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1.
Sci Rep ; 11(1): 16287, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381107

RESUMO

Hyperandrogenic women with PCOS show disrupted decidualization (DE) and placentation. Dihydrotestosterone (DHT) is reported to enhance DE in non-PCOS endometrial stromal cells (eSCCtrl); however, this has not been assessed in PCOS cells (eSCPCOS). Therefore, we studied the transcriptome profile of non-decidualized (non-DE) and DE eSCs from women with PCOS and Ctrl in response to short-term estradiol (E2) and/or progesterone (P4) exposure with/without (±) DHT. The non-DE eSCs were subjected to E2 ± DHT treatment, whereas the DE (0.5 mM 8-Br-cAMP, 96 h) eSCs were post-treated with E2 and P4 ± DHT, and RNA-sequenced. Validation was performed by immunofluorescence and immunohistochemistry. The results showed that, regardless of treatment, the PCOS and Ctrl samples clustered separately. The comparison of DE vs. non-DE eSCPCOS without DHT revealed PCOS-specific differentially expressed genes (DEGs) involved in mitochondrial function and progesterone signaling. When further adding DHT, we detected altered responses for lysophosphatidic acid (LPA), inflammation, and androgen signaling. Overall, the results highlight an underlying defect in decidualized eSCPCOS, present with or without DHT exposure, and possibly linked to the altered pregnancy outcomes. We also report novel factors which elucidate the mechanisms of endometrial dysfunction in PCOS.


Assuntos
Androgênios/metabolismo , Endométrio/metabolismo , Síndrome do Ovário Policístico/metabolismo , Células Estromais/metabolismo , Adulto , Di-Hidrotestosterona/metabolismo , Estradiol/metabolismo , Feminino , Humanos , Gravidez , Progesterona/metabolismo , Transdução de Sinais/fisiologia
2.
Reprod Biol Endocrinol ; 19(1): 115, 2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34289864

RESUMO

BACKGROUND: The period of time when the embryo and the endometrium undergo significant morphological alterations to facilitate a successful implantation-known as "window of implantation"-is a critical moment in human reproduction. Embryo and the endometrium communicate extensively during this period, and lipid bilayer bound nanoscale extracellular vesicles (EVs) are purported to be integral to this communication. METHODS: To investigate the nature of the EV-mediated embryo-maternal communication, we have supplemented trophoblast analogue spheroid (JAr) derived EVs to an endometrial analogue (RL 95-2) cell layer and characterized the transcriptomic alterations using RNA sequencing. EVs derived from non-trophoblast cells (HEK293) were used as a negative control. The cargo of the EVs were also investigated through mRNA and miRNA sequencing. RESULTS: Trophoblast spheroid derived EVs induced drastic transcriptomic alterations in the endometrial cells while the non-trophoblast cell derived EVs failed to induce such changes demonstrating functional specificity in terms of EV origin. Through gene set enrichment analysis (GSEA), we found that the response in endometrial cells was focused on extracellular matrix remodelling and G protein-coupled receptors' signalling, both of which are of known functional relevance to endometrial receptivity. Approximately 9% of genes downregulated in endometrial cells were high-confidence predicted targets of miRNAs detected exclusively in trophoblast analogue-derived EVs, suggesting that only a small proportion of reduced expression in endometrial cells can be attributed directly to gene silencing by miRNAs carried as cargo in the EVs. CONCLUSION: Our study reveals that trophoblast derived EVs have the ability to modify the endometrial gene expression, potentially with functional importance for embryo-maternal communication during implantation, although the exact underlying signalling mechanisms remain to be elucidated.


