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1.
Gene ; 711: 143953, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31269463

RESUMO

Both SMAD4 and miR-126* have been proven to be involved in granulosa cell (GC) apoptosis and even follicular atresia, through commonly regulating follicle-stimulating hormone receptor (FSHR), the FSH-specific transmembrane receptor of GCs. However, the regulatory relationship between them in GCs is still unknown. In this study, we report that SMAD4 suppresses the expression of miR-126* and impairs its function in GCs of the porcine ovary by acting as a transcription factor. A classic SMAD4-binding element (SBE) site was found in the promoter of miR-126* by using in silico methods. Luciferase assay, qRT-PCR, and ChIP assay proved that SMAD4 serves as a transcriptional repressor and directly binds to SBE site within miR-126* gene promoter, which further reduces miR-126* gene expression and inhibits its transcriptional activity in GCs. Furthermore, SMAD4 also controls miR-126*-mediated expression of FSHR (a direct target of miR-126* in GCs). In addition, we prove that SMAD4 induces CYP19A1 expression (encodes aromatase, the key enzyme for oestrogen biosynthesis) and inhibits GC apoptosis through the miR-126*/FSHR axis. Taken together, our findings not only established a direct link between SMAD4 and miRNA-126*, two key factors of GC apoptosis, but also revealed an important way in which the SMAD4 regulates GC function, the miRNA-126*/FSHR axis.


Assuntos
Células da Granulosa/citologia , MicroRNAs/química , MicroRNAs/genética , Proteína Smad4/metabolismo , Animais , Apoptose , Aromatase/metabolismo , Sítios de Ligação , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Células da Granulosa/metabolismo , Regiões Promotoras Genéticas , Receptores do FSH/genética , Suínos
2.
DNA Cell Biol ; 38(6): 549-560, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31120353

RESUMO

Proper course of folliculogenesis and oogenesis have an enormous impact on female fertility. Both processes take place in the ovary and involve not only the maturing germ cell, but also few types of somatic cells that assist the ovarian processes and mediate the dialog with the oocyte. These cells, granulosa and theca, are heavily involved in essential reproductive processes, such as ovulation, fertilization, and embryo implantation. In this study, we have used the expressive microarray approach to analyze the transcriptome of porcine granulosa cells, during short-term in vitro culture. We have further selected differentially expressed gene ontologies, involved in cell proliferation, migration, adhesion, and tissue development, namely, "cell-cell adhesion," "cell motility," "cell proliferation," "tissue development," and "tissue migration" to screen them for the possibility of discovery of new markers of those processes. A total of 303 genes, expression of which varied significantly in different culture periods and belonged to the analyzed ontology groups, were detected, of which 15 that varied the most (between 0 and 48 h of culture) were selected for validation. As the validation confirmed the transcriptomic patterns, 10 genes of biggest changes in expression (CAV1, IGFBP5, ITGB3, FN1, ITGA2, LAMB1, POSTN, FAM83D, KIF14, and CDK1) were analyzed, described, and referred to the context of the study, with the most promising new markers and further proof for the viability of the currently recognized ones detailed. Overall, the study provided valuable insight into the molecular functioning of in vitro granulosa cell cultures.


Assuntos
Adesão Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , Células da Granulosa/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Células da Granulosa/citologia , Células da Granulosa/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Suínos
3.
Chemosphere ; 229: 60-67, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31075703

RESUMO

Bisphenol A (BPA) negatively affects steroid production in human luteinized granulosa cells (GC). This study was designed to address two important questions: (1) whether BPA exerts the same disruptive effect in human cumulus granulosa cells (hCGC) and (2) to reveal the molecular mechanism underlying the BPA's action on steroidogenesis. We used cultured hCGC since these cells exert the properties of GC from early antral follicles. Results showed that BPA at 100 µM decreased estradiol level and CYP19A1 mRNA, but increased progesterone production, steroidogenic acute regulatory protein (STAR) and peroxisome proliferator-activated receptor gamma (PPARγ) mRNA expression after 48 h. Shorter (6 h) exposure to BPA elevated PPARγ mRNA level in hCGC. Addition of ERK1/2 (U0126), EGFR (AG1478) and PPARγ (GW9662) inhibitors prevented the BPA-induced STAR and PPARγ mRNA expression. Western blot analysis showed that BPA induced a rapid EGFR and ERK1/2 activation. The BPA-induced EGFR phosphorylation was prevented by addition of the PPARγ inhibitor, whereas the BPA-induced ERK1/2 activation was prevented by addition of the EGFR or PPARγ inhibitor. These data show that BPA increases the progesterone and decreases the estradiol biosynthetic pathway in hCGC. Augmentation of the progesterone biosynthetic pathway is mediated through the PPARγ-dependent activation of EGFR and ERK1/2, leading to increased expression of STAR mRNA.


