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1.
Gene ; 744: 144591, 2020 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-32220601

RESUMO

Polycystic ovary syndrome (PCOS) is a kind of endocrine disease among women across the global. Recently, many researches have reported circular RNAs can act as significant molecular biomarkers for diseases, especially in tumors. Several Circular RNAs are reported to be aberrantly expressed in PCOS patients. Here, we investigated the biological effects of hsa_circ_0118530 on human granulosa cells, KGN. We observed that hsa_circ_0118530 was greatly elevated in PCOS patients and granulosa cells (including KGN and COV434 cells) compared to normal IOSE80 cells. hsa_circ_0118530 siRNA was transfected into KGN cells. We found that KGN cell viability was repressed, cell apoptosis was induced while cell migration was greatly inhibited. TGF-ß1 was utilized to induce EMT process. As shown, loss of hsa_circ_0118530 significantly enhanced E-cadherin mRNA and protein levels while depressed N-cadherin expression. Furthermore, we indicated that decrease of hsa_circ_0118530 was able to inhibit ROS accumulation, MDA levels while induced SOD activity. Next, it was demonstrated that releases of inflammatory cytokine were suppressed by hsa_circ_0118530 down-regulation. Additionally, miR-136 was predicted and confirmed as the target of hsa_circ_0118530. For another, the functions of hsa_circ_0118530 on KGN cell progression, oxidative stress and inflammation releases were obviously reversed by miR-136 suppression. In conclusion, knockdown of hsa_circ_0118530 repressed PCOS progression via sponging miR-136.


Assuntos
Células da Granulosa/metabolismo , MicroRNAs/metabolismo , Síndrome do Ovário Policístico/genética , RNA Circular/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Células da Granulosa/citologia , Humanos , Mediadores da Inflamação/metabolismo , Estresse Oxidativo/genética , Síndrome do Ovário Policístico/metabolismo , RNA Circular/biossíntese
2.
FASEB J ; 34(1): 571-587, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914586

RESUMO

Beyond the study of its transcriptional target genes, the identification of the various interactors of a transcription factor (TF) is crucial to understand its diverse cellular roles. We focused on FOXL2, a winged-helix forkhead TF important for ovarian development and maintenance. FOXL2 has been implicated in diverse cellular processes, including apoptosis, the control of cell cycle or the regulation of steroid hormone synthesis. To reliably identify partners of endogenous FOXL2, we performed a proteome-wide analysis using co-immunoprecipitation in the murine granulosa cell-derived AT29c and the pituitary-derived alpha-T3 cell lines, using three antibodies targeting different parts of the protein. Following a stringent selection of mass spectrometry data on the basis of identification reliability and protein enrichment, we identified a core set of 255 partners common to both cell lines. Their analysis showed that we could co-precipitate several complexes involved in mRNA processing, chromatin remodeling and DNA replication and repair. We further validated (direct and/or indirect) interactions with selected partners, suggesting an unexpected role for FOXL2 in those processes. Overall, this comprehensive analysis of the endogenous FOXL2 interactome sheds light on its numerous and diverse interactors and unconventional cellular roles.


Assuntos
Proteína Forkhead Box L2/metabolismo , Células da Granulosa/metabolismo , Hipófise/metabolismo , Mapas de Interação de Proteínas , Proteoma/metabolismo , Animais , Células Cultivadas , Feminino , Células da Granulosa/citologia , Camundongos , Hipófise/citologia , Proteoma/análise
3.
Iran Biomed J ; 24(1): 30-8, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31454861

