Actions of sex hormones on the brain.

1. The brain is a target for sex steroid hormones. As a result of sex hormone actions on the brain various behavioral changes are observed in animal and man. This paper gives a brief overview over the multiple central nervous functions that are under modulatory control of sexual hormones and describes the complex sex steroid actions on the brain by giving an example for "activating" and "organizing" effects of estrogens on noradrenergic neurons in the brain of rats. 2. Estradiol-17 beta induced sex specific alterations in the turnover of noradrenaline in the preoptic area and mediobasal hypothalamus showing "female" or "male" responses. 3. Neonatal manipulations of female rat pups by testosterone, estradiol-17 beta or 4-hydroxyestradiol-17 beta defeminized the "female" response of the noradrenaline turnover in the preoptic area. 4. Defeminization was not observed when neonatal females received the non aromatizable sex steroid dihydrotestosterone. 5. Activating and organizing effects of sex steroids on animal brain as shown here for noradrenergic neurons are discussed in relation to the regulation of behavior in man. Special regard is given to psychic disorders that might be associated with abnormalities in the production or metabolism of or the response to gonadal hormones.

Investigation of the corticosteroid receptor system in rat hippocampus by ion exchange fast protein liquid chromatography.

In order to study the receptor system for adrenocortical steroids, hippocampal cytosolic preparations--containing both type I and type II receptors--were subjected to anion exchange fast protein liquid chromatography (FPLC). With running buffer containing Tris, EDTA, and glycerol three peaks (1-3) were eluted from the column at 220, 400 and 560 mM NaCl respectively regardless of whether [3H]corticosterone or [3H]RU 28362 had been used as radiotracer. None of the peaks was caused by serum transcortin as revealed by control studies. However, the sequestering influence of transcortin on receptor binding of corticosterone could be demonstrated by the FPLC technique with mixtures containing serum and hippocampus cytosol. Competition experiments with cytosolic samples revealed that type I receptor was present only in peaks 2 and 3 while type II was found in all three peaks in variable amounts, depending on the presence of molybdate. When molybdate was added to the running buffer only two peaks (2 and 3) were eluted, both containing type I and type II receptors. Peak 1 was attributed to the activated type II receptor while peak 2 represented nonactivated receptors. The origin of peak 3 remains uncertain. The data indicate that molybdate must be present in the cytosolic preparation and in the running buffer to keep type II receptor in its nonactivated form. Type I receptor was probably not transformed into the activated form in the absence of molybdate but lost binding capacity and/or affinity for corticosterone.

Inhibition of synaptosomal high-affinity uptake of dopamine and serotonin by estrogen agonists and antagonists.

High-affinity uptake of dopamine and serotonin into a synaptosomal preparation from rat cerebral cortex was inhibited by a number of estrogen agonists and antagonists in vitro in a stereoselective and competitive manner. The most potent estrogenic inhibitors in the dopaminergic and serotonergic system were ethinylestradiol (KI = 558 nM) and 2-hydroxyethinylestradiol (KI = 226 nM), respectively. Structure-activity relationships are discussed and compared with the effects of estrogens on noradrenaline uptake. However, as all physiologically occurring estrogens inhibited amine uptake only in the micromolar concentration range it seems unlikely that this direct interaction of estrogen with the amine carrier is responsible for the changes in dopamine and serotonin uptake observed during the estrous cycle or after in vivo administration of estrogens and/or progesterone.

Improved model for the induction of proestrus-like gonadotropin surges in long-term ovariectomized rats.

In long-term (greater than 4 wk) ovariectomized rats the positive response of the gonadotropin release apparatus to a priming dose of estradiol is moderate as compared with that of proestrous rats exposed to endogenous estradiol. In the present study, high sensitivity to estrogen was restored in long-term ovariectomized rats by pretreatment with estradiol benzoate (EB, 20 micrograms, day 0) and progesterone (P, 2.5 mg, day 3). Estradiol benzoate (20 micrograms) given on day 5 induced proestrus-like surges of LH and FSH in the afternoon on day 6. Additional administration of P (2.5 mg at noon on day 6) had a facilitatory effect. Stimulation of LH release could be evoked in rats by the described regimen 1, 6 or 50 wk after ovariectomy. The long-term ovariectomized rat injected with EB and P as described might provide a useful model for neuroendocrinological investigations on the gonadotropin surge mechanism.

