碩士 / 國立臺灣大學 / 漁業科學研究所 / 88 / The decapeptide gonadotropin-releasing hormone (GnRH) is a key hormone for the central regulation of reproduction. In order to know the important role of GnRH in fish reproductive physiology, the expression levels of GnRH mRNA in brain and ovary of Mozambique tilapia, Oreochromis mossambicus were observed. Previous studies using reverse transcription- polymerase chain reaction (RT-PCR) and Southern blot analysis have demonstrated that there are cGnRH-II, sbGnRH and sGnRH in both brain and ovary of Mozambique tilapia. In addition, using 5’- and 3’-rapid amplification of cDNA ends (RACE), the full-length of cGnRH-II, sbGnRH and sGnRH cDNAs from mRNA of brain have been cloned with 547, 464 and 455 bp, respectively. In this study, quantitative PCR (QPCR), in situ hybridization histochemistry technique and Northern blot analysis were performed to investigate the dynamic expression, distribution and the molecular size of these three forms of GnRH mRNA in brain and ovary during gonadal development in Mozambique tilapia.
This study had established both QPCR and in situ hybridization histochemistry techniques. In QPCR, the dynamic expression of ■-actin mRNA in brain was constant during all stages, whereas it varies with stages in ovary. Thus, the expression level of ■-actin mRNA was an ideal reference control gene in brain, but not in ovary. Moreover, the data of QPCR were trustworthiness when reactions contained 5~160 ng cDNA, because the QPCR amplification efficiency of■-actin, cGnRH-II, sbGnRH and sGnRH was approximately equal at this time. And the relative expression amounts of GnRH mRNA by QPCR were the first to assess in fish.
Results showed that the expression level of the three GnRH mRNAs in brain were higher than those of ovary. In brain, the relative expression amount of cGnRH-II and sbGnRH mRNA were 1.28 and 1.38 times to that of sGnRH mRNA. The relative expression amounts of the three GnRH mRNAs were the lowest in the early stage and then become higher there after. In ovary, the relative expression amount of cGnRH-II and sbGnRH mRNA were 66.74 and 14.08 times to that of sGnRH mRNA. The relative expression amounts of the three GnRH mRNAs in ovary were higher in the early stage and the stage that occurs dramatic accumulation of the yolk. No significant in situ hybridization signals was detected in ovarian, suggesting that the expression level of GnRH mRNA might be too low to be observed by using digoxigenin-labeled probes. Unexpected results of sbGnRH and sGnRH mRNA were observed in Northern blot analysis. Those are needed to be further confirmed.
| Identifer | oai:union.ndltd.org:TW/088NTU00451006 |
| Date | January 2000 |
| Creators | Chia-Jen Shieh, 謝嘉珍 |
| Contributors | Show-Wan Lou, 羅秀婉 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 0 |
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