Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 55
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Biol Reprod ; 111(1): 11-27, 2024 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-38408205

RESUMO

Profiling bovine blastocyst transcriptome at the single-cell level has enabled us to reveal the first cell lineage segregation, during which the inner cell mass (ICM), trophectoderm (TE), and an undefined population of transitional cells were identified. By comparing the transcriptome of blastocysts derived in vivo (IVV), in vitro from a conventional culture medium (IVC), and in vitro from an optimized reduced nutrient culture medium (IVR), we found a delay of the cell fate commitment to ICM in the IVC and IVR embryos. Developmental potential differences between IVV, IVC, and IVR embryos were mainly contributed by ICM and transitional cells. Pathway analysis of these non-TE cells between groups revealed highly active metabolic and biosynthetic processes, reduced cellular signaling, and reduced transmembrane transport activities in IVC embryos that may lead to reduced developmental potential. IVR embryos had lower activities in metabolic and biosynthetic processes but increased cellular signaling and transmembrane transport, suggesting these cellular mechanisms may contribute to improved blastocyst development compared to IVC embryos. However, the IVR embryos had compromised development compared to IVV embryos with notably over-active transmembrane transport activities that impaired ion homeostasis.


Assuntos
Blastocisto , Linhagem da Célula , Técnicas de Cultura Embrionária , Animais , Bovinos , Blastocisto/metabolismo , Blastocisto/citologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/fisiologia , Feminino , Transcriptoma , Meios de Cultura
2.
Reproduction ; 167(2)2024 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-38301360

RESUMO

In brief: Peroxisome proliferator-activated receptor gamma (PPARG) is a critical regulator of placental function, but earlier roles in preimplantation embryo development and embryonic origins of placental formation have not been established. Results herein demonstrate that PPARG responds to pharmacologic stimulation in the bovine preimplantation embryo and influences blastocyst development, cell lineage specification, and transcripts important for placental function. Abstract: Peroxisome proliferator-activated receptor gamma (PPARG) is a key regulator of metabolism with conserved roles that are indispensable for placental function, suggesting previously unidentified and important roles in preimplantation embryo development. Herein, we report the functional characterization of bovine PPARG to reveal expression beginning on D6 of development with nuclear and ubiquitous patterns. Day 6 PPARG+ embryos have fewer total cells and a lower proportion of trophectoderm cells compared to PPARG- embryos (P < 0.05). Coculture with a PPARG agonist, rosiglitazone (Ros), or antagonist GW9662 (GW), decreases blastocyst development (P < 0.01). Day 7.5 (D7.5) developmentally delayed embryos exposed to Ros express lower transcript abundance of key genes important for placental development and cell lineage formation (CDX2, RXRB, SP1, TFAP2C, SIRT1, and PTEN). In contrast, Ros does not alter transcript abundance in D7.5 blastocysts, but GW treatment lowers RXRA, RXRB, SP1, and NFKB1 expression. Knockout of embryonic PPARG does not alter blastocyst formation and hatching ability but decreases total cell number in D7.5 blastocysts. The decreased embryo development response and affected pathways following targeted pharmacological perturbation vs embryonic knockout of PPARG suggest roles of both maternal and embryonic origins. These data reveal regulatory contributions of PPARG in preimplantation embryo development, cell lineage formation, and regulation of transcripts associated with placental function.


Assuntos
PPAR gama , Placenta , Gravidez , Animais , Bovinos , Feminino , PPAR gama/genética , PPAR gama/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Placenta/metabolismo , Blastocisto/metabolismo , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento
3.
Proc Natl Acad Sci U S A ; 116(45): 22635-22644, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31636193

RESUMO

Single-cell RNA sequencing of cells from cultured human blastocysts has enabled us to define the transcriptomic landscape of placental trophoblast (TB) that surrounds the epiblast and associated embryonic tissues during the enigmatic day 8 (D8) to D12 peri-implantation period before the villous placenta forms. We analyzed the transcriptomes of 3 early placental cell types, cytoTB (CTB), syncytioTB (STB), and migratoryTB (MTB), picked manually from cultured embryos dissociated with trypsin and were able to follow sublineages that emerged from proliferating CTB at the periphery of the conceptus. A unique form of CTB with some features of STB was detectable at D8, while mature STB was at its zenith at D10. A form of MTB with a mixed MTB/CTB phenotype arose around D10. By D12, STB generation was in decline, CTB had entered a new phase of proliferation, and mature MTB cells had begun to move from the main body of the conceptus. Notably, the MTB transcriptome at D12 indicated enrichment of transcripts associated with IFN signaling, migration, and invasion and up-regulation of HLA-C, HLA-E, and HLA-G. The STB, which is distinct from the STB of later villous STB, had a phenotype consistent with intense protein export and placental hormone production, as well as migration and invasion. The studies show that TB associated with human embryos is in rapid developmental flux during peri-implantation period when it must invade, signal robustly to the mother to ensure that the pregnancy continues, and make first contact with the maternal immune system.


