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The Unusual Estrogen-Binding Protein (UEBP) of male rat liver: Structural determinants of ligands

The unusual estrogen-binding protein (UEBP) found in a male rat liver is a sex dependent protein which differs from other known receptor and transport proteins by the high lability of its complexes with estradiol (E2) and also the unique specificity of affinity for hormones. In this work values of relative binding affinity (RBA) of the UEBP for 57 steroids and their analogs were determined. The affinity of steroids was characterised by the amount of the unlabeled compound needed for 50% inhibition of [3H]-E2 binding with the UEBP. A number of derivatives of estrane and androstane possess an ability to interact with this protein, in contrast to the derivatives of pregnane, stilbene and triphenylethane. Characterized by RBA values, natural steroids are found to have the following order: estriol larger than or equal to E2 greater than 16 alpha-hydroxyestrone = 2 alpha-hydroxytestosterone greater than 16-epiestriol greater than or equal to estetrol greater than or equal to 17-epiestriol greater than or equal to 2-methoxyestradiol greater than or equal to 5 alpha-androstane-3 alpha,17 beta-diol greater than or equal to estrone greater than testosterone greater than or equal to 2 beta-hydroxytestosterone greater than 5 alpha-dihydrotestosterone. Affinity of estrogens and androgens for the UEBP diminishes abruptly after removal of 3- and 17-hydroxy groups, masking of these by ether bonds or changing of 17 beta-hydroxyl to 17 alpha. All the investigated 17 oxo-C19-steroids, 5 beta-derivatives of testosterone, its 6 beta- and 16 alpha-hydroxy metabolites as well as 5 alpha-androstane-3 beta,17 beta-diol and 19-nortestosterone exhibit no essential affinity for the protein. On the basis of the results obtained it is suggested that the binding sites for estrogens and androgens in the UEBP molecule overlap but do not completely coincide.

The unusual estrogen-binding protein (UEBP) of rat liver: The role of sex steroids and hypophysis in its regulation

The number of estradiol (E2) binding sites of rat liver unusual estrogen-binding protein (NUEBP) was measured, using a novel modification of the quantitative method of specific UEBP determination. In liver cytosol of mature male and female rats, NUEBP amounted to 6.83 +/- 0.49 and less than 0.05 pmol/mg protein, respectively. Neonatal administration of testosterone-propionate (TP) and TP injections at later periods of ontogenesis increased NUEBP in female rat liver in a similar fashion. The elevated NUEBP was found in the liver of mature ovariectomized females 30 days after cessation of TP injections. Hypophysectomy (but not adrenalectomy or thyroidectomy) prevented TP induction of elevated NUEBP in pubertal females. E2 injections reversibly decreased NUEBP in the liver of all animals under study except of hypophysectomized males. A stimulating regulatory effect of TP on NUEBP in male rat liver was observed only in the case of endogenous androgen deficiency and low NUEBP. TP prevented the E2-dependent decrease of NUEBP upon their simultaneous injections and increased the E2-reduced NUEBP when injected after E2. Hypophysectomy led to a decrease of NUEBP in pubertal males but only slightly affected that in castrated animals. After TP injections to hypophysectomized males, NUEBP returned to a level next to the initial one. It was concluded that estrogen-androgen regulation of the UEBP level led to the maintenance of sex differences in the UEBP content.

In vivo and in vitro estimations of the direct effect of estrogen on rat hepatocytes tested by the changes in the unusual estrogen-binding protein content

