RESUMEN
Liposomes, artificial phospholipid vesicles, have been developed as a non-viral drug delivery system to allow contained agents to be efficiently delivered to target sites via systemic circulation. Liposomes have been used as a gene transfer tool with cultured cells; however, their precise trafficking and processing remain uncertain. Furthermore, liposomes with different surface charges are known to exhibit distinct properties. The purpose of the current study was to elucidate the intracellular trafficking and processing of liposomes with anionic and cationic surface charges from a morphological view point. We found that cationic liposomes (CLs) were more effectively taken by the cells than anionic liposomes (ALs). Confocal laser scanning microscopy and transmission electron microscopy demonstrated distinct intracellular localization and processing patterns of ALs and CLs. ALs and their contents were localized in lysosomes but not in cytosol, indicating that ALs are subjected to the endosome-lysosome system. In contrast, contents of CLs were distributed mainly in the cytosol. CLs appear to disturb the cell membrane and then collapse to release their contents into the cytosol. It is feasible that the contents of CLs enter the cytosol directly rather than via the endosome-lysosome system.
RESUMEN
We have newly developed a system that allows monitoring of the intensity of fluorescent signals from deep brains of rats transgenically modified to express enhanced green fluorescent protein (eGFP) via an optical fiber. One terminal of the optical fiber was connected to a blue semiconductor laser oscillator/green fluorescence detector. The other terminal was inserted into the vicinity of the eGFP-expressing neurons. Since the optical fiber was vulnerable to twisting stresses caused by animal movement, we also developed a cage in which the floor automatically turns, in response to the turning of the rat's head. This relieved the twisting stress on the optical fiber. The system then enabled real-time monitoring of fluorescence in awake and unrestrained rats over many hours. Using this system, we could continuously monitor eGFP-expression in arginine vasopressin-eGFP transgenic rats. Moreover, we observed an increase of eGFP-expression in the paraventricular nucleus under salt-loading conditions. We then performed in vivo imaging of eGFP-expressing GnRH neurons in the hypothalamus, via a bundle consisting of 3000 thin optical fibers. With the combination of the optical fiber bundle connection to the fluorescence microscope, and the special cage system, we were able to capture and retain images of eGFP-expressing neurons from free-moving rats. We believe that our newly developed method for monitoring and imaging eGFP-expression in deep brain neurons will be useful for analysis of neuronal functions in awake and unrestrained animals for long durations.
Asunto(s)
Encéfalo/citología , Rayos Láser , Neuronas/fisiología , Animales , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/biosíntesis , Masculino , Microscopía Fluorescente/instrumentación , Neuronas/citología , Ratas , Ratas Transgénicas , Ratas Wistar , Factores de TiempoRESUMEN
Insufficiency of thyroid hormones inhibits gonadotropin release and results in dysregulation of reproductive function, although the precise mechanism of this disrupted gonadotropin secretion remains unclear. Kisspeptin is a neuropeptide that strongly stimulates gonadotropin secretion and plays an important role in reproductive function. To examine the involvement of kisspeptin in the dysregulation of gonadotropin secretion in hypothyroidism, we investigated Kiss1 mRNA expression and kisspeptin immunoreactivity in the hypothalamus of female rats treated with propylthiouracil (PTU). In the PTU-treated rats, serum thyroxine (T4) was significantly decreased, whereas thyroid stimulating hormone (TSH) levels were significantly increased. In addition, irregular estrus cycles were observed in PTU-treated rats. In situ hybridization and immunohistochemistry revealed significant reductions in the number of Kiss1 mRNA-expressing neurons and kisspeptin-immunoreactive neurons in the arcuate nucleus (ARC) but not in the anteroventral periventricular nucleus (AVPV) of the PTU-treated rats. Although the serum levels of luteinizing hormone (LH) and estradiol (E2) were unaffected, serum prolactin levels were significantly increased after PTU treatment. These data indicate that kisspeptin expression in the ARC is suppressed under thyroid hormone insufficiency, suggesting that the dysregulation of reproductive function in hypothyroidism is caused by inhibition of kisspeptin neurons in the ARC.