Assuntos
Diferenciação Celular , Trofoblastos/citologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Implantação do Embrião , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Placenta/citologia , Placenta/metabolismo , Gravidez , Análise de Sequência de RNA , Análise de Célula Única , Transcriptoma , Trofoblastos/metabolismo
4.
Reprod Biol Endocrinol ; 19(1): 141, 2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34517901

RESUMO

Caudal Type Homeobox 2 (CDX2) is a key regulator of trophectoderm formation and maintenance in preimplantation embryos. We previously demonstrated that supplementation of exogenous follistatin, during in vitro culture of bovine IVF embryos, upregulates CDX2 expression, possibly, via alteration of the methylation status of CDX2 gene. Here, we further investigated the effects of exogenous follistatin supplementation on developmental competence and CDX2 methylation in bovine somatic cell nuclear transfer (SCNT) embryos. SCNT embryos were cultured with or without follistatin for 72h, then transferred into follistatin free media until d7 when blastocysts were collected and subjected to CDX2 gene expression and DNA methylation analysis for CDX2 regulatory regions by bisulfite sequencing. Follistatin supplementation significantly increased both blastocyst development as well as blastocyst CDX2 mRNA expression on d7. Three different CpG rich fragments within the CDX2 regulatory elements; proximal promoter (fragment P1, -1644 to -1180; P2, -305 to +126) and intron 1 (fragment I, + 3030 to + 3710) were identified and selected for bisulfite sequencing analysis. This analysis showed that follistatin treatment induced differential methylation (DM) at specific CpG sites within the analyzed fragments. Follistatin treatment elicited hypomethylation at six CpG sites at positions -1374, -279, -163, -23, +122 and +3558 and hypermethylation at two CpG sites at positions -243 and +20 in promoter region and first intron of CDX2 gene. Motif analysis using MatInspector revealed that differentially methylated CpG sites are putative binding sites for key transcription factors (TFs) known to regulate Cdx2 expression in mouse embryos and embryonic stem cells including OCT1, AP2F, KLF and P53, or TFs that have indirect link to CDX2 regulation including HAND and NRSF. Collectively, results of the present study together with our previous findings in IVF embryos support the hypothesis that alteration of CDX2 methylation is one of the epigenetic mechanisms by which follistatin may regulates CDX2 expression in preimplantation bovine embryos.


Assuntos
Blastocisto/efeitos dos fármacos , Fator de Transcrição CDX2/genética , Metilação de DNA/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Folistatina/farmacologia , Animais , Blastocisto/fisiologia , Fator de Transcrição CDX2/efeitos dos fármacos , Bovinos/embriologia , Células Cultivadas , Clonagem de Organismos/veterinária , Ilhas de CpG/efeitos dos fármacos , Ilhas de CpG/genética , Metilação de DNA/genética , Técnicas de Cultura Embrionária/métodos , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Fertilização in vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Técnicas de Transferência Nuclear/veterinária
5.
Reprod Biomed Online ; 42(6): 1067-1074, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33814309

RESUMO

RESEARCH QUESTION: Is there a risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral exposure and potential cross-contamination from follicular fluid, culture media and vitrification solution within the IVF laboratory using strict patient screening and safety measures? DESIGN: This was a prospective clinical study. All women undergoing transvaginal oocyte retrieval were required to have a negative SARS-CoV-2 RNA test 3-5 days prior to the procedure. Male partners were not tested. All cases used intracytoplasmic sperm injection (ICSI). The first tube of follicular fluid aspirated during oocyte retrieval, drops of media following removal of the embryos on day 5, and vitrification solution after blastocyst cryopreservation were analysed for SARS-CoV-2 RNA. RESULTS: In total, medium from 61 patients, vitrification solution from 200 patients and follicular fluid from 300 patients was analysed. All samples were negative for SARS-CoV-2 viral RNA. CONCLUSIONS: With stringent safety protocols in place, including testing of women and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in follicular fluid, medium or vitrification solution. This work demonstrates the possibility of implementing a rapid laboratory screening assay for SARS-CoV-2 and has implications for safe laboratory operations, including cryostorage recommendations.


