Characterization of different forms of the androgen receptor.

Three forms of the androgen receptor have been characterized at high ionic strength in partially purified cytosol and nuclear fractions of rat ventral prostate, epididymis, testis, and seminal vesicle by ion exchange chromatography on phosphocellulose, gel filtration in Sephadex G-200, and sedimentation in sucrose gradients. The three forms have the following properties, respectively: elution from phosphocellulose at 0.15--0.20, 0.3--0.5, and 0.20--0.32 M KCl; gel filtration radii of 53, 36, and 22 A; sedimentation coefficients of 5.0S, 3.6S, and 3.0S; frictional ratios of 1.65, 1.4, and 1.0; and molecular weights of 115,000, 55,000, and 29,000. In testis and epididymis cytosol, the 53 A, 5S form was more abundant than the 36 A, 3.6S form. In ventral prostate, the 36 A, 3.6S receptor was the predominant form. Variable amounts of all three forms were observed in seminal vesicles. Conversion from 36 A, 3.6S to 22 A, 3.0S was induced by heating ventral prostate cytosol and could be blocked by serine protease inhibitors. The 22 A, 3.0S receptor fragment from heated prostate cytosol was similar in size and symmetry to receptor extracted in 0.5 M KCl from prostate nuclei labeled in vivo. Extracts of epididymis and seminal vesicle nuclei contained both 36 and 22 A forms.     

3,5,3'-triiodothyronine receptor-containing chromatin fragments: production by nuclease digestion

Nuclear thyroid hormone (T3) receptors are nonhistone proteins which are tightly bound to rat liver chromatin. The solubilization of the T3 receptors by micrococcal nuclease was studied using an assay which allows the delection of in vitro hormone binding and which is independent of the state of solubility of the chromatin. Nuclease digestion produces a receptor containing moiety which sediments at a rate of 5--6S. This form of the receptor is different than that released from chromatin at high ionic strength (3.8S) and potentially represents the stable association of the receptor which other elements of chromatin. Partial release of chromatin compaction by the use of dilute buffer solutions increases the rate of nuclease digestion, facilitates the release of the (5--6S) T3-receptor complex, and allows the isolation of sucrose gradient fractions which are enriched with receptor.     

Binding characteristics of the hepatic estrogen receptor of the spotted seatrout, Cynoscion nebulosus

Estrogen receptors were identified in cytosolic and nuclear extracts of livers of adult female spotted seatrout, Cynoscion nebulosus. A single class of high affinity binding sites was found, Kd = 1.26 +/- 0.55 nM (N = 55) for the cytosolic extract and Kd = 1.96 +/- 0.42 nM (N = 8) for the nuclear extract. The Kd did not differ between males and females or between vitellogenic and nonvitellogenic females. The binding in both the cytosolic and nuclear extracts was specific for estrogens (DES greater than E2 much greater than E1 = E3). Receptor concentrations in cytosolic extracts from late vitellogenic females (14.61 +/- 1.07 pmol/g liver, N = 40) were significantly higher than those from nonvitellogenic females (3.91 +/- 0.73 pmol/g liver, N = 7). The nuclear binding capacity of livers from midvitellogenic females (1.12 +/- 0.45 pmol/g liver, N = 10) was significantly higher than the binding capacity in livers from nonvitellogenic females (0.16 +/- 0.07 pmol/g liver, N = 26), but not that of late vitellogenic females (0.80 +/- 0.09 pmol/g liver, N = 77). The concentration of estradiol-binding sites was greatest in the liver (liver much greater than ovary greater than heart greater than spleen greater than muscle greater than brain). No interference from other steroid-binding proteins was detected using a simple dextran-coated charcoal method to separate bound from free hormone. Approximately 14% of the binding in the cytosolic extract had DNA-binding affinity. Estrogen receptor binding activity was maximally extracted from nuclei with buffer containing 0.6 M KCl. Nuclear receptors eluted from gel filtration columns with an apparent molecular weight of 95 kDa.     

