In vitro autoradiographic visualization of occupied estrogen receptors in the rat brain with an iodinated estrogen ligand.
Methods have been developed for the selective measurement of occupied estrogen receptors (ER) in brain tissue sections. Cryostat sections of unfixed tissue were incubated with radiolabeled estrogen at physiological temperatures, displacing endogenous receptor-bound estrogen by radioligand and thereby allowing the receptor complexes to be visualized autoradiographically after washing to remove nonspecifically bound steroid. The resultant autoradiographs were analyzed by computer-assisted densitometry. Synthetic 11 beta-methoxy-substituted radiolabeled estrogens gave the best autoradiographic images, as a result of reduced nonspecific labeling, although [3H]-estradiol was also used successfully. With the synthetic ER ligand 11 beta-methoxy 16 alpha-[125I]-iodo-estradiol, exposure times of less than 24 hr generated acceptable autoradiographs; with 3H-labeled estrogens, exposures of 3 months or more may be required. The method is sufficiently sensitive to detect physiological changes in ER occupation and to allow determination of receptor affinities and saturation binding capacities in discrete cell groups identified in sections from individual animals.
Walters
M J
MJ
Department of Obstetrics and Gynecology, Toronto Hospital, Ontario, Canada.
Brown
T J
TJ
Hochberg
R B
RB
MacLusky
N J
NJ
D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure*; D001923:Brain Chemistry; D016616:Cryoultramicrotomy; D003720:Densitometry; D004958:Estradiol / Q000032:analysis / Q000378:metabolism; D004967:Estrogens / Q000032:analysis* / Q000378:metabolism; D005260:Female; D007457:Iodine Radioisotopes; D008024:Ligands; D051381:Rats; D017208:Rats, Wistar; D011960:Receptors, Estrogen / Q000032:analysis* / Q000378:metabolism; D013997:Time Factors; D014316:Tritium
D004967:Estrogens; D007457:Iodine Radioisotopes; D008024:Ligands; D011960:Receptors, Estrogen; D014316:Tritium; D004958:Estradiol