Assuntos
Meios de Cultura/análise , Fertilização in vitro , Líquido Folicular/virologia , Laboratórios , RNA Viral/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Feminino , Humanos , Recuperação de Oócitos , Segurança do Paciente , Estudos Prospectivos , Injeções de Esperma Intracitoplásmicas , Vitrificação
6.
Biol Reprod ; 102(4): 795-805, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31965149

RESUMO

Characterization of the molecular factors regulating early embryonic development and their functional mechanisms is critical for understanding the causes of early pregnancy loss in monotocous species (cattle, human). We previously characterized a stage specific functional role of follistatin, a TGF-beta superfamily binding protein, in promoting early embryonic development in cattle. The mechanism by which follistatin mediates this embryotropic effect is not precisely known as follistatin actions in cattle embryos are independent of its classically known activin inhibition activity. Apart from activin, follistatin is known to bind and modulate the activity of the bone morphogenetic proteins (BMPs), which signal through SMAD1/5 pathway and regulate several aspects of early embryogenesis in other mammalian species. Present study was designed to characterize the activity and functional requirement of BMP signaling during bovine early embryonic development and to investigate if follistatin involves BMP signaling for its stage specific embryotropic actions. Immunostaining and western blot analysis demonstrated that SMAD1/5 signaling is activated after embryonic genome activation in bovine embryos. However, days 1-3 follistatin treatment reduced the abundance of phosphorylated SMAD1/5 in cultured embryos. Inhibition of active SMAD1/5 signaling (8-16 cell to blastocyst) using pharmacological inhibitors and/or lentiviral-mediated inhibitory SMAD6 overexpression showed that SMAD1/5 signaling is required for blastocyst production, first cell lineage determination as well as mRNA and protein regulation of TE (CDX2) cell markers. SMAD1/5 signaling was also found to be essential for embryotropic actions of follistatin during days 4-7 but not days 1-3 of embryo development suggesting a role for follistatin in regulation of SMAD1/5 signaling in bovine embryos.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Folistatina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Bovinos , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Gravidez , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo
7.
Mol Reprod Dev ; 87(9): 998-1008, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32776625

RESUMO

CDX2 plays a crucial role in the formation and maintenance of the trophectoderm epithelium in preimplantation embryos. Follistatin supplementation during the first 72 hr of in vitro culture triggers a significant increase in blastocyst rates, CDX2 expression, and trophectoderm cell numbers. However, the underlying epigenetic mechanisms by which follistatin upregulates CDX2 expression are not known. Here, we investigated whether stimulatory effects of follistatin are linked to alterations in DNA methylation within key regulatory regions of the CDX2 gene. In vitro-fertilized (IVF) zygotes were cultured with or without 10 ng/ml of recombinant human follistatin for 72 hr, then cultured without follistatin until Day 7. The bisulfite-sequencing analysis revealed differential methylation (DM) at specific CpG sites within the CDX2 promoter and intron 1 following follistatin treatment. These DM CpG sites include five hypomethylated sites at positions -1384, -1283, -297, -163, and -23, and four hypermethylated sites at positions -1501, -250, -243, and +20 in the promoter region. There were five hypomethylated sites at positions +3060, +3105, +3219, +3270, and +3545 in intron 1. Analysis of transcription factor binding sites using MatInspector combined with a literature search revealed a potential association between differentially methylated CpG sites and putative binding sites for key transcription factors involved in regulating CDX2 expression. The hypomethylated sites are putative binding sites for FXR, STAF, OCT1, KLF, AP2 family, and P53 protein, whereas the hypermethylated sites are putative binding sites for NRSF. Collectively, our results suggest that follistatin may increase CDX2 expression in early bovine embryos, at least in part, by modulating DNA methylation at key regulatory regions.


Assuntos
Blastocisto/efeitos dos fármacos , Fator de Transcrição CDX2/genética , Bovinos/embriologia , Metilação de DNA/efeitos dos fármacos , Folistatina/farmacologia , Animais , Blastocisto/metabolismo , Fator de Transcrição CDX2/metabolismo , Bovinos/genética , Células Cultivadas , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Fertilização in vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento
8.
Mol Reprod Dev ; 85(2): 106-116, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29232016