RESUMO

Background: Germ cell development processes are influenced by soluble factors and intercellular signaling events between them and the neighboring somatic cells. More insight into the molecular biology of the germ cell development from embryonic stem (ES) cells and investigation of appropriate factors, specifically those targeting differentiation processes, is of great importance. In this study, we established an in vitro model with higher ES cell differentiation rate to germ cells, using adenylate cyclase activator, forskolin. Methods: ES cells were first cultured for five days, leading to embryoid body (EB) formation. Subsequently, the EB were dissociated and cultured for an additional three days in different forskolin concentrations of 5, 20, and 50 µM, with or without granulosa cells (GC) co-culture. On the 8th day, we analyzed the expressions of 5 germ cell-specific markers using quantitative real-time-PCR technique along with cell viability assay by MTT test. Results: Our results showed that in the GC-free cultures, a 50-µM concentration of forskolin resulted in a significant increase in Mvh, Gdf9, Scp3, and Rec8 expression levels in comparison to the control. However, when the cells were co-cultured with the GCs, 20-µM concentration of forskolin could also increase the expression of those germ cell-specific marker genes. Furthermore, results from the MTT assay showed enhanced cell proliferation and survival at all three concentrations of forskolin, but 20-µM concentration was the most potent one. Conclusion: These data indicate that forskolin can stimulate differentiation and proliferation, dose-dependently; however, the influence of GCs co-culturing should not go unnoticed.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Colforsina/farmacologia , Células da Granulosa/citologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Forma Celular/efeitos dos fármacos , Técnicas de Cocultura , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo
4.
Oxid Med Cell Longev ; 2019: 1076512, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31827667

RESUMO

Oxidative stress is a causal factor and key promoter of all kinds of reproductive disorders related to granulosa cell (GC) apoptosis that acts by dysregulating the expression of related genes. Various studies have suggested that grape seed procyanidin B2 (GSPB2) may protect GCs from oxidative injury, though the underlying mechanisms are not fully understood. Therefore, whether the beneficial effects of GSPB2 are associated with microRNAs, which have been suggested to play a critical role in GC apoptosis by regulating the expression of protein-coding genes, was investigated in this study. The results showed that GSPB2 treatment protected GCs from a H2O2-induced apoptosis, as detected by an MTT assay and TUNEL staining, and increased let-7a expression in GCs. Furthermore, let-7a overexpression markedly increased cell viability and inhibited H2O2-induced GC apoptosis. Furthermore, the overexpression of let-7a reduced the upregulation of Fas expression in H2O2-treated GCs at the mRNA and protein levels. Dual-luciferase reporter assay results indicated that let-7a directly targets the Fas 3'-UTR. Furthermore, the overexpression of let-7a enhanced the protective effects of GSPB2 against GC apoptosis induced by H2O2. These results indicate that GSPB2 inhibits H2O2-induced apoptosis of GCs, possibly through the upregulation of let-7a.


Assuntos
Biflavonoides/farmacologia , Catequina/farmacologia , MicroRNAs/metabolismo , Proantocianidinas/farmacologia , Regulação para Cima/efeitos dos fármacos , Vitis/química , Regiões 3' não Traduzidas , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Extrato de Sementes de Uva/química , Peróxido de Hidrogênio/farmacologia , Ovário/citologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Alinhamento de Sequência , Suínos , Vitis/metabolismo , Receptor fas/química , Receptor fas/genética , Receptor fas/metabolismo
5.
Reprod Biol Endocrinol ; 17(1): 88, 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31690325

RESUMO

BACKGROUND: The antral follicle count (AFC) in mammalian ovaries positively correlates with female fertility. To clarify the causes of differences in fertility between low and high AFC cows, we investigated follicular growth dynamics and hormone concentrations in plasma, follicular fluid, and in vitro growth (IVG) media at different stages of follicular growth. METHODS: Seven cows were divided into high AFC (n = 4, > 30 follicles) and low AFC (n = 3, < 30 follicles) groups based on the peak AFC detected by ultrasonography. These cows were subjected to estrous synchronization, daily ovarian ultrasonography, and blood collection. Their follicular fluid was collected from dominant follicles at different stages (selection, luteal, and ovulatory phases). In another experiment, we cultured oocyte-cumulus-granulosa cell complexes collected from early antral follicles (< 1 mm) for 12 days. Estradiol-17ß (E2), testosterone (T), progesterone (P4), and anti-Müllerian hormone (AMH) concentrations in follicular fluids and plasma were measured. Plasma follicle-stimulating hormone (FSH) concentrations were examined. E2, P4, and AMH concentrations were also measured in IVG media. RESULTS: The numbers of small (< 4 mm) and intermediate (4-8 mm) follicles were larger in the high AFC group than in the low AFC group (P < 0.05). The number of intermediate follicles was stable in the low AFC group, indicating consistent development. However, the number of these follicles fluctuated in the high AFC group. Plasma FSH concentrations were higher, whereas E2 and T concentrations were lower in the low AFC group (P < 0.05). E2 concentrations and the E2/P4 ratio in ovulatory follicles and IVG media on day 8 were higher in the high AFC group (P < 0.05). AMH concentrations in plasma and IVG media (P < 0.01) were higher in the high AFC group. CONCLUSIONS: The weaker response to FSH of granulosa cells caused low E2 production in the low AFC group, resulting in high FSH concentrations and the consistent development of intermediate follicles. Conversely, higher E2 concentrations suppressed FSH secretion in the high AFC group. Granulosa cells in the high AFC group had the ability to produce more AMH than those in the low AFC group throughout IVG culture.


