RESUMO
High-fat diet (HFD) is associated with the regulation of reproductive functions. This study aimed to investigate the effects of short-term HFD on the mRNA expression levels of follicle-stimulating hormone ß subunit (FSHß), luteinizing hormone ß subunit (LHß), gonadotropin-releasing hormone receptor, and long-chain fatty acid receptor, GPR120, in the matured male mouse pituitary gland. Adult male mice were fed either control chow or HFD for 1, 2, 5, 10, 30 and 150 days. Fshb and Gpr120 mRNA expression levels in the pituitary glands were significantly increased during 2 to 30 days of HFD feeding. Gnrh-r mRNA in the 30 days HFD fed group and body weight in the 30 and 150 days HFD fed groups were higher than control. However, there were no significant differences in plasma non-esterified fatty acids or glucose levels during the 150 days of HFD feeding. These results suggest that male mice feeding a short-term HFD induces FSHß synthesis and GPR120 expression in their pituitary gonadotropes.
Assuntos
Dieta Hiperlipídica/métodos , Subunidade beta do Hormônio Folículoestimulante/metabolismo , Expressão Gênica , Hormônio Luteinizante Subunidade beta/metabolismo , Hipófise/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores LHRH/metabolismo , Animais , Subunidade beta do Hormônio Folículoestimulante/genética , Gonadotrofos/metabolismo , Hormônio Luteinizante Subunidade beta/genética , Masculino , Camundongos , Receptores Acoplados a Proteínas G/genética , Receptores LHRH/genética , Fatores de TempoRESUMO
GPR120 is a long-chain fatty acid (LCFA) receptor that is specifically expressed in gonadotropes in the anterior pituitary gland in mice. The aim of this study was to investigate whether GPR120 is activated by free fatty acids in the pituitary of mice and mouse immortalized gonadotrope LßT2 cells. First, the effects of palmitate on GPR120, gonadotropic hormone b-subunits, and GnRH-receptor expression in gonadotropes were investigated in vitro. We observed palmitate-induced an increase in Gpr120 mRNA expression and a decrease in follicle-stimulating hormone b-subunit (Fshb) expression in LßT2 cells. Furthermore, palmitate exposure caused the phosphorylation of ERK1/2 in LßT2 cells, but no significant changes were observed in the expression levels of luteinizing hormone b-subunit (Lhb) and gonadotropin releasing hormone-receptor (Gnrh-r) mRNA and number of GPR120 immunoreactive cells. Next, diurnal variation in Gpr120 mRNA expression in the male mouse pituitary gland was investigated using ad libitum and night-time restricted feeding (active phase from 1900 to 0700 h) treatments. In ad libitum feeding group mice, Gpr120 mRNA expression at 1700 h was transiently higher than that measured at other times, and the peak blood non-esterified fatty acid (NEFA) levels were observed from 1300 to 1500 h. These results were not observed in night-time-restricted feeding group mice. These results suggest that GPR120 is activated by LCFAs to regulate follicle stimulating hormone (FSH) synthesis in the mouse gonadotropes.
