Perinatal exposure to tributyltin affects feeding behavior and expression of hypothalamic neuropeptide Y in the paraventricular nucleus of adult mice.

Organotins such as tributyltin chloride (TBT), are highly diffused environmental pollutants, which act as metabolism disrupting chemicals, i.e. may interfere with fat tissue differentiation, as well as with neuroendocrine circuits, thus impairing the control of energetic balance. We have previously demonstrated that adult exposure to TBT altered the expression of neuropeptides in the hypothalamus. In this study, we orally administered daily a solution containing oil, or TBT (0.25, 2.5, or 25 µg/kg body weight/day) to pregnant females from gestational day 8 until birth, and to their pups from day 0 until post-natal day 21. Our results showed that TBT exposure of female mice during gestation and of pups during lactation permanently altered the feeding efficiency of pups of both sexes and subcutaneous fat distribution in adult males. In addition, the neuropeptide Y system was affected at the level of the paraventricular nucleus, with a decrease in immunoreactivity in both sexes (significant in females for all TBT doses and in males only for intermediate TBT doses), while no effect was observed in other hypothalamic areas (arcuate, ventromedial and dorsomedial nuclei). Metabolic syndrome, as well as obesity and diabetes, which are significant health issues, are considered multifactorial diseases and may be caused by exposure to metabolic disruptors, both in adults and during perinatal life. In addition, our work indicates that TBT doses defined as the tolerably daily intake had a profound and sex-specific long-term effect.

Postnatal genistein administration selectively abolishes sexual dimorphism in specific hypothalamic dopaminergic system in mice.

As demonstrated in previous studies, early postnatal genistein (GEN) administration to mice pups of both sexes, at doses similar to that of infant soy-based formulas, may affect the development of some steroid-sensitive neuronal circuits (i.e. nitrergic and vasopressinergic systems), causing irreversible alterations in adults. Here, we investigated the hypothalamic and mesencephalic dopaminergic system (identified with tyrosine hydroxylase immunohistochemistry). GEN administration (50 mg/kg) to mice of both sexes during the first week of postnatal life specifically affected tyrosine hydroxylase immunohistochemistry in the hypothalamic subpopulation of neurons, abolishing their sexual dimorphism. On the contrary, we did not observe any effects in the mesencephalic groups. Due to the large involvement of dopamine in circuits controlling rodent sexual behavior and food intake, these results clearly indicate that the early postnatal administration of GEN may irreversibly alter the control of reproduction, of energetic metabolism, and other behaviors. These results suggest the need for a careful evaluation of the use of soy products in both human and animal newborns.

Sexually Dimorphic Effect of Genistein on Hypothalamic Neuronal Differentiation in Vitro.

Developmental actions of estradiol in the hypothalamus are well characterized. This hormone generates sex differences in the development of hypothalamic neuronal circuits controlling neuroendocrine events, feeding, growth, reproduction and behavior. In vitro, estradiol promotes sexually dimorphic effects on hypothalamic neuritogenesis. Previous studies have shown that developmental actions of the phytoestrogen genistein result in permanent sexually dimorphic effects in some behaviors and neural circuits in vivo. In the present study, we have explored if genistein, like estradiol, affects neuritogenesis in primary hypothalamic neurons and investigated the estrogen receptors implicated in this action. Hypothalamic neuronal cultures, obtained from male or female embryonic day 14 (E14) CD1 mice, were treated with genistein (0.1 µM, 0.5 µM or 1 µM) or vehicle. Under basal conditions, female neurons had longer primary neurites, higher number of secondary neurites and higher neuritic arborization compared to male neurons. The treatment with genistein increased neuritic arborization and the number of primary neurites and decreased the number of secondary neurites in female neurons, but not in male neurons. In contrast, genistein resulted in a significant increase in primary neuritic length in male neurons, but not in female neurons. The use of selective estrogen receptor antagonists suggests that estrogen receptor α, estrogen receptor ß and G-protein-coupled estrogen receptors are involved in the neuritogenic action of genistein. In summary, these findings indicate that genistein exerts sexually dimorphic actions on the development of hypothalamic neurons, altering the normal pattern of sex differences in neuritogenesis.

Estrogen receptor beta and G protein-coupled estrogen receptor 1 are involved in the acute estrogenic regulation of arginine-vasopressin immunoreactive levels in the supraoptic and paraventricular hypothalamic nuclei of female rats.

