17β estradiol 17β e2

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.

MCF-7 cells were cultured in hormone-free medium (HyClone, Logan, UT, USA) with 10% (V/V) charcoal-stripped fetal bovine serum (HyClone, Logan, UT, USA). There were 5000 cells per well in 96 well plates. Cells were separately exposed to 17β-Estradiol (17β-E2; Sigma, Louis, MO, USA; 1 μM), LA (1, 0.1, 0.001 and 0.0001 mg/mL) and PA (1, 2, 4, 6 and 8 μM) for 24 h. Cell viability was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT; Sigma, Louis, MO, USA).

Reagents used in this study were all of analytical grades. Dimethyl sulfoxide (DMSO), methanol, acetone, hexane, and anhydrous sodium sulfate were obtained from Merck (USA). Glucose, galactose, 17β-estradiol (17β-E2), 4-hydroxytamoxifen (OHT), dihydrotestosterone (DHT), flutamide (FLU), triiodothyronine (T3), 9-cis retinoic acid (9cRA), chlorophenol red-β-D-galactopyranoside (CPRG), o-nitrophenyl-β-D-galactopyranoside (ONPG), and 4-nitroquinoline-1-oxide (4-NQO) were purchased from Sigma (USA).