Deiodination assays and hormone measurements

Assays for type 2 deiodination activity and for T3 and T4 levels were performed as described (8, 18). Pools of dissected eyes, retinae, or cochleae were homogenized in buffer containing 20 mM Tris-HCl (pH 7.6), 5 mM dithiothreitol, and 0.25 mM sucrose. Pools of four to six eyes, six to 10 retinae, and 8 cochleae were analyzed in triplicate. Homogenates were incubated with [¹²⁵I]T4 substrate, 1.2 mM EDTA, 1 mM propylthiouracil, 1 μM T3, and 20 mM dithiothreitol for 1 hour at 37°C, and then products were separated using Bio-Rad AG 50WXG (H⁺) resin to measure [¹²⁵I]I⁻ product. Serum total T4 and T3 values were determined by radioimmunoassay using Coat-A-Count reagents (Diagnostic Systems Laboratories, Webster, TX), as described (8). Hormones were measured for individual adult mice and for pools of two to three pups due to limited volumes of serum obtained.