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1.
Front Endocrinol (Lausanne) ; 13: 818888, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35250874

RESUMO

Female fertility declines with age, and this natural variation culminates in reproductive senescence. Human follicular fluids are rich in low-molecular weight metabolites which are responsible for the maturation of oocytes. The metabolomic approaches are powerful tools to study biochemical markers of oocyte quality in the follicular fluids. It is necessary to identify and quantify the reliable metabolites in follicular fluids reflecting oocyte developmental potential. The goal of this study is to conduct a metabolomic analysis of the follicular fluids in women of different ages and study the metabolomic profile of the follicular fluids in relationship with oocyte quality in assisted reproductive technology (ART) treatment. A total of 30 women seeking for ART treatment at the Women's Hospital, Zhejiang University School of Medicine from October 2014 to April 2015 were recruited for the present study. Fifteen women aged from 39 to 47 were grouped as advanced maternal age, and the other 15 women aged from 27 to 34, as young controls. Ovarian stimulation and oocyte retrieval were conducted using a regular protocol involving mid-luteal pituitary down-regulation and controlled ovarian stimulation. Follicular fluids from mature follicles were collected and centrifuged for analyses. Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectroscopy (GC-MS) were used to perform the quantitative metabolomic analysis. The follicular fluid levels of 311 metabolites and the metabolic significance were assessed. 70 metabolites showed significant differences between women with young and advanced ages. Follicular fluids from women with advanced age showed significantly higher levels of creatine, histidine, methionine, trans-4-hydroxyproline, choline, mevalonate, N2,N2-dimethylguanosine and gamma-glutamylvaline, as compared to those from the young age group. 8 metabolites were found significantly correlated with maternal age positively. Moreover, 3 metabolites were correlated with the number of oocytes retrieved, and 5 metabolites were correlated with cleaved embryo numbers, both negatively. The follicular fluids from women undergoing ART treatment exhibited age-dependent metabolomic profile. Metabolites associated with oocyte quality were identified, suggesting them as potential biomarkers for oocyte maturation and ART outcomes.


Assuntos
Líquido Folicular , Recuperação de Oócitos , Feminino , Líquido Folicular/metabolismo , Humanos , Masculino , Metabolômica , Oócitos/fisiologia , Técnicas de Reprodução Assistida
2.
Adv Sci (Weinh) ; 9(12): e2104140, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35187865

RESUMO

Optogenetics has become a widely used technique in neuroscience research, capable of controlling neuronal activity with high spatiotemporal precision and cell-type specificity. Expressing exogenous opsins in the selected cells can induce neuronal activation upon light irradiation, and the activation depends on the power of incident light. However, high optical power can also lead to off-target neuronal activation or even cell damage. Limiting the incident power, but enhancing power distribution to the targeted neurons, can improve optogenetic efficiency and reduce off-target effects. Here, the use of optical lenses made of polystyrene microspheres is demonstrated to achieve effective focusing of the incident light of relatively low power to neighboring neurons via photonic jets. The presence of microspheres significantly localizes and enhances the power density to the target neurons both in vitro and ex vivo, resulting in increased inward current and evoked action potentials. In vivo results show optogenetic stimulation with microspheres that can evoke significantly more motor behavior and neuronal activation at lowered power density. In all, a proof-of-concept of a strategy is demonstrated to increase the efficacy of optogenetic neuromodulation using pulses of reduced optical power.


Assuntos
Opsinas , Optogenética , Potenciais de Ação , Neurônios/fisiologia , Optogenética/métodos , Fótons
3.
Theranostics ; 12(1): 143-166, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34987639

