Endotoxin-induced apoptosis in ovarian follicles is partially blocked by 2-methylthioATP or 2-chloroATP

The purposes of this study were to determine how early in time endotoxin can trigger apoptosis of bovine ovarian follicles in vitro, and to further characterize if these inductions are mediated via adenine nucleotides and the P2 purinergic receptors. Healthy preantral and early antral follicles (400 and 700 microns) isolated from bovine ovaries were sandwiched between two layers of collagen gel and incubated (39 degrees C, 5% CO2, 95% air) for various time periods up to 72 hr, floating in complete medium with either 2-methylThioATP or with 2-ChloroATP, or with or without LPS (10 or 50 micrograms/ml), or with combinations of LPS with 2-MethylThioATP or 2-ChloroATP. Data from histological examination, and in situ detection of apoptotic DNA cleavage, showed that by 2 hr from start of incubation, both doses of LPS had triggered apoptosis of granulosa cells (P < 0.001), and simultaneously decreased estradiol concentrations to nondetectable levels (P < 0.001), but progesterone values increased (P < 0.001) with time of incubation. Both 2-MethylThioATP and 2-ChloroATP inhibited (P < 0.001) LPS (10 and 50 micrograms/ ml)-induced apoptosis by 30% to 100%. We concluded that adenine nucleotides play a fundamental role in endotoxin-induced apoptosis/atresia of bovine follicles, probably via the P2 purinergic receptors. It is possible that during the first 2 hr of incubation, the apoptotic events associated with LPS-induced follicular atresia might not be detectable with the procedures used in this study.