The ovarian hormone 17ß-estradiol is known to regulate the release, expression and immunoreactivity of arginine-vasopressin (AVP) in the supraoptic and paraventricular hypothalamic nuclei of rodents. Previous studies have shown that estrogen receptor α is involved in the effects of chronic estradiol administration on arginine-vasopressin immunoreactivity in the female rat hypothalamus. In this study we have examined the effect of an acute administration of estradiol or specific agonists for estrogen receptors α, ß and G protein-coupled estrogen receptor 1 on the immunoreactivity of arginine-vasopressin in the hypothalamus of adult ovariectomized female rats. Acute estradiol administration resulted in a significant decrease in the number of arginine-vasopressin immunoreactive neurons in the supraoptic and paraventricular nuclei after 24 h. The effects of the specific estrogen receptors agonists suggest that the action of estradiol on arginine-vasopressin immunoreactivity is mediated in the supraoptic nucleus by G protein-coupled estrogen receptor 1 and in the paraventricular nucleus by both estrogen receptor ß and G protein-coupled estrogen receptor 1. Thus, in contrast to previous studies on the effect of chronic estrogenic treatments, the present findings suggest that estrogen receptor ß and G protein-coupled estrogen receptor 1 mediate the acute effects of estradiol on arginine-vasopressin immunoreactivity in the hypothalamus of ovariectomized rats.

Chronic treatment with tributyltin induces sexually dimorphic alterations in the hypothalamic POMC system of adult mice.

Tributyltin (TBT), an antifouling agent found in boat paints, is a common contaminant of marine and freshwater ecosystems. It is rapidly absorbed by organic materials and accumulated in many aquatic animals. Human exposure may depend on ingestion of contaminated food or by indirect exposure from household items containing organotin compounds. TBT is defined as an endocrine disruptor compound (EDC) because it binds to androgen receptors. Moreover, it is also included on the list of metabolic disruptors. The brain is a known target of TBT and this compound interferes with the orexigenic system, inducing a strong decrease in NPY expression in the hypothalamus. In the present experiment, we investigated the effect of a chronic treatment with TBT on the mouse anorexigenic system in both sexes, to look at the pro-opiomelanocortin (POMC) expression in the paraventricular (PVN), dorsomedial (DMN), ventromedial (VMN), and arcuate (ARC) hypothalamic nuclei. The results show a sexually dimorphic effect of TBT on both systems. TBT induced a significant decrease of POMC-positive structures only in female mice in DMN, ARC, and in PVN for both sexes. Apparently, these results show that TBT may interfere with the anorexigenic system in hypothalamic areas involved in the control of food intake, by inhibiting POMC in a sexually dimorphic way. In conclusion, in addition to having a direct effect on fat tissue, the effects of TBT as metabolic disruptor, may be due to gender-specific actions on both orexigenic and anorexigenic hypothalamic systems.

Perinatal exposure to genistein affects the normal development of anxiety and aggressive behaviors and nitric oxide system in CD1 male mice.

Genistein is a phytoestrogen, particularly abundant in soybeans, that is able to bind estrogen receptors exerting both estrogenic and antiestrogenic activities. Genistein is largely present in the human diet even during pregnancy. Embryos and fetuses are therefore, commonly exposed to genistein during the development and after birth. In the present study, we used a murine model as a test end-point to investigate the effects of early exposure to genistein on adult male behavior and related neural circuits. Daily exposure of dams to genistein (100 µg/g of body weight) during late pregnancy and early lactation, produced in male offspring, when adults, significant changes in anxiety and aggressive behaviors. Moreover, we found statistically significant variations in the number of neuronal nitric-oxide synthase positive cells in the amygdala. In conclusions, these data indicate that early exposure to phytoestrogens may induce life-long effects on the differentiation of brain structures and behaviors.

Synergic effects of estradiol and progesterone on regulation of the hypothalamic neuronal nitric oxide synthase expression in ovariectomized mice.

Nitric oxide (NO) is a gaseous neurotransmitter that plays an important role in the regulation of sexual behavior in rodents. NO is produced, within the central nervous system, by the enzyme neural NO synthase (nNOS) whose expression is influenced by gonadal hormones. In previous studies, we demonstrated that part of the nitrergic hypothalamic and limbic system is influenced, in physiological conditions, by the hormonal fluctuations during the estrous cycle, but we were unable to distinguish among the role played by progesterone (P) or estradiol (E(2)) in inducing these changes. In the present study, we investigated the effects of E(2) and P (alone or together) on the nitrergic system of gonadectomized female mice, following a timing of administration that emulates the different phases of estrous cycle. In parallel, we tested the influence of the two hormones on sexual behavior, confirming that P works in synergistic fashion with E(2) to facilitate female receptivity. The quantitative analysis of nNOS-ir system demonstrated a statistically significant variation in the number of positive cells only in those part of the limbic-hypothalamic nitrergic system that are affected in cycling females, i.e. the bed nucleus of the stria terminalis, the arcuate nucleus and the medial preoptic area, with the highest number of positive neurons observed in E(2)+P group. The variable effects of E(2) and P may depend on the different distribution of their receptors within the analyzed nuclei, but the relationships among variations of estrogen and progesterone levels and in vivo modulation of nNOS expression remain unknown and needed further investigations.

