Exposure to Low Doses of Phthalates in Male Rodents: Effects on Reproductive and Cognitive Behaviors.

BACKGROUND: The nervous system is a sensitive target for exposure to environmental endocrine-disrupting compounds (EDC). This vulnerability is particularly important during the critical windows of development and puberty and lasts even at later stages of life. Among these environmental EDC, phthalates have largely been described for their neurotoxic effects. These effects have been reported for a large majority of studies using high to very high doses of these substances, which are not relevant for environmental exposure. SUMMARY: The aim of this review was to analyze specifically the male rodent studies using low doses of phthalates. This analysis focuses on reproductive and cognitive behaviors, given the described antiandrogenic effects of phthalates and the known regulation of these behaviors by sex steroids. We also analyze the other neural effects in the hypothalamus and hippocampus/cortex, the brain regions governing these behaviors. A particular focus is on the neurovascular unit, which is newly investigated in the field of endocrine disruption. KEY MESSAGES: Exposure to low doses of phthalates can induce modifications in reproductive and cognitive behaviors. Whether these changes are triggered by common initiating cellular and molecular mechanisms in the brain areas controlling these behaviors still needs to be extensively investigated. In this context, given the high sensitivity of the neurovascular unit to sex steroid regulation and its impairment by low doses of phthalates, it could represent a possible initiating trigger for behavioral alterations to assess for phthalate exposure.

Effects and underlying cellular pathway involved in the impairment of the neurovascular unit following exposure of adult male mice to low doses of di(2-ethylhexyl) phthalate alone or in an environmental phthalate mixture.

We have previously shown that adult male mice exposure to low doses of di (2-ethylhexyl)phthalate (DEHP) impacts the blood-brain barrier (BBB) integrity and surrounding parenchyma in the medial preoptic area (mPOA), a key hypothalamic area involved in the male sexual behavior. BBB leakage was associated with a decrease in the endothelial tight junction accessory protein, zona occludens-1, and caveolae protein Cav-1, added to an inflammatory profile including glial activation accompanied by enhanced expression of inducible nitric oxide synthase. As this failure of BBB functionality in the mPOA could participate, at least in part, in reported alteration of sexual behavior following DEHP exposure, we explored the cellular pathway connecting cerebral capillaries and neurons. Two-month-old C57BL/6J male mice were orally exposed for 6 weeks to DEHP alone (5 and 50 µg/kg/day) or to DEHP (5 µg/kg/day) in an environmental phthalate mixture. The presence of androgen receptor (AR) and estrogen receptor-α (ERα) were first evidenced in brain capillaries. Protein levels of AR but not of ERα were reduced in cerebral capillaries after phthalate exposure. The amounts of basement membrane and cell-matrix interaction components were decreased, while matrix metalloprotease MMP-2 and MMP-9 activities were increased. Fluorojade® labelling suggested that exposure to phthalates also lead to a neurodegenerative process in the mPOA. Altogether, the data suggest that environmental exposure to endocrine disruptors such as phthalates, could alter AR/Cav-1 interaction, impacting a Cav-1/nitric oxide/MMP pathway. This would lead to disruption of the glio-neurovascular coupling which is essential to neuronal functioning.

Involvement of Testosterone Signaling in the Integrity of the Neurovascular Unit in the Male: Review of Evidence, Contradictions, and Hypothesis.

Age-related central nervous system function decline and increased susceptibility of females compared to males with respect to prevalence of several neurodegenerative and neuropsychiatric diseases are both based on the principle that hormonal factors could be involved. These cerebral disorders are characterized by an alteration of blood-brain barrier (BBB) properties and chronic neuroinflammation, which lead to disease progression. Neuroinflammation, in turn, contributes to BBB dysfunction. The BBB and its environment, called the neurovascular unit (NVU), are crucial for cerebral homeostasis and neuronal function. Interestingly, sex steroids influence BBB properties and modulate neuroinflammatory responses. To date however, the majority of work reported has focused on the effects of estrogens on BBB function and neuroinflammation in female mammals. In contrast, the effects of testosterone signaling on the NVU in males are still poorly studied. The aim of this review was to summarize and discuss the literature, providing insights and contradictions to highlight hypothesis and the need for further investigations.

Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta.

