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Molecular cloning and characterization of proliferating cell nuclear antigen; its potential involvement in germ cell development of stony corals

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Abstract

To better understand the molecular mechanisms underlying germ cell development in stony corals, this study focused on proliferating cell nuclear antigen (PCNA), which is an auxiliary protein of DNA polymerases and is principally responsible for DNA replication and repair. We performed the molecular identification and characterization of the complementary DNA for the pcna gene in the stony coral Euphyllia ancora. Quantitative reverse transcription-polymerase chain reaction analysis and Western blotting with anti-E. ancora PCNA antibody demonstrated that E. ancora PCNA gene products (transcripts and proteins) were present in all polyp tissues, including the testis and ovaries. Immunohistochemical analysis of spermatogenesis showed that intensive immunoreactivity of PCNA (irPCNA) was detected in the nuclei of spermatogonia. The irPCNA was also weakly detected in spermatocytes, and became almost undetectable in the spermatids and sperm. In contrast, in female germline cells, the irPCNA was detected in the nuclei of oogonia and all stage of oocytes. These results suggest that PCNA plays an important role in both the mitotic and meiotic processes of the coral germ cells. To the best of our knowledge, this study is the first to show the expression profile of the PCNA protein and its subcellular localization in coral germ-cell development.

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Acknowledgments

We gratefully acknowledge the colleagues and divers who helped us over the year to collect samples. This research was funded by grants from the Ministry of Science and Technology, Taiwan (MOST 103-2621-B-019-006-MY3, MOST 106-2313-B-019-007).

Funding

This research was funded by grants from the Ministry of Science and Technology, Taiwan (MOST 103-2621-B-019-006-MY3, MOST 106-2313-B-019-007).

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Authors and Affiliations

  1. Institute of Marine Environment and Ecology, National Taiwan Ocean University, Keelung, Taiwan

    Shinya Shikina

  2. Center of Excellence for the Oceans, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung, 20224, Taiwan

    Shinya Shikina & Ching-Fong Chang

  3. Doctoral Degree Program in Marine Biotechnology, National Taiwan Ocean University, Keelung, Taiwan

    Yi-Ling Chiu

  4. Doctoral Degree Program in Marine Biotechnology, Academia Sinica, Taipei, Taiwan

    Yi-Ling Chiu

  5. Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan

    Man-ru Ye, Jack-I-Chen Yao, Yi-Jou Chung, Chieh-Jhen Chen & Ching-Fong Chang

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  1. Shinya Shikina
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  3. Man-ru Ye
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  4. Jack-I-Chen Yao
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  5. Yi-Jou Chung
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  6. Chieh-Jhen Chen
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  7. Ching-Fong Chang
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Corresponding author

Correspondence to Ching-Fong Chang.

Electronic supplementary material

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Online Resource 1. Evaluation of the anti-proliferating cell nuclear antigen (PCNA) antibody specificity by immunohistochemical analysis.

The antibody specificity was confirmed by immunohistochemical analysis with or without preadsorption of the anti-PCNA antibody with the peptide antigen (a, b). Euphyllia ancora male germ cells (c, d). E. ancora oocyte. Note that the immunoreactivity was eliminated by the preadsorption of the anti-PCNA antibody with the peptide antigens (+preadsorption) in all of the samples we examined (b, d). Serial sections were used for this analysis. a, b Broken lines indicate the spermary periphery. c, d Broken lines in indicate the oocyte periphery. c, d Arrow indicates the nucleus of the oocyte. Scale bar 20 μm. (PDF 19358 kb)

Online Resource 2. Confirmation of the cross-reactivity of the developed anti-PCNA antibody to the other anthozoans (stony corals and sea anemone) by immunohistochemical analysis. a

The Agariciidae family, Leptoseris mycetoseroides (stony coral). b The Acroporidae family, Acropora sp. (stony coral). a, b Arrows indicate the oocytes possessing immunoreactivity of PCNA (irPCNA). c, d The Actiniaria order, Exaiptasia pallida (sea anemone). c irPCNA in female germ cells. Broken lines indicate the periphery of oocytes. Note that nuclei of oocytes exhibited intensive immunoreactivity. d irPCNA in male germ cells. Arrows indicate the spermatogonial clusters. Scale bar 20 μm. (PDF 40062 kb)

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Shikina, S., Chiu, YL., Ye, Mr. et al. Molecular cloning and characterization of proliferating cell nuclear antigen; its potential involvement in germ cell development of stony corals. Fish Sci 84, 765–775 (2018). https://doi.org/10.1007/s12562-018-1210-9

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  • DOI: https://doi.org/10.1007/s12562-018-1210-9

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