Assuntos
Compostos Benzidrílicos/uso terapêutico , Células do Cúmulo/metabolismo , Células da Granulosa/metabolismo , PPAR gama/metabolismo , Fenóis/uso terapêutico , Fosfoproteínas/metabolismo , Compostos Benzidrílicos/farmacologia , Células do Cúmulo/citologia , Receptores ErbB/metabolismo , Feminino , Células da Granulosa/citologia , Humanos , Fenóis/farmacologia
4.
Cell Mol Life Sci ; 76(17): 3311-3322, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31062072

RESUMO

Oxygen deprivation affects human health by modulating system as well as cellular physiology. Hypoxia generates reactive oxygen species (ROS), causes oxidative stress and affects female reproductive health by altering ovarian as well as oocyte physiology in mammals. Hypoxic conditions lead to several degenerative changes by inducing various cell death pathways like autophagy, apoptosis and necrosis in the follicle of mammalian ovary. The encircling somatic cell death interrupts supply of nutrients to the oocyte and nutrient deprivation may result in the generation of ROS. Increased level of ROS could induce granulosa cells as well as oocyte autophagy. Although autophagy removes damaged proteins and subcellular organelles to maintain the cell survival, irreparable damages could induce cell death within intra-follicular microenvironment. Hypoxia-induced autophagy is operated through 5' AMP activated protein kinase-mammalian target of rapamycin, endoplasmic reticulum stress/unfolded protein response and protein kinase C delta-c-junN terminal kinase 1 pathways in a wide variety of somatic cell types. Similar to somatic cells, we propose that hypoxia may induce granulosa cell as well as oocyte autophagy and it could be responsible at least in part for germ cell elimination from mammalian ovary. Hypoxia-mediated germ cell depletion may cause several reproductive impairments including early menopause in mammals.


Assuntos
Autofagia , Células da Granulosa/citologia , Animais , Proteína Beclina-1/metabolismo , Hipóxia Celular , Feminino , Células da Granulosa/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
5.
Reprod Biol Endocrinol ; 17(1): 37, 2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979376

RESUMO

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a common and severe complication for patients undergoing IVF/ICSI-ET. Melatonin widely participates in the regulation of female reproductive endocrine activity. However, whether melatonin participates in the progression of OHSS is largely unknown. This study aims to identify the predictive value of follicular fluid (FF) melatonin for OHSS establishment and the underlying mechanism. METHODS: All participants of this case-control study were enrolled at the Reproductive Medicine Center located in the First Affiliated Hospital of Zhengzhou University in China from January to October in 2017. Quantitative real-time PCR and western blot were used to examine the mRNA and protein levels. Primary granulosa cells were extracted and cultured for in vitro studies. Melatonin concentration was measured by ELISA. Logistic analysis and receiver-operating characteristic (ROC) curves were used to evaluate the predicting value of melatonin on OHSS occurrence. MAIN OUTCOME MEASURES: The expression level of melatonin receptor 2 (MT2), P450 aromatase cytochrome (aromatase), vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS) mRNA in human primary granulosa cells. The concentration of melatonin in FF. The predicting value of melatonin on OHSS and the cut-off value of the prediction. RESULTS: FF melatonin concentrations were significantly higher in patients with OHSS compared to non-OHSS group (35.94 ± 10.18 ng/mL vs 23.93 ± 10.94 ng/mL, p<0.001). The expression of MT2 mRNA (p = 0.0459) and protein in granulosa cells was also significantly higher in the OHSS group. When using a cut-off level of 27.52 ng/ml, the sensitivity and specificity of FF melatonin to predict OHSS was 84.6 and 74.0%, respectively (p < 0.0001). We also found that melatonin could up-regulates aromatase mRNA, VEGF mRNA expression and down-regulates iNOS mRNA expression in the granulosa cells. CONCLUSION: OHSS patients have higher melatonin in the FF as well as higher MT2 expression in the granulosa cells. The melatonin in FF might be used as an effective predictor for the occurrence of OHSS.


Assuntos
Líquido Folicular/metabolismo , Células da Granulosa/metabolismo , Melatonina/metabolismo , Síndrome de Hiperestimulação Ovariana/metabolismo , Receptor MT2 de Melatonina/metabolismo , Adulto , Células Cultivadas , Feminino , Humanos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Receptor MT2 de Melatonina/genética
6.
Toxicol Lett ; 310: 14-22, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30980910