Assuntos
Implantação do Embrião/fisiologia , Embrião de Mamíferos/fisiologia , Endométrio/fisiologia , Circulação Placentária/fisiologia , Transcriptoma/fisiologia , Trofoblastos/fisiologia , Linhagem Celular Tumoral , Embrião de Mamíferos/citologia , Endométrio/citologia , Feminino , Células HEK293 , Humanos , Gravidez
3.
J Mol Med (Berl) ; 99(5): 685-697, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33512581

RESUMO

Embryo-derived extracellular vesicles (EVs) may play a role in mediating the embryo-maternal dialogue at the oviduct, potentially carrying signals reflecting embryo quality. We investigated the effects of bovine embryo-derived EVs on the gene expression of bovine oviductal epithelial cells (BOECs), and whether these effects are dependent on embryo quality. Presumptive zygotes were cultured individually in vitro in culture medium droplets until day 8 while their development was assessed at day 2, 5 and 8. Conditioned medium samples were collected at day 5 and pooled based on embryo development (good quality embryo media and degenerating embryo media). EVs were isolated from conditioned media by size exclusion chromatography and supplemented to primary BOEC monolayer cultures to evaluate the effects of embryo-derived EVs on gene expression profile of BOEC. Gene expression was quantified by RNA-seq and RT-qPCR. A total of 7 upregulated and 18 downregulated genes were detected in the BOECs supplemented with good quality embryo-derived EV compared to the control. The upregulated genes included interferon-τ-induced genes, such as OAS1Y, MX1 and ISG15, which have previously been reported as upregulated in the oviductal epithelial cells in the presence of embryos. Of the upregulated genes, OAS1Y and MX1 were validated with RT-qPCR. In contrast, only one differentially expressed gene was detected in BOECs in response to degenerating embryo-derived EVs, suggesting that oviductal responses are dependent on embryo quality. Our results support the hypothesis that embryo-derived EVs are involved in embryo-maternal communication at the oviduct and the oviductal response is dependant on the embryo quality. KEY MESSAGES: • Extracellular vesicles (EVs) released by individually cultured pre-implantation bovine embryos can alter the gene expression of primary oviductal epithelial cells. • The oviductal response, in terms of gene expression, to the bovine embryo-derived EVs varied depending on the embryo quality. • In vivo, the oviduct may have the ability to sense the quality of the pre-implantation embryos. • The observed effect of embryo-derived EVs on oviductal epithelial cells could serve as a non-invasive method of evaluating the embryo quality.


Assuntos
Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Células Epiteliais/metabolismo , Vesículas Extracelulares/metabolismo , Tubas Uterinas/metabolismo , Transcriptoma/genética , Animais , Bovinos , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Regulação para Baixo/genética , Tubas Uterinas/citologia , Feminino , Fertilização in vitro/métodos , Gravidez , Regulação para Cima/genética , Zigoto/metabolismo
4.
J Cell Commun Signal ; 14(4): 439-451, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32880838

RESUMO

The capability of spermatozoa to directly influence maternal gene expression is already established. Indeed, some of the changes induced by spermatozoa may have a direct functional importance in the pre-conceptional period. Although the mechanisms underlying these sperm-maternal interactions are not well characterized, it is possible that they could involve ligands that are released from the spermatozoa. This study therefore aimed to test whether physical contact between bovine spermatozoa and bovine oviductal epithelial cells (BOECs) is a prerequisite for spermatozoa-induced gene expression changes. We used two co-culture models: a contact co-culture model in which spermatozoa interact directly with BOECs, and a non-contact co-culture model in which an insert with the pore size of 0.4 µm was placed between spermatozoa and BOECs. Messenger RNA sequencing analysis of BOECs by RNA-seq revealed ten differentially expressed genes in contact system and 108 differentially expressed genes in the non-contact system after 10 h of co-culture. Retinol metabolism pathway and ovarian steroidogenesis pathway were significantly enriched in the non-contact co-culture system. Q-PCR analysis revealed that transcriptional responses can be rapid, with increased expression of four genes (DHRS3, CYP1B1, PTGS2, and ATF3) detectable within just 90 min of co-incubation, but with expression levels highly dependent on the type of co-culture system. The findings from our study demonstrate that direct contact with spermatozoa is not necessary to induce changes in gene expression of oviductal epithelial cells, suggesting that spermatozoa may be able to signal to maternal tissues in advance of their arrival.

