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 -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-␣ (ER␣) 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 ER␣ 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 ER␣-to-mGluR1a signaling.

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.

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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.

The brain synthesizes steroids de novo, especially progesterone. Recently estradiol has been shown to stimulate progesterone synthesis in the hypothalamus and enriched astrocyte cultures derived from neonatal cortex. Estradiol-induced hypothalamic progesterone has been implicated in the control of the LH surge. The present studies were undertaken to determine whether hypothalamic astrocytes derived from female neonatal or female postpubertal rats increased production of progesterone in response to an estradiol challenge. Estradiol induced progesterone synthesis in postpubertal astrocytes but not neonatal astrocytes. This estradiol action was blocked by the estrogen receptor antagonist ICI 182,780. Previously we had demonstrated that estradiol stimulates a rapid increase in free cytosolic Ca(2+) ([Ca(2+)](i)) spikes in neonatal cortical astrocytes acting through a membrane estrogen receptor. We now report that estradiol also rapidly increased [Ca(2+)](i) spikes in hypothalamic astrocytes. The membrane-impermeable estradiol-BSA construct also induced [Ca(2+)](i) spikes. Both estradiol-BSA and estradiol were blocked by ICI 182,780. Depleting intracellular Ca(2+) stores prevented the estradiol-induced increased [Ca(2+)](i) spikes, whereas removing extracellular Ca(2+) did not prevent estradiol-induced [Ca(2+)](i) spikes. Together these results indicate that estradiol acts through a membrane-associated receptor to release intracellular stores of Ca(2+). Thapsigargin, used to mimicked the intracellular release of Ca(2+) by estradiol, increased progesterone synthesis, suggesting that estradiol-induced progesterone synthesis involves increases in [Ca(2+)](i). Estradiol treatment did not change levels of steroid acute regulatory protein, P450 side chain cleavage, 3beta-hydroxysteroid dehydrogenase, and sterol carrier protein-2 mRNAs as measured by quantitative RT-PCR, suggesting that in vitro, estradiol regulation of progesterone synthesis in astrocytes does not depend on transcription of new steroidogenic proteins. The present results are consistent with our hypothesis that estrogen-positive feedback regulating the LH surge involves stimulating local progesterone synthesis by hypothalamic astrocytes.

The SRY-related SOX9 gene is involved in both chondrogenesis and the early steps of mammalian sex determination. Mutations in the human SOX9 gene cause campomelic dysplasia, a severe skeletal malformation syndrome associated with male-to-female sex reversal in most, but not all, XY individuals. Here we show that SOX9 contains a dimerization domain, and binds co-operatively as a dimer in the presence of the DNA enhancer element in genes involved in chondrocyte differentiation, such as Col11a2 and Col9a2, but binds as a monomer to the regulatory region of the sex-determining gene SF1. Frameshift SOX9 mutations truncate its two activation domains, while all missense mutations reported to date lie in the high mobility group (HMG) DNA-binding domain. We identify a missense mutation (A76E), the first outside the HMG domain, in an XY patient presenting with campomelic dysplasia but without sex reversal. This mutation disrupts the dimerization capability of SOX9, interfering with both the DNA binding and consequent transactivation of both the Col11a2 and Col9a2 enhancers. Consistent with the patient's phenotype, the A76E mutation does not affect DNA binding and activation of the SF1 enhancer. DNA-dependent cooperative dimerization could represent a novel mechanism to achieve tissue-specific regulation of gene expression by a SOX transcription factor. These results establish that SOX9 cooperative dimerization is required for chondrogenesis but not for sex determination and may explain why campomelic dysplasia need not be associated with XY sex reversal.

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.

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 ␤-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 ␤-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 ␤-actin immunofluorescence and filopodial spines were increased (from 70.57 Ϯ 1.09% to 78.01 Ϯ 1.05%, p Ͻ 0.05). Disruption of estradiol-induced ␤-actin polymerization with cytochalasin D attenuated lordosis behavior, indicating the importance of estradiol-mediated spinogenesis for female sexual receptivity (81.43 Ϯ 7.05 to 35.00 Ϯ 11.76, p Ͻ 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 estradiolinduced spinogenesis in the ARH is an important cellular mechanism for the regulation of female sexual behavior.