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1.
Cells ; 10(7)2021 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206743

RESUMO

UNC-45A (Protein unc-45 homolog A) is a cytoskeletal-associated protein with a dual and non-mutually exclusive role as a regulator of the actomyosin system and a Microtubule (MT)-destabilizing protein, which is overexpressed in human cancers including in ovarian cancer patients resistant to the MT-stabilizing drug paclitaxel. Mapping of UNC-45A in the mouse upper genital tract and central nervous system reveals its enrichment not only in highly proliferating and prone to remodeling cells, but also in microtubule-rich areas, of the ovaries and the nervous system, respectively. In both apparatuses, UNC-45A is also abundantly expressed in the ciliated epithelium. As regulators of actomyosin contractility and MT stability are essential for the physiopathology of the female reproductive tract and of neuronal development, our findings suggest that UNC-45A may have a role in ovarian cancer initiation and development as well as in neurodegeneration.


Assuntos
Genitália/citologia , Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Sistema Nervoso/metabolismo , Animais , Proliferação de Células , Cílios/metabolismo , Tubas Uterinas/metabolismo , Feminino , Camundongos Endogâmicos C57BL , Ovário/metabolismo , Medula Espinal/metabolismo
2.
Sci Rep ; 11(1): 14334, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253763

RESUMO

Sustained adrenergic stimulation by norepinephrine (NE) contributes to ovarian carcinoma metastasis and impairment of chemotherapy response. Although the effect of sustained NE stimulation in cancer progression is well established, less is known about its role in cancer initiation. To determine the extent to which stress hormones influence ovarian cancer initiation, we conducted a long-term (> 3 months; > 40 population doublings) experiment in which normal immortalized fallopian tube secretory (iFTSEC283) and ovarian surface epithelial (iOSE11) cell lines and their isogenic pairs containing a p53 mutation (iFTSEC283p53R175H; iOSE11p53R175H), were continuously exposed to NE (100 nM, 1 µM, 10 µM). Fallopian tube cells displayed a p53-independent increase in proliferation and colony-forming ability in response to NE, while ovarian surface epithelial cells displayed a p53-independent decrease in both assays. Fallopian tube cells with mutant p53 showed a mild loss of chromosomes and TP53 status was also a defining factor in transcriptional response of fallopian tube cells to long-term NE treatment.


Assuntos
Tubas Uterinas/efeitos dos fármacos , Tubas Uterinas/metabolismo , Norepinefrina/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos
3.
Sci Rep ; 11(1): 12041, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103548

RESUMO

Recent evidence supports involvement of the acute phase protein haptoglobin in numerous events during mammalian reproduction. The present study represents an in-depth investigation of haptoglobin expression and secretion in the porcine oviduct and uterus, and assesses its effect on porcine in vitro embryo production. A systematic study was made of sows in different oestrous stages: late follicular, early luteal and late luteal stages. Relative haptoglobin mRNA abundance was quantified by RT-qPCR. In addition, expression of the protein was analysed by immunohistochemistry and the results were complemented by Western-blot and proteomic analyses of the oviductal and uterine fluids. In vitro porcine fertilization and embryo culture were carried out in the presence of haptoglobin. The results indicate that haptoglobin mRNA expression in the porcine oviduct and uterus is most abundant during the late luteal stage of the oestrous cycle. By means of Western blot and proteomic analyses haptoglobin presence was demonstrated in the oviduct epithelium and in the oviductal and uterine fluids in different stages of the oestrous cycle. The addition of haptoglobin during gamete co-incubation had no effect on sperm penetration, monospermy or efficiency rates; however, compared with the control group, blastocyst development was significantly improved when haptoglobin was present (haptoglobin: 64.50% vs. control: 37.83%; p < 0.05). In conclusion, the presence of haptoglobin in the oviduct and uterus of sows at different stages of the oestrous cycle suggests that it plays an important role in the reproduction process. The addition of haptoglobin during in vitro embryo production improved the blastocyst rates.


Assuntos
Estro , Haptoglobinas/química , Suínos/fisiologia , Animais , Blastocisto/química , Desenvolvimento Embrionário , Endométrio/metabolismo , Ciclo Estral/genética , Tubas Uterinas/metabolismo , Feminino , Fertilização In Vitro , Haptoglobinas/metabolismo , Técnicas In Vitro , Fase Luteal , Oviductos/metabolismo , Proteômica/métodos , RNA Mensageiro/metabolismo , Útero/metabolismo
4.
Cells ; 10(5)2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069403

