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1.
PLoS One ; 15(7): e0234795, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645018

RESUMO

Forkhead box L2 (FOXL2) is a single-exon gene encoding a forkhead transcription factor, which is mainly expressed in the ovary, eyelids and the pituitary gland. FOXL2 plays an essential role in ovarian development. To reveal the effects of FOXL2 on the biological process and gene expression of ovarian granulosa cells (GCs), we established stable FOXL2-knockdown GCs and then analysed them using transcriptome sequencing. It was observed that knocking down FOXL2 affected the biological processes of cell proliferation, DNA replication, and apoptosis and affected cell cycle progression. FOXL2 knockdown promoted cell proliferation and DNA replication, decreased cell apoptosis, and promoted mitosis. In addition, by comparing the transcriptome after FOXL2 knockdown, we found a series of DEGs (differentially expressed genes) and related pathways. These results indicated that, through mediating these genes and pathways, the FOXL2 might induce the cell proliferation, cycle, and DNA replication, and play a key role during ovarian development and maintenance.


Assuntos
Proteína Forkhead Box L2/genética , Proteína Forkhead Box L2/metabolismo , Ovário/metabolismo , Animais , Ciclo Celular/genética , Divisão Celular/genética , Proliferação de Células/genética , Galinhas/genética , Replicação do DNA/genética , Feminino , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/genética , Células da Granulosa/metabolismo , Folículo Ovariano/metabolismo , RNA Mensageiro/genética , Transcriptoma , Sequenciamento Completo do Exoma
2.
Anim Sci J ; 91(1): e13385, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32515535

RESUMO

Both oocytes and extracellular vesicles (EV) have emerged as critical regulators of mammalian follicular development; however, the possible interaction between the oocyte-derived paracrine factor (ODPF) and EV signals has never been examined. Therefore, to explore the possibility of an interaction between oocyte and EV signals, the effects of ODPFs on the biogenesis of EVs as well as the expression levels of transcripts related to EV biogenesis in mural granulosa cells (MGCs) were examined using mice. The results showed that, while oocyte coculture has some effects on the expression levels of transcripts related to EV biogenesis, the number of EV particles present in the conditioned medium were not significantly different between ODPF-treated and non-treated MGCs. Therefore, oocytes have no effects on the EV biogenesis by MGCs, at least with respect to the numbers of EV particles.


Assuntos
Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/fisiologia , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Oócitos/fisiologia , Comunicação Parácrina/fisiologia , Animais , Células Cultivadas , Feminino , Camundongos Endogâmicos C57BL , Biogênese de Organelas
3.
Gene ; 754: 144903, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32540374

RESUMO

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders among reproductive-age women. The circRNA-miRNA axis functions in various diseases progression have been partially revealed in the past two decades. However, little is known about the role of the circRNA-miRNA axis in PCOS progression. MicroRNA miR-760, which is characterized by tissue-specific, has been studied in several cancers. Firstly, we found that miR-760 expression was decreased in PCOS tissues insulin treated GCs, KGN and SVOG cells. Secondly, The CCK-8 and apoptosis experiment results suggested that downregulated miR-760 promoted cell proliferation ability and suppressed apoptosis activity in KGN and SVOG cells. Then, the bioinformatic analysis result indicated that circPUM1 was a potential sponge to miR-760. By performing AGO2-RIP, RNA pull-down, Luciferase reporter, and qRT-PCR experiments, we demonstrated that circPUM1 acted as a molecular sponge to miR-760, and decreased miR-760 expression. Moreover, it was found that the promotive effect of circPUM1 was mediated by regulating miR-760. Collectively, our findings suggest that circPUM1 promotes PCOS progression through sponging to miR-760. We may provide a promising therapeutic target for PCOS.


Assuntos
Regulação da Expressão Gênica , Células da Granulosa/patologia , MicroRNAs/genética , Síndrome do Ovário Policístico/patologia , RNA Circular/genética , Apoptose , Proliferação de Células , Células Cultivadas , Progressão da Doença , Feminino , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Humanos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Síndrome do Ovário Policístico/genética , Síndrome do Ovário Policístico/metabolismo
4.
PLoS One ; 15(5): e0232629, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365144

