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1.
Nat Genet ; 39(3): 295-302, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17325680

RESUMO

Therapeutic cloning, whereby somatic cell nuclear transfer (SCNT) is used to generate patient-specific embryonic stem cells (ESCs) from blastocysts cloned by nuclear transfer (ntESCs), holds great promise for the treatment of many human diseases. ntESCs have been derived in mice and cattle, but thus far there are no credible reports of human ntESCs. Here we review the recent literature on nuclear reprogramming by SCNT, including studies of gene expression, DNA methylation, chromatin remodeling, genomic imprinting and X chromosome inactivation. Reprogramming of genes expressed in the inner cell mass, from which ntESCs are derived, seems to be highly efficient. Defects in the extraembryonic lineage are probably the major cause of the low success rate of reproductive cloning but are not expected to affect the derivation of ntESCs. We remain optimistic that human therapeutic cloning is achievable and that the derivation of patient-specific ntESC lines will have great potential for regenerative medicine.


Assuntos
Reprogramação Celular , Clonagem de Organismos , Embrião de Mamíferos/metabolismo , Epigênese Genética , Técnicas de Transferência Nuclear , Animais , Bovinos , Células-Tronco Embrionárias/metabolismo , Feminino , Humanos , Camundongos , Modelos Biológicos
2.
Nat Genet ; 38(11): 1323-8, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17013394

RESUMO

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.


Assuntos
Células-Tronco Adultas/fisiologia , Diferenciação Celular/fisiologia , Clonagem de Organismos/métodos , Células-Tronco Hematopoéticas/citologia , Técnicas de Transferência Nuclear , Células-Tronco Adultas/citologia , Animais , Embrião de Mamíferos/citologia , Feminino , Perfilação da Expressão Gênica , Granulócitos/citologia , Granulócitos/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Modelos Biológicos , Gravidez , Células-Tronco/citologia , Células-Tronco/fisiologia
3.
Nat Genet ; 31(2): 216-20, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12032569

RESUMO

In mammals, epigenetic marks on the X chromosomes are involved in dosage compensation. Specifically, they are required for X chromosome inactivation (XCI), the random transcriptional silencing of one of the two X chromosomes in female cells during late blastocyst development. During natural reproduction, both X chromosomes are active in the female zygote. In somatic-cell cloning, however, the cloned embryos receive one active (Xa) and one inactive (Xi) X chromosome from the donor cells. Patterns of XCIhave been reported normal in cloned mice, but have yet to be investigated in other species. We examined allele-specific expression of the X-linked monoamine oxidase type A (MAOA) gene and the expression of nine additional X-linked genes in nine cloned XX calves. We found aberrant expression patterns in nine of ten X-linked genes and hypomethylation of Xist in organs of deceased clones. Analysis of MAOA expression in bovine placentae from natural reproduction revealed imprinted XCI with preferential inactivation of the paternal X chromosome. In contrast, we found random XCI in placentae of the deceased clones but completely skewed XCI in that of live clones. Thus, incomplete nuclear reprogramming may generate abnormal epigenetic marks on the X chromosomes of cloned cattle, affecting both random and imprinted XCI.


Assuntos
Clonagem de Organismos , Mecanismo Genético de Compensação de Dose , Cromossomo X/genética , Animais , Bovinos , Metilação de DNA , Feminino , Dados de Sequência Molecular , Monoaminoxidase/genética , RNA Longo não Codificante , RNA não Traduzido/genética , Fatores de Transcrição/genética
4.
Proc Natl Acad Sci U S A ; 106(14): 5687-92, 2009 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-19297625

RESUMO

Implantation is crucial for placental development that will subsequently impact fetal growth and pregnancy success with consequences on postnatal health. We postulated that the pattern of genes expressed by the endometrium when the embryo becomes attached to the mother uterus could account for the final outcome of a pregnancy. As a model, we used the bovine species where the embryo becomes progressively and permanently attached to the endometrium from day 20 of gestation onwards. At that stage, we compared the endometrial genes profiles in the presence of an in vivo fertilized embryo (AI) with the endometrial patterns obtained in the presence of nuclear transfer (SCNT) or in vitro fertilized embryos (IVF), both displaying lower and different potentials for term development. Our data provide evidence that the endometrium can be considered as a biological sensor able to fine-tune its physiology in response to the presence of embryos whose development will become altered much later after the implantation process. Compared with AI, numerous biological functions and several canonical pathways with a major impact on metabolism and immune function were found to be significantly altered in the endometrium of SCNT pregnancies at implantation, whereas the differences were less pronounced with IVF embryos. Determining the limits of the endometrial plasticity at the onset of implantation should bring new insights on the contribution of the maternal environment to the development of an embryo and the success of pregnancy.


