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1.
Proc Natl Acad Sci U S A ; 113(51): 14492-14501, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-27940919

RESUMO

A major unresolved issue in the cloning of mammals by somatic cell nuclear transfer (SCNT) is the mechanism by which the process fails after embryos are transferred to the uterus of recipients before or during the implantation window. We investigated this problem by using RNA sequencing (RNA-seq) to compare the transcriptomes in cattle conceptuses produced by SCNT and artificial insemination (AI) at day (d) 18 (preimplantation) and d 34 (postimplantation) of gestation. In addition, endometrium was profiled to identify the communication pathways that might be affected by the presence of a cloned conceptus, ultimately leading to mortality before or during the implantation window. At d 18, the effects on the transcriptome associated with SCNT were massive, involving more than 5,000 differentially expressed genes (DEGs). Among them are 121 genes that have embryonic lethal phenotypes in mice, cause defects in trophoblast and placental development, and/or affect conceptus survival in mice. In endometria at d 18, <0.4% of expressed genes were affected by the presence of a cloned conceptus, whereas at d 34, ∼36% and <0.7% of genes were differentially expressed in intercaruncular and caruncular tissues, respectively. Functional analysis of DEGs in placental and endometrial tissues suggests a major disruption of signaling between the cloned conceptus and the endometrium, particularly the intercaruncular tissue. Our results support a "bottleneck" model for cloned conceptus survival during the periimplantation period determined by gene expression levels in extraembryonic tissues and the endometrial response to altered signaling from clones.


Assuntos
Endométrio/metabolismo , Placenta/metabolismo , Prenhez , Transdução de Sinais , Transcriptoma , Animais , Bovinos , Clonagem de Organismos , Implantação do Embrião , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Inseminação Artificial , Técnicas de Transferência Nuclear , Placentação , Gravidez , Fatores de Tempo , Trofoblastos/metabolismo , Útero/metabolismo
2.
Mol Reprod Dev ; 80(12): 977-87, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24038527

RESUMO

We determined if somatic cell nuclear transfer (SCNT) cloning is associated with WNT-related gene expression in cattle development, and if the expression of genes in the WNT pathway changes during the peri-implantation period. Extra-embryonic and endometrial tissues were collected at gestation days 18 and 34 (d18, d34). WNT5A, FZD4, FZD5, LRP5, CTNNB1, GNAI2, KDM1A, BCL2L1, and SFRP1 transcripts were localized in extra-embryonic tissue, whereas SFRP1 and DKK1 were localized in the endometrium. There were no differences in the localization of these transcripts in extra-embryonic tissue or endometrium from SCNT or artificial insemination (AI) pregnancies. Expression levels of WNT5A were 11-fold greater in the allantois of SCNT than AI samples. In the trophoblast, expression of WNT5A, FZD5, CTNNB1, and DKK1 increased significantly from d18 to d34, whereas expression of KDM1A and SFRP1 decreased, indicating that implantation is associated with major changes in WNT signaling. SCNT was associated with altered WNT5A expression in trophoblasts, with levels increasing 2.3-fold more in AI than SCNT conceptuses from d18 to d34. In the allantois, expression of WNT5A increased 6.3-fold more in SCNT than AI conceptuses from d18 to d34. Endometrial tissue expression levels of the genes tested did not differ between AI or SCNT pregnancies, although expression of individual genes showed variation across developmental stages. Our results demonstrate that SCNT is associated with altered expression of specific WNT-related genes in extra-embryonic tissue in a time- and tissue-specific manner. The pattern of gene expression in the WNT pathway suggests that noncanonical WNT signal transduction is important for implantation of cattle conceptuses.


Assuntos
Implantação do Embrião/genética , Endométrio/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Transferência Nuclear , Via de Sinalização Wnt/genética , Alantoide/metabolismo , Animais , Blastocisto/fisiologia , Bovinos , Clonagem de Organismos , Endométrio/metabolismo , Feminino , Expressão Gênica , Inseminação Artificial , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas Wnt/biossíntese , Proteínas Wnt/metabolismo
3.
Cytokine ; 28(1): 25-8, 2004 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-15341922

RESUMO

We have characterized the expression of six cytokine mRNAs in highly purified B cells from bovine leukemia virus (BLV)-infected cows with persistent lymphocytosis. Selected cytokine mRNAs included those encoding tumor necrosis factor (TNF), lymphotoxin-alpha (LT-alpha), transforming growth factor-beta1 (TGF-beta1), interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and interleukin-10 (IL-10). Fresh B cells from cows with persistent lymphocytosis constitutively transcribed TNF, LT-alpha and TGF-beta1 mRNAs. Although IL-1beta, IL-6 and IL-10 mRNAs were barely detectable in fresh B cells from cows with persistent lymphocytosis, transcripts encoding these cytokines were strongly and rapidly upregulated in B cells after cell culture. Results from this study provide the first evidence that B cells infected with BLV express specific cytokine mRNAs in vivo.


Assuntos
Linfócitos B/imunologia , Citocinas/genética , Leucose Enzoótica Bovina/imunologia , Animais , Sequência de Bases , Bovinos , Primers do DNA , Regulação da Expressão Gênica/imunologia , Interleucinas/genética , Vírus da Leucemia Bovina , RNA Mensageiro/genética
4.
Virology ; 304(1): 1-9, 2002 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-12490398

RESUMO

The role of T-helper (Th) responses in the subclinical progression of bovine leukemia virus (BLV) infection was explored by determining the contribution of CD4+ T cells to the expression of mRNAs encoding interferon-gamma (IFN-gamma), interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10) in BLV-infected cattle. Relative levels of mRNA encoding IFN-gamma, IL-2, IL-4, and IL-10 were measured in fresh and concanavalin A (Con A) activated peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells from cows seronegative to BLV (BLV-), seropositive without persistent lymphocytosis (BLV+PL-), and seropositive with PL (BLV+PL+) using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. The expressions of IFN-gamma, IL-2, and IL-4 mRNAs were significantly reduced in the PBMCs from BLV+PL+ cows as compared to BLV- cows. Reduced levels of IL-2 and IL-4 mRNAs were detected in fresh CD4+ T cells from BLV+PL+ cows. In contrast, Con A stimulated PBMCs and CD4+ T cells did not differ significantly in expression of IFN-gamma, IL-2, IL-10, or IL-4 mRNAs among the BLV infection groups. Using flow-sorted CD4+ T cells and semiquantitative RT-PCR the frequencies of CD4+ T cells transcribing IFN-gamma, IL-2, IL-4, and IL-10 mRNAs in the peripheral blood of BLV-, BLV+PL-, and BLV+PL+ cows were determined. There were no significant differences in the frequencies of CD4+ T cells expressing these cytokine mRNAs among animals in the different BLV infection categories. Thus, the observed differences in IL-2 and IL-4 mRNAs in CD4+ T cells were due to changes in steady-state mRNA levels expressed by individual cells and not to changes in the frequency of cells transcribing IL-2 and IL-4 mRNAs. These results demonstrate that the progression of BLV infection to PL is associated with reduced expression of classical Th1 and Th2 cytokines by CD4+ T cells, thus suggesting aberrant Th regulation in subclinically infected animals.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Leucose Enzoótica Bovina/imunologia , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Linfocitose/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Bovinos , Leucose Enzoótica Bovina/metabolismo , Leucose Enzoótica Bovina/patologia , Leucose Enzoótica Bovina/virologia , Feminino , Expressão Gênica , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Vírus da Leucemia Bovina , RNA Mensageiro/biossíntese
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