The effects of LH ablation/replacement versus steroid ablation/replacement on gene expression in primate corpora lutea

This study was designed to provide a genome-wide analysis of the effects of luteinizing hormone (LH) ablation/replacement versus steroid ablation/replacement on gene expression in the developed corpus luteum (CL) in primates during the menstrual cycle. Naturally cycling, female rhesus monkeys were left untreated (Control; n = 4) or received one of the following treatments for three days beginning on Day 9 of the luteal phase: daily injection of the gonadotropin-releasing hormone (GnRH) antagonist (Antide; n = 5), Antide + recombinant human LH (A+LH; n = 4), Antide + LH + the 3b-HSD antagonist Trilostane (A+LH+TRL; n = 4), and Antide + LH + TRL + progesterone replacement with a synthetic progestin R5020 (A+LH+TRL+ R5020; n = 5). On Day 12 of the luteal phase, CL were removed and samples of RNA from individual CL were fluorescently labeled and hybridized to Affymetrix™ rhesus macaque total genome microarrays. The greatest number of altered transcripts was associated with the ablation/replacement of LH, while ablation/replacement of progestin affected fewer transcripts. Replacement of LH during Antide treatment restored expression of most transcripts to control levels. Real-time PCR validation of a subset of transcripts revealed that most expression patterns were similar between microarray and real-time PCR. Analysis of protein levels were subsequently determined for 2 of the transcripts differentially expressed by real-time PCR. This is the first genome-wide analysis of LH and steroid regulation of gene transcription in the developed primate CL. Further analysis of novel transcripts identified in this data set can clarify the relative role for LH and steroids in CL maintenance and luteolysis. Keywords: LH/steroid ablation/replacement in primate mid-late luteal phase corpora lutea 22 samples from Rhesus Macaque corpus luteum hybridized to individual Rhesus Affymentrix Gene Chip Arrays. 5 treatment groups, with at least 4 replicates per treatment.

本研究旨在开展全基因组分析，探究月经周期中灵长类已形成的黄体（corpus luteum, CL）内，促黄体生成素（luteinizing hormone, LH）敲除/替代与类固醇激素敲除/替代对基因表达的影响。自然发情的雌性恒河猴分为未处理对照组（Control；n=4），以及在黄体期第9天开始连续3天接受以下处理之一的组别：每日注射促性腺激素释放激素（gonadotropin-releasing hormone, GnRH）拮抗剂Antide（n=5）；Antide+重组人LH（A+LH；n=4）；Antide+LH+3β-羟类固醇脱氢酶（3b-HSD）拮抗剂Trilostane（A+LH+TRL；n=4）；Antide+LH+TRL+合成孕激素R5020进行孕酮替代（A+LH+TRL+R5020；n=5）。于黄体期第12天采集黄体组织，提取单个黄体的RNA并进行荧光标记，随后与Affymetrix™恒河猴全基因组微阵列进行杂交。差异表达转录本数量最多的处理组为LH敲除/替代组，而孕激素敲除/替代仅影响较少的转录本。Antide处理期间补充LH可使大多数转录本的表达恢复至对照组水平。对部分转录本开展实时荧光定量PCR（real-time PCR）验证，结果显示微阵列与实时PCR检测的多数表达模式一致。后续对实时PCR筛选出的2个差异表达转录本进行了蛋白水平检测。本研究是首个针对已形成的灵长类黄体中LH与类固醇激素调控基因转录的全基因组分析。对本数据集所鉴定的新型转录本进行进一步分析，可阐明LH与类固醇激素在黄体维持及黄体溶解中的相对作用。关键词：灵长类中晚期黄体期黄体的LH/类固醇敲除/替代；22份恒河猴黄体样本分别与个体恒河猴Affymetrix基因芯片杂交；共设5个处理组，每组至少4个生物学重复。