Assuntos
Hormônio Antimülleriano/metabolismo , Hormônio Foliculoestimulante/metabolismo , Hormônios Esteroides Gonadais/metabolismo , Folículo Ovariano/metabolismo , Animais , Bovinos , Contagem de Células , Células Cultivadas , Estradiol/metabolismo , Feminino , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/crescimento & desenvolvimento , Ovário/citologia , Ovário/metabolismo , Progesterona/metabolismo , Testosterona/metabolismo
6.
Oxid Med Cell Longev ; 2019: 1041583, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31781320

RESUMO

Oxidative stress (OS), a common intracellular phenomenon induced by excess reactive oxygen species (ROS) generation, has been shown to be associated with mammalian ovarian follicular development blockage and granulosa cell (GC) impairment. However, the mechanism involved in these effects remains unknown, and the effect of OS on the transcriptome profiles in porcine GCs has not been fully characterized. In this study, we found that hydrogen peroxide-mediated oxidative stress induced porcine GC apoptosis and impaired cell viability. Moreover, RNA-seq analysis showed that oxidative stress induced dramatic changes in gene expression in porcine GCs. A total of 2025 differentially expressed genes (DEGs) were identified, including 1940 DEmRNAs and 55 DEmiRNAs. Functional annotation showed that the DEGs were mainly associated with cell states and function regulation. In addition, multiple hub genes (FOXO1, SOD2, BMP2, DICER1, BCL2L11, FZD4, ssc-miR-424, and ssc-miR-27b) were identified by constructing protein-protein interaction and DEmiRNA-DEmRNA regulatory networks. Furthermore, a gene-pathway-function coregulatory network was established and demonstrated that these hub genes were enriched in FoxO, TGF-ß, Wnt, PIK3-Akt, MAPK, and cAMP signaling pathways, which play important roles in regulating cell apoptosis, cell proliferation, stress responses, and hormone secretion. The current research provides a comprehensive perspective of the effects of oxidative stress on porcine GCs and also identifies potential therapeutic targets for oxidative stress-induced female infertility.


Assuntos
Regulação da Expressão Gênica , Células da Granulosa/metabolismo , Sistema de Sinalização das MAP Quinases , MicroRNAs/metabolismo , Estresse Oxidativo , RNA Mensageiro/biossíntese , Sistemas do Segundo Mensageiro , Animais , Feminino , Células da Granulosa/citologia , Suínos
7.
J Agric Food Chem ; 67(43): 12117-12128, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31587554