Asunto(s)
Núcleo Arqueado del Hipotálamo/metabolismo , Estro/fisiología , Hipotálamo/metabolismo , Kisspeptinas/metabolismo , Animales , Estradiol/metabolismo , Femenino , Hipotiroidismo/metabolismo , Hormona Luteinizante/metabolismo , Neuronas/metabolismo , RatasRESUMEN
General anesthesia affects the expression of clock genes in various organs. Expression of Per2, a core component of the circadian clock, is markedly and reversibly suppressed by sevoflurane in the suprachiasmatic nucleus (SCN), and is considered to be a biochemical marker of anesthetic effect in the brain. The SCN contains various types of neurons, and this complexity makes it difficult to investigate the molecular mechanisms of anesthesia. Here, we established an in vitro experimental system using a cell line to investigate the mechanisms underlying anesthetic action. Development of the system comprised two steps: first, we developed a system for application of inhalational anesthetics and incubation; next, we established cultures of anesthetic-responsive cells expressing mPer2 promoter-dLuc. GT1-7 cells, derived from the mouse hypothalamus, responded to sevoflurane by reversibly decreasing mPer2-promoter-driven bioluminescence. Interestingly, the suppression of bioluminescence was found only in the serum-starved GT1-7 cells, which showed neuron-like morphology, but not in growing cells, suggesting that neuron-like characteristics are required for anesthetic effects in GT1-7 cells.
Asunto(s)
Anestésicos por Inhalación/farmacología , Anestésicos/farmacología , Línea Celular/citología , Línea Celular/efectos de los fármacos , Éteres Metílicos/farmacología , Animales , Técnicas de Cultivo de Célula , Ritmo Circadiano , Mediciones Luminiscentes , Ratones , Ratas , Sevoflurano , TransgenesRESUMEN
The onset of puberty is initiated by an increase in the release of the gonadotropin-releasing hormone (GnRH) from GnRH neurons in the hypothalamus. However, the precise mechanism that leads to the activation of GnRH neurons at puberty remains controversial. Spines are small protrusions on the surface of dendrites that normally receive excitatory inputs. In this study, we analyzed the number and morphology of spines on GnRH neurons to investigate changes in synaptic inputs across puberty in rats. For morphological estimation, we measured the diameter of the head (DH) of each spine and classified them into small-type (DH < 0.65 µm), large-type (DH > 0.65 µm) and giant-type (DH > 0.9 µm). The greatest number of spines was observed at the proximal dendrite within 50 µm of the soma. At the soma and proximal dendrite, the number of spines was greater in adults than in juveniles in both male and female individuals. Classification of spines revealed that the increase in spine number was due to increases in large- and giant-type spines. To further explore the relationship between spines on GnRH neurons and pubertal development, we next analyzed adult rats neonatally exposed to estradiol benzoate, in which puberty onset and reproductive functions are disrupted. We found a decrease in the number of all types of spines. These results suggest that GnRH neurons become to receive more and greater excitatory inputs on the soma and proximal dendrites as a result of the changes that occur at puberty and that alteration to spines plays a pivotal role in normal pubertal development.
Asunto(s)
Espinas Dendríticas/fisiología , Estradiol/análogos & derivados , Hormona Liberadora de Gonadotropina/metabolismo , Neuronas/metabolismo , Maduración Sexual/efectos de los fármacos , Maduración Sexual/fisiología , Animales , Estradiol/farmacología , Femenino , Hipotálamo/metabolismo , Masculino , Microscopía Confocal , Ratas , Ratas TransgénicasRESUMEN
The inhalation anesthetic sevoflurane suppresses Per2 expression in the suprachiasmatic nucleus (SCN) in rodents. Here, we investigated the intra-SCN regional specificity, time-dependency, and pharmacological basis of sevoflurane-effects. Bioluminescence image was taken from the SCN explants of mPer2 promoter-destabilized luciferase transgenic rats, and each small regions of interest (ROI) of the image was analyzed. Sevoflurane suppressed bioluminescence in all ROIs, suggesting that all regions in the SCN are sensitive to sevoflurane. Clear time-dependency in sevoflurane effects were also observed; application during the trough phase of the bioluminescence cycle suppressed the subsequent increase in bioluminescence and resulted in a phase delay of the cycle; sevoflurane applied during the middle of the ascending phase induced a phase advance; sevoflurane on the descending phase showed no effect. These results indicate that the sevoflurane effect may depend on the intrinsic state of circadian machinery. Finally, we examined the involvement of GABAergic signal transduction in the sevoflurane effect. Co-application of both GABAA and GABAB receptor antagonists completely blocked the effect of sevoflurane on the bioluminescence rhythm, suggesting that sevoflurane inhibits Per2 expression via GABAergic signal transduction. Current study elucidated the anesthetic effects on the molecular mechanisms of circadian rhythm.
