RESUMO
Androgens are steroid hormones crucial for sexual differentiation of the brain and reproductive function. In excess, however, androgens may decrease fertility as observed in polycystic ovary syndrome, a common endocrine disorder characterized by oligo/anovulation and/or polycystic ovaries. Hyperandrogenism may also disrupt energy homeostasis, inducing higher central adiposity, insulin resistance, and glucose intolerance, which may exacerbate reproductive dysfunction. Androgens bind to androgen receptors (ARs), which are expressed in many reproductive and metabolic tissues, including brain sites that regulate the hypothalamo-pituitary-gonadal axis and energy homeostasis. The neuronal populations affected by androgen excess, however, have not been defined. We and others have shown that, in mice, AR is highly expressed in leptin receptor (LepRb) neurons, particularly in the arcuate (ARH) and the ventral premammillary nuclei (PMv). Here, we assessed if LepRb neurons, which are critical in the central regulation of energy homeostasis and exert permissive actions on puberty and fertility, have a role in the pathogenesis of female hyperandrogenism. Prenatally androgenized (PNA) mice lacking AR in LepRb cells (LepRbΔAR) show no changes in body mass, body composition, glucose homeostasis, or sexual maturation. They do show, however, a remarkable improvement of estrous cycles combined with normalization of ovary morphology compared to PNA controls. Our findings indicate that the prenatal androgenization effects on adult reproductive physiology (ie, anestrus and anovulation) are mediated by a subpopulation of LepRb neurons directly sensitive to androgens. They also suggest that the effects of hyperandrogenism on sexual maturation and reproductive function in adult females are controlled by distinct neural circuits.
Assuntos
Anovulação , Hiperandrogenismo , Síndrome do Ovário Policístico , Gravidez , Humanos , Camundongos , Feminino , Animais , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Hiperandrogenismo/genética , Hiperandrogenismo/complicações , Receptores para Leptina/genética , Maturidade Sexual , Androgênios/farmacologia , Síndrome do Ovário Policístico/metabolismo , Virilismo , Ciclo EstralAssuntos
Negro ou Afro-Americano/estatística & dados numéricos , Determinação da Pressão Arterial/estatística & dados numéricos , Doenças Cardiovasculares/diagnóstico , Registros Eletrônicos de Saúde/estatística & dados numéricos , Hipertensão/diagnóstico , Portais do Paciente/estatística & dados numéricos , Idoso , Doenças Cardiovasculares/epidemiologia , Feminino , Humanos , Hipertensão/epidemiologia , Masculino , Medicaid/estatística & dados numéricos , Medicare/estatística & dados numéricos , Pessoa de Meia-Idade , Pennsylvania/epidemiologia , Projetos Piloto , Estados UnidosRESUMO
Neuroendocrine control of reproduction is disrupted in many individuals with polycystic ovary syndrome (PCOS), who present with increased luteinizing hormone (LH), and presumably gonadotropin-releasing hormone (GnRH), release frequency, and high androgen levels. Prenatal androgenization (PNA) recapitulates these phenotypes in primates and rodents. Female offspring of mice injected with dihydrotestosterone (DHT) on gestational days 16-18 exhibit disrupted estrous cyclicity, increased LH and testosterone, and increased GnRH neuron firing rate as adults. PNA also alters the developmental trajectory of GnRH neuron firing rates, markedly blunting the prepubertal peak in firing that occurs in three-week (3wk)-old controls. GnRH neurons do not express detectable androgen receptors and are thus probably not the direct target of DHT. Rather, PNA likely alters GnRH neuronal activity by modulating upstream neurons, such as hypothalamic arcuate neurons co-expressing kisspeptin, neurokinin B (gene Tac2), and dynorphin, also known as KNDy neurons. We hypothesized PNA treatment changes firing rates of KNDy neurons in a similar age-dependent manner as GnRH neurons. We conducted targeted extracellular recordings (0.5-2 h) of Tac2-identified KNDy neurons from control and PNA mice at 3wks of age and in adulthood. About half of neurons were quiescent (<0.005 Hz). Long-term firing rates of active cells varied, suggestive of episodic activity, but were not different among groups. Short-term burst firing was also similar. We thus reject the hypothesis that PNA alters the firing rate of KNDy neurons. This does not preclude altered neurosecretory output of KNDy neurons, involvement of other neuronal populations, or in vivo networks as critical drivers of altered GnRH firing rates in PNA mice.
