Lavranos, G., and Angelopoulou, R. (2006). Molecular patterns of sex determination in the animal kingdom: A comparative study of the biology of reproduction. Reprod. Biol. Endocrinol., 4.","DOI":"10.1186\/1477-7827-4-59"},{"key":"ref_3","unstructured":"Nelson, J.S. (2006). Fishes of the World, John Wiley & Sons, Inc.. [4th ed.]."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"648","DOI":"10.1016\/j.conb.2009.09.015","article-title":"Fish sex: Why so diverse?","volume":"19","author":"Desjardins","year":"2009","journal-title":"Curr. Opin. Neurobiol."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Trukhina, A.V., Lukina, N.A., Wackerow-Kouzova, N.D., and Smirnov, A.F. (2013). The variety of vertebrate mechanisms of sex determination. BioMed Res. Int., 2013.","DOI":"10.1155\/2013\/587460"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"604","DOI":"10.1101\/gr.162172.113","article-title":"Epigenetic modification and inheritance in sexual reversal of fish","volume":"24","author":"Shao","year":"2014","journal-title":"Genome Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/j.gpb.2020.10.004","article-title":"The role of DNA methylation reprogramming during sex determination and transition in zebrafish","volume":"19","author":"Wang","year":"2021","journal-title":"Genom. Proteom. Bioinform."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"146260","DOI":"10.1016\/j.gene.2022.146260","article-title":"DNA methylation differences between male and female gonads of the oyster reveal the role of epigenetics in sex determination","volume":"820","author":"Sun","year":"2022","journal-title":"Gene"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Navarro-Mart\u00edn, L., Vi\u00f1as, J., Ribas, L., D\u00edaz, N., Guti\u00e9rrez, A., Di Croce, L., and Piferrer, F. (2011). DNA methylation of the gonadal aromatase (cyp19a) promoter is involved in temperature-dependent sex ratio shifts in the European sea bass. PLoS Genet., 7.","DOI":"10.1371\/journal.pgen.1002447"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Yang, D., Chen, F., and Ruan, G. (2018). Aquaculture of the paddy Eel, Monopterus albus. Aquaculture in China: Success Stories and Modern Trends, John Wiley & Sons, Inc.","DOI":"10.1002\/9781119120759.ch3_9"},{"key":"ref_11","first-page":"26","article-title":"Current practices of rice fifield eel Monopterus albus (zuiew, 1973) culture in Viet Nam","volume":"15","author":"Khanh","year":"2010","journal-title":"Aquac. Asia Mag."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1016\/j.aquaculture.2018.12.067","article-title":"Effects of different stocking densities on growth performance of Asian swamp eel Monopterus albus, water quality and plant growth of watercress Nasturtium officinale in an aquaponic recirculating system","volume":"503","author":"Nhan","year":"2019","journal-title":"Aquaculture"},{"key":"ref_13","first-page":"1","article-title":"Rudimentary hermaphroditism in the symbranchoid eel, Monopterus javanensis","volume":"15","author":"Liu","year":"1944","journal-title":"Sinensia"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"303","DOI":"10.2307\/1441348","article-title":"Sex reversal as a natural process in the synbranchiform fish Monopterus albus","volume":"2","author":"Liem","year":"1963","journal-title":"Copeia"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1111\/j.1469-7998.1967.tb02868.x","article-title":"The structure of the gonad during natural sex reversal in Monopterus albus (Pisces: Teleostei)","volume":"151","author":"Chan","year":"1967","journal-title":"J. Zool."},{"key":"ref_16","first-page":"45","article-title":"Study on the reproductive biology of Monopterus albus (Zuiew) II female development of Monopterus albus","volume":"18","author":"Xiao","year":"1995","journal-title":"ACTA Sci. Nat. Universtis Norm. Hunanensis"},{"key":"ref_17","first-page":"297","article-title":"Study on the histology in sex changing from intersex to male of Monopterus albus","volume":"19","author":"Xiao","year":"1995","journal-title":"Fish China"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1155","DOI":"10.1007\/s10695-020-00778-9","article-title":"Expression and regulation of Smad2 by gonadotropins in the protogynous hermaphroditic ricefield eel (Monopterus albus)","volume":"46","author":"He","year":"2020","journal-title":"Fish Physiol. Biochem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"giy046","DOI":"10.1093\/gigascience\/giy046","article-title":"Chromosome-scale assembly of the Monopterus genome","volume":"7","author":"Zhao","year":"2018","journal-title":"Gigascience"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"274","DOI":"10.1159\/000074348","article-title":"The rice field eel as a model system for vertebrate sexual development","volume":"101","author":"Cheng","year":"2003","journal-title":"Cytogenet. Genome Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1137","DOI":"10.1016\/j.tig.2021.09.007","article-title":"Swamp eel (Monopterus albus)","volume":"37","author":"Cheng","year":"2021","journal-title":"Trends Genet."