OVARIAN TUMOR NECROSIS FACTOR

肿瘤坏死因子α（TNF）已定位于大鼠卵巢细胞 和巨噬细胞和TNF的不同调节作用已经显示， 卵巢类固醇生成有趣的是，TNF mRNA和免疫活性蛋白 最近在卵母细胞和颗粒细胞中观察到;生物活性TNF 在卵母细胞中也观察到。这项建议将确定是否 卵母细胞和颗粒细胞合成真实的TNF，确定是否 促黄体生成素（LH）、促卵泡激素（FSH）和cAMP 降低卵母细胞和颗粒细胞中TNF基因的表达， TNF的生理刺激因子脂多糖（LPS）的作用 合成，体外对卵巢颗粒和卵泡膜细胞功能的影响， 描述TNF在体内卵巢中的特定功能。第一个目标 将决定卵母细胞是否合成和分泌TNF， 排卵前卵泡的壁细胞和卵丘颗粒细胞。的 卵巢细胞将与35 S-Trans标记（甲硫氨酸和 半胱氨酸）。 掺入的放射性标记将与 特异性TNF抗体并进行凝胶电泳， 确定TNF在分子大小上是否与真正的TNF相似。在 此外，卵母细胞和颗粒细胞分泌结合的能力 将在存在/不存在以下物质的情况下评估放射性标记作为真实TNF LPS。这些结果将有助于了解卵巢细胞是否 合成和分泌TNF。TNF mRNA不存在于卵母细胞中， 促性腺激素激增后排卵前卵泡的颗粒细胞 在发情前期因此，第二个目标将决定 促性腺激素和cAMP对卵母细胞和颗粒细胞TNF基因表达的影响 促性腺激素激增前排卵前卵泡的细胞。的 第三个目的是确定LPS对FSH刺激的cAMP的体外作用， 颗粒细胞产生蛋白多糖和雌二醇， 直接cAMP，LH结合和雄烯二酮合成在卵泡膜中， 间质细胞体外培养。这一目标的基本原理是，在体内， LPS降低卵巢生长、排卵数和雌二醇水平 在未成熟大鼠给予孕马血清促性腺激素。基于 后一观察结果和目标1中的观察结果，如果TNF由卵巢合成， 颗粒细胞并由这些细胞分泌，然后特异性TNF抗体 将用于改变LPS对颗粒的影响。第四个目标 是为了确定TNF反义寡核苷酸在体内的作用， 卵巢肿瘤坏死因子基因表达。此外，TNF的作用 反义寡核苷酸，对健康和闭锁卵泡的数量， 卵巢LH和FSH受体和免疫反应性芳香化酶， granulosa_将确定。这项研究的长期目标是 提供卵巢肿瘤坏死因子功能的深入了解。

Tumor necrosis factor alpha (TNF) has been localized in rat ovarian cells and macrophages and distinct modulatory effects of TNF have been shown on ovarian steroidogenesis. Interestingly, TNF mRNA and immunoactive protein have recently been observed in oocytes and granulosa cells;bioactive TNF has also been observed in oocytes. This proposal will ascertain whether oocytes and granulosa cells synthesize authentic TNF, determine if luteinizing hormone (LH), follicle stimulating hormone (FSH) and cAMP reduce TNF gene expression in oocytes and granulosa, determine specific effects of lipopolysaccharide (LPS), a physiologic stimulator of TNF synthesis, on ovarian granulosa and theca cell function in vitro and delineate specific functions of TNF in the ovary in vivo. The first aim will determine whether TNF, is, synthesized and secreted by the oocyte and mural and cumulus granulosa cells of preovulatory follicles in vitro. The ovarian cells will be incubated with 35S-Trans label (methionine and cysteine). Incorporated radiolabel will be immunoprecipitated with specific TNF antibodies and subjected to gel electrophoresis in order to determine if TNF is similar in molecular size to authentic TNF. In addition, the ability of oocytes and granulosa to secrete incorporated radiolabel as authentic TNF will be assessed in the presence/absence of LPS. These results will provide insight into whether ovarian cells synthesize and secrete TNF. TNF mRNA is not present in oocytes and granulosa cells of preovulatory follicles after the surge of gonadotropins on proestrus. Thus, the second aim will determine the effects of gonadotropins and cAMP on TNF gene expression in oocytes and granulosa cells from preovulatory follicles prior to the gonadotropin surge. The third aim determines the in vitro effects of LPS on FSH-stimulated cAMP, proteoglycan and estradiol production by granulosa cells and on LH- directed cAMP, LH binding and androstenedione synthesis in theca- interstitial cells in vitro. The rationale for this aim is that in vivo, LPS reduced ovarian growth, the number of ova shed, and estradiol levels in immature rats given pregnant mare serum gonadotropin. Based on the latter observations and those in aim 1, if TNF is synthesized by ovarian granulosa cells and secreted by these cells, then specific TNF antibodies will be used to alter the effects of LPS on the granulosa. The fourth aim is to determine the in vivo effects of TNF antisense oligodeoxynucleotides on ovarian TNF gene expression. In addition, the effects of the TNF antisense oligos, on the number of healthy and atretic follicles, on ovarian LH and FSH receptors and on immunoreactive aromatase in the granulosa_will be determined. The long term goal of this research is to provide insight into the function(s) of ovarian TNF.