Ovarian oestradiol is essential for pubertal maturation and adult physiology of the female reproductive axis. It acts at central and peripheral sites through two main oestrogen receptors (ER) α and ß. Here we investigate the role of ERß on central effects of oestradiol, by generating a mouse line specifically lacking the ERß gene in neuronal and glial cells. Central ERß deletion delays the age at vaginal opening and first oestrous and reduces uterine weight without affecting body growth. Analysis of factors necessary for pubertal progression shows reduced levels of Kiss1 transcripts at postnatal (P) day 25 in the preoptic area, but not in the mediobasal hypothalamus (MBH) of mutant females. In agreement with these data, the number of kisspeptin-immunoreactive neurons was decreased by 57-72% in the three subdivisions of the rostral periventricular area of the third ventricle (RP3V), whereas the density of kisspeptin-immunoreactive fibres was unchanged in the arcuate nucleus of mutant mice. These alterations do not involve changes in ERα mRNAs in the preoptic area and protein levels in the RP3V. The number and distribution of GnRH-immunoreactive cells were unaffected, but gonadotropin-releasing hormone (GnRH) transcript levels were higher in the P25 preoptic area of mutants. At adulthood, mutant females have normal oestrous cyclicity, kisspeptin system and exhibit unaltered sexual behaviour. They display, however, reduced ovary weight and increased anxiety-related behaviour during the follicular phase. This argues for the specific involvement of central ERß in the regulation of pubertal onset in female reproduction, possibly through prepubertal induction of kisspeptin expression in the RP3V.

Cognitive and hippocampal effects of adult male mice exposure to environmentally relevant doses of phthalates.

We recently showed that chronic exposure of adult male mice to environmental doses of DEHP alone or in a phthalate mixture altered blood brain barrier integrity and induced an inflammatory profile in the hippocampus. Here, we investigate whether such exposure alters hippocampus-dependent behavior and underlying cellular mechanisms. Adult C57BL/6 J male mice were continuously exposed orally to the vehicle or DEHP alone (5 or 50 µg/kg/d) or to DEHP (5 µg/kg/d) in a phthalate mixture. In the Morris water maze, males showed reduced latencies across days to find the platform in the cue and spatial reference memory tasks, regardless of their treatment group. In the probe test, DEHP-50 exposed males displayed a higher latency to find the platform quadrant. In the temporal order memory test, males exposed to DEHP alone or in a phthalate mixture were unable to discriminate between the most recently and previously seen objects. They also displayed reduced ability to show a preference for the new object in the novel object recognition test. These behavioral alterations were associated with a lowered dendritic spine density and protein levels of glutamate receptors and postsynaptic markers, and increased protein levels of the presynaptic synaptophysin in the hippocampus. Metabolomic analysis of the hippocampus indicated changes in amino acid levels including reduced tryptophan and L-kynurenine and elevated NAD + levels, respectively, a precursor, intermediate and endproduct of the kynurenine pathway of tryptophan metabolism. Interestingly, the protein amounts of the xenobiotic aryl hydrocarbon receptor, a target of this metabolic pathway, were elevated in the CA1 area. These data indicate that chronic exposure of adult male mice to environmental doses of DEHP alone or in a phthalate mixture impacted hippocampal function and structure, associated with modifications in amino acid metabolites with a potential involvement of the kynurenine pathway of tryptophan metabolism.

Behavior, Neural Structure, and Metabolism in Adult Male Mice Exposed to Environmentally Relevant Doses of Di(2-ethylhexyl) Phthalate Alone or in a Phthalate Mixture.