Müllerian inhibiting substance blocks epidermal growth factor receptor phosphorylation in fetal rat lung membranes

Neonatal males develop respiratory distress syndrome more frequently than females for unknown reasons. The fetal testis secretes testosterone and müllerian inhibiting substance (MIS); MIS has been shown to inhibit fetal lung maturation in vitro and in vivo and to block phosphorylation of epidermal growth factor (EGF) receptors in A431 cells. We hypothesized that MIS would also inhibit membrane phosphorylation of EGF receptors in fetal lung, and that ultrastructural study of MIS-exposed lung might complement the biochemical data by assessing the effect of MIS on tissue morphology. Lung membranes were prepared from 19.5-day fetal rats and phosphorylation assays performed with 3 to 4 micrograms of membrane protein, with or without EGF (26 nmol/L), 0.025 mCi AT32P (0.136 mumol/L), and either recombinant human MIS (rhMIS, 30 pmol) from media of Chinese hamster ovary (CHO) cells, rhMIS dialysis buffer, or wild-type CHO media. The 170,000 molecular weight EGF receptor, visualized by autoradiography of polyacrylamide gels, was phosphorylated in both female and male membranes. rhMIS, when added to EGF-stimulated membranes, caused significant inhibition of EGF receptor phosphorylation (females: 32.42% +/- 11.5%; males: 32.3% +/- 19.1%, P less than 0.001; rhMIS-treated v EGF-stimulated state, P = NS, male v female, Cerenkov counting). Electron microscopy (EM) of rhMIS-exposed lung showed decreased lamellar bodies (LB) in both male alveolar spaces and female parenchyma, and, unexpectedly, increased numbers in female alveoli. Immunoabsorption experiments using coincubation of rhMIS with anti-rhMIS IgG polyclonal antibodies or equiprotein normal IgG demonstrated MIS antibody-specific reversal of rhMIS activity in membrane phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of fasting on liver receptors for prolactin and growth hormone

The effects of 3 day fasting on liver prolactin and growth hormone receptors have been investigated in male and female rats. Fasting caused a significant fall in serum immunoreactive insulin (67% decrease), while receptor-reactive somatomedin fell by 82% when measured in whole serum and by 72% when measured in serum fractions following gel chromatography at low pH. Tracer ovine prolactin binding to liver microsomal membranes was reduced by 55% on fasting in females, but unchanged in males. Tracer bovine growth hormone binding fell significantly in both sexes. Analysis of competitive binding curves showed the decreased binding to be due to a loss of prolactin receptors in females, and of high affinity (but not low affinity) growth hormone receptors in males and females. Significant correlations were seen between serum insulin and tracer prolactin (females) and growth hormone (males and females) binding to liver membranes. Correlations between serum insulin and liver high affinity growth hormone binding sites were particulary significant (r = 0.899 in females, r = 0.910 in males). It is proposed that the hypoinsulinemia of fasting causes a loss of high affinity growth hormone receptors in the liver, which could result in a relative hepatic resistance to growth hormone and a decreased hepatic generation of somatomedin.     

Promotion of murine hepatocarcinogenesis by testosterone is androgen receptor-dependent but not cell autonomous.

Tfm (testicular feminization) mutant mice lack functional androgen receptors. By studying liver tumor development in Tfm mice, we have shown that the greater susceptibility of male mice relative to female mice for liver tumor induction by N,N-diethylnitrosamine is androgen receptor-dependent. C57BL/6J normal and Tfm mutant mice were injected at 12 days of age with N,N-diethylnitrosamine (0.2 mumol/g, i.p.), and liver tumors were enumerated in 50-week-old animals. Normal males averaged 20 liver tumors per animal; Tfm males, 0.7; normal females, 0.6; and Tfm/+ heterozygous females, 1.5. The androgen receptor gene and the Tfm mutation are X chromosome linked. Because of random X chromosome inactivation, hepatocytes from Tfm/+ heterozygous female mice are mosaic with respect to the expression of mutant or wild-type receptors. To determine if testosterone acts directly as a liver tumor promoter, through the androgen receptor in preneoplastic hepatocytes, or by an indirect mechanism, we chronically treated these mosaic female mice with testosterone and measured the androgen receptor content of the resulting tumors. B6C3F1 Tfm/+ mosaic and +/+ wild-type female mice were injected i.p. at 12 days of age with N,N-diethylnitrosamine (0.1 mumol/g) and ovariectomized at 8 weeks of age. Half of the mice of each group subsequently received biweekly s.c. injections of testosterone (0.15 mg per mouse) for 30 weeks. Tumor multiplicity was the same for wild-type and Tfm/+ mosaic females treated with testosterone (31-32 tumors per animal at 38 weeks of age) and was increased relative to females not treated with testosterone (13-17 tumors per animal at 50 weeks of age). Testosterone treatment did not significantly increase the percentage of androgen receptor-positive tumors in Tfm/+ mosaic females: 58% of the tumors from Tfm/+ mosaic females treated with testosterone were receptor positive compared to 48% in Tfm/+ females not treated with testosterone and 92% in wild-type females treated with testosterone. Finally, the number of androgen receptors in the majority of liver tumors examined was greatly decreased relative to the surrounding normal liver tissue. We conclude that liver tumor promotion by testosterone requires a functional androgen receptor in the intact animal. However, this promotion is not cell autonomous; that is, the response of the preneoplastic hepatocyte is not dependent on the expression of functional receptor in the target cell.     

