Angiopoietin 1 and 2 are essential for normal folliculogenesis in the prepubertal rat ovary.

DALHIA ABRAMOVICH; IRUSTA GRISELDA; FERNANDA PARBOREL; TESONE MARTA

Simposio; . Frontiers in Reproduction. 13th Annual Symposium; 2010

Introduction: The angiopoietin (ANGPT)-TEK system is involved in blood vessel maturation, being the ANGPT1 essential for vascular bed stability. We have previously shown that the in vivo inhibition of ANGPT1 in gonadotropin-treated rat ovaries caused an increase in the number of atretic follicles and a decrease in the number of early antral (EAF) and preovulatory follicles (POF). These changes were mediated by an increase in ovarian apoptosis due in part to an imbalance in the ratio of antiapoptotic to proapoptotic proteins. The aim of the present study was to evaluate the effect of the in vivo inhibition of ANGPT1 on steroidogenesis, apoptosis, cell proliferation and vascular stability in prepubertal gonadotropin-treated rat ovary. We also analyzed the direct effect of ANGPTs on isolated EAF in culture. Methods: Prepubertal female Sprague-Dawley rats were injected with ANGPT1 neutralizing Ab under the bursa of one ovary and the contralateral ovary was injected with normal goat serum to be used as a control. At the time of the surgery, the animals received 25 IU of eCG. The ovaries were removed 48h after surgery and processed for immunohistochemistry (PCNA, Von Willebrand factor and smooth muscle a-actin), RIA or western blot analysis (PCNA, caspase 3). For in vitro studies, prepubertal female Sprague-Dawley rats were injected subcutaneously with DES daily for three days. The animals were killed and the ovaries removed. EAF were dissected and placed in culture medium either with or without stimulus. Follicles were incubated for 12 h in serum-free medium under different conditions: without stimulus (Basal), with FSH (20 ng/ml), ANGPT1 (100 ng/ml), ANGPT2 (100 ng/ml) or ANGPT1 plus ANGPT2. After the incubation time, the follicles were stored at -70°C until protein extraction or DNA isolation. Results: In vivo inhibition of ANGPT1 significantly decreased the levels of PCNA in granulosa and theca cells and increased the active fragment of caspase 3, p17. In addition, in this group we observed a decrease in the content of ovarian estradiol as well as an increase in the content of androsterone. No changes were found in endothelial cell area but the ANGPT1 Ab decreased the area of perycites. Incubation of isolated EAF with ANGPT1 significantly increased follicular PCNA protein content compared to the control. In addition, ANGPT1 prevented follicle DNA apoptotic cleavage, as well as FSH. While ANGPT2 alone had no effect on follicular cell apoptosis or proliferation, the co-incubation with ANGPT1 interfered with its cytoprotective effect. Conclusions: Intraovarian neutralizing antibody against ANGPT1 produces an increase in ovarian apoptosis interfering with the rat follicular development. This effect is mediated in part by an increase of the ratio of androgen/estrogen steroids and a decrease in granulosa and theca cell proliferation. Furthermore, the results obtained from in vitro experiments demonstrate that, ANGPT/TEK system is not only crucial for the development of functional follicle vasculature but acts directly on follicular cells stimulating follicular growth and preventing the follicles to undergo atresia. Therefore, ANGPT1 would play a critical intraovarian survival role for gonadotropin-dependent folliculogenesis.