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ABSTRACT
Cytosolic androgen receptors from neocortex, hypothalamus and anterior pituitary and ventral prostate glands were analysed by miniature isoelectric focusing and sucrose density-gradient centrifugation before and after precipitation of [3H]dihydrotestosterone (DHT)-bound complexes with ammonium sulphate. In the hypothalamus and neocortex (NH4)2SO4 precipitation appeared to cause heterodisperse peaks, and in the case of the ventral prostate there was a clear shift to a more basic isoelectric point. After sucrose density-gradient centrifugation all cytosols sedimented as large aggregates which appeared to dissociate into subunits in 0·4 m-KCl gradients. The functional significance of these altered forms was tested by nuclear uptake studies of cytosolic [3H]DHT-bound complexes, which could only be detected in brain and pituitary nuclei after prior precipitation with (NH4)2SO4, which also significantly increased extraction of ventral prostate [3H]DHT-bound complexes from the nucleus. The nuclei apparently responded to the (NH4)2SO4-precipitated and redissolved complexes by increased RNA polymerase activity. These results are consistent with the possibility that the neural androgen receptor is altered before interaction with the genome, and this alteration may be necessary for the action of the hormone to be expressed.
J. Endocr. (1984) 102, 181–188
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Available high-affinity binding sites for 5α-dihydrotestosterone (DHT) were measured in cytosols obtained from the amygdala, hypothalamus, anterior pituitary gland and ventral prostate gland of 12-week-old rats at various times after orchidectomy, and in the corresponding tissues of 18-month-old male rats. It is suggested that the lower affinity of the DHT binding reaction in brain and ventral prostatic cytosols after orchidectomy or ageing respectively, may explain, at least in part, the changes in the responsiveness of the tissues to androgens.
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A sensitive, reliable assay of activity of RNA polymerase in purified nuclei from the rat brain is described. The assay measured the incorporation of [3H]guanosine triphosphate into RNA by nuclei. Little incorporation occurred in the presence of α-amanitin (400 ng) suggesting that RNA polymerase B activity was being measured. Preliminary evidence showed that after administration of oestradiol benzoate to 18-day-old female rats RNA polymerase activity was raised in areas of brain known to contain oestrogen receptors.
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Cytosol prepared from rat brains perfused with saline possesses saturable macromolecular components which form unstable complexes with [3H]progesterone in vitro. The components can be distinguished from the serum protein corticosteroid-binding globulin (CBG) which also binds progesterone. Unlike CBG, the cytosol components did not bind corticosterone and were more unstable in the presence of Sephadex LH-20 than was CBG. Furthermore, the components were precipitated by a concentration of ammonium sulphate in which CBG is soluble. Unlabelled progesterone and testosterone competed equipotently with [3H]progesterone, and 5α-pregnane-3,20-dione and deoxycorticosterone competed to a lesser extent, whereas oestradiol, corticosterone and 11β-hydroxy-pregn-4-en-3,20-dione were not bound. Although present in all brain tissues examined and in uterine and anterior pituitary tissue, the saturable binding was not detectable in cytosols from the spleen or diaphragm.
Complete exchange occurred in the incubation at 0 °C between [3H]progesterone and unlabelled progesterone and the saturable binding sites and the reaction attained apparent equilibrium within 2 h at this temperature. An incubation temperature of 30 °C resulted in an almost complete loss of saturable binding. Scatchard plots obtained from binding isotherms were curvilinear and yielded at least three dissociation constants (K d), two of higher affinity (K d ≃ 10−8 mol/l) and one about sixty times lower.
From these results it is concluded that rat brain cytosol possesses progesterone-selective components which fulfil some of the criteria required for steroid hormone receptors.
