Effects of neural estrogen receptor beta deletion on social and mood-related behaviors and underlying mechanisms in male mice.

Estradiol derived from neural aromatization of testosterone plays a key role in the organization and activation of neural structures underlying male behaviors. This study evaluated the contribution of the estrogen receptor (ER) ß in estradiol-induced modulation of social and mood-related behaviors by using mice lacking the ERß gene in the nervous system. Mutant males exhibited reduced social interaction with same-sex congeners and impaired aggressive behavior. They also displayed increased locomotor activity, and reduced or unaffected anxiety-state level in three paradigms. However, when mice were exposed to unescapable stress in the forced swim and tail suspension tests, they spent more time immobile and a reduced time in swimming and climbing. These behavioral alterations were associated with unaffected circadian and restraint stress-induced corticosterone levels, and unchanged number of tryptophan hydroxylase 2-immunoreactive neurons in the dorsal raphe. By contrast, reduced mRNA levels of oxytocin and arginine-vasopressin were observed in the bed nucleus of stria terminalis, whereas no changes were detected in the hypothalamic paraventricular nucleus. The neural ERß is thus involved to different extent levels in social and mood-related behaviors, with a particular action on oxytocin and arginine-vasopressin signaling pathways of the bed nucleus of stria terminalis, yet the involvement of other brain areas cannot be excluded.

Effects of combined exposure of adult male mice to di-(2-ethylexyl)phthalate and nonylphenol on behavioral and neuroendocrine responses.

The present study evaluates the effects of adult exposure to low doses of a mixture of di-(2-ethylexyl)phthalate (DEHP) and nonylphenol (NP) on reproductive neuroendocrine function and behavior. The neural circuitry that processes male sexual behavior is tightly regulated by testosterone and its neural metabolite estradiol. In previous studies, we showed that adult exposure of mice to low doses of each of these widespread environmental contaminants resulted in altered sexual behavior, without any effect on the regulation of the gonadotropic axis. Here, adult C57BL/6J male mice were exposed to DEHP/NP (0.5 or 5 µg/kg body weight/day) for 4 weeks before starting the analyses. Mice treated with DEHP/NP at 0.5 µg/kg/day show altered olfactory preference, and fewer of them emit ultrasonic vocalization compared to the other treatment groups. These mice also exhibit a lower number of mounts and thrusts, increased locomotor activity and unaffected anxiety-state level, along with unaltered testosterone levels and kisspeptin system, a key regulator of the gonadotropic axis. Analysis of the neural circuitry that underlies sexual behavior showed that the number of cells expressing androgen and estrogen receptors is comparable between control and DEHP/NP-exposed males. The comparison of these data with those obtained in males exposed to each molecule separately highlights synergistic effects at the lower dose of contaminants of 0.5 µg/kg/day. In contrast, the effects previously observed for each molecule at 5 µg/kg/day were not detected. A detailed comparison of the effects triggered by separate or combined exposure to DEHP and NP is discussed.

Adult male mice exposure to nonylphenol alters courtship vocalizations and mating.

The neural circuitry processing male sexual behavior is tightly regulated by testosterone and its neural metabolite estradiol. The present study evaluated the effects of adult exposure to low doses of nonylphenol (NP), a widespread environmental contaminant, on the neuroendocrine regulation of testosterone and expression of sexual behavior. Oral exposure of C57BL/6J males to NP (0.5, 5 or 50 µg/kg/day) for 4 weeks did not affect circulating levels of testosterone or the kisspeptin system, a key regulator of the gonadotropic axis. In contrast, mice exposed to NP at 5 µg/kg/day emitted an increased number and duration of ultrasonic vocalizations, took longer to reach ejaculation and showed increased number of mounts, intromissions and thrusts. This was associated with normal olfactory preference and locomotor activity, and increased anxiety level. Analysis of the neural circuitry that underlies sexual behavior showed changes in the number of cells expressing androgen and estrogen receptors in males exposed to NP at 5 µg/kg/day. The neural circuitry underlying sexual behavior is thus highly sensitive to adult exposure to NP. Furthermore, almost all the observed effects were induced at 5 µg/kg/day of NP, indicating that this endocrine disrupter triggers a non-monotonic response in the adult male mouse brain.

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.

Polyhydramnios, Transient Antenatal Bartter's Syndrome, and MAGED2 Mutations.

BACKGROUND: Three pregnancies with male offspring in one family were complicated by severe polyhydramnios and prematurity. One fetus died; the other two had transient massive salt-wasting and polyuria reminiscent of antenatal Bartter's syndrome. METHODS: To uncover the molecular cause of this possibly X-linked disease, we performed whole-exome sequencing of DNA from two members of the index family and targeted gene analysis of other members of this family and of six additional families with affected male fetuses. We also evaluated a series of women with idiopathic polyhydramnios who were pregnant with male fetuses. We performed immunohistochemical analysis, knockdown and overexpression experiments, and protein-protein interaction studies. RESULTS: We identified a mutation in MAGED2 in each of the 13 infants in our analysis who had transient antenatal Bartter's syndrome. MAGED2 encodes melanoma-associated antigen D2 (MAGE-D2) and maps to the X chromosome. We also identified two different MAGED2 mutations in two families with idiopathic polyhydramnios. Four patients died perinatally, and 11 survived. The initial presentation was more severe than in known types of antenatal Bartter's syndrome, as reflected by an earlier onset of polyhydramnios and labor. All symptoms disappeared spontaneously during follow-up in the infants who survived. We showed that MAGE-D2 affects the expression and function of the sodium chloride cotransporters NKCC2 and NCC (key components of salt reabsorption in the distal renal tubule), possibly through adenylate cyclase and cyclic AMP signaling and a cytoplasmic heat-shock protein. CONCLUSIONS: We found that MAGED2 mutations caused X-linked polyhydramnios with prematurity and a severe but transient form of antenatal Bartter's syndrome. MAGE-D2 is essential for fetal renal salt reabsorption, amniotic fluid homeostasis, and the maintenance of pregnancy. (Funded by the University of Groningen and others.).

