Phoebe Dewing

Up-Regulation of WNT-4 Signaling and Dosage-Sensitive Sex Reversal in Humans

Wnt-4, a member of the Wnt family of locally acting secreted growth factors, is the first signaling molecule shown to influence the sex-determination cascade. In mice, a targeted deletion of Wnt-4 causes the masculinization of XX pups. Therefore, WNT-4, the human homologue of murine Wnt-4, is a strong candidate gene for sex-reversal phenotypes in humans. In this article, we show that, in testicular Sertoli and Leydig cells, Wnt-4 up-regulates Dax1, a gene known to antagonize the testis-determining factor, Sry. Furthermore, we elucidate a possible mechanism for human XY sex reversal associated with a 1p31-p35 duplication including WNT-4. Overexpression of WNT-4 leads to up-regulation of DAX1, which results in an XY female phenotype. Thus, WNT-4, a novel sex-determining gene, and DAX1 play a concerted role in both the control of female development and the prevention of testes formation. These observations suggest that mammalian sex determination is sensitive to dosage, at multiple steps in its pathway.

Sexually dimorphic gene expression in mouse brain precedes gonadal differentiation

The classic view of brain sexual differentiation and behavior is that gonadal steroid hormones act directly to promote sex differences in neural and behavioral development. In particular, the actions of testosterone and its metabolites induce a masculine pattern of brain development, while inhibiting feminine neural and behavioral patterns of differentiation. However, recent evidence indicates that gonadal hormones may not solely be responsible for sex differences in brain development and behavior between males and females. Here we examine an alternative hypothesis that genes, by directly inducing sexually dimorphic patterns of neural development, can influence the sexual differences between male and female brains. Using microarrays and RT-PCR, we have detected over 50 candidate genes for differential sex expression, and confirmed at least seven murine genes which show differential expression between the developing brains of male and female mice at stage 10.5 days post coitum (dpc), before any gonadal hormone influence. The identification of genes differentially expressed between male and female brains prior to gonadal formation suggests that genetic factors may have roles in influencing brain sexual differentiation.

Direct regulation of adult brain function by the male-specific factor SRY.

The central dogma of mammalian brain sexual differentiation has contended that sex steroids of gonadal origin organize the neural circuits of the developing brain. Recent evidence has begun to challenge this idea and has suggested that, independent of the masculinizing effects of gonadal secretions, XY and XX brain cells have different patterns of gene expression that influence their differentiation and function. We have previously shown that specific differences in gene expression exist between male and female developing brains and that these differences precede the influences of gonadal hormones. Here we demonstrate that the Y chromosome-linked, male-determining gene Sry is specifically expressed in the substantia nigra of the adult male rodent in tyrosine hydroxylase-expressing neurons. Furthermore, using antisense oligodeoxynucleotides, we show that Sry downregulation in the substantia nigra causes a statistically significant decrease in tyrosine hydroxylase expression with no overall effect on neuronal numbers and that this decrease leads to motor deficits in male rats. Our studies suggest that Sry directly affects the biochemical properties of the dopaminergic neurons of the nigrostriatal system and the specific motor behaviors they control. These results demonstrate a direct male-specific effect on the brain by a gene encoded only in the male genome, without any mediation by gonadal hormones.

Membrane Estrogen Receptor-α Interactions with Metabotropic Glutamate Receptor 1a Modulate Female Sexual Receptivity in Rats