RESUMO

Aims: Hypoxic-ischaemic encephalopathy (HIE) is one of the most serious complications in neonates and infants. Mesenchymal stromal cell (MSC)-based therapy is emerging as a promising treatment avenue for HIE. However, despite its enormous potential, the clinical application of MSCs is limited by cell heterogeneity, low isolation efficiency and unpredictable effectiveness. In this study, we examined the therapeutic effects and underlying mechanisms of human pluripotent stem cell-derived ectomesenchymal stromal cells (hPSC-EMSCs) in a rat model of HIE. Methods: hPSC-EMSCs were induced from either human embryonic stem cells or induced pluripotent stem cells. Stem cells or the conditioned medium (CM) derived from stem cells were delivered intracranially or intranasally to neonatal rats with HIE. Human umbilical cord-derived MSCs (hUC-MSCs) were used as the therapeutic comparison control and phosphate-buffered saline (PBS) was used as a negative control. Lesion size, apoptosis, neurogenesis, astrogliosis and microgliosis were evaluated. The rotarod test and Morris water maze were used to determine brain functional recovery. The PC-12 cell line, rat primary cortical neurons and neural progenitor cells were used to evaluate neurite outgrowth and the neuroprotective and neurogenesis effects of hPSC-EMSCs/hUC-MSCs. RNA-seq and enzyme-linked immunosorbent assays were used to determine the secretory factors that were differentially expressed between hPSC-EMSCs and hUC-MSCs. The activation and suppression of extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB) were characterised using western blotting and immunofluorescent staining. Results: hPSC-EMSCs showed a higher neuroprotective potential than hUC-MSCs, as demonstrated by a more significant reduction in lesion size and apoptosis in the rat brain following hypoxia-ischaemia (HI). Compared with PBS treatment, hPSC-EMSCs promoted endogenous neurogenesis and alleviated astrogliosis and microgliosis. hPSC-EMSCs were more effective than hUC-MSCs. hPSC-EMSCs achieved a greater recovery of brain function than hUC-MSCs and PBS in rats with HIE. CM derived from hPSC-EMSCs had neuroprotective and neurorestorative effects in vitro through anti-apoptotic and neurite outgrowth- and neurogenesis-promoting effects. Direct comparisons between hPSC-EMSCs and hUC-MSCs revealed the significant enrichment of a group of secretory factors in hPSC-EMSCs, including nerve growth factor (NGF), platelet-derived growth factor-AA and transforming growth factor-ß2, which are involved in neurogenesis, synaptic transmission and neurotransmitter transport, respectively. Mechanistically, the CM derived from hPSC-EMSCs was found to potentiate NGF-induced neurite outgrowth and the neuronal differentiation of NPCs via the ERK/CREB pathway. Suppression of ERK or CREB abolished CM-potentiated neuritogenesis and neuronal differentiation. Finally, intranasal delivery of the CM derived from hPSC-EMSCs significantly reduced brain lesion size, promoted endogenous neurogenesis, mitigated inflammatory responses and improved functional recovery in rats with HIE. Conclusion: hPSC-EMSCs promote functional recovery after HI through multifaceted neuromodulatory activities via paracrine/trophic mechanisms. We propose the use of hPSC-EMSCs for the treatment of HIE, as they offer an excellent unlimited cellular source of MSCs.


Assuntos
Hipóxia-Isquemia Encefálica/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Animais , Células Cultivadas , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas , Células-Tronco Mesenquimais , Pessoa de Meia-Idade , Células PC-3 , Cultura Primária de Células , Ratos
4.
Adv Sci (Weinh) ; 9(1): e2103005, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34708571

RESUMO

The neuronal engagement of the peripheral nerve system plays a crucial role in regulating fracture healing, but how to modulate the neuronal activity to enhance fracture healing remains unexploited. Here it is shown that electrical stimulation (ES) directly promotes the biosynthesis and release of calcitonin gene-related peptide (CGRP) by activating Ca2+ /CaMKII/CREB signaling pathway and action potential, respectively. To accelerate rat femoral osteoporotic fracture healing which presents with decline of CGRP, soft electrodes are engineered and they are implanted at L3 and L4 dorsal root ganglions (DRGs). ES delivered at DRGs for the first two weeks after fracture increases CGRP expression in both DRGs and fracture callus. It is also identified that CGRP is indispensable for type-H vessel formation, a biological event coupling angiogenesis and osteogenesis, contributing to ES-enhanced osteoporotic fracture healing. This proof-of-concept study shows for the first time that ES at lumbar DRGs can effectively promote femoral fracture healing, offering an innovative strategy using bioelectronic device to enhance bone regeneration.