5.
Int J Mol Sci ; 21(15)2020 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-32731579

RESUMO

While follicular fluid (FF) is well known to provide an optimal environment for oogenesis, its functional roles following its release into the oviduct during ovulation are currently elusive. We hypothesized that FF and FF-derived extracellular vesicles (EVs) may be conveyors of signals capable of inducing functionally-relevant transcriptional responses in oviductal cells. The aim of this study was, therefore, to evaluate the effect of FF and FF-derived EVs on the transcriptome of primary bovine oviductal epithelial cells (BOECs). We examined the gene expression of BOECs in three conditions: BOECs cultured with FF, FF-derived EVs, and without supplementations. For each condition, cells were cultured for 6 and 24 h. RNA sequencing results revealed that FF had a stronger effect on BOECs gene expression compared to EVs. We detected 488 and 1998 differentially expressed genes (DEGs) with FF treatment in 6 and 24 h, respectively, whereas only 41 DEGs were detected at 6 h following EV treatment. Pathway analysis of the FF-induced DEGs showed that several pathways were highly enriched, notably oxidative phosphorylation, thermogenesis, arachidonic acid metabolism, and steroid hormone biosynthesis. Some of these pathways have a role in sperm survival, fertilization, and early embryo development. In conclusion, the findings of our study demonstrate for the first time that bovine FF and FF-derived EVs can induce changes in the gene expression of the bovine oviductal cells which, although observed in vitro, may be reflective of in vivo responses which may contribute to a favorable periconceptional microenvironment for sperm survival, fertilization, and early embryo development.


Assuntos
Células Epiteliais/metabolismo , Vesículas Extracelulares/metabolismo , Tubas Uterinas/metabolismo , Líquido Folicular/metabolismo , Transcriptoma , Animais , Bovinos , Feminino
6.
Arch Toxicol ; 94(10): 3359-3379, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32638039

RESUMO

Modern living challenges female reproductive health. We are witnessing a rise in reproductive disorders and drop in birth rates across the world. The reasons for these manifestations are multifaceted and most likely include continuous exposure to an ever-increasing number of chemicals. The cause-effect relationships between chemical exposure and female reproductive disorders, however, have proven problematic to determine. This has made it difficult to assess the risks chemical exposures pose to a woman's reproductive development and function. To address this challenge, this review uses the adverse outcome pathway (AOP) concept to summarize current knowledge about how chemical exposure can affect female reproductive health. We have a special focus on effects on the ovaries, since they are essential for lifelong reproductive health in women, being the source of both oocytes and several reproductive hormones, including sex steroids. The AOP framework is widely accepted as a new tool for toxicological safety assessment that enables better use of mechanistic knowledge for regulatory purposes. AOPs equip assessors and regulators with a pragmatic network of linear cause-effect relationships, enabling the use of a wider range of test method data in chemical risk assessment and regulation. Based on current knowledge, we propose ten putative AOPs relevant for female reproductive disorders that can be further elaborated and potentially be included in the AOPwiki. This effort is an important step towards better safeguarding the reproductive health of all girls and women.


Assuntos
Rotas de Resultados Adversos , Segurança Química , Exposição Materna , Ovário/efeitos dos fármacos , Saúde Reprodutiva , Animais , Doenças do Sistema Endócrino/induzido quimicamente , Feminino , Humanos , Camundongos , Doenças Ovarianas/induzido quimicamente , Ovário/fisiopatologia , Gravidez , Medição de Risco , Testes de Toxicidade
7.
Int J Mol Sci ; 21(9)2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32370092

RESUMO

Currently available test methods are not well-suited for the identification of chemicals that disturb hormonal processes involved in female reproductive development and function. This renders women's reproductive health at increasing risk globally, which, coupled with increasing incidence rates of reproductive disorders, is of great concern. A woman's reproductive health is largely established during embryonic and fetal development and subsequently matures during puberty. The endocrine system influences development, maturation, and function of the female reproductive system, thereby making appropriate hormone levels imperative for correct functioning of reproductive processes. It is concerning that the effects of human-made chemicals on the endocrine system and female reproductive health are poorly addressed in regulatory chemical safety assessment, partly because adequate test methods are lacking. Our EU-funded project FREIA aims to address this need by increasing understanding of how endocrine disrupting chemicals (EDCs) can impact female reproductive health. We will use this information to provide better test methods that enable fit-for-purpose chemical regulation and then share our knowledge, promote a sustainable society, and improve the reproductive health of women globally.