RESUMO

PIWI-interacting RNAs (piRNAs) play an important role in gametogenesis, fertility and embryonic development. The current study investigated the effect of different doses of pregnant mare serum gonadotrophin/human chorionic gonadotrophin (PMSG/hCG) and repeated ovarian stimulation (OS) on the expression of the Mili, Miwi, Mael, Tdrd1, Tdrd9, qnd Mitopld genes, which have crucial roles in the biogenesis and function of piRNAs. Here, we found that after treatment with 7.5 I.U. PMSG/hCG and two repeated rounds of OS, both the mRNA and protein levels of Tdrd9, Tdrd1 and Mael showed the greatest decrease in the ovarian tissue, but the plasma E2 levels showed the strongest increases (p<0.05). However, we found that the Mitopld, Miwi and Mili gene levels were decreased significantly after treatment with 12.5 I.U. PMSG/hCG. Our results suggested that exogenous gonadotropin administration leads to a significant decrease in the expression of the Mili, Miwi, Mael, Tdrd1, Tdrd9 and Mitopld genes, which are critically important in the piRNA pathway, and the changes in the expression levels of Tdrd9, Tdrd1 and Mael may be associated with plasma E2 levels. New comprehensive studies are needed to reduce the potential effects of OS on the piRNA pathway, which silences transposable elements and maintains genome integrity, and to contribute to the safety of OS.


Assuntos
Gonadotropina Coriônica/farmacologia , Estradiol/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Gonadotropinas/farmacologia , Ovário/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Animais , Elementos de DNA Transponíveis , Feminino , Fertilização In Vitro , Células da Granulosa/metabolismo , Cavalos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Ovário/metabolismo , Indução da Ovulação , RNA Mensageiro/metabolismo
5.
PLoS One ; 15(5): e0233169, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32407420

RESUMO

In broiler hens, the genetic selection increased susceptibility to metabolic disorders and reproductive dysfunctions. In human ovarian cells, grape seed extracts (GSE) improved steroid production. Here, we investigated the effects of a GSE dietary supplementation on egg production and quality, fertility parameters, Reactive Oxygen Species (ROS) and steroid content in yolk egg associated to plasma adipokines in broiler hens. For this, we designed two in vivo experiments, the first one included three groups of hens: A (control), B and C (supplemented with GSE at 0.5% and 1% of the total diet composition, respectively, since week 4), and the second one used two groups of hens: A (control) and D (supplemented with GSE at 1% of the total diet composition since hatching). We assessed the egg production from 23th to 40th weeks and quality at 33th week. After artificial inseminations, the fertility parameters were calculated. In egg yolk, Reactive Oxygen Species (ROS) level and steroid production were evaluated by Ros-Glo H202 and ELISA assay, respectively. Expression of steroidogenic enzymes and adipokines and their receptors was determined by RT-qPCR in ovarian cells and plasma adipokines (RARRES2, ADIPOQ and NAMPT) were evaluated by specific ELISA assays. The fertility parameters and egg production were unaffected by GSE supplementation whatever the experiment (exp.). However, the rate of double-yolk eggs decreased for all GSE supplemented groups (exp. 1 P <0.01, exp.2, P<0.02). In exp.1, C group eggs were bigger and larger (P<0.0001) and the shell elasticity was higher for both B and C (P<0.0003) as compared to control. In the egg yolk, GSE supplementation in both exp. reduced ROS content and steroidogenesis consistent with a decrease in P450 aromatase and StAR mRNA expression and basal in vitro progesterone secretion in granulosa cells (P<0.001). Interestingly, in both exp. RARRES2 plasma levels were positively correlated while ADIPOQ and NAMPT plasma levels were negatively correlated, with steroids and ROS in yolk (P<0.0001). Taken together, maternal dietary GSE supplementation did not affect egg production and fertility parameters whereas it reduced ROS content and steroidogenesis in yolk egg. Furthermore, it ameliorated egg quality by decreasing the number of double-yolk eggs and by improving the size of normal eggs and the elasticity of the shell. Taken together, our data suggest the possibility of using dietary maternal GSE to improve egg quality.


Assuntos
Galinhas/fisiologia , Suplementos Nutricionais , Fertilidade/efeitos dos fármacos , Extrato de Sementes de Uva/farmacologia , Ovário/metabolismo , Óvulo/metabolismo , Reprodução/efeitos dos fármacos , Esteroides/biossíntese , Adipocinas/sangue , Animais , Galinhas/sangue , Galinhas/genética , Dieta , Gema de Ovo/efeitos dos fármacos , Gema de Ovo/metabolismo , Feminino , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Ovário/efeitos dos fármacos , Oviposição/efeitos dos fármacos , Óvulo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Adipocina/genética , Receptores de Adipocina/metabolismo , Células Tecais/efeitos dos fármacos , Células Tecais/metabolismo
6.
Int. j. morphol ; 38(2): 427-434, abr. 2020. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1056458

