BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERIZATION OF ESTROGEN-RECEPTOR IN THE CYTOSOLIC FRACTION OF GLUTEAL, OMENTAL AND PERIRENAL ADIPOSE TISSUES FROM SHEEP
Gh. Watson et al., BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERIZATION OF ESTROGEN-RECEPTOR IN THE CYTOSOLIC FRACTION OF GLUTEAL, OMENTAL AND PERIRENAL ADIPOSE TISSUES FROM SHEEP, Journal of Endocrinology, 139(1), 1993, pp. 107-115
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.