RESUMO

Zearalenone (ZEA), a pathogenic toxin produced by Fusarium, is widely detected in moldy feed materials. Previous studies have reported that ZEA exerts a harmful influence on animal reproductive systems; however, its effects on the changes of long noncoding RNAs (lncRNAs) remain unclear. Here, tackling this question, we performed RNA sequencing on porcine granulosa cells (GCs) after being exposed to 10 and 30 µM ZEA in vitro. The results showed that ZEA exposure observably changed the expression of lncRNAs in porcine GCs and increased the rate of apoptosis. Furthermore, Gene Ontology analysis showed that ZEA exposure induced variation of the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) signaling pathway in porcine GCs. To verify our bioinformatics analysis, western blotting and immunofluorescence analysis were performed and the results demonstrated that porcine GCs after ZEA exposure increased the expression of key proteins in the JAK2-STAT3 signaling pathway. Further bioinformatics analysis found that MSTRG.22680 and MSTRG.23882 played a pivotal role in activating the JAK2-STAT3 signaling pathway. To summarize, our results throw light on the fact that ZEA exposure dramatically increases the apoptosis of porcine GCs and alters the expression of lncRNAs that play an antiapoptotic role in porcine GCs via activating the JAK2-STAT3 signaling pathway.


Assuntos
Apoptose/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Janus Quinase 2/metabolismo , RNA Longo não Codificante/genética , Fator de Transcrição STAT3/metabolismo , Zearalenona/toxicidade , Animais , Feminino , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Janus Quinase 2/genética , RNA Longo não Codificante/metabolismo , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacos , Suínos
8.
J Agric Food Chem ; 67(36): 10207-10213, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31426637

RESUMO

Forchlorfenuron (FCF) is a synthetic plant cytokine-like growth regulator that is massively used in agriculture to increase fruit size and weight. There is an insufficiency of published data on the safety profile of FCF, especially as it is involved in ovarian function. In our study, a chronic toxicity study on FCF was conducted and designed by feeding at dosage levels of 0, 0.6, and 60 mg/kg body weight in Sprague-Dawley rats for 180 days. During the 180 day FCF administration, no biologically relevant changes were observed in the body weight, clinical signs, food consumption, organ weight, hematology, and clinical biochemistry of the tested animals. However, macroscopic and microscopic evaluations revealed the presence of severe hydrometra in the uterus and pathological changes in the ovaries. In addition, it was found that FCF inhibited the proliferation of granulosa cells (GCs) and H295R cells, as well as downregulated the expression of CYP17A1 and CYP19A1 in estradiol and progesterone production, resulting in decreased steroidogenesis in GCs and H295R cells. Taken together, our findings suggest that FCF has potential adverse effects on the ovaries and on steroidogenesis.


Assuntos
Hormônios Esteroides Gonadais/metabolismo , Compostos de Fenilureia/toxicidade , Reguladores de Crescimento de Planta/toxicidade , Piridinas/toxicidade , Administração Oral , Animais , Aromatase/genética , Aromatase/metabolismo , Peso Corporal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ovário/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Ovário/patologia , Compostos de Fenilureia/administração & dosagem , Piridinas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Fatores de Tempo , Útero/efeitos dos fármacos , Útero/crescimento & desenvolvimento , Útero/metabolismo , Útero/patologia
9.
J Reprod Dev ; 65(5): 423-432, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31378755

RESUMO

Historically, it had been widely accepted that the female mammalian ovary contained a limited number of oocytes that would reduce over time, without the possibility of replenishment. However, recent studies have suggested that female germline stem cells (FGSCs) could replenish the oocyte-pool in adults. The aim of this study was to isolate FGSCs from porcine ovaries and differentiate them into oocyte-like cells (OLCs). The FGSCs were successfully isolated from porcine ovarian tissue and cultured in vitro, in DMEM/F-12 medium supplemented with growth factors (EGF, FGF, GDNF, etc.) and a supplement (N21). These cells possessed spherical morphology and expressed specific germline characteristics (Vasa, Stella, Oct4, c-kit). By evaluating different conditions for in vitro differentiation of FGSCs, co-culturing the isolated FGSCs with MEF cells, under three-dimensional (3D) cell cultures, were shown to be optimal. FGSCs could successfully be differentiated into OLCs and reached about 70 µm in diameter, with a large number of surrounding somatic cells. Importantly, OLCs contained large nuclei, about 25-30 µm, with filamentous chromatin, similar to oocyte morphology, and expressed oocyte-specific markers (Gdf9, Zp2, SCP3, etc.) at the same level as oocytes. In conclusion, we successfully isolated FGSCs from porcine ovarian tissue and differentiated them into oocyte-like cells. This will provide a valuable model for studying a new, alternative source of oocytes.