Assuntos
Endométrio/embriologia , Fertilização in vitro , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Transferência Nuclear , Resultado da Gravidez/genética , Animais , Bovinos , Implantação do Embrião , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Endométrio/metabolismo , Endométrio/fisiologia , Feminino , Fertilização , Gravidez
5.
Reproduction ; 141(1): 79-89, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20926692

RESUMO

Axis specification in mouse is determined by a sequence of reciprocal interactions between embryonic and extra-embryonic tissues so that a few extra-embryonic genes appear as 'patterning' the embryo. Considering these interactions as essential, but lacking in most mammals the genetically driven approaches used in mouse and the corresponding patterning mutants, we examined whether a molecular signature originating from extra-embryonic tissues could relate to the developmental stage of the embryo proper and predict it. To this end, we have profiled bovine extra-embryonic tissues at peri-implantation stages, when gastrulation and early neurulation occur, and analysed the subsequent expression profiles through the use of predictive methods as previously reported for tumour classification. A set of six genes (CALM1, CPA3, CITED1, DLD, HNRNPDL, and TGFB3), half of which had not been previously associated with any extra-embryonic feature, appeared significantly discriminative and mainly dependent on embryonic tissues for its faithful expression. The predictive value of this set of genes for gastrulation and early neurulation stages, as assessed on naive samples, was remarkably high (93%). In silico connected to the bovine orthologues of the mouse patterning genes, this gene set is proposed as a new trait for embryo staging. As such, this will allow saving the bovine embryo proper for molecular or cellular studies. To us, it offers as well new perspectives for developmental phenotyping and modelling of embryonic/extra-embryonic co-differentiation.


Assuntos
Padronização Corporal/genética , Embrião de Mamíferos/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos , Neurulação/genética , Animais , Bovinos , Bases de Dados Genéticas , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Genótipo , Idade Gestacional , Inseminação Artificial , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo
6.
Mol Reprod Dev ; 78(9): 642-50, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21786363

RESUMO

During the past several decades, in vitro fertilization (IVF) has been increasingly used both in animal production and human infertility treatment. Animals derived from in vitro manipulation are occasionally associated with abnormal offspring syndrome (AOS) and other developmental abnormalities. By studying gene expression of in vitro-produced (IVP) embryos/animals, we gain an indicator of how well this procedure mimics the in vivo environment. Most previous studies of this nature have focused on only a few genes at a time or have been limited to studying the pre-implantation stage; a global view of how gene transcription may be influenced by in vitro procedures during fetal development has yet to be ascertained. To this end, we collected liver and placental tissue samples from IVP and in vivo control bovine fetuses at days 90 and 180 of gestation. We used a bovine 13K oligonucleotide microarray to investigate the transcriptional profiles in both tissues from IVP fetuses, and compared them with those of their age-matched in vivo counterparts. Surprisingly, in both liver and placental tissues, the transcriptional profiles between IVP and control fetuses, at either 90 or 180 days of gestation, were indistinguishable. A total of 879 genes were found to be significantly regulated during liver development from 90 to 180 days of gestation, but there were no gene expression changes in the placental tissue during this developmental period. Quantitative real-time RT-PCR on 11 selected genes confirmed these results. Our results have certain implications for IVF technologies, both in agriculture and in human medicine.


