GnRH-agonist Induces Apoptosis of Human Granulosa-luteal Cells Via Caspase-3 and -9 and PARP Cleavage

Park, Eun Joo; Kim, Pyeong Sik; Youm, Yun Hee; Yang, Hyun Won; Park, Won Il; Kang, Byung Moon

Abstract

OBJECTIVE
GnRH-agonist (GnRH-Ag) used in controlled ovarian hyperstimulation (COH) for IVF-ET has been known to affect directly on apoptosis of human ovarian cells, but its mechanism is not clearly understood. Therefore, the purpose of the present study was to investigate whether caspase-3 and -9 activation and poly-(ADP-ribose)-polymerase (PARP) cleavage are involved in the mechanism(s) by which GnRH-Ag induces apoptosis in human granulosa-luteal cells.
METHODS
Human granulosa-luteal cells collected from IVF-ET patients were cultured and treated with 10(-6) M GnRH-Ag or saline as a control. To access apoptosis in the cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay and DNA fragmentation analysis were preformed 24 h after the treatment. Activity of caspase-3 and -9 in the cells was examined by using a fluorogenic substrate. Caspase-3 and -9 activation and poly (ADP-ribose) polymerase (PARP) cleavage were analyzed by Western blotting.
RESULTS
DNA fragmentation in the cells increased in the higher concentration over 10(-6) M GnRH-Ag. In the result of TUNEL assay, the rate of apoptotic cells in GnRH-Ag treatment increased significantly compared with that of saline treatment (p<0.05). The activity of caspase-3 and -9 investigated by using a fluorogenic substrate increased only in the apoptotic cells. In Western blot analysis, the cells treated with GnRH-Ag revealed an increase in active forms of caspase-3 and -9 compared with those of the saline treatment. In addition, cleavage of PARP also increased in the cells treated with GnRH-Ag.
CONCLUSION
These results suggest that activation of caspase-3 and -9 and cleavage of PARP might be involved in apoptosis of human granulosa-luteal cells induced by GnRH-Ag.