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.theriogenology.2021.09.036","article-title":"Crucial role of dead end gene for primordial germ cell survival in rice field eel (Monopterus albus)","volume":"176","author":"Hu","year":"2021","journal-title":"Theriogenology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"736543","DOI":"10.1016\/j.aquaculture.2021.736543","article-title":"Blood cell identification and hematological analysis during natural sex reversal in rice field eel (Monopterus albus)","volume":"538","author":"Chen","year":"2021","journal-title":"Aquaculture"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"113871","DOI":"10.1016\/j.ygcen.2021.113871","article-title":"Nr5a homologues in the ricefield eel Monopterus albus: Alternative splicing, tissue-specific expression, and differential roles on the activation of cyp19a1a promoter in vitro","volume":"312","author":"Yan","year":"2021","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Wang, X., Lai, F., Xiong, J., Zhu, W., Yuan, B., Cheng, H., and Zhou, R. (2020). DNA methylation modification is associated with gonadal differentiation in Monopterus albus. Cell Biosci., 10.","DOI":"10.1186\/s13578-020-00490-4"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Hu, Q., Lian, Z., Xia, X., Tian, H., and Li, Z. (2022). Integrated chromatin accessibility and DNA methylation analysis to reveal the critical epigenetic modification and regulatory mechanism in gonadal differentiation of the sequentially hermaphroditic fish, Monopterus albus. Biol. Sex Differ., 13.","DOI":"10.1186\/s13293-022-00484-6"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1093\/nar\/gkj079","article-title":"SMART 5: Domains in the context of genomes and networks","volume":"34","author":"Letunic","year":"2006","journal-title":"Nucleic Acids Res."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Walker, J.M. (2005). Protein identification and analysis tools on the expasy server. The Proteomics Protocols Handbook, Humana Press.","DOI":"10.1385\/1592598900"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"W296","DOI":"10.1093\/nar\/gky427","article-title":"SWISS-MODEL: Homology modelling of protein structures and complexes","volume":"46","author":"Waterhouse","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1870","DOI":"10.1093\/molbev\/msw054","article-title":"MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets","volume":"33","author":"Kumar","year":"2016","journal-title":"Mol. Biol. Evol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1935","DOI":"10.1093\/bioinformatics\/bts295","article-title":"CodonLogo: A sequence logo-based viewer for codon patterns","volume":"28","author":"Sharma","year":"2012","journal-title":"Bioinformatics"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1093\/nar\/gkp335","article-title":"MEME SUITE: Tools for motif discovery and searching","volume":"37","author":"Bailey","year":"2009","journal-title":"Nucleic Acids Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"D700","DOI":"10.1093\/nar\/gks1156","article-title":"Genomicus: Five genome browsers for comparative genomics in eukaryota","volume":"41","author":"Louis","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.fsi.2021.11.021","article-title":"Characterization of reference genes for qRT-PCR normalization in rice-field eel (Monopterus albus) to assess differences in embryonic developmental stages, the early development of immune organs, and cells infected with rhabdovirus","volume":"120","author":"Liu","year":"2022","journal-title":"Fish Shellfish Immunol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1101","DOI":"10.1038\/nprot.2008.73","article-title":"Analyzing real-time PCR data by the comparative C(T) method","volume":"3","author":"Schmittgen","year":"2008","journal-title":"Nat. Protoc."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1089\/152791600459894","article-title":"Primer premier 5","volume":"1","author":"Lalitha","year":"2000","journal-title":"Biotech Softw. Internet Rep."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1427","DOI":"10.1093\/bioinformatics\/18.11.1427","article-title":"MethPrimer: Designing primers for methylation PCRs","volume":"18","author":"Li","year":"2002","journal-title":"Bioinformatics"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Rohde, C., Zhang, Y., Reinhardt, R., and Jeltsch, A. (2010). BISMA\u2014Fast and accurate bisulfite sequencing data analysis of individual clones from unique and repetitive sequences. BMC Bioinform., 11.","DOI":"10.1186\/1471-2105-11-230"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"D165","DOI":"10.1093\/nar\/gkab1113","article-title":"JASPAR 2022: The 9th release of the open-access database of transcription factor binding profiles","volume":"50","author":"Rauluseviciute","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Mustapha, U.F., Assan, D., Huang, Y.Q., Li, G.L., and Jiang, D.N. (2022). High polymorphism in the dmrt2a gene is incompletely sex-linked in spotted 395 scat, Scatophagus argus. Animals, 12.","DOI":"10.3390\/ani12050613"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1038\/nature08298","article-title":"The avian Z-linked gene DMRT1 is required for male sex determination in the chicken","volume":"461","author":"Smith","year":"2009","journal-title":"Nature"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"404","DOI":"10.1016\/j.cub.2013.12.039","article-title":"FOXL2 is a female sex-determining gene in the goat","volume":"24","author":"Boulanger","year":"2014","journal-title":"Curr. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1042\/bj3490247","article-title":"Definition of Munc13-homology-domains and characterization of a novel ubiquitously expressed Munc13 isoform","volume":"349","author":"Koch","year":"2000","journal-title":"Biochem. J."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"25273","DOI":"10.1074\/jbc.270.42.25273","article-title":"Mammalian homologues of Caenorhabditis elegans unc-13 gene define novel family of C2-domain proteins","volume":"270","author":"Brose","year":"1995","journal-title":"J. Biol. Chem."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"463","DOI":"10.1016\/j.cmet.2006.04.012","article-title":"Munc13-1 is required for the sustained release of insulin from pancreatic beta cells","volume":"3","author":"Kang","year":"2006","journal-title":"Cell Metab."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"731","DOI":"10.1091\/mbc.e04-10-0923","article-title":"Munc13-4 is an effector of rab27a and controls secretion of lysosomes in hematopoietic cells","volume":"16","author":"Neeft","year":"2005","journal-title":"Mol. Biol. Cell."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1016\/S0092-8674(03)00855-9","article-title":"Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3)","volume":"115","author":"Feldmann","year":"2003","journal-title":"Cell"},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Pang, W.K., Amjad, S., Ryu, D.Y., Adegoke, E.O., Rahman, M.S., Park, Y.J., and Pang, M.G. (2022). Establishment of a male fertility prediction model with sperm RNA markers in pigs as a translational animal model. J. Anim. Sci. Biotechnol., 13.","DOI":"10.1186\/s40104-022-00729-9"},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Pang, W.K., Park, Y.J., and Pang, M.G. (2023). Development of a biomolecular approach to identify sperm functions and fertility using sperm RNAs. Front. Cell Dev. Biol., 11.","DOI":"10.3389\/fcell.2023.1308167"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/j.ydbio.2016.12.008","article-title":"Dmrt1 is necessary for male sexual development in zebrafish","volume":"422","author":"Webster","year":"2017","journal-title":"Dev. Biol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"916","DOI":"10.1136\/jmg.39.12.916","article-title":"Evolution and expression of FOXL2","volume":"39","author":"Cocquet","year":"2002","journal-title":"Med. Genet."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1419","DOI":"10.1007\/s10695-015-0096-z","article-title":"Molecular identification and expression of the Foxl2 gene during gonadal sex differentiation in northern snakehead Channa argus","volume":"41","author":"Wang","year":"2015","journal-title":"Fish Physiol. Biochem."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1016\/j.ygcen.2011.08.015","article-title":"Cloning and differential expression of FOXL2 during ovarian development and recrudescence of the catfish, Clarias gariepinus","volume":"174","author":"Sridevi","year":"2011","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1016\/j.bbrc.2004.05.133","article-title":"Molecular cloning and gene expression of Foxl2 in the Nile tilapia, Oreochromis niloticus","volume":"320","author":"Wang","year":"2004","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"933","DOI":"10.1242\/dev.00969","article-title":"The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance","volume":"131","author":"Schmidt","year":"2004","journal-title":"Development"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"1130","DOI":"10.1016\/j.cell.2009.11.021","article-title":"Somatic sex reprogramming of adult ovaries to testes by foxl2 ablation","volume":"139","author":"Uhlenhaut","year":"2009","journal-title":"Cell"}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/15\/2\/122\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T10:24:25Z","timestamp":1759919065000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/15\/2\/122"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1,7]]},"references-count":56,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2025,1]]}},"alternative-id":["ani15020122"],"URL":"https:\/\/doi.org\/10.3390\/ani15020122","relation":{},"ISSN":["2076-2615"],"issn-type":[{"type":"electronic","value":"2076-2615"}],"subject":[],"published":{"date-parts":[[2025,1,7]]}}}