Assuntos
Bovinos/embriologia , Bovinos/genética , Fertilização in vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento , Transcriptoma/genética , Animais , Bovinos/metabolismo , Feminino , Perfilação da Expressão Gênica , Fígado/embriologia , Fígado/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Placenta/metabolismo , Placentação , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes
7.
Reprod Biomed Online ; 22(2): 184-91, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21196132

RESUMO

During vitrification, the glass-like solidification is the phase-transition process from liquid to solid. Phase transition is one of the major factors suspected to affect the physiology of the oocyte, such as the structure of the meiotic spindle. Therefore, it is very important to investigate the systematic and morphological alterations of the metaphase-II spindle and chromosome arrangement during complete course of a vitrification and warming process. B6D2F1 (C57BL/6 X DBA/2) mouse oocytes were cryopreserved by minimum volume cooling (MVC) method of vitrification in a solution with 15% ethylene glycol, 15% dimethylsulphoxide and 0.5 mol/l sucrose. To examine the spindle, oocytes were fixed before, during and after vitrification and were analysed by immunocytochemistry and confocal microscopy. It was shown that spindles in all oocytes could be maintained through the vitrification and warming process, even though they were exposed to extreme temperature and two rounds of phase transition. According to the sequential observations, chromosome alignment was maintained throughout the complete course of vitrification, warming and post-warming stage. The impact of phase transition was barely detectable when the oocyte was exposed to the vitrification and warming process. The oocyte spindle was able to recover immediately after warming.


Assuntos
Criopreservação , Oócitos , Transição de Fase/efeitos dos fármacos , Fuso Acromático/ultraestrutura , Animais , Cromossomos/efeitos dos fármacos , Crioprotetores/farmacologia , Feminino , Camundongos , Fuso Acromático/efeitos dos fármacos
8.
Biol Reprod ; 83(2): 177-84, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20410454

RESUMO

Tetraploid (4N) complementation assay is regard as the most stringent characterization test for the pluripotency of embryonic stem (ES) cells. The technology can generate mice fully derived from the injected ES cell (ES-4N) with 4N placentas. However, it remains a very inefficient procedure owing to a lack of information on the optimal conditions for ES incorporation into the 4N embryos. In the present study, we injected ES cells from embryos of natural fertilization (fES) and somatic cell nuclear transfer (ntES) into 4N embryos at various stages of development to determine the optimal stage of ES cells integration by comparing the efficiency of full-term ES-4N mouse generation. Our results demonstrate that fES/ntES cells can be incorporated into 4N embryos at 2-cell, 4-cell and blastocyst stages and full-term mice can be generated. Interestingly, ntES cells injected into the 4-cell group resulted in the lowest efficiency (5.6%) compared to the 2-cell (13.8%, P > 0.05) and blastocyst (16.7%, P < 0.05) stages. Because 4N embryos start to form compacted morulae at the 4-cell stage, we investigated whether the lower efficiency at this stage was due to early compaction by injecting ntES cells into artificially de-compacted embryos treated with calcium free medium. Although the treatment changed the embryonic morphology, it did not increase the efficiency of ES-4N mice generation. Immunochemistry of the cytoskeleton displayed microtubule and microfilament polarization at the late 4-cell stage in 4N embryos, which suggests that de-compaction treatment cannot reverse the polarization process. Taken together, we show here that a wide developmental range of 4N embryos can be used for 4N complementation and embryo polarization and compaction may restrict incorporation of ES cells into 4N embryos.


Assuntos
Embrião de Mamíferos , Desenvolvimento Embrionário , Células-Tronco Embrionárias/fisiologia , Poliploidia , Animais , Blastocisto , Diploide , Transferência Embrionária , Células-Tronco Embrionárias/transplante , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Transferência Nuclear , Células-Tronco Pluripotentes/fisiologia , Células-Tronco Pluripotentes/transplante , Pseudogravidez , Quimeras de Transplante
9.
J Biomed Biotechnol ; 2010: 527539, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20589073

RESUMO

Imprinted in placenta and liver (IPL) gene has been identified as an imprinted gene in the mouse and human. Its sequence and imprinting status, however, have not been determined in the domestic pigs. In the present study, a 259 base pair-specific sequence for IPL gene of the domestic pig was obtained and a novel SNP, a T/C transition, was identified in IPL exon 1. The C allele of this polymorphism was found to be the predominant allele in Landrace,Yorkshire, and Duroc. The frequency of CC genotype and C allele are different in Duroc as compared with Yorkshire (P = .038 and P = .005, resp.). Variable imprinting status of this gene was observed in different developmental stages. For example, it is imprinted in 1-day old newborns (expressed from the maternal allele), but imprinting was lost in 180-day-old adult (expressed from both parental alleles). Real-time PCR analysis showed the porcine IPL gene is expressed in all tested eight organ/tissues. The expression level was significantly higher in spleen, duodenum, lung, and bladder of 180-day-old Lantang adult compared to that in 1-day-old newborns Lantang pigs (P < .05). In conclusion, the imprinting of the porcine IPL gene is developmental stage and tissue specific.


