AJP - Endocrinology and Metabolism, Vol 244, Issue 5 494-E498, Copyright © 1983 by American Physiological Society
Monoclonal antibody recognition of estrogen receptor in rabbit corpus luteum
J. A. Holt, M. A. Lorincz and W. J. King
We demonstrated previously by administering [3H]estradiol and using
autoradiography that, in vivo, the exogenous estradiol that will sustain
progesterone production is sequestered to nuclei of the luteal cells in
corpora lutea of pseudopregnant rabbits. Our objective in the present
experiments was to use an immunological method to demonstrate that estrogen
receptor of the rabbit corpus luteum is associated with this uptake. For
this purpose, we prepared nuclear extracts from corpora lutea removed from
anesthetized pseudopregnant rabbits 10 min after arterial infusion of
125I-estradiol. These extracts were then analyzed by sucrose density
gradient centrifugation in the presence and absence of monoclonal antibody
against human breast cancer estrogen receptor. We found that the
125I-estradiol-binding component extracted with 0.5 M KCl from the nuclear
pellet sediments in the 3-4S region in high-salt gradients; in the presence
of monoclonal antibody (D-547 Sp2 gamma), the entire estradiol-binding
component is shifted to the 8-9S region, indicating the formation of
immunocomplex with the 125I-estradiol-receptor moiety. The principal
estradiol-binding component that can be observed in the 3-4S region of
high-salt gradients of cytosol of rabbit corpora lutea reacts with this
antibody regardless of whether 125I- or [3H]estradiol is given in vivo or
in vitro. In both cytosol and nuclear extracts of luteal tissue, unlabeled
diethylstibestrol totally inhibits the binding of radiolabeled estrogen to
this antibody-shifted 125I- or [3H]estradiol-binding moiety; neither
progesterone nor testosterone inhibit binding of radiolabeled estrogen to
the antibody-shifted moiety. From these findings we conclude that
sequestering of estradiol by the rabbit corpus luteum in vivo is
accomplished by the steroid-specific estrogen receptor that shares
characteristics of the estrogen receptor found in human breast cancers,
including a) antigenic features and b) the capacity to translocate
estrogens from the cytosol to the nuclear fraction.