Assuntos
Kisspeptinas , Efeitos Tardios da Exposição Pré-Natal , Androgênios/farmacologia , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Kisspeptinas/genética , Kisspeptinas/metabolismo , Camundongos , Neurônios/metabolismo , GravidezRESUMO
Here, we present an in-depth protocol for extracting ribosome-bound mRNAs in low-abundance cells of hypothalamic nuclei. mRNAs are extracted from the micropunched tissue using refined translating ribosome affinity purification. Isolated RNAs can be used for sequencing or transcript quantification. This protocol enables the identification of actively translated mRNAs in varying physiological states and can be modified for use in any neuronal subpopulation labeled with a ribo-tag. We use leptin receptor-expressing neurons as an example to illustrate the protocol. For complete details on the use and execution of this protocol, please refer to Han et al. (2020).
Assuntos
Cromatografia de Afinidade/métodos , Hipotálamo/metabolismo , RNA Mensageiro/isolamento & purificação , Ribossomos/metabolismo , Animais , Proteínas de Fluorescência Verde/genética , Camundongos , Neurônios/metabolismo , RNA Mensageiro/metabolismoRESUMO
Epidemiological and genome-wide association studies (GWAS) have shown high correlation between childhood obesity and advance in puberty. Early age at menarche is associated with a series of morbidities, including breast cancer, cardiovascular diseases, type 2 diabetes, and obesity. The adipocyte hormone leptin signals the amount of fat stores to the neuroendocrine reproductive axis via direct actions in the brain. Using mouse genetics, we and others have identified the hypothalamic ventral premammillary nucleus (PMv) and the agouti-related protein (AgRP) neurons in the arcuate nucleus (Arc) as primary targets of leptin action in pubertal maturation. However, the molecular mechanisms underlying leptin's effects remain unknown. Here we assessed changes in the PMv and Arc transcriptional program during leptin-stimulated and typical pubertal development using overlapping analysis of bulk RNA sequecing, TRAP sequencing, and the published database. Our findings demonstrate that dynamic somatodendritic remodeling and extracellular space organization underlie leptin-induced and typical pubertal maturation in female mice.
RESUMO
Polycystic ovary syndrome (PCOS) is the most common form of infertility in women. The causes of PCOS are not yet understood and both genetics and early-life exposure have been considered as candidates. With regard to the latter, circulating androgens are elevated in mid-late gestation in women with PCOS, potentially exposing offspring to elevated androgens in utero; daughters of women with PCOS are at increased risk for developing this disorder. Consistent with these clinical observations, prenatal androgenization (PNA) of several species recapitulates many phenotypes observed in PCOS. There is increasing evidence that symptoms associated with PCOS, including elevated luteinizing hormone (LH) (and presumably gonadotropin-releasing hormone [GnRH]) pulse frequency emerge during the pubertal transition. We utilized translating ribosome affinity purification coupled with ribonucleic acid (RNA) sequencing to examine GnRH neuron messenger RNAs from prepubertal (3 weeks) and adult female control and PNA mice. Prominent in GnRH neurons were transcripts associated with protein synthesis and cellular energetics, in particular oxidative phosphorylation. The GnRH neuron transcript profile was affected more by the transition from prepuberty to adulthood than by PNA treatment; however, PNA did change the developmental trajectory of GnRH neurons. This included families of transcripts related to both protein synthesis and oxidative phosphorylation, which were more prevalent in adults than in prepubertal mice but were blunted in PNA adults. These findings suggest that prenatal androgen exposure can program alterations in the translatome of GnRH neurons, providing a mechanism independent of changes in the genetic code for altered expression.
Assuntos
Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Área Pré-Óptica/efeitos dos fármacos , Virilismo , Androgênios/efeitos adversos , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurogênese/genética , Neurônios/metabolismo , Neurônios/fisiologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Área Pré-Óptica/citologia , Área Pré-Óptica/crescimento & desenvolvimento , Área Pré-Óptica/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores Sexuais , Virilismo/induzido quimicamente , Virilismo/genética , Virilismo/fisiopatologiaRESUMO
Changes in gonadotropin-releasing hormone (GnRH) release frequency from the brain help drive reproductive cycles. In polycystic ovary syndrome (PCOS), persistent high GnRH/luteinizing hormone (LH) frequency disrupts cycles and exacerbates hyperandrogenemia. Adult prenatally-androgenized (PNA) mice exhibit increased GnRH neuron firing rate, elevated ovarian androgens, and disrupted cycles, but before puberty, GnRH neuron activity is reduced in PNA mice compared with controls. We hypothesized that ovarian feedback mediates the age-dependent change in GnRH neuron firing rate in PNA vs control mice. Extracellular recordings of green fluorescent protein (GFP)-identified GnRH neurons were made 5 to 7 days after sham-surgery, ovariectomy (OVX), or, in adults, after OVX plus replacement of sub-male androgen levels with dihydrotestosterone implants (OVXâ +â DHT). In 3-week-old mice, OVX did not affect GnRH neuron firing rate in either group. In adult controls, OVX increased GnRH neuron firing rate, which was further enhanced by DHT. In adult PNA mice, however, OVX decreased GnRH neuron firing rate, and DHT restored firing rate to sham-operated levels. In contrast to the differential effects of ovarian feedback on GnRH neuron firing rate, serum LH increased after OVX in both control and PNA mice and was not altered by DHT. Pituitary gene expression largely reflected changes expected with OVX, although in PNA but not control mice, DHT treatment increased Lhb expression. These results suggest prenatal androgen exposure programs marked changes in GnRH neuron regulation by homeostatic steroid feedback. PNA lowers GnRH neuron activity in low-steroid states (before puberty, OVX), and renders activity in adulthood dependent upon ongoing exposure to elevated ovarian androgens.