Assuntos
Ácidos Graxos não Esterificados/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Gonadotrofos/metabolismo , Ácido Palmítico/farmacologia , Hipófise/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Linhagem Celular , Subunidade beta do Hormônio Folículoestimulante/genética , Subunidade beta do Hormônio Folículoestimulante/metabolismo , Gonadotrofos/efeitos dos fármacos , Hormônio Luteinizante Subunidade beta/genética , Hormônio Luteinizante Subunidade beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fosforilação/efeitos dos fármacos , Hipófise/efeitos dos fármacos , Receptores Acoplados a Proteínas G/genética , Receptores LHRH/genética , Receptores LHRH/metabolismoRESUMO
Hindbrain ependymocytes are postulated to have a glucose-sensing role in regulating gonadal functions. Previous studies have suggested that malnutrition-induced suppression of gonadotropin secretion is mediated by noradrenergic inputs from the A2 region in the solitary tract nucleus to the paraventricular nucleus (PVN), and by corticotropin-releasing hormone (CRH) release in the hypothalamus. However, no morphological evidence to indicate the neural pathway from the hindbrain ependymocytes to hypothalamic kisspeptin neurons, a center for reproductive function in mammals, currently exists. The present study aimed to examine the existence of a neuronal pathway from the hindbrain ependymocytes to kisspeptin neurons in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). To determine this, wheat-germ agglutinin (WGA), a trans-synaptic tracer, was injected into the fourth ventricle (4V) in heterozygous Kiss1-tandem dimer Tomato (tdTomato) rats, where kisspeptin neurons were visualized by tdTomato fluorescence. 48 h after the WGA injection, brain sections were taken from the forebrain, midbrain and hindbrain and subjected to double immunohistochemistry for WGA and dopamine ß-hydroxylase (DBH) or CRH. WGA immunoreactivities were found in vimentin-immunopositive ependymocytes of the 4V and the central canal (CC), but not in the third ventricle. The WGA immunoreactivities were detected in some tdTomato-expressing cells in the ARC and AVPV, DBH-immunopositive cells in the A1-A7 noradrenergic nuclei, and CRH-immunopositive cells in the PVN. These results suggest that the hindbrain ependymocytes have neuronal connections with the kisspeptin neurons, most probably via hindbrain noradrenergic and CRH neurons to relay low energetic signals for regulation of reproduction.
Assuntos
Epêndima , Hipotálamo , Kisspeptinas/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Rombencéfalo , Animais , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Núcleo Arqueado do Hipotálamo/metabolismo , Epêndima/citologia , Epêndima/efeitos dos fármacos , Epêndima/metabolismo , Estradiol/metabolismo , Estradiol/farmacologia , Feminino , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Kisspeptinas/genética , Vias Neurais/citologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Neurônios/efeitos dos fármacos , Ovariectomia , Núcleo Hipotalâmico Paraventricular/citologia , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/metabolismo , Ratos , Ratos Transgênicos , Rombencéfalo/citologia , Rombencéfalo/efeitos dos fármacos , Rombencéfalo/metabolismo , Aglutininas do Germe de Trigo/metabolismoRESUMO
GPR120 is a G-protein-coupled receptor that is activated by long-chain fatty acids. In our previous study, GPR120 expression was detected in gonadotrophs of the mouse anterior pituitary gland. It is well known that the function of anterior pituitary cells is largely under the influence of circulating sex steroids. Thus, in the present study, we investigated the modulatory roles of the ovarian hormones, estrogen (E2) and progesterone (P), on the expression levels of GPR120 mRNA in mouse pituitary glands. GPR120 mRNA expression levels in the pituitary gland were increased after ovariectomy or P treatment, and were decreased after the administration of E2. Simultaneous injection of E2 and P interfered with the action of E2 on GPR120 mRNA expression. The GnRH antagonist, Cetrotide, did not inhibit the increase in GPR120 expression in ovariectomized (OVX) animals. In addition, immunohistochemistry revealed that more than 95.4% of GPR120 immunoreactive cells colocalized with the luteinizing hormone ß (LHß) in the anterior pituitary gland of intact, ovariectomized (OVX), estradiol-primed OVX (OVX+E2), or progesterone-primed OVX (OVX+P) animals. Furthermore, GPR120 mRNA expression levels were not significantly different in the pituitary gland of females throughout the ovarian cycle. It is suggested that low levels of P may mask the inhibitory effect of estradiol on the synthesis of GPR120 in the estrous stage in intact animals. These results demonstrate that ovarian hormones may directly regulate GPR120 expression in the reproductive cycle at the pituitary level.
Assuntos
Estradiol/farmacologia , Gonadotrofos/efeitos dos fármacos , Gonadotrofos/metabolismo , Progesterona/farmacologia , Receptores Acoplados a Proteínas G/genética , Animais , Ciclo Estral/efeitos dos fármacos , Ciclo Estral/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios Esteroides Gonadais/metabolismo , Hormônios Esteroides Gonadais/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Ovário/metabolismo , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Reprodução/efeitos dos fármacos , Reprodução/genéticaRESUMO
Figure 3(a) have been corrected.
