E2 benzoate

Male gonads were left intact and all females were subjected to OVX at least 7 d before each experiment. Rimadyl (4–5 mg/kg, s.c.) was given immediately after surgery for relief of postoperative pain. Females received either an injection of sesame oil (50 μl, s.c.; Sigma-Aldrich) or a priming dose (0.125 μg/50 μl sesame oil, s.c.) of E2 benzoate (Sigma-Aldrich) on the Friday morning following surgery. In addition, oil or a low (0.25 μg) and then a high (1.5 μg) dose of E2 benzoate was administered in the morning of the 2 d preceding experiments. Circulating levels of E2 were verified by the uterine weights (<25 mg for OVX and >95 mg for E2 treated) at the time of hypothalamic slice preparation (between 8:30 and 10:30 A.M.).

All sexual behavioral experiments were performed during the dark phase of the dark/light cycle, 1 hour after lights off. Tests were filmed with an infra-red light in a dark room. Two weeks before W0, male mice were placed for a week with a receptive female (in oestrus phase) to gain sexual experience. At W4, males were tested in an open field (50 x 50 cm) with a receptive female for 30 minutes. Females were ovariectomized and implanted with Silastic implants (Dow Corning, Saint Denis, France) containing 50 μg of E2-benzoate (Sigma-Aldrich). Four hours before the tests, females were given a subcutaneous injection of 1 mg of progesterone (Sigma-Aldrich, P6149, St. Quentin Fallavier, France) diluted in 100 μL of oil to induce receptivity (36 (link)). The latencies and frequencies of mounts and intromissions were recorded. The sexual preference of males was also evaluated. Males were placed in the centre of a three-compartment chamber separated by Plexiglas with an opening at the base permitting olfactory and visual contact. Following a 10 min period of habituation, a receptive female (detected by a vaginal smear) and an unfamiliar male were each placed in one of the side compartments of the chamber. The time spent by the tested male mouse near each compartment was recorded for 10 min.

E2, E2 benzoate (EB), and P4 (Sigma-Aldrich; St. Louis, MO) were independently dissolved in ethanol prior to dissolution in sesame oil (Sigma-Aldrich) at a concentration of 600 μg/ml (E2 or EB) and 15 mg/ml (P4). Equivalent volumes of ethanol without hormone were added to sesame oil to serve as the vehicle control for each hormone. Body weights were obtained prior to feeding each day and used to determine the appropriate volume of hormone to administer to each animal. Each day the appropriate volume of hormone or vehicle was mixed into approximately 4 g of peanut butter (Skippy Natural Peanut Butter) placed on 2x2 inch squares of parchment paper. Each animal was offered two allotments of peanut butter to achieve the appropriate treatment combination e.g. E2 and vehicle, P4 and vehicle, E2 and P4, or vehicle and vehicle. The animals had access to the treatment until the next day when old papers were removed and new treatments were given. Hormone consumption was evaluated each morning before administering new treatments based on visualization of whether the paper was completely devoid of peanut butter (all), peanut butter was partially consumed (partial), or appeared the same as it had when hormones were introduced the previous day (none).