Assuntos
Regeneração Óssea/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Estimulação Elétrica/instrumentação , Estimulação Elétrica/métodos , Consolidação da Fratura/fisiologia , Gânglios Espinais/metabolismo , Fraturas por Osteoporose/terapia , Animais , Modelos Animais de Doenças , Fraturas por Osteoporose/metabolismo , Ratos
5.
Endocr Connect ; 11(1)2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-34825893

RESUMO

Objective: The beneficial effect of angiotensin(1-7) (Ang(1-7)), via the activation of its receptor, MAS-1, has been noted in diabetes treatment; however, how Ang(1-7) or MAS-1 affects insulin secretion remains elusive and whether the endogenous level of Ang(1-7) or MAS-1 is altered in diabetic individuals remains unexplored. We recently identified an important role of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, in the regulation of insulin secretion. Here, we tested the possible involvement of CFTR in mediating Ang(1-7)'s effect on insulin secretion and measured the level of Ang(1-7), MAS-1 as well as CFTR in the blood of individuals with or without type 2 diabetes. Methods: Ang(1-7)/MAS-1/CFTR pathway was determined by specific inhibitors, gene manipulation, Western blotting as well as insulin ELISA in a pancreatic ß-cell line, RINm5F. Human blood samples were collected from 333 individuals with (n = 197) and without (n = 136) type 2 diabetes. Ang(1-7), MAS-1 and CFTR levels in the human blood were determined by ELISA. Results: In RINm5F cells, Ang(1-7) induced intracellular cAMP increase, cAMP-response element binding protein (CREB) activation, enhanced CFTR expression and potentiated glucose-stimulated insulin secretion, which were abolished by a selective CFTR inhibitor, RNAi-knockdown of CFTR, or inhibition of MAS-1. In human subjects, the blood levels of MAS-1 and CFTR, but not Ang(1-7), were significantly higher in individuals with type 2 diabetes as compared to those in non-diabetic healthy subjects. In addition, blood levels of MAS-1 and CFTR were in significant positive correlation in type-2 diabetic but not non-diabetic subjects. Conclusion: These results suggested that MAS-1 and CFTR as key players in mediating Ang(1-7)-promoted insulin secretion in pancreatic ß-cells; MAS-1 and CFTR are positively correlated and both upregulated in type 2 diabetes.

6.
Bioact Mater ; 8: 95-108, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34541389

RESUMO

Magnesium metal and its alloys are being developed as effective orthopedic implants; however, the mechanisms underlying the actions of magnesium on bones remain unclear. Cystic fibrosis, the most common genetic disease in Caucasians caused by the mutation of CFTR, has shown bone disorder as a key clinical manifestation, which currently lacks effective therapeutic options. Here we report that implantation of magnesium-containing implant stimulates bone formation and improves bone fracture healing in CFTR-mutant mice. Wnt/ß-catenin signaling in the bone is enhanced by the magnesium implant, and inhibition of Wnt/ß-catenin by iCRT14 blocks the magnesium implant to improve fracture healing in CFTR-mutant mice. We further demonstrate that magnesium ion enters osteocytes, increases intracellular cAMP level and activates ATF4, a key transcription factor known to regulate Wnt/ß-catenin signaling. In vivo knockdown of ATF4 abolishes the magnesium implant-activated ß-catenin in bones and reverses the improved-fracture healing in CFTR-mutant mice. In addition, oral supplementation of magnesium activates ATF4 and ß-catenin as well as enhances bone volume and density in CFTR-mutant mice. Together, these results show that magnesium implantation or supplementation may serve as a potential anabolic therapy for cystic fibrosis-related bone disease. Activation of ATF4-dependent Wnt/ß-catenin signaling in osteocytes is identified as a previously undefined mechanism underlying the beneficial effect of magnesium on bone formation.

7.
Biochim Biophys Acta Mol Basis Dis ; 1867(12): 166242, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34389474

RESUMO

Endoplasmic reticulum (ER) stress, with adaptive unfolded protein response (UPR), is a key link between obesity, insulin resistance and type 2 diabetes, all of which are often present in the most common endocrine-metabolic disorder in women of reproductive age, polycystic ovary syndrome (PCOS), which is characterized with hyperandrogenism. However, the link between excess androgen and endoplasmic reticulum (ER) stress/insulin resistance in patients with polycystic ovary syndrome (PCOS) is unknown. An unexpected role of kisspeptin was reported in the regulation of UPR pathways and its involvement in the androgen-induced ER stress in hypothalamic neuronal cells. To evaluate the relationship of kisspeptin and ER stress, we detected kisspeptin and other factors in blood plasm of PCOS patients, rat models and hypothalamic neuronal cells. We detected higher testosterone and lower kisspeptin levels in the plasma of PCOS than that in non-PCOS women. We established a PCOS rat model by dihydrotestosterone (DHT) chronic exposure, and observed significantly downregulated kisspeptin expression and activated UPR pathways in PCOS rat hypothalamus compared to that in controls. Inhibition or knockdown of kisspeptin completely mimicked the enhancing effect of DHT on UPR pathways in a hypothalamic neuronal cell line, GT1-7. Kp10, the most potent peptide of kisspeptin, effectively reversed or suppressed the activated UPR pathways induced by DHT or thapsigargin, an ER stress activator, in GT1-7 cells, as well as in the hypothalamus in PCOS rats. Similarly, kisspeptin attenuated thapsigargin-induced Ca2+ response and the DHT- induced insulin resistance in GT1-7 cells. Collectively, the present study has revealed an unexpected protective role of kisspeptin against ER stress and insulin resistance in the hypothalamus and has provided a new treatment strategy targeting hypothalamic ER stress and insulin resistance with kisspeptin as a potential therapeutic agent.