BACKGROUND: We have previously shown that chronic exposure of adult male mice to low doses of di(2-ethylhexyl) phthalate (DEHP) altered male sexual behavior and induced down-regulation of the androgen receptor (AR) in the neural circuitry controlling this behavior. OBJECTIVES: The cellular mechanisms induced by chronic exposure of adult male mice to low doses of DEHP alone or in an environmental phthalate mixture were studied. METHODS: Two-month-old C57BL/6J males were exposed orally for 8 wk to DEHP alone (0, 5, or 50µg/kg/d) or to DEHP (50µg/kg/d) in a phthalate mixture. Behavior, dendritic density per 50-µm length, pre-/postsynaptic markers, synapse ultrastructure, and bioenergetic activity were analyzed. RESULTS: Mice exposed to DEHP either alone or in a phthalate mixture differed in mating, emission of ultrasonic vocalizations, and the ability to attract receptive females in urinary preference tests from control mice. Analyses in the medial preoptic area, the key hypothalamic region involved in male sexual behavior, showed lower dendritic spine density and protein levels of glutamate receptors and differences in other postsynaptic components and presynaptic markers between the treated groups. Ultrastructural observation of dendritic synapses by electron microscopy showed comparable morphology between the treated groups. Metabolic analyses highlighted differences in hypothalamic metabolites of males exposed to DEHP alone or in a phthalate mixture compared to control mice. These differences included lower tryptophan and higher NAD+ levels, respectively, a precursor and end product of the kynurenine pathway of tryptophan metabolism. The protein amounts of the xenobiotic aryl hydrocarbon receptor, one of the targets of this metabolic pathway and known negative regulator of the AR, were higher in the medial preoptic area of exposed male mice. DISCUSSION: Differences in behavior of male mice exposed to environmental doses of phthalates were associated with differences in neural structure and metabolism, with possibly a key role of the kynurenine pathway of tryptophan metabolism in the effects mediated by these substances. https://doi.org/10.1289/EHP11514.

Differential involvement of noradrenaline and nitric oxide in the regulation of vasopressin and oxytocin expression in rat supraoptic nucleus.

The hydroosmotic balance of the body is controlled by supraoptic nuclei and paraventricular nuclei of the hypothalamo-posthypophyseal complex. In response to a physiological stimulation such as an osmotic stress, the supraoptic nuclei (SON) and the paraventricular nuclei undergo remarkable neurochemical and morphological changes. Therefore, the neuroendocrine hypothalamus is a particularly relevant model for studying the molecular and cellular mechanisms that govern these plasticity phenomena. Slices of rat hypothalamus maintained ex vivo by perfusion were used to study the short-term involvement of noradrenaline (NA) and nitric oxide (NO) in the mechanisms of chemical plasticity of the SON. NA is involved early in the regulation of the expression of neuropeptides, including vasopressin (AVP) and oxytocin (OT). NO appears to be a key molecule in noradrenergic control of the chemical plasticity of the endocrine neurons: in the SON, NO is involved in the signaling pathway regulating the expression of AVP but not that of OT.

Architecture of the hypothalamo-posthypophyseal complex is controlled by monoamines.

The hypothalamo-neurohypophyseal system displays significant plasticity when subjected to physiological stimuli, such as dehydration, parturition, or lactation. This plasticity arises at the neurochemical and electrophysiological levels but also at a structural level. Several studies have demonstrated the role of monoaminergic afferents in controlling neurochemical and electrophysiological plasticity of the supraoptic nucleus (SON) and of the neurohypophysis (NH), but little is known about how the changes in structural plasticity are triggered. We used Tg8 mice, disrupted for the monoamine oxidase A gene, to study monamine involvement in the architecture of the SON and of the NH. SON astrocytes in Tg8 mice displayed an active status, characterized by an increase in S100ß expression and a significant decrease in vimentin expression, with no modification in glial fibrillary acidic protein (GFAP) levels. Astrocytes showed a decrease in glutamate dehydrogenase (GDH) levels, whereas glutamine synthetase (GS) levels remained constant, suggesting a reduction in astrocyte glutamate catabolism. Tenascin C and polysialic acid-neural cell adhesion molecule (PSA-NCAM) expressions were also elevated in the SON of Tg8 mice, suggesting an increased capacity for structural remodelling in the SON. In the NH, similar date were obtained with a stability in GFAP expression and an increase in PSA-NCAM immunostaining. These results establish monoamine (serotonin and noradrenaline) involvement in SON and NH structural arrangement. Monoamines therefore appear to be crucial for the coordination of the neurochemical and structural aspects of neuroendocrine plasticity, allowing the hypothalamo-neurohypopyseal system to respond appropriately when stimulated.

Disruption of the blood-brain barrier and its close environment following adult exposure to low doses of di(2-ethylhexyl)phthalate alone or in an environmental phthalate mixture in male mice.