Assuntos
Técnicas de Cultura de Células , Oócitos/citologia , Células-Tronco de Oogônios/citologia , Ovário/citologia , Animais , Diferenciação Celular , Proliferação de Células , Cromatina/metabolismo , Criopreservação , Proteínas de Ligação a DNA/metabolismo , Feminino , Células da Granulosa/citologia , Fator 9 de Diferenciação de Crescimento/metabolismo , Óvulo/citologia , Suínos , Glicoproteínas da Zona Pelúcida/metabolismo
10.
Int J Mol Sci ; 20(16)2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31443152

RESUMO

Nowadays, science has a lot of knowledge about the physiology of ovarian processes, especially folliculogenesis, hormone production and ovulation. However, the molecular basis for these processes remains largely undiscovered. The cell layer surrounding the growing oocyte-granulosa cells-are characterized by high physiological capabilities (e.g., proliferation, differentiation) and potential for growth in primary cultures, which predisposes them for analysis in the context of possible application of their cultures in advanced methods of assisted reproduction. In this study, we have used standard molecular approaches to analyze markers of these processes in primarily in vitro cultured porcine granulosa, subjected to conditions usually applied to cultures of similar cells. The material for our research came from commercially slaughtered pigs. The cells were obtained by enzymatic digestion of tissues and in vitro culture in appropriate conditions. The obtained genetic material (RNA) was collected at specific time intervals (0 h-before culture; reference, 48, 98, 144 h) and then analyzed using expression microarrays. Genes that showed a fold change greater than |2| and an adjusted p value lower than 0.05 were described as differentially expressed. Three groups of genes: "Cell morphogenesis", "cell differentiation" and "cell development" were analyzed. From 265 differently expressed genes that belong to chosen ontology groups we have selected DAPL1, CXCL10, NEBL, IHH, TGFBR3, SCUBE1, DAB1, ITM2A, MCOLN3, IGF1 which are most downregulated and PDPN, CAV1, TMOD1, TAGLN, IGFBP5, ITGB3, LAMB1, FN1, ITGA2, POSTN genes whose expression is upregulated through the time of culture, on which we focused in downstream analysis. The results were also validated using RT-qPCR. The aim of our work was to conduct primary in vitro culture of granulosa cells, as well as to analyze the expression of gene groups in relation to the proliferation of follicular granulosa cells in the model of primary culture in real time. This knowledge should provide us with a molecular insight into the processes occurring during the in vitro cultures of porcine granulosa cells, serving as a basic molecular entry on the extent of the loss of their physiological properties, as well as gain of new, culture-specific traits.


Assuntos
Células da Granulosa/citologia , Células da Granulosa/metabolismo , Folículo Ovariano/metabolismo , Ovário/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Feminino , Morfogênese/genética , Morfogênese/fisiologia , Suínos , Transcriptoma/genética
11.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(6): 518-525, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31292056

RESUMO

Objective To investigate the expression of microRNA-383 (miR-383) in viability classification of growing follicles and its biological function on proliferation and cycle of granulosa cells. Methods The expression of miR-383 in ovaries tissues, follicles and granulosa cells extracted from Kunming mice after birth of 12, 21 days, 21 days [after injection of pregnant mare serum gonadotropin (PMSG) for 48 hours] and 21 days [after injection of PMSG for 48 hours and (human chorionic gonadotrophin) hCG for 6 hours] were detected by real-time quantitative PCR. The miR-383 mimics or miR-383 inhibitor were transfected into granulosa cells extracted from Kunming mice after birth of 21 days, respectively as miR-383 mimics group and miR-383 inhibitor group. Meanwhile, we established negative control (NC) group. MTT assay was used to detect the proliferation and flow cytometry was used to detect cell cycle of granulosa cells. And the levels of cyclin D1, cyclin B, CDK1, CDK2, and CDK4 protein were tested by Western blot analysis. Results Compared with small preantral follicles, miR-383 level in big preantral follicles and big antral follicles were both lower, but miR-383 in small antral follicles was higher. Similarly, miR-383 of granulosa cells in big preantral follicles and small antral follicles were lower than that of granulosa cells in small preantral follicles, but up-regulated in granulosa cells of big antral follicles. Compared with granulosa cells in the NC group, the proliferation activity of granulosa cells in miR-383 mimics group was lower, and there was more granulosa cells in G0/G1 phase, less in G2/M phase in miR-383 mimics group, besides cyclin D1, cyclinB, CDK1, CDK2, CDK4 proteins in miR-383 mimics group were all lower. Meanwhile, the proliferation activity of granulosa cells in miR-383 inhibitor group was higher, and there was less granulosa cells in G0/G1 phase, more in G2/M phase in miR-383 inhibitor group, besides cyclinD1, cyclinB, CDK1, CDK2, CDK4 proteins in miR-383 inhibitor group were all higher than those proteins in the NC group. Conclusion miR-383 inhibits the proliferation of granulosa cells by down-regulating of cycle-related proteins and blocking granulosa cells in G0/G1 phase.


