{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,16]],"date-time":"2026-02-16T11:13:52Z","timestamp":1771240432715,"version":"3.50.1"},"reference-count":42,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,2,15]],"date-time":"2026-02-15T00:00:00Z","timestamp":1771113600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key R&D Program of China","doi-asserted-by":"publisher","award":["2022YFD2400405"],"award-info":[{"award-number":["2022YFD2400405"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Taishan Scholars Program","award":["tsqn202408298"],"award-info":[{"award-number":["tsqn202408298"]}]},{"name":"Shandong Key Research and Development Program for Academician team","award":["2023ZLYS02"],"award-info":[{"award-number":["2023ZLYS02"]}]},{"name":"Central Public interest Scientific Institute Basal Research Fund, CAFS","award":["2023TD20"],"award-info":[{"award-number":["2023TD20"]}]},{"name":"State Key Laboratory of Mariculture Biobreeding and Sustainable Goods","award":["BRESG202303"],"award-info":[{"award-number":["BRESG202303"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Chinese tongue sole (Cynoglossus semilaevis) is an important aquaculture species in China. Under high-temperature conditions, genetically female fish can undergo sex reversal and develop into phenotypic males (pseudomale fish). Previous studies have demonstrated that apoptosis might function in sex differentiation. Based on this, we identified and characterized the functions of six caspase genes (caspase1-like, caspase3a, caspase6, caspase8, caspase8-like, caspase9) in Chinese tongue sole. These six caspase genes were expressed in all analyzed tissues of both males and females. They were detected to be expressed in the gonads at various developmental stages, with expression levels peaking between 7 months and 2 years of age. In situ hybridization (ISH) analysis showed that the caspase genes were mainly localized in spermatocytes and oocytes. Promoter activity analysis indicated that with the exception of caspase3a, the remaining five caspase genes exhibited promoter activity and were regulated by transcription factors, including sp1 and gata4. High-temperature stimulation can significantly affect the expression of caspases in the gonads of both male and female fish, with female fish showing a more pronounced response. An siRNA-mediated knockdown experiment revealed that following caspase knockdown, the expression of sex differentiation-related genes, heat shock transcription factors (hsf), and heat shock proteins (hsp) in Chinese tongue sole was significantly altered. Based on these findings, we speculate that caspases play an important role in the sex differentiation process by responding to temperature stimuli.<\/jats:p>","DOI":"10.3390\/ijms27041864","type":"journal-article","created":{"date-parts":[[2026,2,16]],"date-time":"2026-02-16T10:14:32Z","timestamp":1771236872000},"page":"1864","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Functional Study of the Apoptosis Caspase Gene Family in the Sex Differentiation of Chinese Tongue Sole (Cynoglossus semilaevis)"],"prefix":"10.3390","volume":"27","author":[{"given":"Lijun","family":"Wang","sequence":"first","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture, College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, 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"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-2731-0586","authenticated-orcid":false,"given":"Haipeng","family":"Yan","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture, College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, 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"}]},{"given":"Xuexue","family":"Sun","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture, College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, 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"}]},{"given":"Mingyue","family":"He","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China"},{"name":"Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao 266071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8142-8927","authenticated-orcid":false,"given":"Zhen","family":"Meng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China"},{"name":"Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao 266071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2772-0908","authenticated-orcid":false,"given":"Xihong","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China"},{"name":"Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao 266071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2135-090X","authenticated-orcid":false,"given":"Na","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China"},{"name":"Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao 266071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7898-5223","authenticated-orcid":false,"given":"Zhongdian","family":"Dong","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture, College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China"},{"name":"State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China"},{"name":"Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao 266071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3887-9973","authenticated-orcid":false,"given":"Wenteng","family":"Xu","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture, College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China"},{"name":"State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China"},{"name":"Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao 266071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,15]]},"reference":[{"key":"ref_1","first-page":"447","article-title":"Histological study of the organogenesis of the digestive system of the tonguefish, Cynoglossus semilaevis","volume":"29","author":"Qing","year":"2005","journal-title":"J. Fish. China"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Wang, Q., Liu, K., Feng, B., Zhang, Z., Wang, R., Tang, L., Li, W., Li, Q., Piferrer, F., and