性類固醇激素誘發吳郭魚仔魚基因差異性表現之探討

Sex steroidal hormones induce differential gene expressions in Mozambique tilapia fry (Oreochromis mossambicus)

朱啟璟(Chi-Ching Ju)

指導教授 ： 李士傑

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01516

摘要

本研究主要利用減差基因選殖的技術篩選出吳郭魚早期性別分化關鍵時期可受性類固醇激素所調控的基因群，進一步分析其位於組織內之表現情形，期望可找到可專一地表現在精、卵巢的基因，以暸解這些基因跟魚類性別分化機制之關係。我們使用莫三比克吳郭魚（Oreochromis mossambicus）為實驗動物，在取得口孵仔魚之後，將仔魚分成三群，分別餵食添加甲基睪固酮（17?methyltestosterone, MT）、餵食添加雌二醇（17β-estradiol, E2）飼料或無添加任何激素飼料，而得到較高雌雄比之仔魚群，以提供進行減差基因選殖實驗。結果顯示因MT處理而增加表現的已知基因有八個，受MT處理降低表現的已知基因有六個，受E2刺激增加表現的已知基因有兩個，受E2刺激而降低表現的已知基因有一個。由其中先挑選可因MT處理而增加表現的基因transferrin和parvalbumin來進一步分析其在組織中之表現，我們發現transferrin主要表現在成魚肝臟中，其次是在眼睛、腦、心臟，而在卵巢中則有鴩B選殖出一段較長之transferrin片段，認為它是屬於粒腺體中之transferrin前驅物；而parvalbumin在肌肉中表現較多，其次是腦和表皮組織，其序列相似高。利用原位雜合技術進一步地分析transferrin和parvalbumin在組織中之表現情形，發現parvalbumin會在大腦內側之海馬迴區域及肌肉中的肌質網有表現；transferrin則是會在腦室間連結之脈絡叢上表現。未來，將繼續尋找可專一性表現在生殖組織內且又可受性類固醇激素表現的基因群，藉以探討魚類性別分化的複雜分子機制；另外，繼續探討MT在仔魚早期促使parvalbumin和transferrin表現量增加所代表之特殊生理意義。

關鍵字

性別分化 ； 性類固醇激素 ； 仔魚

並列摘要

The development and growth of sex organs is mainly mediated by sex hormones, including estrogen and testosterone, which govern the female and male development, respectively. Exogenous sex steroids have a remarkable influence on phenotypic sex. It is well demonstrated that the exogenous steroids affect sex differentiation in developing fry（Baroiller et al., 1999）. However, the changes in gene expression upon sex hormone treatments in fry are less clear. To approach this question, we examine the changes in gene expression pattern in fry of tilapia, Oreochromis mossambicus, under the influences of exogenous steroids, 17?methyltestosterone (MT) and 17β- estradiol（E2）. Mozambique tilapia is an excellent model for studying steroidogenic actions during gonadal sex differentiation since the sex can be altered by sex hormones irregardless of its genetic sex. Therefore, we had adapted a method from Tsai et al. (2000) to prepare all male or all female tilapia. We then performed PCR subtracted hybridization on those homo-sex fry and identified some sex steroid-induced differentially expressed genes in developing tilapia fry. Among those genes, we found parvalbumin and transferrin were up-regulated by MT during sex-sensitive periods in tilapia. We have cloned the full open reading frame of parvalbumin. Parvalbumin expressed in brain, muscle and skin as tissue-specific isoforms, respectively. From their amino acid sequences, we suggest that one or two amino acid shift could decrease the sequence similarity of calcium-binding motif. We have also cloned a transferrin with partial sequence. Transferrin expressed in liver, heart, eye and brain. We also have cloned a longer transferrin fragment（753 bp）in the ovary. The up-regulation of parvalbumin by MT were confirmed by quantitative-PCR. Using in-situ hybridization, we found that parvalbumin was expressed in the hippocampus and sarcoplasmic reticulum, and transferrin was expressed in the brain choroids plexus. The significance of up-regulation of parvalbumin and transferrin by MT is still under investigation.

並列關鍵字

Sex steroidal hormones ； sex differentiation ； fry

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