β-arrestin regulates estradiol membrane-initiated signaling in hypothalamic neurons.

Estradiol (E2) action in the nervous system is the result of both direct nuclear and membrane-initiated signaling (EMS). E2 regulates membrane estrogen receptor-α (ERα) levels through opposing mechanisms of EMS-mediated trafficking and internalization. While ß-arrestin-mediated mERα internalization has been described in the cortex, a role of ß-arrestin in EMS, which underlies multiple physiological processes, remains undefined. In the arcuate nucleus of the hypothalamus (ARH), membrane-initiated E2 signaling modulates lordosis behavior, a measure of female sexually receptivity. To better understand EMS and regulation of ERα membrane levels, we examined the role of ß-arrestin, a molecule associated with internalization following agonist stimulation. In the present study, we used an immortalized neuronal cell line derived from embryonic hypothalamic neurons, the N-38 line, to examine whether ß-arrestins mediate internalization of mERα. β-arrestin-1 (Arrb1) was found in the ARH and in N-38 neurons. In vitro, E2 increased trafficking and internalization of full-length ERα and ERαΔ4, an alternatively spliced isoform of ERα, which predominates in the membrane. Treatment with E2 also increased phosphorylation of extracellular-signal regulated kinases 1/2 (ERK1/2) in N-38 neurons. Arrb1 siRNA knockdown prevented E2-induced ERαΔ4 internalization and ERK1/2 phosphorylation. In vivo, microinfusions of Arrb1 antisense oligodeoxynucleotides (ODN) into female rat ARH knocked down Arrb1 and prevented estradiol benzoate-induced lordosis behavior compared with nonsense scrambled ODN (lordosis quotient: 3 ± 2.1 vs. 85.0 ± 6.0; p < 0.0001). These results indicate a role for Arrb1 in both EMS and internalization of mERα, which are required for the E2-induction of female sexual receptivity

The Neurosteroid Progesterone Underlies Estrogen Positive Feedback of the LH Surge

Our understanding the steroid regulation of neural function has rapidly evolved in the past decades. Not long ago the prevailing thoughts were that peripheral steroid hormones carried information to the brain which passively responded to these steroids. These steroid actions were slow, taking hours to days to be realized because they regulated gene expression. Over the past three decades, discoveries of new steroid receptors, rapid membrane-initiated signaling mechanisms, and de novo neurosteroidogenesis have shed new light on the complexity of steroids actions within the nervous system. Sexual differentiation of the brain during development occurs predominately through timed steroid-mediated expression of proteins and long term epigenetic modifications. In contrast across the estrous cycle, estradiol release from developing ovarian follicles initially increases slowly and then at proestrus increases rapidly. This pattern of estradiol release acts through both classical genomic mechanisms and rapid membrane-initiated signaling in the brain to coordinate reproductive behavior and physiology. This review focuses on recently discovered estrogen receptor-α membrane signaling mechanisms that estradiol utilizes during estrogen positive feedback to stimulate de novo progesterone synthesis within the hypothalamus to trigger the luteinizing hormone (LH) surge important for ovulation and estrous cyclicity. The activation of these signaling pathways appears to be coordinated by the rising and waning of estradiol throughout the estrous cycle and integral to the negative and positive feedback mechanisms of estradiol. This differential responsiveness is part of the timing mechanism triggering the LH surge

Hormonal regulation of female reproduction

Reproduction is an event that requires the coordination of peripheral organs with the nervous system to ensure that the internal and external environments are optimal for successful procreation of the species. This is accomplished by the hypothalamic-pituitary-gonadal axis that coordinates reproductive behavior with ovulation. The primary signal from the central nervous system is gonadotropin-releasing hormone (GnRH), which modulates the activity of anterior pituitary gonadotropes regulating follicle stimulating hormone (FSH) and luteinizing hormone (LH) release. As ovarian follicles develop they release estradiol, which negatively regulates further release of GnRH and FSH. As estradiol concentrations peak they trigger the surge release of GnRH, which leads to LH release inducing ovulation. Release of GnRH within the central nervous system helps modulate reproductive behaviors providing a node at which control of reproduction is regulated. To address these issues, this review focuses on several critical questions. How is the HPG axis regulated in species with different reproductive strategies? What internal and external conditions modulate the synthesis and release of GnRH? How does GnRH modulate reproductive behavior within the hypothalamus? How does disease shift the activity of the HPG axis.Fil: Christensen, A.. University of California at Los Angeles; Estados UnidosFil: Bentley, G. E.. University of California at Berkeley; Estados UnidosFil: Cabrera Kreiker, Ricardo Jorge. Universidad de Mendoza; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Litoral; ArgentinaFil: Perfito, N.. University of California at Berkeley; Estados UnidosFil: Wu, T. J.. Uniformed Services University Of The Health Sciences; Estados UnidosFil: Micevych, P.. University of California at Los Angeles; Estados Unido