We have previously shown that adult male mice exposure to low doses of di(2-ethylhexyl)phthalate (DEHP) alters neural function and behaviour. Whether such exposure also affects the integrity and function of the blood-brain barrier (BBB) remained to be explored. The impact of adult exposure to low doses of DEHP alone or in an environmental phthalate mixture on the BBB integrity and surrounding parenchyma was studied in male mice. Two-month-old C57BL/6J males were orally exposed for 6 weeks to DEHP alone (5, and 50 µg/kg/day) or to DEHP (5 µg/kg/day) in an environmental phthalate mixture. BBB permeability, glial activation and neuroinflammation were investigated in the hypothalamic medial preoptic area (mPOA) and hippocampus involved, respectively on the reproductive and cognitive functions. Exposure to DEHP alone or in a phthalate mixture increased BBB permeability and affected the endothelial accessory tight junction protein zona occludens-1 and caveolae protein Cav-1 in the mPOA and the hippocampal CA1 and CA3 areas. This was associated with an inflammatory profile including astrocyte activation accompanied by enhanced expression of inducible nitric oxide synthase in the mPOA, and a microglial activation in the mPOA and the hippocampal CA1 and CA3 areas. The protein levels of the inflammatory molecule cyclooxygenase-2 were increased in activated microglial cells of the exposed mPOA. None of the major effects induced by DEHP alone or in a mixture was detected in the hippocampal dendate gyrus. The data highlight that environmental exposure to endocrine disruptors such as phthalates, could represent a risk factor for the cerebrovascular function.

Carbon Black Nanoparticles Selectively Alter Follicle-Stimulating Hormone Expression in vitro and in vivo in Female Mice.

Toxic effects of nanoparticles on female reproductive health have been documented but the underlying mechanisms still need to be clarified. Here, we investigated the effect of carbon black nanoparticles (CB NPs) on the pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which are key regulators of gonadal gametogenesis and steroidogenesis. To that purpose, we subjected adult female mice to a weekly non-surgical intratracheal administration of CB NPs at an occupationally relevant dose over 4 weeks. We also analyzed the effects of CB NPs in vitro, using both primary cultures of pituitary cells and the LßT2 gonadotrope cell line. We report here that exposure to CB NPs does not disrupt estrous cyclicity but increases both circulating FSH levels and pituitary FSH ß-subunit gene (Fshb) expression in female mice without altering circulating LH levels. Similarly, treatment of anterior pituitary or gonadotrope LßT2 cells with increasing concentrations of CB NPs dose-dependently up-regulates FSH but not LH gene expression or release. Moreover, CB NPs enhance the stimulatory effect of GnRH on Fshb expression in LßT2 cells without interfering with LH regulation. We provide evidence that CB NPs are internalized by LßT2 cells and rapidly activate the cAMP/PKA pathway. We further show that pharmacological inhibition of PKA significantly attenuates the stimulatory effect of CB NPs on Fshb expression. Altogether, our study demonstrates that exposure to CB NPs alters FSH but not LH expression and may thus lead to gonadotropin imbalance.

Glucocorticoids stimulate hypothalamic dynorphin expression accounting for stress-induced impairment of GnRH secretion during preovulatory period.

Stress-induced reproductive dysfunction is frequently associated with increased glucocorticoid (GC) levels responsible for suppressed GnRH/LH secretion and impaired ovulation. Besides the major role of the hypothalamic kisspeptin system, other key regulators may be involved in such regulatory mechanisms. Herein, we identify dynorphin as a novel transcriptional target of GC. We demonstrate that only priming with high estrogen (E2) concentrations prevailing during the late prooestrus phase enables stress-like GC concentrations to specifically stimulate Pdyn (prodynorphin) expression both in vitro (GT1-7 mouse hypothalamic cell line) and ex vivo (ovariectomized E2-supplemented mouse brains). Our results indicate that stress-induced GC levels up-regulate dynorphin expression within a specific kisspeptin neuron-containing hypothalamic region (antero-ventral periventricular nucleus), thus lowering kisspeptin secretion and preventing preovulatory GnRH/LH surge at the end of the prooestrus phase. To further characterize the molecular mechanisms of E2 and GC crosstalk, chromatin immunoprecipitation experiments and luciferase reporter gene assays driven by the proximal promoter of Pdyn show that glucocorticoid receptors bind specific response elements located within the Pdyn promoter, exclusively in presence of E2. Altogether, our work provides novel understanding on how stress affects hypothalamic-pituitary-gonadal axis and underscores the role of dynorphin in mediating GC inhibitory actions on the preovulatory GnRH/LH surge to block ovulation.