Asunto(s)
Anestésicos por Inhalación/farmacología , Éteres Metílicos/farmacología , Proteínas Circadianas Period/metabolismo , Núcleo Supraquiasmático/efectos de los fármacos , Animales , Antagonistas de Receptores de GABA-A/farmacología , Antagonistas de Receptores de GABA-B/farmacología , Mediciones Luminiscentes , Masculino , Imagen Molecular , Proteínas Circadianas Period/genética , Ratas Transgénicas , Sevoflurano , Núcleo Supraquiasmático/metabolismoRESUMEN
Nutrition has significant influences on the development of reproductive functions. Post-weaning manipulation of nutritional status has been shown to alter puberty onset accompanied by changes in the expression of kisspeptin, a neuropeptide encoded by the Kiss1 gene which plays important roles in pubertal development. However, information about the influence of overnutrition during early development is sparse. In this study, we examined pubertal development and Kiss1 mRNA expression in female pups reared by dams fed a high-fat diet (HFD) during lactation. Maternal HFD significantly increased body weight and accelerated puberty onset of female offspring. The number of Kiss1-expressing neurons in the arcuate nucleus (ARC) at weaning was significantly greater in pups of HFD-fed dams than in pups of dams fed a normal diet (ND), whereas no significant difference was observed in the anteroventral periventricular nucleus, the other Kiss1-expressing nucleus. Because adipocyte size and serum leptin level were increased in HFD offspring, we examined the effects of exogenous leptin during the pre-weaning period on Kiss1 expression. Unexpectedly, exogenous leptin had no effect on Kiss1 expression. In summary, we demonstrate that a maternal HFD during the early postnatal period induces increased Kiss1 expression in the ARC and early puberty onset in female offspring.
Asunto(s)
Núcleo Arqueado del Hipotálamo/metabolismo , Dieta Alta en Grasa , Kisspeptinas/metabolismo , Lactancia , Fenómenos Fisiologicos Nutricionales Maternos , Pubertad , Tejido Adiposo/metabolismo , Animales , Femenino , Leptina/administración & dosificación , Leptina/sangre , Neuronas/metabolismo , Embarazo , ARN Mensajero/metabolismo , Ratas , DesteteRESUMEN
The neuropeptide kisspeptin plays an important role in fertility and the onset of puberty, stimulating gonadotropin-releasing hormone (GnRH) neurons to activate the hypothalamic-pituitary-gonadal axis. Several studies have demonstrated a morphological interaction between kisspeptin- and GnRH-expressing neurons; however, few have addressed the interaction of kisspeptin with other neuronal subtypes. We recently showed that fibers immunoreactive for kisspeptin were densely distributed in the dorsal part of the arcuate nucleus. These fibers were found in close proximity to GnRH and tuberoinfundibular dopamine (TIDA) neurons. In the present study, using biotinylated kisspeptin, we established a visualization method for identifying kisspeptin binding sites on TIDA neurons. Biotinylated kisspeptin bound to the cell bodies of TIDA neurons and surrounding fibers, suggesting that TIDA neurons express sites of action for kisspeptin. Our assay also detected biotinylation signals from kisspeptin binding to GnRH fibers in the median eminence, but not to cell bodies of GnRH neurons in the medial preoptic area. Positive signals were completely eliminated by addition of excess non-labeled kisspeptin. This method enabled us to detect kisspeptin binding sites on specific neural structures and neuronal fibers.
RESUMEN
BACKGROUND: We previously reported that sevoflurane anesthesia reversibly suppresses the expression of the clock gene, Period2 (Per2), in the mouse suprachiasmatic nucleus (SCN). However, the molecular mechanisms underlying this suppression remain unclear. In this study, we examined the possibility that sevoflurane suppresses Per2 expression via epigenetic modification of the Per2 promoter. METHODS: Mice were anesthetized with a gas mixture of 2.5% sevoflurane/40% oxygen at a 6 L/min flow for 1 or 4 h. After termination, brains were removed and samples of SCN tissue were derived from frozen brain sections. Chromatin immunoprecipitation (ChIP) assays using anti-acetylated-histone antibodies were performed to investigate the effects of sevoflurane on histone acetylation of the Per2 promoter. Interaction between the E'-box (a cis-element in the Per2 promoter) and CLOCK (the Clock gene product) was also assessed by a ChIP assay using an anti-CLOCK antibody. The SCN concentration of nicotinamide adenine dinucleotide (NAD(+)), a CLOCK regulator, was assessed by liquid chromatography-mass spectrometry. RESULTS: Acetylation of histone H4 in the proximal region of the Per2 promoter was significantly reduced by sevoflurane. This change in the epigenetic profile of the Per2 gene was observed prior to suppression of Per2 expression. Simultaneously, a reduction in the CLOCK-E'-box interaction in the Per2 promoter was observed. Sevoflurane treatment did not affect the concentration of NAD(+) in the SCN. CONCLUSIONS: Independent of NAD(+) concentration in the SCN, sevoflurane decreases CLOCK binding to the Per2 promoter E'-box motif, reducing histone acetylation and leading to suppression of Per2 expression.
