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

前列腺素（PG） F2_< α >参与妊娠期发情周期黄体细胞凋亡和黄体细胞功能的维持。我们之前的数据清楚地表明PGF2_< α >受体（FP）的mRNA表达在发情周期黄体成熟和回归过程中受到关键调控。为了阐明FP基因在牛黄体发情周期中的表达调控机制，我们分离并鉴定了FP基因的转录启动子区。双启动子之一A区在黄体牛发情中期制备的原代细胞中表现出较强的启动子活性。凝胶阻滞实验和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结合到A区（ATG上游-249—861 ntd）。此外，荧光素酶测定表明，A区在发情中期制备的牛黄体细胞中具有较强的启动子活性。此外，为了鉴定在黄体细胞循环中特异性表达并参与黄体细胞凋亡的新mRNA，采用不同发情期制备的总rna进行了差异显示实验。结果，从中期黄体cDNA库中分离到催化超氧还原的Mn型超氧歧化酶（Mn- SOD） cDNA片段。为了进一步鉴定在循环黄体中特异性表达的mrna，我们对Cu/Zn-SOD、GPX和过氧化氢酶进行了RT-PCR和northern blot分析。在发情周期中，除了sod和过氧化氢酶的表达升高外，GPx也显著降低，表明H2O2的过量积累可诱导黄体细胞凋亡。本研究结果表明，卵磷脂和抗氧化酶基因表达的严格调控在发情周期中对黄体细胞的维持和凋亡起着关键作用。

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.