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ABSTRACT
Cytosols were incubated from the hypothalamus and mid-brain and from the uterus, and incubated with [3H]progesterone alone or in the presence of excess radioinert steroid to reveal saturable binding sites. Bound and free hormone were separated by gel filtration. Scatchard analysis of the binding sites yielded evidence for only one class of binding sites of high affinity and limited capacity. The binding components in the hypothalamus and uterus appeared to fluctuate during the oestrous cycle, attaining a nadir at metoestrus, while those in the mid-brain were apparently unchanged. During pregnancy hypothalamic [3H]progesterone-binding sites appeared to lose affinity for the steroid while in the uterus the affinity for the steroid was unchanged but the absolute numbers of binding sites were greatly increased at day 10. It is concluded, both from studies of the properties intrinsic to the binding reaction and from endocrine correlates, that the macromolecular progesterone-binding components in the brain may be receptors for the hormone and that there may be differences between the properties of progesterone receptors in different tissues. Furthermore, during pregnancy there may be qualitative changes in the neural progesterone receptors which are not mediated by oestradiol.
J. Endocr. (1985) 107, 159–162
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ABSTRACT
The rat brain is sexually dimorphic with respect to structure and function, and there is evidence that these differences are effected in the fetus through changes in protein synthesis, some of which may result from the intervention of gonadal steroids. To investigate this, messenger RNA (mRNA) from the limbic system and cerebellum of neonatal rats was prepared, translated in a rabbit reticulocyte system in vitro and the products were analysed by two-dimensional electrophoresis and fluorography. Some of the results were further analysed using image analysis. There was a striking sexual dimorphism in the patterns of incorporation of [35S]methionine into proteins using mRNA from the limbic system, in that groups of proteins were apparently present in male-but not in female-derived fluorograms and vice versa. One protein, tentatively identified from its coordinates as α-tubulin, was more abundant in male-derived fluorograms. Although there were no clear-cut qualitative sex differences using mRNA derived from the cerebellum, that derived from the male cerebellum appeared to be consistently more active. These results provide direct evidence for a sexual dimorphism at the transcriptional level in the neonatal limbic system of the rat.
J. Endocr. (1986) 109, 23–28
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There is sexual dimorphism of specific species of mRNA in the neonatal rat brain and this sexual dimorphism may be imprinted by steroids of testicular origin during the perinatal period. According to current theories, only aromatizable androgens may cause sexual differentiation of sexual behaviour and function in the adult. The effects of oestradiol benzoate on mRNA synthesis in the neonatal female limbic system were therefore studied. In addition, cytosolic and nuclear oestrogen receptors were measured after administration of testosterone propionate, oestradiol benzoate or dihydrotestosterone (DHT). An attempt was made to distinguish between the brain oestrogen receptor and the plasma oestrogen-binding protein, alphafoetoprotein (AFP) by isoelectric focussing. After injection of 50 μg oestradiol benzoate s.c. to neonatal female rats, the expression of mRNA coding for sexually dimorphic proteins appeared to be changed to a male-type pattern. The overall density of labelling was noticeably greater and specific changes in labelled proteins were observed. These effects were observed within 3 h of injection. Both testosterone and oestradiol caused a marked depletion of cytosolic oestrogen receptors in the limbic system whereas DHT was ineffective in this respect. Nuclear receptors were present in equal abundance in male- and female-derived nuclei and only oestradiol was able to cause a significant (P < 0·025) increase in nuclear oestrogen receptors. The receptor and AFP could be distinguished by isoelectric focussing, since the pI of the receptor was 7·05, while that of AFP was 4·5. These results are consistent with the possibility that oestradiol alters transcription in the neonatal rat brain and may do this through the oestrogen receptor. Nevertheless, it is also possible that oestradiol could alter post-transcriptional events such as the stability of mRNA or the binding of tRNA to the polysomal complex.
Journal of Endocrinology (1989) 120, 83–88
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ABSTRACT
The effects of several steroids on the regenerating thymus in ageing male rats have been studied. Rats aged from 12 to 15 months were orchidectomized and 7 days later implanted s.c. with silicone elastomer tubing containing 25 mg testosterone, 5α-dihydrotestosterone (DHT), oestradiol, progesterone or corticosterone. One group of rats received an empty implant. Thirty days later the rats were killed and the thymus, spleen, ventral prostate and seminal vesicles weighed and retained for histology. Whole blood was taken for total and differential white cell counts; plasma was prepared for radioimmunoassay of testosterone, oestradiol, progesterone and corticosterone.