RESUMO

Granulosa cells (GCs) are essential components of follicles and play a role in regulating follicle development. The aim of this study was to investigate certain cellular components involved in the proliferation, differentiation and functional characteristics of granulosa cells in the success of fertilization of human oocytes during invitro fertilization (IVF) via immunohistochemical techniques. In this study, 30 patients who were diagnosed as primary or secondary infertility, polycystic ovary syndrome in the IVF center of Memorial Hospital, Department of Obstetrics and Gynecology were included. The amount of Anti Müllerian Hormone (AMH) in blood and granulosa cell diameter and cell core diameter were measured in 20 cells collected from each patient. In addition, degeneration scoring and BAX, ADAMTS-1, IL-10 expressions in granulosa cells were evaluated (p <0.01). It was thought that apoptosis induced by human GCs might be an indicator of egg quality. Moderate expression of ADAMTS-1 was thought to be related to failure of ovulation, deterioration of oocyte quality and decreased fertilization rate. This decrease in AMH levels may be associated with defects in granulosa cells. Therefore, significantly lower AMH secretion and increase in IL10 expression levels in healthy people can be explained by the increase of granulocyte cells.


Las células de la granulosa (GC) son componentes esenciales de los folículos y tienen un papel en la regulación del desarrollo de éste. El objetivo del estudio fue investigar ciertos componentes celulares involucrados en la proliferación, diferenciación y características funcionales de las células de la granulosa en el éxito de la fertilización de los ovocitos humanos durante la fertilización in vitro (FIV) a través de técnicas inmunohistoquímicas. En este estudio, se incluyeron 30 pacientes diagnosticados con infertilidad primaria o secundaria, síndrome de ovario poliquístico en el centro de FIV del Departamento de Obstetricia y Ginecología del Hospital Memorial. La cantidad de Hormona Anti Mülleriana (AMH) en la sangre, el diámetro de las células de la granulosa y el diámetro del núcleo celular se midieron en 20 células obtenidas de cada paciente. Además, se evaluó la puntuación de degeneración y las expresiones BAX, ADAMTS-1, IL-10 en células de granulosa (p <0,01). Se estimó que la apoptosis inducida por los GC humanos podría ser un indicador de la calidad del huevo. Se estimó que la expresión moderada de ADAMTS-1 estaba relacionada con el fracaso de la ovulación, el deterioro de la calidad de los ovocitos y la disminución de la tasa de fertilización. La disminución en los niveles de AMH puede estar asociada con defectos en las células de la granulosa. Por lo tanto, el aumento de las células de granulocitos puede explicar la disminución significativa de la secreción de AMH y el aumento de los niveles de expresión de IL10 en personas sanas.


Assuntos
Humanos , Feminino , Fertilização In Vitro/métodos , Interleucina-10/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína ADAMTS1/metabolismo , Células da Granulosa/metabolismo , Imuno-Histoquímica
7.
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
8.
Ecotoxicol Environ Saf ; 194: 110401, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32143102

RESUMO

Zearalenone (ZEA), a toxic substance produced by Fusarium fungi, accumulated in cereals grain and animal feed, causes injury to humans and animals. ZEA can induce obvious reproductive toxicity with the ovarian granulosa cells (GCs) as the main target. However, the study on exploring the protective compounds against ZEA-induced mouse primary ovarian GCs damage remains less. In the current study, the protective effect of 20 compounds derived from traditional Chinese medicines (TCMs) on the injury of mouse GCs caused by ZEA were evaluated using MTT assay and the cell morphology. Our results showed that chlorogenic acid (250, 500, and 1000 µg/mL) significantly suppress ZEA-induced GCs death. Western blot analysis suggested chlorogenic acid could rescue the up-regulated apoptosis of GCs induced by ZEA via attenuating the protein expression of cleaved caspase-3, the ratio of Bax/Bcl-2 and cleaved-PARP. Our results provide strong evidence that chlorogenic acid warrants further optimization for more potent and safer compounds for against the ZEA lead toxicity to humans and animals.


Assuntos
Apoptose/efeitos dos fármacos , Ácido Clorogênico/farmacologia , Células da Granulosa/efeitos dos fármacos , Zearalenona/toxicidade , Animais , Caspase 3/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Células da Granulosa/metabolismo , Células da Granulosa/patologia , Camundongos , Camundongos Endogâmicos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
9.
Nucleic Acids Res ; 48(8): 4480-4491, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32112110

RESUMO

The genetic etiology of premature ovarian insufficiency (POI) has been well established to date, however, the role of long noncoding RNAs (lncRNAs) in POI is largely unknown. In this study, we identified a down-expressed lncRNA HCP5 in granulosa cells (GCs) from biochemical POI (bPOI) patients, which impaired DNA damage repair and promoted apoptosis of GCs. Mechanistically, we discovered that HCP5 stabilized the interaction between YB1 and its partner ILF2, which could mediate YB1 transferring into the nucleus of GCs. HCP5 silencing affected the localization of YB1 into nucleus and reduced the binding of YB1 to the promoter of MSH5 gene, thereby diminishing MSH5 expression. Taken together, we identified that the decreased expression of HCP5 in bPOI contributed to dysfunctional GCs by regulating MSH5 transcription and DNA damage repair via the interaction with YB1, providing a novel epigenetic mechanism for POI pathogenesis.


