Elucidation of physiological roles of prostaglandin F_<2alpha> and its regulatory mechanism in the reproductive system

二十多年来，人们已经认识到前列腺素（PG） F_<2alpha>在许多物种的黄体退化中起着至关重要的作用。我们成功克隆了一个编码PGF_<2alpha>受体的cDNA。我们最近发现，在整个发情周期的黄体期和妊娠早中期，PGF_<2alpha>在卵巢黄体中大量表达，提示PGF_<2alpha>可能不仅参与黄体溶解，还参与黄体功能。今年我们重点阐明了PGF_<2alpha>受体mRNA表达和PG生成的调控机制，得到了以下结果。1)通过RNA印迹分析发现，促性腺激素（PMSG、hCG、FSH）和霍乱毒素可激活腺苷酸环化酶，使cAMP水平升高，1-16 h小鼠血清孕酮水平和卵巢PGF_<2alpha>受体mRNA表达升高。我们还证实了这些药物诱导PGF_<2alpha>受体mRNA在预先存在的小鼠卵巢黄体细胞中的表达增加。这些结果支持了PGF_<2alpha>可能不仅参与黄体溶解，而且参与黄体功能的观点。2)成功克隆了PGF_<2alpha>受体基因，全长超过30kb，由3个外显子组成。启动子区域包含转录因子的几个结合基序，并被TPA和forskolin激活。3)为了阐明PG产生的调控机制，我们首先通过气相色谱和质谱法在小鼠尿液中鉴定了3种PGF_<2alpha>的代谢产物。我们检测了其中一种代谢物的变化，发现代谢物在妊娠期增加，提示PGF_<2alpha>参与分娩。4)用抗PGF合成酶抗血清进行Western blot分析，发现PGF合成酶蛋白在妊娠期升高，可能与PGF_<2alpha>的产生有关。少

It has been recognized for overe two decades that prostaglandin (PG) F_<2alpha> is of prime importance in initiating luteal regression in many species. We succeeded in the cloning of a cDNA encoding PGF_<2alpha> receptor. We recently demonstrated that its mRNA and receptor protein are abundantly expressed in corpus luteum of the ovary throughout the luteal phase of the estrous cycle and early and middle pregnancy, suggesting that PGF_<2alpha> may be involved in not only luteolysis but also luteal functions. This year we focused on the elucidation of the regulatory mechanisms of PGF_<2alpha> receptor mRNA expression and PG production, and obtained following results. 1) We revealed by RNA blot analyzes that serum progesterone level and expression of PGF_<2alpha> receptor mRNA in mouse ovary increased 1-16 h after administration of gonadotropins (PMSG,hCG,FSH) and cholera toxin, which activate adenylate cyclase and increase cAMP level. We also demonstrated that these agents induced the ex … More pression of PGF_<2alpha> receptor mRNA in luteal cells of preexisting mouse ovary. These results supports the above-mentioned notion that PGF_<2alpha> may be involved in not only luteolysis but also luteal funciton. 2) We succeeded in the cloning of PGF_<2alpha> receptor gene and showed that it spans over 30 kb and consists of 3 exons. The promoter region contains several binding motifs for transcription factors and was activated by TPA and forskolin. 3) In order to elucidate the regulatory mechanism of PG production, we first identified three urinary metabolites of PGF_<2alpha> in mice by gas chromatography and mass spectrometry. We examined the change of one of the metabolites and showed that the metabolite increased at the term of pregnancy, suggesting the involvement of PGF_<2alpha> in delivery. 4) We also demonstrated by Western blot analysis with anti-PGF synthase antisera that PGF synthase protein increased at the term of pregnancy and suggested that it may be responsible for the PGF_<2alpha> production. Less