Neural Androgen Receptor Deletion Impairs the Temporal Processing of Objects and Hippocampal CA1-Dependent Mechanisms.

We studied the role of testosterone, mediated by the androgen receptor (AR), in modulating temporal order memory for visual objects. For this purpose, we used male mice lacking AR specifically in the nervous system. Control and mutant males were gonadectomized at adulthood and supplemented with equivalent amounts of testosterone in order to normalize their hormonal levels. We found that neural AR deletion selectively impaired the processing of temporal information for visual objects, without affecting classical object recognition or anxiety-like behavior and circulating corticosterone levels, which remained similar to those in control males. Thus, mutant males were unable to discriminate between the most recently seen object and previously seen objects, whereas their control littermates showed more interest in exploring previously seen objects. Because the hippocampal CA1 area has been associated with temporal memory for visual objects, we investigated whether neural AR deletion altered the functionality of this region. Electrophysiological analysis showed that neural AR deletion affected basal glutamate synaptic transmission and decreased the magnitude of N-methyl-D-aspartate receptor (NMDAR) activation and high-frequency stimulation-induced long-term potentiation. The impairment of NMDAR function was not due to changes in protein levels of receptor. These results provide the first evidence for the modulation of temporal processing of information for visual objects by androgens, via AR activation, possibly through regulation of NMDAR signaling in the CA1 area in male mice.

Loss of Maged1 results in obesity, deficits of social interactions, impaired sexual behavior and severe alteration of mature oxytocin production in the hypothalamus.

MAGED1, NECDIN and MAGEL2 are members of the MAGE gene family. The latter two of these genes have been involved in Prader-Willi syndrome (PWS), which includes hyperphagia, repetitive and compulsive behaviors, and cognitive impairment. Here, we show that Maged1-deficient mice develop progressive obesity associated with hyperphagia and reduced motor activity. Loss of Maged1 also results in a complex behavioral syndrome that includes reduced social interactions and memory, deficient sexual behavior, as well as increased anxiety and self-grooming. Oxytocin (OT), which is produced in the hypothalamus, can act as a neurotransmitter that reduces anxiety, promotes social behaviors and regulates food intake. Growing evidences indicate that OT is involved in autism. We found that Maged1 mutants showed a severe reduction in the levels of mature OT, but not of its precursors, in the hypothalamus. Moreover, the administration of OT rescued the deficit in social memory of these mice. We conclude that Maged1 is required for OT processing or stability. A decrease in mature OT levels in Maged1 mutants affects social interactions and possibly other behavioral processes. Our observations suggest that, in human, MAGED1 could play a role in autism or cause a neurodevelopmental condition that is reminiscent of the PWS.

Systems genetic analysis of osteoblast-lineage cells.

The osteoblast-lineage consists of cells at various stages of maturation that are essential for skeletal development, growth, and maintenance. Over the past decade, many of the signaling cascades that regulate this lineage have been elucidated; however, little is known of the networks that coordinate, modulate, and transmit these signals. Here, we identify a gene network specific to the osteoblast-lineage through the reconstruction of a bone co-expression network using microarray profiles collected on 96 Hybrid Mouse Diversity Panel (HMDP) inbred strains. Of the 21 modules that comprised the bone network, module 9 (M9) contained genes that were highly correlated with prototypical osteoblast maker genes and were more highly expressed in osteoblasts relative to other bone cells. In addition, the M9 contained many of the key genes that define the osteoblast-lineage, which together suggested that it was specific to this lineage. To use the M9 to identify novel osteoblast genes and highlight its biological relevance, we knocked-down the expression of its two most connected "hub" genes, Maged1 and Pard6g. Their perturbation altered both osteoblast proliferation and differentiation. Furthermore, we demonstrated the mice deficient in Maged1 had decreased bone mineral density (BMD). It was also discovered that a local expression quantitative trait locus (eQTL) regulating the Wnt signaling antagonist Sfrp1 was a key driver of the M9. We also show that the M9 is associated with BMD in the HMDP and is enriched for genes implicated in the regulation of human BMD through genome-wide association studies. In conclusion, we have identified a physiologically relevant gene network and used it to discover novel genes and regulatory mechanisms involved in the function of osteoblast-lineage cells. Our results highlight the power of harnessing natural genetic variation to generate co-expression networks that can be used to gain insight into the function of specific cell-types.