Effects of estrous cycle and xenoestrogens expositions on mice nitric oxide producing system.

Nitric oxide (NO)-containing neurons are widely distributed within the central nervous system, including regions involved in the control of reproduction and sexual behavior. Nitrergic neurons may co-localize with gonadal hormone receptors and gonadal hormones may influence neuronal NO synthase expression in adulthood as well as during development. In rodents, the female, in physiological conditions, is exposed to short-term changes of gonadal hormones levels (estrous cycle). Our studies, performed in mouse hypothalamic and limbic systems, reveal that the expression of neuronal NO synthase may vary according to the rapid variations of hormonal levels that take place during the estrous cycle. This is in accordance with the hypothesis that gonadal hormone activation of NO-cGMP pathway is important for mating behavior. NO-producing system appears particularly sensitive to alterations of endocrine balance during development, as demonstrated by our experiments utilizing perinatal exposure to bisphenol A, an endocrine disrupting chemical. In fact, significant effects were detected in adulthood in the medial preoptic nucleus and in the ventromedial subdivision of the bed nucleus of the stria terminalis. Therefore, alteration of the neuronal NO synthase expression may be one of the causes of the important behavioral alterations observed in bisphenol-exposed animals.

Neuroactive steroids: an update of their roles in central and peripheral nervous system.

After five editions, the congress on "Steroids and Nervous System" held in Torino, Italy, represents an important international event for researchers involved in this field aimed to recapitulate mechanisms, physiological and pharmacological effects of neuroactive steroids. The present review introduces manuscripts collected in this supplement issue which are based on new interesting findings such as the influence of sex steroids on cannabinoid-regulated biology, the role of steroids in pain, the importance of co-regulators in steroidal mechanisms and the understanding of new non classical mechanism, the emerging role of vitamin D as a neuroactive steroid and the pathogenetic mechanisms mediated by glucocorticoid receptors. Finally, we have integrated these aspects with an update on some of the several and important observations recently published on this hot topic.

Estrous cycle influences the expression of neuronal nitric oxide synthase in the hypothalamus and limbic system of female mice.

BACKGROUND: Nitric oxide plays an important role in the regulation of male and female sexual behavior in rodents, and the expression of the nitric oxide synthase (NOS) is influenced by testosterone in the male rat, and by estrogens in the female. We have here quantitatively investigated the distribution of nNOS immunoreactive (ir) neurons in the limbic hypothalamic region of intact female mice sacrificed during different phases of estrous cycle. RESULTS: Changes were observed in the medial preoptic area (MPA) (significantly higher number in estrus) and in the arcuate nucleus (Arc) (significantly higher number in proestrus). In the ventrolateral part of the ventromedial nucleus (VMHvl) and in the bed nucleus of the stria terminalis (BST) no significant changes have been observed. In addition, by comparing males and females, we observed a stable sex dimorphism (males have a higher number of nNOS-ir cells in comparison to almost all the different phases of the estrous cycle) in the VMHvl and in the BST (when considering only the less intensely stained elements). In the MPA and in the Arc sex differences were detected only comparing some phases of the cycle. CONCLUSION: These data demonstrate that, in mice, the expression of nNOS in some hypothalamic regions involved in the control of reproduction and characterized by a large number of estrogen receptors is under the control of gonadal hormones and may vary according to the rapid variations of hormonal levels that take place during the estrous cycle.

Sex-dependent regulation of hypothalamic neuropeptide Y-Y1 receptor gene expression in moderate/high fat, high-energy diet-fed mice.

In this study we investigated whether long-term consumption of a moderate/high fat (MHF), high-energy diet can affect the gene expression of the Y(1) receptor (Y(1)R) for neuropeptide Y (NPY) in the dorsomedial (DMH), ventromedial (VMH), arcuate (ARC) and paraventricular (PVN) hypothalamic nuclei of male and female Y(1)R/LacZ transgenic mice, carrying the murine Y(1)R promoter linked to the LacZ gene. MHF diet-fed male mice showed an increased consumption of metabolizable energy that was associated with a significant increase in body weight as compared with chow-fed controls. In parallel, consumption of a MHF diet for 8 weeks significantly decreased Y(1)R/LacZ transgene expression in the DMH and VMH of male mice whereas no changes were found in the ARC and PVN. Leptin treatment reduced body weight of both MHF diet- and chow-fed male mice but failed to prevent the decrease in Y(1)R/LacZ transgene expression apparent in the DMH and VMH of male mice after 8 weeks of MHF diet intake. Conversely, no significant changes of metabolizable energy intake, body weight or hypothalamic beta-galactosidase expression were found in MHF diet-fed female Y(1)R/LacZ transgenic mice. A gender-related difference of Y(1)R/LacZ transgenic mice was also observed in response to leptin treatment that failed to decrease body weight of both MHF diet- and chow-fed female mice. Results herein demonstrate that Y(1)R/LacZ FVB mice show a sexual dimorphism both on energy intake and on nucleus-specific regulation of the NPY Y(1)R system in the hypothalamus. Overall, these results provide new insights into the mechanism by which diet composition affects the hypothalamic circuit that controls energy homeostasis.