RESUMO

Autocrine/paracrine factors generated in response to 17ß-estradiol (E2) within the fallopian tube (FT) facilitate fertilization and early embryo development for implantation. Since cyclic AMP (cAMP) plays a key role in reproduction, regulation of its synthesis by E2 may be of biological/pathophysiological relevance. Herein, we investigated whether cAMP production in FT cells (FTCs) is regulated by E2 and environmental estrogens (EE's; xenoestrogens and phytoestrogens). Under basal conditions, low levels of extracellular cAMP were detectable in bovine FTCs (epithelial cells and fibroblasts; 1:1 ratio). Treatment of FTCs with forskolin (AC; adenylyl cyclase activator), isoproterenol (ß-adrenoceptor agonist) and IBMX (phosphodiesterase (PDE) inhibitor) dramatically (>10 fold) increased cAMP; whereas LRE1 (sAC; soluble AC inhibitor) and 2',5'-dideoxyadenosine (DDA; transmembrane AC (tmAC)) inhibitor decreased cAMP. Comparable changes in basal and stimulated intracellular cAMP were also observed. Ro-20-1724 (PDE-IV inhibitor), but not milrinone (PDE-III inhibitor) nor mmIBMX (PDE-I inhibitor), augmented forskolin-stimulated cAMP levels, suggesting that PDE-IV dominates in FTCs. E2 increased cAMP levels and CREB phosphorylation in FTCs, and these effects were mimicked by EE's (genistein, 4-hydroxy-2',4',6'-trichlorobiphenyl, 4-hydroxy-2',4',6'-dichlorobiphenyl). Moreover, the effects of E2 and EE were blocked by the tmAC inhibitor DDA, but not by the ERα/ß antagonist ICI182780. Moreover, BAPTA-AM (intracellular-Ca2+ chelator) abrogated the effects of E2, but not genistein, on cAMP suggesting differential involvement of Ca2+. Treatment with non-permeable E2-BSA induced cAMP levels and CREB-phosphorylation; moreover, the stimulatory effects of E2 and EEs on cAMP were blocked by G15, a G protein-coupled estrogen receptor (GPER) antagonist. E2 and IBMX induced cAMP formation was inhibited by LRE1 and DDA suggesting involvement of both tmAC and sAC. Our results provide the first evidence that in FTCs, E2 and EE's stimulate cAMP synthesis via GPER. Exposure of the FT to EE's and PDE inhibitors may result in abnormal non-cyclic induction of cAMP levels which may induce deleterious effects on reproduction.


Assuntos
AMP Cíclico/metabolismo , Disruptores Endócrinos/farmacologia , Células Epiteliais/efeitos dos fármacos , Estrogênios/farmacologia , Tubas Uterinas/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Bovinos , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Epiteliais/metabolismo , Estradiol/farmacologia , Tubas Uterinas/citologia , Tubas Uterinas/metabolismo , Feminino , Fibroblastos/metabolismo , Genisteína/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Fosforilação , Bifenilos Policlorados/farmacologia , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo
5.
Cells ; 10(6)2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073739

RESUMO

The functions of the female reproductive tract not only encompass sperm migration, storage, and fertilization, but also support the transport and development of the fertilized egg through to the birth of offspring. Further, because the tract is open to the external environment, it must also provide protection against invasive pathogens. In biophysics, sperm are considered "pusher microswimmers", because they are propelled by pushing fluid behind them. This type of swimming by motile microorganisms promotes the tendency to swim along walls and upstream in gentle fluid flows. Thus, the architecture of the walls of the female tract, and the gentle flows created by cilia, can guide sperm migration. The viscoelasticity of the fluids in the tract, such as mucus secretions, also promotes the cooperative swimming of sperm that can improve fertilization success; at the same time, the mucus can also impede the invasion of pathogens. This review is focused on how the mammalian female reproductive tract and sperm interact physically to facilitate the movement of sperm to the site of fertilization. Knowledge of female/sperm interactions can not only explain how the female tract can physically guide sperm to the fertilization site, but can also be applied for the improvement of in vitro fertilization devices.


Assuntos
Tubas Uterinas/metabolismo , Fertilização/fisiologia , Motilidade Espermática/fisiologia , Espermatozoides/citologia , Animais , Feminino , Genitália Feminina/metabolismo , Humanos , Masculino
6.
Sci Rep ; 11(1): 9294, 2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33927274

RESUMO

Transforming growth factor ß (TGFß) signaling plays critical roles in reproductive development and function. TGFß ligands signal through the TGFß receptor type 2 (TGFBR2)/TGFBR1 complex. As TGFBR2 and TGFBR1 form a signaling complex upon ligand stimulation, they are expected to be equally important for propagating TGFß signaling that elicits cellular responses. However, several genetic studies challenge this concept and indicate that disruption of TGFBR2 or TGFBR1 may lead to contrasting phenotypic outcomes. We have shown that conditional deletion of Tgfbr1 using anti-Mullerian hormone receptor type 2 (Amhr2)-Cre causes oviductal and myometrial defects. To determine the functional requirement of TGFBR2 in the female reproductive tract and the potential phenotypic divergence/similarity resulting from conditional ablation of either receptor, we generated mice harboring Tgfbr2 deletion using the same Cre driver that was previously employed to target Tgfbr1. Herein, we found that conditional deletion of Tgfbr2 led to a similar phenotype to that of Tgfbr1 deletion in the female reproductive tract. Furthermore, genetic removal of Tgfbr1 in the Tgfbr2-deleted uterus had minimal impact on the phenotype of Tgfbr2 conditional knockout mice. In summary, our results reveal the functional similarity between TGFBR2 and TGFBR1 in maintaining the structural integrity of the female reproductive tract.


