Characterization of foxl2, sox9, and wt1a genes and their expression pattern as putative targets for sex identification in Astronotus ocellatus (Agassiz, 1831)
DOI:
https://doi.org/10.5380/avs.v30i3.98358Palavras-chave:
Wilm’s tumor suppressor gene, Sex determination, Sex identification, Molecular markers.Resumo
Abstract: Using molecular markers is an effective method for sex identification in fish. In this regard, autosomal genes related to sex determination and differentiation may be of great interest. This study aimed to evaluate the expression pattern of Foxl2, Sox9, and Wt1a as molecular markers for sex identification in Oscar (Astronotus ocellatus). Molecular characterization and gene expression profiles for forkhead box l2 (Foxl2), SRY-box 9 (Sox9), and Wilm’s tumor suppressor gene (Wt1a) were performed in male and female gonads. After euthanasia, histological and molecular analyses were conducted using ovarian and testis fragments of adult A. ocellatus. Histology was used for sex confirmation. The isolation of partial cDNA sequences encoding Foxl2, Sox9, and Wt1a from A. ocellatus was obtained by PCR using primers designed from conserved regions of the corresponding coding-domain sequence in other species. Sequencing of A. ocellatus Foxl2, Sox9, and Wt1a cDNA fragments allowed us to perform RT-qPCR assays in male and female gonadal tissue to analyze the gene expression profile. RT-qPCR revealed markedly gender-influenced gene expression patterns for all target genes. Foxl2 mRNA levels were significantly higher in the ovary than in the testes. At the same time, Sox9 and Wt1a were upregulated considerably, and mRNA levels for Sox9 and Wt1a were 8- and 9-fold higher in the testis compared to the ovary. Our findings indicate that Foxl2 expression may serve as a molecular marker for the identification of males. At the same time, the genes Sox9 and Wt1a are effective in identifying females in A. ocellatus gonads up to 12 months of age.
Referências
Begum S, Gnanasree SM, Anusha N, et al. Germ cell markers in fishes: A review. Aquaculture and Fisheries, 7:(5);540-552, 2022. (DOI: 10.1016/j.aaf.2022.03.015)
Bertho S; Pasquier J; Pan Q, et al. Foxl2 and its relatives are evolutionarily conserved players in gonadal sex differentiation. Sexual Development, 10:(3);111-129, 2016. (DOI: 10.1159/000447611)
Bhat IA, Rather MA, Saha R, et al Expression analysis of Sox9 genes during annual reproductive cycles in gonads and after nanodelivery of LHRH in Clarias batrachus. Research in Veterinary Science, 106;100-106, 2016a. (DOI: 10.1016/j.rvsc.2016.03.022)
Bhat IA, Rather MA, Dar JY, et al. Molecular cloning, computational analysis and expression pattern of forkhead box l2 (Foxl2) gene in catfish. Computational Biology and Chemistry, 64;09-18, 2016b. (DOI: 10.1016/j.compbiolchem.2016.05.001)
Bollig F, Mehringer R, Perner B, et al. Identification and comparative expression analysis of a second wt1 gene in zebrafish. Developmental Dynamics, 235:(2);554-561, 2006. (DOI: 10.1002/dvdy.20645)
Caburet S, Georges A, L’Hôte D, et al. The transcription factor FOXL2: At the crossroads of ovarian physiology and pathology. Molecular and Cellular Endocrinology, 356:(1-2);55-64, 2006. (DOI: 10.1016/j.mce.2011.06.019)
Castro JP, Hattori RS, Yoshinaga TT, et al. Differential Expression of Genes Related to Sexual Determination Can Modify the Reproductive Cycle of Astyanax scabripinnis (Characiformes: Characidae) in B Chromosome Carrier Individuals. Genes (Basel), 10:(11);909, 2019. (DOI: 10.3390/genes10110909)
Chen JJ, Xia XH, Wang LF, et al. Identification and comparison of gonadal transcripts of testis and ovary of adult common carp Cyprinus carpio using suppression subtractive hybridization. Theriogenology, 83:(9);1416-1427, 2015. (DOI: 10.1016/j.theriogenology.2015.01.001)
