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Mechanism of Sex Differentiation in Chicken:Screening for Female and Male Specific Genes in Chicken Early Embryo

鸡性别分化机制：鸡早期胚胎雌雄特异性基因的筛选

基本信息

批准号：
03660073
负责人：
NISHIMORI Katsuhiko
金额：
$ 1.41万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

We are looking for the gene which has essential roles in determination of sexes and developmental control of reproductive organs in chick early embryo. In avian species, sex steroid hormones, especially estrogen in early embryo was reported to act a crucial function to determine the sex and in gonadal development. To obtain a clue to mechanism which regulate the steroidogenic genes, we have cloned some cDNAs and a 5'-upstream sequence of a gene which code enzymes catalyzing successive reactions in sex-steroid hormone's biosynthesis.We have previously cloned a chicken cytochrome P-450c17 cDNA. This enzyme has two kinds of activities, 17-alpha hydroxylase activity and 17,20-lyase activity. I recently discovered that an amino acid at c-terminal region of chicken P-450c17 had important role especially in its 17,20-lyase activity. And I observed an incidental conversion of an amino acid in this region led to limited loss of only 17,20-lyase activity of this enzyme. Now,I am carrying out experiments to confirm whether this 'mutated' enzyme actually exists in vivo and has a role in steroid metabolism in chicken, or not.Other works relating to sex-steroid hormone's biosynthesis are as follows: The cDNA of chicken 3-beta HSD had been cloned and its activity was confirmed by introduction of cDNA- expression vector into cos7 cells. cDNA cloning of chicken 17-beta HSD is now on the way.Complete length cDNA of chicken FSH-receptor was cloned, and specific activity binding to human FSH hormone was ascertained by expression of this cDNA in human 293 cells.A project to elucidate the cis-elements which regulate the expression of cytochrome P- 450arom gene has been recently progressed to localize at least two or more cis regulatory regions, one of which facilitates the expression of P-450arom gene in primarily cultured theca cells prepared from chicken ovarian follicles, and the other represent repressive control for the same gene's expression in chicken embryonic fibroblast cells.

本研究旨在寻找在鸡早期胚胎性别决定和生殖器官发育调控中起重要作用的基因。在鸟类中，性类固醇激素，特别是早期胚胎中的雌激素，在鸟类性别决定和性腺发育中起着重要作用。为了获得类固醇基因调控机制的线索，我们克隆了一些cDNA和一个编码性类固醇激素生物合成中催化连续反应的酶的基因的5 '上游序列，我们以前克隆了鸡细胞色素P-450 c17 cDNA。该酶具有两种活性，17-α羟化酶活性和17，20-裂解酶活性。我最近发现鸡P-450 c17蛋白C端的一个氨基酸在其17，20-裂解酶活性中起重要作用。我观察到这个区域的一个氨基酸的偶然转化导致了这种酶的17，20-裂解酶活性的有限损失。现在，我正在进行实验，以确认这种“突变”的酶是否真的存在于体内，并在鸡的类固醇代谢中发挥作用。其他与性类固醇激素生物合成有关的工作如下：鸡3-β HSD的cDNA已经克隆，并通过将cDNA表达载体导入cos 7细胞证实了其活性。鸡17-β-HSD基因的cDNA克隆工作正在进行中。已克隆了鸡FSH受体的全长cDNA，并通过在人293细胞中表达，确定了与人FSH激素结合的特异性活性。最近，阐明细胞色素P-450 arom基因表达调控的顺式元件的计划已取得进展，确定了至少两个或多个顺式调控区，其中一个促进P-450 arom基因在鸡卵泡膜细胞中的表达，另一个抑制该基因在鸡胚胎成纤维细胞中的表达。