Peripheral and central mechanisms involved in hormonal control of male and female reproduction

Reproduction involves the integration of hormonal signals acting across multiple systems togenerate a synchronized physiological output. A critical component of reproduction is the luteinizinghormone (LH) surge, which is mediated by estradiol (E2) and neuroprogesterone interacting tostimulate kisspeptin release in the rostral periventricular nucleus of the third ventricle in rats. Recentevidence has shown that both classical and membrane E2 and progesterone signaling is involved inthis pathway. A metabolite of gonadotropin-releasing hormone (GnRH), GnRH-(1-5), has been shownto stimulate GnRH expression, secretion, and has a role in the regulation of lordosis. Additionally,gonadotropin-inhibitory hormone (GnIH) projects to and influences the activity of GnRH neurons inbirds. Stress-induced changes in GnIH have been shown to alter breeding behaviors in birds,demonstrating another molecular control of reproduction. Peripherally, paracrine and autocrineactions within the gonad have been suggested as therapeutic targets for infertility in both males andfemales. Dysfunction of testicular prostaglandin synthesis is a possible cause of idiopathic maleinfertility. Indeed, local production of melatonin and corticotropin-releasing hormone (CRH) couldinfluence spermatogenesis via immune pathways in the gonad. In females, vascular endothelialgrowth factor A (VEGF-A) has been implicated in an angiogenic process that mediates developmentof the corpus luteum and thus fertility via the Notch signaling pathway. Age-induced decreases infertility involve ovarian kisspeptin and its regulation of ovarian sympathetic innervation. Finally,morphological changes in the arcuate nucleus of the hypothalamus influence female sexualreceptivity in rats. The processes mediating these morphological changes have been shown toinvolve rapid effects of E2 controlling synaptogenesis in this hypothalamic nucleus. Together, thisreview highlights new research in these areas, focusing on recent findings in the molecularmechanisms of central and peripheral hormonal control of reproduction.Fil: Rudolph, L. M.. University of California at Los Angeles; Estados UnidosFil: Bentley, G. E.. University of California Berkeley; Estados UnidosFil: Calandra, Ricardo Saul. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Paredes, A. H.. Universidad de Chile; ChileFil: Tesone, Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Wu, T. J.. Uniformed Services University; Estados UnidosFil: Micevych, P. E.. University of California at Los Angeles; Estados Unido

Sex differences in hypothalamic astrocyte response to estradiol stimulation

<p>Abstract</p> <p>Background</p> <p>Reproductive 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.</p> <p>Methods</p> <p>Primary 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 (<it>Sry</it>) deleted and inserted into an autosome. Astrocytes were analyzed for <it>Sry </it>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.</p> <p>Results</p> <p>Estradiol stimulated both [Ca²⁺]_irelease and progesterone synthesis in hypothalamic astrocytes from adult female mice. Male astrocytes had a significantly elevated [Ca²⁺]_iresponse 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⁺]_irelease. 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^-<it>Sry </it>and XX<it>Sry</it>).</p> <p>Conclusions</p> <p>Astrocytes 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.</p

Exploiting macrophage autophagy-lysosomal biogenesis as a therapy for atherosclerosis