RESUMO

Histone variant H3.3 is encoded by two distinct genes, H3F3A and H3F3B, that are closely associated with actively transcribed genes. H3.3 replacement is continuous and essential for maintaining correct chromatin structure during mouse oogenesis. Upon fertilization, H3.3 is incorporated to parental chromatin, and is required for blastocyst formation in mice. The H3.3 exchange process is facilitated by the chaperone HIRA, particularly during zygote development. We previously demonstrated that H3.3 is required for bovine early embryonic development; here, we explored the mechanisms of its functional requirement. H3F3A mRNA abundance is stable whereas H3F3B and HIRA mRNA are relatively dynamic during early embryonic development. H3F3B mRNA quantity is also considerably higher than H3F3A. Immunofluorescence analysis revealed an even distribution of H3.3 between paternal and maternal pronuclei in zygotes, and subsequent stage-specific localization of H3.3 in early bovine embryos. Knockdown of H3.3 by targeting both H3F3A and H3F3B dramatically decreased the expression of NANOG (a pluripotency marker) and CTGF (Connective tissue growth factor; a trophectoderm marker) in bovine blastocysts. Additionally, we noted that Histone H3 lysine 36 dimethylation and linker Histone H1 abundance is reduced in H3.3-deficient embryos, which was similar to effects following knockdown of CHD1 (Chromodomain helicase DNA-binding protein 1). By contrast, no difference was observed in the abundance of Histone H3 lysine 4 trimethylation, Histone H3 lysine 9 dimethylation, or Splicing factor 3 B1. Collectively, these results established that H3.3 is required for correct epigenetic modifications and H1 deposition, dysregulation of which likely mediate the poor development in H3.3-deficient embryos.


Assuntos
Blastocisto/metabolismo , Bovinos , Chaperonas de Histonas/genética , Chaperonas de Histonas/fisiologia , Histonas/genética , Animais , Bovinos/embriologia , Bovinos/genética , Linhagem da Célula/genética , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Masculino , Gravidez , Zigoto/metabolismo
9.
Reprod Biol Endocrinol ; 16(1): 1, 2018 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-29310676

RESUMO

BACKGROUND: TGF-ß signaling pathways regulate several crucial processes in female reproduction. AKT is a non-SMAD signaling pathway regulated by TGF-ß ligands essential for oocyte maturation and early embryonic development in the mouse, but its regulatory role in bovine early embryonic development is not well established. Previously, we demonstrated a stimulatory role for follistatin (a binding protein for specific members of TGF-ß superfamily) in early bovine embryonic development. The objectives of the present studies were to determine the functional role of AKT signaling in bovine early embryonic development and embryotrophic actions of follistatin. METHODS: We used AKT inhibitors III and IV as pharmacological inhibitors of AKT signaling pathway during the first 72 h of in vitro embryo culture. Effects of AKT inhibition on early embryonic development and AKT phosphorylation were investigated in the presence or absence of exogenous follistatin. RESULTS: Pharmacological inhibition of AKT signaling resulted in a significant reduction in early embryo cleavage, and development to the 8- to 16-cell and blastocyst stages (d7). Treatment with exogenous follistatin increased AKT phosphorylation and rescued the inhibitory effect of AKT inhibitors III and IV on AKT phosphorylation and early embryonic development. CONCLUSIONS: Collectively, results suggest a potential requirement of AKT for bovine early embryonic development, and suggest a potential role for follistatin in regulation of AKT signaling in early bovine embryos.


Assuntos
Bovinos/embriologia , Desenvolvimento Embrionário , Folistatina/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Animais , Bovinos/metabolismo , Feminino , Folistatina/metabolismo , Folistatina/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo
10.
J Cell Biochem ; 118(5): 959-961, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27813176

RESUMO

Within the last two decades, the advancement in cellular and cancer biology research can be defined by a few foundational pillars-one of the most important ones being the advent of small-molecule inhibitors (SMIs). Due to their several advantages over contemporary cytotoxic drugs, these engineered molecules have not only shed light on a much deeper understanding of the cellular microenvironment and signal transduction pathways, they have also provided hope of achieving clinical benefits of better patient tolerability while surpassing the efficacy of chemotherapeutic agents. It is, therefore, no surprise that research and development of SMIs has witness a steady surge amongst pharmaceutical industries and research institutions. It is, however, safe to assume that the current applications of SMIs are the mere tip of a yet-inconceivable iceberg. J. Cell. Biochem. 118: 959-961, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/uso terapêutico , Antineoplásicos/farmacologia , Aprovação de Drogas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/metabolismo , Medicina de Precisão , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia
11.
J Cell Biochem ; 118(7): 1712-1721, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-27935102