Assuntos
Genitália Feminina/anormalidades , Genitália Feminina/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Animais , Endométrio/anormalidades , Endométrio/metabolismo , Endométrio/patologia , Tubas Uterinas/anormalidades , Tubas Uterinas/metabolismo , Tubas Uterinas/patologia , Feminino , Técnicas de Inativação de Genes , Genitália Feminina/patologia , Hiperplasia , Camundongos , Camundongos Endogâmicos C57BL , Miométrio/anormalidades , Miométrio/metabolismo , Miométrio/patologia , Fenótipo , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
7.
Cell Prolif ; 54(5): e13029, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33768671

RESUMO

High-grade serous carcinoma (HGSC) is the most common and malignant histological type of epithelial ovarian cancer, the origin of which remains controversial. Currently, the secretory epithelial cells of the fallopian tube are regarded as the main origin and the ovarian surface epithelial cells as a minor origin. In tubal epithelium, these cells acquire TP53 mutations and expand to a morphologically normal 'p53 signature' lesion, transform to serous tubal intraepithelial carcinoma and metastasize to the ovaries and peritoneum where they develop into HGSC. This shifting paradigm of the main cell of origin has revolutionarily changed the focus of HGSC research. Various cell lines have been derived from the two cellular origins by acquiring immortalization via overexpression of hTERT plus disruption of TP53 and the CDK4/RB pathway. Malignant transformation was achieved by adding canonical driver mutations (such as gain of CCNE1) revealed by The Cancer Genome Atlas or by noncanonical gain of YAP and miR181a. Alternatively, because of the extreme chromosomal instability, spontaneous transformation can be achieved by long passage of murine immortalized cells, whereas in humans, it requires ovulatory follicular fluid, containing regenerating growth factors to facilitate spontaneous transformation. These artificially and spontaneously transformed cell systems in both humans and mice have been widely used to discover carcinogens, oncogenic pathways and malignant behaviours in the development of HGSC. Here, we review the origin, aetiology and carcinogenic mechanism of HGSC and comprehensively summarize the cell models used to study this fatal cancer having multiple cells of origin and overt genomic instability.


Assuntos
Carcinoma/patologia , Modelos Biológicos , Neoplasias Ovarianas/patologia , Animais , Carcinoma/metabolismo , Transformação Celular Neoplásica , Tubas Uterinas/citologia , Tubas Uterinas/metabolismo , Tubas Uterinas/patologia , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Telomerase/genética , Telomerase/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
8.
J Clin Endocrinol Metab ; 106(7): 1929-1955, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-33755733

RESUMO

CONTEXT: The ability of ovarian steroids to modify ovarian cancer (OC) risk remains controversial. Progesterone is considered to be protective; recent studies indicate no effect or enhanced OC risk. Knowledge of progesterone receptor (PR) signaling during altered physiology that typifies OC development is limited. OBJECTIVE: This study defines PR-driven oncogenic signaling mechanisms in p53-mutant human fallopian tube epithelia (hFTE), a precursor of the most aggressive OC subtype. METHODS: PR expression in clinical samples of serous tubal intraepithelial carcinoma (STIC) lesions and high-grade serous OC (HGSC) tumors was analyzed. Novel PR-A and PR-B isoform-expressing hFTE models were characterized for gene expression and cell cycle progression, emboli formation, and invasion. PR regulation of the DREAM quiescence complex and DYRK1 kinases was established. RESULTS: STICs and HGSC express abundant activated phospho-PR. Progestin promoted reversible hFTE cell cycle arrest, spheroid formation, and invasion. RNAseq/biochemical studies revealed potent ligand-independent/-dependent PR actions, progestin-induced regulation of the DREAM quiescence complex, and cell cycle target genes through enhanced complex formation and chromatin recruitment. Disruption of DREAM/DYRK1s by pharmacological inhibition, HPV E6/E7 expression, or DYRK1A/B depletion blocked progestin-induced cell arrest and attenuated PR-driven gene expression and associated OC phenotypes. CONCLUSION: Activated PRs support quiescence and pro-survival/pro-dissemination cell behaviors that may contribute to early HGSC progression. Our data support an alternative perspective on the tenet that progesterone always confers protection against OC. STICs can reside undetected for decades prior to invasive disease; our studies reveal clinical opportunities to prevent the ultimate development of HGSC by targeting PRs, DREAM, and/or DYRKs.


