I'~IUTTE RWQ RT H l~rlE I N E M A N N Papers In vitro labeling of gonadal steroid hormone receptors in brain tissue sections Theodore J. Brown,*'t Monika Sharma,* Lawrence E. Heisler,* Naznin Karsan,$ Michael J. Waiters,§ and Neil J. MacLusky*'~ Division of Reproductive Science,* The Toronto Hospital Research Institute; Departments of Obstetrics and Gynecology,* Zoology, 4- and Physiology, ¢ UniversiO, of Toronto, Toronto, Ontario; and Imaging Research, Inc.§ St. Catharines, Ontario, Canada Autoradiographic methods" have been developed Jor measurement ql gonadal steroid receptors in situ in brain tissue sections. Based on principles established previously ft." estrogen receptors in the rat brain using a 1251-labeled ligand, procedures have been developed fi)r in vitro labeling of estrogen, androgen, and progestin receptors with commercially available tritiated ligands. Addition of protamine sul)ate to the incubation buffer precipitates the receptors in situ in the tissue sections, allowing them to be detected autoradiographically after incubation with labeled steroid and subsequent washing to remove unbound and nonspecifically bound ligand. Occupied and unoccupied estrogen receptors can be measured selectively using appropriately modified incu- bation conditions. In the case of androgen and progestin receptors, unoccupied receptors are readih, detected by in vitro labeling of tissue sections, but occupied receptors do not appear to label efficiently. Preliminao' data suggest that these methods should be equally applicable to a variety q[ laboratoo, animals, including the rat, mouse, guinea pig. and monkey. (Steroids" 60:726-737. 1995) Keywords: estrogen receptor; progesterone receptor; androgen receptor: autoradiography:brain Introduction Studies using biochemical receptor assay methods and in situ hybridization techniques for quantitation of receptor mRNAs have clearly demonstrated that control of steroid receptor biosynthesis may play an important role in the regulation of hormone sensitivity. ~-6 Problems associated with measurement of the receptor proteins have, however, limited attempts to correlate regional analysis of steroid receptor concentrations with behavioral and neuroendocrine responsiveness to steroid hormone exposure. Conventional methods for receptor assay lack the necessary sensitivity and anatomical resolution to allow determination of recep- tor levels in precisely defined neuronal cell groups (re- viewed in references 7 and 8). While imnmnocytochemical and in vivo autoradiographic methods do provide sufficient resolution, the former lacks the necessary quantitative pre- cision while the latter requires relatively large quantities of isotope to saturate the receptors in vivo and cannot be used Address reprint requests to Theodore J. Brown, Ph.D., Division of Re- productive Science, CCRW 3-825, The Toronto Hospital Research Insti- tute, 585 UniversityAvenue, Toronto, Ontario M5G 2C4, Canada. Received February 27, 1995; accepted June 2, 1995 Steroids 60:726-737, 1995 © 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 under physiological conditions because of the requirement to eliminate competition from endogenous steroids by go- nadectomy. Recently, we introduced an in vitro labeling procedure Ii~r the estrogen receptor which circumvented many of the problems historically associated with receptor binding as- says. ~ Using a synthetic estrogen receptor ligand (1 113- methoxy-16e~-iodoestradiol) labeled with J25I to very high specific activity (-2,200 Ci/mmol, [125I]MIE2) and wash- ing to remove free and nonspecifically bound steroid, re- ceptor-bound [12511MIE 2 was visualized autoradiographi- cally alter a brief exposure (l 8-24 h) against photosensitive film We speculated at the time of this study s that similar methodology might be applicable to other classes of steroid hormone receptor. However, widespread application of this and related in vitro autoradiographic techniques in neural tissues has not yet been possible. While ~25I-labeled ligands offer enormous advantages in terms of the speed with which data can be obtained---exposure times typically being only about 1/100th of the duration required for tritiated ligands-- in vitro autoradiography with iodinated steroids is not yet feasible in the majority of laboratories. Although iodinated steroids have been developed for use in binding assays with estrogen,9' ~0 androgen, ~ ~'t 2 and progestin 13 receptors, the 0039-128X/95/$10.00 SSDI 0039-128X(95)00107-2