Asunto(s)
Epigénesis Genética/efectos de los fármacos , Éteres Metílicos/farmacología , Proteínas Circadianas Period/genética , Núcleo Supraquiasmático/metabolismo , Factores de Transcripción ARNTL/metabolismo , Acetilación/efectos de los fármacos , Anestésicos por Inhalación/farmacología , Animales , Sitios de Unión/genética , Encéfalo/metabolismo , Proteínas CLOCK/metabolismo , Metilación de ADN/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/genética , Histonas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , NAD/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sevoflurano , Factores de TiempoRESUMEN
BACKGROUND: Our previous studies revealed that application of the inhalation anesthetic, sevoflurane, reversibly repressed the expression of Per2 in the mouse suprachiasmatic nucleus (SCN). We aimed to examine whether sevoflurane directly affects the SCN. METHODS: We performed in vivo and in vitro experiments to investigate rat Per2 expression under sevoflurane-treatment. The in vivo effects of sevoflurane on rPer2 expression were examined by quantitative in situ hybridization with a radioactively-labeled cRNA probe. Additionally, we examined the effect of sevoflurane anesthesia on rest/activity rhythms in the rat. In the in vitro experiments, we applied sevoflurane to SCN explant cultures from Per2-dLuc transgenic rats, and monitored luciferase bioluminescence, representing Per2 promoter activity. Bioluminescence from two peripheral organs, the kidney cortex and the anterior pituitary gland, were also analyzed. RESULTS: Application of sevoflurane in rats significantly suppressed Per2 expression in the SCN compared with untreated animals. We observed no sevoflurane-induced phase-shift in the rest/activity rhythms. In the in vitro experiments, the intermittent application of sevoflurane repressed the increase of Per2-dLuc luminescence and led to a phase delay in the Per2-dLuc luminescence rhythm. Sevoflurane treatment did not suppress bioluminescence in the kidney cortex or the anterior pituitary gland. CONCLUSION: The suppression of Per2-dLuc luminescence by sevoflurane in in vitro SCN cultures isolated from peripheral inputs and other nuclei suggest a direct action of sevoflurane on the SCN itself. That sevoflurane has no such effect on peripheral organs suggests that this action might be mediated through a neuron-specific cellular mechanism or a regulation of the signal transduction between neurons.
Asunto(s)
Anestésicos por Inhalación/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Éteres Metílicos/farmacología , Proteínas Circadianas Period/genética , Núcleo Supraquiasmático/efectos de los fármacos , Núcleo Supraquiasmático/metabolismo , Animales , Temperatura Corporal/efectos de los fármacos , Ritmo Circadiano/efectos de los fármacos , Ritmo Circadiano/fisiología , Masculino , Oxígeno/metabolismo , Ratas , Ratas Wistar , Descanso/fisiología , Sevoflurano , Núcleo Supraquiasmático/fisiologíaRESUMEN
Kisspeptin, a neuropeptide encoded by Kiss1 gene, plays pivotal roles in the regulation of reproductive function. Recently various stressors and stress-induced molecules such as corticotropin-releasing hormone (CRH) and corticosterone have been shown to inhibit Kiss1 expression in rat hypothalamus. To determine whether CRH and glucocorticoids directly act on kisspeptin neurons, we examined the colocalization of CRH receptor (CRH-R) and glucocorticoid receptor (GR) in kisspeptin neurons in the female rat hypothalamus. Double-labeling immunohistochemistry revealed that most kisspeptin neurons in the anteroventral periventricular nucleus and periventricular nucleus continuum (AVPV/PeN), and arcuate nucleus (ARC) expressed CRH-R. We also observed a few close appositions of CRH immunoreactive fibers on some of kisspeptin neurons in AVPV/PeN and ARC. On the other hand, most kisspeptin neurons in AVPV/PeN expressed GR, whereas only a few of kisspeptin neurons in ARC expressed GR. Altogether, our study provides neuroanatomical evidence of the direct modulation of kisspeptin neurons by CRH and glucocorticoids and suggests that stress-induced CRH and glucocorticoids inhibit gonadotropin secretion via the kisspeptin system.