Assuntos
Processos de Crescimento Celular/genética , Cistadenocarcinoma Seroso/genética , Neoplasias das Tubas Uterinas/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Receptores de Progesterona/metabolismo , Proteínas Repressoras/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Tubas Uterinas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Ovarianas/genética , Fenótipo , Proteína Supressora de Tumor p53/metabolismo
9.
Sci Rep ; 11(1): 6270, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33737539

RESUMO

Malignant transformation of fallopian tube secretory epithelial cells (FTSECs) is a key contributing event to the development of high-grade serous ovarian carcinoma (HGSOC). Our recent findings implicate oncogenic transformative events in chronic iron-exposed FTSECs, including increased expression of oncogenic mediators, increased telomerase transcripts, and increased growth/migratory potential. Herein, we extend these studies by implementing an integrated transcriptomic and mass spectrometry-based proteomics approach to identify global miRNA and protein alterations, for which we also investigate a subset of these targets to iron-induced functional alterations. Proteomic analysis identified > 4500 proteins, of which 243 targets were differentially expressed. Sixty-five differentially expressed miRNAs were identified, of which 35 were associated with the "top" proteomic molecules (> fourfold change) identified by Ingenuity Pathway Analysis. Twenty of these 35 miRNAs are at the 14q32 locus (encoding a cluster of 54 miRNAs) with potential to be regulated by DNA methylation and histone deacetylation. At 14q32, miR-432-5p and miR-127-3p were ~ 100-fold downregulated whereas miR-138-5p was 16-fold downregulated at 3p21 in chronic iron-exposed FTSECs. Combinatorial treatment with methyltransferase and deacetylation inhibitors reversed expression of these miRNAs, suggesting chronic iron exposure alters miRNA expression via epigenetic alterations. In addition, PAX8, an important target in HGSOC and a potential miRNA target (from IPA) was epigenetically deregulated in iron-exposed FTSECs. However, both PAX8 and ALDH1A2 (another IPA-predicted target) were experimentally identified to be independently regulated by these miRNAs although TERT RNA was partially regulated by miR-138-5p. Interestingly, overexpression of miR-432-5p diminished cell numbers induced by long-term iron exposure in FTSECs. Collectively, our global profiling approaches uncovered patterns of miRNA and proteomic alterations that may be regulated by genome-wide epigenetic alterations and contribute to functional alterations induced by chronic iron exposure in FTSECs. This study may provide a platform to identify future biomarkers for early ovarian cancer detection and new targets for therapy.


Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Tubas Uterinas/efeitos dos fármacos , Tubas Uterinas/metabolismo , Compostos Férricos/farmacologia , Loci Gênicos , MicroRNAs/genética , Proteoma/genética , Compostos de Amônio Quaternário/farmacologia , Transcriptoma/efeitos dos fármacos , Azacitidina/farmacologia , Biomarcadores Tumorais/genética , Linhagem Celular Transformada , Transformação Celular Neoplásica/genética , Regulação para Baixo/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Proteína do Locus do Complexo MDS1 e EVI1/genética , Proteína do Locus do Complexo MDS1 e EVI1/metabolismo , MicroRNAs/metabolismo , Neoplasias Ovarianas/genética , Proteômica/métodos , Transfecção , Vorinostat/farmacologia
10.
Methods Mol Biol ; 2273: 219-238, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33604857

RESUMO

Intercellular communication can be carried out by circulating systemic and/or locally released extracellular vesicles (EVs), produced by nearly every cell type and tissue, and are involved in physiological and pathological processes. In recent years, EVs have been identified in reproductive tissues, such as oviduct and uterus, and have been shown to be related to several events important for reproductive success. The understanding of their functions in reproduction has important implications for assisted reproductive technologies, for the treatment of infertility in humans and improvement of reproduction efficiency in animals. To study such EVs, it is necessary to isolate and concentrate them from fluid samples, which in the case of reproductive tissues, are usually of limited volume. Several methods for EV isolation are available such as chromatography, ultracentrifugation, polymer-based precipitation, and immunoaffinity.Outcomes can be variable in terms of the amount and quality of isolated EVs, due to the type of isolation method. The choice of method, or a different combination of methods, may depend on the type of sample and scientific question to be addressed in a given study. In this chapter, we describe a method for isolation of EVs from bovine oviductal and uterine fluids for use in functional studies. The method combines size exclusion chromatography and ultracentrifugation. We also describe the different protocols for characterization of isolated EVs (transmission electron microscopy, nanoparticle tracking analysis, and western blot), as well as the isolation of RNA content in EVs, and their miRNAs profiling for functional studies.


