ABSTRACT

Determination of the presence and characterization of oestrogen receptors (ERs) in subcutaneous and internal fat depots were performed and compared with ERs in the uterus using ligand binding and immunological techniques. Successful and consistent measurement of ERs in ovine adipose tissue could only be accomplished in animals depleted of endogenous sex steroids by combined ovariectomy and adrenalectomy. Scatchard, sucrose gradient and Western blot analyses all confirmed the presence of ERs in the cytosolic fractions of various adipose and uterine tissues from ovariectomized-adrenalectomized ewes. The approximate K _d values of 0·1–0·4 nmol/l for oestradiol binding in cytosolic fractions of gluteal, omental and perirenal adipose tissues were similar to the expected high affinity binding of K _d 0·35 nmol/l observed in uterine tissue. The binding was specific for oestrogens, as unlabelled diethylstilboestrol and oestradiol effectively competed with labelled hormone for receptor sites and progesterone, R5020, testosterone and dexamethasone all failed to compete. Mean (± s.e.m.) concentrations of ERs, expressed as fmol specific binding sites per mg protein, were much lower (P<0·05) in adipose tissues than in uterine tissue (975 ± 33). However, the content of ERs was greater (P<0·05) in subcutaneous gluteal fat (11·5 ± 0·8) than in the internal omental or perirenal fat (5 ± 0·6) depots. ERs from adipose and uterine tissues both migrated as moieties of 8S on 5–20% sucrose gradients. Western blot analysis of ERs from uterine and adipose tissues in the presence of protease inhibitors demonstrated an immunostaining band with a molecular mass of 67 kDa. In the absence of protease inhibitors, the adipose ER was degraded into two smaller bands with molecular masses of 45 kDa and 36 kDa.

In conclusion, these results support the presence of specific ERs in adipose tissues of sheep with characteristics similar to those of the ERs of the uterus. Furthermore, their presence is supportive of sex steroid-dependent effects on adipose accretion and metabolism.

Journal of Endocrinology (1993) 139, 107–115

SUMMARY

The binding of ¹²⁵I-labelled human growth hormone (HGH) to the 'lactogenic' binding sites of rat liver membranes has been shown to be highly dependent on the oestrogen and androgen status of the animal from which the membranes were prepared. Oestradiol treatment of either male or female rats induced a highly significant rise in HGH binding. The minimum effective dose used was 2–5 μg/day and the rise in HGH binding was apparent after 4 days of treatment. Following cessation of oestradiol treatment of male rats HGH binding declined with a half-time of approximately 9 days. In contrast to the stimulatory effect of oestrogen, treatment of female rats with testosterone propionate (minimum effective dose 100–200 μg/day) led to a marked reduction in HGH binding. The influence of both oestrogens and androgens was confirmed following the removal of endogenous sex steroids by adrenalectomy–ovariectomy of female rats and castration of male rats. Scatchard analysis showed that, with the possible exception of adrenalectomy–ovariectomy, all pharmacologically and physiologically induced changes in HGH specific binding reflected changes in binding site capacity; there were no changes in binding affinity. While earlier studies have indicated that the oestrogen effect is primarily indirect and is mediated by the pituitary gland, the mode of action of the androgens is currently unknown. The relatively slow response of HGH binding to hormonal changes would support an indirect action for both the sex steroids. The stimulatory effect of oestrogens and the inhibitory effect of androgens may provide an explanation for the marked sex difference in HGH binding to rat liver membranes.

Apart from the role of sex steroids in reproduction, sex steroids are also important regulators of the immune system. 17β-estradiol (E2) represses T and B cell development, but augments B cell function, possibly explaining the different nature of immune responses in men and women. Both E2 and selective estrogen receptors modulators (SERM) act via estrogen receptors (ER). Activating functions (AF)-1 and 2 of the ER bind to coregulators and thus influence target gene transcription and subsequent cellular response to ER activation. The importance of ERαAF-1 and AF-2 in the immunomodulatory effects of E2/SERM has previously not been reported. Thus, detailed studies of T and B lymphopoiesis were performed in ovariectomized E2-, lasofoxifene- or raloxifene-treated mice lacking either AF-1 or AF-2 domains of ERα, and their wild-type littermate controls. Immune cell phenotypes were analyzed with flow cytometry. All E2 and SERM-mediated inhibitory effects on thymus cellularity and thymic T cell development were clearly dependent on both ERαAFs. Interestingly, divergent roles of ERαAF-1 and ERαAF-2 in E2 and SERM-mediated modulation of bone marrow B lymphopoiesis were found. In contrast to E2, effects of lasofoxifene on early B cells did not require functional ERαAF-2, while ERαAF-1 was indispensable. Raloxifene reduced early B cells partly independent of both ERαAF-1 and ERαAF-2. Results from this study increase the understanding of the impact of ER modulation on the immune system, which can be useful in the clarification of the molecular actions of SERMs and in the development of new SERM.

