BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERIZATION OF ESTROGEN-RECEPTOR IN THE CYTOSOLIC FRACTION OF GLUTEAL, OMENTAL AND PERIRENAL ADIPOSE TISSUES FROM SHEEP

Determination of the presence and characterization of oestrogen recept ors (ERs) in subcutaneous and internal fat depots were performed and c ompared with ERs in the uterus using ligand binding and immunological techniques. Successful and consistent measurement of ERs in ovine adip ose tissue could only be accomplished in animals depleted of endogenou s sex steroids by combined ovariectomy and adrenalectomy. Scatchard, s ucrose gradient and Western blot analyses all confirmed the presence o f ERs in the cytosolic fractions of various adipose and uterine tissue s from ovariectomized-adrenalectomized ewes. The approximate K-d value s of 0.1-0.4 nmol/l for oestradiol binding in cytosolic fractions of g luteal, omental and perirenal adipose tissues were similar to the expe cted high affinity binding of K-d 0.35 nmol/l observed in uterine tiss ue. The binding was specific for oestrogens, as unlabelled diethylstil boestrol and oestradiol effectively competed with labelled hormone for receptor sites and progesterone, R5020, testosterone and dexamethason e all failed to compete. Mean (+/- S.E.M.) concentrations of ERs, expr essed as fmol specific binding sites per mg protein, were much lower ( P<0.05) in adipose tissues than in uterine tissue (975 +/- 33). Howeve r, the content of ERs was greater (P<0.05) in subcutaneous gluteal fat (11.5 +/- 0.8) than in the internal omental or perirenal fat (5 +/- 0 .6) depots. ERs from adipose and uterine tissues both migrated as moie ties of 8S on 5-20% sucrose gradients. Western blot analysis of ERs fr om uterine and adipose tissues in the presence of protease inhibitors demonstrated an immunostaining band with a molecular mass of 67 kDa. I n the absence of protease inhibitors, the adipose ER was degraded into two smaller bands with molecular masses of 45 kDa and 36 kDa. In conc lusion, these results support the presence of specific ERs in adipose tissues of sheep with characteristics similar to those of the ERs of t he uterus. Furthermore, their presence is supportive of sex steroid-de pendent effects on adipose accretion and metabolism.