Assuntos
Bovinos/genética , Vesículas Extracelulares/genética , Tubas Uterinas/metabolismo , MicroRNAs/genética , Animais , Western Blotting/métodos , Cromatografia em Gel/métodos , Feminino , Perfilação da Expressão Gênica/métodos , MicroRNAs/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcriptoma , Ultracentrifugação/métodos , Útero/metabolismo
11.
Int J Mol Sci ; 22(3)2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33499129

RESUMO

Long non-coding RNA's (lncRNA) are RNA sequences that do not encode proteins and are greater than 200 nucleotides in length. They regulate complex cellular mechanisms and have been associated with prognosis in various types of cancer. We aimed to identify lncRNA sequences that are associated with high grade serous ovarian cancer (HGSC) and assess their impact on overall survival. RNA was extracted from 112 HGSC patients and 12 normal fallopian tube samples from our Biobank tissue repository. RNA was sequenced and the Ultrafast and Comprehensive lncRNA detection and quantification pipeline (UClncR) was used for the identification of lncRNA sequences. Univariate logistic and multivariate lasso regression analyses identified lncRNA that was associated with HGSC. Univariate and multivariate Cox proportional hazard ratios were used to evaluate independent predictors of survival. 1943 of 16,325 investigated lncRNA's were differentially expressed in HGSC as compared to controls (p < 0.001). Nine of these demonstrated association with cancer after multivariate lasso regression. Our multivariate analysis of survival identified four lncRNA's associated with survival in HGSC. Three out of these four were found to be independently significant after accounting for all clinical covariates. Lastly, seven lncRNAs were independently associated with initial response to chemotherapy; four portended a worse response, while three were associated with improved response. More research is needed, but there is potential for these lncRNAs to be used as biomarkers of HGSC or predictors of treatment outcome in the future.


Assuntos
Neoplasias Ovarianas/genética , RNA Longo não Codificante/genética , Antineoplásicos/farmacologia , Bancos de Espécimes Biológicos , Biomarcadores Tumorais/metabolismo , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/mortalidade , Estudos de Casos e Controles , Tubas Uterinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Genômica , Humanos , Estimativa de Kaplan-Meier , Análise Multivariada , Neoplasias Ovarianas/mortalidade , Modelos de Riscos Proporcionais , RNA-Seq , Estudos Retrospectivos , Fatores de Tempo , Resultado do Tratamento
12.
Int J Mol Sci ; 22(2)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33477993

RESUMO

Information on molecular mechanisms through which sex-steroids regulate oviductal function to support early embryo development is lacking. Here, we hypothesized that the periovulatory endocrine milieu affects the miRNA processing machinery and miRNA expression in bovine oviductal tissues. Growth of the preovulatory follicle was controlled to obtain cows that ovulated a small follicle (SF) and subsequently bore a small corpus luteum (CL; SF-SCL) or a large follicle (LF) and large CL (LF-LCL). These groups differed in the periovulatory plasmatic sex-steroid's concentrations. Ampulla and isthmus samples were collected on day four of the estrous cycle. Abundance of DROSHA, DICER1, and AGO4 transcripts was greater in the ampulla than the isthmus. In the ampulla, transcription of these genes was greater for the SF-SCL group, while the opposite was observed in the isthmus. The expression of the 88 most abundant miRNAs and 14 miRNAs in the ampulla and 34 miRNAs in isthmus were differentially expressed between LF-LCL and SF-SCL groups. Integration of transcriptomic and miRNA data and molecular pathways enrichment showed that important pathways were inhibited in the SF-SCL group due to miRNA control. In conclusion, the endocrine milieu affects the miRNA expression in the bovine oviduct in a region-specific manner.


Assuntos
Bovinos , Tubas Uterinas/efeitos dos fármacos , Hormônios Esteroides Gonadais/farmacologia , MicroRNAs , Animais , Bovinos/genética , Bovinos/metabolismo , Ciclo Estral/efeitos dos fármacos , Ciclo Estral/genética , Tubas Uterinas/metabolismo , Feminino , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica/efeitos dos fármacos , MicroRNAs/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Ovulação/efeitos dos fármacos , Ovulação/genética , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/genética , Transcriptoma/efeitos dos fármacos
13.
Cancer Lett ; 501: 224-233, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33221455