Assuntos
Proteínas de Ciclo Celular/genética , Insuficiência Ovariana Primária/genética , RNA Longo não Codificante/metabolismo , Ativação Transcricional , Proteína 1 de Ligação a Y-Box/metabolismo , Adulto , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Regulação para Baixo , Epigênese Genética , Feminino , Células da Granulosa/metabolismo , Humanos , Insuficiência Ovariana Primária/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/fisiologia
10.
Nanotoxicology ; 14(5): 683-695, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32189538

RESUMO

The aim of this survey is to explore the possible effects of unsupported and supported copper nanoparticles (CuNPs) of different morphologies on basic ovarian cell functions. For this purpose, we have compared the activity of unsupported spherical, triangular, and hexagonal CuNPs, as well as of spherical CuNPs supported on titania, zeolite Y and activated charcoal (0, 1, 10, or 100 ng/mL) on cultured porcine ovarian granulosa cells. Cell viability, proliferation (accumulation of proliferating cell nuclear antigen, PCNA), apoptosis (accumulation of Bcl-2-associated X protein, bax) and release of steroid hormones progesterone, testosterone, and 17ß-estradiol have been analyzed by the Trypan blue test, quantitative immunocytochemistry, and ELISA, respectively. Cell viability decreased after treatment with hexagonal CuNPs, whilst all the other CuNPs increased it. Unsupported spherical and hexagonal CuNPs, and spherical CuNPs/titania reduced PCNA accumulation; in contrast, an increase was noted for unsupported triangular CuNPs and CuNPs/zeolite Y. Bax accumulation was not affected by hexagonal CuNPs, whereas CuNPs/zeolite Y promoted it and all the other CuNPs depleted it. The release of all steroid hormones was inhibited by CuNPs/titanium dioxide and stimulated by CuNPs/charcoal, whilst CuNPs/zeolite Y promoted the testosterone and 17ß-estradiol output, but not that of progesterone. These results demonstrate the direct, mainly stimulatory, impact of CuNPs on basic ovarian cell functions. The character of the CuNPs' action depends on their shape and support. Therefore, CuNPs with appropriate chemical modification could be potentially useful for the control of reproductive processes and treatment of reproductive disorders.


Assuntos
Proliferação de Células/efeitos dos fármacos , Cobre/química , Cobre/toxicidade , Hormônios Gonadais/metabolismo , Células da Granulosa/efeitos dos fármacos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Carvão Vegetal/química , Relação Dose-Resposta a Droga , Feminino , Células da Granulosa/metabolismo , Células da Granulosa/fisiologia , Ovário/metabolismo , Ovário/patologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Propriedades de Superfície , Suínos , Titânio/química , Zeolitas/química
11.
Arch Gynecol Obstet ; 301(4): 963-971, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32193602

RESUMO

PURPOSE: Circular RNAs (circRNAs) are widely expressed noncoding RNAs which play important roles in various processes. The present study aimed to explore the effect of maternal PCOS on the expression of circRNAs in fetus and assessed the potential role of circRNA in human ovarian granulosa cell proliferation. METHODS: Total RNA was extracted from the fetal side of placental tissues from maternal PCOS (n = 3) and healthy puerpera (n = 3) for circRNA microarray. Real-time reverse transcriptase quantitative PCR (RT-qPCR) was used to validate the microarray data in fetal side of placental tissues from puerpera with (n = 18) and without (n = 30) PCOS. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were applied to predict the functions and pathways of circ_0023942 host genes. The circRNA-miRNA-mRNA network was constructed through bioinformatics prediction. Circ_0023942 overexpression vector was transiently transfected into human ovarian granulosa cell lines KGN and COV434. Cell proliferation was detected by cell counting kit-8. The protein expression level was determined by western blot. RESULTS: Compared with healthy puerpera, 14 circRNAs were significantly upregulated and 101 circRNAs were significantly downregulated in the fetal side of placenta from maternal PCOS according to the microarray data. Six differentially expressed circRNAs were selected for validation by RT-qPCR, and the expression patterns of circ_0023942, circ_0002151, circ_0001274, and circ_0008514 were consistent with the microarray data. Circ_0023942 was chosen for further investigation. GO and KEGG analysis predicted that circ_0023942 participated in the regulation of developmental process and the MAPK signaling pathway. Seven miRNAs were predicted to be the targets of circ_0023942. Overexpression of circ_0023942 inhibited human ovarian granulosa cell proliferation and suppressed the expression of CDK-4. CONCLUSION: Maternal PCOS impairs circ_0023942 expression in fetus. Overexpression of circ_0023942 inhibits human ovarian granulosa cell proliferation possibly via regulating CDK-4.