RESUMO
G-protein-coupled receptor 120 (GPR120) has been known to be a receptor of long-chain fatty acids. Here, we investigated GPR120 expression in the mouse pituitary gland via real-time PCR, in situ hybridization, and immunohistochemistry. GPR120 mRNA was abundantly expressed in the pituitary gland of ad-lib fed animals. In situ hybridization and immunohistochemistry revealed GPR120 expression in the gonadotropes of the anterior pituitary gland, but not in thyrotropes, somatotropes, lactotropes, corticotropes, melanotropes, and the posterior pituitary gland. Furthermore, 24 h of fasting induced an increase in GPR120 mRNA expression in the pituitary gland. These results demonstrate that GPR120 in mouse pituitary gonadotropes is upregulated by fasting and that it may play a role in controlling gonadotropin secretion.
Assuntos
Gonadotrofos/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Animais , Gonadotrofos/citologia , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos ICR , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
This study aims to determine the epigenetic mechanism regulating Kiss1 gene expression in the anteroventral periventricular nucleus (AVPV) to understand the mechanism underlying estrogen-positive feedback action on gonadotropin-releasing hormone/gonadotropin surge. We investigated estrogen regulation of the epigenetic status of the mouse AVPV Kiss1 gene locus in comparison with the arcuate nucleus (ARC), in which Kiss1 expression is down-regulated by estrogen. Histone of AVPV Kiss1 promoter region was highly acetylated, and estrogen receptor α was highly recruited at the region by estrogen. In contrast, the histone of ARC Kiss1 promoter region was deacetylated by estrogen. Inhibition of histone deacetylation up-regulated in vitro Kiss1 expression in a hypothalamic non-Kiss1-expressing cell line. Gene conformation analysis indicated that estrogen induced formation of a chromatin loop between Kiss1 promoter and the 3' intergenic region, suggesting that the intergenic region serves to enhance estrogen-dependent Kiss1 expression in the AVPV. This notion was proved, because transgenic reporter mice with a complete Kiss1 locus sequence showed kisspeptin neuron-specific GFP expression in both the AVPV and ARC, but the deletion of the 3' region resulted in greatly reduced GFP expression only in the AVPV. Taken together, these results demonstrate that estrogen induces recruitment of estrogen receptor α and histone acetylation in the Kiss1 promoter region of the AVPV and consequently enhances chromatin loop formation of Kiss1 promoter and Kiss1 gene enhancer, resulting in an increase in AVPV-specific Kiss1 gene expression. These results indicate that epigenetic regulation of the Kiss1 gene is involved in estrogen-positive feedback to generate the gonadotropin-releasing hormone/gonadotropin surge.
Assuntos
Núcleos Anteriores do Tálamo/metabolismo , Epigênese Genética/fisiologia , Estrogênios/metabolismo , Retroalimentação Fisiológica/fisiologia , Regulação da Expressão Gênica/fisiologia , Kisspeptinas/metabolismo , Acetilação , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Sequência de Bases , DNA Intergênico/metabolismo , Epigênese Genética/genética , Hormônio Liberador de Gonadotropina/metabolismo , Proteínas de Fluorescência Verde/genética , Histonas/metabolismo , Kisspeptinas/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Accumulating evidence suggests that the arcuate nucleus (ARC) kisspeptin/neurokinin B (NKB)/dynorphin (KNDy) neurons play a role in estrogen negative feedback action on pulsatile gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release. The present study aimed to determine if dynorphin (Dyn) is involved in estrogen negative feedback on pulsatile GnRH/LH release. The effect of the injection of nor-binaltorphimine (nor-BNI), a kappa-opioid receptor (KOR) antagonist, into the third cerebroventricle (3V) on LH pulses was determined in ovariectomized (OVX) adult female rats with/without replacement of negative feedback levels of estradiol (low E2). The mean LH concentrations and baseline levels of LH secretion in nor-BNI-injected, low E2-treated rats were significantly higher compared with vehicle-treated controls. On the other hand, the nor-BNI treatment failed to affect any LH pulse parameters in OVX rats without low E2 treatment. These results suggest that Dyn is involved in the estrogen negative feedback regulation of pulsatile GnRH/LH release. The low E2 treatment had no significant effect on the numbers of ARC Pdyn (Dyn gene)-,Kiss1- and Tac2 (NKB gene)-expressing cells. The treatment also did not affect mRNA levels of Pdyn and Oprk1 (KOR gene) in the ARC-median eminence region, but significantly increased the ARC kisspeptin immunoreactivity. These findings suggest that the negative feedback level of estrogen suppresses kisspeptin release from the ARC KNDy neurons through an unknown mechanism without affecting the Dyn and KOR expressions in the ARC. Taken together, the present result suggests that Dyn-KOR signaling is a part of estrogen negative feedback action on GnRH/LH pulses by reducing the kisspeptin release in female rats.