Photoaffinity labeling with [3H]RU 28362: a powerful tool for the study of rat brain glucocorticoid receptors.

In order to study the receptor system for adrenocortical steroids in rat brain the synthetic glucocorticoid RU 28362 (11 beta, 17 beta-dihydroxy-6-methyl-17 alpha-(1-propynyl) androsta-1,4,6-trien-3-one) has been used for photoaffinity labeling. Competition and dissociation studies revealed a single class of binding sites for RU 28362 in rat brain cytosol. Photoaffinity labeling was performed by u.v.-irradiation for 2 min with a coupling efficiency of about 25%. The high efficiency permitted investigation of crude cytosolic preparations under denaturating conditions. Sodium dodecyl sulfate (SDS) and high resolution two-dimensional gel electrophoresis confirmed the high specificity of the photoaffinity labeling. The molecular weight (93 kD) as well as the isoelectric point (5.6) evaluated by these methods corresponded well to data reported for the classical glucocorticoid receptor in rat liver.

Time-dependent effects of oestradiol and progesterone on hypothalamic catecholamine turnover in ovariectomized rats.

Long-term ovariectomized rats received a single injection of 20 micrograms oestradiol benzoate (OB) which reduced the serum levels of LH for at least 3 days. The inhibitory effects were accompanied by time-dependent alterations of noradrenaline and dopamine turnover rates in the mediobasal hypothalamus (MBH) and the preoptic-anterior hypothalamic brain area (POAH). Oestradiol markedly interfered with the time-dependent variations of noradrenaline and dopamine turnover seen in the MBH of untreated ovariectomized animals during daylight hours. In the POAH the turnover rate of noradrenaline decreased 2 days after priming with OB and then increased in the afternoon of day 3. The increase of noradrenaline turnover in the POAH was accompanied by a low afternoon turnover rate of dopamine in the MBH and by an increased sensitivity of the LH secretory system to progesterone. Dopamine and noradrenaline turnover involve a time element. While the negative feedback actions of oestradiol do not seem to be associated with changes in dopamine or noradrenaline turnover, the results support the view that the induction of LH afternoon surges depends upon an increase of stimulatory noradrenergic inputs to the POAH and a decrease of inhibitory dopaminergic inputs in the MBH.

Antioestrogen inhibition of oestradiol-induced alterations in hypothalamic noradrenaline turnover.

Long-term (4-6 weeks) ovariectomized rats were injected with either oestradiol benzoate (OB; 20 micrograms s.c.) or monohydroxytamoxifen (MTAM; 0.2 mg i.p.) plus OB. Oestradiol benzoate was administered at 12.00 h on day 0 and MTAM was given immediately before OB, followed by further injections twice daily to maintain sufficiently high antioestrogen levels. When given alone, OB reduced the serum levels of LH during the morning (08.00-09.00 h) and afternoon (17.30-18.30 h) hours of day 3 after priming. The feedback actions of OB on LH release were accompanied by time-dependent alterations of noradrenaline turnover in the preoptic-anterior hypothalamic brain area (POAH). On day 3 after priming the noradrenaline turnover rate was reduced in the morning and increased in the afternoon. The increase correlated with an enhanced sensitivity of the LH secretory system to progesterone. The antioestrogen MTAM blocked the OB-induced sensitization of LH release to the stimulatory action of progesterone and interfered with the stimulatory long-term effect of oestradiol on hypothalamic noradrenaline turnover. The data strongly support the view that the oestrogen-induced afternoon increase of noradrenaline turnover in the POAH represents a prerequisite for the induction of LH surges. The stimulatory effect of oestradiol on hypothalamic noradrenaline turnover seems to be mediated by a classical oestrogen receptor mechanism.

Sex-specific effects of estradiol on hypothalamic noradrenaline turnover in gonadectomized rats.