The direct effect of estradiol (E2) on the hepatocytes of mature male rats has been examined by measuring the changes in the unusual estrogen-binding protein (UEBP) content and parallel measuring the level of liver estrogen receptors (ER). The content of UEBP (NUEBP) and ER (NER) in the liver were determined using the quantitative methods for differential specific determination of the E2-binding sites of these proteins. It has been shown that the administration of E2 in vivo induced a considerable decrease in hepatic NUEBP not only in intact males, but also in hypophysectomized males during the initial period after the operation (when the content of hepatic ER was still high) and produced no effect in hypophysectomized males during the later period (when liver ER were depleted). Repeated administration of human growth hormone (hGH) (twice a day) resulted in a considerable increase in NER in hypophysectomized males and restored the sensitivity to the subsequent inhibitory effect of E2 on UEBP. We also used rat hepatocytes after a 4-day primary culturing. These cells had a stable morpho-functional status, high ER level, and sex-differentiated UEBP content. Culturing of mature male rat hepatocytes in the medium containing E2 at concentrations close to physiological levels (10(-10)-10(-7) M) decreased NUEBP in a dose-dependent manner. Hexestrol (10(-7) M) but not cholesterol (10(-5) M) also exhibited a direct effect on NUEBP in cultured rat hepatocytes. The effect of E2 was reversible: statistically significant increase in NUEBP was observed 3 days after 10(-9) M E2 had been removed from the culturing medium. It was concluded that hepatocytes may be a primary target for E2 under physiological conditions and that GH may modulate the direct effect of E2 at the hepatic level by modifying the content of liver ER.

Evidence for direct action of testosterone on rat liver cells: In vivo and in vitro induction of unusual estrogen-binding protein

We demonstrate that on the rat liver, testosterone (T) induced differentiated functions and enhanced unusual estrogen-binding protein (UEBP) content through mechanisms dependent on cell activation by androgens, the presence of growth hormone (GH) and the hormonal status of the animal. To determine whether liver cells are a target for androgens, we measured T effects on UEBP in gonadectomized adult male and female rats in vivo and in vitro. In ovariectomized rats, T increased 8- to 9-fold UEBP levels that remained constant during 10 days. Also in vitro, using hepatocytes from ovariectomized rats, T alone increased UEBP levels 3-fold in a dose-response pattern. Combining a fixed low dose of GH with different concentrations of T increased UEBP 2-fold above T alone. Whereas GH alone had no effects in ovariectomized rats, hepatocytes were responsive to GH, in a dose dependent pattern that was abolished when T was used together with GH. On the other hand, T alone had no effect in hypophysectomized-ovariectomized animals. The latter group was rendered T responsive after the simultaneous injection of GH with T that increased UEBP content 6.6-fold in vivo. Castrated males revealed a marked responsiveness to T and GH in vivo and in vitro, when added separately or in combination. The results obtained suggest a complex regulatory system and we conclude that T acts directly on rat liver as: (1) an inducer of sex differentiation; and (2) a regulator of UEBP production in males. In addition, liver regeneration studies in castrated-hypophysectomized males revealed the UEBP phenotype in daughter cells in the absence of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Inheritance of androgen program of male-specific expression of unusual estrogen-binding protein by daughter hepatocytes at rat liver regeneration.

A possibility of inheritance of androgen and basic genetic programs at the level of unusual estrogen-binding protein (UEBP) by daughter hepatocytes was investigated. Liver regeneration after partial (2/3) hepatectomy or after selective poisoning of hepatocytes of the central zone of hepatic lobules with CCl4 in adult rats were used as models of total and zonal proliferation of hepatocytes, respectively. UEBP content and the pattern of its tissue expression in the course of liver regeneration were monitored by radioligand and immunocytochemical technique. In animals of all groups possessing the androgen program of UEBP expression (intact, castrated and/or hypophysectomized males, and ovariectomized females treated with androgen) UEBP content was shown to be similarly high before initiation and after completion of liver regeneration. Unlike in males, in androgenized females a transient 4-fold increase of UEBP concentration on day 4 after partial hepatectomy was observed. In animals with a basic genetic program at the level of this protein (ovariectomized females, neonatally castrated males) only trace amounts of UEBP were observed in intact as well as in regenerated liver. The data were confirmed by immunocytochemical technique. A gradient mode of distribution of UEBP-contained cells within hepatic lobules with the highest specific staining around central veins was found by immunocytochemical technique in males. Specific staining of centrolobular and periportal hepatocytes was 7- to 10-fold in intact, and 4- to 6-fold in castrated and/or hypophysectomized males. In intact females specific staining was distributed uniformly at extremely low levels similar to that in periportal hepatocytes of males. Androgen administration to ovariectomized females stimulated a significant and stable increase of UEBP content in two layers of hepatocytes surrounding the central vein. Profiles of specific staining of hepatocytes within the hepatic lobules similar to that in control animals were observed after the completion of liver regeneration of different groups of rats. The results obtained suggest all the hepatocytes to be targets for androgen programming, natural in males or experimental in females, while the extent of expression of this program depends on the position of a hepatocyte within the liver lobules and the sex of the animal.

