Sex hormone levels in the brain of d-aspartate-treated rats

d-Aspartate (d-Asp) is an endogenous amino acid present in the central nervous system and endocrine glands of various animal taxa. d-Asp is implicated in neurotransmission, physiology of learning, and memory processes. In gonads, it plays a crucial role in sex hormone synthesis. We have investigated the effects of chronic (30 days d-Asp drinking solution) and acute (i.p. injection of 2 μmol/g bw d-Asp) treatments on sex steroid synthesis in rat brain. Furthermore, to verify the direct effect of d-Asp on neurosteroidogenic enzyme activities, brain homogenates were incubated with different substrates (cholesterol, progesterone, or testosterone) with or without the addition of d-Asp. Enzyme activities were measured by evaluating the in vitro conversion rate of (i) cholesterol to progesterone, testosterone, and 17β-estradiol, (ii) progesterone to testosterone and 17β-estradiol, (iii) testosterone to 17β-estradiol. We found that d-Asp oral administration produced an increase of approximately 40% in progesterone, 110% in testosterone, and 35% in 17β-estradiol. Similarly, the results of the acute experiment showed that at 30 min after d-Asp treatment, the progesterone, testosterone, and 17β-estradiol levels increased by 29–35%, and at 8 h they further increased by a 100% increment. In vitro experiments demonstrate that the addition of d-Asp to brain homogenate + substrate induces a significant increase in progesterone, testosterone and 17β-estradiol suggesting that the amino acid upregulates the local activity of steroidogenic enzymes.     

Hypothalamic receptors of sex hormones in the mechanism of the sexual differentiation of the brain in rats

Studies have been made on the content of receptors of estradiol (E2) and testosterone (T) in cytoplasmic and nuclear fractions of the hypothalamus of male and female rats during neonatal development, as well as in adult females after androgenization in neonatal period and adult males castrated within 3 days of postnatal life. It was shown that both E2 and T are present in the blood serum of male and female newborn rats. In female hypothalamus, only E2 receptors were found, whereas in males both types of receptors were revealed, their content being higher than in females. In adult animals subjected to changes in the level of sex hormones in the blood during early neonatal period, changes in concentration of the receptors in the hypothalamic centres of regulation of tonic and cyclic secretion of gonadotropins were found. The data obtained presumably reveal the role of receptors of sex hormones in sex differentiation of the brain.     

Sex hormones and neurotransmitters as mediators for sexual differentiation of the brain

Sexual differentiation of the brain is regarded as a model for environment-dependent brain development mediated by systemic hormones and neurotransmitters. Abnormal concentrations of systemic hormones and/or neurotransmitters, if occurring during a critical period of brain development, can lead to permanent developmental disabilities of fundamental processes of life. Such developmental disabilities appear to be avoidable, at least in part, by improving the external, i.e. psychosocial and natural environment, or by correcting abnormalities in the internal, i.e. metabolic and hormonal environment and, particularly, by correcting abnormal neurotransmitter concentrations (and/or turnover rates) during brain development.     

Sex hormone-dependent brain maturation and sexual behaviour in rats.

In male and female rats the endogenous steroid and gonadotrophin secretion was inhibited by injecting high doses of chlormadinone acetate (CmAc) from day 14 to 24 of life, i. e. during the period of brain maturation. In adulthood the males treated prepubertally with CmAc exhibited reduced sexual activity and fertility, whereas the females did not differ from the controls. More complete sex hormone deficiency during brain maturation was achieved by castration on day 14 of life. Controls were castrated at normal puberty time (40--60 days). Both groups were then substituted with androgens or oestrogens. In the females castrated on day 14 no impairment of sexual behaviour was observed as compared to the later castrated controls. In contrast, the early castrated males showed delayed onset of mounting behaviour. At autopsy, the weights of their sex organs were found to be lower than in the controls despite equal testosterone replacement for several months. These findings speak in favour of a permanently diminished responsiveness to androgens in males having been exposed to more or less severe androgen deficiency during sex specific brain maturation. Hence, the maturation of a male hypothalamus as well as the differentiation appears to depend at least in part on the presence of androgens, whereas in females it runs without hormonal influence.     

Dynamics of the changes in the content of testosterone and estradiol receptors in the hypothalamus of male rats in the course of sexual development

The content of receptors to testosterone and estradiol in hypothalamus of the male rats was studied during their sexual maturation (7, 14, 21, 28, 35 and 42 days). In all the age groups of animals the concentration of receptors to testosterone in the cytoplasmic and nuclear fractions of hypothalamus was at a relatively constant level, except in 7 day old males in which the minimal concentration of cytoplasmic and the maximal concentration of nuclear receptors were noted. The highest values of estradiol-binding sites in cytosol of hypothalamus were observed on the 7th and 14th days and in the nuclear fraction on the 28th, 35th and 42nd days of life. The binding of both the hormones with their receptors is a specific process characterized by a high affinity. A suggestion is put forward that receptors both to androgens and estrogens take part in the brain sexual differentiation.     