Abstract

Obesity is a heterogeneous condition and not every obese patient is at increased risk of cardiovascular diseases (CVD). It is now well established that the regional distribution of body fat is a critical correlate of the metabolic complications of obesity. Studies that have assessed adipose tissue distribution by imaging techniques such as computed tomography have demonstrated the importance of the intra-abdominal (visceral) fat depot as a marker of a cluster of metabolic abnormalities which include glucose intolerance, insulin resistance, hyperinsulinemia, hypertriglyceridemia, elevated number of apo B-carrying lipoproteins as well as hypoalphalipoproteinemia. Although the association between visceral obesity and metabolic complications can hardly be questioned, it has been suggested that it may not necessarily represent a causal relationship. For instance, concomitant alterations in sex steroid levels have been found in both men and women with abdominal (visceral) obesity which have also been reported to be significantly correlated with the insulin resistant-dyslipidemic state found in abdominal obese subjects. In women, abdominal obesity is associated with increased free testosterone concentrations and reduced sex hormone binding globulin (SHBG) levels, whereas in men this condition is associated with reduced testosterone and adrenal C₁₉ steroid (dehydroepiandrosterone, androstenedione, androstene-3β, 17β-diol) levels as well as decreased SHBG concentrations. These altered steroid and SHBG levels have been reported to be independent correlates of the metabolic complications of visceral obesity although they cannot solely account for the increased CVD risk found in these patients. In this regard, intervention studies are clearly warranted to better quantify the respective contribution of excess visceral adipose tissue and of the concomitant alterations in sex steroid levels as modulators of metabolic disturbances increasing CVD risk in obesity.

Journal of Endocrinology (1996) 150, S155–S164

Significant levels of estrogen and androgens circulate in men and women, and both play an important role in bone metabolism. While it is well established that either estrogen or androgen replacement therapy is effective at ameliorating bone loss associated with hypogonadism, recent evidence nevertheless suggests that estrogen and androgens have distinct molecular actions on the skeleton. In this study, we have employed normal rat calvarial osteoblast cultures to characterize relative expression profiles of estrogen (ERalpha and ERbeta) and androgen receptors (AR) during osteoblast differentiation. Normal osteoblast cultures can proceed through in vitro differentiation with distinct stages of proliferation, matrix maturation and mineralization in the appropriate differentiation medium containing ascorbic acid. Expression profiles of AR, ERalpha and ERbeta in primary cultures during osteoblast differentiation were characterized both by semi-quantitative relative RT-PCR and by Western analysis. In cultures induced to differentiate by growth in the presence of ascorbic acid, the expression profile for each receptor was unique during the course of differentiation. ERalpha levels were elevated during matrix maturation and then declined during mineralization. ERbeta expression was relatively constant throughout differentiation, exhibiting more constitutive expression. In contrast, AR levels were lowest during proliferation, and then increased throughout differentiation with highest levels in the most mature mineralizing cultures. Since steroid hormone action is generally mediated by specific cognate receptors, these results suggest that androgen actions may target cells during the mineralization stage of osteoblast differentiation, while estrogen action through either receptor isoform is more likely to affect osteoblasts earlier during matrix maturation. Interestingly, sex steroid receptor expression profiles did not exhibit the same patterns of regulation if osteoblast cultures were grown without ascorbic acid in medium that did not support extracellular matrix deposition. Thus, sex steroids may distinctly influence skeletal health by differential modulation of function during osteoblast differentiation.

