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

Developmental and functional effects of steroid hormones on the neuroendocrine axis and spinal cord

This review highlights the principal effects of steroid hormones at central and peripheral levels in the neuroendocrine axis. The data discussed highlight the principal role of oestrogens and testosterone in hormonal programming in relation to sexual orientation, reproductive and metabolic programming, and the neuroendocrine mechanism involved in the development of polycystic ovary syndrome phenotype. Moreover, consistent with the wide range of processes in which steroid hormones take part, we discuss the protective effects of progesterone on neurodegenerative disease and the signalling mechanism involved in the genesis of oestrogen-induced pituitary prolactinomas.Fil: Zubeldia Brenner, Lautaro. 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: Roselli, C. E.. Oregon Health and Science University Portland; Estados UnidosFil: Recabarren, S. E.. Universidad de Concepción; ChileFil: Gonzalez Deniselle, Maria Claudia. 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: Lara, H. E.. Universidad de Chile; Chil

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

Non-Neuronal Cells in the Hypothalamic Adaptation to Metabolic Signals

Although the brain is composed of numerous cell types, neurons have received the vast majority of attention in the attempt to understand how this organ functions. Neurons are indeed fundamental but, in order for them to function correctly, they rely on the surrounding “non-neuronal” cells. These different cell types, which include glia, epithelial cells, pericytes, and endothelia, supply essential substances to neurons, in addition to protecting them from dangerous substances and situations. Moreover, it is now clear that non-neuronal cells can also actively participate in determining neuronal signaling outcomes. Due to the increasing problem of obesity in industrialized countries, investigation of the central control of energy balance has greatly increased in attempts to identify new therapeutic targets. This has led to interesting advances in our understanding of how appetite and systemic metabolism are modulated by non-neuronal cells. For example, not only are nutrients and hormones transported into the brain by non-neuronal cells, but these cells can also metabolize these metabolic factors, thus modifying the signals reaching the neurons. The hypothalamus is the main integrating center of incoming metabolic and hormonal signals and interprets this information in order to control appetite and systemic metabolism. Hence, the factors transported and released from surrounding non-neuronal cells will undoubtedly influence metabolic homeostasis. This review focuses on what is known to date regarding the involvement of different cell types in the transport and metabolism of nutrients and hormones in the hypothalamus. The possible involvement of non-neuronal cells, in particular glial cells, in physiopathological outcomes of poor dietary habits and excess weight gain are also discussed.The authors are funded by grants from the Spanish Ministry of Science and Innovation (BFU2014-51836-C2-2 to JAC and BFU2014-51836-C2-1 to LG-S), Spanish Ministry of Education, Culture and Sports (university training grant FPU13/00909 to AF-R), Fondo de Investigación Sanitaria (PI-1302195, PI-1600485, and CIBEROBN to JA and CIBERFES to LG-S) and Fondos FEDER.Peer reviewedPeer Reviewe

Analysis of the roles of kisspeptins and the tachykinin receptor, Tacr2, in the control of Reproduction: novel interactive pathways, regulatory mechanisms and metabolic implications