Assuntos
Células da Granulosa/metabolismo , Síndrome do Ovário Policístico/genética , RNA Circular/metabolismo , Proliferação de Células , Feminino , Humanos , Gravidez , Transfecção
12.
J Endocrinol ; 245(2): 291-300, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32171180

RESUMO

Polycystic ovary syndrome (PCOS), one of the most common female endocrine disorder, is a prevalent cause of infertility. Hyperandrogenism is a key feature in PCOS and is correlated with increased expression of VEGF and cytokines in the ovaries. We have previously shown that pigment epithelium-derived factor (PEDF), an endogenous protein, presents potent anti-angiogenic and anti-inflammatory activities in the ovary and negates the effects of cytokines and VEGF. Additionally, PEDF plays a role in both pathophysiology and treatment of ovarian-hyperstimulation syndrome (OHSS), frequently seen in PCOS patients. We established hyperandrogenic-PCOS models, both in vivo, using mice exposed prenatally to dihydrotestosterone (DHT) and, in vitro, using human primary granulosa cells (hpGCs) and human granulosa cell line (KGN). In PCOS-induced mice, the mRNA levels of I l-6, V egf and Amh were higher than those of control; yet, treatment with rPEDF decreased these levels. Moreover, treating OHSS-induced PCOS-mice with rPEDF alleviated all OHSS symptoms. Stimulation of hpGCs with DHT resulted in downregulation of PEDF mRNA expression, concomitantly with a significant increase in IL-6 and IL-8 mRNAs expression. However, co-stimulation of DHT with rPEDF attenuated the increase in cytokines expression. The anti-inflammatory effect of PEDF was found to be mediated via PPARγ pathway. Our findings suggest that rPEDF treatment may normalize the ovarian angiogenic-inflammatory imbalance, induced by PCOS-associated hyperandrogenism. Moreover, the therapeutic potency of PEDF in preventing OHSS symptomes offers a rationale for using PEDF as novel physiological treatment for PCOS sequels.


Assuntos
Anti-Inflamatórios/metabolismo , Proteínas do Olho/metabolismo , Hiperandrogenismo/metabolismo , Fatores de Crescimento Neural/metabolismo , Síndrome do Ovário Policístico/metabolismo , Serpinas/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Di-Hidrotestosterona , Modelos Animais de Doenças , Feminino , Células da Granulosa/metabolismo , Humanos , Hiperandrogenismo/induzido quimicamente , Hiperandrogenismo/complicações , Camundongos , Ovário/metabolismo , Síndrome do Ovário Policístico/induzido quimicamente
13.
Ann Endocrinol (Paris) ; 81(1): 18-27, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32127169

RESUMO

BACKGROUND: We aimed to identify key genes and microRNAs (miRNAs) associated with the development of polycystic ovary syndrome (PCOS). METHODS: GSE84376 mRNA microarray data (15 PCOS granulosa cells and 13 control granulosa cells) and GSE34526 mRNA microarray data (7 PCOS granulosa cells and 3 control granulosa cells) were downloaded from the Gene Expression Omnibus (GEO) database. First, differentially expressed gene (DEG) analysis, gene set enrichment analysis (GSEA) for differentially expressed mRNAs, and protein-protein interaction (PPI) network analysis were conducted. Next, miRNA-target genes were analyzed and functions predicted, and a competing endogenous RNA (ceRNA) network was constructed. Finally, the relationship between miR-486-5p and PRELID2 was experimentally validated. RESULTS: Spleen tyrosine kinase (SYK), major histocompatibility complex, class II, DR alpha (HLA-DRA), and interleukin 10 (IL-10) were important nodes in the PPI network. Interestingly, HLA-DRA was significantly enriched in phagosomes mediated by Staphylococcus aureus infection, and in IL-10 enriched during S. aureus infection. One miRNA (miR-486-5p) and a single target gene (PRELID2) were obtained from the ceRNA network. Further experiments showed that miR-486-5p is upregulated and PRELID2 is downregulated in PCOS patient granulosa cells, and that miR-486-5p targets the PRELID2 3'UTR. Topological property analysis showed that hsa-miR-4687-5p downregulation and hsa-miR-4651 upregulation determined the levels of most mRNAs. Levels of the hsa-miR-4651 target gene were significantly enriched in the leukocyte transendothelial migration pathway. CONCLUSIONS: Our results suggest that HLA-DRA and IL-10 may contribute to PCOS progression via phagosome enriched by S. aureus infection, while miR-486-5p may be implicated in follicular development in PCOS by targeting PRELID2. Besides, miR-4651 may be involved in inflammation via leukocyte transendothelial migration, by regulating its target gene. These findings may indicate new directions and constitute a breakthrough in studying the pathophysiology of PCOS.


