Biochemical and immunological characterization of oestrogen receptor in the cytosolic fraction of gluteal, omental and perirenal adipose tissues from sheep

Abstract

ABSTRACT Determination of the presence and characterization of oestrogen receptors (ERs) in subcutaneous and internal fat depots were performed and compared with ERs in the uterus using ligand binding and immunological techniques. Successful and consistent measurement of ERs in ovine adipose tissue could only be accomplished in animals depleted of endogenous sex steroids by combined ovariectomy and adrenalectomy. Scatchard, sucrose gradient and Western blot analyses all confirmed the presence of ERs in the cytosolic fractions of various adipose and uterine tissues from ovariectomized-adrenalectomized ewes. The approximate Kd values of 0·1–0·4 nmol/l for oestradiol binding in cytosolic fractions of gluteal, omental and perirenal adipose tissues were similar to the expected high affinity binding of Kd 0·35 nmol/l observed in uterine tissue. The binding was specific for oestrogens, as unlabelled diethylstilboestrol and oestradiol effectively competed with labelled hormone for receptor sites and progesterone, R5020, testosterone and dexamethasone all failed to compete. Mean (± s.e.m.) concentrations of ERs, expressed as fmol specific binding sites per mg protein, were much lower (P<0·05) in adipose tissues than in uterine tissue (975 ± 33). However, 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 moieties of 8S on 5–20% sucrose gradients. Western blot analysis of ERs from uterine and adipose tissues in the presence of protease inhibitors demonstrated an immunostaining band with a molecular mass of 67 kDa. In the absence of protease inhibitors, the adipose ER was degraded into two smaller bands with molecular masses of 45 kDa and 36 kDa. In conclusion, these results support the presence of specific ERs in adipose tissues of sheep with characteristics similar to those of the ERs of the uterus. Furthermore, their presence is supportive of sex steroid-dependent effects on adipose accretion and metabolism. Journal of Endocrinology (1993) 139, 107–115