Oocytic tumor necrosis factor alpha: localization in the neonatal ovary and throughout follicular development in the adult rat.

Tumor necrosis factor alpha (TNF alpha) has previously been immunolocalized within mouse oocytes. Our first objective was to examine TNF alpha immunolocalization in ovaries of adult, fetal, and neonatal rats. Our second objective was to examine TNF alpha mRNA in ovaries by Northern blot analysis and in oocytes by reverse-transcriptase polymerase chain reaction (RT-PCR). Our final objective was to determine whether oocytes contained bioactive TNF alpha. Ovaries and oviducts were collected throughout the estrous cycle in adult rats, fetal ovaries were obtained 1 day before expected delivery, and neonatal ovaries were collected 2 days after birth. TNF alpha was localized in tissues by a biotin-avidin immunocytochemical procedure. Immunocytochemistry in adult ovaries showed that the ooplasm of the oocyte was the primary site of TNF alpha localization within the follicle. Immunostaining was present in all oocytes in the adult, including ovulated oocytes within the oviduct. Oocytic TNF alpha immunostaining was also present within oocytes in the neonate; however, fetal oocytes did not contain immunoreactive TNF alpha. Northern blots showed that ovaries in the adult, neonate, and fetus all contained TNF alpha mRNA. RT-PCR analysis of oocytes collected from preovulatory follicles generated a cDNA band of 500 bp, corresponding to the predicted size for amplified TNF alpha cDNA. Subsequent Southern blot analysis showed that the 500-bp band hybridized to the TNF alpha probe, indicating that preovulatory oocytes contain TNF alpha mRNA. Preovulatory oocytes were used in TNF alpha cytotoxicity assays with L929 cells. Oocytes contained TNF alpha bioactivity that was similar to that of recombinant murine TNF alpha in the bioassay. Our results provide evidence for the identification of immunoreactive and bioactive TNF alpha within oocytes in the rat, which is further supported by the presence of TNF alpha mRNA within the oocyte. These studies also indicate that TNF alpha may appear in the oocyte around the time of birth.