Assuntos
Proteínas de Ciclo Celular/genética , Células da Granulosa/citologia , MicroRNAs/genética , Animais , Regulação para Baixo , Feminino , Camundongos , Folículo Ovariano , Gravidez
12.
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
13.
Eur J Pharmacol ; 860: 172560, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31344364

RESUMO

Plants, fruits, and vegetables containing the bioflavonoid quercetin are widely used in food, beverages, and medicines; however, the effects of quercetin on reproductive processes and the possible mechanisms of quercetin action require extensive investigation. The aim of our study was to examine the direct effects of quercetin on basic ovarian cell functions and their response to follicle-stimulating hormone (FSH) and insulin-like growth factor I (IGF-I), known hormonal stimulators of reproduction. We analyzed the effects of quercetin alone (0, 1, 10, and 100 ng/ml) on cultured porcine ovarian granulosa cells or isolated ovarian follicles; or of quercetin (10 ng/ml) in combination with FSH (0, 0.01, 0.1, or 1 IU/ml) or IGF-I (0, 1, 10, or 100 ng/ml) on cultured porcine granulosa cells. The expression of proliferative (PCNA, cyclin B1) and apoptotic (BAX) markers, as well as markers for release of progesterone (P4), testosterone (T), and leptin (L), were measured by quantitative immunocytochemistry, Western immunoblotting, RT-qPCR, and EIA/RIA. Addition of quercetin reduced the accumulation of PCNA and cyclin B1, as well as their transcript levels, promoted the accumulation of BAX, decreased the release of P4 and L, and increased the release of T in cultured granulosa cells. In ovarian follicles, quercetin reduced the levels of both P4 and T. Exposure to FSH stimulated PCNA and decreased BAX accumulation, and increased the release of P4, T, and L. Quercetin inhibited and even reversed the effects of FSH. Like FSH, IGF-I also promoted granulosa cell proliferation and suppressed apoptosis. Quercetin did not modify IGF-I effects. These data suggest that the plant molecule quercetin can directly down-regulate basal ovarian cell functions (proliferation, apoptosis, and release of ovarian steroid and peptide hormones) and their response to the stimulatory activity of the upstream hormonal stimulator FSH.


Assuntos
Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Quercetina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina B1/metabolismo , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Leptina/metabolismo , Progesterona/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , RNA Mensageiro/genética , Suínos , Testosterona/metabolismo
14.
Acta Biochim Biophys Sin (Shanghai) ; 51(8): 845-855, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31287492