RESUMO

Mechanism of PAH mixtures, using granulosa tumour cells, was investigated. Cells were exposed to a mixture of all 16 priority PAHs (M1) or a mixture of five PAHs not classified as human carcinogens (M2). The effect of siAHR, siAHRR and siNFKB2 on the expression of CYP1A1, CYP1B1, GSTM1, ERα, AR and cell proliferation was described. M1 decreased AhR and CYP1A1, while increased AhRR and ARNT expression. M2 also decreased AhR and CYP1A1 but had no effect on AhRR expression. siAHRR reversed the inhibitory effect of M1 on AhR and CYP1A1,while inhibitory effect of M2 was still observed. siNFKB2 reversed inhibitory effect of both mixtures on AhR and CYP1A1 expression and stimulatory effect of M1 on AhRR expression. siAHR reversed stimulatory effect of both mixtures on ERα expression. Stimulatory effect of M1 on cell proliferation was not observed in siAHR, was still observed in siESR1 cells. M2 had no effect on cell proliferation, however stimulatory effect was appeared in siAHR and siESR1cells. In conclusion: M1 by activation of AhRR and NFkB p52, but M2 only by activation of NFκB attenuated AhR signalling and ligand-induced CYP1A1 expression. Interaction between AhR and ER following M1 and M2 exposure is primarily initiated through AhR.


Assuntos
Misturas Complexas/toxicidade , Tumor de Células da Granulosa , Células da Granulosa/efeitos dos fármacos , Neoplasias Ovarianas , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Metabolismo Energético/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Tumor de Células da Granulosa/genética , Tumor de Células da Granulosa/metabolismo , Tumor de Células da Granulosa/patologia , Células da Granulosa/metabolismo , Células da Granulosa/patologia , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
7.
Pol J Vet Sci ; 22(1): 83-90, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30997768

RESUMO

The purpose of the study was to study the activity of the phytoestrogen genistein (GEN) act- ing on FSHR and LHR in rat ovaries with polycystic ovary syndrome (PCOS). Sixty rats were di- vided into six groups. Rats in the dose group received genistein at a concentration of either 5 (low genistein dose group, L-gen), 10 (middle genistein dose group, M-Gen) or 20 (high genistein dose group, H-Gen) mg per kg of body weight per day. Estrogen group (EG, received 0.5 mg/kg Dieth- ylstilbestrol). Concentration of sex hormones in serum was quantified by enzyme-linked immuno- sorbent assay (ELISA). Expressions of follicle-stimulating hormone receptor (FSHR) and lutein- izing hormone receptor (LHR) protein were determined by immunohistochemistry. Treatment with genistein resulted in a strong stimulation of the concentration of sex hormone in serum. The concentration of progesterone and FSH was significantly higher in H-Gen when compared to the PCOS model control group (MG) (P ⟨ 0.01). In contrast, the concentration of testosterone, LH and the ratio of LH/FSH decreased in GEN treatment groups compared to MG, the effect was statistically significant, tested by the ANOVA test (p⟨0.01). For hormone receptor activity, treat- ment with genistein resulted in an improvement of ovarian function with LHR protein expression being enhanced and FSHR protein expression being suppressed. Our results demonstrate that Genistein played a significant role in regulating FSH and LH receptor expression to improve perimenopausal ovarian and uterine function.


Assuntos
Genisteína/farmacologia , Síndrome do Ovário Policístico/metabolismo , Receptores do FSH/metabolismo , Receptores do LH/metabolismo , Animais , Feminino , Hormônio Foliculoestimulante/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Hormônio Luteinizante , Fitoestrógenos , Síndrome do Ovário Policístico/patologia , Ratos , Ratos Wistar
8.
Food Chem Toxicol ; 128: 256-266, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30959089

RESUMO

Prenatal nicotine exposure (PNE) could induce ovarian dysplasia in offspring. This study aimed to confirm its intrauterine origin and explore a programming mechanism of ovarian dysplasia caused by PNE. Pregnant Wistar rats were injected subcutaneously with nicotine (2 mg/kg.d) from gestation day (GD) 9 to GD20. Serum of female offspring was obtained for hormone assays and ovarian tissues were collected. The results showed that PNE impaired ovarian development, and inhibited estradiol production and cytochrome P450 aromatase (P450arom) expression before and after birth. Moreover, the nicotinic acetylcholine receptors (nAChRs) expression was increased in utero, while histone 3 lysine 9 acetylation (H3K9ac) and H3K27ac levels in the P450arom promoter region were decreased persistently in PNE group before and after birth. In vitro, nicotine decreased P450arom expression and estradiol production in human granulosa cell line KGN. Furthermore, nicotine treatment up-regulated nAChRα6 and α9 expression and down-regulated the H3K9ac and H3K27ac levels of the P450arom promoter region. Non-specific nAChRs inhibitor vecuronium bromide reversed these effects. These results suggest that PNE could induce ovarian dysplasia and inhibit estradiol synthesis in the female offspring rats, which was related to the decreased H3K9ac and H3K27ac levels in the promotor region of the P450arom via the nAChRs.


