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

  • Shinya Shikina
  • Yi-Ling Chiu
  • Man-ru Ye
  • Jack-I-Chen Yao
  • Yi-Jou Chung
  • Chieh-Jhen Chen
  • Ching-Fong Chang
Original Article Biology

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.

Keywords

Euphyllia ancora DNA replication DNA repair Gametogenesis Reverse transcription-polymerase chain reaction Western blot 

Notes

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).

Supplementary material

12562_2018_1210_MOESM1_ESM.pdf (18.9 mb)
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)
12562_2018_1210_MOESM2_ESM.pdf (39.1 mb)
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)

References

  1. Bauer GA, Burgers PM (1990) Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene. Nucleic Acids Res 18:261–265CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bravo R, Macdonald-Bravo H (1987) Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell Biol 105:1549–1554CrossRefPubMedGoogle Scholar
  3. Bravo R, Frank R, Blundell PA, MacDonald-Bravo H (1987) Cyclin/PCNA is the auxiliary protein of DNA polymerase. Nature 326:515–517CrossRefPubMedGoogle Scholar
  4. Celis JE, Celis A (1985) Cell cycle-dependent variations in the distribution of the nuclear protein cyclin proliferating cell nuclear antigen in cultured cells: subdivision of S phase. Proc Natl Acad Sci USA 82:3262–3266CrossRefPubMedGoogle Scholar
  5. Chapman DL, Wolgemuth DJ (1994) Expression of proliferating cell nuclear antigen in the mouse germ line and surrounding somatic cells suggests both proliferation-dependent and -independent modes of function. Int J Dev Biol 38:491–497PubMedGoogle Scholar
  6. Chen CJ, Shikina S, Chen WJ, Chung YJ, Chiu YL, Bertrand JA, Lee YH, Chang CF (2016) A novel female-specific and sexual reproduction-associated Dmrt gene discovered in the Stony Coral, Euphyllia ancora. Biol Reprod 94:40PubMedGoogle Scholar
  7. D’Angelo C, Smith EG, Oswald F, Burt J, Tchernov D, Wiedenmann J (2012) Locally accelerated growth is part of the innate immune response and repair mechanisms in reef-building corals as detected by green fluorescent protein (GFP)-like pigments. Coral Reefs 31:1045–1056CrossRefGoogle Scholar
  8. Foley JF, Dietrich DR, Swenberg JA, Maronpot RR (1991) Detection and evaluation of proliferating cell nuclear antigen (PCNA) in rat tissue by an improved immunohistochemical procedure. J Histotech 14:237–241CrossRefGoogle Scholar
  9. Franco A, Jouaux A, Mathieu M, Sourdaine P, Lelong C, Kellner K, Heude Berthelin C (2010) Proliferating cell nuclear antigen in gonad and associated storage tissue of the Pacific oyster Crassostrea gigas: seasonal immunodetection and expression in laser microdissected tissues. Cell Tissue Res 340:201–210CrossRefPubMedGoogle Scholar
  10. Hanselmann R, Smolowitz R, Gibson D (2000) Identification of proliferating cells in hard clams. Biol Bull 199:199–200CrossRefPubMedGoogle Scholar
  11. Harrison PL (2011) Sexual reproduction in scleractinian corals. In: Dubinsky Z (eds) Coral reefs: ecosystem in transition. Springer, Netherlands, pp 59–85CrossRefGoogle Scholar
  12. Harrison PL, Wallace CC (1990) Reproduction, dispersal and recruitment of scleractinian corals. In: Dubinsky Z (ed) Ecosystems of the world 25. Coral reefs. Elsevier, Amsterdam, pp 133–207Google Scholar
  13. Kelman Z (1997) PCNA: structure, functions and interactions. Oncogene 14:629–640CrossRefPubMedGoogle Scholar
  14. Korfsmeier KH (2002) PCNA in the ovary of zebrafish (Brachydanio rerio, Ham.-Buch.). Acta Histochem 104:73–76CrossRefPubMedGoogle Scholar
