Five-month-old mice were superovulated by intraperitoneal injection of 5 IU pregnant mare's serum gonadotropin (PMSG) and, 48 h later, the mice were additionally injected with 5 IU human chorionic gonadotropin (hCG). The mice were euthanized 22 h after the injection of hCG for quantification of ovulated oocytes as previously described [26 (link)]. GnRH stimulation was performed in 5-month-old WT and Kiss1-PgrKO by intraperitoneal injection of 200 ng/kg GnRH peptide (Sigma–Aldrich) in 100 μl saline. Blood was collected 10 min later via cardiac puncture immediately following euthanasia [27 (link)]. Estrogen treatment was performed as previously described [28 (link)]. Briefly, mice were ovariectomized, and 1 cm SILASTIC (Dow Corning Corp., Midland, MI) brand capsules (0.04”ID, 0.085” OD; American Scientific Products, McGaw Park, IL) containing 2.5 μg of 17β-estradiol (17β-E2, Sigma Chemical Co., St. Louis, MO) mixed into Silicone Type A Medical Adhesive (Dow Corning Corp.) were placed subcutaneously under one flank. Controls were implanted with empty capsules. Following 6 days, mice were injected subcutaneously with estradiol benzoate (EB, 1 μg in 0.1 ml sesame oil) or vehicle at 0900 h. On the evening of the seventh day following ovariectomy at 1900 h, blood was collected via cardiac puncture immediately following euthanasia.
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