Immunohistochemial Study on Effectors of Ovary Medulla Blood Vessel in Pseudopregnant Rabbit

Background: The ovarian blood flow provides oxygen-rich blood and nutrients which are necessary for the growth and secretory activities of follicles and corpora lutea (CL) in cycling and pregnant animals. The blood flow of the ovary is finely regulated by local and systemic mechanisms that integrate nervous, endocrine, and metabolic signals. In the rabbit ovary, administration of prostaglandin F2 alpha (PGF2 alpha) to pseudopregnant or pregnant animals affects specific vascular mechanisms regulating luteolytic process, in particular, the regulatory role of progesterone on luteal function through direct and uterine-mediated mechanisms. Thus, the main objective of the present work was to evaluate, at the level of ovarian blood vessels, the immunopresence of the receptors for progesterone (PR-R), PGF2 alpha (FP) and GnRH (GnRH-R), and that of the enzymes endothelial nitric oxide synthase (eNOS), cyclooxygenase 1 (COX1), COX2, and prostaglandin E2-9-ketoreductase (PGE2-9-K). Materials, Methods & Results: Sexually mature New Zealand White female rabbits were used for all experiments. The animals were housed individually, in an indoor facility under controlled conditions of light and temperature, and feed ad libitum. The rabbits were treated with equine chorionic gonadotropin followed 2 days later by an intramuscular injection of a GnRH analogue to induce pseudopregnancy. Five rabbits were sacrificed by cervical dislocation, at early, mid and late stages of pseudopregnancy, for the immunohistochemical detection of GnRH-R, COX1, COX2, PGE2-9-K, eNOS, FP, and PR- R. The ovaries were immersed for fixation in formaldehyde and subsequently processed for embedding in paraffin. Serial sections were cut and individually mounted on slides. Background labelling was prevented by incubating the sections with normal goat serum (for COX1, COX2, eNOS, GnRH-R, PR- R, and FP) and with normal rabbit serum (for PGE2-9-K). Subsequently, the sections were incubated overnight with the following primary antisera: mouse monoclonal anti-COX1, anti-COX2, anti-eNOS, anti-GnRH-R, and antiPR-R, rabbit polyclonal anti-FP, and goat polyclonal anti-PGE2-9-K (CBR1). The next day, the slides were treated again with normal goat or rabbit serum and then incubated with biotin goat anti-mouse (for COX1, COX2, eNOS, PR- R, and GnRH-R), biotin goat anti-rabbit (for FP) or biotin rabbit anti-goat (for PGE2-9-K) secondary antibodies diluted 1: 200 in TBS for 30 min at room temperature. The slides were exposed to the avidin-biotin complex and the peroxidase activity sites were visualized using the 3,3-diaminobenzidine tetrachloride kit as chromogen. At all stages of pseudopregnancy, the veins and arteries, within the ovary hilus and medulla, showed a variable immunoreactivity to anti-COX2, -PGE2-9-K, -eNOS and -FP antibodies, whereas anti-GnRH-R, -COX1 and -PR-R did not produce immunosignals. In particular, COX2 immunopresence was strongly localized only in the arterial endothelial cells but not in the veins. PGE2-9-K immunosignals were moderately detected in the nuclei of endothelial, smooth muscle and stromal cells of both veins and arteries. The eNOS immunopositivity was moderately evidenced in the nuclei and cytoplasm of endothelial, smooth muscle and stromal cells of both veins and arteries. Strong positive immune reaction for FP was detected in the endothelial cells of ovarian arteries and veins; the arterial muscle cells were negative. Discussion: The present results suggest that the ovarian blood flow of rabbits is dynamically regulated as a consequence of exogenous treatments for the synchronization of estrous and the induction of ovulation. These haemodynamic changes are likely due to combined actions by several intraovarian factors, such as prostaglandins and correlated enzymes, steroids, and other autocrine/paracrine factors (GnRH, PGF2 alpha, NO).