Assuntos
Dinorfinas/metabolismo , Estrogênios/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Kisspeptinas/metabolismo , Hormônio Luteinizante/metabolismo , Receptores Opioides kappa/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Ventrículos Cerebrais/metabolismo , Retroalimentação Fisiológica , Feminino , Hibridização In Situ , Neurocinina B/metabolismo , Neurônios/metabolismo , Ovariectomia , Precursores de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Radioimunoensaio , Ratos , Ratos Wistar , Transdução de Sinais , Taquicininas/metabolismoRESUMO
Puberty in mammals is timed by an increase in gonadotropin-releasing hormone (GnRH) secretion. Previous studies have shown involvement of the two neuropeptides, kisspeptin and neurokinin B (NKB), in controlling puberty onset. Little is known about the role of the other key neuropeptide, dynorphin, in controlling puberty onset, although these three neuropeptides colocalize in the arcuate kisspeptin neurons. The arcuate kisspeptin neuron, which is also referred to as the KNDy neuron, has recently been considered to play a role as an intrinsic source of the GnRH pulse generator. The present study aimed to determine if attenuation of inhibitory dynorphin-kappa-opioid receptor (KOR) signaling triggers the initiation of puberty in normal developing female rats. The present study also determined if stimulatory NKB-neurokinin 3 receptor (NK3R) signaling advances puberty onset. Female Wistar-Imamichi rats were weaned and intraperitoneally implanted with osmotic minipumps filled with nor-binaltorphimine (nor-BNI), a KOR antagonist, or senktide, a NK3R agonist, at 20 days of age. Fourteen days of intraperitoneal infusion of nor-BNI or senktide advanced puberty onset, manifested as vaginal opening and the first vaginal estrus in female rats. Frequent blood sampling showed that nor-BNI significantly increased luteinizing hormone (LH) pulse frequency at 29 days of age compared with vehicle-treated controls. Senktide tended to increase this frequency, but its effect was not statistically significant. The present results suggest that the inhibitory input of dynorphin-KOR signaling plays a role in the prepubertal restraint of GnRH/LH secretion in normal developing female rats and that attenuation of dynorphin-KOR signaling and increase in NKB-NK3R signaling trigger the onset of puberty in female rats.
Assuntos
Dinorfinas/metabolismo , Hormônio Luteinizante/metabolismo , Neurocinina B/metabolismo , Ovário/metabolismo , Receptores da Neurocinina-3/metabolismo , Receptores Opioides kappa/metabolismo , Maturidade Sexual , Animais , Implantes de Medicamento , Dinorfinas/antagonistas & inibidores , Feminino , Hormônio Luteinizante/sangue , Naltrexona/administração & dosagem , Naltrexona/análogos & derivados , Naltrexona/toxicidade , Antagonistas de Entorpecentes/administração & dosagem , Antagonistas de Entorpecentes/toxicidade , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neurocinina B/agonistas , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ovário/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/toxicidade , Puberdade Precoce/sangue , Puberdade Precoce/induzido quimicamente , Puberdade Precoce/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Receptores da Neurocinina-3/agonistas , Receptores Opioides kappa/antagonistas & inibidores , Maturidade Sexual/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Substância P/administração & dosagem , Substância P/análogos & derivados , Substância P/toxicidade , DesmameRESUMO
Sensitive and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), frequently performed using direct polymerase chain reaction (PCR), is essential for restricting the spread of coronavirus disease 2019 (COVID-19). However, studies evaluating accurate detection are still required. This study evaluated the quantitativeness and sensitivity of the Ampdirect™ 2019-nCoV detection kit, a direct PCR method. Using saliva with or without Tris-buffered saline (TBS) dilution, linearity, and limits of the N1 and N2 regions of SARS-CoV-2 genomic RNA were assessed using EDX SARS-CoV-2 RNA standard dissolved in RNase-free water (RFW). Fluorescence intensities in non-diluted saliva were higher than those in TBS-diluted samples. Linear regression analysis of detected quantification cycle values and spiked standard RNA concentrations showed that the coefficient of determination of the N1 and N2 genes was 0.972 and 0.615 in RFW and 0.947 and 0.660 in saliva, respectively. N1- and N2-positive detection rates in saliva were 46% (6/13 tests) and 0% (0/12 tests) at one copy/reaction, respectively. These results indicate good quantitativeness and sensitivity for N1 but not for N2. Therefore, our findings reveal that the Ampdirect™ 2019-nCoV system, especially targeting the N1 gene, enables rapid and convenient quantification of SARS-CoV-2 RNA in saliva at one copy/reaction.