Assuntos
Estresse do Retículo Endoplasmático/genética , Kisspeptinas/sangue , Neurônios/metabolismo , Síndrome do Ovário Policístico/genética , Androgênios/efeitos adversos , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Hipotálamo/metabolismo , Hipotálamo/patologia , Resistência à Insulina/genética , Kisspeptinas/genética , Neurônios/patologia , Obesidade/metabolismo , Obesidade/patologia , Síndrome do Ovário Policístico/sangue , Síndrome do Ovário Policístico/patologia , Ratos , Testosterona/sangue , Resposta a Proteínas não Dobradas/genética
8.
Mol Brain ; 14(1): 78, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33962650

RESUMO

Reconsolidation has been considered a process in which a consolidated memory is turned into a labile stage. Within the reconsolidation window, the labile memory can be either erased or strengthened. Manipulating acid-sensing ion channels (ASICs) in the amygdala via carbon dioxide (CO2) inhalation enhances memory retrieval and its lability within the reconsolidation window. Moreover, pairing CO2 inhalation with retrieval bears the reactivation of the memory trace and enhances the synaptic exchange of the calcium-impermeable AMPA receptors to calcium-permeable AMPA receptors. Our patch-clamp data suggest that the exchange of the AMPA receptors depends on the ubiquitin-proteasome system (UPS), via protein degradation. Ziram (50 µM), a ubiquitination inhibitor, reduces the turnover of the AMPA receptors. CO2 inhalation with retrieval boosts the ubiquitination without altering the proteasome activity. Several calcium-dependent kinases potentially involved in the CO2-inhalation regulated memory liability were identified using the Kinome assay. These results suggest that the UPS plays a key role in regulating the turnover of AMPA receptors during CO2 inhalation.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Tonsila do Cerebelo/metabolismo , Dióxido de Carbono/farmacologia , Ativação do Canal Iônico , Consolidação da Memória , Proteólise , Sinapses/metabolismo , Administração por Inalação , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Dióxido de Carbono/administração & dosagem , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Consolidação da Memória/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Receptores de AMPA/metabolismo , Sinapses/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos
9.
Biomaterials ; 238: 119828, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32045781

RESUMO

Magnesium (Mg)-based biometal attracts clinical applications due to its biodegradability and beneficial biological effects on tissue regeneration, especially in orthopaedics, yet the underlying anabolic mechanisms in relevant clinical disorders are lacking. The present study investigated the effect of magnesium (Mg) and vitamin C (VC) supplementation for preventing steroid-associated osteonecrosis (SAON) in a rat experimental model. In SAON rats, 50 mg/kg Mg, or 100 mg/kg VC, or combination, or water control was orally supplemented daily for 2 or 6 weeks respectively. Osteonecrosis was evaluated by histology. Serum Mg, VC, and bone turnover markers were measured. Microfil-perfused samples prepared for angiography and trabecular architecture were evaluated by micro-CT. Primary bone marrow cells were isolated from each group to evaluate their potentials in osteoblastogenesis and osteoclastogenesis. The mechanisms were tested in vitro. Histological evaluation showed SAON lesions in steroid treated groups. Mg and VC supplementation synergistically reduced the apoptosis of osteocytes and osteoclast number, and increased osteoblast surface. VC supplementation significantly increased the bone formation marker PINP, and the combination significantly decreased the bone resorption marker CTX. TNFα expression and oxidative injury were decreased in bone marrow in Mg/VC/combination group. Mg significantly increased the blood perfusion in proximal tibia and decreased the leakage particles in distal tibia 2 weeks after SAON induction. VC significantly elevated the osteoblast differentiation potential of marrow cells and improved the trabecular architecture. The combination supplementation significantly inhibited osteoclast differentiation potential of marrow cells. In vitro study showed promoting osteoblast differentiation effect of VC, and anti-inflammation and promoting angiogenesis effect of Mg with underlying mechanisms. Mg and VC supplementation could synergistically alleviate SAON in rats, indicating great translational potentials of metallic minerals for preventing SAON.