Asunto(s)
Hipotálamo/metabolismo , Kisspeptinas/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Receptores de Glucocorticoides/metabolismo , Animales , Femenino , Hipotálamo/anatomía & histología , Inmunohistoquímica/métodos , Neuronas/metabolismo , Ratas , Ratas WistarRESUMEN
Some anesthetics can affect gene expression. Previously, we reported that sevoflurane anesthesia drastically and reversibly repressed the expression of mouse Per2 (mPer2), a core clock gene in the suprachiasmatic nucleus (SCN). In the current study, we examined the time-dependent effect of sevoflurane on mPer2 expression and its interactions with the circadian rest/activity rhythm of mice. During certain hours of the day, mice were anesthetized with 2.5% sevoflurane in 40% oxygen for 4h. The expression level of mPer2 in the SCN was measured by in situ hybridization using a radiolabeled cRNA probe. Anesthesia during the morning hours showed the greatest repressive effect on mPer2 expression. Sevoflurane anesthesia repressed mPer2 expression during the conditions of light/dark and constant dark, and the light conditions modified the repression rate under anesthesia. Moreover, anesthesia in the morning also repressed mPer2 expression the following day. This dominant effect of anesthesia in the morning indicates that sevoflurane anesthesia affects the onset of mPer2 transcription. Behavior analysis revealed that the anesthetic treatment also induced a phase-delay in the rest/activity rhythm. However, no time-dependent effects of anesthesia on the circadian rest/activity rhythm were observed. Further investigation into the molecular events caused by anesthesia are required to explain atypical clinical signs observed in patients after surgical procedures, such as fatigue, sleep disorder, mood alteration and delirium.
Asunto(s)
Anestésicos por Inhalación/farmacología , Ritmo Circadiano , Éteres Metílicos/farmacología , Proteínas Circadianas Period/antagonistas & inhibidores , Núcleo Supraquiasmático/efectos de los fármacos , Anestésicos por Inhalación/efectos adversos , Animales , Masculino , Éteres Metílicos/efectos adversos , Ratones , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Proteínas Circadianas Period/metabolismo , Descanso , Sevoflurano , Núcleo Supraquiasmático/metabolismo , Factores de TiempoRESUMEN
Kisspeptins, encoded by Kiss1 gene, play pivotal roles in the regulation of reproduction. Recently, several studies reported a sex difference in Kiss1 expression in the arcuate nucleus (ARC) during the neonatal period. In this study, we investigated the effect of gonadal steroid manipulation on the sex difference in Kiss1 expression in ARC of rats. At neonatal and prepubertal stages, females had a greater number of Kiss1 neurons than the males. Gonadectomy at those stages resulted in significant increases in the Kiss1 neuron number and the sex differences disappeared. We also confirmed the expression of estrogen receptor α in kisspeptin neurons in neonates. Altogether, our results indicate that ARC Kiss1 expression is negatively regulated by gonadal steroids from early postnatal stages, and that the sex difference in ARC Kiss1 expression is attributed to the difference in circulating gonadal steroid levels. We also found that neonatal estrogenization inhibits Kiss1 expression and impairs negative feedback system.
