O-204: Angiotensin II (Ang II) regulates apoptosis in human granulosa-lutein cells (GL)

Abstract

The human ovary has a complete and functional gonadotrophin-sensitive renin-angiotensin system and both types of AngII receptors, AT1 and AT2, are present in ovarian cells. We have shown that AngII is present in high concentrations in granulosa cells and follicular fluid of human preovulatory follicles and in luteal cells. AngII regulates ovulation and modulates steroidogenesis in rat luteal cells and human GL cells. Other investigators have shown that AngII regulates luteal cell apoptosis. Recently, we have shown that apoptosis of human GL cells correlates with several indicators of IVF outcome, such as embryo fragmentation and pregnancy rate, and is regulated by intraovarian growth factors. The aim of the present study was to test whether AngII could induce in vitro apoptosis of gonadotrophin-exposed human IVF GL cells. Following AngII exposure, apoptosis was assessed by caspace expression. Controls included the non-specific angiotensin receptor blocker Saralasin and the specific AT2 receptor (AT2-R) blocker CGP-42112A. GL cells from 87 IVF-intra-cytoplasmic sperm injection (ICSI) cycles were analyzed. Ovarian stimulation was performed using recombinant FSH and LH or HMG. The pregnancy rate for these cycles was 56.32%. Following oocyte recovery GL cells were collected from pooled follicular fluid (FF), washed and purified using a 50% percoll gradient and anti-CD45-coated magnetic beads. Cells were cultured without serum for 48 hours in the presence or absence of 10-11, 10-9, 10-7M AngII, with and without Saralasin10-5 or CGP-42112A 10-5M. The percentage of apoptotic cells was determined under a fluorescence microscope by staining with 10μm caspace FITV-VAD-FMK (a fluorescent marker for activated caspaces) and 0.5μg/ml propidium iodide. Statistical analysis was carried out using the student-t test. AngII in concentrations present in human follicle fluid caused increased apoptosis of GL cells compared to untreated control cells (10-11M p=0.312; 10-9M p<0,05; 10-7M p<0,05, respectively). This effect was partially inhibited by both Saralasin and CGP-42112A (18.15% and 33.01%, respectively, p<0,05). In these preliminary studies on human GL cells Ang II at concentrations that are present in FF from human IVF cycles (J Soc Gynecol Investig. 1994 1:118-27) is proapoptotic. The partial blockade by angiotensin receptor antagonists is consistent with a receptor-mediated action. Ovarian AII has been linked to fertility, steroidogenesis and polycystic ovarian syndrome. Further studies are underway to determine the relevance of these results to the outcome of IVF cycles and other clinical scenarios.