EXPRESSION OF CYCLOOXYGENASE 1 & 2 GENES IN PRIMATE OVULATORY FOLLICLES

对促性腺激素刺激的分子事件知之甚少 在导致排卵和黄体化的颗粒细胞中 灵长类动物的毛囊。一种新的消减杂交策略是 用于鉴定颗粒细胞中表达增强的基因产物 细胞对排卵期黄体生成素激增的反应。颗粒细胞 来自接受重组人的恒河猴(n=3/组) 促性腺激素(Ares Advanced Technology，Inc.)为了刺激 多个大卵泡发育，或之前(非黄体化) 或在(黄体化)给药排卵量后27小时 促性腺激素。制备了可重复使用的单链cDNA文库 从未黄体化和黄体化的颗粒细胞反转 寡核苷酸脱氧核糖核糖核酸与磁性核糖核酸杂交的转录 珠子。用随机引发法制备的寡核苷酸探针 黄素化颗粒细胞cDNA库与 非黄体化颗粒细胞c DNA文库。未杂交的 寡核苷酸(代表至少8倍增强的基因 黄体化颗粒细胞与非黄体化颗粒细胞表达的比较) 用来筛选猕猴黄体cDNA文库。到目前为止，cDNA 从37个阳性斑块中获得的序列与 可用的数据库。几个cDNA与任何已知序列都不匹配， 这表明这些基因代表了新的基因产物 排卵和黄体生成。胸腺肽颗粒细胞的表达 B-10、泛素以及各种线粒体和核糖体蛋白基因 也得到了增强。环氧合酶(COX)-1和COX-2的cDNA分别为 获得并增强颗粒中COX-1和COX-2的表达 促性腺激素激增后的细胞被证实 免疫细胞化学。由于颗粒细胞表达环氧合酶基因 细胞在排卵期促性腺激素激增后得到增强 啮齿动物和家畜，这些数据证实了成功 促性腺激素刺激基因的选择及其支持作用 灵长类动物排卵过程中的前列腺素(PG)未来 研究将检验排卵和排卵之间的时间关系。 促性腺激素激增，启动COX-1和COX-2的表达，以及 灵长类卵泡颗粒细胞产生PG。假说 促性腺激素直接作用于颗粒细胞刺激COX 表达并提高相关生化标记物的水平 黄体化和卵泡破裂将使用 体外和体内技术相结合。这些研究是 适用于以下两种情况的治疗： 不孕症，如黄素化未破裂卵泡综合征 因为新避孕药的开发会阻止排卵。 上述数据构成了一项关于校外活动的建议的基础 资金问题。

Little is known about the gonadotropin-stimulated molecular events in granulosa cells which lead to ovulation and luteinization of the follicle in primates. A novel subtractive hybridization strategy was used to identify gene products with enhanced expression in granulosa cells in response to the ovulatory LH surge. Granulosa cells were harvested from rhesus monkeys (n=3/group) receiving recombinant human gonadotropins (Ares Advanced Technology, Inc.) to stimulate the development of multiple large follicles, either before (nonluteinized) or 27 hours after (luteinized) administration of an ovulatory dose of gonadotropin. A reusable single-stranded cDNA library was prepared from nonluteinized and luteinized granulosa cells by reverse transcription of polyA+ RNA hybridized to oligo dT-linked magnetic beads. Oligonucleotide probes prepared by random priming of the luteinized granulosa cell cDNA library were allowed to hybridize with the nonluteinized granulosa cell cDNA library. Unhybridized oligonucleotides (representing genes with at least 8-fold enhanced expression by luteinized compared to nonluteinized granulosa cells) were used to screen a monkey luteal cDNA library. To date, cDNA sequences obtained from 37 positive plaques have been compared to available databases. Several cDNAs did not match any known sequences, suggesting that these represent novel gene products involved in ovulation and luteinization. Granulosa cell expression of thymosin b-10, ubiquitin, and various mitochondrial and ribosomal protein genes was also enhanced. cDNAs for cyclooxygenase (COX)-1 and COX-2 were obtained, and enhanced expression of both COX-1 and COX-2 in granulosa cells following the gonadotropin surge was verified by immunocytochemistry. Since the expression of COX genes by granulosa cells is enhanced following the ovulatory gonadotropin surge in rodents and domestic animals, these data confirm the successful selection of gonadotropin-stimulated genes and support a role for prostaglandins (PG) in periovulatory processes in primates. Future studies will examine the temporal relationship between the ovulatory gonadotropin surge, the initiation of COX-1 and COX-2 expression, and PG production by granulosa cells of primate follicles. The hypothesis that gonadotropin act directly at granulosa cells to stimulate COX expression and enhance the levels of biochemical markers related to luteinization and rupture of the follicle will be tested using a combination of in vitro and in vivo techniques. These studies are applicable to both the treatment of conditions contributing to infertility, such as luteinized unruptured follicle syndrome, as well as the development of new contraceptives which would block ovulation. The data described above forms the basis for a proposal for extramural funding.