An unusual sex steroid-binding protein in mature male rat liver cytosol.

Mature male rat liver cytosol contains a moderate affinity and capacity estrogen-binding protein in at least a 200-fold higher level than mature female or immature male rat liver cytosol. Binding of estradiol to this protein is very rapid, is stabilized by EDTA, and is inhibited by divalent cations. This is the major binding protein for [3H]estradiol ([3H]E2) in mature male rat liver cytosol, and it has properties clearly distinguishing it from putative liver or uterine estrogen receptors. In addition to binding [3H]E2, this protein seems to rapidly bind a [3H]5alpha-dihydrotestosterone ([3H]DHT) metabolite at the same binding site. The binding of this androgen metabolite is stabilized by EDTA and is inhibited by divalent cations. The binding properties of the [3H]DHT metabolite suggest that these binding sites are not classical androgen receptors. Cytosol binding levels of both the [3H]E2 and the [3H]DHT metabolites change in a similar direction in resonse to endocrine manipulation. The putative liver estrogen receptor level, determined after partial purification (in a redissolved 30% ammonium sulfate-precipitated fraction), seems to change in an opposite direction in response to these same endocrine manipulations.     

Role of protein biosynthetic processes in realizing the action of sex steroids on the level of specific estrogen-binding protein in the rat liver

The authors have assessed the life-span of special estrogen-binding rat liver protein (SEBP) through inhibition of it by long-term administration of cyclohexymid (C1) and have examined how C1 affects the efficiency with which sex steroids influence the level of SEBP. The level of SEBP synthesis was assessed by the changes in the number of SEBP estradiol (E2)-binding sites. The long-term C1 administration in the dose of 100 micrograms suppressed SEBP synthesis by an average of 82.4% with the half-life of SEBP being about 18 hr. Testosterone propionate (TP) and 5 alpha-dihydrotestosterone, but not E2, were found able to mediate enhanced SEBP levels in the liver of ovariectomized female rats. A single 200 micrograms dose of C1 administered 30 min prior to hormone injection caused significant decreases in the efficiency of SEBP-mediated androgen activities in the liver of ovariectomized female rats, in TP-induced stimulation of the SEBP level in the liver of castrated male rats and in E2 inhibitory effect on the SEBP level in the liver of mature males. It is concluded that de novo protein synthesis is necessary for the early manifestation of all the above mentioned effects of sex hormones on the SEBP level. Based on the data regarding the duration of SEBP life and the rate with which it affects the activity of sex steroids it is supposed that in the early stages of hormonally induced manifestations there are changes in biosynthesis of regulating protein rather than in SEBP itself.     

Prolactin receptor in rat liver: sex difference in estrogenic stimulation and imprinting of the responsiveness to estrogen by neonatal androgen in male rats

Rat hepatic prolactin receptor is regulated by sex steroids. A high level of the receptor was found in female rats but the level was nearly undetectable in males. Gonadectomy reduced the receptor level in females but increased the level in males. Administration of estradiol benzoate (0.05 μmoles/kg on alternate days subcutaneously for 9 days) to adult gonadectomized females increased the receptor level by 473% whereas the same treatment in adult gonadectomized males produced a more modest 276% increase. This sexually dimorphic pattern in the responsiveness to estrogen stimulation in adult rats appeared to be determined neonatally. Neonatal gonadectomy of male rats changed the hepatic response system to a more female pattern in adulthood. Replacement of testosterone (1.45 μmoles at days 1 and 3 after birth) to these neonatally gonadectomized male rats restored the male pattern. Diethylstilbestrol replacement (1.45μmoles at days 1 and 3 after birth) to the neonatally gonadectomized male rats showed the same effect as neonatally administered testosterone. Scatchard analysis revealed that the observed changes in binding are related to changes in binding capacity but not affinity. Desaturation by 4 M MgCl₂ indicated that the amount of endogenously bound hormone was negligible in our membrane preparations.     