In long-term gonadectomized rats of either sex a single injection of estradiol-17 beta 3-benzoate acutely decreased the turnover rates of noradrenaline in the preoptic-anterior hypothalamic brain area (POAH) and reduced the serum concentrations of luteinizing hormone (LH). On the afternoon of day 3, between 72 and 78 h after estrogen priming, an increase of noradrenaline turnover was observed in female rats, whereas the turnover remained low in males. The increase of noradrenergic activity in female rat brain occurred at the time when LH release could be stimulated by progesterone. On the other hand, the low noradrenergic activity in the POAH of male rats correlated with the lack of stimulatory progesterone effects on LH secretion. The data indicate that estradiol induces a sex-specific increase of noradrenaline turnover in the POAH. This increase appears to be a prerequisite for the induction of LH surges.

Interaction of non-steroidal antiestrogens with dopamine receptor binding.

The ability of various estrogen antagonists and agonists to compete with [3H]spiroperidol, [3H]domperidone, [3H]dihydroalprenolol, [3H]dihydroergocryptine, [3H]dopamine or [3H]5-hydroxytryptamine for binding to membrane preparations from rat brain tissue was tested. The non-steroidal triphenylethylene-type antiestrogens with an amine side chain--enclomiphene, nitromifene, tamoxifen and zuclomiphene--were found to be competitive inhibitors of [3H]spiroperidol (Kd = 0.12 nM; Bmax = 101 fmol/mg protein) and [3H]domperidone (Kd = 0.62 nM; Bmax = 86 fmol/mg protein) binding to striatal membranes. The Ki values ranged from 4-12 microM. Estradiol-17 beta (Ki = 480 microM) or diethylstilbestrol (Ki = 63 microM) were much less effective inhibitors exhibiting noncompetitive interaction with the in vitro binding of [3H]spiroperidol. The pharmacological relevance of the antiestrogen interactions with dopamine receptor binding is discussed with respect to adverse effects of the in vivo administered compounds such as nausea and vomiting.

Preferential uptake of norepinephrine into dopaminergic terminals of a synaptosomal preparation from rat cerebral cortex.

In a synaptosomal preparation from male rat cerebral cortex only 34% of total norepinephrine (NE) uptake could be inhibited by nanomolar concentrations of desmethylimipramine (DMI) with an apparent IC50 value of 0.37 nM. The residual uptake was efficiently inhibited by micromolar concentrations of DMI (IC50 = 4.0 microM). In synaptosomes from the hypothalamus, 74% of total NE uptake could be blocked by DMI with an IC50 of 0.1 nM whereas in synaptosomes from the striatum the IC50 for DMI inhibition was 3.8 microM. It is concluded that in synaptosomes from rat cerebral cortex only 34%, and in synaptosomes from the hypothalamus 74% of total NE are taken up by noradrenergic nerve terminals whereas the residual NE uptake occurs in dopaminergic nerve endings.

Effects of fasting on the distribution of cytoplasmic and nuclear oestrogen receptors in rat liver, uterus, pituitary and hypothalamus before and after exogenous oestrogen administration.

The accumulation of oestrogen receptors in the liver cell nuclei of intact female rats 45 min after administration of 100 micrograms 17 alpha-ethynyloestradiol-17 beta i.p., decreased progressively during a 72-h fast from 2550 +/- 860 to 257 +/- 67 fmol/mg DNA, a level not significantly different from that in uninjected animals. Cytoplasmic oestrogen receptor concentrations also decreased, but only to about 60% of the original level (from 84.1 +/- 27.5 to 50.3 +/- 2.09 fmol/mg protein during the fast). Similar differences were found when these parameters were examined in normally fed and 72-h-fasted ovariectomized rats. On the other hand these parameters were unaffected in uterus, pituitary and hypothalamus. Uterine cytoplasmic receptor concentrations remained at about 500 fmol/mg protein during the fasting period, those in the pituitary and hypothalamus at about 230 and 30 fmol/mg protein, respectively. Nor was in vivo translocation in these organs affected by fasting. Regardless of nutritional status, the nuclear oestrogen receptor concentrations in uterus rose from about 500 to 2000 fmol/mg DNA after ethynyloestradiol administration, those in the pituitary and hypothalamus from approximately 250 to 2000 and from 250 to 500 fmol/mg DNA respectively.

Interactions of methylestrogens with cytoplasmic and nuclear estrogen receptors in rat pituitary gland, hypothalamus and uterus.

