Rats then received either 0.1 or 0.5 ig of Trap in 5 il of PBS with 0.1% BSA under the bursa of one ovary (Trap ovary). The contralateral ovary was injected with 5 il of vehicle (PBS with 0.1% BSA, control ovary). Taking into account that the VEGF Trap protein is a chimera that contains the Fc region of human immunoglobulin G (IgG), an additional control was designed in which one ovary was injected with 0.65 ig of human IgG, and the contralateral ovary was injected with vehicle. No significant differences were found in the number of follicles at any stage between the human IgG and the vehicle-injected ovary.
Therefore, PBS with 0.1% BSA was chosen as the control. After injection, ovaries were replaced, and the incision was closed with skin adhesive. Subsequently, rats were injected s.c. with 0.1 ml of eCG (25 IU per rat). Rats were killed 12, 24, or 48 h after surgery by CO2 asphyxiation. The ovaries were removed and cleaned of adhering tissue in culture medium for subsequent assays.
The experimental protocols were approved by the Animal Experimentation Committee of the IByME.