Assuntos
MicroRNAs/genética , Síndrome do Ovário Policístico/genética , RNA Mensageiro/genética , Adulto , Estudos de Casos e Controles , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Estudos de Associação Genética , Predisposição Genética para Doença , Células da Granulosa/metabolismo , Humanos , MicroRNAs/metabolismo , Análise em Microsséries , Síndrome do Ovário Policístico/patologia , RNA Mensageiro/metabolismo
14.
Mol Immunol ; 119: 154-158, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32035362

RESUMO

A genome-wide profiling of microRNA (miRNA) in endotoxin tolerant buffalo granulosa cells identified miR-326 amongst top-10 upregulated miRNAs. In this study, we have elucidated the role of miR-326 in granulosa cells in vitro. In-silico analysis revealed that miR-326 have binding site for 3'UTR of TLR-4 (Toll-like receptor 4). Transfection experiments showed that there was a significant inhibition of TLR-4 when miR-326 mimic was transfected to buffalo granulosa cells. Furthermore, when miR-326 transfected granulosa cells were exposed to LPS, followed by expression analysis of TLR-4 complex genes (TLR-4 and MyD88) and pro-inflammatory cytokines, we found a decreased expression of both TLR-4 complex and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß). We also found that the expression of anti-inflammatory (IL-10) gene was upregulated. To the best of our knowledge, this is the first study that showed the regulation of TLR-4 using miR-326. The present findings on regulation of TLR-4 are important and would help in understanding innate immunity regulation under different patho-physiology.


Assuntos
Células da Granulosa/metabolismo , MicroRNAs/metabolismo , Receptor 4 Toll-Like/metabolismo , Regiões 3' não Traduzidas , Animais , Búfalos , Células Cultivadas , Feminino , Receptor 4 Toll-Like/genética , Regulação para Cima
15.
DNA Cell Biol ; 39(4): 563-571, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32073892

RESUMO

Growth differentiation factor (GDF) 9 gene is involved in regulating reproductive traits in animals, but little is known about the promoter, single-nucleotide polymorphisms (SNPs), transcription factor binding sites, and regulating mechanism of GDF9 gene. In this study, the SNPs in the GDF9 promoter region were explored and their transcription mechanisms in regulating GDF9 expression were analyzed. Ear tissues of 267 Hu ewes were collected, and genomic DNA was extracted. GDF9 promoter region was amplified by PCRs, and identified SNPs genotyped by sequencing. SPSS16.0 software was used to analyze the association between genotypes and litter sizes. Flow cytometry assay was used to detect cell apoptosis, and dual-luciferase reporter assay was used to discover the promoter activity. A length of 1789 bp promoter region of GDF9 in Hu sheep was obtained by PCR amplification, and luciferase activity assay showed that there was a negative regulatory element in the region within -725 to -309 bp and a positive regulatory element in the region within -309 to +43 bp. Three complete linkage SNPs at -534A/G, -407T/G, and -332C/T were detected, resulting in three genotypes (namely, AA, AB, and BB). The association analysis indicated that the AA genotype ewes had larger litter size at average parity than those with the BB genotype. The -534A/G mutation created a novel binding site for the octamer transcription factor 1 (OCT1), and the Annexin V FITC/PI flow cytometry assay showed OCT1 promoted cell apoptosis in sheep ovarian granulosa cells. Overexpression of OCT1 considerably inhibited the luciferase activity of both genotypes and the inhibition effect of pGL3-BB was higher than that of pGL3-AA. Three complete linkage SNPs of the GDF9 gene regulate the litter size in Hu sheep probably via inhibition of the promoter activity by binding with OCT1 at -534 GG genotype and forming a complex between OCT1 and CCAAT/enhancer-binding protein (C/EBP).