RESUMO

High grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy with a need for better understanding the disease pathogenesis. The biologically active thyroid hormone, T3, is considered a tumor suppressor by promoting cell differentiation and mitochondrial respiration. Tumors evolved a strategy to avoid these anticancer actions by expressing the T3 catabolizing enzyme, Deiodinase type 3 (DIO3). This stimulates cancer proliferation and aerobic glycolysis (Warburg effect). We identified DIO3 expression in HGSOC cell lines, tumor tissues from mice and human patients, fallopian tube (FT) premalignant lesion and secretory cells of normal FT, considered the disease site-of-origin. Stable DIO3 knockdown (DIO3-KD) in HGSOC cells led to increased T3 bioavailability and demonstrated induced apoptosis and attenuated proliferation, migration, colony formation, oncogenic signaling, Warburg effect and tumor growth in mice. Proteomics analysis further indicated alterations in an array of cancer-relevant proteins, the majority of which are involved in tumor suppression and metabolism. Collectively this study establishes the functional role of DIO3 in facilitating tumorigenesis and metabolic reprogramming, and proposes this enzyme as a promising target for inhibition in HGSOC.


Assuntos
Cistadenocarcinoma Seroso/patologia , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Neoplasias Ovarianas/patologia , Regulação para Cima , Aerobiose , Animais , Linhagem Celular Tumoral , Proliferação de Células , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Tubas Uterinas/metabolismo , Tubas Uterinas/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glicólise , Humanos , Camundongos , Gradação de Tumores , Transplante de Neoplasias , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo
14.
Cell Tissue Res ; 383(3): 1191-1202, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33242172

RESUMO

Prosaposin (PSAP) has two forms: a precursor and a secreted form. The secreted form has neurotrophic, myelinotrophic, and myotrophic properties. The precursor form is a precursor protein of saposins A-D. Although the distribution of PSAP in male reproductive organs is well known, its distribution in female reproductive organs, especially in the oviduct, is unclear. Immunoblots and immunohistochemistry of oviducts showed that oviductal tissues contain PSAP proteins, and a significant increase in PSAP was observed in the estrus-metestrus phase compared to the diestrus-proestrus phase in the ampulla. To identify PSAP trafficking in cells, double-immunostaining was performed with antibodies against PSAP in combination with sortilin, mannose 6 phosphate receptor (M6PR), or low-density lipoprotein receptor-related protein 1 (LRP1). PSAP and sortilin double-positive reactions were observed near the nuclei, as well as in the apical portion of microvillous epithelial cells, whereas these reactions were only observed near the nuclei of ciliated epithelial cells. PSAP and M6PR double-positive reactions were observed near the nuclei of microvillous and ciliated epithelial cells. PSAP and M6PR double-positive reactions were also observed in the apical portion of microvillous epithelial cells. PSAP and LRP1 double-positive reactions were observed in the plasma membrane and apical portion of both microvillous and ciliated epithelial cells. Immunoelectron staining revealed PSAP immunoreactive small vesicles with exocytotic features at the apical portion of microvillous epithelial cells. These findings suggest that PSAP is present in the oviductal epithelium and has a pivotal role during pregnancy in providing an optimal environment for gametes and/or sperm in the ampulla.


Assuntos
Células Epiteliais , Ciclo Estral/metabolismo , Tubas Uterinas , Receptor IGF Tipo 2/metabolismo , Saposinas/metabolismo , Animais , Membrana Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Tubas Uterinas/citologia , Tubas Uterinas/metabolismo , Feminino , Gravidez , Ratos , Ratos Wistar
15.
PLoS One ; 15(12): e0243959, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33315943

RESUMO

There has been significant concern regarding fertility and reproductive outcomes during the SARS-CoV2 pandemic. Recent data suggests a high concentration of SARS-Cov2 receptors, ACE2 or TMPRSS2, in nasal epithelium and cornea, which explains person-to-person transmission. We investigated the prevalence of SARS-CoV2 receptors among reproductive tissues by exploring the single-cell sequencing datasets from uterus, myometrium, ovary, fallopian tube, and breast epithelium. We did not detect significant expression of either ACE2 or TMPRSS2 in the normal human myometrium, uterus, ovaries, fallopian tube, or breast. Furthermore, none of the cell types in the female reproductive organs we investigated, showed the co-expression of ACE2 with proteases, TMPRSS2, Cathepsin B (CTSB), and Cathepsin L (CTSL) known to facilitate the entry of SARS2-CoV2 into the host cell. These results suggest that myometrium, uterus, ovaries, fallopian tube, and breast are unlikely to be susceptible to infection by SARS-CoV2.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Catepsina B/genética , Catepsina L/genética , SARS-CoV-2/genética , Serina Endopeptidases/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Mama/metabolismo , Mama/virologia , COVID-19/epidemiologia , COVID-19/transmissão , COVID-19/virologia , Epitélio/metabolismo , Epitélio/virologia , Tubas Uterinas/metabolismo , Tubas Uterinas/virologia , Feminino , Fertilidade/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Miométrio/metabolismo , Miométrio/virologia , Ovário/metabolismo , Ovário/virologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Infecções do Sistema Genital/genética , Infecções do Sistema Genital/virologia , SARS-CoV-2/patogenicidade , Serina Endopeptidases/metabolismo , Análise de Célula Única , Útero/metabolismo , Útero/virologia
16.
Mol Reprod Dev ; 87(11): 1133-1140, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33022130

