Effect of the Methoxychlor Metabolite HPTE on the Rat Ovarian Granulosa Cell Transcriptome in Vitro

Ovarian granulosa cells play a central role in steroidogenesis, which is critical for female reproduction. Follicle-stimulating hormone (FSH) promotes cAMP–mediated signaling to regulate granulosa cell steroidogenesis. We have shown previously that 2, 2-bis-(p-hydroxyphenyl)-1, 1, 1-trichloroethane (HPTE) inhibits FSH- and dibutyryl cAMP-stimulated steroidogenesis, and affects the mRNA levels of steroidogenic pathway enzymes in rat granulosa cells. However, HPTE showed a differential effect in FSH- and cAMP-stimulated cells in that HPTE more completely blocked FSH- when compared to cAMP-driven steroidogenesis. The objective of this study was to analyze the effects of HPTE on global gene expression profiles in untreated granulosa cells and those challenged with FSH or cAMP. Granulosa cells from immature rats were cultured with 0, 1, 5, or 10 µM HPTE in the presence and absence of either 3 ng FSH/ml or 1 mM cAMP for 48 h. Total RNA was isolated for microarray analysis using the GeneChip Rat Genome 230 2.0 and ArrayAssist Microarray Suite. An investigation of changes in gene expression across all HPTE treatments showed that HPTE altered more genes in FSH- (~670 genes) than in cAMP-stimulated cells (~366 genes). Analysis confirmed that HPTE more effectively inhibited FSH- than cAMP-induced steroid pathway gene expression and steroidogenesis. Furthermore, expression patterns of novel genes regulating signal transduction, transport, cell cycle, adhesion, differentiation, motility and growth, apoptosis, development, and metabolism were all altered by HPTE. This study further established that HPTE exerts differential effects within the granulosa cell steroidogenic pathway, and revealed that these effects include broader changes in gene expression.卵巢颗粒细胞（Ovarian granulosa cells）在类固醇生成过程中发挥核心作用，而类固醇生成对雌性生殖功能至关重要。促卵泡激素（Follicle-stimulating hormone, FSH）可通过激活环磷酸腺苷（cAMP）介导的信号通路，调控颗粒细胞的类固醇生成过程。本团队既往研究表明，2,2-双(对羟基苯基)-1,1,1-三氯乙烷（HPTE）可抑制大鼠颗粒细胞中FSH及二丁酰cAMP诱导的类固醇生成过程，并影响类固醇生成通路相关酶的mRNA表达水平。然而，HPTE在FSH与cAMP刺激的细胞中表现出差异化调控效应：相较于cAMP介导的类固醇生成，HPTE可更完全地阻断FSH诱导的类固醇生成。 本研究旨在分析HPTE对未处理颗粒细胞，以及经FSH或cAMP刺激的颗粒细胞的全局基因表达谱的影响。实验中将未成熟大鼠的颗粒细胞进行体外培养，分别添加0、1、5或10 μM的HPTE，并设置是否添加3 ng/ml FSH或1 mM cAMP的组别，培养时长为48小时。随后提取总RNA，使用GeneChip Rat Genome 230 2.0芯片与ArrayAssist Microarray Suite进行微阵列分析。 对所有HPTE处理组的基因表达变化进行全面分析后发现，HPTE在FSH刺激的细胞中可调控约670个基因的表达，而在cAMP刺激的细胞中仅调控约366个基因，前者调控的基因数量显著更多。进一步分析证实，HPTE对FSH诱导的类固醇通路基因表达及类固醇生成的抑制效果，显著优于其对cAMP诱导过程的抑制作用。此外，HPTE还可改变调控信号转导、物质转运、细胞周期、细胞黏附、分化、迁移、增殖、凋亡、发育及代谢等多种生物学过程的新型基因的表达模式。 本研究进一步证实，HPTE在颗粒细胞的类固醇生成通路中发挥差异化调控作用，并揭示其调控效应涉及更为广泛的基因表达改变。