Assuntos
Androgênios/farmacologia , Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/fisiologia , Ovário/metabolismo , Animais , Di-Hidrotestosterona/farmacologia , Eletrofisiologia , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ovariectomia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Maturidade Sexual/fisiologiaRESUMO
The brain regulates fertility through gonadotropin-releasing hormone (GnRH) neurons. Estradiol induces negative feedback on pulsatile GnRH/luteinizing hormone (LH) release and positive feedback generating preovulatory GnRH/LH surges. Negative and positive feedbacks are postulated to be mediated by kisspeptin neurons in arcuate and anteroventral periventricular (AVPV) nuclei, respectively. Kisspeptin-specific ERα knockout mice exhibit disrupted LH pulses and surges. This knockout approach is neither location-specific nor temporally controlled. We utilized CRISPR-Cas9 to disrupt ERα in adulthood. Mice with ERα disruption in AVPV kisspeptin neurons have typical reproductive cycles but blunted LH surges, associated with decreased excitability of these neurons. Mice with ERα knocked down in arcuate kisspeptin neurons showed disrupted cyclicity, associated with increased glutamatergic transmission to these neurons. These observations suggest that activational effects of estradiol regulate surge generation and maintain cyclicity through AVPV and arcuate kisspeptin neurons, respectively, independent from its role in the development of hypothalamic kisspeptin neurons or puberty onset.
Assuntos
Hipotálamo/fisiologia , Neurônios/fisiologia , Reprodução , Comportamento Sexual Animal , Animais , Estradiol/metabolismo , Receptor alfa de Estrogênio/deficiência , Feminino , Técnicas de Inativação de Genes , Kisspeptinas/análise , Camundongos Knockout , Neurônios/químicaRESUMO
Gonadotropin-releasing hormone (GnRH) neurons are a nexus of fertility regulation. We used translating ribosome affinity purification coupled with RNA sequencing to examine messenger RNAs of GnRH neurons in adult intact and gonadectomized (GDX) male and female mice. GnRH neuron ribosomes were tagged with green fluorescent protein (GFP) and GFP-labeled polysomes isolated by immunoprecipitation, producing one RNA fraction enhanced for GnRH neuron transcripts and one RNA fraction depleted. Complementary DNA libraries were created from each fraction and 50-base, paired-end sequencing done and differential expression (enhanced fraction/depleted fraction) determined with a threshold of >1.5- or <0.66-fold (false discovery rate P ≤ 0.05). A core of â¼840 genes was differentially expressed in GnRH neurons in all treatments, including enrichment for Gnrh1 (â¼40-fold), and genes critical for GnRH neuron and/or gonadotrope development. In contrast, non-neuronal transcripts were not enriched or were de-enriched. Several epithelial markers were also enriched, consistent with the olfactory epithelial origins of GnRH neurons. Interestingly, many synaptic transmission pathways were de-enriched, in accordance with relatively low innervation of GnRH neurons. The most striking difference between intact and GDX mice of both sexes was a marked downregulation of genes associated with oxidative phosphorylation and upregulation of glucose transporters in GnRH neurons from GDX mice. This may suggest that GnRH neurons switch to an alternate fuel to increase adenosine triphosphate production in the absence of negative feedback when GnRH release is elevated. Knowledge of the GnRH neuron translatome and its regulation can guide functional studies and can be extended to disease states, such as polycystic ovary syndrome.