RESUMO
Ependymocytes are one of the energy-sensing cells that regulate animal reproduction through their responsiveness to changes in extracellular glucose levels and the expression of pancreatic-type glucokinase and glucose transporter 2, which play a critical role in sensing blood glucose levels in pancreatic ß-cells. Molecular mechanisms underlying glucose sensing in the ependymocytes remain poorly understood. The AMP-activated protein kinase (AMPK), a serine/threonine kinase highly conserved in all eukaryotic cells, has been suggested to be an intracellular fuel gauge that detects cellular energy status. The present study aims to clarify the role AMPK of the lower brainstem ependymocytes has in sensing glucose levels to regulate reproductive functions. First, we will show that administration of 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, an AMPK activator, into the 4th ventricle suppressed pulsatile LH release in female rats. Second, we will demonstrate the presence of AMPK catalytic subunit immunoreactivities in the rat lower brainstem ependymocytes. Third, transgenic mice were generated to visualize the ependymocytes with Venus, a green fluorescent protein, expressed under the control of the mouse vimentin promoter for further in vitro study. The Venus-labeled ependymocytes taken from the lower brainstem of transgenic mice revealed that AMPK activation by 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, an AMPK activator, increased in vitro intracellular calcium concentrations. Taken together, malnutrition-induced AMPK activation of ependymocytes of the lower brainstem might be involved in suppression of GnRH/LH release and then gonadal activities.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Tronco Encefálico/citologia , Tronco Encefálico/metabolismo , Reprodução/efeitos dos fármacos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Glicemia/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Tronco Encefálico/efeitos dos fármacos , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Ratos , Ribonucleosídeos/farmacologiaRESUMO
Sexual dimorphism of the behaviors or physiological functions in mammals is mainly due to the sex difference of the brain. A number of studies have suggested that the brain is masculinized or defeminized by estradiol converted from testicular androgens in perinatal period in rodents. However, the mechanisms of estrogen action resulting in masculinization/defeminization of the brain have not been clarified yet. The large-scale analysis with microarray in the present study is an attempt to obtain the candidate gene(s) mediating the perinatal estrogen effect causing the brain sexual differentiation. Female mice were injected with estradiol benzoate (EB) or vehicle on the day of birth, and the hypothalamus was collected at either 1, 3, 6, 12, or 24 h after the EB injection. More than one hundred genes down-regulated by the EB treatment in a biphasic manner peaked at 3 h and 12-24 h after the EB treatment, while forty to seventy genes were constantly up-regulated after it. Twelve genes, including Ptgds, Hcrt, Tmed2, Klc1, and Nedd4, whose mRNA expressions were down-regulated by the neonatal EB treatment, were chosen for further examination by semiquantitative RT-PCR in the hypothalamus of perinatal intact male and female mice. We selected the genes based on the known profiles of their potential roles in brain development. mRNA expression levels of Ptgds, Hcrt, Tmed2, and Nedd4 were significantly lower in male mice than females at the day of birth, suggesting that the genes are down-regulated by estrogen converted from testicular androgen in perinatal male mice. Some genes, such as Ptgds encoding prostaglandin D2 production enzyme and Hcrt encording orexin, have been reported to have a role in neuroprotection. Thus, Ptgds and Hcrt could be possible candidate genes, which may mediate the effect of perinatal estrogen responsible for brain sexual differentiation.