Assuntos
Magnésio , Osteonecrose , Animais , Ácido Ascórbico , Suplementos Nutricionais , Osteonecrose/induzido quimicamente , Osteonecrose/tratamento farmacológico , Ratos , Esteroides
10.
iScience ; 21: 375-390, 2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31704649

RESUMO

Epigenomic changes and stem cell deterioration are two hallmarks of aging. Accumulating evidence suggest that senescence of mesenchymal stromal cells (MSCs) perpetuates aging or age-related diseases. Here we report that two H3K9 demethylases, KDM3A and KDM4C, regulate heterochromatin reorganization via transcriptionally activating condensin components NCAPD2 and NCAPG2 during MSC senescence. Suppression of KDM3A or KDM4C by either genetic or biochemical approach leads to robust DNA damage response and aggravates cellular senescence, whereas overexpression of KDM3A/KDM4C or NCAPD2 promotes heterochromatin reorganization and blunts DNA damage response. Moreover, MSCs derived from Kdm3a-/- mice exhibit defective chromosome organization and exacerbated DNA damage response, which are associated with accelerated bone aging. Consistently, analysis of human bone marrow MSCs and transcriptome database reveals inverse correlation of KDM3A/KDM4C and/or NCAPD2/NCAPG2 with aging. Taken together, the present finding unveils that H3K9 demethylases function as a surveillance mechanism to restrain DNA damage accumulation in stem cells during aging.

11.
Theranostics ; 9(17): 5049-5064, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31410201

RESUMO

Rationale: Abnormal Wnt/ß-catenin signaling in the endometrium can lead to both embryo implantation failure and severe pathogenic changes of the endometrium such as endometrial cancer and endometriosis. However, how Wnt/ß-catenin signaling is regulated in the endometrium remains elusive. We explored possible regulation of Wnt/ß-catenin signaling by multi-drug resistance protein 4 (MRP4), a potential target in cancer chemotherapy, and investigated the mechanism. Methods: Knockdown of MRP4 was performed in human endometrial cells in vitro or in a mouse embryo-implantation model in vivo. Immunoprecipitation, immunoblotting and immunofluorescence were used to assess protein interaction and stability. Wnt/ß-catenin signaling was assessed by TOPflash reporter assay and quantitative PCR array. Normal and endometriotic human endometrial tissues were examined. Data from human microarray or RNAseq databases of more than 100 participants with endometriosis, endometrial cancer or IVF were analyzed. In vitro and in vivo tumorigenesis was performed. Results: MRP4-knockdown, but not its transporter-function-inhibition, accelerates ß-catenin degradation in human endometrial cells. MRP4 and ß-catenin are co-localized and co-immunoprecipitated in mouse and human endometrium. MRP4-knockdown in mouse uterus reduces ß-catenin levels, downregulates a series of Wnt/ß-catenin target genes and impairs embryo implantation, which are all reversed by blocking ß-catenin degradation. Analysis of human endometrial biopsy samples and available databases reveals significant and positive correlations of MRP4 with ß-catenin and Wnt/ß-catenin target genes in the receptive endometrium in IVF, ectopic endometriotic lesions and endometrial cancers. Knockdown of MRP4 also inhibits in vitro and in vivo endometrial tumorigenesis. Conclusion: A previously undefined role of MRP4 in stabilizing ß-catenin to sustain Wnt/ß-catenin signaling in endometrial cells is revealed for both embryo implantation and endometrial disorders, suggesting MRP4 as a theranostic target for endometrial diseases associated with Wnt/ß-catenin signaling abnormality.


