Mariangela Martini

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

BackgroundNitric 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.ResultsChanges 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.ConclusionThese 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.

Neuropeptides and Enzymes are Targets for the Action of Endocrine Disrupting Chemicals in the Vertebrate Brain

Endocrine-disrupting chemicals (EDC) are molecules that interfere with endocrine signaling pathways and produce adverse consequences on animal and human physiology, such as infertility or behavioral alterations. Some EDC act through binding to androgen or/and estrogen receptors primarily operating through a genomic mechanism regulating gene expression. This mechanism of action may induce profound developmental adverse effects, and the major targets of the EDC action are the gene products, i.e., mRNAs inducing the synthesis of various peptidic molecules, which include neuropeptides and enzymes related to neurotransmitters syntheses. Available immunohistochemical data on some of the systems that are affected by EDC in lower and higher vertebrates are detailed in this review.

Effects of Xenoestrogens on the Differentiation of Behaviorally Relevant Neural Circuits in Higher Vertebrates

Several environmental chemicals have the capability of impacting endocrine function (endocrine disrupting chemicals [EDCs]), and therefore they may have long‐term consequences, especially if exposure occurs during embryonic development. In this study we present data relative to two widely used animal models: the Japanese quail and the mouse. These two species have been used to understand neural, neuroendocrine, and behavioral components of reproduction and are optimal models to understand how these components are altered by precocious exposure to EDCs. In particular, we discuss the effects of embryonic exposure to diethylstilbestrol, genistein, or ethylene,1,1‐dichloro‐2,2‐bis(p‐chlorophenyl) on the sexually dimorphic parvocellular vasotocin system and male copulatory behavior in quail and the effects of bisphenol A on the nitrinergic and kisspeptin systems and their behavioral impact in the mouse. In both models the exposure to EDCs during the critical period (early embryonic period in birds, perinatal period in rodents) alters the differentiation of relevant sexually dimorphic pathways, often inducing the appearance of a sex‐reversed neurochemical phenotype that is the most probable cause of the final alteration of sexually differentiated behaviors in the adult animal. In conclusion, the data presented here should stimulate a critical reanalysis of the way to determine the “safe” exposure levels to EDCs for wild species and humans, considering behavior and related neural circuits among the factors to be analyzed.

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.

Rapid changes on nitrinergic system in female mouse hippocampus during the ovarian cycle

Fluctuating levels of estradiol and progesterone during the estrous cycle may induce structural changes in several brain nuclei including the hippocampus, where some neurons express estrogen receptors. Nitric oxide plays a wide range of functions in the nervous system generally by acting as a neurotransmitter-like molecule. It has been demonstrated that long-term treatments with estradiol in ovariectomized females and with testosterone in castrated males induce neuronal nitric oxide synthase (nNOS) expression in rat hypothalamus, whereas changes in nNOS immunoreactivity or in associated NADPH-diaphorase activity were observed both in hypothalamus and in amygdala during different phases of estrous cycle. Estradiol could induce nNOS expression in several brain regions in rodents. Therefore, to clarify if the hippocampal NO producing system is a target for gonadal hormones in physiological conditions, we have investigated the effects of estrous cycle in the expression of nNOS immunoreactivity on two-month-old intact female mice. Immunoreactive cells were observed in all hippocampal subregions: the higher number was detected in the pyramidal layer of CA1 region and in polymorph layer of dentate gyrus. The number of nNOS positive neurons fluctuates during the estrous cycle, reaching its peak during proestrus and metaestrus, and these variations were statistically significant in CA1, CA2 and CA3 regions. These results suggest that the nitrinergic system is a target for estrogen action in the hippocampus, and that this action may take place in physiological conditions according to the short-term variations of gonadal hormones during the estrous cycle.

Effects of estrous cycle and sex on the expression of neuropeptide Y Y1 receptor in discrete hypothalamic and limbic nuclei of transgenic mice.