Experimental colorectal liver metastases. Influence of sex, immunological status and liver regeneration

The liver is the most frequent site of metastases from colon cancer. To improve our knowledge of liver metastases and to develop new adjuvant therapies, a good animal model is necessary. The aims of this study were to obtain a model of liver metastases with intraportal injection of colon adrenocarcinoma cell aggregates (DHDK12 cell line) and to study the effect of various factors, i.e., sex, liver regeneration and immunosuppression, on the development of liver metastasis. Cell aggregates were injected into the portal vein of 59 syngenic male and female BD IX rats following randomization into three groups. Group 1, (control 12 males and 10 females) received only cell aggregates; group 2 (12 males and 10 females) underwent a 70% hepatectomy before cell injection; group 3 (15 males and 10 females) received cyclosporin A injections at a dose of 10 mg/kg per day for 28 days following cell injection. Autopsy was performed at 10 weeks. Liver metastases were more frequent in the male rats in group 3 than in those in group 1 (80% vs. 30%, p less than 0.04). The rate of liver metastases in females was not increased by immunosuppression (22.2% vs. 12.5%, N.S.). Liver resection (group 2) did not significantly modify the incidence of liver metastasis. No female had liver metastases in this group. This relatively simple model rapidly produces liver metastasis with a high yield, but only in male rats. Besides sexual factors, immunosuppression also increased the rate of experimental liver metastasis, while liver regeneration failed to do so.     

Characterization of the calf uterine androgen receptor and its activation to the deoxyribonucleic acid-binding state

The androgen receptor (AR) from calf uterine cytosol has been studied in terms of steroid-binding affinity, hormone dissociation kinetics, and DNA-cellulose-binding capacity. The binding affinity for three androgens, analyzed under conditions where binding to progesterone receptor did not occur, decreased in the order: methyltrienolone greater than 5 alpha-dihydrotestosterone greater than testosterone. Activation of the receptor to the DNA-binding state involved the following changes of the receptor: decrease in dissociation rate for the steroid, disaggregation of the receptor, and increase in affinity for DNA. Dissociation studies with methyltrienolone and 5 alpha-dihydrotestosterone revealed that the AR can exist in two affinity states which differ 13- to 30-fold in their affinity for the steroid. Molybdate (10-20 mM) prevented the formation of the high affinity state. The high affinity state receptor was formed in the absence of molybdate or after ammonium sulfate precipitation (0-40% saturation) of the molybdate-stabilized low affinity state receptor. During formation of the high affinity state, the sedimentation coefficient of the receptor in low ionic strength buffer decreased from 8-9S to 4.5S, indicating receptor disaggregation. DNA-cellulose binding capacity increased from 3 to 65% upon formation of the high affinity state. The DNA-binding form could be eluted from DNA-cellulose at 0.14 M NaCl. After elution the DNA-binding form maintained its sedimentation coefficient of 4.5S and chromatographed as a protein with a Stokes radius of 44 A. From these results it can be concluded that the activated, DNA-binding form of the AR in calf uterus is a protein with a molecular mass of approximately 85,000, which acquires a higher affinity for the ligand upon its formation.     

Hyperlipoproteinemia in angiographically documented coronary artery disease.

Plasma lipids and lipoprotein levels were measured in 100 patients who underwent coronary angiography for chest pains. The coronary arteries were normal in 33 patients (22 males and 11 females), moderately pathological (30--60% narrowing) in 4, and markedly pathological (greater than 60% narrowing) in 63 (53 males and 10 females). Plasma cholesterol and triglyceride levels and very low and low density lipoprotein cholesterol levels were higher by 20--40% in both males and females with pathological coronary arteries as compared to normals. The high density plasma lipoprotein cholesterol level was lower in females with pathological arteries than in normals, but did not differ between the male groups. 67% of the males with pathological arteries had hyperlipoproteinemia as compared to 26% of the normals, and hyperlipoproteinemia was as frequent among males emigrating to Isreal from Europe or America as among those born in Asia or Africa. Hyperlipoproteinemia types IIA, IIB and IV were encountered among the patients at similar rates.     