Chen M, Wang X, Wang Y, et al. Wt1 is involved in Leydig cell steroid hormone biosynthesis by regulating paracrine factor expression in mice. Biology of Reproduction, 90:(4);71-72, 2014. (DOI: 10.1095/biolreprod.113.114702)
Crespo B, Lan-Chow-Wing O, Rocha A, et al. foxl2 and foxl3 are two ancient paralogs that remain fully functional in teleosts. General and Comparative Endocrinology, 194;81-93, 2013. (DOI: 10.1016/j.ygcen.2013.08.016)
Dai S, Qi S, Wei X, et al. Germline sexual fate is determined by the antagonistic action of dmrt1 and foxl3/foxl2 in tilapia. Development, 148:(8), 2021. (DOI: 10.1242/dev.199380)
Godinho HP, Santos JE, Formagio PS, et al Gonadal morphology and reproductive traits of the Amazonian sh. Acta Zoologica, 86;289-294, 2005. (DOI: 10.1111/j.1463-6395.2005.00213.x)
Gonen N, Lovell-Badge R. The regulation of Sox9 expression in the gonad. Curr Top Dev Biol, 134;223-252, 2019. (DOI: 10.1016/bs.ctdb.2019.01.004)
Hu Y, Wang B, Du H. A review of Sox genes in fish. Reviews in Aquaculture, 13:(4);1986-2003, 2021. (DOI: 10.1111/raq.12554)
Ijiri S, Kaneko H, Kobayashi T, et al. Sexual Dimorphic Expression of Genes in Gonads During Early Differentiation of a Teleost Fish, the Nile Tilapia Oreochromis niloticus. Biology of Reproduction, 78:(2);333-341, 2008. (DOI: 10.1095/biolreprod.107.064246)
Jiang D, Chen J, Fan Z, et al. CRISPR/Cas9-induced disruption of wt1a and wt1b reveals their different roles in kidney and gonad development in Nile tilapia. Developmental Biology, 428:(1);63-73, 2017. (DOI: 10.1016/j.ydbio.2017.05.017)
Jiang D, Jia S, Chen J, et al. Timing of gonadal development and dimorphic expression of sex-related genes in gonads during early sex differentiation in the Yellow River carp. Aquaculture, 518;734825, 2020. (DOI: 10.1016/j.aquaculture.2019.734825)
Jiang D, Yang Y, Zhao D, et al. Effects of sexual steroids on the expression of foxl2 in Gobiocypris rarus. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 160:(4);187-193, 2011. (DOI: 10.1016/j.cbpb.2011.08.005)
Klüver N, Herpin A, Braasch I, et al. The regulatory backup circuit of medaka Wt1 co-orthologs ensures PGC maintenance. Developmental Biology, 325:(1);179-188, 2009. (DOI: 10.1016/j.ydbio.2008.10.009)
Li X, Yu H, Wang Y, et al. Roles of two Sox9 genes during gonadal development in Japanese Flounder: sex differentiation, spermatogenesis and gonadal function maintenance. International Journal of Molecular Sciences, 19:(2);512, 2018. (DOI: 10.3390/ijms19020512)
Liu H, Mu X, Gui L, et al. Characterization and gonadal expression of FOXL2 relative to Cyp19a genes in spotted scat Scatophagus argus. Gene, 561:(1);6-14, 2015. (DOI: 10.1016/j.gene.2014.12.060)
Luo YS, Hu W, Liu XC, et al. Molecular cloning and mRNA expression pattern of Sox9 during sex reversal in orange-spotted grouper (Epinephelus coioides). Aquaculture, 306:(1-4);322-328, 2010. (DOI: 10.1016/j.aquaculture.2010.06.019)
Meinsohn MC, Smith OE, Bertolin K, et al. The orphan nuclear receptors steroidogenic factor-1 and liver receptor homolog-1: structure, regulation, and essential roles in mammalian reproduction. Physiological reviews, 99:(2);1249-1279, 2019. (DOI: 10.1152/physrev.00019.2018)
Murugananthkumar R, Senthilkumaran B. Expression analysis and localization of wt1, ad4bp/sf-1 and gata4 in the testis of catfish, Clarias batrachus: Impact of wt1-esiRNA silencing. Molecular and Cellular Endocrinology, 431;164-176, 2016. (DOI: 10.1016/j.mce.2016.05.006)
Nagahama Y, Chakraborty T, Paul-Prasanth B, et al. Sex determination, gonadal sex differentiation, and plasticity in vertebrate species. Physiol Rev, 101:(3);1237-1308, 2021. (DOI: 10.1152/physrev.00044.2019)
Nakamoto M, Matsuda M, Wang DS, et al. Molecular cloning and analysis of gonadal expression of Foxl2 in the medaka, Oryzias latipes. Biochemical and Biophysical Research Communications, 344:(1);353-361, 2006. (DOI: 10.1016/j.bbrc.2006.03.137)
