A polyclonal antibody to the rat oestrogen receptor expressed in Escherichia coli: characterization and application to immunohistochemistry

Abstract

A rat oestrogen receptor-beta-galactosidase fusion protein was expressed using a pEX2/rat oestrogen receptor cDNA construct. Scatchard analysis of [3H]oestradiol-17 beta binding to the cell lysate revealed that the fusion protein had functional binding sites specific for oestradiol with a dissociation constant of 1.49 nmol/l. The relative molecular weight (M(r)) of the fusion protein was determined as 180,000 by immunoblot analysis of the cell lysate employing a monoclonal antibody to the human oestrogen receptor. The protein was isolated by means of SDS-PAGE and subsequent electroblotting. By immunization with the purified materials on nitrocellulose membrane, a polyclonal antibody to the rat oestrogen receptor was raised in a rabbit. Binding of [3H]oestradiol to the oestrogen receptor from the rat uterus was inhibited by the antibody in a dose-dependent manner. The antibody was also able to recognize the oestrogen receptor occupied by [3H]oestradiol. Thus, the antibody could react with both forms of the receptor molecule, either occupied or unoccupied by the hormone. In immunoblot analysis of the cytosol fraction of the rat uterus, a single band of M(r) 67,000, the size of the oestrogen receptor, was detected by the antibody. Moreover, when the antibody was applied to immunohistochemical examination of paraffin-embedded pituitary and brain sections of the rat, immunostaining was observed in cells of the anterior pituitary and in neurones in specific regions of the brain. The immunoreactivity was restricted exclusively to cell nuclei in both tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical Localization of Estrogen Receptor in Various Anterior Pituitary Hormone Cells of Adult Male and Female Rats.

“…Sen et al (23) have reported that, in the female rat anterior pituitary gland at proestrus, both the concentration of unoccupied and occupied ER and the total estradiol binding in the cytosol decreased markedly with a concomitant marked increase in the concentration of ER in the cell nucleus, to which it was translocated from the cytosol as an ER-estradiol complex. In this experiment, we detected all of the ER in a section, since the polyclonal antibody against rat ER we used is able to react with both forms of the receptor molecule, whether unoccupied or occupied by estradiol (21). The present data supported the study by Sen et al (23) and revealed that the high ER level was due to an increase in that of gonadotrophs and lactotrophs.…”

“…However, this rat monoclonal antibody may not be suitable for detecting ER in rat organs, as it produces widespread background staining (3). More recently, Okamura et al (21) produced a highly specific polyclonal antiserum against ER expressed in Escherichia coli by utilizing the cDNA which encodes the B-through F-domains of the full length of the rat ER protein (15), and demonstrated the ER in the rat brain (5, 21, 32) and pituitary (21). However, cellular identification of ER-bearing pituitary parenchymal cells has not yet been done with the antiserum.…”

Temporal expression of estrogen receptor α in the hypothalamus and medulla oblongata during fasting: a role of noradrenergic neurons

“…Using immunoblot analysis, the characterization and specificity of the rabbit antiserum against rat ERa used in the present study have been previously described (Okamura et al 1992). Specifically, immunocytochemistry recognizes ERa in the pituitary and neurons in specific brain regions (Okamura et al 1992(Okamura et al , 1994.…”

“…Rats were OVX under ether anesthesia to eliminate endogenous estrogens. The antibody used in this study has been reported to recognize both occupied and unoccupied ERa (Okamura et al 1992). However, one study using the same antibody shows a downregulation of ERa expression in the presence of estrogen (Okamura et al 1994).…”

“…The AS 409 anti-rat ERa used in this study was kindly supplied by Dr Hayashi (Yokohama City University, Yokohama, Japan) and its specificity has been reported elsewhere (Okamura et al 1992).…”