  15. Kurki P, Vanderlaan M, Dolbeare F, Gray J, Tan EM (1986) Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle. Exp Cell Res 166:209–219CrossRefPubMedGoogle Scholar
  16. Leibovici M, Gusse M, Bravo R, Méchali M (1990) Characterization and developmental expression of Xenopus proliferating cell nuclear antigen. Dev Biol 141:183–192CrossRefPubMedGoogle Scholar
  17. Leung AY, Leung JC, Chan LY, Ma ES, Kwan TT, Lai KN, Meng A, Liang R (2005) Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis. Histochem Cell Biol 124:105–111CrossRefPubMedGoogle Scholar
  18. Lin S, Corstjens PLAM (2002) Molecular cloning and expression of the proliferating cell nuclear antigen gene from the coccolithophorid Pleurochrysis carterae (Haptophyceae). J Phycol 38:164–173CrossRefGoogle Scholar
  19. Maga G, Hübscher U (2003) Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J Cell Sci 116:3051–3060CrossRefPubMedGoogle Scholar
  20. Miura C, Miura T, Kudo N, Yamashita M, Yamauchi K (1999) cDNA cloning of a stage-specific gene expressed during HCG-induced spermatogenesis in the Japanese eel. Dev Growth Differ 41:463–471CrossRefPubMedGoogle Scholar
  21. Miura C, Miura T, Yamashita M (2002) PCNA protein expression during spermatogenesis of the Japanese eel (Anguilla japonica). Zool Sci 19:87–91CrossRefPubMedGoogle Scholar
  22. Moldovan GL, Pfander B, Jentsch S (2007) PCNA, the maestro of the replication fork. Cell 129:665–679CrossRefPubMedGoogle Scholar
  23. Ohkura H (2015) Meiosis: an overview of key differences from mitosis. Cold Spring Harb Perspect Biol 7:a015859CrossRefPubMedPubMedCentralGoogle Scholar
  24. Orii H, Sakurai T, Watanabe K (2005) Distribution of the stem cells (neoblasts) in the planarian Dugesia japonica. Dev Genes Evol 215:143–157CrossRefPubMedGoogle Scholar
  25. Prelich G, Tan CK, Kostura M, Mathews MB, So AG, Downey KM, Stillman B (1987) Functional identity of proliferating cell nuclear antigen and a DNA polymerase-auxiliary protein. Nature 326:517–520CrossRefPubMedGoogle Scholar
  26. Roos G, Landberg G, Huff JP, Houghten R, Takasaki Y, Tan EM (1993) Analysis of the epitopes of proliferating cell nuclear antigen recognized by monoclonal antibodies. Lab Invest 68:204–210PubMedGoogle Scholar
  27. Ruike T, Takeuchi R, Takata K, Oshige M, Kasai N, Shimanouchi K, Kanai Y, Nakamura R, Sugawara F, Sakaguchi K (2006) Characterization of a second proliferating cell nuclear antigen (PCNA2) from Drosophila melanogaster. FEBS J 273:5062–5073CrossRefPubMedGoogle Scholar
  28. Shikina S, Chang CF (2016) Sexual reproduction in stony corals and insight into the evolution of oogenesis in Cnidaria. In: Goffredo S (eds) The Cnidaria, past, present and future. Springer, Switzerland, pp 249–268CrossRefGoogle Scholar
  29. Shikina S, Chen CJ, Liou JY, Shao ZF, Chung YJ, Lee YH, Chang CF (2012) Germ cell development in the scleractinian coral Euphyllia ancora (Cnidaria, Anthozoa). PLOS One 7:e41569CrossRefPubMedPubMedCentralGoogle Scholar
  30. Shikina S, Chen CJ, Chung YJ, Shao ZF, Liou JY, Tseng HP, Lee YH, Chang CF (2013) Yolk formation in a stony coral Euphyllia ancora (Cnidaria, Anthozoa): insight into the evolution of vitellogenesis in nonbilaterian animals. Endocrinology 154:3447–3459CrossRefPubMedGoogle Scholar
  31. Shikina S, Chung YJ, Wang HM, Chiu YL, Shao ZF, Lee YH, Chang CF (2015a) Localization of early germ cells in a stony coral, Euphyllia ancora: potential implications for a germline stem cell system in coral gametogenesis. Coral Reefs 34:639–653CrossRefGoogle Scholar