Determination of the content of specific estrogen-binding protein in the rat liver cytosol

A method of quantitative determination of the areas of binding of a peculiar estrogen-binding protein (PEBP) in the cytosol of the rat liver by means of ion-exchange sorption on DEAE-cellulose is suggested. For differential determination of the PEBP contribution to the total estradiol (E2) binding with cytosol proteins a unique capacity of PEBP to form complexes with 3H-E2, which were rapidly decomposed on addition of an excess of unlabeled E2, was used. Theoretical prerequisites and experimental substantiation of the suggested method of the PEBP testing are considered. The content of the binding PEBP areas in the cytosol of the liver of male rats, measured by the mentioned method, constituted (5.8 +/- 0.7).10(-12) mol per 1 mg of protein (M +/- m, according to the data of 18 determinations). No analogous areas of E2 binding were revealed in the cytosol of the liver in female rats. It was also demonstrated that the cytosol of the liver of male rats contained specific areas of testosterone (T) binding, forming highly labile complexes with 3H-T, rapidly decomposing in the presence of an excess of T and E2.     

Effect of liver regeneration on hepatic cytochrome P450 isozymes and serum sex steroids in the male rat

Partial hepatectomy in male adult rats results in raised serum estrogen levels and demasculinization of certain aspects of hepatic metabolism. Some constitutive forms of hepatic cytochrome P450 are sex-dependent and we have previously demonstrated demasculinization of cytochrome P450 isozyme distribution in a rat model of cirrhosis. As liver regeneration is an integral component of cirrhosis, the present study was performed to ascertain the effects of regeneration on hepatic cytochrome P450 isozyme composition and serum sex steroid concentrations. Adult male rats were subjected to 65% partial hepatectomy or sham-operation. The position-specific hydroxylation of androstenedione was used as a probe for isozyme activity. Serum sex steroids, hepatic enzymes, and hepatic deoxyribonucleic acid synthesis were measured in groups of animals at 0, 6, 24, 48, and 72 h. By 72 h total microsomal cytochrome P450 in partially hepatectomized animals had fallen to 66% of that in nonoperated animals. In both partially hepatectomized and sham-operated animals, androstenedione 7 alpha- and 16 beta-hydroxylase activity returned to preoperative levels by 48 h. However, the male-specific androstenedione 16 alpha- and 6 beta-hydroxylase activities and aromatase activity remained suppressed in partially hepatectomized liver. Serum estradiol increased eightfold in partially hepatectomized rats and peaked at 6 h followed by a gradual fall to control values. No change in serum estradiol was observed in sham-operated animals. We conclude that demasculinization of hepatic oxidative metabolism occurs in regenerating rat liver. The early rise in serum estradiol is consistent with a role for this hormone in the changes in cytochrome P450 observed, and possibly the process of liver regeneration.     

Augmented transport and metabolism of sex steroids in lymphoid neoplasia in the rat

Sex steroid hormones have been shown to influence a number of biological properties of lymphoid neoplastic tissue. Since receptor occupancy is a function of the pool size of cellular exchangeable hormone, it is important to understand the mechanisms regulating hormone transport from the microcirculation and hormone metabolism, since these two pathways are the dominant factors controlling cellular exchangeable hormone. In the present studies, steroid hormone transport and metabolism were investigated in control and neoplastic lymph nodes after transplanting control rats with the WR-6 leukemic line. Steroid hormone transport and metabolism were studied after pulse labeling the nodal tissue in vivo with arterial bolus injections of [3H]testosterone. Residual vascular radioactivity was monitored by simultaneously injecting 113mindium chelated to bovine transferrin. Both testosterone and estradiol were partially available for transport through the capillary barriers of control and neoplastic lymph nodes from the circulating albumin-bound pool. Estradiol was readily available for transport from the circulating sex hormone-binding globulin-bound pool in both control and neoplastic lymph nodes. Testosterone was not available for transport from the sex hormone-binding globulin-bound pool in control lymph nodes, but was readily available for transport in metastatic lymph nodes. Thaw-mount autoradiography and physiological measurements showed that plasma proteins such as albumin or transferrin were confined to the microcirculation compartment. Therefore, the transport of protein-bound hormones into lymph node represents a mechanism of enhanced steroid hormone dissociation from the binding protein without the plasma protein per se significantly exiting the microcirculation compartment. Metabolic studies showed no measurable metabolism of [3H]testosterone in the control lymph nodes by 60 sec after arterial injection. However, testosterone was extensively metabolized in metastatic lymph nodes; the major metabolites formed from testosterone comigrated on a two-dimensional TLC system with epiandrosterone/androsterone and dihydrotestosterone. In conclusion, these studies indicate that both steroid hormone transport and metabolism are augmented in the lymphoid neoplastic state, and both processes may alter the concentration of cellular exchangeable hormone and, thus, steroid hormone receptor occupancy in lymphoid neoplasms.     