Assuntos
Aromatase/genética , Estradiol/biossíntese , Células da Granulosa/efeitos dos fármacos , Histonas/metabolismo , Exposição Materna , Nicotina/toxicidade , Ovário/enzimologia , Regiões Promotoras Genéticas , Acetilação , Animais , Feminino , Células da Granulosa/metabolismo , Humanos , Ovário/metabolismo , Gravidez , Ratos , Ratos Wistar , Receptores Nicotínicos/metabolismo
9.
J Biol Regul Homeost Agents ; 33(2): 461-468, 2019 Mar-Apr,.
Artigo em Inglês | MEDLINE | ID: mdl-30968676

RESUMO

Endocrinal interactions are one of the most crucial regulatory mechanisms that maintain the state of homeostasis in humans. Processes such as oogenesis, folliculogenesis, menstruation and pregnancy remain under hormonal control. A key role in folliculogenesis is played by granulosa cells. Moreover, granulosa cells take part in corpus luteum formation after ovulation. Because of that, it is important to understand the ways in which the granulosa cells, associated with those processes, respond to hormonal stimulus. In the present study, a transcriptomic analysis of human granulosa cells (GCs) was carried out with the use of expression microarrays. The results were validated by RT-qPCR. The total RNA was isolated after 1st, 7th, 15th and 30th days of long-term primary cultures. The main focus of this work was placed on the genes belonging to "Response to estradiol", "Response to follicle-stimulating-hormone", "Cellular response to hormone stimulus", "Cellular hormone metabolic process" and "Hormone biosynthetic process" gene ontology groups. These groups of genes have been associated with GC hormone metabolism and cellular response to hormones. Eighty genes belonging to these groups were identified. Those that were members of more than one of the analyzed gene ontology groups, or exhibited unique expression patterns, were selected for further analysis. All of the selected genes were described, with their expression patterns detailed. In this manuscript, two gene expression patterns have been described. The first one showed large downregulation of genes in the later stages of culture, with the second one presenting upregulation of expression after day 1 of IVC. The present research was focused on six genes found to be the most important for steroidogenesis: STAR, POR, CYP11A1, ADM, GCLC, IL1B, as well as three genes of higher expression at the later stages of long-term in vitro culture: NR2F2, BMP4, COL1A1. The main goal of the presented study was to select genes involved in response to hormonal stimulus and hormone metabolism in GC long-term in vitro culture.


Assuntos
Estradiol/genética , Hormônio Foliculoestimulante/genética , Células da Granulosa/metabolismo , Folículo Ovariano/crescimento & desenvolvimento , Células Cultivadas , Feminino , Humanos , Oogênese , Ovulação , Gravidez
10.
Anim Reprod Sci ; 204: 140-151, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30948244

RESUMO

This study was conducted with the aim to understand the roles of apoptosis signal-regulating kinase (ASK1) and transcription factor tumor suppressor protein TP53, as well as the possible interrelationships, in the control of healthy ovarian cell functions. Rabbit ovarian granulosa cells were transfected with constructs encoding ASK1, TP53, or TP53 + ASK1 and cultured with or without insulin-like growth factor 1 (IGF1). The accumulation of ASK1, the cytoplasmic apoptosis regulators BAX and BCL2, and proliferating cell nuclear antigen (PCNA, a cell proliferation marker), as well as progesterone release, were evaluated by quantitative immunocytochemistry and radioimmunoassay. Results indicate both ASK1 and TP53 promoted the accumulation of BAX, but suppressed that of BCL2 and PCNA. Progesterone release was inhibited by ASK1 and promoted by TP53, while TP53 also stimulated ASK1 accumulation. Additionally, IGF1 stimulated PCNA and reduced progesterone release, but did not affect ASK1. Transfection with ASK1, TP53, or TP53 + ASK1 could modify IGF1 activity, however, there was no cumulative effect with co-transfection of TP53 and ASK1. This is the first results that indicate there is ASK1 suppression of healthy ovarian granulosa cell functions, including promoting apoptosis, inhibiting proliferation, and alter progesterone release. There was also TP53 actions in rabbit ovarian granulosa cells, where it stimulated ASK1, apoptosis, and progesterone release, thus suppressing proliferation and responses to IGF1. The similarity of ASK1 and TP53 effects on apoptosis and proliferation, lack of cumulative action of these molecules, and capacity of TP53 to promote ASK1 accumulation suggest that TP53 can suppress some ovarian granulosa cell functions through ASK1 stimulation.


Assuntos
Células da Granulosa/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Coelhos , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/fisiologia , Biomarcadores , Sobrevivência Celular , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde , Transtornos do Crescimento , Perda Auditiva Neurossensorial , Fator de Crescimento Insulin-Like I/deficiência , Fator de Crescimento Insulin-Like I/farmacologia , MAP Quinase Quinase Quinase 5/genética , Progesterona , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
11.
Int J Mol Sci ; 20(7)2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30986945