The 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) family of proteins regulates the levels of the active 17 beta-hydroxy forms of sex steroids. The expression of 17 beta-HSD type IV is induced by peroxisome proliferator chemicals (PPC) in rat liver. In order to characterize more generally the impact of PPC on 17 beta-HSD expression, we determined (1) if expression of other members of the 17 beta-HSD family was coordinately induced by PPC exposure, (2) the tissues in which 17 beta-HSD was induced by PPC, and (3) whether the induction of 17 beta-HSD by PPC was dependent on the peroxisome proliferator-activated receptor alpha (PPAR alpha), the central mediator of PPC effects in the mouse liver. The mRNA levels of 17 beta-HSD I, II, and III were not altered in the liver, kidney, and testis or uterus of rats treated with PPC. The mRNA or 80 kDa a full-length protein levels of 17 beta-HSD IV were strongly induced in liver and kidney, but not induced in adrenals, brown fat, heart, testis, and uterus of rats treated with diverse PPC. In liver and kidneys from treated rats, additional proteins of 66 kDa, 56 kDa, and 32 kDa were also induced which reacted with the anti-17 beta-HSD IV antibodies and were most likely proteolytic fragments of 17 bega-HSD IV. Treatment of mice which lack a functional form of PPAR alpha with PPC, demonstrated that PPC-inducibility of 17 beta-HSD IV mRNA or the 80 kDa protein was dependent on PPAR alpha expression in liver and kidney. Our results demonstrate that 17 beta-HSD IV is induced by PPC through a PPAR alpha-dependent mechanism and support the hypothesis that exposure to PPC leads to alterations in sex steroid metabolism.

ABSTRACT

We studied 24-h hormone profiles and hormonal responses to insulin-induced hypoglycaemia prospectively in 23 children of similar age and pubertal stage, nine of whom had received prior cranial irradiation (group 1) and fourteen of whom had not (group 2), before and 6–12 months after total body irradiation (TBI) for bone marrow transplantation in leukaemia.

Fourier transformation demonstrated that group 1 children had a faster periodicity of GH secretion before TBI than group 2 children (160 vs 200 min) but the amplitude of their GH peaks was similar. There were no differences between the groups in circadian cortisol rhythm, serum concentrations of insulin-like growth factor-I (IGF-I), sex steroids and basal thyroxine (T₄). The peak serum GH concentrations observed after insulin-induced hypoglycaemia were similar between the two groups but the majority of patients had blunted responses.

TBI increased the periodicity of GH secretion in both groups (group 1 vs group 2; 140 vs 180 min), but the tendency to attenuation of amplitude was not significant. There were no significant changes in the peak serum GH concentration response to insulin-induced hypoglycaemia which remained blunted. Serum IGF-I, sex steroid, cortisol or T₄ concentrations were unchanged.

Low-dose cranial irradiation has an effect on GH secretion affecting predominantly frequency modulation leading to fast frequency, normal amplitude GH pulsatility. This change is accentuated by TBI. In patients with leukemia, there is a marked discordance between the peak serum GH response to insulin-induced hypoglycaemia compared with the release of GH during 24-h studies, irrespective of the therapeutic regimen used. Pharmacological assessment of GH reserve needs to be interpreted with caution in such situations.

Journal of Endocrinology (1993) 136, 331–338

Abstract

Differing risk factors between men and women for a number of vascular and metabolic diseases have been linked to regional obesity. The differences in the distribution of adipose tissues between men (abdominal or upper-body obesity) and women (gluteal/femoral or lower body obesity) suggest a role for sex steroids in the regional distribution of fat. Previous work from this laboratory has shown the presence of oestrogen receptor (ER) in gluteal, perirenal and omental adipose tissues of ewes with similar physical characteristics to the ER in uterine tissue. The concentration profile for adipose ER was gluteal> perirenal>omental. In this report, we determined the physiological significance of adipose ERs by showing an up-regulation of the progesterone receptor (PR) in adipose tissues after oestrogen treatment in a fashion similar to that seen in a major responsive tissue such as uterus. Using PR antibodies (PR-6 and C-262), Western blot analysis of PR from oestrogen-treated sheep indicated that PR was induced in uterus>>>gluteal adipose>perirenal adipose consistent with the concentration of ER contained in these tissues. PR could not be detected by Western blotting in omental adipose tissue from oestrogen-treated animals or in gluteal, perirenal and omental adipose tissues from untreated animals. Sucrose gradient profiles of progestin (R-5020) binding from uterus and gluteal adipose tissues of oestrogen-treated ewes showed specific binding in both the 5S and 9S regions of the gradient, while perirenal and omental adipose tissue had only the 5S peak. The amount of specific binding was increased with oestrogen treatment in all the tissues. When gluteal adipose tissue cytosol was preincubated with PR antibody (C-262) to prevent binding of ligand and subjected to sucrose gradient analysis, both the 5S and 9S regions were diminished, suggesting that both peaks contained PR. Dilution of uterine cytosol resulted in an increase in the ratio of the 5S to the 9S peak, indicating that the 9S PR complex dissociates at low concentrations; this may be the reason why only the 5S peak was observed in perirenal and omental adipose tissues. These data offer further support for a direct role of sex steroids in regional adipose accretion and metabolism.

