Centre de recherche en reproduction animale and the département de biomedicine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada.
Biol Reprod. 2013 Oct 24;89(4):98. doi: 10.1095/biolreprod.113.111849. Print 2013 Oct.
Vanin-2 (VNN2) is known to be involved in inflammation and leukocyte migration, but its regulation in follicles remains unknown. The objectives of this work were to study the regulation of VNN2 transcripts in bovine follicles prior to ovulation and to characterize the control of its expression in bovine granulosa cells. VNN2 expression was studied using total RNA extracted from granulosa cells of small follicles (2-4 mm in diameter), dominant follicles obtained on Day 5 of the estrous cycle, ovulatory follicles obtained 0-24 h after human chorionic gonadotropin (hCG), and corpora lutea on Day 5 of the cycle. The results from RT-PCR analyses showed that levels of VNN2 mRNA were high in ovulatory follicles 24 h post-hCG but low in the other tissues. In ovulatory follicles, levels of VNN2 mRNA were low at 0 h but significantly up-regulated 12-24 h post-hCG. To determine factors controlling VNN2 gene expression, established primary cultures of granulosa cells isolated from bovine dominant follicles were used. Treatment with forskolin elevated VNN2 mRNA expression as observed in vivo. Mutation studies identified the minimal region conferring basal and forskolin-stimulated VNN2 promoter activities, which were dependent on chicken ovalbumin upstream promoter-transcription factor (COUP-TF), GATA, and Ebox cis-elements. Electrophoretic mobility shift assays identified COUP-TF, GATA4, and upstream stimulating factor proteins as key factors interacting with these elements. Chromatin immunoprecipitation assays confirmed basal and forskolin-induced interactions between these proteins and the VNN2 promoter in bovine granulosa cell cultures. VNN2 promoter activity and mRNA expression were markedly stimulated by forskolin and overexpression of the catalytic subunit of PKA, but inhibited by PKA and ERK1/2 inhibitors. Collectively, the findings from this study describe for the first time the gonadotropin/forskolin-dependent up-regulation of VNN2 transcripts in granulosa cells of preovulatory follicles and provide insights into some of the molecular bases of VNN2 gene expression in follicular cells.
Vanin-2（VNN2）已知参与炎症和白细胞迁移，但在卵泡中的调节尚不清楚。本工作的目的是研究排卵前牛卵泡中 VNN2 转录物的调节，并研究其在牛颗粒细胞中的表达调控。使用从小卵泡（直径 2-4mm）、发情周期第 5 天获得的优势卵泡、人绒毛膜促性腺激素（hCG）后 0-24 小时获得的排卵卵泡和周期第 5 天的黄体中提取的总 RNA 研究 VNN2 的表达。RT-PCR 分析结果表明，排卵卵泡 24 小时 hCG 后 VNN2 mRNA 水平较高，但其他组织中水平较低。在排卵卵泡中，VNN2 mRNA 水平在 0 小时较低，但在 hCG 后 12-24 小时显著上调。为了确定控制 VNN2 基因表达的因素，使用从牛优势卵泡中分离的颗粒细胞建立了原代培养。与体内观察到的一样，佛司可林处理可升高 VNN2 mRNA 的表达。突变研究鉴定了赋予基础和佛司可林刺激 VNN2 启动子活性的最小区域，该区域依赖于鸡卵清蛋白上游启动子转录因子（COUP-TF）、GATA 和 Ebox 顺式元件。电泳迁移率变动分析鉴定了 COUP-TF、GATA4 和上游刺激因子蛋白作为与这些元件相互作用的关键因子。染色质免疫沉淀分析证实了在牛颗粒细胞培养物中这些蛋白与 VNN2 启动子之间的基础和佛司可林诱导的相互作用。VNN2 启动子活性和 mRNA 表达被佛司可林和蛋白激酶 A（PKA）催化亚基的过表达显著刺激，但被 PKA 和 ERK1/2 抑制剂抑制。总之，本研究首次描述了促性腺激素/佛司可林依赖性上调排卵前卵泡中颗粒细胞 VNN2 转录物，并为卵泡细胞中 VNN2 基因表达的一些分子基础提供了见解。
