Odau Simone, Gabler Christoph, Holder Christoph, Einspanier Ralf
Institute of Veterinary-Biochemistry, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.
J Endocrinol. 2006 Oct;191(1):263-74. doi: 10.1677/joe.1.06761.
The aim of the present study was to investigate the enzymes for the local prostaglandin (PG) biosynthesis present in the bovine oviduct during the estrous cycle to influence early reproductive events. Bovine oviducts were classified into four phases: pre-ovulatory, post-ovulatory, early-to-mid luteal, and late luteal phase, subdivided further into ipsi- or contralateral site and separated into ampulla or isthmus. Oviductal cells were gained by flushing the oviductal regions. Quantitative real-time reverse transcriptase-PCR was performed for the secretory and cytosolic phospholipases A(2) (sPLA(2)IB, cPLA(2)alpha, and cPLA(2)beta) and cyclooxygenases (COX-1 and COX-2) as the first step enzymes of PG synthesis. COX-1 and cPLA(2)beta showed significant highest mRNA expression around and before ovulation compared with the luteal phase respectively. sPLA(2)IB and cPLA(2)alpha mRNA expression was unregulated during the estrous cycle. Regional differences in mRNA content were found for sPLA(2)IB with higher mRNA expression in the ampulla than in the isthmus. Western blot analysis revealed the highest COX-1 protein content in the early-to-mid luteal phase. Immunohistochemistry demonstrated that COX-1 was localized in epithelial and smooth muscle cells, whereas COX-2 was only localized in epithelial cells. COX-2 showed a differential distribution within the epithelial cell layer suggesting a regulation on a cellular level, although the COX-2 mRNA and protein amounts did not vary throughout the estrous cycle. A COX activity assay of oviductal cells revealed that COX activity originated predominantly from COX-1 than from COX-2. Treatment of primary oviductal cells with 10 pg/ml 17beta-estradiol or 10 ng/ml progesterone resulted in a higher expression of COX-2 and cPLA(2)alpha, but not of the other enzymes. The expression pattern of these enzymes suggests that an estrous-cycle dependent and region-specific PG synthesis in the bovine oviduct may be required for a successful reproduction.
本研究的目的是调查发情周期中存在于牛输卵管内的参与局部前列腺素（PG）生物合成的酶，以影响早期生殖事件。牛输卵管被分为四个阶段：排卵前、排卵后、黄体早期至中期和黄体晚期，进一步细分为同侧或对侧部位，并分为壶腹部或峡部。通过冲洗输卵管区域获取输卵管细胞。对分泌型和胞质型磷脂酶A2（sPLA2IB、cPLA2α和cPLA2β）以及环氧化酶（COX-1和COX-2）进行定量实时逆转录聚合酶链反应，它们是PG合成的第一步酶。与黄体期相比，COX-1和cPLA2β分别在排卵前后显示出显著最高的mRNA表达。sPLA2IB和cPLA2α的mRNA表达在发情周期中无规律变化。发现sPLA2IB的mRNA含量存在区域差异，壶腹部的mRNA表达高于峡部。蛋白质印迹分析显示黄体早期至中期COX-1蛋白含量最高。免疫组织化学表明COX-1定位于上皮细胞和平滑肌细胞，而COX-2仅定位于上皮细胞。COX-2在上皮细胞层内显示出差异分布，表明在细胞水平上受到调控，尽管COX-2的mRNA和蛋白量在整个发情周期中没有变化。输卵管细胞的COX活性测定表明，COX活性主要来源于COX-1而非COX-2。用10 pg/ml 17β-雌二醇或10 ng/ml孕酮处理原代输卵管细胞导致COX-2和cPLA2α的表达升高，但其他酶没有升高。这些酶的表达模式表明，牛输卵管中依赖发情周期和区域特异性的PG合成可能是成功繁殖所必需的。