Chronic depletion of gonadal testosterone leads to blood-brain barrier dysfunction and inflammation in male mice.

A dysfunction in the blood-brain barrier (BBB) is associated with many neurological and metabolic disorders. Although sex steroid hormones have been shown to impact vascular tone, endothelial function, oxidative stress, and inflammatory responses, there are still no data on the role of testosterone in the regulation of BBB structure and function. In this context, we investigated the effects of gonadal testosterone depletion on the integrity of capillary BBB and the surrounding parenchyma in male mice. Our results show increased BBB permeability for different tracers and endogenous immunoglobulins in chronically testosterone-depleted male mice. These results were associated with disorganization of tight junction structures shown by electron tomography and a lower amount of tight junction proteins such as claudin-5 and ZO-1. BBB leakage was also accompanied by activation of astrocytes and microglia, and up-regulation of inflammatory molecules such as inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), interleukin 1 beta (IL-1ß), and tumor necrosis factor (TNF). Supplementation of castrated male mice with testosterone restored BBB selective permeability, tight junction integrity, and almost completely abrogated the inflammatory features. The present demonstration that testosterone transiently impacts cerebrovascular physiology in adult male mice should help gain new insights into neurological and metabolic diseases linked to hypogonadism in men of all ages.

Carbon Black Nanoparticles Inhibit Aromatase Expression and Estradiol Secretion in Human Granulosa Cells Through the ERK1/2 Pathway.

Secretion of 17-ß-estradiol (E2) by human granulosa cells can be disrupted by various environmental toxicants. In the current study, we investigated whether carbon black nanoparticles (CB NPs) affect the steroidogenic activity of cultured human granulosa cells. The human granulosa cell line KGN and granulosa cells from patients undergoing in vitro fertilization were treated with increasing concentrations of CB NPs (1 to 100 µg/mL) together or not with follicle-stimulating hormone (FSH). We observed that CB NPs are internalized in KGN cells without affecting cell viability. CB NPs could be localized in the cytoplasm, within mitochondria and in association with the outer face of the endoplasmic reticulum membrane. In both cell types, CB NPs reduced in a dose-dependent manner the activity of aromatase enzyme, as reflected by a decrease in E2 secretion. A significant decrease was observed in response to CB NPs concentrations from 25 and 50 µg/mL in KGN cell line and primary cultures, respectively. Furthermore, CB NPs decreased aromatase protein levels in both cells and reduced aromatase transcript levels in KGN cells. CB NPs rapidly activated extracellular signal-regulated kinase 1 and 2 in KGN cells and pharmacological inhibition of this signaling pathway using PD 98059 significantly attenuated the inhibitory effects of CB NPs on CYP19A1 gene expression and aromatase activity. CB NPs also inhibited the stimulatory effect of FSH on aromatase expression and activity. Altogether, our study on cultured ovarian granulosa cells reveals that CB NPs decrease estrogens production and highlights possible detrimental effect of these common NPs on female reproductive health.

Neural Mechanisms Underlying the Disruption of Male Courtship Behavior by Adult Exposure to Di(2-ethylhexyl) Phthalate in Mice.

