OVARIAN ADMINISTRATION OF ANGIOPOIETIN-1 Ab INCREASES FOLLICULAR APOPTOSIS MEDIATED IN PART BY AN IMBALANCE IN THE RATIO OF ANDROGENS/ESTROGENS AND A DECREASE IN THE GRANULOSA AND THECA CELL PROLIFERATION

PARBORELL F; ABRAMOVICH D; RODRIGUEZ CELIN A; TESONE M

Hawaii, USA

Congreso; Society for Studies of Reproduction (SSR); 2008

Society for Studies of Reproduction (SSR)

The angiopoietin (ANGPT)-receptor (TEK) system plays a crucial role in blood vessel development and regression. Previously, we showed that the inhibition of ANGPT1 caused an increase in the number of atretic follicles and a decrease in the number of both antral follicles (AF) and preovulatory follicles in gonadotropin-treated rat ovaries. The aim of this study was to evaluate the effect of local administration of anti-ANGPT1 antibody (ANGPT1-Ab) on steroidogenesis, apoptosis, cellular proliferation and vascular development, in ovarian follicles from prepubertal eCG-treated rats. Female immature rats superovulated with eCG (25 IU/rat) were injected with an antibody for ANGPT 1 (10ng/ovary) in 5 ul of saline under the bursa of one ovary (ANGPT1 Ab ovary). The contralateral ovary was injected with the same volume and concentration of normal goat IgG (Control ovary). Rats were killed 48 hours after surgery by CO2 asphyxiation. The ovaries were removed, cleaned off adhering tissue in culture medium and AF (250- 400 um) were dissected under a stereoscopic microscope and used for steroid extractions. Treatment with ANGPT1 Ab resulted in a significant increase in follicular androsterone levels (C: 59.93 ± 7.5 vs ANGPT1 Ab: 85.65 ±6.7 ng/ovary, p<0.05, n=7), whereas a significant decrease in estradiol was observed (C: 12.13 ± 2.3 vs ANGPT1 Ab: 8.7 ± 2.3 ng/ovary, p<0.05, n=7). However, follicular progesterone levels were unchanged. As granulosa cells (gCs) of the follicle die by apoptosis during the process of follicular atresia, the TUNEL technique was performed on histological ovarian slides from ovaries obtained 48 h after injection of ANGPT1 Ab. Specific staining was observed in gCs and ANGPT1 Ab treatment caused a significant increase in the number of apoptotic cells in AF (C: 2.29 ± 0.36 vs ANGPT1 Ab: 6.08 ± 0.84 apoptotic cells/field, p<0.05). To identify proliferating cells, slides were incubated with anti-PCNA Ab. A proliferation index (PCNA-positive cells expressed as a percentage of the total number of cells) was calculated in the theca and granulosa compartments for each antral follicle. PCNA protein expression decreased significantly in gCs (C: 63.98 ± 3.03 vs ANGPT1 Ab: 46.81 ± 4.02; p<0.01) as well as cTs (C: 59.64 ± 3.7 vs ANGPT1 Ab: 41.37 ± 46; p<0.01) from AF in ANGPT1 Ab-treated ovaries. In order to evaluate the endothelial cell (eC) density, histological ovarian slides were immunostained using an antibody raised against the Von Willebrand Factor. No significant differences were found in the % of endothelial cell area between control and ANGPT1 Ab-treated ovaries In conclusion, intraovarian neutralizing antibody against ANGPT1 interferes with the rat follicular development associated with an increase in ovarian apoptosis mediated in part by an imbalance in the ratio of androgen/estrogen steroids and a decrease in granulosa and theca cell proliferation. Therefore, ANGPT1 would play a critical intraovarian survival role for gonadotropin-dependent folliculogenesis. Supported by: UBA (EX237), CONICET (PIP 5471), ANPCYT (PICT 5-26047) and Roemmers Foundation.