RESUMO

Nrf2 is a redox sensitive transcription factor regulating the expression of antioxidant genes as defense mechanism against various stressors. The aim of this study is to investigate the potential role of noncoding miRNAs as endogenous and quercetin as exogenous regulators of Nrf2 pathway in bovine granulosa cells. For this cultured granulosa cells were used for modulation of miRNAs (miR-28, 153 and miR-708) targeting the bovine Nrf2 and supplementation of quercentin to investigate the regulatory mechanisms of the Nrf2 antioxidant system. Moreover, cultured cells were treated with hydrogen peroxide to induce oxidative stress in those cells. Our results showed that, oxidative stress activated the expression of Nrf2 as a defense mechanism, while suppressing the expression of those miRNAs. Overexpression of those miRNAs resulted in downregulation of Nrf2 expression resulted in higher ROS accumulation, reduced mitochondrial activity and cellular proliferation. Quercetin supplementation showed its protective role against oxidative stress induced by H2O2 by inducing the expression of antioxidant enzymes. In conclusion, this study highlighted the involvement of miR-153, miR-28 and miR-708 in regulatory network of Nrf2 mediated antioxidant system in bovine granulosa cells function. Furthermore, quercetin at a low dose played a protective role in bovine granulosa cells against oxidative stress damage.


Assuntos
Células da Granulosa/metabolismo , Células da Granulosa/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Ovário/patologia , Ovário/fisiopatologia , Estresse Oxidativo , Animais , Antioxidantes/metabolismo , Sequência de Bases , Bovinos , Proliferação de Células , Feminino , Técnicas de Silenciamento de Genes , Peróxido de Hidrogênio/toxicidade , MicroRNAs/genética , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Quercetina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
Environ Toxicol ; 34(7): 844-852, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30951242

RESUMO

Our goals were to investigate whether environmentally relevant doses of T-2 toxin can affect human ovarian granulosa cells' function and to reveal the potential mechanism of T-2 toxin's action. Results showed that T-2 toxin strongly attenuated luteinizing hormone/choriogonadotropin receptor (LHCGR) mRNA expression in follicle-stimulating hormone (FSH)-stimulated human cumulus granulosa cells. Addition of human chorionic gonadotropin was not able to elicit maximal response of ovulatory genes amphiregulin, epiregulin, and progesterone receptor. T-2 toxin reduced mRNA levels of CYP19A1 and steroidogenic acute regulatory protein (STAR) and lowered FSH-stimulated estradiol and progesterone production. Mechanistic experiments demonstrated that T-2 toxin decreased FSH-stimulated cyclic adenosine monophosphate (cAMP) production. Addition of total PDE inhibitor 3-isobutyl-1-methylxanthine prevented T-2 toxin's action on LHCGR, STAR, and CYP19A1 mRNA expression in FSH-stimulated human cumulus granulosa cells. Furthermore, T-2 toxin partially decreased 8-bromoadenosine 3'5'-cyclic monophosphate (8-Br-cAMP)-stimulated LHCGR and STAR, but did not affect 8-Br-cAMP-stimulated CYP19A1 mRNA expression in human cumulus granulosa cells. Overall, our data indicate that environmentally relevant dose of T-2 toxin decreases steroidogenesis and ovulatory potency in human cumulus granulosa cells probably through activation of PDE, thus posing a significant risk for female fertility.


Assuntos
Aromatase/genética , Células do Cúmulo/efeitos dos fármacos , AMP Cíclico/metabolismo , Hormônios Esteroides Gonadais/biossíntese , Fosfoproteínas/genética , Receptores do LH/genética , Toxina T-2/farmacologia , Adulto , Aromatase/metabolismo , Células Cultivadas , Gonadotropina Coriônica/metabolismo , Células do Cúmulo/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Estradiol/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Humanos , Fosfoproteínas/metabolismo , Progesterona/metabolismo , RNA Mensageiro/metabolismo , Receptores do LH/metabolismo , Adulto Jovem
13.
J Biochem Mol Toxicol ; 33(7): e22329, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30934154

RESUMO

Lipopolysaccharide (LPS) can cause ovarian dysfunction and infertility in mammals. The purpose of this study was to investigate the effects of LPS on the accumulation of lipid droplets (LDs), proliferation, and steroidogenesis in goat luteinized granulosa cells (LGCs). GCs isolated from the ovarian follicles were spontaneously luteinized under media with fetal bovine serum, resulting in increased progesterone and shifted shape from spherical to star with multiple prolongations. Then, LGCs were treated with LPS (0-10 µg/mL) for 0-48 hours. Oil Red O staining was performed to observe LDs accumulation and commercial kit was applied to detect intracellular triglyceride (TG) content. The cell proliferation were detected by cell counting kit-8. Expressions of cell-cycle-related genes were determined by real-time polymerase chain reaction. Estradiol (E 2 ) and progesterone (P 4 ) from cell supernatants were determined by enzyme-linked immunosorbent assay, and expressions of STAR, P450scc, 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and CYP19A1 were detected by Western blot. Results showed that LPS treatment significantly increased LDs accumulation after 24 hours, and 5 µg/mL LPS increased TG content ( P < 0.05). LPS treatment for 24 hours stimulated the LGCs activities ( P<0.05), which was confirmed by the increases in the expressions of proliferating cell nuclear antigen (PCNA), cyclinB1 and cyclinD1, while 48 hours treatment had no effect. LPS treatment suppressed E 2 and P 4 output of LGCs ( P < 0.05). Western blot results showed that 10 µg/mL LPS decreased the protein expression of 3ß-HSD in LGCs ( P < 0.05). In conclusion, LPS increased LDs accumulation and cell proliferation, and LPS-mediated P 4 reduction could be attributed to the decreased 3ß-HSD protein expression, which provide new information for the regulation of ovarian function in goats.