Assuntos
Expressão Gênica , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/metabolismo , Neurônios/metabolismo , Animais , Feminino , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Análise de Sequência de RNARESUMO
Estradiol feedback regulates gonadotropin-releasing hormone (GnRH) neurons and subsequent luteinizing hormone (LH) release. Estradiol acts via estrogen receptor α (ERα)-expressing afferents of GnRH neurons, including kisspeptin neurons in the anteroventral periventricular (AVPV) and arcuate nuclei, providing homeostatic feedback on episodic GnRH/LH release as well as positive feedback to control ovulation. Ionotropic glutamate receptors are important for estradiol feedback, but it is not known where they fit in the circuitry. Estradiol-negative feedback decreased glutamatergic transmission to AVPV and increased it to arcuate kisspeptin neurons; positive feedback had the opposite effect. Deletion of ERα in kisspeptin cells decreased glutamate transmission to AVPV neurons and markedly increased it to arcuate kisspeptin neurons, which also exhibited increased spontaneous firing rate. KERKO mice had increased LH pulse frequency, indicating loss of negative feedback. These observations indicate that ERα in kisspeptin cells is required for appropriate differential regulation of these neurons and neuroendocrine output by estradiol.SIGNIFICANCE STATEMENT The brain regulates fertility through gonadotropin-releasing hormone (GnRH) neurons. Ovarian estradiol regulates the pattern of GnRH (negative feedback) and initiates a surge of release that triggers ovulation (positive feedback). GnRH neurons do not express the estrogen receptor needed for feedback (estrogen receptor α [ERα]); kisspeptin neurons in the arcuate and anteroventral periventricular nuclei are postulated to mediate negative and positive feedback, respectively. Here we extend the network through which feedback is mediated by demonstrating that glutamatergic transmission to these kisspeptin populations is differentially regulated during the reproductive cycle and by estradiol. Electrophysiological and in vivo hormone profile experiments on kisspeptin-specific ERα knock-out mice demonstrate that ERα in kisspeptin cells is required for appropriate differential regulation of these neurons and for neuroendocrine output.
Assuntos
Estradiol/farmacologia , Glutamatos/fisiologia , Hipotálamo/citologia , Hipotálamo/fisiologia , Kisspeptinas/fisiologia , Neurônios/fisiologia , Receptores de Estrogênio/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Animais , Núcleo Arqueado do Hipotálamo/fisiologia , Dinorfinas/farmacologia , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Hipotálamo/efeitos dos fármacos , Hormônio Luteinizante/fisiologia , Camundongos , Núcleos da Linha Média do Tálamo/fisiologia , Neurônios/efeitos dos fármacos , Hipófise/efeitos dos fármacos , Hipófise/fisiologia , Proestro/fisiologia , Receptores Ionotrópicos de Glutamato/efeitos dos fármacos , Receptores Ionotrópicos de Glutamato/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
During the female reproductive cycle, estradiol exerts negative and positive feedback at both the central level to alter gonadotropin-releasing hormone (GnRH) release and at the pituitary to affect response to GnRH. Many studies of the neurobiologic mechanisms underlying estradiol feedback have been done on ovariectomized, estradiol-replaced (OVX+E) mice. In this model, GnRH neuron activity depends on estradiol and time of day, increasing in estradiol-treated mice in the late afternoon, coincident with a daily luteinizing hormone (LH) surge. Amplitude of this surge appears lower than in proestrous mice, perhaps because other ovarian factors are not replaced. We hypothesized GnRH neuron activity is greater during the proestrous-preovulatory surge than the estradiol-induced surge. GnRH neuron activity was monitored by extracellular recordings from fluorescently tagged GnRH neurons in brain slices in the late afternoon from diestrous, proestrous, and OVX+E mice. Mean GnRH neuron firing rate was low on diestrus; firing rate was similarly increased in proestrous and OVX+E mice. Bursts of action potentials have been associated with hormone release in neuroendocrine systems. Examination of the patterning of action potentials revealed a shift toward longer burst duration in proestrous mice, whereas intervals between spikes were shorter in OVX+E mice. LH response to an early afternoon injection of GnRH was greater in proestrous than diestrous or OVX+E mice. These observations suggest the lower LH surge amplitude observed in the OVX+E model is likely not attributable to altered mean GnRH neuron activity, but because of reduced pituitary sensitivity, subtle shifts in action potential pattern, and/or excitation-secretion coupling in GnRH neurons.