In rats, female sexual behavior is regulated by a well defined limbic–hypothalamic circuit that integrates sensory and hormonal information. Estradiol activation of this circuit results in μ-opioid receptor (MOR) internalization in the medial preoptic nucleus, an important step for full expression of sexual receptivity. Estradiol acts through both membrane and intracellular receptors to influence neuronal activity and behavior, yet the mechanism(s) and physiological significance of estradiol-mediated membrane responses in vivo have remained elusive. Recent in vitro evidence found that stimulation of membrane-associated estrogen receptor-α (ERα) led to activation of metabotropic glutamate receptor 1a (mGluR1a). Furthermore, mGluR1a signaling was responsible for the observed downstream effects of estradiol. Here we present data that show that ERα and mGluR1a directly interact to mediate a rapid estradiol-induced activation of MOR in the medial preoptic nucleus, leading to female sexual receptivity. In addition, blockade of mGluR1a in the arcuate nucleus of the hypothalamus resulted in a significant attenuation of estradiol-induced MOR internalization, leading to diminished female sexual behavior. These results link membrane-initiated estradiol actions to neural events modulating behavior, demonstrating the physiological importance of ERα-to-mGluR1a signaling.

Membrane-initiated estradiol signaling induces spinogenesis required for female sexual receptivity.

Estrogens have profound actions on the structure of the nervous system during development and in adulthood. One of the signature actions of estradiol is to alter the morphology of neural processes. In the hippocampus, estradiol modulates spines and cellular excitability that affect cognitive behaviors. In the hypothalamus, estradiol increases spine density in mediobasal hypothalamic nuclei that regulate reproduction. The hypothalamic arcuate nucleus (ARH), an important site for modulation of female sexual receptivity, has a sexual dimorphism in dendritic spine density that favors females. In the present study, we used both β-actin immunostaining and Golgi staining to visualize estradiol-induced changes in spine density in Long–Evans rats. Golgi impregnation was used to visualize spine shape, and then β-actin immunoreactivity was used as a semiquantitative measure of spine plasticity since actin forms the core of dendritic spines. At 4 h after estradiol treatment, both β-actin immunofluorescence and filopodial spines were increased (from 70.57 ± 1.09% to 78.01 ± 1.05%, p < 0.05). Disruption of estradiol-induced β-actin polymerization with cytochalasin D attenuated lordosis behavior, indicating the importance of estradiol-mediated spinogenesis for female sexual receptivity (81.43 ± 7.05 to 35.00 ± 11.76, p < 0.05). Deactivation of cofilin, an actin depolymerizing factor is required for spinogenesis. Membrane-initiated estradiol signaling involving the metabotropic glutamate receptor 1a was responsible for the phosphorylation and thereby deactivation of cofilin. These data demonstrate that estradiol-induced spinogenesis in the ARH is an important cellular mechanism for the regulation of female sexual behavior.

Protein Kinase C Signaling in the Hypothalamic Arcuate Nucleus Regulates Sexual Receptivity in Female Rats

Rapid membrane-mediated estradiol signaling regulating sexual receptivity requires the interaction of the estrogen receptor (ER)-alpha and the metabotropic glutamate receptor 1a (mGluR1a). A cell signaling antibody microarray revealed that estradiol activated 42 proteins in the arcuate nucleus of the hypothalamus (ARH). To begin an analysis of various signaling pathways, protein kinase A and protein kinase C (PKC)-theta, whose signaling pathways have been implicated in the estradiol regulation of sexual receptivity, were examined. In the ARH sample, the increase in phospho-protein kinase A could not be confirmed by Western blotting, in either cytosolic or membrane fractions. However, the increase in phosphorylated PKCtheta seen with the pathway array was verified by Western blotting. To study whether rapid estradiol activation of PKC regulates the ARH-medial preoptic nucleus pathway regulating lordosis, mu-opioid receptor (MOR) internalization and lordosis reflex were tested. Blocking PKC in ARH with 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]3-(1H-indol-3-yl) maleimide significantly attenuated estradiol-induced MOR internalization. Furthermore, disruption of PKC signaling within the ARH at the time of estradiol treatment significantly diminished the lordosis reflex. Moreover, blocking PKC prevented MOR internalization when the circuit was activated by the mGluR1a agonist, (RS)-3,5-dihydroxyphenylglycine. Activation of PKC with phorbol 12, 13-dibutyrate induced MOR internalization, indicating that PKC was a critical step for membrane ERalpha-initiated mGluR1a-mediated cell signaling and phorbol 12, 13-dibutyrate significantly facilitated the lordosis reflex. Together these findings indicate that rapid membrane ERalpha-mGluR1a interactions activate PKCtheta cell signaling, which regulates female sexual receptivity.