Assuntos
Neoplasias do Endométrio/metabolismo , Endometriose/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Gravidez/metabolismo , Via de Sinalização Wnt , Adulto , Animais , Linhagem Celular Tumoral , Endométrio/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos ICR , Camundongos Nus , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , beta Catenina/metabolismo
12.
J Clin Med ; 8(7)2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31336927

RESUMO

Human cardiac progenitor cells isolated from the same host may have advantages over other sources of stem cells. The aim of this study is to establish a new source of human progenitor cells collected from a waste product, pericardiac effusion fluid, after open-heart surgery in children with congenital heart diseases. The fluid was collected every 24 h for 2 days after surgery in 37 children. Mononuclear cells were isolated and expanded in vitro. These pericardial effusion-derived progenitor cells (PEPCs) exhibiting cardiogenic lineage markers, were highly proliferative and enhanced angiogenesis in vitro. Three weeks after stem cell transplantation into the ischemic heart in mice, cardiac ejection fraction was improved significantly without detectable progenitor cells. Gene expression profiles of the repaired hearts revealed activation of several known repair mechanisms including paracrine effects, cell migration, and angiogenesis. These progenitor cells may have the potential for heart regeneration.

13.
Fertil Steril ; 111(1): 157-167, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30477916

RESUMO

OBJECTIVE: To measure blood and follicular antimüllerian hormone (AMH) levels in women with polycystic ovary syndrome (PCOS) undergoing assisted reproductive technologies (ART) and to examine the direct action of insulin on AMH expression in human granulosa cells. DESIGN: Prospective clinical and experimental study. SETTING: University Hospital-based laboratory. PATIENT(S): Women with (n = 86) and without (n = 172) PCOS in ART. INTERVENTION(S): Blood, follicular fluid, and luteinized granulosa cells were collected from PCOS and non-PCOS women in ART. MAIN OUTCOME MEASURE(S): Hormone levels in blood and fluid, and gene expression in granulosa cells. RESULT(S): Serum levels of AMH were elevated and inversely correlated with embryo cleavage rate in PCOS women in ART. Significant higher levels of AMH were also found in small and large follicles collected from PCOS women compared with non-PCOS women. Luteinized granulosa cells from PCOS women showed higher expression of AMH and its receptor AMHR2. Direct effect of insulin in increasing the expression of AMH in the isolated luteinized granulosa cells was observed, with the PCOS granulosa cells responding to a high dose of insulin. Cotreatment with AMH attenuated insulin-induced aromatase expression in the luteinized granulosa cells. CONCLUSION(S): These results suggest that insulin may contribute to AMH elevation in PCOS and that AMH counteracts insulin-promoted aromatase expression in granulosa cells.


Assuntos
Hormônio Antimülleriano/metabolismo , Células da Granulosa/metabolismo , Insulina/administração & dosagem , Síndrome do Ovário Policístico/metabolismo , Técnicas de Reprodução Assistida , Adulto , Hormônio Antimülleriano/sangue , Biomarcadores/sangue , Biomarcadores/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Células da Granulosa/efeitos dos fármacos , Humanos , Indução da Ovulação/métodos , Indução da Ovulação/tendências , Síndrome do Ovário Policístico/diagnóstico , Estudos Prospectivos , Técnicas de Reprodução Assistida/tendências , Adulto Jovem
14.
EMBO Mol Med ; 10(10)2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30154237

RESUMO

The shift of cytokine profile from anti- to pro-inflammatory is the most recognizable sign of labor, although the underlying mechanism remains elusive. Here, we report that the epithelial sodium channel (ENaC) is upregulated and activated in the uterus at labor in mice. Mechanical activation of ENaC results in phosphorylation of CREB and upregulation of pro-inflammatory cytokines as well as COX-2/PGE2 in uterine epithelial cells. ENaC expression is also upregulated in mice with RU486-induced preterm labor as well as in women with preterm labor. Interference with ENaC attenuates mechanically stimulated uterine contractions and significantly delays the RU486-induced preterm labor in mice. Analysis of a human transcriptome database for maternal-fetus tissue/blood collected at onset of human term and preterm births reveals significant and positive correlation of ENaC with labor-associated pro-inflammatory factors in labored birth groups (both term and preterm), but not in non-labored birth groups. Taken together, the present finding reveals a pro-inflammatory role of ENaC in labor at term and preterm, suggesting it as a potential target for the prevention and treatment of preterm labor.