In the present study we used a transgenic mouse model, carrying the neuropeptide Y (NPY) Y1 receptor gene promoter linked to the LacZ reporter gene (Y1R/LacZ mice) to test the hypothesis of its up-regulation by gonadal hormones. Y1 receptor gene expression was detected by means of histochemical procedures and quantitative image analysis in the paraventricular nucleus, arcuate nucleus, medial preoptic nucleus, ventromedial nucleus and bed nucleus of stria terminalis of two-month-old female mice at different stages of estrous cycle. Qualitative and quantitative analyses showed that Y1R/LacZ transgene expression was higher in the paraventricular, arcuate, and ventromedial nuclei of proestrus mice as compared to mice in the other stages of the estrous cycle. In addition, we performed a comparison with a group of sexually active males. In this comparison a significant difference (less in males) was observed between males and proestrus females in the same nuclei. In conclusion, these data indicate that fluctuations in circulating levels of gonadal hormones, depending by estrous cycle, are paralleled by changes in the expression of NPY Y1 receptor in the hypothalamic nuclei involved in the control of both energy balance and reproduction.

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.

Kisspeptin system in ovariectomized mice: estradiol and progesterone regulation

The kisspeptin system is clustered in two main groups of cell bodies (the periventricular region, RP3V and the arcuate nucleus, ARC) that send fibers mainly to the GnRH neurons and in a few other locations, including the paraventricular nucleus, PVN. In physiological conditions, gonadal hormones modulate the kisspeptin system with expression changes according to different phases of the estrous cycle: the highest being in estrus phase in RP3V and PVN (positive feedback), and in ARC during the diestrus phase (negative feedback). In this work we wanted to study these hormonal fluctuations during the estrous cycle, investigating the role played by progesterone (P) or estradiol (E2), alone or together, on the kisspeptin system. Gonadectomized CD1 female mice were treated with P, E2 or both (E2 + P), following a timing of administration that emulates the different phases of estrous cycle, for two cycles of 4 days. As expected, the two cell groups were differentially affected by E2; the RP3V group was positively influenced by E2 (alone or with the P), whereas in the ARC the administration of E2 did not affect the system. However P (alone) induced a rise in the kisspeptin immunoreactivity. All the treatments significantly affected the kisspeptin innervation of the PVN, with regional differences, suggesting that these fibers arrive from both RP3V and ARC nuclei.

Modification of nitric oxide synthase immunoreactivity in female mouse limbic system during the estrous cycle

Nitric oxide (NO) is a gaseous neuronal messenger, synthesized by the enzyme nitric oxide synthase (NOS), and implicated in the regulation of several physiological and behavioral functions. NO-producing neurons have been localized in numerous regions of the mammalian and non-mammalian central nervous system. In rodents, NOS-immunoreactive neurons and fibers were described in hypothalamic and limbic nuclei belonging to neural circuits implicated in the control of reproductive behavior. Fluctuating levels of estradiol and progesterone during the estrous cycle may induce structural changes in several brain nuclei, where some neurons express estrogen receptors. Various reports indicate that the expression of nNOS is influenced by estrogens in the female. Therefore, to clarify if the nitrergic system is a target for gonadal hormones in physiological conditions, we have investigated the effects of estrous cycle in the expression of nNOS immunoreactivity in different nuclei of the limbic system on two-month-old intact female mice. Changes were observed in the medial preoptic area (significantly higher number in estrus) and in the arcuate nucleus (significantly higher number in proestrus). In the ventrolateral part of the ventromedial nucleus and in the bed nucleus of the stria terminalis no significant changes have been observed. In hippocampus the number of nNOS positive neurons fluctuates during the estrous cycle, reaching its peak during proestrus and metaestrus, and these variations were statistically significant. These results suggest that, in mice, the expression of nNOS in some limbic 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 short-term variations of hormonal levels that take place during the estrous cycle.? This study has been supported by grants from MURST and University of Torino (SG and GCP).