Membrane-initiated estradiol signaling regulating sexual receptivity

Estradiol has profound actions on the structure and function of the nervous system. In addition to nuclear actions that directly modulate gene expression, the idea that estradiol can rapidly activate cell signaling by binding to membrane estrogen receptors (mERs) has emerged. Even the regulation of sexual receptivity, an action previously thought to be completely regulated by nuclear ERs, has been shown to have a membrane-initiated estradiol signaling (MIES) component. This highlighted the question of the nature of mERs. Several candidates have been proposed, ERα, ERβ, ER-X, GPR30 (G protein coupled estrogen receptor), and a receptor activated by a diphenylacrylamide compound, STX. Although each of these receptors has been shown to be active in specific assays, we present evidence for and against their participation in sexual receptivity by acting in the lordosis-regulating circuit. The initial MIES that activates the circuit is in the arcuate nucleus of the hypothalamus (ARH). Using both activation of μ-opioid receptors (MOR) in the medial preoptic nucleus and lordosis behavior, we document that both ERα and the STX-receptor participate in the required MIES. ERα and the STX-receptor activation of cell signaling are dependent on the transactivation of type 1 metabotropic glutamate receptors (mGluR1a) that augment progesterone synthesis in astrocytes and protein kinase C (PKC) in ARH neurons. While estradiol-induced sexual receptivity does not depend on neuroprogesterone, proceptive behaviors do. Moreover, the ERα and the STX-receptor activation of medial preoptic MORs and augmentation of lordosis were sensitive to mGluR1a blockade. These observations suggest a common mechanism through which mERs are coupled to intracellular signaling cascades, not just in regulating reproduction, but in actions throughout the neuraxis including the cortex, hippocampus, striatum, and dorsal root ganglias.

Sex differences in hypothalamic astrocyte response to estradiol stimulation

BackgroundReproductive functions controlled by the hypothalamus are highly sexually differentiated. One of the most dramatic differences involves estrogen positive feedback, which leads to ovulation. A crucial feature of this positive feedback is the ability of estradiol to facilitate progesterone synthesis in female hypothalamic astrocytes. Conversely, estradiol fails to elevate hypothalamic progesterone levels in male rodents, which lack the estrogen positive feedback-induced luteinizing hormone (LH) surge. To determine whether hypothalamic astrocytes are sexually differentiated, we examined the cellular responses of female and male astrocytes to estradiol stimulation.MethodsPrimary adult hypothalamic astrocyte cultures were established from wild type rats and mice, estrogen receptor-α knockout (ERKO) mice, and four core genotype (FCG) mice, with the sex determining region of the Y chromosome (Sry) deleted and inserted into an autosome. Astrocytes were analyzed for Sry expression with reverse transcription PCR. Responses to estradiol stimulation were tested by measuring free cytoplasmic calcium concentration ([Ca2+]i) with fluo-4 AM, and progesterone synthesis with column chromatography and radioimmunoassay. Membrane estrogen receptor-α (mERα) levels were examined using surface biotinylation and western blotting.ResultsEstradiol stimulated both [Ca2+]i release and progesterone synthesis in hypothalamic astrocytes from adult female mice. Male astrocytes had a significantly elevated [Ca2+]i response but it was significantly lower than in females, and progesterone synthesis was not enhanced. Surface biotinylation demonstrated mERα in both female and male astrocytes, but only in female astrocytes did estradiol treatment increase insertion of the receptor into the membrane, a necessary step for maximal [Ca2+]i release. Regardless of the chromosomal sex, estradiol facilitated progesterone synthesis in astrocytes from mice with ovaries (XX and XY-), but not in mice with testes (XY-Sry and XXSry).ConclusionsAstrocytes are sexually differentiated, and in adulthood reflect the actions of sex steroids during development. The response of hypothalamic astrocytes to estradiol stimulation was determined by the presence or absence of ovaries, regardless of chromosomal sex. The trafficking of mERα in female, but not male, astrocytes further suggests that cell signaling mechanisms are sexually differentiated.

