Characterization of 11 beta-methoxy-16 alpha-[125I]iodoestradiol binding: neuronal localization of estrogen-binding sites in the developing rat brain.

The binding properties of the gamma-emitting 125I-labeled 11 beta-methoxy analog of 16 alpha-iodoestradiol, 11 beta-methoxy-16 alpha-iodoestradiol (MIE2), were characterized for its use in vivo as a ligand for the measurement and localization of estrogen-binding sites. In binding displacement studies, MIE2 bound to rat, rabbit, and human estrogen receptors with high affinity. Association of MIE2 with uterine cytosol estrogen receptors reached maximum values within 30 min at 25 C. At 0-4 C, association was much slower, with maximum binding values not achieved until 16-24 h after the start of the incubation. Once formed, the MIE2-estrogen receptor complex was quite stable at 0-4 C (t1/2 much greater than 24 h). At 25 C, dissociation of MIE2-estrogen receptor complexes occurred nearly 3 times more slowly than that of E2-estrogen receptor complexes (t1/2, 3.3 vs. 1.2 h). The iodinated estrogen was highly specific for the estrogen receptor and did not bind appreciably to androgen, progestin, or glucocorticoid receptors or to either human sex hormone-binding globulin or rat alpha-fetoprotein. MIE2 is also not a ligand for human sex hormone-binding globulin. Dose-dependent uptake of [125I]MIE2 into pituitary and brain cell nuclei was observed after its in vivo administration to 25-day-old female rats. In 10-micron brain sections from immature female rats treated with [125I]MIE2 (7.5 microCi/g BW), regional localization of estrogen-sensitive brain areas could be obtained by autoradiography using LKB Ultrofilm with an exposure time of only 16 h. In comparison, after an identical dose of 16 alpha-[125I]iodoestradiol, an exposure time of 72 h was required to achieve an image of similar density. Combined autoradiographic and immunocytochemical studies in 5- to 11-day-old female rats demonstrated nuclear binding of [125I]MIE2 in cells immunoreactive for neurofilament protein but not glial fibrillary acidic protein, indicating that estrogen receptors in the developing postnatal brain are restricted to neurons and are not present in astroglial cells. The biological characteristics of [125I]MIE2 combined with its high specific activity make it an estrogenic probe with a wide range of possible uses for the study of estrogen action in the developing brain as well as other estrogen target tissues.