RESUMO

Female germ cell and its intricate milieu regulate key processes of folliculogenesis and early embryonic development. However, the composition and dynamics of the oocyte transcriptome defines its future fertilizing ability which in turn depends on a number of oocyte specific genes whose identities are still unknown. In this context, the construction of buffalo oocyte specific subtracted cDNA library has raised fresh challenges of defining the importance of a battery of oocyte expressed transcripts in oocyte maturation. The present study tried to characterize these hitherto unknown transcripts and further to assess their expression dynamics in buffalo oocytes of different quality. For this purpose, three ESTs were selected from the library and subjected to 5' and 3' RACE for generating their full length sequences. These constructed full length sequences were validated by amplifying them in oocytes. Further these sequences were extensively analyzed for their coding potential and possible role using coding potential calculator and miRNA database. Besides, their expression was monitored during in vitro maturation in good (BCB+) and poor quality (BCB-) oocytes which was interestingly found to be differing significantly. All the three sequences under study were interpreted as long intergenic non-coding RNAs with the possibility of two of them acting as a miRNA precursors. Also, their differential expression trends in competitively diverse oocytes hints at their possible involvement in oocyte maturation and future embryonic development which needs to be explored further. J. Cell. Biochem. 118: 1712-1721, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Búfalos/embriologia , Búfalos/genética , RNA Longo não Codificante/genética , Animais , Biologia Computacional , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Feminino , Biblioteca Gênica , Células Germinativas/citologia , Células Germinativas/metabolismo , Oócitos/citologia , Oócitos/metabolismo
12.
Biol Reprod ; 94(6): 140, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27170440

RESUMO

The CHD family of proteins is characterized by the presence of chromodomains and SNF2-related helicase/ATPase domains, which alter gene expression by modification of chromatin structure. Chd1-null embryos arrest at the peri-implantation stage in mice. However, the functional role of CHD1 during preimplantation development remains unclear, given maternal-derived CHD1 may mask the essential role of CHD1 during this stage in traditional knockout models. The objective of this study was to characterize CHD1 expression and elucidate its functional role in preimplantation development using the bovine model. CHD1 mRNA was elevated after meiotic maturation and remained increased through the 16-cell stage, followed by a sharp decrease at morula to blastocyst stage. Similarly, immunoblot analysis indicated CHD1 protein level is increased after maturation, maintained at high level after fertilization and declined sharply afterwards. CHD1 mRNA level was partially decreased in response to alpha-amanitin (RNA polymerase II inhibitor) treatment, suggesting that CHD1 mRNA in eight-cell embryos is of both maternal and zygotic origin. Results of siRNA-mediated silencing of CHD1 in bovine early embryos demonstrated that the percentages of embryos developing to the 8- to 16-cell and blastocyst stages were both significantly reduced. However, expression of NANOG (inner cell mass marker) and CDX2 (trophectoderm marker) were not affected in CHD1 knockdown blastocysts. In addition, we found that histone variant H3.3 immunostaining is altered in CHD1 knockdown embryos. Knockdown of H3.3 using siRNA resulted in a similar phenotype to CHD1-ablated embryos. Collectively, our results demonstrate that CHD1 is required for bovine early development, and suggest that CHD1 may regulate H3.3 deposition during this period.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário , Histonas/metabolismo , Animais , Bovinos , Feminino
13.
J Cell Biochem ; 116(5): 743-53, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25501625

RESUMO

In this study genome-wide di-methylated H3K4 (H3K4me2) and tri-methylated H3K27 (H3K27me3) modification profiles were analyzed in spermatozoa of buffalo bulls having wide fertility differences. The custom designed 4 × 180 K buffalo (Bubalus bubalis) ChIP-on-chip array was fabricated by employing array-based sequential hybridization using bovine and buffalo genomic DNA for comparative hybridization. The buffalo specific array developed had 177,440 features assembled from Coding sequences, Promoter and CpG regions comprising 2967 unique genes. A total of 84 genes for H3K4me2 and 80 genes for H3K27me3 were found differentially enriched in mature sperm of high and sub-fertile buffalo bulls. Gene Ontology analysis of these genes revealed their association with different cellular functions and biological processes. Genes identified as differentially enriched between high and sub-fertile bulls were found to be involved in the processes of germ cell development, spermatogenesis and embryonic development. This study presents the first genome-wide H3K4me2 and H3K27me3 profiling of buffalo bull sperm. Results provide a list of specific genes which could be made responsible for differential bull fertility.


Assuntos
Búfalos/metabolismo , Fertilidade , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Espermatozoides/metabolismo , Animais , Biomarcadores/metabolismo , Bovinos , Masculino
14.
Biol Reprod ; 93(4): 86, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26289443