For sake of clarity, this Doctoral Thesis has been divided in 3 main experimental sets: In the Experimental Set 1, a comprehensive series of phenotypical, pharmacological, behavioral and histological analyses were conducted in a novel mouse line with congenital ablation of Tacr2, namely, the Tacr2 KO mouse, to unravel the physiological role of neurokinin 2 receptor (NK2R) signaling, and its eventual interplay with kisspeptins, in the control of the HPG axis. Our initial pharmacological studies documented the capacity of the agonist of NK2R to evoke acute LH responses after icv administration in control mice, which was comparable with those elicited by the other TAC agonists as well as kisspeptin (Kp-10). Similar pharmacological studies in Tacr2 KO mice revealed that the LH responses to the NK1R and NK3R receptor agonists and Kp-10 were not affected by Tacr2 ablation. On the contrary, the LH response to a NK2R agonist was sexually dimorphic, so that Tacr2 KO female mice displayed an attenuated LH response while, despite effective ablation of NK2R, Tacr2 KO male mice displayed a grossly conserved LH responsiveness to NK2R agonist, which, nonetheless, was abrogated after blockade of NK3R. Next, we carried out a thorough reproductive characterization of Tacr2 null mice of both sexes, which documented that the congenital lack of NK2R signaling does not compromise the timing of puberty onset or the fertility either in males or females. Yet, a trend for increased breeding intervals together with partially alteration in LH pulsatility were observed in Tacr2 KO female mice in adulthood; the latter consisting in the suppression of basal LH levels, but no changes in the number of LH pulses. On the other hand, histological analyses suggested the existence of a defective function of Sertoli cells in their interaction with spermatids in adult Tacr2 KO male, but no gross morphological alterations in the ovary or the uterus of females KO mice. In addition, Tacr2 KO female mice failed to display a significant increase of LH levels at 2 days after ovariectomy (OVX), although no differences were detected at later periods, nor did OVX Tacr2 KO female mice showed differences in terms of tail skin temperature as compared with littermate controls. On the other hand, LH levels in Tacr2 KO mice subjected to conditions of energy deficit caused by 24-h fasting were significantly lower than in controls. A three-chamber social sex preference and female urine sniffing tests were also applied to adult male and female null mice and their controls. Yet, no differences in any of the behavioral parameters explored, including velocity, distance moved and time spent in the vicinity of the same or opposite-sex mouse, were noted between control and KO mice. Additionally, to assess the impact of the lack of NK2R signaling on key metabolic parameters, we monitored body weight gain in both adult female and male Tacr2 KO mice, which was similar to that of control littermates. Likewise, body composition analyses demonstrated similar fat and lean mass in Tacr2 KO and control mice; energy expenditure and respiratory quotient, total and nocturnal locomotor activity, and the day/night feeding patterns during 24 hours were also similar in control and Tacr2 KO mice. Moreover, measurement of blood pressure demonstrated that Tacr2 KO mice display values of systolic blood pressure similar to their control littermates. Finally, metabolic analyses addressing glucose homeostasis after challenge mice with an obesogenic diet revealed a significant increase in the basal glucose levels in Tacr2 KO males. In the Experimental Set 2, a series of proteomic, molecular and immunohistochemical analyses were implemented using genetically modified mouse lines to explore novel targets mediating kisspeptins actions. We performed a first proteomic analysis in samples from the preoptic area (POA) of Kiss1 KO male mice icv injected with Kp-10, which pointed out a set of 30 differentially-expressed proteins involved in cellular metabolism and energy balance, cell signaling, protein folding and synaptic plasticity; the later revealed that glial fibrillary acidic protein (GFAP), an astrocyte marker, was modified in response to Kp-10. In a second proteomic analysis using a next-generation technology, we found that astrocyte markers including GFAP were again modified in response to Kp-10; a finding that was also confirmed by RT-qPCR and western blot analyses. These findings led us to analyze and demonstrate for the first time the expression of Gpr54 and the presence of functional kisspeptin receptors in astrocytes. On the latter, we found in both primary astrocyte cultures from neonatal rats and mice the activation of downstream signaling pathways in response to kisspeptin. Our data also demonstrated the co-expression of Gpr54 in GFAP-positive cells in mouse brain tissue, with a substantial enrichment in the % of co-location in the hypothalamic regions of the vascular organ of lamina terminalis (VOLT) and anteroventral periventricular nucleus (AVPV). In addition, in two models of congenital ablation of Gpr54, we observed changes in astrocytic GFAP labelling by immunohistochemistry in the arcuate nucleus (ARC) of these mouse models, although immunoreactivity of S100 calcium binding protein B (S100β), another astrocytic marker protein, was not affected. Finally, in the Experimental Set 3, a series of phenotypic and pharmacological analyses were conducted in a novel mouse line