Evans A C O, Ireland J L H, Winn M E, Lonergan P, Smith G W, Coussens P M, Ireland J J
Department of Animal Science and Production and the Centre for Integrative Biology, Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
Biol Reprod. 2004 May;70(5):1475-84. doi: 10.1095/biolreprod.103.025114. Epub 2004 Jan 21.
We hypothesize that granulosa and theca cells from growing dominant follicles, with relatively high intrafollicular concentrations of estradiol, have a greater expression of genes involved in inhibiting apoptosis pathways and lower expression of genes involved in apoptosis pathways than growing subordinate follicles with lower estradiol concentrations. Using the well-characterized bovine dominant follicle model, we collected granulosa and theca cells from individual dominant and the largest subordinate follicle 3 days after initiation of a follicular wave in four animals. Based on ultrasound analysis, both follicle types were in the growth phase at the time of ovariectomy. However, dominant follicles were larger (9.8 +/- 1.0 versus 7.6 +/- 0.6 mm in diameter, P < 0.05) and had greater intrafollicular concentrations of estradiol (132.2 +/-3 8.5 versus 24.1 +/- 12.1 ng/ml, P < 0.05), compared with the largest subordinate follicles. We used bovine cDNA microarrays, which contained a total of 1400 genes, including a subset of 53 genes known to be involved in apoptosis pathways, to determine which apoptosis and marker genes from each of the four dominant versus subordinate follicles were potentially differentially expressed. Using a low stringency-screening criterion, 22 genes were identified. Quantitative real-time polymerase chain reaction confirmed that 16 of these genes were differentially expressed. Our novel results demonstrate that the high intrafollicular concentrations of estradiol in growing dominant follicles were positively associated with enhanced expression of mRNAs in granulosa cells for aromatase, LH receptor, estradiol receptor beta, DICE-1, and MCL-1, compared with granulosa cells from subordinate follicles (all survival-associated genes). In contrast, the relatively low intrafollicular concentrations of estradiol in growing subordinate follicles were positively associated with enhanced expression of mRNAs in granulosa cells for beta glycan, cyclo-oxygenase-1, tumor necrosis factor alpha, caspase-activated DNase, and DRAK-2, and in theca cells for beta glycan, caspase 13, P58(IPK), Apaf-1, BTG-3, and TS-BCLL, compared with granulosa or theca cells from dominant follicles (genes that are all associated with cell death and/or apoptosis). We suggest that that these genes may be candidate estradiol target genes and that they may be early markers for the final stages of follicle differentiation or initiation of apoptosis and thus selection of dominant follicles during follicular waves.
我们推测，与雌二醇浓度较低的生长中的次级卵泡相比，来自生长中的优势卵泡的颗粒细胞和卵泡膜细胞，其卵泡内雌二醇浓度相对较高，参与抑制凋亡途径的基因表达更高，而参与凋亡途径的基因表达更低。利用已充分表征的牛优势卵泡模型，我们在四只动物卵泡波启动3天后，从单个优势卵泡和最大的次级卵泡中收集了颗粒细胞和卵泡膜细胞。根据超声分析，在切除卵巢时，两种卵泡均处于生长阶段。然而，与最大的次级卵泡相比，优势卵泡更大（直径分别为9.8±1.0毫米和7.6±0.6毫米，P<0.05），卵泡内雌二醇浓度更高（分别为132.2±38.5纳克/毫升和24.1±12.1纳克/毫升，P<0.05）。我们使用了包含总共1400个基因的牛cDNA微阵列，其中包括已知参与凋亡途径的53个基因的子集，以确定来自四个优势卵泡与次级卵泡的每个凋亡和标记基因是否存在潜在的差异表达。使用低严格度筛选标准，鉴定出22个基因。定量实时聚合酶链反应证实其中16个基因存在差异表达。我们的新结果表明，与次级卵泡的颗粒细胞相比（所有与存活相关的基因），生长中的优势卵泡内高浓度的雌二醇与颗粒细胞中芳香化酶、促黄体生成素受体、雌激素受体β、DICE-1和MCL-1的mRNA表达增强呈正相关。相反，与优势卵泡的颗粒细胞或卵泡膜细胞相比（所有与细胞死亡和/或凋亡相关的基因），生长中的次级卵泡内相对较低的雌二醇浓度与颗粒细胞中β聚糖、环氧化酶-1、肿瘤坏死因子α、半胱天冬酶激活的脱氧核糖核酸酶和DRAK-2的mRNA表达增强呈正相关，在卵泡膜细胞中与β聚糖、半胱天冬酶13、P58(IPK)、凋亡蛋白酶激活因子-1、BTG-3和TS-BCLL的mRNA表达增强呈正相关。我们认为，这些基因可能是雌二醇的候选靶基因，并且它们可能是卵泡分化最后阶段或凋亡启动的早期标志物，从而在卵泡波期间成为优势卵泡选择的标志物。