Differential responses of an invariant region in the ectodomain of three glycoprotein hormone receptors to mutagenesis and assay conditions

The glycoprotein hormone receptors—luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), and thyroid-stimulating hormone receptor (TSHR)—are G-protein-coupled receptors with an invariant 10-amino acid residue sequence in the ectodomain proximal to transmembrane helix 1. A Glu-ASP, located at the midpoint of this conserved sequence, has been suggested to be important in ligand-mediated signaling of LHR and/or receptor expression or stability, but not binding. One goal of this study was to expand the studies on LHR and determine whether the invariant Glu and Asp residues were functional in FSHR and TSHR as well. Another goal was to investigate systematically the role of ionic strength, particularly Na⁺, which appears to have enigmatic functions in the three receptors regarding ligand binding and receptor activation, and to ascertain whether any of the purported effects of Na⁺ could involve the conserved pair of acidic side chains in the ectodomain. COS-7 cells were transiently transfected with cDNAs to the wild-type (WT) receptor (rat) and identical single and double mutants of each (Glu→Ala, Asp; Asp→Ala, Glu; and Glu-Asp→Asp-Glu), followed by characterization of cognate ligand binding and signaling (basal and hormone mediated) in two commonly used buffer systems: Waymouth’s medium, containing a near-physiologic concentration of Na⁺ (132 mM); a low ionic strength buffer with a 1 mM concentration of Na⁺. The three receptors exhibited differential responses to mutagenesis and the two buffers. Notably, a comparison of basal cyclic adenosine monophosphate (cAMP) production showed that the buffer of lower ionic strength resulted in increased basal cAMP production in WT TSHR but not LHR and FSHR; that the maximal ligand-mediated cAMP production was greatest in the buffer of higher ionic strength for the three WT receptors; that functionality of the conserved Glu and Asp residues in ligand-mediated signaling was buffer dependent in LHR, whereas it did not appear to be particularly important in FSHR and TSHR signaling; and that apparent ligand binding in WT and mutant TSHRs seemed to be particularly diminished in the buffer of higher ionic strength. These results demonstrate that identical amino acid residues in homologous receptors can exhibit distinct functions; moreover, the role of ionic strength (Na⁺) on signaling differs in the three receptors.     

性激素及其受体在肝脏脂类代谢中的作用机制研究进展

肝脏是调节体内脂类代谢最重要的器官之一。肝脂肪变性是代谢综合征的主要表现,与脂类物质合成和分解之间的不平衡有关。脂肪肝的患病率存在性别和年龄差异,提示性激素在其中起着至关重要的作用。本文总结了目前有关雌激素和雄激素通过雌激素受体和雄激素受体来调控肝脏脂类代谢机制的文献。于雌性而言,雌二醇与雌激素受体结合降低肝脏脂肪生成及脂肪酸摄取,同时增强脂肪分解和胆固醇分泌。在雄性体内,睾丸激素通过雄激素受体减少脂肪生成并促进脂肪分解。这些研究结果表明,性激素及其受体可以作为预防肝脂肪变性的潜在靶标。     

Identification of cytoplasmic estrogen receptors in the accessory sex organs of the rabbit and their comparison to the cytoplasmic estrogen receptor in the epididymis

Estrogen receptors are present in cytosol prepared from the accessory sex organs (vesicular gland, proprostate, prostate, bulbourethral gland) of sexually immature and of sexually mature rabbits. The receptor in these organs from animals of both age groups has a sedimentation coefficient of 8-10S on low ionic strength (0.01 M KCl) sucrose gradients. Under high ionic strength (0.4 M KCl) conditions, the receptor sediments at approximately 4S. The cytoplasmic estrogen receptor from the epididymis shows age-dependent changes in its sedimentation coefficient. It is 8S under low ionic strength conditions when prepared from immature rabbits and 4S under identical conditions when prepared from sexually mature animals. Although the dissociation constant of the cytoplasmic estrogen receptor in the immature and mature epididymis and accessory sex organs remains constant during development (approximately 0.1 nM), the number of available cytoplasmic estrogen binding sites declines from about 160 fmoles/mg cytosol protein in the immature rabbit to about 40 fmoles/mg cytosol protein in the adult animal. The estrogen receptor in the accessory sex organs is highly specific, the relative affinities of various potential competitors being: estradiol and estrone = 1, diethylstilbestrol = 0.3, estriol = 0.2, tamoxifen = 0.08, testosterone = 0.0004 and 5 alpha-DHT = 0.00005. Changes with age in the physicochemical characteristics of the estrogen receptor and in the concentration of binding sites suggest that the estrogen receptor may be involved in the development and physiological regulation of the male reproductive tract.     