RESUMO

Copulation produces different stimuli in the female reproductive tract in camelids, which lead to ovulation. Expression of ß-nerve growth factor (ß-NGF) and its specific receptor, tropomyosin receptor kinase A (TrKA), was studied comparing the oviductal microenvironment of mated and nonmated llamas. ß-NGF and TrKA were expressed in the llama ampulla, isthmus, and utero-tubal-junction (UTJ), and they were mainly colocalized in the apical region of the oviductal mucosa. A TrKA immunosignal was also found in muscle cells and blood vessels, with the highest mark in UTJ muscle cells of copulated females. Both ß-NGF and TrKA transcripts were expressed in the three oviductal segments. Relative TrKA abundance did not differ between mated and nonmated females, but relative ß-NGF abundance was higher in the UTJ of copulated females (p < .05). ß-NGF might not be secreted into the oviductal fluid (OF) since the protein was not found in the OF of mated or nonmated females. Therefore, it can be concluded that the llama oviduct expresses the ß-NGF/TrKA system and that an increase in ß-NGF gene expression in the UTJ 24 h after copulation along with an increase in TrKA protein expression may indicate an important role in the gamete transport and fertilization process in llamas.


Assuntos
Camelídeos Americanos/fisiologia , Copulação/fisiologia , Tubas Uterinas/metabolismo , Regulação da Expressão Gênica , Fator de Crescimento Neural/biossíntese , RNA Mensageiro/biossíntese , Receptor trkA/biossíntese , Animais , Líquidos Corporais/metabolismo , Camelídeos Americanos/genética , Feminino , Fator de Crescimento Neural/genética , RNA Mensageiro/genética , Receptor trkA/genética
17.
Sci Rep ; 10(1): 16522, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020549

RESUMO

To date sperm-oviduct interactions have largely been investigated under in vitro conditions. Therefore we set out to characterize the behaviour of bovine spermatozoa within the sperm reservoir under near in vivo conditions and in real-time using a novel live cell imaging technology and a newly established fluorescent sperm binding assay. Sperm structure and tubal reactions after sperm binding were analysed using scanning and transmission electron microscopy and histochemistry. As a model to specify the impact of stress on sperm-oviduct interactions, frozen-thawed conventional and sex-sorted spermatozoa from the same bulls (n = 7) were co-incubated with oviducts obtained from cows immediately after slaughter. Our studies revealed that within the oviductal sperm reservoir agile (bound at a tangential angle of about 30°, actively beating undulating tail), lagging (bound at a lower angle, reduced tail movement), immotile (absence of tail movement) and hyperactivated (whip-like movement of tail) spermatozoa occur, the prevalence of which changes in a time-dependent pattern. After formation of the sperm reservoir, tubal ciliary beat frequency is significantly increased (p = 0.022) and the epithelial cells show increased activity of endoplasmic reticula. After sex sorting, spermatozoa occasionally display abnormal movement patterns characterized by a 360° rotating head and tail. Sperm binding in the oviduct is significantly reduced (p = 0.008) following sexing. Sex-sorted spermatozoa reveal deformations in the head, sharp bends in the tail and a significantly increased prevalence of damaged mitochondria (p < 0.001). Our results imply that the oviductal cells specifically react to the binding of spermatozoa, maintaining sperm survival within the tubal reservoir. The sex-sorting process, which is associated with mechanical, chemical and time stress, impacts sperm binding to the oviduct and mitochondrial integrity affecting sperm motility and function.


Assuntos
Oviductos/fisiologia , Capacitação Espermática/fisiologia , Espermatozoides/fisiologia , Animais , Bovinos , Tubas Uterinas/metabolismo , Feminino , Masculino , Microscopia Eletrônica de Transmissão/métodos , Oviductos/metabolismo , Motilidade Espermática/fisiologia , Espermatozoides/metabolismo
18.
BMC Cancer ; 20(1): 1020, 2020 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087072