Assuntos
Estradiol/fisiologia , Hormônio Liberador de Gonadotropina/fisiologia , Hormônio Luteinizante/sangue , Hipófise/fisiologia , Proestro/fisiologia , Potenciais de Ação , Animais , Feminino , Camundongos Endogâmicos C57BL , Neurônios/metabolismoRESUMO
Leptin signals energy sufficiency to the reproductive hypothalamic-pituitary-gonadal (HPG) axis. Studies using genetic models have demonstrated that hypothalamic neurons are major players mediating these effects. Leptin receptor (LepR) is also expressed in the pituitary gland and in the gonads, but the physiological effects of leptin in these sites are still unclear. Female mice with selective deletion of LepR in a subset of gonadotropes show normal pubertal development but impaired fertility. Conditional deletion approaches, however, often result in redundancy or developmental adaptations, which may compromise the assessment of leptin's action in gonadotropes for pubertal maturation. To circumvent these issues, we adopted a complementary genetic approach and assessed if selective reexpression of LepR only in gonadotropes is sufficient to enable puberty and improve fertility of LepR null female mice. We initially assessed the colocalization of gonadotropin-releasing hormone receptor (GnRHR) and LepR in the HPG axis using GnRHR-IRES-Cre (GRIC) and LepR-Cre reporter (tdTomato or enhanced green fluorescent protein) mice. We found that GRIC and leptin-induced phosphorylation of STAT3 are expressed in distinct hypothalamic neurons. Whereas LepR-Cre was observed in theca cells, GRIC expression was rarely found in the ovarian parenchyma. In contrast, a subpopulation of gonadotropes expressed the LepR-Cre reporter gene (tdTomato). We then crossed the GRIC mice with the LepR null reactivable (LepR(loxTB)) mice. These mice showed an increase in FSH levels, but they remained in a prepubertal state. Together with previous findings, our data indicate that leptin-selective action in gonadotropes serves a role in adult reproductive physiology but is not sufficient to allow pubertal maturation in mice.
Assuntos
Fertilidade/fisiologia , Hormônio Foliculoestimulante/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Puberdade/fisiologia , Receptores LHRH/metabolismo , Receptores para Leptina/metabolismo , Animais , Células Cultivadas , Camundongos , Camundongos Knockout , Receptores para Leptina/genéticaRESUMO
Gonadal steroids regulate the pattern of GnRH secretion. Arcuate kisspeptin (kisspeptin, neurokinin B, and dynorphin [KNDy]) neurons may convey steroid feedback to GnRH neurons. KNDy neurons increase action potential firing upon the activation of neurokinin B receptors (neurokinin-3 receptor [NK3R]) and decrease firing upon the activation of dynorphin receptors (κ-opioid receptor [KOR]). In KNDy neurons from intact vs castrated male mice, NK3R-mediated stimulation is attenuated and KOR-mediated inhibition enhanced, suggesting gonadal secretions are involved. Estradiol suppresses spontaneous GnRH neuron firing in male mice, but the mediators of the effects on firing in KNDy neurons are unknown. We hypothesized the same gonadal steroids affecting GnRH firing pattern would regulate KNDy neuron response to NK3R and KOR agonists. To test this possibility, extracellular recordings were made from KNDy neurons in brain slices from intact, untreated castrated or castrated adult male mice treated in vivo with steroid receptor agonists. As observed previously, the stimulation of KNDy neurons by the NK3R agonist senktide was attenuated in intact vs castrated mice and suppression by dynorphin was enhanced. In contrast to observations of steroid effects on the GnRH neuron firing pattern, both estradiol and DHT suppressed senktide-induced KNDy neuron firing and enhanced the inhibition caused by dynorphin. An estrogen receptor-α agonist but not an estrogen receptor-ß agonist mimicked the effects of estradiol on NK3R activation. These observations suggest the steroid modulation of responses to activation of NK3R and KOR as mechanisms for negative feedback in KNDy neurons and support the contribution of these neurons to steroid-sensitive elements of a GnRH pulse generator.