The development of gonadotropin and steroid hormone patterns in male and female hamsters from birth to puberty.

Male (1--60 days old) and female (1--30 days old) hamsters were decapitated and serum levels of LH, FSH, PRL, progesterone, androgens (males), and estradiol (females) were measured by RIA. Males and females had similar levels of LH until 15 days of age and of FSH until 12 days of age, at which times gonadotropin levels increased significantly in females. Peak levels for females occurred on days 19--21 for LH and on days -2--24 for FSH, later than the times reported for female rats. Adjusting female gonadotropin peaks for gestation length places these peaks for hamsters and rats at the same time in postmating age. In female hamsters, large variations occur in LH between 16--25 days of age, as reported for female rats. Males reached peak serum levels of LH and FSH on day 40, just before the first motile epididymal sperm. Serum PRL levels were identical in male and female hamsters until at least day 30. PRL levels sharply increased in both sexes after day 18 and remained elevated until at least day 30. In males, serum androgens were low until 30 days of age, in contrast to high levels reported for infantile rats. Androgens rose sharply in male hamsters after day 30 to peak levels on day 50. Progesterone in males also remained low until after day 30. Serum estradiol in females did not attain the extremely high elevations seen in rats. Some fluctuations occurred between 10--30 days of age, which presumably represent maturational changes in the ovary. Serum progesterone in females followed a pattern of development similar to estradiol.     

Induction of somatogenic receptors in livers of hypersomatotropic rats

Male and female Wistar-Furth rats bearing the pituitary tumor MtT/W15 had serum GH and PRL levels several hundred-fold higher, and immunoreactive somatomedin levels 3-fold higher, than those of controls. The presence of tumor appeared to have no effect on specific binding of radioiodinated bovine GH to liver microsomal membranes in male animals, and to decrease specific binding by 78% in females. However, after desaturating receptors by treatment with 3.2 M MgCl2 for 5 min, bovine GH binding to membranes from tumor-bearing males was 3-fold higher than in normal males, while in tumor-bearing females binding was more than twice as high as in normal females. Competitive binding curves showed the induced receptors to be specific for growth hormones, and thus somatogenic in nature. This study indicates that a high degree of occupancy of GH receptors in hypersomatotropic rat liver does not cause their down-regulation. The mechanism of receptor induction is unknown.     

Differential expression of three estrogen receptor subtype mRNAs in gonads and liver from embryos to adults of the medaka, Oryzias latipes

In fish, estradiol-17β (E2) regulates various reproductive processes by acting through estrogen receptors (ERs). Here, we cloned three ER subtypes from medaka and examined their developmental expression in the gonads and liver of genetically females and males from embryos to adults. During embryogenesis, marked increases in the expression of ERβ2, but not either ERα or ERβ1, were found in genetically female embryos during sex differentiation. E2 treatment induced marked up-regulation of ERβ2 expression in genetically male embryos. In adult ovaries, ERα levels were high in follicles (granulosa cells) during oocyte growth. In the testis, ERβ1 expression exhibited a distinct peak at 10 days post hatching (dph). In the liver, very high levels of ERβ2 were found in both females and males throughout the sampling period with significantly higher levels in females during 30-50 dph. These findings suggest each action of E2 to be mediated by different types of ERs.     

Water-soluble gonadotropin receptors of the rat ovary

Soluble receptors for LH/hCG were characterized in aqueous and low ionic strength buffer extracts of the luteinized rat ovary. These receptors are proteins with high affinity (KA = 10(10) M-1) and specificity for hCG- and LH-like gonadotropins. The water-soluble receptors are stable and retained 60-80% of their binding capacity after lyophilization. Resolution of the water-soluble receptors by gel electrophoresis demonstrated five binding species, with molecular weights of 165,000, 81,000, 48,000, 24,000 and 12,000. These findings have demonstrated that a proportion (5-10%) of ovarian LH/hCG membrane receptors can be rendered water soluble with preservation of their binding properties and that the specific binding site for LH/hCG is present in proteins much smaller than the predominant 6.5S (mol wt, 194,000) form extracted by nonionic detergents. The stability of these small LH-binding sites is of value for further purification and analysis of structural determinants for gonadotropin binding.     

