"Orbital blood was obtained from PND30/PND60 offspring and GD20 dams under pentobarbital (3 mg/100 ml, IP)–induced anesthesia. Blood was collected from PND1 offspring via decapitation. The serum was separated by centrifugation at 4 °C and stored at °80 °C until assay. Mice brains were removed quickly under pentobarbital (3 mg/100 ml, IP)–induced anesthesia. The hypothalamus was microdissected and homogenized in phosphate buffered saline (pH 7.1, 400 ll) and then centrifuged at 5000 rpm/4 min at 4°C. These hypothalamus samples were stored at °80 °C until assay. Total thyroxine (T4) and 3,30,5-triiodothyronine (T3), free T4, thyroid-stimulating hormone (TSH), estrogen (E2), luteinizing hormone (LH), progesterone (P4), and gonadotropin-releasing hormone (GnRH) levels were measured using commercial ELISA kits (Uscn Life Science Inc., Houston), according to the manufacturer’s instructions. The sensitivities were 1.4 ng/ml for total T4, 47.1 pg/ml for total T3, 0.2 ng/dl for free T4, 19.3 pg/ml for TSH, 2.0 pg/ml for E2, 0.2 ng/ml for LH, 5.2 pg/ml for GnRH, and 0.2 ng/ml for P4. The intra- and interassay. coefficients of variation were 4.3% and 7.5% for total T4, 4.5% and 7.2% for total T3, 4.6% and 6.3% for free T4, 3.2% and 9.5% for TSH, 6.0% and 5.8% for E2, 5.5% and 8.9% for LH, 2.1% and 6.3% for GnRH, and 5.8% and 8.4% for P4."

Estrogen

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