Assuntos
Citocinas/metabolismo , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Trabalho de Parto , Animais , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Modelos Animais , Gravidez , Útero/fisiologia
15.
Biochim Biophys Acta Mol Cell Res ; 1865(4): 605-615, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29326073

RESUMO

Retinoic acid (RA) plays a pivotal role in many cellular processes; however, the signaling mechanisms mediating the effect of RA are not fully understood. Here, we show that RA transcriptionally upregulates cystic fibrosis transmembrane conductance regulator (Cftr) by promoting the direct binding of its receptor RARα to Cftr promoter in mouse spermatogonia and embryonic stem (ES) cells. The RA/CFTR pathway is involved in the differentiation of spermatogonia and organogenesis during the embryo development of Xenopus laevis. Loss of CFTR by siRNA-mediated knockdown blunts the RA-induced spermatogonial differentiation. Overexpression of CFTR mimics the effect of RA on the induction of spermatogonial differentiation or restores the developmental defects induced by the knockdown of RARα in spermatogonial cells and Xenopus laevis. Analysis of the human database shows that the expression of CFTR positively correlates with RARα in brain tissues, stem cells as well as cancers, supporting the role of RA/CFTR pathway in various developmental processes in humans. Together, our study discovers an essential role of CFTR in mediating the RA-dependent signaling for stem cell differentiation and embryonic development.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Desenvolvimento Embrionário/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/metabolismo , Transcrição Genética/efeitos dos fármacos , Tretinoína/farmacologia , Xenopus laevis/embriologia , Animais , Sequência de Bases , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Receptor alfa de Ácido Retinoico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espermatogônias/citologia , Células-Tronco/efeitos dos fármacos , Xenopus laevis/metabolismo , beta Catenina/metabolismo
16.
Front Physiol ; 8: 835, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29204121

RESUMO

Glucagon, produced by islet α cells, functions to increase blood glucose. Abnormal glucose levels are often seen in cystic fibrosis (CF), a systematic disease caused by mutations of the CF transmembrane conductance regulator (CFTR), and in polycystic ovarian syndrome (PCOS), an endocrine disorder featured with hyperandrogenism affecting 5-10% women of reproductive age. Here, we explored the role of CFTR in glucagon production in α cells and its possible contribution to glucagon disturbance in CF and PCOS. We found elevated fasting glucagon levels in CFTR mutant (DF508) mice compared to the wildtypes. Glucagon and prohormone convertase 2 (PC2) were also upregulated in CFTR inhibitor-treated or DF508 islets, as compared to the controls or wildtypes, respectively. Dihydrotestosterone (DHT)-induced PCOS rats exhibited significantly lower fasting glucagon levels with higher CFTR expression in α cells compared to that of controls. Treatment of mouse islets or αTC1-9 cells with DHT enhanced CFTR expression and reduced the levels of glucagon and PC2. The inhibitory effect of DHT on glucagon production was blocked by CFTR inhibitors in mouse islets, and mimicked by overexpressing CFTR in αTC1-9 cells with reduced phosphorylation of the cAMP/Ca2+ response element binding protein (p-CREB), a key transcription factor for glucagon and PC2. These results revealed a previously undefined role of CFTR in suppressing glucagon production in α-cells, defects in which may contribute to glucose metabolic disorder seen in CF and PCOS.

17.
Oncotarget ; 8(45): 78520-78529, 2017 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-29108246

RESUMO

Multi-drug resistance protein 4 (MRP4), a potential chemotherapeutic target as well as a transporter for endogenous signaling molecules (e.g. prostaglandins), is known to be expressed in the endometrium, although its possible role(s) in the physiology of the endometrium remains unknown. Here, we show that MRP4 is upregulated at implantation window and localized to the basolateral membrane of the endometrial epithelium, the interface between the epithelium and stroma in mice. In human endometrial epithelial cells, MRP4 expression is upregulated by ENaC activation and the inhibition of MRP4 blocks ENaC-dependent PGE2 release as well as phosphorylation of CREB. Intrauterine injection of MRP4 inhibitor in mice prior to implantation significantly downregulated implantation markers COX-2, Claudin4 and Lif, and reduced implantation rate. These results in together have revealed a previously undefined role of MRP4 in mediating ENaC-dependent CREB/COX-2/PGE2 signaling essential to embryo implantation with implication in cancer progression as well.