Shao, C. (2019). Transcriptome of Gonads From High Temperature Induced Sex Reversal During Sex Determination and Differentiation in Chinese Tongue Sole, Cynoglossus semilaevis. Front. Genet., 10.","DOI":"10.3389\/fgene.2019.01128"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"109119","DOI":"10.1016\/j.fsi.2023.109119","article-title":"Establishment and identification of a skin cell line from Chinese tongue sole (Cynoglossus semilaevis) and analysis of the changes in its transcriptome upon LPS stimulation","volume":"142","author":"Gong","year":"2023","journal-title":"Fish Shellfish Immunol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1352","DOI":"10.1016\/j.immuni.2019.05.020","article-title":"Caspases in Cell Death, Inflammation, and Disease","volume":"50","author":"Lamkanfi","year":"2019","journal-title":"Immunity"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1038\/s41421-025-00791-3","article-title":"Caspases: Structural and molecular mechanisms and functions in cell death, innate immunity, and disease","volume":"11","author":"Nadendla","year":"2025","journal-title":"Cell Discov."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1459","DOI":"10.1038\/s42003-024-07184-4","article-title":"PI3K-AKT-mediated phosphorylation of Thr260 in CgCaspase-3\/6\/7 regulates heat-induced activation in oysters","volume":"7","author":"Wang","year":"2024","journal-title":"Commun. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"831","DOI":"10.1038\/nature04002","article-title":"Structure of the CED-4-CED-9 complex provides insights into programmed cell death in Caenorhabditis elegans","volume":"437","author":"Yan","year":"2005","journal-title":"Nature"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"949","DOI":"10.1038\/s41419-021-04240-3","article-title":"A long way to go: Caspase inhibitors in clinical use","volume":"12","author":"Dhani","year":"2021","journal-title":"Cell Death Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"487","DOI":"10.1007\/s10815-013-9938-8","article-title":"Caspase signalling pathways in human spermatogenesis","volume":"30","author":"Almeida","year":"2013","journal-title":"J. Assist. Reprod. Genet."},{"key":"ref_10","first-page":"622","article-title":"Advances in the study of relationship between Caspases and innate immunity","volume":"50","author":"Yinghua","year":"2019","journal-title":"J. China Pharm. Univ."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"725","DOI":"10.1016\/j.ceb.2003.10.009","article-title":"Mechanisms of caspase activation","volume":"15","author":"Boatright","year":"2003","journal-title":"Curr. Opin. Cell Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"a008656","DOI":"10.1101\/cshperspect.a008656","article-title":"Caspase functions in cell death and disease","volume":"5","author":"McIlwain","year":"2013","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1016\/j.immuni.2016.01.020","article-title":"Caspases Connect Cell-Death Signaling to Organismal Homeostasis","volume":"44","author":"Galluzzi","year":"2016","journal-title":"Immunity"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1016\/j.gene.2018.05.099","article-title":"Oocyte-G1 promotes male germ cell apoptosis through activation of Caspase-3","volume":"670","author":"Hou","year":"2018","journal-title":"Gene"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1038\/cdd.2012.156","article-title":"Caspase-8 cleaves its substrates from the plasma membrane upon CD95-induced apoptosis","volume":"20","author":"Beaudouin","year":"2013","journal-title":"Cell Death Differ."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1146\/annurev-immunol-073119-095439","article-title":"Caspases in Cell Death, Inflammation, and Pyroptosis","volume":"38","author":"Kesavardhana","year":"2020","journal-title":"Annu. Rev. Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"He, Z., Chen, Q., He, L., Xiong, J., Gao, K., Lai, B., Zheng, L., Pu, Y., Jiao, Y., and Ma, Z. (2022). Cyt-C Mediated Mitochondrial Pathway Plays an Important Role in Oocyte Apoptosis in Ricefield Eel (Monopterus albus). Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms231810555"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Liu, J., Liu, X., Jin, C., Du, X., He, Y., and Zhang, Q. (2019). Transcriptome Profiling Insights the Feature of Sex Reversal Induced by High Temperature in Tongue Sole Cynoglossus semilaevis. Front. Genet., 10.","DOI":"10.3389\/fgene.2019.00522"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1534\/g3.116.036822","article-title":"Single Locus Maintains Large Variation of Sex Reversal in Half-Smooth Tongue Sole (Cynoglossus semilaevis)","volume":"7","author":"Jiang","year":"2017","journal-title":"G3"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1151","DOI":"10.1007\/s11427-021-2236-4","article-title":"Single-cell-resolution transcriptome map revealed novel genes involved in testicular germ cell progression and somatic cells specification in Chinese tongue sole with sex reversal","volume":"66","author":"Wang","year":"2023","journal-title":"Sci. China Life Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"340","DOI":"10.1016\/j.fsi.2016.08.047","article-title":"Molecular characterization reveals involvement of four caspases in the antibacterial immunity of tongue sole (Cynoglossus semilaevis)","volume":"57","author":"Long","year":"2016","journal-title":"Fish Shellfish Immunol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1007\/s10695-012-9632-2","article-title":"Survival of ovarian somatic cells during sex change in the protogynous wrasse, Halichoeres trimaculatus","volume":"39","author":"Nozu","year":"2013","journal-title":"Fish Physiol. Biochem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"762","DOI":"10.1086\/590219","article-title":"Warm water induces apoptosis, gonadal degeneration, and germ cell loss in subadult pejerrey Odontesthes bonariensis (Pisces, Atheriniformes)","volume":"81","author":"Ito","year":"2008","journal-title":"Physiol. Biochem. Zool."