Assuntos
Disruptores Endócrinos/farmacologia , Reprodução/efeitos dos fármacos , Saúde Reprodutiva , Animais , Sistema Endócrino/efeitos dos fármacos , Exposição Ambiental , Poluentes Ambientais/efeitos adversos , Feminino , Humanos , Puberdade/efeitos dos fármacos , Medição de Risco , Fatores de Risco
8.
Reprod Biomed Online ; 39(5): 725-736, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31519421

RESUMO

RESEARCH QUESTION: How does mucin MUC20 expression change during the menstrual cycle in different cell types of human endometrium? DESIGN: Study involved examination of MUC20 expression in two previously published RNA-seq datasets in whole endometrial tissue (n = 10), sorted endometrial epithelial (n = 44) or stromal (n = 42) cell samples. RNA-Seq results were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in whole tissue (n = 10), sorted epithelial (n = 17) and stromal (n = 17) cell samples. MUC20 protein localization and expression were analysed in human endometrium by immunohistochemical analysis of intact endometrial tissue (n = 6) and also Western blot of cultured stromal and epithelial cells (n = 2). RESULTS: MUC20 is differentially expressed in the endometrium between the pre-receptive and receptive phases. We show that MUC20 is predominantly expressed by epithelial cells of the receptive endometrium, both at the mRNA (RNA-Seq, P = 0.005; qRT-PCR, P = 0.039) and protein levels (Western blot; immunohistochemistry, P = 0.029). CONCLUSION: Our results indicate MUC20 as a novel marker of mid-secretory endometrial biology. We propose a model of MUC20 function in the hepatocyte growth factor (HGF)-activated mesenchymal-epithelial transition (MET) receptor signalling specifically in the receptive phase. Further investigations should reveal the precise function of MUC20 in human endometrium and the possible connection between MUC20 and HGF-activated MET receptor signalling. MUC20 could potentially be included in the list of endometrial receptivity markers after further clinical validation.


Assuntos
Endométrio/metabolismo , Regulação da Expressão Gênica , Ciclo Menstrual/metabolismo , Mucinas/metabolismo , Adulto , Biópsia , Índice de Massa Corporal , Citoplasma/metabolismo , Implantação do Embrião , Células Epiteliais/metabolismo , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Imuno-Histoquímica , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA-Seq
9.
Gene ; 533(1): 173-9, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24103479

RESUMO

Mesenchymal stem cells (MSCs) possess a multi-lineage differentiation capacity that makes them important players in the field of regenerative medicine. MSC populations derived from different tissues or donors have been shown to exhibit variable gene expression patterns. Further, it is widely acknowledged that MSC isolates are heterogeneous mixtures of cells at different developmental stages. However, the heterogeneity of expression of lineage regulators has not been linked to differentiation potential of different MSC populations towards mesenchymal lineages. Here, we analyzed variation of expression of differentiation markers across whole population and between single differentiating cells of multipotent stromal cell populations derived from adipose tissue (AdMSCs) and skin (FBs) of seven donors. The results of the analyses show that all cell populations exhibit similar differentiation potential towards adipocyte, osteoblast and chondrocyte lineages despite tissue type- and donor-specific variations of expression of differentiation-associated genes. Further, we detected variable expression of lineage regulators in individual differentiating cells. Together, our data indicate that single cells of stromal cell populations could use distinct molecular mechanisms to reach a common cell fate.


Assuntos
Diferenciação Celular , Linhagem da Célula , Células-Tronco Mesenquimais/citologia , Células Cultivadas , Humanos , Reação em Cadeia da Polimerase em Tempo Real
10.
PLoS One ; 7(6): e38833, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22723894

