ATP‐induced apoptotic cell death in porcine ovarian theca cells through P2X7 receptor activation

Abstract

Folliculogenesis modulation via distinct neurotransmitters is a well-documented phenomenon. Intraovarian purinergic signaling mechanisms have been identified previously in different species. However, the molecular elements involved and the physiological role of this purinergic signaling remain to be elucidated. Here, studies using RT-PCR amplification, immunoblotting, and immunofluorescence microscopy showed that murine and porcine ovaries express the P2X7 subtype receptor, a cationic receptor-channel operated by ATP. Using immunofluorescence it was demonstrated that P2X7 protein expression, in both mouse and pig, occurs specifically in the theca cells from antral follicles. Isolated porcine theca cells maintained in primary cultures and tested with 1 mM ATP or 250 microM Bz-ATP, a specific agonist of P2X7, responded with an increase in intracellular calcium concentration, as demonstrated in cells loaded with fluo-4 as calcium indicator. This strongly suggested that P2X7 receptors in theca cells are functional. Moreover, application for 24 hr of 1 mM ATP or 250 microM Bz-ATP induced apoptotic cell death as indicated by the DNA fragmentation pattern, positive TUNEL test, and annexin V binding. This ATP effect was antagonized by 300 microM PPADS and 200 microM oxidized ATP. Also, addition of 5 mM EGTA in the external medium to chelate free Ca++ decreased death cell to 24% of that produced by 200 microM Bz-ATP, suggesting that Ca++ influx participates in the phenomenon. The highly specific and functional expression of P2X7 receptors in theca cells suggest a role for ATP in modulating follicular physiology.