Assuntos
Androgênios/farmacologia , Di-Hidrotestosterona/farmacologia , Estradiol/farmacologia , Estrogênios/farmacologia , Kisspeptinas/metabolismo , Neurônios/efeitos dos fármacos , Receptores da Neurocinina-3/agonistas , Receptores Opioides kappa/agonistas , Animais , Núcleo Arqueado do Hipotálamo , Dinorfinas/metabolismo , Receptor alfa de Estrogênio/agonistas , Receptor beta de Estrogênio/agonistas , Hormônio Liberador de Gonadotropina , Masculino , Camundongos , Neurocinina B/metabolismo , Neurônios/metabolismo , Orquiectomia , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/farmacologia , Receptores de Esteroides/agonistas , Substância P/análogos & derivados , Substância P/farmacologiaRESUMO
Prenatal androgen (PNA) exposure in mice produces a phenotype resembling lean polycystic ovary syndrome. We studied effects of voluntary exercise on metabolic and reproductive parameters in PNA vs vehicle (VEH)-treated mice. Mice (8 wk of age) were housed individually and estrous cycles monitored. At 10 weeks of age, mice were divided into groups (PNA, PNA-run, VEH, VEH-run, n = 8-9/group); those in the running groups received wheels allowing voluntary running. Unexpectedly, PNA mice ran less distance than VEH mice; ovariectomy eliminated this difference. In ovary-intact mice, there was no difference in glucose tolerance, lower limb muscle fiber types, weight, or body composition among groups after 16 weeks of running, although some mitochondrial proteins were mildly up-regulated by exercise in PNA mice. Before running, estrous cycles in PNA mice were disrupted with most days in diestrus. There was no change in cycles during weeks 1-6 of running (10-15 wk of age). In contrast, from weeks 11 to 16 of running, cycles in PNA mice improved with more days in proestrus and estrus and fewer in diestrus. PNA programs reduced voluntary exercise, perhaps mediated in part by ovarian secretions. Exercise without weight loss improved estrous cycles, which if translated could be important for fertility in and counseling of lean women with polycystic ovary syndrome.
Assuntos
Androgênios/farmacologia , Ciclo Estral/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Condicionamento Físico Animal , Síndrome do Ovário Policístico , Efeitos Tardios da Exposição Pré-Natal , Virilismo , Animais , Composição Corporal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Diestro , Feminino , Teste de Tolerância a Glucose , Extremidade Inferior , Camundongos , Proteínas Mitocondriais/metabolismo , GravidezRESUMO
Acquisition of a mature pattern of gonadotropin-releasing hormone (GnRH) secretion from the CNS is a hallmark of the pubertal process. Little is known about GnRH release during sexual maturation, but it is assumed to be minimal before later stages of puberty. We studied spontaneous GnRH secretion in brain slices from male mice during perinatal and postnatal development using fast-scan cyclic voltammetry (FSCV) to detect directly the oxidation of secreted GnRH. There was good correspondence between the frequency of GnRH release detected by FSCV in the median eminence of slices from adults with previous reports of in vivo luteinizing hormone (LH) pulse frequency. The frequency of GnRH release in the late embryonic stage was surprisingly high, reaching a maximum in newborns and remaining elevated in 1-week-old animals despite low LH levels. Early high-frequency GnRH release was similar in wild-type and kisspeptin knock-out mice indicating that this release is independent of kisspeptin-mediated excitation. In vivo treatment with testosterone or in vitro treatment with gonadotropin-inhibitory hormone (GnIH) reduced GnRH release frequency in slices from 1-week-old mice. RF9, a putative GnIH antagonist, restored GnRH release in slices from testosterone-treated mice, suggesting that testosterone inhibition may be GnIH-dependent. At 2-3 weeks, GnRH release is suppressed before attaining adult patterns. Reduction in early life spontaneous GnRH release frequency coincides with the onset of the ability of exogenous GnRH to induce pituitary LH secretion. These findings suggest that lack of pituitary secretory response, not lack of GnRH release, initially blocks downstream activation of the reproductive system.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Hipófise/metabolismo , Animais , Células Cultivadas , Hormônio Liberador de Gonadotropina/genética , Kisspeptinas/genética , Kisspeptinas/metabolismo , Hormônio Luteinizante/genética , Hormônio Luteinizante/metabolismo , Masculino , Camundongos , Hipófise/efeitos dos fármacos , Hipófise/embriologia , Hipófise/crescimento & desenvolvimento , Hormônios Inibidores da Liberação de Hormônio Hipofisário/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Maturidade Sexual , Testosterona/farmacologiaRESUMO
Pulsatile GnRH release is essential to fertility and is modulated by gonadal steroids, most likely via steroid-sensitive afferents. Arcuate neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) are steroid-sensitive and have been postulated to both generate GnRH pulses and mediate steroid feedback on pulse frequency. KNDy neurons are proposed to interact with one another via NKB and dynorphin to activate and inhibit the KNDy network, respectively, and thus alter kisspeptin output to GnRH neurons. To test the roles of NKB and dynorphin on KNDy neurons and the steroid sensitivity of these actions, targeted extracellular recordings were made of Tac2(NKB)-GFP-identified neurons from castrate and intact male mice. Single-cell PCR confirmed most of these cells had a KNDy phenotype. The neurokinin 3 receptor (NK3R) agonist senktide increased action potential firing activity of KNDy neurons. Dynorphin reduced spontaneous KNDy neuron activity, but antagonism of κ-opioid receptors (KOR) failed to induce firing activity in quiescent KNDy neurons. Senktide-induced activation was greater in KNDy neurons from castrate mice, whereas dynorphin-induced suppression was greater in KNDy neurons from intact mice. Interactions of dynorphin with senktide-induced activity were more complex; dynorphin treatment after senktide had no consistent inhibitory effect, whereas pretreatment with dynorphin decreased senktide-induced activity only in KNDy neurons from intact but not castrate mice. These data suggest dynorphin-mediated inhibition of senktide-induced activity requires gonadal steroid feedback. Together, these observations support the hypotheses that activation of NK3R and KOR, respectively, excites and inhibits KNDy neurons and that gonadal steroids modulate these effects.