with selective ablation of Gpr54 in GFAP-positive cells, namely, the G-KiRKO mouse (for GFAP cell-specific Kisspeptin Receptor KO), to evaluate the physiological role of kisspeptin signaling in astrocytes in the control of the reproductive axis. First, we carried out a set of molecular and immunohistochemical analyses that involved the use of a reporter mouse line and primary astrocyte cultures for the validation of our GKiRKO model. These analyses confirmed effective ablation of Gpr54 in astrocytes. However, despite the effective disruption of kisspeptin signaling in astrocytes, characterization of GKiRKO mice revealed normal pubertal timing, and preserved LH levels and estrous cyclicity in the adulthood of G-KiRKO mice. Yet, challenging of G-KiRKO mice with icv injection of Kp-10 demonstrated an enhancement of LH responses, suggesting a repressive role of astrocyte signaling in mediating kisspeptin actions in terms of gonadotropin secretion. The measurement of the metabolic parameters (i.e., body weight gain and body composition analysis) did not show differences in G-KiRKO vs. control mice. Nevertheless, the glucose tolerance test (GTT) revealed a subtle improvement of the response to a glucose bolus in GKiRKO mice, without showing alterations in insulin sensitivity. On the other hand, obesogenic conditions defined by chronic exposure to HFD evidenced a delay in the vaginal opening (VO), external marker of puberty in female rodents, in HFD-fed G-KiRKO mice in comparison to HFD-fed control mice. In contrast, no differences were detected in term of first estrus, indirect marker of first ovulation, neither in the age of balano-preputial separation (BSP), external marker of puberty in male rodents, in HFD-fed KO mice versus controls. In adulthood, only HFD-fed G-KiRKO female mice displayed the LH hyper-response to Kp-10, as observed in lean conditions. In addition, high-fat diet exposure induced estrous cycle irregularities in GKiRKO female mice, as evidenced by longer cycle length and a higher number of days in diestrus and reduced number of days in estrus. The metabolic parameters, such as body weight gain and body composition remained unaltered in G-KiRKO mice. In HFD-fed G-KiRKO males, the levels of glucose during the GTT were lower than those observed for control mice, as already registered in conditions of normal feeding. In contrast, no differences in terms of insulin sensitivity (assessed by ITT) were detected. In the case of HFD-fed G-KiRKO female mice, no differences in basal glucose levels, neither in the response to glucose or insulin boluses, were detected. Finally, GFAP immunoreactivity was not different in the ARC nucleus of G-KiRKO male mice fed HFD in comparison to their controls. By contrast, GFAP immunoreactivity responses to acute exposure to HFD were significantly lower in the ARC of G-KiRKO male mice.Esta Tesis Doctoral se ha dividido en tres bloques experimentales principales: En el Bloque Experimental 1, se ha realizado una caracterización fenotípica, farmacológica y comportamental exhaustiva, incluyéndose también análisis histológicos, del modelo de ratón con ablación congénita de Tacr2, denominado Tacr2 KO, para desvelar la implicación de la señalización por NK2R, y su interacción con kisspeptinas, en el control de la función reproductiva. Los estudios farmacológicos iniciales documentaron la capacidad del agonista de NK2R para evocar una respuesta en términos de secreción de LH tras su administración icv en ratones control, en la misma medida que lo hacen los otros agonistas de taquiquininas y la kisspeptina (Kp-10). Estudios farmacológicos similares en el modelo Tacr2 KO no mostraron alteraciones en la respuesta de LH a los agonistas de NK1R y NK3R así como de Kp-10. No obstante, en la respuesta de LH al agonista de NK2R se observó un dimorfismo sexual; esto es, mientras que en hembras la respuesta apareció parcialmente atenuada, en los machos Tacr2 KO esta respuesta estuvo prácticamente conservada, siendo esta última suprimida tras el bloqueo de la señalización por NK3R. La caracterización reproductiva de este modelo reveló que el inicio de la pubertad y la fertilidad no están comprometidas por la ausencia de señalización por NK2R, aunque sí se detectó una tendencia al incremento en el intervalo de cría, así como un deterioro en la función en las células de Sertoli en su interacción con las espermátides en los ratones macho adultos Tacr2 KO, aunque ninguna alteración en ovario ni útero en el caso de las hembras KO. Por otro lado, los ratones hembra Tacr2 KO presentaron niveles basales de secreción pulsátil de LH significativamente bajos, sin cambios aparentes en el número de pulsos de LH. Además, en las hembras Tacr2 KO, no se observó el incremento de los niveles de LH tras dos días después de la ovariectomía (OVX) que sí es detectado en las hembras controles. En estas mismas hembras Tacr2 KO ovariectomizadas, el perfil de temperatura de la cola monitorizado no reveló diferencias con respecto a los controles. En el caso de los machos Tacr2 KO, en condiciones de déficit energético consecuencia de un ayudo de 24 horas de duración, se registraron niveles de LH significativamente más bajos que los de los controles. Por otro lado, los estudios de comportamiento de preferencia por el sexo opuesto y de orina de hembras, en los que se incluye la monitorización de la velocidad y distancia recorrida durante el test, no mostraron ninguna respuesta diferencial entre los ratones