OVARIAN TUMOR NECROSIS FACTOR

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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.