Functional Assay of Prostaglandin F_<2alpha> Receptor for Luteal Cell Apoptosis in Bovine Estrous Cycle

前列腺素(PG)参与了动情周期中黄体细胞的凋亡和妊娠黄体细胞功能的维持。我们以前的研究表明，在发情周期黄体成熟和退化过程中，PGF2α受体(FP)的mRNA表达受到关键调控。为了阐明发情周期黄体中FP基因表达的调控机制，我们分离并鉴定了FP基因转录启动子区。在发情中期的牛黄体原代细胞中，A区是转录的双重启动子之一，具有很强的启动子活性。凝胶滞留分析和DNasel足迹分析表明，特定的核蛋白可以以发情周期依赖的方式与A区(ATG上游的-249--861 NTD)结合。此外，荧光素酶分析表明，A区在发情中期的牛黄体细胞中表现出很强的启动子活性。此外，为了筛选在周期黄体细胞中特异表达的与黄体细胞凋亡相关的新基因，对不同发情期的总RNA进行了差异显示分析。因此，从中期黄体cDNA库中分离到了催化超氧还原的Mn型超氧化物歧化酶(Mn-SOD)的cDNA片段。为了进一步寻找在周期黄体中特异表达的mRNAs，采用RT-PCR和Northern印迹分析了铜/锌-超氧化物歧化酶、谷丙转氨酶和过氧化氢酶。在发情周期中，除了超氧化物歧化酶和过氧化氢酶的表达升高外，GPx的表达也显著降低，这表明过氧化氢的过度积累可以诱导黄体细胞的凋亡。本研究的结果表明，在发情周期中，对FP和抗氧化酶基因表达的严格调控对黄体细胞的维持和凋亡起关键作用。

It is well understood that prostaglandin (PG) F2_<alpha> involved in luteal celI apoptosis in estrous cycle and maintenance of luteal cell function in pregnancy. Our previous data clealy indicated that mRNA expression of PGF2_<alpha> receptor (FP) is critically regulated during the course of maturation and regression of corpus luteum in estrous cycle.In order to elucidate the regulation mechanism of gene expression of FP in bovine corpus luteum in estrous cycle, we isolated and characterized transcriptional promoter regions of FP gene. One of dual promoters for transcription, so called A region, represented strong promoter activity in primary cells which prepared from bovine corpora lutea in middle estrous cycle. The gel retardation assay and DNasel foot print analysis revealed that specific nuclear proteins could bind to A region (-249--861 ntd upstream of ATG) in estrous cycle dependent manner. In addition, luciferase assay indicated that A region represented strong promoter activity in bovine luteal cells prepared from mid-estrous phase. Futhermore, to identify novel mRNA which specifically expressed in cycling luteal cells and involved in luteal cell apoptosis, differential display assay was performed using total RNAs prepared from different estrous phases. As a result, the cDNA fragment of Mn-type Super Oxyde Dismutase (Mn- SOD) which catalyzes superoxyde reduction, was isolated from mid-phase luteal cDNA pool. To further identify mRNAs which specifically expressed in cycling corpora lutea, RT-PCR and northern blot analysis were performed for Cu/Zn-SOD, GPX and Catalase. Besides the elevated expression of SODs and Catalase during the progress of estrous cycle, GPx was dramatically reduced indicating that excess accumulation of H2O2 could induce luteal cell apoptosis.As the results of this study, we concluded that strict regulation of gene expression of FP and anti- oxidative enzymes critically controlled luteal cell maintenance and apoptosis in estrous cycle.