Indirect mechanism of the positive action of estrogens on corticosteroid-binding globulin level in rats

: The mechanism of estrogen action on CBG level in blood serum was studied in the experiments on white rats. It was shown that estradiol injection at the dose 100 micrograms for 3 weeks induced the increasing of CBG serum level in intact males, but it was not changed in the gonadectomized males and females. The presence of the positive effect of exogenic estrogens only in the intact males was connected with the estrogen depression of testis function. It suggests the absence of direct positive estrogens action on the level of CBG in the rats. The estrogen effect is not mediated by adrenal corticosteroids, and has independent negative action on CBG level in the rat. Thus, estrogen does not participate in the endocrine regulation of CBG content in the mature rats.

EFFECT OF SOME ENDOCRINE FACTORS ON THE CONTENT OF A SPECIFIC ESTROGEN-BINDING PROTEIN IN RAT-LIVER

As a result of the development of a method of differential quantitative determination of SEBP among other liver proteins specifically binding estradiol, it was possible in the present investigation to approach the study of the role of androgens in the induction and regulation of the content of this protein in liver cells, a matter of great importance in connection with determination of the biological role of SEBP,

PROGRAMMING ACTION OF ANDROGENS ON FORMATION OF SEXUAL DIMORPHISM OF THE CORTICOSTEROID-BINDING GLOBULIN LEVEL IN RATS

Corticosteroid-binding globulin (CBG) is a plasma protein produced by hepatocytes that specifically binds coorticosteroids and progestins circulating in the blood stream. Although the physiological role of this protein has not yet been completely explained, CBG evidently plays an important part in the regulation of activity and metabolism of the steroids which it binds [5]. The CBG level in rats exhibits sexual differentiation [1, 4]: its level in females is about 2.5 times higher than in males. This fact enables the CBG level to be used as a model object for the study of principles governing the formation of sexual dimorphism of liver functions. The study of this problem would shed light on the biological importance of the existence of a definite sex-specific level of integration of adaptive and reproductive processes, which is performed by CBG at the blood stream level, by binding corticosteroids and progestins circulating in the blood. Previously [1] the writers showed that a lower concentration of CBG in male rats is due, on the one hand, to its irreversible negative programming by androgens in the prepubertal period, and on the other hand, to the presence of a negative regulatory effect of endogenous androgens in sexually mature males [1]. However, it is not yet clear in what period sensitivity of the CBG level to the determining effect of androgens first appears, and when it disappears. Likewise the problem of the boundaries of the period of formation of the sex-dependent CBG level in physiological rather than model conditions likewise remains unexplained; the possibility that the negative programming effect of androgens on the CBG content may be mediated indirectly through estrogens likewise has not been investigated. The aim of the present investigation was to study these problems.

Role of certain hormones with general metabolic action in the expression of sexual differentiation of the rat liver with respect to a special estrogen-binding protein.

Conclusions1.Administration of insulin does not significantly affect the level of SEBP in the livers of male rats.2.Adrenalectomy and administration of dexamethasone do not cause any pronounced changes in the sexual differentiation of the SEBP content in the liver.3.Administration of physiological doses of T3 produces a dose-dependent and reversible decrease in the level of SEBP in the livers of sexually mature male rats, and also decreases the degree of induction of this protein by androgens in the livers of females.4.The action of T3 does not significantly depend on the sex hormones and pituitary factors.5.Administration of STH raises the level of SEBP in the liver only in hypophysectomized males but not in intact or castrated males.

Effect of androgens and growth hormone on expression of the unusual estrogen-binding protein level in hepatocytes

Sexual differntiation of various aspects of liver metabolism is now well known [3]. It has been shown that sexual dependence of liver functions is formed on the basis of the fundamental genetic program, under the irreversible determinant action of androgens (AD) in the early period of postnatal ontogeny of males. An important role in the realization of the effects of sex hormones on the liver is played by the pituitary gland; the action of sex and pituitary hormones of the growth hormone (GH) family is closely interconnected [5, 7].