The dynamics of testosterone and dihydrotestosterone metabolism in the adult male rhesus monkey.

MCRs of testosterone (T) and 5 alpha-dihydrotestosterone (DHT) were determined in the morning and evening in four chronically chaired adult rhesus monkeys (Macaca mulatta) by means of steady state infusion of [4-14C]testosterone and [1,2-3H]dihydrotestosterone. Morning and evening MCRs were comparable for both T and DHT, averaging 169 and 212 ml/min for T and 93 and 108 ml/min for DHT, respectively, indicating that temporal differences in blood steroid concentrations are the result of changes in glandular secretion and/or synthesis and not differences in MCRs. Blood transfer factors for the conversion of T to DHT were also similar at these two times (0.024 and 0.022). MCRs were determined in one monkey by the single injection of [14C]T and [3H]DHT. From the rates at which the hormones disappeared from the circulation, MCRT and MCRDHT were calculated to be 172 ml/min for T and 92.2 ml/min for DHT. The inner volumes of hormone distribution were 4.04 liters for T and 3.67 liters for DHT.     

Role of various hormones of total metabolic action in the expression of sex differentiation of the rat liver by a specific estrogen-binding protein

The effect of some hormones of total metabolic action on a degree of sex differentiation of the liver by a specific estrogen-binding protein (SEBP) was investigated. Insulin administration did not change the SEBP level of the male rat liver. Adrenalectomy and dexamethasone administration caused no significant changes of sex differentiation of the SEBP level in the male and female rat liver. Neither did thyroidectomy cause significant changes in the SEBP level. Dose-related T3 administration decreased reversibly the SEBP level in the liver of adult male rats. The effect of T3 on the SEBP level was preserved after castration or hypophysectomy of males. T3 administration decreased a degree of determination of a raised level of SEBP in the female rat liver by androgens. STH injections were effective mainly in hypophysectomized male rats resulting, on the one hand, in an increase in the level of SEBP and creating conditions, on the other hand, for a decrease in its level caused by estradiol. It has been concluded that thyroid hormones and STH are able to be modulators of adaptive changes of sex differences of the liver by SEBP.     

Role of estrogens in the induction and regulation of a specific estrogen-binding protein in the rat liver

A study was made of the role of estrogens (E) in the induction and regulation of the level of specific estrogen binding protein (SEBP) in the rat liver using a differentiated quantitative method of its determination. It was shown that E could not replace androgens (A) in the primary determination of the SEPB level, neither did they prevent its A-dependent induction. Multiple administration of 0.4 microgram of estradiol (E2) caused a significant decrease in the SEPB level in intact and castrated male rats as well as in ovariectomized females with the A-induced SEPB level. Multiple administration of even 10 micrograms of E2 did not influence the SEPB level in hypophysectomized males. The rate of development of the E2 effect on the SEPB level of the mature male liver increased with the growth of the dose and duration of hormone administration. The inhibitory effect of E2 was also revealed in a single administration of 10 micrograms of the hormone. In that case the effect was observed after a 3-day lag period, and a maximum decrease in the SEPB level occurred in 6 days. The regulatory negative effect of E2 was reversible. Complete regeneration of the initial SEPB content took place 10-12 days after the development of the maximum effect of E2.     

Organizational role for testosterone and estrogen on adult hypothalamic-pituitary-adrenal axis activity in the male rat

Organizational effects of testosterone during a critical period of neonatal life have major irreversible effects on adult sexual behavior. We have investigated whether perinatal androgen changes also affect another major sexually differentiated system, the hypothalamo-pituitary-adrenal axis. This was assessed in male rats who had been exposed to perinatal flutamide or 1,4,6-androstatriene-3,17-dione (ATD). Once the animals reached adulthood, an automated sampling system was used to collect blood from freely moving animals at 10-min intervals over 24 h, followed by a noise stress and then the administration of lipopolysaccharide (LPS). Perinatal flutamide- and ATD-treated rats not only had higher mean corticosterone levels and increased frequency and amplitude of corticosterone pulses over the 24 h compared with vehicle-injected controls, but they also showed markedly increased corticosterone responses to both noise and LPS. All parameters of increased hypothalamo-pituitary-adrenal activity resembled the normal physiological state of the intact adult female rather than that of the intact adult male rat. Furthermore, 3 h after LPS administration, both flutamide- and ATD-treated animals had markedly higher levels of corticotropin-releasing factor mRNA in the parvocellular paraventricular nucleus (PVN) and proopiomelanocortin mRNA in the adenohypophysis. Flutamide-treated rats also had a greater level of PVN arginine vasopressin mRNA. PVN glucocorticoid receptor mRNA levels were significantly lower in both the flutamide- and the ATD-treated male rats. These data highlight the importance of perinatal exposure to both testosterone and estrogen(s) on the development of a masculinized circadian corticosterone profile and stress-induced hypothalamo-pituitary-adrenal axis activity in the adult male rat.     