Macrophages specialize in removing lipids and debris present in the atherosclerotic plaque. However, plaque progression renders macrophages unable to degrade exogenous atherogenic material and endogenous cargo including dysfunctional proteins and organelles. Here we show that a decline in the autophagy-lysosome system contributes to this as evidenced by a derangement in key autophagy markers in both mouse and human atherosclerotic plaques. By augmenting macrophage TFEB, the master transcriptional regulator of autophagy-lysosomal biogenesis, we can reverse the autophagy dysfunction of plaques, enhance aggrephagy of p62-enriched protein aggregates and blunt macrophage apoptosis and pro-inflammatory IL-1β levels, leading to reduced atherosclerosis. In order to harness this degradative response therapeutically, we also describe a natural sugar called trehalose as an inducer of macrophage autophagy-lysosomal biogenesis and show trehalose's ability to recapitulate the atheroprotective properties of macrophage TFEB overexpression. Our data support this practical method of enhancing the degradative capacity of macrophages as a therapy for atherosclerotic vascular disease

Osp/Claudin-11 Forms a Complex with a Novel Member of the Tetraspanin Super Family and β1 Integrin and Regulates Proliferation and Migration of Oligodendrocytes

Oligodendrocyte-specific protein (OSP)/claudin-11 is a major component of central nervous system myelin and forms tight junctions (TJs) within myelin sheaths. TJs are essential for forming a paracellular barrier and have been implicated in the regulation of growth and differentiation via signal transduction pathways. We have identified an OSP/claudin-11–associated protein (OAP)1, using a yeast two-hybrid screen. OAP-1 is a novel member of the tetraspanin superfamily, and it is widely expressed in several cell types, including oligodendrocytes. OAP-1, OSP/claudin-11, and β1 integrin form a complex as indicated by coimmunoprecipitation and confocal immunocytochemistry. Overexpression of OSP/claudin-11 or OAP-1 induced proliferation in an oligodendrocyte cell line. Anti–OAP-1, anti–OSP/claudin-11, and anti–β1 integrin antibodies inhibited migration of primary oligodendrocytes, and migration was impaired in OSP/claudin-11–deficient primary oligodendrocytes. These data suggest a role for OSP/claudin-11, OAP-1, and β1 integrin complex in regulating proliferation and migration of oligodendrocytes, a process essential for normal myelination and repair

Gene Expression Profiles of Intracellular and Membrane Progesterone Receptor Isoforms in the Mediobasal Hypothalamus During Pro-Oestrus

Progesterone action is mediated by its binding to specific receptors. Two progesterone receptor (PR) isoforms (PRA and PRB), three membrane progesterone receptor (mPR) subtypes (mPRα, mPRβ and mPRγ) and at least one progesterone membrane-binding protein [PR membrane component 1 (PRmc1)] have been identified in reproductive tissues and brain of various species. In the present study, we examined gene expression patterns for PR isoforms, mPR subtypes and PRmc1 in the rat mediobasal hypothalamus (MBH) during pro-oestrus. The mRNA level for each receptor subtype was quantified by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) at the time points: 13.00 h on dioestrous day 2; 09.00, 13.00, 17.00 and 22.00 h on pro-oestrus; and 13.00 h on oestrus. For PR, one primer set amplified PRA+PRB, whereas a second primer set amplified PRB. As expected, PRA+PRB mRNA expression was greater than PRB in MBH tissue. PRB mRNA levels increased throughout the day on pro-oestrus, with the highest levels being observed at 17.00 h. PRB mRNA levels in the MBH were increased by 2.4- and 3.0-fold at 13.00 and 17.00 h, respectively, on pro-oestrus compared to 13.00 h on dioestrous day 2. There were differential mRNA expression levels for mPRs and PRmc1 in the MBH, with the highest expression for PRmc1 and the lowest for mPRγ. The mPRα mRNA contents at 13.00 and 17.00 h on pro-oestrus were increased by 1.5-fold compared to that at 13.00 h on dioestrous day 2. The mPRβ mRNA levels at 13.00 and 17.00 h on pro-oestrus were 2.5- and 2.4-fold higher compared to that at 13.00 h on dioestrous day 2, respectively. PRA+PRB, mPRγ and PRmc1 mRNA levels did not vary on pro-oestrus. These findings suggest that the higher expression of PRB, mPRα and mPRβ in the MBH on pro-oestrous afternoon may influence both genomic and nongenomic mechanisms of progesterone action during the critical pre-ovulatory period