Estrogen-responsive genes in the parenchyma and fat pad of the bovine mammary gland by microarray analysis

Characterizing estrogen-responsive genes is an essential step towards fully understanding mechanisms of estrogen action during mammary gland development and function. To catalogue these genes, sixteen prepubertal heifers were used in a 2 x 2 factorial with ovarian status (intact or ovariectomized) as the first factor and estrogen treatment as the second (control or estradiol). Heifers were ovariectomized at approximately 4.5 months of age and estrogen treatments were initiated one month later. After 3 days of treatment, gene expression in the parenchyma and fat pad of the bovine mammary gland was analyzed using a high-density oligonucleotide microarray. This microarray contained probes representing 40,808 Tentative Consensus sequences from the TIGR Bos taurus Gene Index and 4,575 singletons derived from libraries of pooled mammary gland and gut tissues. Microarray data were analyzed using the SAS Mixed Procedure with permutation testing. A total of 125 estrogen-responsive genes were identified using an experiment-wide permutation-based significance level of P < 0.1. Among these genes are known estrogen-targeted genes such as stanniocalcin 1, alpha-1-antiproteinase, progesterone receptor, nucleobindin 2, insulin-like growth factor 1, and tissue factor pathway inhibitor. However, the majority of the genes identified were not previously reported to be estrogen-responsive. In silico mapping and an estrogen response element (ERE) search indicated potential EREs in the promoter regions of some of these novel estrogen-responsive genes. The distinctive expression patterns regulated by estrogen in parenchyma and fat pad suggest mechanisms of action and reciprocal signaling between cell types. Keywords: Cell type comparision, two class unpaired design Overall design: Sixteen prepubertal heifers were used in a 2 x 2 factorial with ovarian status (intact or ovariectomized) as the first factor and estrogen treatment as the second (control or estradiol). Two tissues (parenchyma and fat pad) from each heifer were sampled. 32 single-channel microarray hybridization were performed.

鉴定雌激素应答基因是全面理解雌激素在乳腺发育与功能过程中作用机制的关键步骤。为编制此类基因目录，本研究选取了十六头青春期前母牛，采用2x2析因设计，其中卵巢状态（完整或去势）作为第一因素，雌激素处理作为第二因素（对照组或雌二醇组）。母牛于约4.5月龄时进行去势手术，并在一个月后开始雌激素处理。经过3天的处理后，利用高密度寡核苷酸微阵列对牛乳腺实质和脂肪垫中的基因表达进行了分析。该微阵列含有代表TIGR Bos taurus 基因索引中40,808个假共识序列的探针，以及从乳腺和肠道组织库中获得的4,575个单克隆探针。使用SAS混合程序和置换检验对微阵列数据进行分析。通过全实验范围内的置换检验显著性水平P < 0.1，共鉴定出125个雌激素应答基因。其中包含已知的雌激素靶基因，如骨形态发生蛋白1、α-1-抗蛋白酶、孕酮受体、核结合蛋白2、胰岛素样生长因子1和组织因子途径抑制剂。然而，大部分鉴定出的基因之前并未被报道为雌激素应答基因。通过计算机模拟映射和雌激素反应元件（ERE）搜索，表明这些新型雌激素应答基因启动子区域存在潜在的ERE。雌激素调节的实质和脂肪垫中的独特表达模式提示了细胞类型之间的作用机制和相互信号传递。关键词：细胞类型比较，两分类非配对设计。整体设计：十六头青春期前母牛被用于2x2析因设计，其中卵巢状态（完整或去势）作为第一因素，雌激素处理作为第二因素（对照组或雌二醇组）。每头母牛的实质和脂肪垫各采样一次。共进行32次单通道微阵列杂交。