碩士 / 國立臺灣海洋大學 / 水產養殖學系 / 104 / Currently, interest is continuing to grow in stony (scleractinian) coral biology and ecology because of the increasing human concerns about reef degradation and the importance of the conservation of coral reefs. A deeper understanding of both environmental and intrinsic factors that control sexual reproduction in stony corals would allow us to examine the restoration of corals from a new perspective, such as the propagation of target scleractinians by using sperm and eggs obtained in aquaculture. To date, various aspects of the sexual reproduction, such as broadcast spawning, gametogenesis, and sexuality, have been studied in many species and locations worldwide during the last three decades, primarily from ecological perspective. However, the intrinsic mechanisms underlying the sexual reproduction remain largely unknown. To get a better understanding of its mechanism, in the present study, we established of a tool for detecting cell proliferation activity, and development of techniques for in vitro tissue culture systems in the stony coral Euphyllia ancora.
As a tool for detecting cell proliferation in corals, we focused on a gene proliferating cell nuclear antigen (Pcna), and succeeded in cloning the full length of Pcna-like cDNA from E. ancora (EaPcna). Quantitative RT-PCR (RT-qPCR) analysis and Western blotting with anti-EaPcna antibody demonstrated that Pcna product (transcripts and proteins) distributed all the tissues we examined, such as tentacle, mesenterial filaments, and gonads in both sexes. The subsequent immunohistochemical analysis revealed that EaPcna are localized in nuclei of various kinds of cells in the polyp. The anti-Pcna antibody that we produced could be used for detecting Pcna in other corals and sea anemone. Furthermore, It was shown that significantly large numbers of Pcna-positive cells were detected in the regenerating tissues, which have active cell proliferation, compared to intact tissues. These results strongly suggested that Pcna could be used as a cell proliferation marker in corals, and would be a useful tool to investigate the effects of these intrinsic factor on the cell proliferation.
To establish a gonad culture system in coral, firstly, we developed several basal techniques such as 1) isolation of ovary and testis, 2) sterilization of the isolated tissues. Secondary, we found a basal culture medium that is suitable culture ovary. Under this culture medium, the isolated ovary could survive more than 3 days with a state of health, as assessed by histological analysis. Using this ovarian culture system, we investigated the effects of 17β-Estradiol on the mRNA expression levels of yolk proteins (vitellogenin, egg protein, and euphy) in the ovary by RT-qPCR analysis. We found that the expression levels of these genes tend to slightly increase under the presence of E2. We believe that the established culture system would be a promising tool for investigating the effects of intrinsic factors on germ cell development.
Thus, we succeeded in developing two techniques as research tools to identify the find intrinsic factors underlying sexual reproduction in future studies.
| Identifer | oai:union.ndltd.org:TW/104NTOU5086042 |
| Date | January 2016 |
| Creators | Ye, Man-Ru, 葉蔓儒 |
| Contributors | Chang, Ching-Fong, 張清風 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 72 |
Page generated in 0.0015 seconds