The in vitro affinities to cytoplasmic estrogen receptors of the methylestrogens 2-methylestradiol-17 beta, 4-methylestradiol-17 beta and 4-hydroxy-2-methylestradiol-17 beta, which are useful probes to test the biological importance of 2- or 4-hydroxylation of estradiol-17 beta (catecholestrogen formation), have been determined in hypothalamic, pituitary and uterine tissue of the ovariectomized rat. Moreover, the in vivo capacity of these compounds to translocate estrogen receptors into the cell nucleus of pituitary and uterine tissue has been studied. Methylestrogens exhibited estrogen receptor affinities which were not significantly different from the binding affinity of estradiol-17 beta. When given at a high dose (100 micrograms/animal) their nuclear translocation capacity was equal (4-methylestradiol-17 beta) or even higher (2-methylestradiol-17 beta) than that of estradiol-17 beta. However, at a low dose (5 micrograms/animal) 4-methylestradiol was completely ineffective in both the pituitary gland and the uterus, and 2-methylestradiol-17 beta was less potent than estradiol-17 beta in the pituitary gland.

Re-establishment of stimulatory estradiol effects on luteinizing hormone secretion in long term ovariectomized rats.

The ability of a single injection of estradiol benzoate (EB, 20 micrograms) to induce proestrus-like surges of luteinizing hormone (LH) on the day following EB administration was tested in long term ovariectomized rats which had been pretreated with EB (20 micrograms) and/or progesterone (P, 2.5 mg). LH afternoon surges could be evoked only in those animals which had previously received both ovarian hormones, EB and P. Pretreatment with a single hormone was ineffective. Moreover, in rats preinjected only with P the LH surge was shifted from afternoon to morning hours.

Estrogen agonistic and antagonistic action of 8 non-steroidal antiestrogens on progestin receptor induction in rat pituitary gland and uterus.

All 8 non-steroidal antiestrogens tested considerably increased progestin receptor concentration in the uterus and, to a lesser extent, in the pituitary of ovariectomized rats. However, the pituitary was more sensitive than the uterus to the estrogen antagonistic action of these compounds, in that monohydroxytamoxifen, LY 117,018, enclomiphene, nitromifen, nafoxidine and trans-tamoxifen completely blocked progestin receptor induction by estradiol benzoate. In these tissues the order of the in vitro binding affinity of antiestrogens to cytoplasmic estrogen receptors was not correlated with either their in vivo estrogen agonistic or antagonistic potency.

Competition by monophenolic estrogens and catecholestrogens for high-affinity uptake of [3H](-)-norepinephrine into synaptosomes from rat cerebral cortex and hypothalamus.

High affinity uptake of [3H](-)-norepinephrine (NE) was investigated in synaptosomes from rat cerebral cortex (Km = 360 +/- 30 nM) and hypothalamus (Km = 307 +/- 90 nM). Estrogens but not androgens, glucocorticoids or progestin interfered competitively with NE uptake. Ethinylestradiol was the most effective competitor tested, its Ki value being 200 nM in the cortex and 144 nM in the hypothalamus. Stereospecificity of the inhibitory effect of estradiol-17 beta with a preference for the 17 beta-hydroxy group was indicated by the ineffectiveness of estradiol-17 alpha and estrone as competitors. A-ring substitution of estradiol-17 beta or ethinylestradiol by hydroxyl groups in positions 2 and 4 (yielding catecholestrogens) or methyl substitution in positions 2 and 4 (yielding methylestrogens) significantly reduced the inhibitory potency of the estrogen. Methoxylation in positions 2, 4 or 11 beta completely abolished the competitive action of estradiol-17 beta or ethinylestradiol on NE uptake.

Do catecholestrogens interact with the in vitro binding of radioligands to catecholamine receptors?

Using a competitive binding assay the effects of 2-hydroxyestradiol-17 beta, 4-hydroxyestradiol-17 beta, estradiol-17 beta and progesterone on the binding of tritiated catecholaminergic ligands to membrane preparations from rat brain and pituitary gland were studied. Up to a concentration of 10(-5) M none of the steroids tested was able to displace [3H]spiroperidol, [3H]dihydroergocryptine or [3H]dihydroalprenolol. The data suggest that the catecholestrogens do not interfere directly with the binding of catecholaminergic ligands to dopaminergic, alpha-adrenergic or beta-adrenergic receptors in the central nervous system. The view that a catechol structure is not essential for the interaction with dopaminergic receptors was further supported by the results obtained from additional studies on the competition of O-methylated and deaminated dopamine metabolites with [3H]spiroperidol binding.

Estrogen induction of progestin receptors in pituitary, hypothalamic and uterine cytosol of androgenized female rats.

Female rats treated neonatally with a single dose (1.25 mg/animal) of testosterone propionate and ovariectomized when adult did not respond to a priming dose (20 micrograms/animal) of estradiol-17 beta 3-benzoate and subsequent application of progesterone (2.5 mg/animal) 72 h later with an afternoon surge of luteinizing hormone, which could be induced by the same hormonal regimen in neonatally oil-treated long-term ovariectomized female rats. However, both treatment groups responded equally well to the estrogen stimulus with an increase in cytosolic progestin receptors in hypothalamic and pituitary, as well as uterine tissue. It therefore seems unlikely that the observed loss of sensitivity of the gonadotropin release mechanism in neonatally androgenized, estrogen-primed female rats to a progesterone stimulus can be explained by a loss of progestin receptor induction capacity of estrogen/progestin target tissues involved in gonadotropin secretion.

Effects of oestradiol benzoate and progesterone on luteinizing hormone release and catecholamine turnover in the preoptic-hypothalamic brain area of ovariectomized rats.

At noon, long-term (4-6 weeks) ovariectomized rats were exposed for 6-78 h to a single subcutaneous injection of oestradiol benzoate (20 micrograms) which significantly reduced the serum levels of LH over the whole time-interval investigated. The negative feedback action of oestradiol was accompanied by reduced turnover of both noradrenaline and dopamine in the preoptic-anterior hypothalamic brain area (POAH), but not in the mediobasal hypothalamus, 6, 68 and 72 h after administration of the hormone. Between 72 and 78 h after oestradiol-priming an afternoon increase of noradrenaline turnover was observed in the POAH. In rats primed with oestradiol benzoate for 72 h, short-term exposure (6 h) to progesterone (2.5 mg) induced a marked surge of serum LH and FSH in the late afternoon. In the POAH of these rats progesterone did not interfere with the afternoon increase of noradrenaline turnover induced by oestradiol-priming. However, it markedly increased the dopamine turnover rate of primed rats, thus reversing the inhibitory action of oestradiol benzoate on the dopaminergic system of the POAH. It is concluded that both the noradrenergic and the dopaminergic neurones of the POAH are involved in the negative and positive feedback actions of oestradiol and progesterone on LH and FSH release. The paper discusses whether the oestradiol-induced afternoon increase in noradrenaline turnover represents a prerequisite for the induction of LH surges by progesterone.

Neonatal defeminization of the luteinizing hormone release mechanism by catecholestrogens: different potencies of 2- and 4-hydroxyestradiol.

Female rats were neonatally treated with estradiol-17 beta-benzoate or the long-acting dibenzoate esters of the isomeric catecholestrogens, 2-hydroxyestradiol-17 beta and 4-hydroxyestradiol-17 beta. Estrogen benzoates were administered subcutaneously from day 1 to 5 of life at doses of 0.05, 0.10, 0.50 and 1.00 micrograms/day. All rats were ovariectomized as adults and, 4 weeks later, the luteinizing hormone (LH) response to progesterone (2.5 mg) was tested after priming with estradiol-17 beta-benzoate (20 micrograms). At a dose of 0.5 micrograms/day, estradiol-17 beta-benzoate and 4-hydroxyestradiol-17 beta-dibenzoate were equally effective in neonatally defeminizing the LH surge mechanism. In contrast, up to a dose of 1.00 micrograms/day, 2-hydroxyestradiol-17 beta-dibenzoate did not interfere with the LH response in adult life. In the pituitary gland and uterus of the neonatally defeminized rats estrogen responsiveness of cytosolic progestin receptor induction was unimpaired. Moreover, in the uterus of these rats nuclear translocation of cytosolic progestin receptors was intact.