  32. Shikina S, Chiu YL, Lee YH, Chang CF (2015b) From somatic cells to oocytes: a novel yolk protein produced by ovarian somatic cells in a stony coral, Euphyllia ancora. Biol Reprod 93:1–10CrossRefGoogle Scholar
  33. Shikina S, Chung YJ, Chiu YL, Huang YJ, Lee YH, Chang CF (2016a) Molecular cloning and characterization of a steroidogenic enzyme, 17β-hydroxysteroid dehydrogenase type 14, from the stony coral Euphyllia ancora (Cnidaria, Anthozoa). Gen Comp Endocrinol 228:95–104CrossRefPubMedGoogle Scholar
  34. Shikina S, Chiu YL, Chung YJ, Chen CJ, Lee YH, Chang CF (2016b) Oocytes express an endogenous red fluorescent protein in a stony coral, Euphyllia ancora: a potential involvement in coral oogenesis. Sci Rep 6:25868CrossRefPubMedPubMedCentralGoogle Scholar
  35. Shikina S, Chiu YL, Chen CJ, Yang SH, Yao JIC, Chen CC, Wei JD, Shao ZF, Chang CF (2017) Immunodetection of acetylated alpha-tubulin in stony corals: evidence for the existence of flagella in coral male germ cells. Mol Rep Dev 84:1285–1295CrossRefGoogle Scholar
  36. Steger K, Aleithe I, Behre H, Bergmann M (1998) The proliferation of spermatogonia in normal and pathological human seminiferous epithelium: an immunohistochemical study using monoclonal antibodies against Ki-67 protein and proliferating cell nuclear antigen. Mol Hum Reprod 4:227–233CrossRefPubMedGoogle Scholar
  37. Szmant-Froelich A, Yevich P, Pilson MEQ (1980) Gametogenesis and early development of the temperate coral Astrangia danae (Anthozoa: Scleractinia). Biol Bull 158:257–269CrossRefGoogle Scholar
  38. Szmant-Froelich A, Reutter M, Riggs L (1985) Sexual reproduction of Favita fragum (Esper): lunar patterns of gametogenesis, embryogenesis and planulation in Puerto Rico. Bull Mar Sci 37:880–892Google Scholar
  39. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefPubMedPubMedCentralGoogle Scholar
  40. Tománek M, Chronowska E (2006) Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary. Folia Histochem Cytobiol 44:269–274PubMedGoogle Scholar
  41. Tsai YJ, Lee MF, Chen CY, Chang CF (2011) Development of gonadal tissue and aromatase function in the protogynous orange-spotted grouper Epinephelus coioides. Zool Stu 50:693–704Google Scholar
  42. Waseem NH, Lane DP (1990) Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA). Structural conservation and the detection of a nucleolar form. J Cell Sci 96:121–129PubMedGoogle Scholar
  43. Wrobel KH, Bickel D, Kujat R (1996) Immunohistochemical study of seminiferous epithelium in adult bovine testis using monoclonal antibodies against Ki-67 protein and proliferating cell nuclear antigen (PCNA). Cell Tissue Res 283:191–201CrossRefPubMedGoogle Scholar
  44. Xie Y, Wang B, Li F, Jiang H, Xiang J (2008) Molecular cloning and characterization of proliferating cell nuclear antigen (PCNA) from Chinese shrimp Fenneropenaeus chinensis. Comp Biochem Physiol B: Biochem Mol Biol 151:225–229CrossRefGoogle Scholar

Copyright information

© Japanese Society of Fisheries Science 2018

Authors and Affiliations

  1. 1.Institute of Marine Environment and EcologyNational Taiwan Ocean UniversityKeelungTaiwan
  2. 2.Center of Excellence for the OceansNational Taiwan Ocean UniversityKeelungTaiwan
  3. 3.Doctoral Degree Program in Marine BiotechnologyNational Taiwan Ocean UniversityKeelungTaiwan
  4. 4.Doctoral Degree Program in Marine BiotechnologyAcademia SinicaTaipeiTaiwan
  5. 5.Department of AquacultureNational Taiwan Ocean UniversityKeelungTaiwan

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