Assuntos
Dinorfinas/metabolismo , Kisspeptinas/metabolismo , Neurocinina B/metabolismo , Neurônios/metabolismo , Receptores da Neurocinina-3/metabolismo , Receptores Opioides kappa/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/metabolismo , Benzenoacetamidas/farmacologia , Dinorfinas/genética , Dinorfinas/farmacologia , Hormônio Liberador de Gonadotropina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Kisspeptinas/genética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Neurocinina B/genética , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Orquiectomia , Fragmentos de Peptídeos/farmacologia , Pirrolidinas/farmacologia , Receptores da Neurocinina-3/agonistas , Receptores da Neurocinina-3/genética , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/genética , Substância P/análogos & derivados , Substância P/farmacologia , Fatores de TempoRESUMO
AIM: To determine if a Clinical Reasoning Protocol assisted occupational therapists to consistently choose casting as an intervention in the context of moderate/severe upper limb hypertonia and possible contracture. METHODS: Sixty-four intervention decisions (including strength/movement training, splinting and/or casting) were drawn retrospectively from initial reports at a community clinic. Associations between identified upper limb characteristics, stated clinical aims and intervention decisions were analysed using logistic regression. RESULTS: Casting was statistically significantly likely to be chosen in the presence of moderate (CI(95) 1.88-39.80, p=0.01) or severe hypertonicity (CI(95) 1.34-135.98, p=0.03), and if the stated clinical aim was to reduce hypertonicity (CI(95) 2.01-18.10, p=0.001) or contracture (CI(95) 1.31-12.73, p=0.02). When reports included both these clinical aims, there was a highly significant association with the decision to cast (CI(95) 5.67-9.13, p=0.001). Where casting was indicated as appropriate, but not chosen as an intervention, mitigating factors included older age (70-95 years), limited personal support and a clinical aim of comfort/hygiene maintenance. CONCLUSION: Occupational therapists using the Protocol consistently chose casting as an upper limb intervention for adults who demonstrated moderate/severe hypertonicity, contracture or limited functional ability. Prospective research is required to determine intervention outcomes following use of the Protocol.
Assuntos
Moldes Cirúrgicos , Terapia por Exercício/métodos , Hipertonia Muscular/reabilitação , Extremidade Superior/fisiopatologia , Atividades Cotidianas , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Hipertonia Muscular/fisiopatologia , Estudos Retrospectivos , Resultado do TratamentoRESUMO
The issue of how rapid frequency GnRH pulses selectively stimulate LH transcription is not fully understood. The rat LHß promoter contains two GnRH-responsive regions: the proximal region has binding elements for SF1, and the distal site contains a CArG box, which binds SRF. This study determined whether GnRH stimulates pituitary SF1, DAX1 (an endogenous SF1 inhibitor), and SRF transcription in vivo, and whether regulation is frequency dependent. Male rats were pulsed with 25 ng GnRH i.v. every 30 min or every 240 min for 1-24 h, and primary transcripts (PTs) and mRNAs were measured by real time PCR. Fast frequency GnRH pulses (every 30 min) increased SF1 PT (threefold) within 1 h, and then declined after 6 h. SF1 mRNA also increased within 1 h and remained elevated through 24 h. Fast frequency GnRH also stimulated a transient increase in DAX1 PT (twofold after 1 h) and mRNA (1.7-fold after 6 h), while SRF mRNA rose briefly at 1 h. Slow frequency pulses did not affect gene expression of SF1, DAX1, or SRF. These findings support a mechanistic link between SF1 in the frequency regulation of LHß transcription by pulsatile GnRH.