Thyroid hormone nuclear receptors and their role in the metabolic action of the hormone

Thyroid hormone nuclear receptor molecules have been characterized as proteins of approximately 49,000 molecular weight existing in cells attached to chromatin and with 4000-8000 copies per nucleus. They bind T3 with Ka of 0.2 X 10(10) l/mol and show microheterogeneity on isoelectric focusing. Hormone responsiveness varies with receptor content in the nucleus and occupancy of receptor by T3. Recent investigations have shown that the receptors are part of the v-erbA related super family of nuclear hormone receptors. At least two types of T3 receptors (TR) exist, one coded by a gene on chromosome 3 (TR beta) and a second coded on chromosome 17 (hTR alpha). Receptors are low in the fetus and, in the adult, are dramatically reduced by starvation, illness and glucagon. Receptors function through binding of T3 or other hormone analogs to a domain in the carboxyl portion of the protein, and binding of the receptor-T3 complex through 'DNA-fingers' to specific response elements as enhancers and located in the 5'-flanking DNA of thyroid hormone responsive genes. Extensive studies on regulation of rat growth hormone have suggested binding of receptor or associated factors to several positions in the 5'-flanking DNA, and recent studies suggest that a crucial area may be a 15 bp segment between bases -179 and -164. Abnormal receptors are believed to be responsible for the syndrome of generalized resistance to thyroid hormone action, but it is yet unclear as to which form (or forms) of the receptor is abnormal in this syndrome.     

Decrease of mu opioid receptors in the brain and in the hypothalamus of the aged male rat

Experiments have been designed in order to analyze whether the binding capability of mu opioid receptors in the brain of the male rat is modified by age. In a first experiment, the number of receptors (Bmax) and the constant of affinity (Ka) for the mu ligand 3H-dihydromorphine (3H-DHM) have been measured in the whole brain of male rats of 2, 15 and 22 months of age. In a second experiment the Bmax and the Ka for 3H-DHM have been evaluated in the hypothalamus of male rats of 2 and 22 months of age. In this experiment it was also investigated whether the administration of exogenous testosterone modifies the number and/or the affinity of the hypothalamic mu receptors. Serum levels of LH, FSH, prolactin and testosterone have been measured by specific RIAs. The results obtained show that: serum testosterone levels are significantly decreased in aged rats, while serum LH and FSH show only a small decline; serum prolactin is higher in old than in young animals; the number of mu receptors in the whole brain of 15 and 22 month old animals and in the hypothalamus of 22 month old rats is significantly lower than in the same tissues of young animals; the administration to old animals of testosterone, in doses able to bring back towards normal serum levels of testosterone, induces a decrease of LH and FSH, but has no effect on serum prolactin titers. Testosterone administration does not modify the number of hypothalamic mu opioid receptors, indicating that the decline of brain mu receptors in old animals is not the consequence of the physiological decline of testosterone secretion; in no instance the Ka for the mu ligand is significantly affected.     

Sex determination and sexual differentiation in the avian model

The sex of birds is determined by the inheritance of sex chromosomes (ZZ male and ZW female). Genes carried on one or both of these sex chromosomes control sexual differentiation during embryonic life, producing testes in males (ZZ) and ovaries in females (ZW). This minireview summarizes our current understanding of avian sex determination and gonadal development. Most recently, it has been shown that sex is cell autonomous in birds. Evidence from gynandromorphic chickens (male on one side, female on the other) points to the likelihood that sex is determined directly in each cell of the body, independently of, or in addition to, hormonal signalling. Hence, sex-determining genes may operate not only in the gonads, to produce testes or ovaries, but also throughout cells of the body. In the chicken, as in other birds, the gonads develop into ovaries or testes during embryonic life, a process that must be triggered by sex-determining genes. This process involves the Z-linked DMRT1 gene. If DMRT1 gene activity is experimentally reduced, the gonads of male embryos (ZZ) are feminized, with ovarian-type structure, downregulation of male markers and activation of female markers. DMRT1 is currently the best candidate gene thought to regulate gonadal sex differentiation. However, if sex is cell autonomous, DMRT1 cannot be the master regulator, as its expression is confined to the urogenital system. Female development in the avian model appears to be shared with mammals; both the FOXL2 and RSPO1/WNT4 pathways are implicated in ovarian differentiation.     

Lordotic behavior in male rats: genetic and hormonal regulation of sexual differentiation

The male offspring of Long-Evans rats treated with the aromatization inhibitor ATD (1,4,6-androstatriene-3,17-dione) during pregnancy show high levels of lordotic behavior when treated with estrogen and progesterone in adulthood. The male offspring of Sprague-Dawley dams treated in the same way show only a slight facilitation of lordotic potential. These strain differences could reflect strain differences in gestation length and therefore differences in the timing of the sensitive period of sexual differentiation; they could reflect differences in the sensitivity to the defeminizing actions of gonadal hormones; or they could reflect differences in the sensitivity to ATD treatment. We therefore directly compared the effects of prenatal and early postnatal treatment with ATD on the potential of male Long-Evans and Sprague-Dawley rats to show lordosis when given estrogen and progesterone in adulthood. In both strains ATD treatment facilitated adult lordotic behavior. Treatment appeared to have a greater effect in the Long-Evans strain. However, control Long-Evans males were substantially more responsive to hormone treatment in adulthood than were Sprague-Dawley males. In the Long-Evans strain short-term ATD treatment (Days 20-23 of pregnancy) was as effective as long-term treatment (Days 10-23). In the Sprague-Dawley strain, ATD treatment was most effective when given prenatally and postnatally. Strain differences in hormonal sensitivity best account for the present findings.     