Effects of early embryonic exposure to genistein on male copulatory behavior and vasotocin system of Japanese quail.

Genistein is a phytoestrogen, particularly abundant in soybeans that can bind estrogen receptors and sex hormone binding proteins, exerting both estrogenic and antiestrogenic activity. In this study we used the Japanese quail embryo as a test end-point to investigate the effects of early embryonic exposure to genistein on male copulatory behavior and on vasotocin parvocellular system. Both differentiate by the organizational effects of estradiol during development and may therefore represent an optimal model to study the effects of xenoestrogens. We injected two doses of genistein (100 and 1000 microg) into the yolk of 3-day-old Japanese quail eggs. Other eggs were treated with either 25 microg of estradiol benzoate or sesame oil as positive and negative controls. At the age of 6 weeks, behavioral tests revealed a significant decrease of all aspects of copulatory behavior (in comparison to the control group) in estradiol-treated birds. In contrast, genistein-treated animals demonstrated various degrees of decrease in the mean frequencies of some aspects of the sexual behavior. The computerized analysis of vasotocin innervation in medial preoptic, stria terminalis and lateral septum nuclei revealed a statistically significant decreased immunoreactivity in treated animals compared to control ones. These results demonstrate that genistein, similarly to estradiol, has an organizational effect on quail parvocellular vasotocin system and on copulatory behavior. In conclusion, present results confirm, in this avian model, that embryonic exposure to phytoestrogens may have life-long effects on sexual differentiation of brain structures and behaviors.

Early embryonic administration of xenoestrogens alters vasotocin system and male sexual behavior of the Japanese quail.

The copulatory behavior and the parvocellular vasotocin (VT) system of the nucleus of the stria terminalis (BST) are sexually dimorphic in the Japanese quail. Embryonic administration of estradiol benzoate (EB) induces an organizational effect determining the disappearance of such a dimorphism (male shows behavior and cerebral phenotype of the female). The VT parvocellular system can therefore be considered an accurate marker of the sexual differentiation of brain circuits and a very sensitive indicator of the activity of estrogen-like substances on neural circuits. To test this hypothesis we administered diethylstilbestrol (DES), a powerful synthetic xenoestrogen, genistein (GEN), a phytoestrogen produced by soy, and bisphenol A (BPA). After 3 days of incubation, quail eggs were injected with vehicle, EB, DES, GEN or BPA. Administration of BPA caused an early blockage of development and no further analyses were done on the BPA groups. At puberty, the copulatory behavior of EB- or DES-treated male quail was totally abolished, whereas only the highest doses of GEN determined a significant decrease of the behavior. After the tests, the animals were sacrificed and perfused. The fractional area (FA) covered by VT immunoreactivity was analyzed in BST, medial preoptic nucleus, and lateral septum by computerized image analysis. The FA was significantly reduced after treatment with EB, DES and GEN at high doses. These results confirm that the sexually dimorphic VT system of the Japanese quail is a sensible indicator of the effects of xenoestrogens at the level of the central nervous system.

Evidence of altered neuropeptide Y content and neuropeptide Y1 receptor gene expression in the hypothalamus of pregnant transgenic mice.

Alterations in the density of neuropeptide Y (NPY)-immunoreactive fibers and of NPY1 receptor gene expression in the hypothalamus of Y1R/LacZ transgenic pregnant mice were investigated. In the paraventricular nucleus of mice on the 18th d of pregnancy NPY immunoreactivity was significantly decreased, and NPY1 receptor gene expression, as measured by histochemical staining of beta-galactosidase and in situ hybridization of NPY1 receptor mRNA, was significantly increased compared with those in estrous mice. Conversely, pregnant transgenic mice displayed a significant induction of NPY immunoreactivity and a reduction of NPY1 receptor gene expression in the ventromedial nucleus. A significant increase in Y1R/LacZ transgene expression and NPY1 receptor mRNA, but no changes in NPY immunoreactivity were observed in the arcuate nucleus of mice on the 18th d of pregnancy. These results suggest that the elevated expression of NPY in the ventromedial nucleus may contribute to the state of leptin resistance that occurs during pregnancy.