RESUMO

The TGF-beta-SMAD signaling pathway is involved in regulation of various aspects of female reproduction. However, the intrinsic functional role of SMADs in early embryogenesis remains poorly understood. Previously, we demonstrated that treatment with follistatin, an activin (TGF-beta superfamily ligand)-binding protein, is beneficial for bovine early embryogenesis and specific embryotropic actions of follistatin are dependent on SMAD4. Because SMAD4 is a common SMAD that can bind both SMAD2/3 and SMAD1/5, the objective of this study was to further determine the intrinsic role of SMAD2/3 in the control of early embryogenesis and delineate if embryotropic actions of follistatin in early embryos are SMAD2/3 dependent. By using a combination of pharmacological and small interfering RNA-mediated inhibition of SMAD2/3 signaling in the presence or absence of follistatin treatment, our results indicate that SMAD2 and SMAD3 are both required for bovine early embryonic development and stimulatory actions of follistatin on 8- to 16-cell and that blastocyst rates, but not early cleavage, are muted when SMAD2/3 signaling is inhibited. SMAD2 deficiency also results in reduced expression of the bovine trophectoderm cell-specific gene CTGF. In conclusion, the present work provides evidence supporting a functional role of SMAD2/3 in bovine early embryogenesis and that specific stimulatory actions of follistatin are not observed in the absence of SMAD2/3 signaling.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Folistatina/farmacologia , Proteína Smad2/genética , Proteína Smad3/genética , Animais , Bovinos , Fator de Crescimento do Tecido Conjuntivo/genética , Técnicas de Cultura Embrionária , Feminino , Fertilização in vitro , Gravidez , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo
15.
Reproduction ; 149(2): 203-12, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25385722

RESUMO

Upstream stimulating factor 1 (USF1) is a basic helix-loop-helix transcription factor that specifically binds to E-box DNA motifs, known cis-elements of key oocyte expressed genes essential for oocyte and early embryonic development. However, the functional and regulatory role of USF1 in bovine oocyte and embryo development is not understood. In this study, we demonstrated that USF1 mRNA is maternal in origin and expressed in a stage specific manner during the course of oocyte maturation and preimplantation embryonic development. Immunocytochemical analysis showed detectable USF1 protein during oocyte maturation and early embryonic development with increased abundance at 8-16-cell stage of embryo development, suggesting a potential role in embryonic genome activation. Knockdown of USF1 in germinal vesicle stage oocytes did not affect meiotic maturation or cumulus expansion, but caused significant changes in mRNA abundance for genes associated with oocyte developmental competence. Furthermore, siRNA-mediated depletion of USF1 in presumptive zygote stage embryos demonstrated that USF1 is required for early embryonic development to the blastocyst stage. A similar (USF2) yet unique (TWIST2) expression pattern during oocyte and early embryonic development for related E-box binding transcription factors known to cooperatively bind USF1 implies a potential link to USF1 action. This study demonstrates that USF1 is a maternally derived transcription factor required for bovine early embryonic development, which also functions in regulation of JY1, GDF9, and FST genes associated with oocyte competence.


Assuntos
Bovinos/embriologia , Desenvolvimento Embrionário/fisiologia , Oócitos/crescimento & desenvolvimento , Fatores Estimuladores Upstream/fisiologia , Animais , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos/química , Embrião de Mamíferos/fisiologia , Feminino , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/química , Oogênese/fisiologia , RNA Mensageiro/análise , RNA Interferente Pequeno/farmacologia , Proteína 1 Relacionada a Twist/análise , Proteína 1 Relacionada a Twist/genética , Fatores Estimuladores Upstream/análise , Fatores Estimuladores Upstream/genética
16.
Mol Reprod Dev ; 82(3): 251-64, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25704641

RESUMO

Brilliant cresyl blue (BCB) is a super-vital stain that has been used to select competent oocytes in different species. One objective of the present study was to assess the relationship between BCB staining, which correlates with an oocyte's developmental potential, and the transcript abundance for select TGFß-superfamily components, SMAD2/3 and SMAD1/5 phosphorylation levels, and oocyte (JY1) and cumulus-cell (CTSB, CTSK, CTSS, and CTSZ) transcript markers in bovine oocytes and/or adjacent cumulus cells. The capacity of exogenous follistatin or JY1 supplementation or cathepsin inhibitor treatment to enhance development of embryos derived from low-quality oocytes, based on BCB staining, was also determined. Cumulus-oocyte complexes (COCs) from abattoir-derived ovaries were subjected to BCB staining, and germinal-vesicle-stage oocytes and cumulus cells were harvested from control, BCB+, and BCB- (low-quality oocyte) groups for real-time PCR or Western-blot analysis. Remaining COCs underwent in vitro maturation, in vitro fertilization, and embryo culture in the presence or absence of the above exogenous supplements. Levels of FST, JY1, BMP15, and SMAD1, 2, 3, and 5 transcripts were higher in BCB+ oocytes whereas CTSB, CTSK, CTSS, and CTSZ mRNA abundance was higher in cumulus cells surrounding BCB- oocytes. Western-blot analysis revealed higher SMAD1/5 and SMAD2/3 phosphorylation in BCB+ than BCB- oocytes. Embryo-culture studies demonstrated that follistatin and cathepsin inhibitor treatment, but not JY-1 treatment, improve the developmental competence of BCB- oocytes. These results contribute to a better understanding of molecular indices of oocyte competence.