In Vitro Conversion of Estradiol-Receptor Protein to Its Nuclear Form: Dependence on Hormone and DNA

Early events in the action of 17-beta-estradiol can be studied in soluble extracts of rat uterus by exposure of the estradiol-receptor protein to a DNA-cellulose matrix. After complexing with [(3)H]estradiol, the 4S receptor protein binds to the DNA, and it can be eluted with buffer of high ionic strength as a more tightly binding, 5S form. This parallels the in vivo situation, where migration of the receptor to the nucleus follows addition of hormone and is concomitant with a similar increase in sedimentation rate to 5 S. In both cases, the formation of a 5S receptor requires the presence of 17-beta-estradiol. The rate at which 5S receptor forms is sensitive to extract concentration in a way that suggests that this receptor is a complex created by addition of a second subunit to the hormone-binding 4S component; physical studies on both in vivo and in vitro 5S receptors also support this view. These results are interpreted in terms of a model for action of estrogen in which the hormone potentiates binding of receptor to DNA, and in turn, the DNA-binding process triggers the cell response.     

Intraspecific hybridization of Anopheles minimus (Diptera: Culicidae) species A and C in Thailand

Hybridization tests of laboratory-raised, isolines of Anopheles minimus, species A and C were conducted by induced copulation. The three isolines were established based on three morphological variants of wild-caught, fully engorged females and two distinct types of metaphase chromosomes. They were An. minimus species A: V form (X1,Y1), M form (X2,Y1); species C: P form (X3,Y2). The results of reciprocal and back crosses indicated that the two morphologically variant forms of species A were genetically compatible, providing viable progeny and completely synaptic salivary gland polytene chromosomes, whereas they were genetically incompatible with species C and/or the P form. Hybrid progeny was only obtained from both forms of species A females x species C males, but asynaptic salivary gland polytene chromosomes on 3L and partial development of ovarian follicles in females were seen. Back crosses of F1 hybrid males with parental species A females provided viable progeny, while back crosses of F1 hybrid females with parental species C males provided progeny of low viability and adult males with abnormal spermatozoa, suggesting the partial reproductive isolation of An. minimus species A and C.     

Androgen receptors in two androgen-mediated, sexually dimorphic characters of frogs

The presence/absence of androgen receptors is examined in two sexually dimorphic features of frogs: the nuptial pad and the external oblique muscle. Immunohistochemistry reveals that both males and females possess androgen receptors in these tissues. Males have a higher density of immunopositive nuclei in the oblique muscle than do females. The presence of androgen receptors in both male and female tissues is consistent with results from hormone experiments in which androgen supplements induce the expression of a nuptial pad and enlarge the external oblique muscles in castrated males and ovariectomized females.     

Sex-steroid receptors in the diethylnitrosamine model of hepatocarcinogenesis: modifications by gonadal ablation and steroid replacement therapy

The results of this study confirm our previous report of increased androgen receptor expression in livers of female SUAH Wistar rats during development of liver tumours induced by diethylnitrosamine (DENA). In adult female rats not treated with DENA, removal of the ovary increased liver androgen receptor levels but testosterone did not further enhance the androgen receptor status of ovariectomized rats. In normal adult males the testis and/or testosterone maintained high levels of androgen receptors but oestrogen reduced them in castrated rats. Oestrogen receptor levels were not significantly changed in either males or females by gonadectomy. Treatment of female rats with DENA for 10 and 16 weeks increased liver androgen receptors but oestrogen receptors were only reduced by 16 weeks of DENA treatment, whether the rats were intact or ovariectomized. Concentrations of liver androgen receptors were increased in intact and castrated male rats by 10 and 16 weeks of DENA treatment, an increase not seen in the previous experiments. Oestrogen appeared to inhibit both the increases in liver androgen receptor expression and liver tumour development in rats treated with the weakly carcinogenic dose of 10 weeks of DENA. However, the full carcinogenic dose of 16 weeks of DENA increased liver androgen receptors and decreased oestrogen receptors in female rats regardless of sex-steroid status. Development of malignant hepatocellular carcinoma (HCC) was associated with both an increase in liver androgen receptors and a decrease in oestrogen receptors. Maintenance of relatively high levels of liver oestrogen receptors appeared to protect the liver against development of HCC.