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

我们正在寻找在鸡早期胚胎中对性别决定和生殖器官发育控制具有重要作用的基因。在鸟类中，性类固醇激素，特别是早期胚胎中的雌激素，被认为在决定性别和性腺发育中起着至关重要的作用。为了获得调控类固醇激素基因的机制的线索，我们克隆了一些基因的cDNA和一个基因的5‘上游序列，该基因编码催化类固醇激素生物合成的连续反应的酶。该酶具有两种活性，即17-α羟基酶活性和17，20-裂解酶活性。我最近发现鸡肉P-450c17的c-末端的一个氨基酸在其17，20-裂解酶的活性中起着重要的作用。我观察到，这个区域的一个氨基酸的偶然转换导致了这种酶的17，20-裂解酶活性的有限损失。目前，我正在进行实验，以证实这种突变的酶是否在体内确实存在，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相关的工作如下：克隆了鸡3-βHSD的cDNA，并将其表达载体导入COS7细胞，证实了其活性。鸡17-β-HSD基因的克隆目前正在进行中。鸡FSH受体的全长cDNA被克隆，并通过在人293细胞中的表达确定了与人FSH激素结合的特异性活性。最近，一个旨在阐明调控细胞色素P-450arom基因表达的顺式元件的项目已经取得进展，定位至少两个或两个以上的顺式调控区，其中一个促进鸡卵巢原代培养的卵泡膜细胞中P-450arom基因的表达，另一个代表对同一基因在鸡胚胎成纤维细胞中表达的抑制控制。

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.