Assuntos
Proliferação de Células/efeitos dos fármacos , Estradiol/metabolismo , Células da Granulosa/metabolismo , Gotículas Lipídicas/metabolismo , Lipopolissacarídeos/toxicidade , Progesterona/metabolismo , Animais , Células Cultivadas , Feminino , Cabras , Células da Granulosa/patologia , Gotículas Lipídicas/patologia
14.
C R Biol ; 342(3-4): 90-96, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028003

RESUMO

The objective of our study was to elucidate the role of the transcription factor CREB-1 in controlling ovarian cell proliferation, apoptosis, and hormone release and the significance of CREB-1 phosphorylation in these processes. Human ovarian granulosa cells were transfected with a gene construct encoding wild-type CREB-1 (CREB-1 WT) or CREB-1 nonphosphorylatable mutant (CREB-1 M1). The expression of total and phosphorylated CREB-1, markers of proliferation (PCNA) and apoptosis (bax), as well as the release of progesterone, oxytocin, prostaglandin F2 alpha (PGF2), prostaglandin E2 (PGE2), and insulin-like growth factor I (IGF-I) were compared by immunocytochemistry, enzyme immunoassay (EIA), and immunoradiometric assay (IRMA). Transfection with CREB-1 WT or CREB-1 M1 increased total CREB-1 expression and proliferation and decreased the release of oxytocin, PGE2, and IGF-I by ovarian cells. CREB-1 M1, not CREB-1 WT, promoted apoptosis and inhibited progesterone output. PGF2 release was inhibited by CREB-1 WT but stimulated by CREB-1 M1 construct. Phosphorylated CREB-1 was undetected in any cell group. These observations confirm the involvement of CREB-1 in the control of ovarian cell proliferation, apoptosis, and steroid hormone release. This is the first demonstration of the involvement of CREB-1 in the regulation of the ovarian non-steroidal hormones such as oxytocin, PGF2, PGE2, and IGF-I. The absence of CREB-1 phosphorylation, similar effects exerted by CREB-1 WT and CREB-1 M1 on cell proliferation and release of oxytocin, PGE2, and IGF-I, and the influence of CREB-1 M1 on apoptosis and progesterone suggest that phosphorylation plays no role in the action of CREB-1 on the majority of analyzed functions of human ovarian cells.


Assuntos
Proliferação de Células/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ovário/fisiologia , Fosforilação/fisiologia , Adulto , Animais , Apoptose/fisiologia , Células Cultivadas , Feminino , Células da Granulosa/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Ocitocina/metabolismo , Progesterona/metabolismo
15.
Theriogenology ; 131: 89-95, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30965208

RESUMO

Oxidative stress-induced apoptosis of granulosa cells (GCs) is believed to be an important cause of follicular atresia. Our previous work showed that the c-Jun N-terminal kinase (also known as JNK) might promote apoptosis in GCs during oxidative stress. The aim of this study was to investigate the upstream signaling required for JNK-mediated GCs apoptosis during oxidative stress. Since PKCδ and ASK1 have been suggested to regulate JNK activity in some types of cells, we hypothesized that PKCδ and ASK1 might contribute to JNK-dependent apoptosis in GCs suffering oxidative stimulation. To test this assumption, porcine GCs obtained from healthy follicles were treated with H2O2 alone, or together with inhibitors against PKCδ and JNK, and then collected for cell viability assay, TUNEL staining, immunoprecipitation, western blotting, or JNK activity detection in vitro. The current results showed that the cell viability loss, DNA fragmentation, morphological shrinkage, and nuclear condensation in H2O2-treated porcine GCs was correlated with enhanced activation of JNK. Although ASK1 was supposed to be a JNK activator, we found no definite role of ASK1 in JNK-induced GCs apoptosis during oxidative stress. Further investigations revealed that H2O2-mediated PKCδ activation was required for the apoptotic death of porcine GCs. Particularly, the pro-apoptotic effects of PKCδ on porcine GCs might be achieved by activating the mitochondrial pathway. Importantly, we found that p-PKCδ acts as an upstream activator of JNK in H2O2-treated porcine GCs. However, JNK has no regulatory effect on PKCδ activity. Taken together, our findings provided a novel model of GCs apoptosis involving the activation of PKCδ/JNK/mitochondrial apoptosis axis during oxidative stress.