RESUMO

Autoimmune ovarian disease (AOD) is considered to be a major cause of premature ovarian failure (POF). The immunomodulatory properties of human amniotic epithelial cells (hAECs) have been studied in many disease models. We previously reported that hAECs restored ovarian function in chemotherapy-induced POF mice, but the immunomodulatory mechanism of hAECs is still unclear. To investigate the effect of hAECs on recipient mice, especially on regulatory Treg cells, hAECs and hAEC-conditioned medium (hAEC-CM) were intravenously injected into AOD mice immunized with zona pellucida protein 3 peptides (pZP3). Ovarian function was evaluated through estrous cycle, hormone secretion, follicle development, and cell apoptosis analysis. Immune cells including CD3, CD4, CD8 and Treg cells in the spleens were tested by flow cytometry. To elucidate the effect of hAEC-CM on macrophage function, inflammation model in vitro was established in RAW264.7 cells induced by lipopolysaccharide (LPS). hAECs and hAEC-CM regulated estrous cycles, promoted follicle development, ameliorated cell apoptosis and fibrosis in ovaries of AOD mice. In addition, hAECs significantly reversed the decrease of pZP3-induced Treg cells in the spleens. In vitro, hAEC-CM significantly inhibited the inflammatory reaction induced by LPS in RAW264.7 cells via up-regulating the expression of M2 macrophage genes. Further study demonstrated that hAEC-secreted transforming growth factor-beta and macrophage inhibitory factor played important roles in the macrophage polarization and migration under inflammatory stimulation. Taken together, hAECs restored ovarian function by up-regulating Treg cells in the spleens and reduced the inflammatory reaction via modulating the activated macrophage function in a paracrine manner in the ovaries of AOD mice.


Assuntos
Âmnio/citologia , Doenças Autoimunes/terapia , Células Epiteliais/citologia , Doenças Ovarianas/terapia , Insuficiência Ovariana Primária/terapia , Animais , Apoptose , Movimento Celular , Meios de Cultivo Condicionados/química , Modelos Animais de Doenças , Feminino , Células da Granulosa/citologia , Humanos , Imuno-Histoquímica , Inflamação , Oxirredutases Intramoleculares/metabolismo , Lipopolissacarídeos/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Macrófagos/metabolismo , Camundongos , Células RAW 264.7 , Baço/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Glicoproteínas da Zona Pelúcida/metabolismo
15.
Zygote ; 27(4): 203-213, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31296276

RESUMO

The present study investigated if the presence of encircling granulosa cells protected against di(2-ethylhexyl)phthalate (DEHP)-induced oxidative stress in rat oocytes cultured in vitro. Denuded oocytes and cumulus-oocyte complexes (COCs) were treated with or without various doses of DEHP (0.0, 25.0, 50.0, 100, 200, 400 and 800 µM) in vitro. Morphological apoptotic changes, levels of oxidative stress and reactive oxygen species (ROS), mitochondrial membrane potential, and expression levels of apoptotic markers (Bcl2, Bax, cytochrome c) were analyzed. Our results showed that DEHP induced morphological apoptotic changes in a dose-dependent manner in denuded oocytes cultured in vitro. The effective dose of DEHP (400 µg) significantly (P>0.05) increased oxidative stress by elevating ROS levels and the mitochondrial membrane potential with higher mRNA expression and protein levels of apoptotic markers (Bax, cytochrome c). Encircling granulosa cells protected oocytes from DEHP-induced morphological changes, increased oxidative stress and ROS levels, as well as increased expression of apoptotic markers. Taken together our data suggested that encircling granulosa cells protected oocytes against DEHP-induced apoptosis and that the presence of granulosa cells could act positively towards the survival of oocytes under in vitro culture conditions and may be helpful during assisted reproductive technique programmes.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Células da Granulosa/fisiologia , Oócitos/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Células Cultivadas , Dietilexilftalato/toxicidade , Feminino , Expressão Gênica/efeitos dos fármacos , Células da Granulosa/citologia , Potencial da Membrana Mitocondrial/fisiologia , Oócitos/citologia , Oócitos/metabolismo , Estresse Oxidativo/fisiologia , Plastificantes/toxicidade , Ratos , Espécies Reativas de Oxigênio/metabolismo
16.
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
17.
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
18.
J Ovarian Res ; 12(1): 43, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31077207

