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 immunoloca lized within mouse oocytes. Our first objective was to examine TNF alp ha immunolocalization in ovaries of adult, fetal, and neonatal rats. O ur second objective was to examine TNF alpha mRNA in ovaries by Northe rn blot analysis and in oocytes by reverse-transcriptase polymerase ch ain reaction (RT-PCR). Our final objective was to determine whether oo cytes contained bioactive TNF alpha. Ovaries and oviducts were collect ed throughout the estrous cycle in adult rats, fetal ovaries were obta ined 1 day before expected delivery, and neonatal ovaries were collect ed 2 days after birth. TNF alpha was localized in tissues by a biotin- avidin immunocytochemical procedure. Immunocytochemistry in adult ovar ies 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 oviduc t. Oocytic TNF alpha immunostaining was also present within oocytes in the neonate; however, fetal oocytes did not contain immunoreactive TN F alpha. Northern blots showed that ovaries in the adult, neonate, and fetus all contained TNF alpha mRNA. RT-PCR analysis of oocytes collec ted froth preovulatory follicles generated a cDNA band of 500 bp, corr esponding to the predicted size for amplified TNF alpha cDNA. Subseque nt Southern blot analysis showed that the 500-bp band hybridized to th e TNF alpha probe, indicating that preovulatory oocytes contain TNF al pha mRNA. Preovulatory oocytes were used in TNF alpha cytotoxicity ass ays with L929 cells. Oocytes contained TNF alpha bioactivity that was similar to that of recombinant murine TNF alpha in the bioassay. Our r esults provide evidence for the identification of immunoreactive and b ioactive TNF alpha within oocytes in the rat, which is further support ed 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.