KO y los controles. Adicionalmente, se analizaron indicadores metabólicos para valorar el impacto de la ausencia de señalización por NK2R sobre la homeostasis metabólica. Entre ellos la ganancia de peso corporal fue similar entre ratones Tacr2 KO y sus controles. Del mismo modo, los análisis de composición corporal indicaron que no había diferencias en el porcentaje de masa grasa y magra entre KO y controles, ni tampoco en el gasto energético, coeficiente respiratorio, actividad locomotora total y nocturna, ni en los patrones de ingesta durante el día y la noche registrados durante 24 horas. La medida de la presión sanguínea tampoco evidenció diferencias entre animales controles y KO. Finalmente, se observaron niveles basales de glucosa significativamente más elevados en los ratones macho Tacr2 KO tras la exposición crónica de estos a una dieta obesogénica. En el Bloque Experimental 2, se llevaron a cabo una serie de análisis proteómicos, moleculares e inmunohistoquímicos en modelos de ratón modificados genéticamente dirigidos a identificar nuevas dianas de acción de las kisspeptinas. En el primer estudio proteómico realizado en muestras del área preóptica del hipotálamo de ratones deficientes para kisspeptina, Kiss1 KO, inyectados intracerebroventriculamente (icv) con Kp-10, se observó la expresión diferencial de 30 proteínas implicadas en el metabolismo celular y balance energético, señalización celular, plegamiento de proteínas y plasticidad sináptica; identificándose en esta última categoría a la proteína acídica fibrilar glial (GFAP), marcador de astrocitos. En el segundo estudio proteómico, en el que se empleó una tecnología de nueva generación, se detectó de nuevo que la expresión de GFAP y otros indicadores de astrocitos eran modificados en respuesta a la administración de kisspeptina; efecto que fue confirmado también por análisis de RT-qPCR y western blot. A partir de estas evidencias, implementamos una serie de análisis que permitieron demostrar por primera vez en cultivos primarios de astrocitos de rata y ratón la expresión del receptor de kisspeptina, Gpr54. Además, se comprobó que la señalización por kisspeptinas era funcional en estas células. In vivo se detectó la co-expresión de Gpr54 y GFAP en cerebro de ratón, registrándose un enriquecimiento del porcentaje de co-localización Gpr54- GFAP en las áreas hipotalámicas del órgano vascular de la lámina terminal (VOLT) y del área anteroventral periventricular (AVPV). Por último, mediante análisis por inmunohistoquímica se observaron cambios en el marcaje de GFAP en el núcleo arcuato (ARC) en dos modelos de ratón con eliminación congénita de Gpr54, si bien dichos cambios no fueron extensibles a la proteína S100B de unión a calcio, otro marcador de astrocitos. Finalmente, en el Bloque Experimental 3, se llevaron a cabo una serie de estudios fenotípicos y farmacológicos en un modelo novedoso de ratón con deleción selectiva del receptor Gpr54 en células GFAP-positivas, modelo de ratón denominado como G-KiRKO (del inglés GFAP cell-specific Kisspeptin Receptor KO), a fin de caracterizar el papel fisiológico de la señalización por kisspeptinas en astrocitos en el control de la función reproductiva. Primero, se validó el modelo mediante análisis moleculares e inmunohistoquímicos empleando una línea reportera de ratón y cultivos primarios de astrocitos; análisis que confirmaron la eficiente eliminación de Gpr54 de astrocitos. Sin embargo, la eliminación efectiva de la señalización de kisspeptina en astrocitos en el modelo G-KiRKO, no afectó la edad de llegada a la pubertad, ni modificó la secreción de LH ni alteró el ciclo estral en la edad adulta. En cambio, los ratones G-KiRKO presentaron respuestas significativamente aumentadas de LH tras la administración icv de Kp-10. Por otro lado, los ratones G-KiRKO tampoco presentaron alteraciones en parámetros metabólicos como son el peso y composición corporal, pero sí se registró una respuesta glucémica mejorada en el test de sobrecarga de glucosa, sin verse afectada la respuesta a insulina. A su vez, los ratones hembra G-KiRKO mostraron una atenuación del efecto de una dieta obesogénica (HFD) sobre el adelanto de la edad de apertura vaginal, marcador externo de llegada a la pubertad en roedores hembra. No obstante, no se afectó la edad de primer estro, indicador de la primera ovulación en roedores, ni tampoco la edad de la separación balanoprepucial en el caso de los ratones macho. En la edad adulta, solo en el caso de las hembras GKiRKO se detectó un aumento excesivo de la respuesta de LH tras la administración con Kp- 10, que previamente se había observado en condicionales basales. Además, con la exposición a la dieta rica en grasas, el ciclo estral en hembras KO se vio comprometido, registrándose ciclos más largos, con un mayor número de días en la fase de diestro y menos en estro. Por otro lado, los valores de parámetros metabólicos como el peso y la composición corporal en los ratones G-KiRKO fueron comparables a los de los controles. Bajo esta condición de estrés metabólico, solo los machos G-KiRKO mostraron una respuesta glucémica mejorada, similar a la observada en condiciones fisiológicas, sin alteraciones en la respuesta a insulina, y sin diferencias en la respuesta de glucosa ni de insulina entre las hembras controles y G-KiRKO. Por otro lado, los ratones macho G-KiRKO presentaron una respuesta disminuida en términos de inmunoreactividad de GFAP en el ARC tras exposición aguda a HFD

