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Investigation of Sex Steroid Induced Gene Expression in Juvenile Mozambique Tilapia (Oreochromis mossambicus)

探討性荷爾蒙對莫三比克吳郭魚(Oreochromis mossambicus)仔魚早期發育之基因調控

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摘要


本篇研究取用本土水產養殖中的經濟性魚種,莫三比克吳郭魚(Mozambique tilapia),當作實驗動物。主要目的是想探討性荷爾蒙對仔魚早期發育時的基因調控,鑑定出可受性荷爾蒙所開啓或抑制的基因以及這些基因在仔魚早期性別分化關鍵時期所扮演的角色探討。我們根據中山大學蔡錦玲博士在2000年發表的報告,自製添加有性荷爾蒙(甲基睪固酮或雌性素)的飼料,在授精後10至20天投餵甲基睪固酮(Methyltestosterone, MT)飼料;在授精後7至20天投餵雌性素(17β-estradiol, E2)飼料。我們將經過MT、E2飼料投餵過和投餵一般飼料的三組仔魚收集,分成頭部與軀幹部分,分開萃取組織RNA,採用減差基因組方法(PCR-select cDNA subtraction kit),篩選出可受性荷爾蒙所調控的基因。將這些基因片段送入pGEM-T aesy載體中,送去做定序及基因片段大小鑑定,將得到之序列輸入NCBI資料庫做比對,已找出一些有興趣的基因。初步想先探討可受MT所屬表現量增加的基因Parvalbumin和Transferrin,以RT-PCR和石蠟切片原位雜交法(Section in situ hybridization)分析這些基因的表現情形,發現Parvalbumin會在成魚肌肉、心臟、鰓、腎臟、腦和卵巢組織有表現;Transferrin則會在腦、肝、卵巢及精巢組織有表現,細部來看,Parvalbumin會在成魚腦部大腦內側接近海馬迴(hippocampus)位置會有表現,基因的表現分析工作還在進行中。未來的目標是在探討這些基因受性荷爾蒙調控後,在仔魚早期性別分化關鍵時期之角色扮演。

並列摘要


The development and growth of sex organs is mainly mediated by sex ormones, including estrogen and testosterone, which govern the female and male evelopment, respectively. Exogenous sex steroids have a remarkable influence on phenotypic sex. It is well demonstrated that the exogenous steroids affects ex differentiation in developing fries (Baroiller et al., 1999). However, the hanges in gene expression upon sex hormone treatments in juvenile fries are less clear. To approach this question, we examine the changes in gene xpression pattern in juvenile tilapia, Oreochromis mossambicus, under the nfluences of exogenous steroids, methyltestosterone (MT) and 17-b estradiol. Mozambique tilapia is an excellent model for studying steroidogenic actions uring gonadal sex differentiation since the sex can be altered by sex hormones rregardless of its genetic sex. Therefore, we have adapted a method from Tsai t al. (2000) to prepare all male or all female tilapia. We then performed PCR subtracted hybridization on those homo-sex fries and identified some sex steroid-induced differentially expressed genes in developing tilapia fries. Among those genes, we found parvalbumin and transferrin are up-regulated by MT during sex-sensitive periods in tilapia. Parvalbumin is a calcium binding protein in muscle (Pechere et al., 1971). We have cloned full open reading frame of parvalbumin. Parvalbumin expressed in gill, brain, ovary, kidney, muscle and heart. We have also cloned partial sequences of transferrin. Transferrin was expressed in liver, testis, ovary and brain. The up-regulation of both parvalbumin and transferrin by MT were confirmed by quantitative-PCR. Using in-situ hybridization, we also demonstrated parvalbumin is expressed at both brain and muscle. The changes in expression of parvalbumin and transferrin by MT imply they may involve in sex differentiation. The functional analysis of both genes in sex differentiation is underway.

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