Assuntos
Apoptose , Células da Granulosa/metabolismo , Sistema de Sinalização das MAP Quinases , Ovário/metabolismo , Proteína Quinase C-delta/fisiologia , Suínos , Animais , Feminino , Células da Granulosa/citologia , Marcação In Situ das Extremidades Cortadas , Ovário/citologia , Estresse Oxidativo , Proteína Quinase C-delta/metabolismo
16.
J Environ Sci Health B ; 54(6): 533-537, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30947605

RESUMO

Quercetin is a dietary bioflavonoid used widely as a food supplement and is generally recognized as safe. The aim of this in vitro study was to examine the steroid hormone (progesterone and 17- ß estradiol) release, proliferation (PCNA and cyclin B1) and apoptosis (caspase 3 and p53) of porcine ovarian granulosa cells after the addition of quercetin at concentrations 0.01, 0.1, 1, 10 and 100 µmol L-1. Progesterone release was stimulated at the concentration 10 µmol L-1. Quercetin neither had any impact on 17-ß estradiol secretion nor on the presence of PCNA. However, a significant enhancement of the occurrence of cyclin B1 was noted except for the lowest concentration 0.01 µmol L-1. Quercetin did not have any influence on the number of granulosa cells containing caspase 3, but at the concentration 10 µmol L-1 it inhibited p53 occurrence. Results confirm the safety of quercetin in porcine ovarian granulosa cell model and further suggest its possible concentration-dependent influence on ovarian functions through pathway that may involve progesterone, cyclin B1 and p53.


Assuntos
Células da Granulosa/efeitos dos fármacos , Quercetina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ciclina B1/metabolismo , Suplementos Nutricionais , Estradiol/metabolismo , Feminino , Células da Granulosa/metabolismo , Progesterona/metabolismo , Quercetina/administração & dosagem , Suínos
17.
BMC Genomics ; 20(1): 273, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30953450

RESUMO

BACKGROUND: Previously, we could show that L-lactate affects cultured bovine granulosa cells (GC) in a specific manner driving the cells into an early pre-ovulatory phenotype. Here we studied genome wide effects in L-lactate-treated GC to further elucidate the underlying mechanisms that are responsible for the L-lactate induced transformation. Cultured estrogen producing GC treated either with L-lactate or vehicle control were subjected to mRNA microarray analysis. RESULTS: The analysis revealed 487 differentially expressed clusters, representing 461 annotated genes. Of these, 333 (= 318 genes) were identified as up- and 154 (= 143 genes) as down-regulated. As the top up-regulated genes we detected TXNIP, H19 and AHSG as well as our previously established marker transcripts RGS2 and PTX3. The top down-regulated genes included VNN1, SLC27A2 and GFRA1, but also MYC and the GC marker transcript CYP19A1. Pathway analysis with differentially expressed genes indicated "cAMP-mediated signaling" and "Axon guidance signaling" among the most affected pathways. Furthermore, estradiol, progesterone and Vegf were identified as potential upstream regulators. An effector network analysis by IPA provided first hints that processes of "angiogenesis" and "vascularization", but also "cell movement" appeared to be activated, whereas "organismal death" was predicted to be inhibited. CONCLUSIONS: Our data clearly show that L-lactate alters gene expression in cultured bovine GC in a broad, but obviously specific manner. Pathway analysis revealed that the mode of L-lactate action in GC initiates angiogenic processes, but also migratory events like cell movement and axonal guidance signaling, thus supporting the transformation of GC into an early luteal phenotype.


Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células da Granulosa/metabolismo , Lactatos/farmacologia , Transcriptoma , Animais , Bovinos , Feminino , Redes Reguladoras de Genes , Genoma , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos
18.
Ann Clin Lab Sci ; 49(2): 175-182, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31028061

RESUMO

In recent years, environmental endocrine disruptors (EEDs) have received extensive attention because of their hormone-like or anti-hormone effects. Dibutyl phthalate (DBP) is not only one of the most widely-used phthalates but also a member of EEDs with the estrogenic property. Although some studies have revealed the negative effect of DBP on the reproductive system, the underlying mechanisms are still elusive. Here the effect of DBP on P450 aromatase, a rate-limiting enzyme stimulated by FSH in the estradiol synthesis, was investigated in human granulosa cell line KGN. Cultured cells were treated with FSH and various doses of DBP (0.1µM, 1µM, 10µM, 50µM, or 100µM) for 24hr. Then the expression of aromatase was assessed, and the synthesis of estradiol was detected to reflect aromatase activity. As shown by the results, all concentrations of DBP could up-regulate the mRNA as well as protein levels of aromatase, and 0.1µM DBP increased the production of estradiol significantly. Furthermore, the ovary-specific promoter of aromatase, promoter II, was activated by 0.1µM DBP, and the expression of FSH receptor (FSHR) was increased by DBP from 0.1µM to 100µM. The study results show that DBP can affect aromatase from both quantitative and functional aspects, and this process may involve the activation of aromatase promoter II and upregulation of FSHR in KGN. Additionally, low-concentration DBP, near human serum concentration, has a more robust effect. This study suggests that DBP may affect the steroidogenic capacity in human ovaries and contributes to our understanding of the effects of DBP on the female reproductive system.