RELEASE OF ORPHANIN FQ/NOCICEPTIN IN THE MEDIAL PREOPTIC NUCLEUS AND VENTROMEDIAL NUCLEUS OF THE HYPOTHALAMUS FACILITATES LORDOSIS

Opioid regulation of reproduction has been widely studied. However, the role of opioid receptor-like 1 receptor (NOP; also referred to as ORL-1 and OP4) and its endogenous ligand orphanin FQ/nociceptin (OFQ/N) have received less attention despite their extensive distribution throughout nuclei of the limbic-hypothalamic system, a circuit that regulates reproductive behavior in the female rat. Significantly, the expression of both receptor and ligand is regulated in a number of these nuclei by estradiol and progesterone. Activation of NOP in the ventromedial nucleus of the hypothalamus (VMH) of estradiol-primed nonreceptive female rats facilitates lordosis. NOPs are also expressed in the medial preoptic nucleus (MPN), however, their roles in reproductive behavior have not been studied. The present experiments examined the role of NOP in the regulation of lordosis in the MPN and tested whether endogenous OFQ/N in the MPN and VMH mediates reproductive behavior. Activation of NOP by microinfusion of OFQ/N in the MPN facilitated lordosis in estradiol-primed sexually nonreceptive female rats. Passive immunoneutralization of OFQ/N in either the MPN or the VMH reduced lordosis in estradiol-primed females, but had no effect on lordosis in estradiol+progesterone-primed sexually receptive rats. These studies suggest that OFQ/N has a central role in estradiol-only induced sexual receptivity, and that progesterone appears to involve additional circuits that mediate estradiol+progesterone sexual receptivity.

Modulation of the arcuate nucleus–medial preoptic nucleus lordosis regulating circuit: A role for GABAB receptors☆

Estradiol rapidly activates a microcircuit in the arcuate nucleus of the hypothalamus (ARH) that is needed for maximal female sexual receptivity. Membrane estrogen receptor-α complexes with and signals through the metabotropic glutamate receptor-1a stimulating NPY release within the ARH activating proopiomelanocortin (POMC) neurons. These POMC neurons project to the medial preoptic nucleus (MPN) and release β-endorphin. Estradiol treatment induces activation/internalization of MPN μ-opioid receptors (MOR) to inhibit lordosis. Estradiol membrane action modulates ARH gamma-aminobutyric acid receptor-B (GABAB) activity. We tested the hypothesis that ARH GABAB receptors mediate estradiol-induced MOR activation and facilitation of sexual receptivity. Double-label immunohistochemistry revealed expression of GABAB receptors in NPY, ERα and POMC expressing ARH neurons. Approximately 70% of POMC neurons expressed GABAB receptors. Because estradiol initially activates an inhibitory circuit and maintains activation of this circuit, the effects of blocking GABAB receptors were evaluated before estradiol benzoate (EB) treatment and after at the time of lordosis testing. Bilateral infusions of the GABAB receptor antagonist, CGP52432, into the ARH prior to EB treatment of ovariectomized rats prevented estradiol-induced activation/internalization of MPN MOR, and the rats remained unreceptive. However, in EB-treated rats, bilateral CGP52432 infusions 30 min before behavior testing attenuated MOR internalization and facilitated lordosis. These results indicated that GABAB receptors were located within the lordosis-regulating ARH microcircuit and are necessary for activation and maintenance of the estradiol inhibition of lordosis behavior. Although GABAB receptors positively influence estradiol signaling, they negatively regulate lordosis behavior since GABAB activity maintains the estradiol-induced inhibition.