性釋素在莫三比克吳郭魚腦部及卵巢的表現

Abstract

本研究以莫三比克吳郭魚(Oreochromis mossambious)為模式魚，觀察GnRH在腦部及卵巢中的表現，探討 GnRH在魚類生殖生理所扮演之角色。本實驗室之前已利用RT-PCR及Southern blot的方式初步確定吳郭魚腦部及卵巢皆有sGnRH、cGnRH-Ⅱ及sbGnRH存在，並以3'及5'RACE (rapid amplification of cDNA ends)的方法得到腦部三種GnRH之cDNA序列全長依序為547、464和455 bp。因此為進一步窺探三種GnRH mRNA在吳郭魚生殖週期間於腦部及卵巢內之動態變化，乃以QPCR (Quantitative PCR)偵測此三者之相對量。由於吳郭魚卵巢中之卵胞屬於部分同步發育類型(group synchronism)，且GnRH mRNA在魚類卵巢中表現的位置未知，乃以in situ hybridization的方式確立GnRH mRNA在卵胞之表現時期及表現位置。另外以北方雜合法確認三者GnRH mRNA在腦部及卵巢的分子大小。 本研究確立了QPCR之相對定量方法及非放射性之原位雜合定位方法，尤其於QPCR方面，確認腦部β-actin在各生殖時期之表現量不變，可作為吳郭魚腦部各基因表現之reference control，但卵巢中β-actin在各時期之表現量並非維持恆定，非一理想之reference control，另外檢測出β-actin、cGnRH-Ⅱ、sbGnRH及sGnRH四組引子在 cDNA含量為5至160 ng的範圍內，產物增幅效率一致，偵測值可信度最佳。本研究為首次應用QPCR方法於魚類中觀測出GnRH mRNA之相對表現量。 QPCR結果發現三種GnRH mRNA在腦部的相對表現量較卵巢為高，腦部的三者相對表現量cGnRH-Ⅱ及sbGnRH分別為sGnRH之1.28倍及1.38倍，而卵巢的三者相對表現量cGnRH-Ⅱ及sbGnRH分別為sGnRH的66.74倍及14.08倍。在探求 GnRH mRNA表現與卵胞發育之關連性方面，隨著生殖週期中卵胞發育，腦部各GnRH mRNA在不同生殖週期的相對表現量皆於初期為最低而後增高，於卵巢GnRH mRNA則皆在初期及大量卵黃生成期間之相對表現量較高。在in situ hybridization的結果顯示，因GnRH mRNA表現量極低，目前以digoxigenin標識探針的方法並未偵測到。北方雜合結果顯示，sbGnRH mRNA及sGnRH mRNA於腦部及卵巢中出現其他非預期大小之產物，此有待日後更進一步確認。

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, usings 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 dynanuc expression of β-actin mRNA in brain was constant during all stages, whereas it varies with stages in ovary. Thus, the expression level of β-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 β-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 thlee 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 sbGllRH InRNA 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.