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Tumor necrosis factor-alpha (TNF alpha) was localized to follicular and luteal compartments of the human ovary using a biotin strept-avidin immunocytochemical technique with polyclonal antibodies to human recombinant TNF alpha. Immunoreactive TNF alpha (I-TNF) was observed in granulosa cells of healthy antral and atretic follicles and appeared to be secreted by the granulosa cells, since it was present in the follicular fluid surrounding antral and degenerating granulosa. Preantral follicles did not exhibit I-TNF. I-TNF was in the cytoplasm of large granulosa-lutein cells and small paraluteal cells. Granulosa cells cultured on an Immobilon membrane exhibited I-TNF in the cytoplasm and I-TNF released onto the membrane, which appeared as a dark-red stain after immunolocalization. Granulosa cells cultured on plastic slides exhibited I-TNF throughout the cytoplasm. To determine if I-TNF was secreted by granulosa cells, granulosa cell-conditioned medium was examined for the presence of TNF by a double sandwich enzyme-linked immunosorbant assay using both monoclonal and polyclonal antibodies to human recombinant TNF alpha. Conditioned medium contained up to 800 pg TNF/500,000 cells after 24 h of culture. I-TNF was also found (100-170 pg/mL) in human follicular fluid. Immunoblot assay of I-TNF in cultured granulosa revealed approximately 240 pg TNF/250,000 cells. The results indicate that the human ovary contains I-TNF, and thus, a paracrine and/or autocrine role of TNF in human ovarian follicular function seems likely.

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