RESUMO

BACKGROUND: Loss of the genomic stability jeopardize genome stability and promote malignancies. A fraction of ovarian cancer (OvCa) arises from pathological mutations of DNA repair genes that result in highly mutagenic genomes. However, it remains elusive why the ovarian epithelial cells are particularly susceptible to the malfunction of genome surveillance system. METHODS: To explore the genotoxic responses in the unique context of microenvironment for ovarian epithelium that is periodically exposed to high-level steroid hormones, we examined estrogen-induced DNA damage by immunofluorescence in OvCa cell lines, animal and human samples. RESULTS: We found that OvCa cells are burdened with high levels of endogenous DNA damage that is not correlated with genomic replication. The elevation of damage burden is attributable to the excessive concentration of bioactive estrogen instead of its chemomimetic derivative (tamoxifen). Induction of DNA lesions by estrogen is dependent on the expression of hormone receptors, and occurs in G1 and non-G1 phases of cell cycle. Moreover, depletion of homologous recombination (HR) genes (BRCA1 and BRCA2) exacerbated the genotoxicity of estrogen, highlighting the role of HR to counteract hormone-induced genome instability. Finally, the estrogen-induced DNA damage was reproduced in the epithelial compartments of both ovarian and fallopian tubes. CONCLUSIONS: Taken together, our study disclose that estrogen-induced genotoxicity and HR deficiency perturb the genome stability of ovarian and fallopian epithelial cells, representing microenvironmental and genetic risk factors, respectively.


Assuntos
Dano ao DNA , Estrogênios/toxicidade , Tubas Uterinas/efeitos dos fármacos , Neoplasias Ovarianas/genética , Ovário/efeitos dos fármacos , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Tubas Uterinas/metabolismo , Feminino , Recombinação Homóloga , Humanos , Camundongos , Neoplasias Experimentais , Especificidade de Órgãos , Neoplasias Ovarianas/tratamento farmacológico , Ovário/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo
19.
Future Oncol ; 16(25): 1921-1930, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32915667

RESUMO

Aim: To explore FBXW7 protein-coding transcript isoform (α, ß and γ) expression, their functions and prognostic value in ovarian serous cystadenocarcinoma (OSC). Materials & methods: FBXW7 transcript data were collected from The Cancer Genome Atlas and the Genotype-Tissue Expression project. IOSE, A2780 and SKOV3 cells were used for in vitro and in vivo studies. Results: FBXW7α and FBXW7γ are dominant protein-coding transcripts that were downregulated in OSC. FBXW7γ overexpression reduced the protein expression of c-Myc, Notch1 and Yap1 and suppressed OSC cell growth in vitro and in vivo. FBXW7γ expression was an independent indicator of longer disease-specific survival (HR: 0.588; 95% CI: 0.449-0.770) and progression-free survival (HR: 0.708; 95% CI: 0.562-0.892). Conclusion: FBXW7γ is a tumor-suppressive and might be the only prognosis-related FBXW7 transcript in OSC.


Assuntos
Processamento Alternativo , Biomarcadores Tumorais , Cistadenocarcinoma Seroso/etiologia , Cistadenocarcinoma Seroso/mortalidade , Proteína 7 com Repetições F-Box-WD/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/etiologia , Animais , Linhagem Celular Tumoral , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Modelos Animais de Doenças , Tubas Uterinas/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Camundongos , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Prognóstico , Isoformas de RNA , RNA Mensageiro/genética , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Mol Reprod Dev ; 87(10): 1059-1069, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32914493

RESUMO

We previously reported that sperm binding to cultured bovine oviduct epithelial cells induces an anti-inflammatory immune response. Now we have developed a differentiated explant model to focus on the oviductal ampulla, where fertilization occurs, and to study the effect of sperm capacitation on the immune response. We used heparin to stimulate bovine sperm capacitation. Fluorescence imaging showed that 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide-labeled sperm pretreated with (Hep(+) ) or without (Hep( -) ) heparin rapidly attached to the explant ciliated epithelium in similar numbers. However, only Hep(+) sperm upregulated explant messenger RNA (mRNA) transcription of TLR2, IL8, TGFB1, and PGES, without changes in TNFA and IL-10 expression, while Hep( -) sperm only upregulated PGES. The responses were primarily anti-inflammatory, with a greater response produced by Hep(+) sperm, which also produced a substantial increase in TLR2 protein expression in the epithelium. The addition of TLR1/2 (toll-like receptor 1/2) antagonist to the Hep(+) and (Hep( -) ) sperm-explant coincubations reduced sperm attachment to the epithelium and inhibited TLR2 protein expression and some of the Hep(+) sperm-induced mRNA transcription. Our observations suggest that the ampullar epithelium immunologically reacts more strongly to sperm that have undergone heparin stimulation of capacitation. This anti-inflammatory response could serve to protect capacitated sperm as they approach the oocyte in the ampulla.


Assuntos
Comunicação Celular/imunologia , Tubas Uterinas , Espermatozoides/metabolismo , Receptor 2 Toll-Like/fisiologia , Animais , Bovinos , Comunicação Celular/genética , Células Cultivadas , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Tubas Uterinas/imunologia , Tubas Uterinas/metabolismo , Feminino , Imunidade/fisiologia , Masculino , Capacitação Espermática/fisiologia , Espermatozoides/imunologia
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