Asunto(s)
Núcleo Arqueado del Hipotálamo/fisiología , Hormonas Esteroides Gonadales/metabolismo , Kisspeptinas/biosíntesis , Kisspeptinas/genética , ARN Mensajero/genética , Animales , Núcleo Arqueado del Hipotálamo/metabolismo , Receptor alfa de Estrógeno/metabolismo , Femenino , Masculino , Neuronas/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Reproducción , Caracteres SexualesRESUMEN
Kisspeptin is a pivotal regulator of the onset of puberty and the estrus cycle, but may also take part in pregnancy and lactation. Kisspeptin neurons and their fibers are distributed abundantly throughout the arcuate nucleus (ARC) of the hypothalamus, but the targets of the fiber projections in the ARC have not been fully investigated. The present study followed the projection of kisspeptin fibers to tuberoinfundibular dopaminergic (TIDA) neurons in the ARC, pivotal endocrine neurons that control prolactin secretion. Immunoreactive fibers of kisspeptin or neurokinin B, a peptide coexpressed in kisspeptin neurons, were abundantly found adjacent to TIDA neurons in female rats, but few were observed in male rats. The immunoreactivities of both peptides adjacent to TIDA neurons were significantly reduced in estradiol-primed ovariectomized rats. Precise 3D analysis of the attachment of kisspeptin-immunoreactive fibers to TIDA neurons was achieved using a synaptic marker that indicated synaptic connection. Finally, double-labeling immunoelectron microscopy confirmed the synaptic connections of kisspeptin-immunoreactive fibers to the cell body and fibers of TIDA neurons. These findings indicate that in female rats, kisspeptin/NKB fibers may directly affect TIDA neurons that regulate prolactin secretion, and that they are more likely to be activated during low estradiol status.
Asunto(s)
Núcleo Arqueado del Hipotálamo/metabolismo , Núcleo Arqueado del Hipotálamo/ultraestructura , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/ultraestructura , Kisspeptinas/metabolismo , Neuroquinina B/metabolismo , Prolactina/metabolismo , Animales , Estradiol/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Vías Nerviosas/metabolismo , Vías Nerviosas/ultraestructura , Ovariectomía , Ratas , Ratas Wistar , Caracteres SexualesRESUMEN
Changes in the expression of estrogen-related substances in monkeys' brains at the menopausal transition, when estrogen deficit starts to occur, have not yet been examined thoroughly. In the present study, we immunohistochemically investigated the expression levels of estrogen receptor beta (ERß) and aromatase (local estrogen synthesizing enzyme) in the hippocampal formation of premenopausal, menopausal, and ovariectomized premenopausal monkeys. In all monkeys tested, ERß immunoreactivity was observed in interneurons located in the subiculum and the Ammon's horn, and most of these ERß-immunoreactive neurons coexpressed a GABAergic neuron marker, parvalbumin. In the menopausal monkeys who exhibited a decline in estrogen concentration, hippocampal ERß was highly upregulated, while aromatase expression was not markedly changed. By contrast, aromatase in the ovariectomized monkeys was significantly upregulated, while ERß expression was not changed. In the brains of ovariectomized and menopausal monkeys, depletion of ovary-derived estrogen brought about different reactions which may be attributed to the senescence of brain aging.
Asunto(s)
Aromatasa/metabolismo , Receptor beta de Estrógeno/metabolismo , Hipocampo/metabolismo , Menopausia/metabolismo , Ovariectomía , Animales , Femenino , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Macaca , RadioinmunoensayoRESUMEN
Kisspeptin is a family of neuropeptides and the natural ligands of G protein-coupled receptor (GPR)-54. Kisspeptin/GPR-54 system is known to play a pivotal role in puberty onset and in the regulation of reproductive functions. To clarify the postnatal ontogeny of kisspeptin neurons in rat hypothalamus, we analyzed the expression patterns of kisspeptin mRNA from neonate to adult by in situ hybridization. In anteroventral periventricular nucleus (AVPV), kisspeptin mRNA were first detected at postnatal day (PND) 7 and postnatal week 3 in males and females, respectively, and the number of kisspeptin mRNA-expressing neurons increased during development in both sexes. In the arcuate nucleus (ARC), kisspeptin mRNA was present from PND3. In males, the number of kisspeptin mRNA-expressing neurons gradually increased during development. In females, the number of kisspeptin mRNA-expressing neurons in neonates was greater than in males; it significantly decreased at juvenile stages and then increased toward adulthood. These results indicate that the increase in kisspeptin mRNA expression in ARC across puberty might be involved in the onset of puberty. We also demonstrate that the kisspeptin mRNA-expressing neurons in ARC appear earlier than those in AVPV and show clear sex differences in their numbers during neonatal period.