Assuntos
Fator 9 de Diferenciação de Crescimento/genética , Tamanho da Ninhada de Vivíparos/genética , Fator 1 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas/genética , Animais , Apoptose/fisiologia , Sítios de Ligação/genética , Células COS , Linhagem Celular , Chlorocebus aethiops , Feminino , Células da Granulosa/metabolismo , Fator 1 de Transcrição de Octâmero/genética , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , Gravidez , Sequências Reguladoras de Ácido Nucleico/genética , Ovinos
16.
Cell ; 180(3): 585-600.e19, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32004457

RESUMO

Molecular mechanisms of ovarian aging and female age-related fertility decline remain unclear. We surveyed the single-cell transcriptomic landscape of ovaries from young and aged non-human primates (NHPs) and identified seven ovarian cell types with distinct gene-expression signatures, including oocyte and six types of ovarian somatic cells. In-depth dissection of gene-expression dynamics of oocytes revealed four subtypes at sequential and stepwise developmental stages. Further analysis of cell-type-specific aging-associated transcriptional changes uncovered the disturbance of antioxidant signaling specific to early-stage oocytes and granulosa cells, indicative of oxidative damage as a crucial factor in ovarian functional decline with age. Additionally, inactivated antioxidative pathways, increased reactive oxygen species, and apoptosis were observed in granulosa cells from aged women. This study provides a comprehensive understanding of the cell-type-specific mechanisms underlying primate ovarian aging at single-cell resolution, revealing new diagnostic biomarkers and potential therapeutic targets for age-related human ovarian disorders.


Assuntos
Envelhecimento/genética , Ovário/fisiologia , Análise de Célula Única/métodos , Transcriptoma , Idoso , Animais , Antioxidantes/metabolismo , Apoptose/fisiologia , Atlas como Assunto , Biomarcadores , Linhagem Celular Tumoral , Feminino , Células da Granulosa/metabolismo , Humanos , Macaca fascicularis , Oócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia
17.
Endocrinology ; 161(2)2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32020188

RESUMO

Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism, and we previously found that androgens activate endoplasmic reticulum (ER) stress in granulosa cells from patients with PCOS. In addition, recent studies demonstrated the accumulation of advanced glycation end products (AGEs) in granulosa cells from PCOS patients, which contribute to the pathology. Therefore, we hypothesized that androgens upregulate the receptor for AGEs (RAGE) expression in granulosa cells by activating ER stress, thereby increasing the accumulation of AGEs in these cells and contributing to the pathology. In the present study, we show that testosterone increases RAGE expression and AGE accumulation in cultured human granulosa-lutein cells (GLCs), and this is reduced by pretreatment with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor in clinical use. Knockdown of the transcription factor C/EBP homologous protein (CHOP), an unfolded protein response factor activated by ER stress, inhibits testosterone-induced RAGE expression and AGE accumulation. The expression of RAGE and the accumulation of AGEs are upregulated in granulosa cells from PCOS patients and dehydroepiandrosterone-induced PCOS mice. Administration of the RAGE inhibitor FPS-ZM1 or TUDCA to PCOS mice reduces RAGE expression and AGE accumulation in granulosa cells, improves their estrous cycle, and reduces the number of atretic antral follicles. In summary, our findings indicate that hyperandrogenism in PCOS increases the expression of RAGE and accumulation of AGEs in the ovary by activating ER stress, and that targeting the AGE-RAGE system, either by using a RAGE inhibitor or a clinically available ER stress inhibitor, may represent a novel approach to PCOS therapy.


Assuntos
Estresse do Retículo Endoplasmático , Produtos Finais de Glicação Avançada/metabolismo , Células da Granulosa/metabolismo , Hiperandrogenismo/metabolismo , Síndrome do Ovário Policístico/etiologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Animais , Benzamidas/uso terapêutico , Estudos de Casos e Controles , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Síndrome do Ovário Policístico/tratamento farmacológico , Síndrome do Ovário Policístico/metabolismo , Ácido Tauroquenodesoxicólico/uso terapêutico , Testosterona
18.
Mol Cell Biochem ; 465(1-2): 187-197, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31894528