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.ygcen.2018.11.015","article-title":"Roles of estrogens in fish sexual plasticity and sex differentiation","volume":"277","author":"Li","year":"2019","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Nanjidsuren, T., and Min, K.S. (2014). The transcription factor Ap-1 regulates monkey 20\u03b1-hydroxysteroid dehydrogenase promoter activity in CHO cells. BMC Biotechnol., 14.","DOI":"10.1186\/1472-6750-14-71"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1080\/25785826.2021.1872838","article-title":"Regulation of T cell differentiation by the AP-1 transcription factor JunB","volume":"44","author":"Katagiri","year":"2021","journal-title":"Immunol. Med."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e2024067118","DOI":"10.1073\/pnas.2024067118","article-title":"Sexual fate of murine external genitalia development: Conserved transcriptional competency for male-biased genes in both sexes","volume":"118","author":"Kajioka","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","first-page":"929","article-title":"Effects of Different Temperature Stresses on Gill Apoptosis of Medaka (Oryzias latipes)","volume":"36","author":"Hu","year":"2021","journal-title":"J. Dalian Ocean. Univ."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1016\/0092-8674(82)90249-5","article-title":"A regulatory upstream promoter element in the Drosophila hsp 70 heat-shock gene","volume":"30","author":"Pelham","year":"1982","journal-title":"Cell"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1038\/nrm.2017.73","article-title":"Regulation of heat shock transcription factors and their roles in physiology and disease","volume":"19","author":"Burchfiel","year":"2018","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"929","DOI":"10.1038\/s41419-022-05373-9","article-title":"HSP90 mediates the connection of multiple programmed cell death in diseases","volume":"13","author":"Peng","year":"2022","journal-title":"Cell Death Dis."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1038\/nature05378","article-title":"Hsp70 regulates erythropoiesis by preventing caspase-3-mediated cleavage of GATA-1","volume":"445","author":"Ribeil","year":"2007","journal-title":"Nature"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1038\/s41419-022-04766-0","article-title":"HspBP1 is a dual function regulatory protein that controls both DNA repair and apoptosis in breast cancer cells","volume":"13","author":"Youn","year":"2022","journal-title":"Cell Death Dis."},{"key":"ref_34","first-page":"2634","article-title":"Mutation of foxl2 or cyp19a1a Results in Female to Male Sex Reversal in XX Nile Tilapia","volume":"158","author":"Zhang","year":"2017","journal-title":"Endocrinology"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Galotta, M., Dam, A.T., Eto, Y., Toyoda, A., Itoh, T., Mohapatra, S., Ogino, Y., Saitou, M., Matsuyama, M., and Chakraborty, T. (2024). Insights into epigenetic regulation of cyp19a via comparative analysis using the scombrid chub mackerel as model. Front. Mar. Sci., 11.","DOI":"10.3389\/fmars.2024.1408561"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"3237","DOI":"10.1210\/en.2002-0095","article-title":"Etiology of ovarian failure in blepharophimosis ptosis epicanthus inversus syndrome: FOXL2 is a conserved, early-acting gene in vertebrate ovarian development","volume":"144","author":"Loffler","year":"2003","journal-title":"Endocrinology"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"966","DOI":"10.1093\/biolre\/ioaa146","article-title":"Aberrant and constitutive expression of FOXL2 impairs ovarian development and functions in mice","volume":"103","author":"Nicol","year":"2020","journal-title":"Biol. Reprod."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1016\/j.gene.2016.07.062","article-title":"Ubiquitin ligase gene neurl3 plays a role in spermatogenesis of half-smooth tongue sole (Cynoglossus semilaevis) by regulating testis protein ubiquitination","volume":"592","author":"Xu","year":"2016","journal-title":"Gene"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1016\/j.fsi.2010.02.021","article-title":"\u03b2-Actin is a useful internal control for tissue-specific gene expression studies using quantitative real-time PCR in the half-smooth tongue sole Cynoglossus semilaevis challenged with LPS or Vibrio anguillarum","volume":"29","author":"Li","year":"2010","journal-title":"Fish Shellfish Immunol."},{"key":"ref_40","first-page":"787","article-title":"SCAR\u2014Transformation of Sex\u2014Specific SSR Marker and Its Application in Half\u2014Smooth Tongue Sole (Cynoglossus semiliaevis)","volume":"22","author":"Liu","year":"2014","journal-title":"J. Agric. Biotechnol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"452","DOI":"10.7150\/ijbs.7.452","article-title":"Establishment and characterization of a testicular cell line from the half-smooth tongue sole, Cynoglossus semilaevis","volume":"7","author":"Zhang","year":"2011","journal-title":"Int. J. Biol. Sci."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1111\/jfb.12535","article-title":"Establishment and characterization of an ovarian cell line from half-smooth tongue sole Cynoglossus semilaevis","volume":"86","author":"Sun","year":"2015","journal-title":"J. Fish Biol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/4\/1864\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,16]],"date-time":"2026-02-16T10:24:41Z","timestamp":1771237481000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/4\/1864"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,15]]},"references-count":42,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["ijms27041864"],"URL":"https:\/\/doi.org\/10.3390\/ijms27041864","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,15]]}}}