18.
Endocrinology ; 158(10): 3188-3199, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28977595

RESUMO

The secretion of glucagon by islet α cells is normally suppressed by high blood glucose, but this suppressibility is impaired in patients with diabetes or cystic fibrosis (CF), a disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a cyclic adenosine monophosphate-activated Cl- channel. However, precisely how glucose regulates glucagon release remains controversial. Here we report that elevated glucagon secretion, together with increased glucose-induced membrane depolarization and Ca2+ response, is found in CFTR mutant (DF508) mice/islets compared with the wild-type. Overexpression of CFTR in AlphaTC1-9 cells results in membrane hyperpolarization and reduced glucagon release, which can be reversed by CFTR inhibition. CFTR is found to potentiate the adenosine triphosphate-sensitive K+ (KATP) channel because membrane depolarization and whole-cell currents sensitive to KATP blockers are significantly greater in wild-type/CFTR-overexpressed α cells compared with that in DF508/non-overexpressed cells. KATP knockdown also reverses the suppressive effect of CFTR overexpression on glucagon secretion. The results reveal that by potentiating KATP channels, CFTR acts as a glucose-sensing negative regulator of glucagon secretion in α cells, a defect of which may contribute to glucose intolerance in CF and other types of diabetes.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Glucose/farmacologia , Canais KATP/fisiologia , Animais , Cálcio/análise , Linhagem Celular , Cloretos/metabolismo , Fibrose Cística/complicações , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Glucagon/antagonistas & inibidores , Glucagon/sangue , Células Secretoras de Glucagon/fisiologia , Intolerância à Glucose/complicações , Camundongos , Camundongos Mutantes , Mutação
19.
Endocrinology ; 158(11): 4000-4016, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28938460

RESUMO

Spermatogenesis is sustained by a heterogeneous population of spermatogonia that includes the spermatogonial stem cells. However, the mechanisms underlying their establishment from gonocyte embryonic precursors and their maintenance thereafter remain largely unknown. In this study, we report that inactivation of the ubiquitin ligase Huwe1 in male germ cells in mice led to the degeneration of spermatogonia in neonates and resulted in a Sertoli cell-only phenotype in the adult. Huwe1 knockout gonocytes showed a decrease in mitotic re-entry, which inhibited their transition to spermatogonia. Inactivation of Huwe1 in primary spermatogonial culture or the C18-4 cell line resulted in cell degeneration. Degeneration of Huwe1 knockout spermatogonia was associated with an increased level of histone H2AX and an elevated DNA damage response that led to apparent mitotic catastrophe but not apoptosis or senescence. Blocking this increase in H2AX prevented the degeneration of Huwe1-depleted cells. Taken together, these results reveal a previously undefined role of Huwe1 in orchestrating the physiological DNA damage response in the male germline that contributes to the establishment and maintenance of spermatogonia.


Assuntos
Diferenciação Celular/genética , Dano ao DNA/genética , Espermatogênese/genética , Espermatogônias/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Células Cultivadas , Regulação para Baixo/genética , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Knockout , Proteínas Supressoras de Tumor
20.
Hum Genet ; 136(2): 227-239, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27896428

RESUMO

Mechanisms underlying female gonadal dysgenesis remain unclarified and relatively unstudied. Whether X-chromosome inactivation (XCI)-escaping genes and microRNAs (miRNAs) contribute to this condition is currently unknown. We compared 45,X Turner Syndrome women with 46,XX normal women, and investigated differentially expressed miRNAs in Turner Syndrome through plasma miRNA sequencing. We found that miR-320a was consistently upregulated not only in 45,X plasma and peripheral blood mononuclear cells (PBMCs), but also in 45,X fetal gonadal tissues. The levels of miR-320a in PBMCs from 45,X, 46,XX, 46,XY, and 47,XXY human subjects were inversely related to the expression levels of XCI-escaping gene KDM5C in PBMCs. In vitro models indicated that KDM5C suppressed miR-320a transcription by directly binding to the promoter of miR-320a to prevent histone methylation. In addition, we demonstrated that KITLG, an essential gene for ovarian development and primordial germ cell survival, was a direct target of miR-320a and that it was downregulated in 45,X fetal gonadal tissues. In conclusion, we demonstrated that downregulation of miR-320a by the XCI-escaping gene KDM5C contributed to ovarian development by targeting KITLG.


Assuntos
Histona Desmetilases/genética , MicroRNAs/genética , Ovário/crescimento & desenvolvimento , Síndrome de Turner/genética , Inativação do Cromossomo X/genética , Adolescente , Adulto , Sequência de Aminoácidos , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Regulação para Baixo , Feminino , Regulação da Expressão Gênica , Ontologia Genética , Células HEK293 , Humanos , Leucócitos Mononucleares/metabolismo , MicroRNAs/sangue , Regiões Promotoras Genéticas , Análise de Sequência de RNA , Regulação para Cima , Adulto Jovem
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