RESUMO

BACKGROUND: Palmitic acid (PA), the main component of dietary saturated fat, causes apoptosis in many cell types, including mouse granulosa cell. Melatonin, an important endogenous hormone, has beneficial effects on female reproductive processes. Since elevated PA levels are present in follicular fluid (FF) of patients with infertility and are shown to be toxic for granulosa cells, we investigated the molecular mechanisms of PA toxicity in mouse granulosa cells and explored the effects of melatonin on PA-induced apoptosis. METHODS: Granulosa cells from immature female mice were cultured for 24 h in medium containing PA and/or melatonin. Then, the effects of PA alone or combined with melatonin on viability, apoptosis and endoplasmic reticulum (ER) stress in granulosa cells were detected by methyl thiazolyl tetrazolium (MTT) assay, flow cytometry assay and western blot. After 48 h of PA and/or melatonin treatment, the concentrations of estradiol (E2) and progesterone (P4) in the culture supernatants were measured with ELISA kits. RESULTS: In this study, we explored the effects of melatonin on cell viability and apoptosis in PA-treated mouse granulosa cells and uncovered the signaling pathways involved in these processes. Our results showed that 200-800 µM PA treatment reduces cell viability, induces cell apoptosis, enhances the expression of apoptosis-related genes (Caspase 3 and B-cell lymphoma-2 (BCL-2) associated X protein (BAX)), and activates the expression of ER stress marker genes (glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)). Melatonin treatment (1-10 µM) suppresses 400 µM PA-induced cell viability decrease, cell apoptosis, Caspase 3 activation, and BAX, CHOP, and GRP78 expression. In addition, we found that 10 µM melatonin successfully attenuated the 400 µM PA-induced estrogen (E2) and progesterone (P4) decreases. CONCLUSIONS: This study suggests that PA triggers cell apoptosis via ER stress and that melatonin protects cells against apoptosis by inhibiting ER stress in mouse granulosa cells.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Melatonina/farmacologia , Ácido Palmítico/farmacologia , Animais , Antioxidantes/farmacologia , Proliferação de Células/efeitos dos fármacos , Feminino , Células da Granulosa/citologia , Humanos , Camundongos , Modelos Animais
19.
Cell Biol Int ; 43(8): 899-909, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31081266

RESUMO

Previous studies have shown that the ovarian failure in autoimmune-induced premature ovarian failure (POF) mice could be improved by the transplantation of human placenta-derived mesenchymal stem cells (hPMSCs); however, the protective mechanism of hPMSCs transplantation on ovarian dysfunction remains unclear. Ovarian dysfunction is closely related to the apoptosis of granulosa cells (GCs). To determine the effects of hPMSCs transplantation on GCs apoptosis, an autoimmune POF mice model was established with zona pellucida glycoprotein 3 (ZP3) peptide. It is reported that the inositol-requiring enzyme 1α (IRE1α) and its downstream molecules play a central role in the endoplasmic reticulum (ER) stress-induced apoptosis pathway. So the aim of this study is to investigate whether hPMSCs transplantation attenuated GCs apoptosis via inhibiting ER stress IRE1α signaling pathway. The ovarian dysfunction, follicular dysplasia, and GCs apoptosis were observed in the POF mice. And the IRE1α pathway was activated in ovaries of POF mice, as demonstrated by, increased X-box binding protein 1 (XBP1), up-regulated 78 kDa glucose-regulated protein (GRP78) and caspase-12. Following transplantation of hPMSCs, the ovarian structure and function were significantly improved in POF mice. In addition, the GCs apoptosis was obviously attenuated and IRE1α pathway was significantly inhibited. Transplantation of hPMSCs suppressed GCs apoptosis-induced by ER stress IRE1α signaling pathway in POF mice, which might contribute to the hPMSCs transplantation-mediating ovarian function recovery.


Assuntos
Apoptose/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células-Tronco Mesenquimais/imunologia , Placenta/citologia , Insuficiência Ovariana Primária , Animais , Caspase 12/metabolismo , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/metabolismo , Feminino , Células da Granulosa/citologia , Proteínas de Choque Térmico/metabolismo , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Ovário/metabolismo , Gravidez , Insuficiência Ovariana Primária/metabolismo , Insuficiência Ovariana Primária/terapia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismo , Glicoproteínas da Zona Pelúcida/metabolismo
20.
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
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