Assuntos
Receptor Nuclear Órfão DAX-1/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/administração & dosagem , Hormônio Luteinizante Subunidade beta/genética , Hipófise/metabolismo , Fator de Resposta Sérica/genética , Animais , Receptor Nuclear Órfão DAX-1/análise , Masculino , Periodicidade , Hipófise/química , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Fator de Resposta Sérica/análise , Fator Esteroidogênico 1/análise , Fator Esteroidogênico 1/genética , Transcrição GênicaRESUMO
Pulsatile GNRH regulates the gonadotropin subunit genes in a differential manner, with faster frequencies favoring Lhb gene expression and slower frequencies favoring Fshb. Early growth response 1 (EGR1) is critical for Lhb gene transcription. We examined GNRH regulation of EGR1 and its two corepressors, Ngfi-A-binding proteins 1 and 2 (NAB1 and NAB2), both in vivo and in cultured rat pituitary cells. In rats, fast GNRH pulses (every 30 min) stably induced Egr1 primary transcript (PT) and mRNA 2-fold (P < 0.05) for 1-24 h. In contrast, slow GNRH pulses (every 240 min) increased Egr1 PT at 24 h (6-fold; P < 0.05) but increased Egr1 mRNA 4- to 5-fold between 4 and 24 h. Both GNRH pulse frequencies increased EGR1 protein 3- to 4-fold. In cultured rat pituitary cells, GNRH pulses (every 60 min) increased Egr1 (PT, 2.5- to 3-fold; mRNA, 1.5- to 2-fold; P < 0.05). GNRH pulses had little effect on Nab1/2 PT/mRNAs either in vivo or in vitro. We also examined specific intracellular signaling cascades activated by GNRH. Inhibitors of mitogen-activated protein kinase 8/9 (MAPK8/9 [also known as JNK]; SP600125) and MAP Kinase Kinase 1 (MAP2K1 [also known as MEK1]; PD98059) either blunted or totally suppressed the GNRH induction of Lhb PT and Egr1 PT/mRNA, whereas the MAPK14 (also known as p38) inhibitor SB203580 did not. In summary, pulsatile GNRH stimulates Egr1 gene expression and protein in vivo but not in a frequency-dependent manner. Additionally, GNRH-induced Egr1 gene expression is mediated by MAPK8/9 and MAPK1/3, and both are critical for Lhb gene transcription.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Hormônio Liberador de Gonadotropina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Hormônio Luteinizante Subunidade beta/genética , MAP Quinase Quinase 1/metabolismo , Hipófise/metabolismo , Análise de Variância , Animais , Antracenos/farmacologia , Western Blotting , Células Cultivadas , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Flavonoides/farmacologia , Hormônio Liberador de Gonadotropina/administração & dosagem , Imidazóis/farmacologia , Hormônio Luteinizante Subunidade beta/metabolismo , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Hipófise/citologia , Hipófise/efeitos dos fármacos , Piridinas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Pulsatile GnRH (GNRH) differentially regulates LH and FSH subunit genes, with faster frequencies favoring Lhb transcription and slower favoring Fshb. Various intracellular pathways mediate the effects of GNRH, including CaMK II (CAMK2), ERK, and JNK. We examined whether activation of these pathways is regulated by GNRH pulse frequency in vivo. GNRH-deficient rats received GNRH pulses (25 ng i.v. every 30 or 240 min for 8 h, vehicle to controls). Pituitaries were collected 5 min after the last pulse, bisected, and one half processed for RNA (to measure beta subunit primary transcripts [PTs]) and the other for protein. Phosphorylated CAMK2 (phospho-CAMK2), ERK (mitogen-activated protein kinase 1/3 [MAPK1/3], also known as p42 ERK2 and p44 ERK1, respectively), and JNK (MAPK8/9, also known as p46 JNK1 and p54 JNK2, respectively) were determined by Western blotting. The 30-min pulses maximally stimulated Lhb PT (8-fold), whereas 240 min was optimal for Fshb PT (3-fold increase). Both GNRH pulse frequencies increased phospho-CAMK2 4-fold. Activation of MAPK1/3 was stimulated by both 30- and 240-min pulses, but phosphorylation of MAPK3 was significantly greater following slower GNRH pulses (240 min: 4-fold, 30 min: 2-fold). MAPK8/9 activation was unchanged by pulsatile GNRH in this paradigm, but as previous results showed that GNRH-induced activation of MAPK8/9 is delayed, 5 min after GNRH may not be optimal to observe MAPK8/9 activation. These data show that CAMK2 is activated by GNRH, but not in a frequency-dependant manner, whereas MAPK3 is maximally stimulated by slow-frequency GNRH pulses. Thus, the ERK response to slow pulse frequency is part of the mechanisms mediating Fhb transcriptional responses to GNRH.