Interaction of gonadal steroids and growth factors in brain sex differentiation

Sex hormones have developmental trophic actions on neurons and glial cells and activational effects in the adult brain. It has been proposed that sex steroids may interact with peptide trophic factors to induce part of their biological effects in the nervous system. The first evidence of such an interaction was provided by Toran-Allerand et al (Brain Research 1980; 184: 517-524), showing that in explant cultures of fetal rodent hypothalamus, estrogen and insulin have synergistic effects on neurite growth, an effect probably mediated by insulin-like growth factor-1 receptors. Recent data indicate that estrogen and insulin-like growth factor-1 signaling pathways interact on hypothalamic neurons to regulate survival and differentiation and that sex steroids interact with a variety of different trophic signals in vivo to regulate neuroendocrine events. These findings suggest that trophic factors may be involved in the genesis of sex differences in the developing brain and in the maintenance of a sexually differentiated brain function in the adult.Biomedical Reviews 1997; 7: 67-74

Steroid metabolism and effects in central and peripheral glial cells

Hormonal steroids participate in the control of a large number of functions of the central nervous system (CNS); recent data show that they may also intervene at the level of the peripheral nervous system (PNS). Both the CNS and the PNS metabolize endogenous as well as exogenous steroids; one of the major enzymatic system is represented by the 5alpha-reductase-3alpha-hydroxysteroid complex. This is a versatile system, since every steroid possessing the delta 4-3keto configuration (e.g., testosterone, progesterone, deoxycorticosterone) may be a substrate. High levels of 5alpha-reductase are found in the white matter of the CNS and in purified myelin. The observation that, in addition to neurons, glia may be a target for steroid action is an important recent finding. The effects of progesterone, testosterone, corticoids, and their respective 5alpha and 3alpha-5alpha derivatives on the expression of glial genes are presented and discussed. It has also been found that progesterone and/or its 5alpha-reduced metabolites increase the mRNA for the two major proteins of peripheral myelin, the glycoprotein Po and the peripheral myelin protein 22, in the sciatic nerve of normal and aged animals and in Schwann cells. The hypothesis has been put forward that glycoprotein Po might be under the control of progestagens acting mainly via the progesterone receptor, and that peripheral myelin protein 22 might be controlled via an interaction of steroids with the gamma-aminobutyric acid (GABA)ergic system. It is known that tetrahydroprogesterone, the 3alpha-5alpha-reduced metabolite of progesterone, interacts with the GABA(A) receptor. Our recent data show that several subunits of this receptor are present in sciatic nerve as well as in Schwann cells that reside in this nerve. These data open multiple possibilities for new therapeutic approaches to demyelinating diseases

Nuclear Progesterone Receptors Are Up-Regulated by Estrogens in Neurons and Radial Glial Progenitors in the Brain of Zebrafish

In rodents, there is increasing evidence that nuclear progesterone receptors are transiently expressed in many regions of the developing brain, notably outside the hypothalamus. This suggests that progesterone and/or its metabolites could be involved in functions not related to reproduction, particularly in neurodevelopment. In this context, the adult fish brain is of particular interest, as it exhibits constant growth and high neurogenic activity that is supported by radial glia progenitors. However, although synthesis of neuroprogestagens has been documented recently in the brain of zebrafish, information on the presence of progesterone receptors is very limited. In zebrafish, a single nuclear progesterone receptor (pgr) has been cloned and characterized. Here, we demonstrate that this pgr is widely distributed in all regions of the zebrafish brain. Interestingly, we show that Pgr is strongly expressed in radial glial cells and more weakly in neurons. Finally, we present evidence, based on quantitative PCR and immunohistochemistry, that nuclear progesterone receptor mRNA and proteins are upregulated by estrogens in the brain of adult zebrafish. These data document for the first time the finding that radial glial cells are preferential targets for peripheral progestagens and/or neuroprogestagens. Given the crucial roles of radial glial cells in adult neurogenesis, the potential effects of progestagens on their activity and the fate of daughter cells require thorough investigation

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