BACKGROUND: Courtship behavior plays a critical role in attracting females and reproduction success. However, the effects of exposure to a ubiquitous contaminant di(2-ethylhexyl) phthalate (DEHP) on these behaviors and, in particular, on courtship vocalizations have not been examined. OBJECTIVE: The effects of adult exposure to DEHP on courtship and mating behaviors and gonadotropic axis and neural mechanisms involved in DEHP-induced effects were analyzed in male mice. METHODS: Adult C57BL/6J males were orally exposed to DEHP (0, 0.5, 5, and 50µg/kg/d) for 4 wk. Olfactory preference, ultrasonic vocalizations (USVs), partner preference and mating, as well as locomotor activity and motor coordination, were measured. The kisspeptin system and testosterone levels were analyzed. Proteomic and molecular studies were conducted on the hypothalamic preoptic nucleus, the key region involved in sexual motivation to vocalize and mate. RESULTS: DEHP at 50µg/kg/d reduced the emission of USVs, whereas lower doses changed the ratio of syllable categories. This was associated with diminished sexual interest of female partners toward males exposed to 5 or 50µg/kg/d and increased latency to mate, despite normal olfactory preference. The kisspeptin system and circulating testosterone levels were unaffected. In DEHP-exposed males, proteomic analysis of the preoptic nucleus identified differentially expressed proteins connected to the androgen receptor (AR). Indeed, exposure to 5 or 50µg/kg/d of DEHP induced selective AR downregulation in this nucleus and upstream chemosensory regions. The involvement of AR changes in the observed alterations was further supported by the reduced emission of courtship vocalizations in males with disrupted neural AR expression. CONCLUSIONS: These data demonstrate the critical role of neural AR in courtship vocalizations and raises the possibility that the vulnerability of this signaling pathway to exposure to endocrine disrupters may be detrimental for courtship communication and mating in several species. https://doi.org/10.1289/EHP1443.

Reactive oxygen species are physiological mediators of the noradrenergic signaling pathway in the mouse supraoptic nucleus.

Free radicals are essential for the vasopressin (AVP) response to plasmatic hyperosmolarity. Noradrenergic afferents are the major projections on the supraoptic nucleus (SON) of the hypothalamus and stimulate the expression of AVP via a nitric oxide (NO) pathway. In this study, we investigated the mechanisms linking free radicals and noradrenaline (NA)-induced regulation of AVP. Analysis of Tg8 transgenic mice, invalidated for the monoamine oxidase-A gene and with consequently high levels of brain monoamines and AVP in the SON, showed that free radicals are more abundant in their SON than in that of wild-type mice (WT). Antioxidant superoxide dismutase 1 and 2 and catalase enzyme activities were also higher in these mice than in WT. This may explain the observed absence of cytotoxicity that would otherwise be associated with such high level of free radicals. Treatment of Tg8 mice with α-MPT, a blocking agent for NA synthesis, decreased both the production of free radicals and the AVP levels in the SON. Furthermore, incubation of ex vivo slices including the SON with NA increased the production of free radicals and AVP levels in wild-type mice. When NA was associated with α-lipoic acid, an antioxidant blocking the production of free radicals, AVP remained at its control level, indicating that free radicals are required for the effect of NA on the expression of AVP. In slices incubated with SNP, a producer of NO, free radicals and AVP levels increased. When NA was associated with L-NAME (a NO synthase blocker), the levels of free radicals and AVP were the same as in controls. Thus, the noradrenaline-NO pathway, which stimulates the expression of vasopressin, involves free radicals. This study provides further evidence of the physiological importance of free radicals, which should no longer be considered solely as cytotoxic factors.

Vulnerability of the neural circuitry underlying sexual behavior to chronic adult exposure to oral bisphenol a in male mice.

There are human reproduction concerns associated with extensive use of bisphenol A (BPA)-containing plastic and, in particular, the leaching of BPA into food and beverages. In this context, it remains unclear whether and how exposure to BPA interferes with the developmental organization and adult activation of male sexual behavior by testosterone. We evaluated the developmental and adult exposure to oral BPA at doses equivalent to the no-observed-adverse-effect-level (5 mg/kg body weight per day) and tolerable daily intake (TDI) (50 µg/kg body weight per day) on mouse sexual behavior and the potential mechanisms underlying BPA effects. Adult exposure to BPA reduced sexual motivation and performance at TDI dose only. Exposed males took longer to initiate mating and reach ejaculation despite normal olfactory chemoinvestigation. This deficiency was not restored by sexual experience and was associated with unchanged circulating levels of testosterone. By contrast, developmental exposure to BPA at TDI or no-observed-adverse-effect-level dose did not reduce sexual behavior or alter the neuroanatomical organization of the preoptic area. Disrupting the neural androgen receptor resulted in behavioral and neuroanatomical effects similar to those induced by adult exposure to TDI dose. Moreover, adult exposure of mutant males to BPA at TDI dose did not trigger additional alteration of sexual behavior, suggesting that BPA and neural androgen receptor mutation share a common mechanism of action. This shows, for the first time, that the neural circuitry underlying male sexual behavior is vulnerable to chronic adult exposure to low dose of BPA and suggests that BPA could act in vivo as an antiandrogenic compound.