Journal of Endocrinology (1996) 148, 19–25

ABSTRACT

This study aimed at determining the relationship of sex steroids, particularly in the perinatal period, to the pubertal insulin-like growth factor-I (IGF-I) surge in male mice. We used hypogonadal (hpg) mice, which have a major deletion in the gonadotrophin-releasing hormone (GnRH) gene, in order to have a model lacking all GnRH-induced gonadotrophin and sex steroid secretion throughout pre- and postnatal life. Cross-sectional data on body weights and weights of testes, seminal vesicles, kidneys, liver and spleen from 9 to 77 days of age were obtained in male hpg, heterozygous (Hz) and homozygous normal (N/N) littermates (n = 75–78/group). These data did not reveal any difference between Hz and N/N mice. Hpg mice had decreased body weights which by 70–77 days of age were approximately 18% less than normal controls. Testes and seminal vesicles of hpg mice did not demonstrate any significant postnatal growth. Relative to body weight, kidney weights were also markedly reduced in hpg mice (P<0·0001), deviating significantly from normal by 28–35 days of age, reflecting the impact of androgen deficiency on a non-reproductive organ. From the cross-sectional data it was concluded that puberty commenced soon after weaning (21 days) in the male and that maturity was achieved within 4–5 weeks. Longitudinal study showed that, compared with normal controls, untreated hpg mice had an exaggerated pubertal IGF-I surge (P<0·005) which peaked in mid-puberty. This, together with their reduced body weights (P<0·05), were normalized by treatment from 21 to 70 days of age with two 1 cm s.c. implants of testosterone (n=6) or dihydrotestosterone (n=7). There was no difference in IGF-I levels or in weights of testes, seminal vesicles, kidney, liver or spleen between testosterone and dihydrotestosterone treatments (P>0·05). Prolonged high levels of androgen also restored testicular and seminal vesicle weights to 40% of phenotypically normal controls, while kidney, liver and spleen weights were also significantly increased. The pubertal IGF-I surge in mice does not, therefore, require androgens in either the pre- or postnatal periods, and it is exaggerated in androgen-deficient male mice and dampened to normal regardless of aromatization.

Journal of Endocrinology (1993) 139, 57–65

ABSTRACT

While intrasexual competition for mates is generally considered to be an androgen-dependent characteristic of reproductively active males, in the Wilson's phalarope (Phalaropus tricolor) it is the female that acquires the brighter nuptial plumage and aggressively competes for access to the less aggressive males. Despite this pronounced sex-role reversal, circulating sex steroid hormones of breeding phalaropes are similar to those of avian species displaying traditional male–female reproductive roles. To investigate whether these behavioural and morphological steroid-dependent differences may be due to differences in target organ metabolism of circulating androgen, [³H]androstenedione in the presence of an NADPH-generating system was incubated with homogenates of brain, pituitary and skin of male and female Wilson's phalaropes collected from a naturally breeding population. Oestrone, 5α-androstanedione and 5β-androstanedione were measured as endpoints of aromatization, 5α-reduction and 5β-reduction respectively. Aromatase activity in the anterior hypothalamus/preoptic area (AHPOA) and posterior hypothalamus was greater in breeding males with high circulating concentrations of testosterone than in females, and activity in the AHPOA was greater in breeding than in non-breeding males (with low circulating testosterone). Aromatase levels did not differ in septum, archistriatum, hyperstriatum or pituitary. 5α- and 5β-reductase were detected in all neuroendocrine tissues sampled and although there were no significant male–female differences, 5α-reductase was greater in the AHPOA of breeding than of non-breeding males. We infer from this that the behavioural sex-role reversal of phalaropes is unlikely to be accounted for by differences in androgen metabolism in neural targets, although the capacity to form greater quantities of oestrogenic and 5α-reduced metabolites in the AHPOA of breeding males may be linked to the expression of masculine copulatory behaviours. Aromatase activity was not detected in skin containing a sexually dimorphic feather tract; however, 5α- and 5β-reductase activities were significantly higher in females than in males and may account for the brighter nuptial plumage of females. These data suggest that alternate determinants of neural responsiveness such as sex-steroid receptor abundance or neural circuitry may underlie atypical sexual behaviours in phalaropes.

Journal of Endocrinology (1989) 122, 573–581