Assuntos
Biomarcadores/metabolismo , Células do Cúmulo/fisiologia , Desenvolvimento Embrionário/fisiologia , Oócitos/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Análise de Variância , Animais , Western Blotting , Bovinos , Células do Cúmulo/metabolismo , Primers do DNA/genética , Técnicas de Cultura Embrionária , Fertilização in vitro , Folistatina/farmacologia , Perfilação da Expressão Gênica , Técnicas de Maturação in Vitro de Oócitos , Oócitos/metabolismo , Oxazinas , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Smad/metabolismo
17.
J Biol Chem ; 288(51): 36733-40, 2013 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-24217247

RESUMO

Aldo-keto reductase 1B10 (AKR1B10) protein is a new tumor biomarker in humans. Our previous studies have shown that AKR1B10 is secreted through a lysosome-mediated nonclassical pathway, leading to an increase in the serum of breast cancer patients. This study illuminates the regulatory mechanism of AKR1B10 secretion. The cytosolic AKR1B10 associates with and is translocated to lysosomes by heat shock protein 90α (HSP90α), a chaperone molecule. Ectopic expression of HSP90α significantly increased the secretion of endogenous AKR1B10 and exogenous GFP-AKR1B10 fusion protein when cotransfected. Geldanamycin, a HSP90α inhibitor, dissociated AKR1B10-HSP90α complexes and significantly reduced AKR1B10 secretion in a dose-dependent manner. We characterized the functional domain in AKR1B10 and found that helix 10 (amino acids 233-240), located at the C terminus, regulates AKR1B10 secretion. Targeted point mutations recognized that amino acids Lys-233, Glu-236, and Lys-240 in helix 10 mediate the interaction of AKR1B10 with HSP90α. Together, our data suggest that HSP90α mediates AKR1B10 secretion through binding to its helix 10 domain. This finding is significant in exploiting the use of AKR1B10 in cancer clinics.


Assuntos
Aldeído Redutase/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Lisossomos/metabolismo , Vesículas Secretórias/metabolismo , Aldeído Redutase/química , Aldeído Redutase/genética , Aldo-Ceto Redutases , Benzoquinonas/farmacologia , Linhagem Celular Tumoral , Células HEK293 , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Lactamas Macrocíclicas/farmacologia , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico
18.
Mol Reprod Dev ; 81(4): 350-62, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24436206

RESUMO

Associations between abnormal methylation of spermatozoan DNA with male infertility have been sought in recent years to identify a molecular explanation of differential spermatozoan function. The present work was undertaken to investigate the methylation profile of differentially methylated regions (DMRs) in the IGF2-H19 locus of Bos taurus X Bos indicus crossbred bull spermatozoa. Bulls having more than at least 100 insemination records over a period of 12 years were classified into two groups of five bulls each belonging to low- and high-fertility groups. The IGF2 and H19 DMR sequences in B. indicus cattle were observed to be in absolute homology with B. taurus cattle. The DNA of crossbred bull spermatozoa was isolated, bisulfite treated, and amplified for specific DMR regions using methylation-change-specific primers. The overall degree of methylation at IGF2-H19 DMRs was not found to be significantly different among two groups of bulls. The sixth CTCF binding site (CCCTC) identified in H19 DMR, however, had a significant methylation difference between the high- and low-fertility bulls. It was concluded that alteration of the methylation levels at IGF2-H19 DMRs might not be responsible for the fertility difference of crossbred bulls, although the role played by the specific CTCF binding sites at this locus, which could influence IGF2 expression during spermatogenesis and early embryonic development, deserves further attention.


Assuntos
Doenças dos Bovinos/genética , Bovinos/fisiologia , Metilação de DNA , Hibridização Genética , Infertilidade Masculina/veterinária , Fator de Crescimento Insulin-Like II/genética , RNA Longo não Codificante/genética , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Bovinos/genética , Cruzamentos Genéticos , Criopreservação , DNA/genética , Feminino , Infertilidade Masculina/genética , Fator de Crescimento Insulin-Like II/metabolismo , Masculino , Gravidez , Taxa de Gravidez , RNA Longo não Codificante/metabolismo , Proteínas Repressoras/metabolismo , Análise do Sêmen , Preservação do Sêmen , Homologia de Sequência do Ácido Nucleico , Espermatogênese/genética , Espermatozoides/fisiologia , Espermatozoides/ultraestrutura
19.
Reprod Biol Endocrinol ; 12: 67, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25027287