Reactive oxygen species are required for the hypothalamic osmoregulatory response.

Free radicals, or reactive oxygen species (ROS), are highly reactive byproducts of oxygen degradation. They are well known for their cellular toxicity, but few studies have analyzed their potential role in homeostatic processes. We investigated ROS production and function during the arginine vasopressin (AVP) hypothalamic response to hyperosmolarity. Six-week-old male C3H/HeJ mice were subjected to salt loading for 2 or 8 d. The osmotic axis was progressively activated and reached a new steady-state status at 8 d as demonstrated by monitoring of plasmatic osmolality and c-Fos and AVP expression in the supraoptic nucleus (SON). Free radicals, visualized by dihydroethidine staining and measured by 2'-7'dichlorofluorescein diacetate assays, were detected after 2 d of salt loading. The activity and expression of superoxide dismutase 2 and catalase were concomitantly up-regulated in the SON, suggesting that free radicals are detoxified by endogenous antioxidant systems, thereby avoiding their deleterious effects. The early phase of the osmoregulatory response has been investigated using an acute hyperosmotic model; free radicals were produced 45 min after an ip injection of 1.5 m NaCl. This was followed by an increase in c-Fos and AVP expression and an increase in superoxide dismutase 2 and catalase activities. α-Lipoic acid, a ROS scavenger, administrated during the 3 d before the hypertonic ip injection, abolished the increase of AVP. These findings establish that hyperosmolarity causes ROS production in the SON, which is essential for AVP increase. This demonstrates the importance of free radicals as physiological signaling molecules in the regulation of body-fluid balance.

Direct evidence for the co-expression of URP and GnRH in a sub-population of rat hypothalamic neurones: anatomical and functional correlation.

Urotensin-II-related peptide (URP) is an eight amino-acid neuropeptide recently isolated from rat brain and considered as the endogenous ligand for the GPR14 receptor. Using single and double immunohistochemical labelling, in situ hybridization and ultrastructural immunocytochemistry, we explored the cellular and subcellular localization of URP in the male rat brain. URP peptide was detected in numerous varicose fibres of the median eminence (ME) and organum vasculosum laminae terminalis (OVLT) as well as in neuronal cell bodies of the medial septal nucleus and diagonal band of Broca where corresponding mRNA were also detected. Combining in situ hybridization with immunohistochemistry, we showed that cell bodies of the rat anterior hypothalamus contained both URP mRNA and GnRH peptide. In addition, double ultrastructural immunodetection of URP and GnRH peptides clearly revealed, in the median eminence, the co-localization of both peptides in the same neuronal processes in the vicinity of fenestrated portal vessels. This remarkable cellular and subcellular distribution led us to test the effect of URP on the GnRH-induced gonadotrophins release in the anterior pituitary, and to discuss its putative role at the level of the median eminence.

Cellular and subcellular aquaporin-4 distribution in the mouse neurohypophysis and the effects of osmotic stimulation.

Water channel aquaporin-4 (AQP4) is the most abundant water channel in the rodent brain and is mainly expressed in cerebral areas involved in central osmoreception and osmoregulation. The neurohypophysis is the release site of hypothalamic neurohormones vasopressin and oxytocin, which are involved in the regulation of the water balance. The authors investigated the cellular and subcellular distribution of AQP4 in the mouse neurohypophysis before and after chronic osmotic stimulation, using immunofluorescence microscopy and immunoperoxidase electron microscopy. They showed that AQP4 was abundant in the mouse hypophysis, mainly in the neural lobe. AQP4 was discontinuously distributed along pituicytes plasma membranes, in the dense neurosecretory granules and microvesicles of nerve endings and fibers, and along the luminal and abluminal membranes of fenestrated capillary endothelial cells. After chronic osmotic stimulation, AQP4 immunolabeling was enhanced. Taken together, these results suggest that AQP4 could be involved in the pituicyte sensor effect during osmoregulation, the modification and/or maturation mechanism of neurosecretory granules during neurohormone release, and the blood perfusion of the hypophysis.