Assuntos
Aromatase/genética , Dibutilftalato/toxicidade , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/metabolismo , Aromatase/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células da Granulosa/efeitos dos fármacos , Humanos , PPAR alfa/genética , PPAR alfa/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do FSH/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
19.
J Ovarian Res ; 12(1): 21, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30819231

RESUMO

BACKGROUND: The cumulus expansion process is one of the LH mediated ovulatory processes. Hyaluronan synthase 2 (HAS2) regulates the synthesis of hyaluronic acid, the main component of the cumulus expansion process. Recently, the lncRNA HAS2 antisense RNA 1 (HAS2-AS1) was identified in our global transcriptome RNA-sequencing of novel ovulation associated genes. The role of HAS2-AS1 in HAS2 regulation w.as studied previously with contradictive results in different models but not in the ovary. Taken together the induction of HAS2-AS1 and the important role of HAS2 in the cumulus expansion process, we hypothesize that HAS2-AS1 regulate HAS2 expression and function in the ovary. Therefore we undertook to study the expression, regulation, and possible functional role of HAS2-AS1 in the human ovary. RESULTS: HAS2-AS1, located within the HAS2 gene that was highly regulated in our library. We found that HAS2-AS1 express mainly in cumulus cells (CCs). Furthermore, HAS2-AS1 showed low expression in immature CCs and a significant increase expression in mature CCs. Functional studies reveal that inhibition of HAS2-AS1 by siRNA caused decrease expression of HAS2. Furthermore, inhibition of HAS2-AS1 by siRNA results in decrease migration of granulosa cells. CONCLUSIONS: Our results suggest that HAS2-AS1 is an LH/hCG target gene that plays a positive role in HAS2 expression and thus might play a role in regulating cumulus expansion and migration.


Assuntos
Gonadotropina Coriônica/farmacologia , Células do Cúmulo/citologia , Regulação da Expressão Gênica , Hialuronan Sintases/genética , RNA Longo não Codificante/metabolismo , Movimento Celular , Células Cultivadas , Gonadotropina Coriônica/administração & dosagem , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Humanos , Hialuronan Sintases/metabolismo , Ovário/metabolismo , Ovulação/efeitos dos fármacos , Ovulação/genética , Ovulação/fisiologia , RNA Longo não Codificante/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo
20.
Biomed Res Int ; 2019: 6545210, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30834271

RESUMO

The physiological processes that drive the development of ovarian follicle, as well as the process of oogenesis, are quite well known. Granulosa cells are major players in this occurrence, being the somatic element of the female gamete development. They participate directly in the processes of oogenesis, building the cumulus-oocyte complex surrounding the ovum. In addition to that, they have a further impact on the reproductive processes, being a place of steroid sex hormone synthesis and secretion. It is known that the follicle development creates a major need for angiogenesis and blood vessel development in the ovary. In this study, we use novel molecular approaches to analyze markers of these processes in porcine granulosa cultured primarily in vitro. The cells were recovered from mature sus scrofa specimen after slaughter. They were then subjected to enzymatic digestion and culture primarily for a short term. The RNA was extracted from cultures in specific time periods (0h, 24h, 48h, 96h, and 144h) and analyzed using expression microarrays. The genes that exhibited fold change bigger than |2|, and adjusted p-value lower than 0.05, were considered differentially expressed. From these, we have chosen the members of "angiogenesis," "blood vessel development," "blood vessel morphogenesis," "cardiovascular system development," and "vasculature development" for further selection. CCL2, FGFR2, SFRP2, PDPN, DCN, CAV1, CHI3L1, ITGB3, FN1, and LOX which are upregulated, as well as CXCL10, NEBL, IHH, TGFBR3, SCUBE1, IGF1, EDNRA, RHOB, PPARD, and SLITRK5 genes whose expression is downregulated through the time of culture, were chosen as the potential markers, as their expression varied the most during the time of culture. The fold changes were further validated with RT-qPCR. The genes were described, with special attention to their possible function in GCs during culture. The results broaden the general knowledge about GC's in vitro molecular processes and might serve as a point of reference for further in vivo and clinical studies.


Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Células da Granulosa/citologia , Neovascularização Fisiológica/genética , Folículo Ovariano/crescimento & desenvolvimento , Animais , Vasos Sanguíneos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Células da Granulosa/metabolismo , Humanos , Morfogênese/genética , Oócitos/crescimento & desenvolvimento , Oogênese/genética , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Cultura Primária de Células , Biossíntese de Proteínas/genética , Suínos
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