Nakamoto M, Suzuki A, Matsuda M, et al. Testicular type Sox9 is not involved in sex determination but might be in the development of testicular structures in the medaka, Oryzias latipes. Biochemical and Biophysical Research Communications, 333:(3);729-736, 2005. (DOI: 10.1016/j.bbrc.2005.05.158)
Raghuveer K, Senthilkumaran B. Isolation of sox9 duplicates in catfish: localization, differential expression pattern during gonadal development and recrudescence, and hCG-induced up-regulation of sox9 in testicular slices. Reproduction, 140:(3);477-487, 2010. (DOI: 10.1530/REP-10-0200)
Rodríguez-Hernández ME, Martínez-Castellanos G, López-Méndez MC, et al. Production Costs and Growth Performance of Tilapia (Oreochromis niloticus) in Intensive Production Systems: A Review. Sustainability, 17:(4);1745, 2025. (DOI: 10.3390/su17041745)
Rodríguez-Marí A, Yan YL, Bremiller RA, et al. Characterization and expression pattern of zebrafish anti-Müllerian hormone (amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development. Gene Expression Patterns, 5:(5);655-667, 2005. (DOI: 10.1016/j.modgep.2005.02.008)
Si Y, Ding Y, He F, et al. DNA methylation level of cyp19a1a and Foxl2 gene related to their expression patterns and reproduction traits during ovary development stages of Japanese flounder (Paralichthys olivaceus). Gene, 575:(2);321-330, 2016. (DOI: 10.1016/j.gene.2015.09.006)
Smith EK, Guzmán JM, Luckenbach AJ, et al. Molecular cloning, characterization, and sexually dimorphic expression of five major sex differentiation-related genes in a Scorpaeniform fish, sablefish (Anoplopoma fimbria). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 165:(2);125-137, 2013. (DOI: 10.1016/j.cbpb.2013.03.011)
Sreenivasan R, Gonen N, Sinclair A. SOX Genes and Their Role in Disorders of Sex Development. Sexual Development, 16:(2-3);80-91, 2022. (DOI: 10.1159/000524453)
Tucker, Elena J. The genetics and biology of FOXL2. Sexual Development, 16:(2-3);184-193, 2022. (DOI: 10.1159/000519836)
Vidal VP, Chaboissier MC, Rooij DG, et al. Sox9 induces testis development in XX transgenic mice. Nature Genetics, 28:(3);216-217, 2001. (DOI: 10.1038/90046)
Wagner S, Whiteley SL, Castelli M, et al. Gene expression of male pathway genes sox9 and amh during early sex differentiation in a reptile departs from the classical amniote model. BMC Genomics, 24:(243); 2023. (DOI: 10.1186/s12864-023-09334-0)
Wallace RA, Selman K. Cellular and Dynamic Aspects of Oocyte Growth in Teleosts. Integrative and Comparative Biology, 21:(2);325-343, 1981. (DOI: 10.1093/icb/21.2.325)
Wang D, Kobayashi T, Zhou L, et al. Molecular cloning and gene expression of Foxl2 in the Nile tilapia, Oreochromis niloticus. Biochemical and Biophysical Research Communications, 320:(1);83-89, 2004. (DOI: 10.1016/j.bbrc.2004.05.133)
Wang X, Meng K, Wang Y, et al. Wilms' tumor (WT1) (±KTS) variants decrease the progesterone secretion of bovine ovarian theca cells. Domestic Animal Endocrinology, 74: 106521, 2021. (DOI: 10.1016/j.domaniend.2020.106521)
Wei L, Yang C, Tao W, et al. Genome-wide identification and transcriptome-based expression profiling of the Sox gene family in the Nile tilapia (Oreochromis niloticus). International journal of molecular sciences, 17:(3);270, 2016. (DOI: 10.3390/ijms17030270)
Wilm B, Muñoz-Chapuli R, et al. The role of WT1 in embryonic development and normal organ homeostasis. The Wilms' Tumor (WT1) Gene: Methods and Protocols, 1467;23-39, 2016. (DOI: 10.1007/978-1-4939-4023-3_3)
Yang YJ, Wang Y, Li Z, et al. Sequential, Divergent, and Cooperative Requirements of Foxl2a and Foxl2b in Ovary Development and Maintenance of Zebrafish. Genetics, 205:(4);1551-1572, 2017. (DOI: 10.1534/genetics.116.199133)
Zhang X, Li M, Ma H, et al. Mutation of foxl2 or cyp19a1a results in female to male sex reversal in XX Nile tilapia. Endocrinology, 158:(8);2634-2647, 2017. (DOI: 10.1210/en.2017-00127)
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