RESUMO

BACKGROUND: We previously demonstrated embryotrophic actions of maternal (oocyte-derived) follistatin during bovine early embryogenesis. Classical actions of follistatin are attributed to inhibition of activity of growth factors including activins and bone morphogenetic proteins (BMP). However, temporal changes in BMP mRNA in early bovine embryos and the effects of exogenous BMP on embryo developmental progression are not understood. The objectives of present studies were to characterize mRNA abundance for select BMP, BMP receptors and BMP receptor associated SMADs during bovine oocyte maturation and early embryogenesis and determine effects of addition of exogenous BMP protein on early development. METHODS: Relative abundance of mRNA for BMP2, BMP3, BMP7, BMP10, SMAD1, SMAD5, ALK3, ALK6, ALK2, BMPR2, ACVR2A and ACVR2B was determined by RT-qPCR analysis of germinal vesicle (GV) and in vitro matured metaphase II (MII) oocytes and in vitro produced embryos collected at pronuclear, 2-cell (C), 4C, 8C, 16C, morula and blastocyst stages. Effects of addition of recombinant human BMP2 (0, 1, 10 and 100 ng/ml) during initial 72 h of embryo culture on early cleavage (within 30 h post insemination), total cleavage, development to 8C-16C and blastocyst stages and blastocyst mRNA abundance for markers of inner cell mass (NANOG) and trophectoderm (CDX2) were also determined. RESULTS: Abundance of mRNA for BMP2, BMP10, SMAD1, SMAD5, ALK3, ALK2, BMPR2 and ACVR2B was elevated in MII oocytes and/or pronuclear stage embryos (relative to GV) and remained elevated through the 8C -16C stages, whereas BMP3, BMP7 and ALK2 mRNAs were transiently elevated. Culture of embryos to the 8C stage in the presence of α-amanitin resulted in increased abundance for all of above transcripts examined relative to untreated 8C embryos. Effects of addition of exogenous BMP2 on early cleavage rates and rates of development to 8C-16C and blastocyst stages were not observed, but BMP2 treatment increased blastocyst mRNA for CDX2 and NANOG. CONCLUSIONS: Abundance of maternally derived mRNAs for above BMP system components are dynamically regulated during oocyte maturation and early embryogenesis. Exogenous BMP2 treatment does not influence progression to various developmental endpoints, but impacts characteristics of resulting blastocysts. Results support a potential role for BMPs in bovine early embryogenesis.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Ectogênese , RNA Mensageiro/metabolismo , Proteínas Smad/metabolismo , Regulação para Cima , Alfa-Amanitina/farmacologia , Animais , Biomarcadores/metabolismo , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Proteína Morfogenética Óssea 2/genética , Receptores de Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/genética , Bovinos , Fase de Clivagem do Zigoto/citologia , Fase de Clivagem do Zigoto/efeitos dos fármacos , Fase de Clivagem do Zigoto/metabolismo , Ectogênese/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Técnicas de Maturação in Vitro de Oócitos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Smad/genética , Regulação para Cima/efeitos dos fármacos
20.
Front Reprod Health ; 6: 1330161, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38406668

RESUMO

Mitogen-activated protein kinases (MAPKs) represent widely expressed and evolutionarily conserved proteins crucial for governing signaling pathways and playing essential roles in mammalian male reproductive processes. These proteins facilitate the transmission of signals through phosphorylation cascades, regulating diverse intracellular functions encompassing germ cell development in testis, physiological maturation of spermatozoa within the epididymis, and motility regulation at ejaculation in the female reproductive tract. The conservation of these mechanisms appears prevalent across species, including humans, mice, and, to a limited extent, livestock species such as bovines. In Sertoli cells (SCs), MAPK signaling not only regulates the proliferation of immature SCs but also determines the appropriate number of SCs in the testes at puberty, thereby maintaining male fertility by ensuring the capacity for sperm cell production. In germ cells, MAPKs play a crucial role in dynamically regulating testicular cell-cell junctions, supporting germ cell proliferation and differentiation. Throughout spermatogenesis, MAPK signaling ensures the appropriate Sertoli-to-germ cell ratio by regulating apoptosis, controlling the metabolism of developing germ cells, and facilitating the maturation of spermatozoa within the cauda epididymis. During ejaculation in the female reproductive tract, MAPKs regulate two pivotal events-capacitation and the acrosome reaction essential for maintaining the fertility potential of sperm cells. Any disruptions in MAPK pathway signaling possibly may disturb the testicular microenvironment homeostasis, sperm physiology in the male body before ejaculation and in the female reproductive tract during fertilization, ultimately compromising male fertility. Despite decades of research, the physiological function of MAPK pathways in male reproductive health remains inadequately understood. The current review attempts to combine recent findings to elucidate the impact of MAPK signaling on male fertility and proposes future directions to enhance our understanding of male reproductive functions.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA