Abstract
The homeodomain transcription factor Nanog plays a crucial role in the embryonic and gonadal development and the maintenance of embryonic stem cells (ESCs), interacting with transcription factors such as Oct4 and Sox2 in mammals. Nevertheless, its pathways to molecular mechanisms remain unclear as to teleosts. This study investigates the role of the Nanog gene in gonadal development and sex reversal of pearlscale angelfish (Centropyge vrolikii). To understand the expression pattern of gonadal development, we identified the Nanog gene of C. vrolikii, which we named Cv-Nanog. The full-length cDNA sequence of Cv-Nanog was 2,136 bp in length and encoded a homeodomain protein of 436 amino acid residues. The gene structure and western blot prove results that Cv-Nanog was homologous to the Nanog gene of mammalians. The protein sequence comparison demonstrates that the Cv-Nanog shared a high degree of similarity with orthologs from other vertebrates in the conserved homeodomain. The Cv-Nanog gene was substantially expressed in gonads, and the expression was significantly higher in the ovaries than in the testis, according to quantitative real-time PCR (qRT-PCR) and western blot analyses. In situ hybridization reveals that the transcripts were located in the cytoplasm and membrane of the oocytes in the ovaries and testes. The expression of Cv-Nanog mRNA was weak in Sertoli cells but strong in germ cells. After overexpression of Cv-Nanog, the expression levels of pluripotent factors Sox2 and Oct4 increased significantly with 21.5-fold and 12.2-fold, respectively. Simultaneously, the TGF-beta signaling pathway was activated, and the gonadal cell growth was promoted. The expression of ovary-bias genes Cyp19a and Foxl2 was upregulated, and the expression of testis-bias genes Sox9 and Dmrt1 was downregulated to promote ovarian development. These results imply that the Nanog gene might play a crucial role in the process of gonadal development and sexual reversion in C. vrolikii. This study provides new insight to understand the molecular regulatory mechanism of the Nanog gene further and important clues for the future studies in gonadal development.
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Zhong ZW, Ao LL, Wang YL, Wang SH, Zhao LP, Ma SW, Jiang YH (2021) Comparison of differential expression genes in ovaries and testes of Pearlscale angelfish Centropyge vrolikii based on RNA-seq analysis. Fish Physiol Biochem 47:00997. https://doi.org/10.1007/s10695-021-00977-y
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Thanks to Prof. Jian-guang Qin, Flinders University (Australia), for polishing and revising the English of this manuscript.
Funding
This project was supported by the Natural Science Foundation of Fujian Province (2018J01451); Fujian Provincial Department of Education (JAT190350); Fujian Engineering Research Center of Aquatic Breeding and Healthy Aquaculture (DF201905); and Innovation Training Program for College Students of Fujian Province (202110390039; 202110390043).
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ZW Zhong is responsible for sampling, the proposal of research scheme, carrying out the experiments, bioinformatics analysis, and manuscript writing. Y Xu, Y Feng, and LL Ao are responsible for sampling, cell interference experiments, and quantitative analysis. YH Jiang is responsible for the task proposal, funding and technology, the research proposal, and implementation support and critically edited the manuscript. All authors read and approved the final manuscript.
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All experimental protocols involved in this study were approved by the Regulations for the Administration of Affairs Concerning Experimental Animals for the Science and Technology Bureau of China. The sample collection and experimental protocols were approved by the Animal Care and Use Committee of the Fisheries College of Jimei University (Animal Ethics No. 1067). All animal handling and methods were performed according to the relevant guidelines.
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Highlights
• The Cv-Nanog gene was found to be highly expressed in the gonads.
• The expression level of Cv-Nanog was significantly higher in the ovaries than in the testis.
• In the conserved homeodomain, the Cv-Nanog shared a high degree of similarity with orthologs of other vertebrates.
• In situ hybridization revealed that the transcript was located in the germ cells of the gonads.
• After overexpression of Cv-Nanog in the gonads, the TGF-beta signaling pathway was activated, and the gonadal cells’ growth was promoted.
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Zhong, Z., Xu, Y., Feng, Y. et al. Characterization of the Nanog gene involved in the gonadal development in pearlscale angelfish (Centropyge vrolikii). Fish Physiol Biochem 48, 303–319 (2022). https://doi.org/10.1007/s10695-022-01054-8
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DOI: https://doi.org/10.1007/s10695-022-01054-8