Sex steroids and their receptors: molecular actions on brain cells

Sex steroids exert actions of paramount importance on brain cells. They contribute to shape the central nervous system during embryo development. They modulate the formation and the turnover of the interconnections between neurons. They control the function of glial cells. And they do it through a signaling machinery that is apparently simple, but that hides a level of complexity that has been unveiled only in part. Different receptor isoforms, different interactions between receptors and co-regulators, chains of events originating at the cell membrane and leading to effects in the nucleus (or the other way around) all interact to determine selective modulations of brain cells. All these actions end up in phenomenal effects on brain function that change through adolescence, pregnancy, adulthood, up to menopause and ageing. Many of these actions are relevant for degenerative processes and research may offer soon new strategies to counteract these diseases.     

Effect of alcohol on the content of sex steroid receptors in the hypothalamus and hypophysis of male rats

In experimental dipsomania model (formation of physical dependence by method of intensive alcoholization) we have studied receptor binding of testosterone (T) and estradiol (E2) in the hypothalamus and pituitary body of mature male rats. Administration (at 10 and 16 h) of 25% ethanol-saline solution at a dose of 7.5 g/kg of body weight in the course of 5 days significantly decreased serum T level but did not change serum LH and FSH levels. Essential reduction of the nuclear androgen receptors in the preoptic-anterior hypothalamic area (POA), mediobasal hypothalamus (MBH) and adenohypophysis was noted in alcohol-treated rats. Unlike androgen receptors the number of the nuclear E2-binding sites in PaO was significantly increased in these males. Thus the results of the present paper demonstrate that multiple administration of ethanol stipulates deficit of serum T, androgen receptors in MBH and pituitary body that possibly results in separation of negative feedback mechanism between the gonads and pituitary body. Increase of specific binding of E2 to nuclear receptors in PoA might appear to explain feminization of alcohol-treated rats.     

Age-related changes in sexual function and steroid-hormone receptors in the medial preoptic area of male rats

Testosterone is the main circulating steroid hormone in males, and acts to facilitate sexual behavior via both reduction to dihydrotestosterone (DHT) and aromatization to estradiol. The mPOA is a key site involved in mediating actions of androgens and estrogens in the control of masculine sexual behavior, but the respective roles of these hormones is not fully understood. As males age they show impairments in sexual function, and a decreased facilitation of behavior by steroid hormones compared to younger animals. We hypothesized that an anatomical substrate for these behavioral changes is a decline in expression and/or activation of hormone receptor-sensitive cells in the mPOA. We tested this by quantifying and comparing numbers of AR- and ERα-containing cells, and Fos as a marker of activated neurons, in the mPOA of mature (4–5 months) and aged (12–13 months) male rats, assessed one hour after copulation to one ejaculation. Numbers of AR- and ERα cells did not change with age or after sex, but the percentage of AR- and ERα-cells that co-expressed Fos were significantly up-regulated by sex, independent of age. Age effects were found for the percentage of Fos cells that co-expressed ERα (up-regulated in the central mPOA) and the percentage of Fos cells co-expressing AR in the posterior mPOA. Interestingly, serum estradiol concentrations positively correlated with intromission latency in aged but not mature animals. These data show that the aging male brain continues to have high expression and activation of both AR and ERα in the mPOA with copulation, raising the possibility that differences in relationships between hormones, behavior, and neural activation may underlie some age-related impairments.     

Distribution of kappa opioid receptors in the brain of young and old male rats

The experiments to be described have been designed in order to: (a) provide new information on the concentrations of opioid kappa receptors in different regions of the brain of the male rats; and (b) to analyze whether the density of brain kappa receptors might be modified by the process of aging. The concentration of kappa receptors was investigated in the hypothalamus, amygdala, mesencephalon, corpus striatum, hippocampus, thalamus, frontal poles, anterior and posterior cortex collected from male rats of 2 and 19 months of age. 3H-bremazocine (BRZ) was used as the ligand of kappa receptors, after protection of mu and delta receptors respectively with dihydromorphine and d-ala-d-leu-enkephalin. The results obtained show that: (1) in young male rats, the number of kappa opioid receptors is different in the various brain areas examined: the hypothalamus and the striatum have a concentration of kappa binding sites which is significantly higher than that found in the mesencephalon and in the amygdala; much lower concentrations of kappa binding sites have been found in the thalamus, the frontal poles, the hippocampus, the anterior and posterior cerebral cortex. (2) Aging exerts little influence on the number of kappa receptors in the majority of the brain structures considered. However in the amygdala and in the thalamus the number of kappa receptors was increased in old animals. To the authors' knowledge, the data here presented are the first ones which suggest that age may increase rather than decrease the number of neurotransmitter receptors in the brain.