OVARIAN TUMOR NECROSIS FACTOR

肿瘤坏死因子α(TNF)定位于大鼠卵巢细胞 巨噬细胞和肿瘤坏死因子的明显调节作用已被证实。 卵巢类固醇激素的生成。有趣的是，肿瘤坏死因子mRNA和免疫活性蛋白 最近在卵母细胞和颗粒细胞中观察到；生物活性肿瘤坏死因子 在卵母细胞中也观察到了这种现象。这项提议将确定是否 卵母细胞和颗粒细胞合成真正的肿瘤坏死因子，确定是否 黄体生成素、卵泡刺激素和cAMP 降低卵母细胞和颗粒细胞中肿瘤坏死因子基因的表达，确定特异性 肿瘤坏死因子生理刺激物--脂多糖的作用 体外合成对卵巢颗粒和膜细胞功能的影响 描述肿瘤坏死因子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肿瘤坏死因子，以及 体外培养的排卵前卵泡的壁细胞和卵丘颗粒细胞。这个 卵巢细胞将与35S-反式标记(蛋氨酸和 半胱氨酸)。掺入的放射性标签将与 特异性肿瘤坏死因子抗体，并进行凝胶电泳法检测 确定肿瘤坏死因子在分子大小上是否与正品肿瘤坏死因子相似。在……里面 此外，卵母细胞和颗粒细胞分泌联合的能力 放射性标记作为真实的肿瘤坏死因子将在有/没有的情况下进行评估 LP。这些结果将为卵巢细胞 合成和分泌肿瘤坏死因子。在卵母细胞中不存在肿瘤坏死因子mRNA， 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肿瘤坏死因子基因表达的影响 促性腺激素激增之前排卵前卵泡中的细胞。这个 第三个目的是确定脂多糖对FSH刺激的cAMP的体外作用， 颗粒细胞和黄体生成素分泌蛋白多糖和雌二醇 膜上直接cAMP、黄体生成素结合和雄烯二酮的合成 间质细胞体外培养。这个目标的基本原理是，在活体内， 脂多糖降低卵巢生长、排卵数和雌二醇水平 给予孕马血清促性腺激素的未成熟大鼠。基于 后者的观察和目标1中的观察，如果肿瘤坏死因子是由卵巢合成的 颗粒细胞，并由这些细胞分泌，然后特异性的肿瘤坏死因子抗体 将被用来改变内毒素对颗粒的影响。第四个目标 目的是确定肿瘤坏死因子反义寡核苷酸的体内效应 对卵巢肿瘤坏死因子基因表达的影响。此外，肿瘤坏死因子的作用 反义寡核苷酸，对健康和闭锁卵泡的数量，on 卵巢黄体生成素和卵泡刺激素受体及其对芳香化酶免疫反应的影响 颗粒_将被测定。这项研究的长期目标是 洞察卵巢肿瘤坏死因子的功能(S)。

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.