RESUMO

BACKGROUND: Tissue regeneration and recovery in the adult body depends on self-renewal and differentiation of stem and progenitor cells. Mesenchymal stem cells (MSCs) that have the ability to differentiate into various cell types, have been isolated from the stromal fraction of virtually all tissues. However, little is known about the true identity of MSCs. MSC populations exhibit great tissue-, location- and patient-specific variation in gene expression and are heterogeneous in cell composition. METHODOLOGY/PRINCIPAL FINDINGS: Our aim was to analyze the dynamics of differentiation of two closely related stromal cell types, adipose tissue-derived MSCs (AdMSCs) and dermal fibroblasts (FBs) along adipogenic, osteogenic and chondrogenic lineages using multiplex RNA-seq technology. We found that undifferentiated donor-matched AdMSCs and FBs are distinct populations that stay different upon differentiation into adipocytes, osteoblasts and chondrocytes. The changes in lineage-specific gene expression occur early in differentiation and persist over time in both AdMSCs and FBs. Further, AdMSCs and FBs exhibit similar dynamics of adipogenic and osteogenic differentiation but different dynamics of chondrogenic differentiation. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that stromal stem cells including AdMSCs and dermal FBs exploit different molecular mechanisms of differentiation to reach a common cell fate. The early mechanisms of differentiation are lineage-specific and are similar for adipogenic and osteogenic differentiation but are distinct for chondrogenic differentiation between AdMSCs and FBs.


Assuntos
Tecido Adiposo/citologia , Diferenciação Celular/genética , Derme/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Transcriptoma , Adipogenia/genética , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Especificidade de Órgãos/genética , Osteogênese/genética , Análise de Sequência de RNA
11.
Int J Oncol ; 41(1): 299-309, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22576689

RESUMO

The Wnt/ß-catenin pathway regulates key cellular processes such as differentiation, proliferation, apoptosis; and its activation promotes development of several cancer types. Expression of CD43 (leukosialin), the predominant leukocyte transmembrane sialoglycoprotein, has been detected in many tumors of non-hematopoietic origin. CD43 participates in cell adhesion and regulates intracellular signal transduction pathways involved in cell proliferation and survival. The cytoplasmic domain of CD43 has been reported to translocate to the nucleus, interact with ß-catenin and affect its target gene expression, but the impact of this action on cell fate is still unknown. We demonstrate, here, by colony formation assay and siRNA-mediated gene silencing that CD43 and ß-catenin co-operate in promoting cell growth. Moreover, in cells with down-regulated ß-catenin expression the activation of p53 in response to CD43 overexpression is significantly impaired. In addition, the presence of both CD43 and ß-catenin is required for the TCF/LEF-mediated transcription. Presumably, the full-length CD43 participates in this transcriptional regulation. We show that the mature CD43 localizes to the nucleus, where it binds chromatin, co-localizes and co-immunoprecipitates with ß-catenin, and enhances the reporter gene expression regulated by ß-catenin. These observations provide clear evidence linking CD43 to the Wnt/APC/ß-catenin signaling pathway and supporting our hypothesis according to which CD43 plays a role in tumor development.


Assuntos
Proliferação de Células , Leucossialina/metabolismo , beta Catenina/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Cromatina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Reporter , Humanos , Leucossialina/genética , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Regiões Promotoras Genéticas , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-mdm2/genética , Interferência de RNA , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Via de Sinalização Wnt , beta Catenina/genética
12.
Stem Cells Dev ; 20(8): 1327-36, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21142453

RESUMO

Human dermal fibroblasts (FBs) express mesenchymal stem cell (MSC)-specific cell surface markers and differentiate into several cell types under appropriate conditions. Molecular mechanisms controlling the early stages of differentiation of dermal FBs and MSCs isolated from different sources have not been well studied. Here, we have analyzed the cell type-specific changes of adipose tissue-derived mesenchymal stem cells (AdMSCs) and dermal FBs in the process of differentiation into adipocytes and osteoblasts. Analysis of gene expression in the course of adipogenic differentiation of AdMSCs and FBs isolated from the same individuals revealed a time lag in the induction of adipogenesis-related genes in FBs compared with AdMSCs, a phenomenon not previously described. Further, preliminary evidence suggests that delayed adipogenesis of FBs is related to the delayed induction of preadipocyte transcription factor ZNF423 in FBs. These findings clearly show that AdMSCs and FBs have similar developmental potential but different molecular control mechanisms of initial stages of adipogenic differentiation.


Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Diferenciação Celular , Fibroblastos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Adipócitos/citologia , Adipogenia/genética , Tecido Adiposo/citologia , Células da Medula Óssea/citologia , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteoblastos/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/citologia
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