Comparison of the metabolism of testosterone undecanoate and testosterone in the gastrointestinal wall of the rat in vitro and in vivo

The metabolism of testosterone undecanoate (TU) and testosterone (T) is studied in the gastrointestinal wall of the rat in vitro. A comparison is made with the in vivo metabolism of these compounds in the rat. The major metabolite first appearing during incubation of TU with the small intestine is T. Incubation of TU or T with the small intestine reveals a great similarity between the metabolite patterns obtained. This is also the case with the patterns derived from portal vein plasma upon oral administration of TU and T. Incubation of different parts of the gastrointestinal tract with TU or T shows that the greatest metabolic activity is located in the wall of the small intestine. Unlike T, TU is metabolized only to a small extent in the wall of the stomach and the large intestine.     

Inhibition of steroid 5 alpha-reductase in vivo: effect on suppression of luteinizing hormone and stimulation of accessory sex organs by testosterone in the orchidectomized rat.

An inhibitor of 5 alpha-reductase, the 17 beta-carboxylic acid derivative of testosterone (testosterone-17 beta CA), has been used to evaluate the importance of the 5 alpha-reduction of testosterone in its action on the suppression of LH secretion in male rats. The potential of testosterone-17 beta CA to inhibit the formation of 5 alpha-dihydrotestosterone (DHT) was first demonstrated in vitro. When homogenates of hypothalami or anterior pituitary glands were incubated with [3H]Testosterone in the presence of a 50-fold excess of testosterone-17 beta CA, the formation of labelled DHT was inhibited by more than 80%. Adult male rats that had been castrated for 1-2 months were fitted with chronic intravenous catheters and implanted with silicone elastomer sheets: one group received one sheet, 0.5-2.0 cm2 in size containing 1.6% testosterone, a second group received one 50 cm2 sheet containing 1.6% testosterone-17 beta CA and a third group received two sheets, one sheet 50 cm2 in size containing 1.6% testosterone-17 beta CA and the second ranging in size from 0.5 to 2.0 cm2 and containing 1.6% testosterone. Blood was withdrawn daily from each rat over a 4-5 day period after implantation of the steroids and the level of LH in the plasma was measured by radioimmunoassay. The seminal vesicles and the ventral prostate gland were removed at autopsy on day 4 or 5; the weights of these organs were shown to have increased progressively as the size of the implant of testosterone increased. In contrast, the level of LH in the plasma was suppressed to a comparable extent by implants of testosterone between 0.6 and 2 cm2, whereas a 0.5 cm2 implant of testosterone had no effect. Implants of testosterone-17 beta CA alone did not influence the weight of the accessory organs or the level of LH. When testosterone-17 beta CA and testosterone were implanted together, the growth-promoting effect of the latter on the accessory sex organs was significantly reduced. The effectiveness of testosterone in suppressing the level of LH in the plasma of these animals was not influenced by the presence of testosterone-17 beta CA and in certain instances the level was raised.     