RESUMO

Polycystic ovary syndrome (PCOS) is a hormonal disorder common among women of reproductive age. Although much is understood concerning the pathology of PCOS, further investigation into the influence of microribonucleic acids (miRNAs) on the proliferation of ovarian granulosa cells (GCs) is needed. This study investigated the role of specific miRNAs in ovarian dysfunction of PCOS and its effect on the proliferation of GCs. Initially, miRNA profiling was performed on the ovarian cortexes of 15 rats in which PCOS had been induced and 15 rats without PCOS (non-PCOS). This mechanical study was performed on ovarian GCs extracted from human chorionic gonadotrophin (hCG)-induced rats. Insulin was used to treat GCs to establish the PCOS cell model. Increased Equus caballus mir-9119 expression was observed and confirmed in the insulin-induced model of PCOS in GCs (GC-PCOS) as well as in the hCG-induced rats when compared to non-PCOS rats and cells. Observation and confirmation were carried out through both miRNA array and quantitative PCR. In contrast, downregulation of the nuclear factor kappa B (NFκB) p65 was observed in the PCOS cell model. Additionally, annexin V, FITC, and propidium iodide flow cytometry showed overexpression of miR-9119-induced apoptosis. In this study, we revealed that miR-9119 inhibition regulates p65 expression levels in insulin-treated GCs by binding to the 3'-untranslated of p65. Additionally, regulation of p65 expression was positively correlated with the expression of the double-stranded RNA endoribonuclease DICER. Moreover, RNA silencing/overexpression of p65 affected the functional role of miR-9119. In conclusion, GCs of PCOS, the expression of miR-9119, and targeted NFκB/p65-DICER axis are upregulated in order to maintain cell viability and prevent apoptosis, thereby promoting Anti-Müllerian hormone production in GCs. This study may provide a new understanding of the mechanism of GC dysfunction.


Assuntos
Regulação Enzimológica da Expressão Gênica , Células da Granulosa/metabolismo , MicroRNAs/metabolismo , Síndrome do Ovário Policístico/metabolismo , Ribonuclease III/biossíntese , Animais , Hormônio Antimülleriano/metabolismo , Sobrevivência Celular , Feminino , Células da Granulosa/patologia , Cavalos , Humanos , Síndrome do Ovário Policístico/induzido quimicamente , Síndrome do Ovário Policístico/patologia , Ratos , Fator de Transcrição RelA/metabolismo
19.
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
20.
J Steroid Biochem Mol Biol ; 199: 105608, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31996328

RESUMO

Ovarian granulosa cells, known to be endocrine cells, have well active TLR4-/NFKB signalling mediated innate immune capabilities. We have previously shown that endotoxin not only transiently regulates proinflammatory cytokines but cells become tolerant on repeated exposure to endotoxin and impaired granulosa cells functions, which includes downregulation of CYP19A1 gene. To understand further endotoxin tolerance and impaired granulosa cells function, genome-wide transcriptomic profiling in endotoxin tolerant buffalo granulosa cells (bGCs) identified miR-326 as upregulated amongst top 5 DE miRNAs [unpublished data] and qPCR validation confirmed its upregulation during endotoxin tolerance. In silico analyses showed that miR-326 targets CYP19A1 gene. Therefore, in the present study, we elucidated the role of miR-326 in buffalo granulosa cells (bGCs). We first validated its expression vis-à-vis CYP19A1 gene expression in bGCs, both in vivo and in vitro. Results showed an inverse relationship between miR-326 and CYP19A1 expression. Similarly, transcription factors, known to be involved in CYP19A1 gene regulation, CREB and C/EBP-ß expression was also found to be decreased in granulosa cells mimicking pre-ovulatory follicular stage. Further, miR-326 mimic was transfected to bGCs in culture and expression of CYP19A1 and CREB & C/EBP-ß and genes encoding other enzymes of steroidogenesis pathway were also analyzed. The present study results showed that miR-326 significantly inhibits the expression of CYP19A1 gene while expression of transcription factors CREB and C/EBP-ß was found to be upregulated. The expression of STAR and CYP11A1 was found to be unaffected. To elucidate the molecular mechanism of miR-326 mediated downregulation of CYP19A1, binding analyses of RNA polymerase II and CEBP-ß to CYP19A1 gene promoter II was analyzed. The result also showed decreased binding of RNA polymerase II with increased binding of CEBP-ß to CYP19A1 gene promoter II in bGCs, transfected with miR-326 as compared to control. In summary, our results suggest that miR-326 upregulate CREB and CREB may activate C/EBP-ß and later inhibited the transcription of CYP19A1 and decreased estradiol-17b production. The miR-326 mediated down-regulation of the CYP19A1 gene involving CREB-C/EBP-ß can be exploited in developing strategies to attenuate endotoxin-mediated tolerance induced impaired granulosa cells function to ensure proper fertility in females.


Assuntos
Aromatase/genética , Estradiol/genética , Estrogênios/genética , MicroRNAs/genética , Animais , Búfalos/genética , Búfalos/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Bovinos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Estradiol/biossíntese , Estrogênios/biossíntese , Feminino , Regulação da Expressão Gênica/genética , Células da Granulosa/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais
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