Asunto(s)
Hipotálamo/crecimiento & desarrollo , Hipotálamo/metabolismo , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/metabolismo , Animales , Femenino , Humanos , Hipotálamo/citología , Hibridación in Situ , Kisspeptinas , Masculino , Neuronas/citología , Neuronas/metabolismo , Embarazo , Pubertad/fisiología , ARN Mensajero/genética , Ratas , Ratas WistarRESUMEN
To investigate the reported discrepancy regarding the immunohistochemical expression of kisspeptin neurons, we produced a new antibody against synthetic peptide containing the same amino acid residual sequence as rat kisspeptin10. Although the antibody showed cross-reactivities against neurons other than kisspeptin neurons, these cross-reactivities were excluded by preabsorption with neuropeptide FF (NPFF). Immunohistochemistry using the antibody preabsorbed with NPFF showed specific kisspeptin immunoreactivities (IRs) in the arcuate nucleus (Arc), the inner layer of the median eminence, and the infundibulum in the rat hypothalamus. IRs were more intense in the adult female rats than in the males. This sexual dimorphism became observable at the 7th day after birth. These IRs intensified with age. Ovariectomy enhanced the IRs in the Arc in the female rats. In contrast, regarding IRs in the anteroventral periventricular nucleus (AVPV), only a few immunoreactive fibers were detected in the adult rats. We applied this antibody for the investigation of the interaction between kisspeptin fibers and gonadotropin-releasing hormone (GnRH) neurons. No direct morphological interaction between the cell bodies of GnRH neurons and kisspeptin fibers was observed in the medial preoptic area. Many projections of kisspeptin fibers were found close to the GnRH fibers in the lateral part of the median eminence. However, we did not observe any direct contact between kisspeptin fibers and the GnRH fibers. These results suggest that kisspeptin neurons regulate GnRH neurons not via the synaptic contact but via another information transmission process that is not synapse-dependent, such as volume transmission.
Asunto(s)
Anticuerpos/inmunología , Hormona Liberadora de Gonadotropina/metabolismo , Inmunohistoquímica/métodos , Neuronas/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Hipotálamo/anatomía & histología , Hipotálamo/metabolismo , Hibridación in Situ , Kisspeptinas , Masculino , Datos de Secuencia Molecular , Neuronas/citología , Péptidos/genética , Péptidos/inmunología , Isoformas de Proteínas/genética , Proteínas/genética , Ratas , Ratas Wistar , Caracteres SexualesRESUMEN
The specific and efficient activation of mitogen-activated protein kinase (MAPK) signaling modules is mediated, at least in part, by scaffold proteins. c-Jun NH(2)-terminal kinase (JNK)-associated leucine zipper protein (JLP) was identified as a scaffold protein for JNK and p38 MAPK signaling modules. JLP is expressed nearly ubiquitously and is involved in intracellular signaling pathways, such as the G(alpha13) and Cdo-mediated pathway, in vitro. To date, however, JLP expression has not been analyzed in detail, nor are its physiological functions well understood. Here we investigated the expression of JLP in the mouse testis during development. Of the tissues examined, JLP was strongest in the testis, with the most intense staining in the elongated spermatids. Since the anti-JLP antibody used in this study can recognize both JLP and sperm-associated antigen 9 (SPAG9), a splice variant of JLP that has been studied extensively in primates, we also examined its expression in macaque testis samples. Our results indicated that in mouse and primate testis, the isoform expressed at the highest level was JLP, not SPAG9. We also investigated the function of JLP by disrupting the Jlp gene in mice, and found that the male homozygotes were subfertile. Taken together, these observations may suggest that JLP plays an important role in testis during development, especially in the production of functionally normal spermatozoa.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Eliminación de Gen , Infertilidad Masculina/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos , Fibroblastos/metabolismo , Homocigoto , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación , ARN Mensajero/análisis , Recuento de Espermatozoides , Espermatozoides/metabolismo , Testículo/metabolismoRESUMEN
Based on morphological and physiological characteristics, the mammalian neocortex is divided into various neocortical areas and its diversity is prominent in the primates including humans. These neocortical areas are constructed during development, but the details of the developmental events remain unclear, especially at the molecular level. We measured the mRNA and protein levels of neurotrophins in various neocortical areas of developing rhesus monkeys. The expression patterns of both the neurotrophin-3 (NT-3) mRNA and the protein showed area differences. In the sensory and motor areas, NT-3 mRNA and protein levels had started to decline by a week after birth. In contrast, the levels declined after the third postnatal week in the association neocortical areas. The level of brain-derived neurotrophic factor (BDNF) protein changed in an area-dependent manner during development, but that of mRNA did not. The decline of the BDNF protein level started earlier in the sensory and motor neocortical areas than in the association neocortical areas, suggesting that sensory and motor neocortical areas develop earlier than the association areas in terms of the developmental changes in neurotrophins.