Androgen induction of specific estrogen-binding protein of rat liver: relationship between the stage of ontogenesis and various endocrine factors

A new modification of specific estrogen-binding protein (SEBP) assay in the liver was used to measure the content of this protein at different stages of ontogenesis in male and female rats after administration of testosterone propionate (TP). It was shown that liver capability to form SEBP could be initially programmed by androgens. The crucial period of the SEBP-determining action of androgens in females is not restricted, since potential capability of androgens of primary induction of SEBP may be realized both neonatally and in the course of pubertation. The emergence of SEBP in the liver of males may be the result of natural pre(neo)natal androgenization. Androgens do not play any decisive role in the control of the level of induced SEBP, inasmuch the content of SEBP in the liver of non-pubertal and pubertal males does not appreciably change after TP administration but grows with pubescence. SEBP is capable of long-term existence in the absence of androgens. The presence of the ovaries produces an inhibitory effect on the level of androgen-induced SEBP and the time of its existence after TP discontinuation. Hypophysectomy does not provoke SEBP emergence in pubertal intact and ovariectomized females but prevents its induction with androgens. Adrenal- and thyroectomy produce no influence on the SEBP-programming effect of TP or promote its spontaneous appearance in the same groups of female rats.     

Transmission of "hormonal memory" of a unusual estrogen-binding protein during rat hepatocyte proliferation in the process of regeneration

The content of unusual estrogen-binding protein (UEBP), the level of DNA and common protein as well as the organ mass were studied over time during regeneration of the rat liver of intact males and gonadectomized males and females. In 21 days after partial hepatectomy of males with intact testes complete restoration of the UEBP content was observed simultaneously with restoration of the DNA and protein levels and the organ mass. There was no temporary decline of the UEBP content per DNA unit at the early stages of regeneration. The UEBP content in the liver grows during regeneration with the elevation of the DNA level not only in males with intact testes but also in preliminarily castrated males. Different UEBR programs were revcaled by the 21st day of liver regeneration in preliminarily gonadectomized animals: the androgen program of the elevated UEBP content in males, and the basal genetic program of the low level of UEBP expression in females. A conclusion has been made that the hormonal memory of a high UEBP content may be transferred to a new generation of hepatocytes in the absence of androgens. It has been assumed that androgens cause the real determination of masculinization of the EUBP content in liver cells.     

The characteristics of the estrogen-binding protein from the pancreas of female and male rats

Hormone-binding and some other physicochemical properties of an estrogen-binding protein (EBP) from the female and male rat pancreas in partially purified specimens were studied. Kinetic parameters of the hormone-protein interaction were found to be the same for males and females. However the concentration of binding sites for estradiol in females was higher than that in males. The female and male EBP exhibited nearly the same specificity for hormonal compounds except for some estrogens and androgens, whose competitive efficiency was higher in males. These sex differences were found not to be ascribed to diverse intensity of hormonal metabolism during incubation with male and female EBP specimens. The data obtained are suggestive of sex differentiation in the quantity and/or quality of low molecular "accessory" factor modifying EBP hormone-binding properties.     

Regional sex differences in cell nuclear estrogen-binding capacity in the rat hypothalamus and preoptic area

Estrogen binding was compared in cell nuclear KCl extracts from microdissected brain regions of gonadectomized-adrenalectomized male and female rats treated with a near-saturating dose of 17 beta-estradiol. Injection of 3.6 or 36.0 micrograms 17 beta-estradiol/kg BW, iv, 1 h before death resulted in a higher level of estrogen binding in the periventricular preoptic area (PVPOA), medial preoptic area, and ventromedial nucleus of the hypothalamus (VMN) of the female than in comparable tissue samples from the male. No significant sex differences in nuclear estrogen binding were observed in the arcuate-median eminence region, bed nucleus of the stria terminalis, or corticomedial amygdala. Scatchard analysis of saturation binding data revealed that the sex differences in cell nuclear estrogen binding in the PVPOA, medial preoptic area, and VMN reflect a difference in binding capacity rather than binding affinity. These in vitro biochemical findings were confirmed by autoradiographic studies. Gonadectomized-adrenalectomized animals were injected with 125I-labeled 11 beta-methoxy-16 alpha-iodoestradiol (2.0 micrograms/kg BW). Thin frozen sections (10 microns) through the preoptic area and hypothalamus were thaw-mounted onto microscope slides, then exposed against LKB Ultrofilm for 21 days. The autoradiographic images exhibited similar silver distributions and densities in males and females in the arcuate-median eminence region bed nucleus of the stria terminalis, and amygdala. However, 11 beta-[125I]methoxy-16 alpha-iodoestradiol uptake was lower in males than in females in the PVPOA and VMN. These results suggest that sex differences in responsiveness to estrogen stimulation in the rat may be due in part to sex differences in estrogen-binding capacity in specific regions of the hypothalamus that play important roles in the control of pituitary function and reproductive behaviors.