After orchidectomy only, a multilobular thymus was present, and histologically the tissue appeared healthy. In testosterone- and oestradiol-treated rats, thymus weight was reduced to about 50% of that in untreated animals. Histologically, much of the thymus taken at autopsy was fat and what remained was poorly organized and contained a much lower density of thymocytes. The total white cell count was significantly reduced in these animals, the effect appearing to be predominantly on lymphocytes. Although treatment with DHT also resulted in a lower mean thymus weight than that of orchidectomized animals, histologically the tissue appeared similar to that of the untreated castrated animals. In rats treated with DHT, the total white cell count was significantly higher than in testosterone-implanted rats. Both progesterone and corticosterone implants resulted in significantly smaller mean thymus weights, although these steroids were not as potent as testosterone or oestradiol. Corticosterone, but not progesterone, appeared to cause a significant reduction in circulating lymphocytes. Dihydrotestosterone possessed only half the potency of testosterone in restoring the weights of the accessory sex organs. Serum concentrations of testosterone in orchidectomized old rats were 0·33 ± 0·02 nmol/l and in testosterone-implanted rats 4·8 ± 0·4 nmol/l. These results raise the possibility that testosterone and oestradiol may have caused atrophy of the thymus, while DHT may have retarded regeneration of the thymus without any atrophic effect. It remains to be seen whether the different responses between testosterone and DHT, in both the thymus and accessory sex organs, are due to differences in intrinsic action or differences in the metabolism of the steroids.
J. Endocr. (1987) 113, 51–55
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ABSTRACT
The hypothesis was tested that the inhibitory action of testosterone on thymus growth is mediated by its metabolism to oestradiol. Immature female rats were given s.c. implants of silicone elastomer tubing containing 5 or 20 mg testosterone alone, or together with 25 or 50 mg of an aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD). Some rats received implants containing 5 mg oestradiol or 5 mg dihydrotestosterone (DHT). After 14 days the thymus was removed and weighed. Body weight gain was similar in animals treated with empty implants, or 5 mg testosterone or DHT, or with ATD alone. The combination of testosterone and ATD significantly increased body weight gain, and oestradiol significantly decreased it. Thymus growth was inhibited by both doses of testosterone and by oestradiol, but not by DHT. ATD alone did not inhibit thymus growth, nor did the lower dose of ATD inhibit the action of testosterone. The higher dose of ATD did, however, significantly reduce the inhibitory action of testosterone on the thymus. The inhibitory action of testosterone on the growing thymus may be due, at least in part, to its conversion to oestradiol.
J. Endocr. (1988) 119, 65–67
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ABSTRACT
It has been shown that the thymus can be regenerated in intact old rats by implanting s.c. a stable analogue of LHRH. Old male rats were given s.c. implants of osmotic pumps containing a solution in citrate buffer of the analogue which was given at a rate of 1 μg/h for 28 days. Some animals were given pumps containing buffer alone, and another group of rats was orchidectomized. The animals were killed after 28 days and the tissues weighed and taken for histology. Serum testosterone was measured by radioimmunoassay.
Sham-treated rats had small fatty thymuses, which were poorly organized with a very narrow band of cortex. Animals treated with the analogue of LHRH and those which had been orchidectomized had relatively large thymuses which were multi-lobed in drug-treated rats, and atrophied accessory sex organs. The testes were grossly atrophied in analogue-treated rats. Histologically, the thymus looked healthy, having a wide, thymocyte-filled cortex and a clearly defined corticomedullary junction. Serum testosterone concentrations were similar in orchidectomized and analogue-treated rats.
It is concluded that it is possible to regenerate the thymus in old rats treated with an analogue of LHRH, but the effect is accompanied by chemical castration. It is also clear that the old pituitary gland is susceptible to the desensitizing action of an LHRH analogue.
J. Endocr. (1987) 112, 345–350