Assuntos
Animais Recém-Nascidos , Impressão Genômica/genética , Proteínas Nucleares/genética , Polimorfismo Genético , Fatores Etários , Animais , Animais Recém-Nascidos/genética , Animais Recém-Nascidos/crescimento & desenvolvimento , Animais Recém-Nascidos/metabolismo , Clonagem Molecular , Frequência do Gene , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Suínos
10.
Nat Biotechnol ; 25(1): 77-83, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17211406

RESUMO

Research on, and commercialization of, cloned cattle has been conducted for more than 20 years. Early techniques relied on the physical splitting of embryos or using embryo cells for nuclear transfer to generate cloned animals. Milk and meat from these animals entered into the human food market with no evidence of problems. With the advent of nuclear transfer, which enables the direct transference and preservation of high-value meat- and milk-producing genotypes to offspring, concerns have been raised about whether the products from somatic cell nuclear transfer-produced animals are safe for human consumption. Studies on the biochemical properties of food products from cloned and noncloned animals have thus far not detected any differences. All data to date indicate no significant differences in the measured parameters between animals created by nuclear transfer and normally bred animals. Public acceptance of cloned animal products depends upon forthcoming US Food and Drug Administration approval along with convincing safety data.


Assuntos
Animais Geneticamente Modificados , Clonagem de Organismos , Alimentos Geneticamente Modificados , Carne/análise , Leite/química , Medição de Risco/métodos , Fatores de Risco , Animais , Qualidade de Produtos para o Consumidor , Análise de Alimentos/métodos
11.
Physiol Genomics ; 39(1): 14-27, 2009 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-19622795

RESUMO

At implantation the endometrium undergoes modifications necessary for its physical interactions with the trophoblast as well as the development of the conceptus. We aim to identify endometrial factors and pathways essential for a successful implantation in the caruncular (C) and the intercaruncular (IC) areas in cattle. Using a 13,257-element bovine oligonucleotide array, we established expression profiles at day 20 of the estrous cycle or pregnancy (implantation), revealing 446 and 1,295 differentially expressed genes (DEG) in C and IC areas, respectively (false discovery rate = 0.08). The impact of the conceptus was higher on the immune response function in C but more prominent on the regulation of metabolism function in IC. The C vs. IC direct comparison revealed 1,177 and 453 DEG in cyclic and pregnant animals respectively (false discovery rate = 0.05), with a major impact of the conceptus on metabolism and cell adhesion. We selected 15 genes including C11ORF34, CXCL12, CXCR4, PLAC8, SCARA5, and NPY and confirmed their differential expression by quantitative RT-PCR. The cellular localization was analyzed by in situ hybridization and, upon pregnancy, showed gene-specific patterns of cell distribution, including a high level of expression in the luminal epithelium for C11ORF34 and MX1. Using primary cultures of bovine endometrial cells, we identified PTN, PLAC8, and CXCL12 as interferon-tau (IFNT) target genes and MSX1 and CXCR7 as IFNT-regulated genes, whereas C11ORF34 was not an IFNT-regulated gene. Our transcriptomic data provide novel molecular insights accounting for the biological functions related to the C or IC endometrial areas and may contribute to the identification of potential biomarkers for normal and perturbed early pregnancy.


Assuntos
Bovinos/genética , Implantação do Embrião/genética , Endométrio/metabolismo , Perfilação da Expressão Gênica , Animais , Implantação do Embrião/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Ciclo Estral/efeitos dos fármacos , Ciclo Estral/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Interferon Tipo I/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Proteínas da Gravidez/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo
12.
Mol Reprod Dev ; 76(1): 38-47, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18449896

RESUMO

In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture on bovine embryos, we compared the expression profiles of single blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF); (2) in vivo maturation, fertilization and in vitro culture (IVD); and (3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367, and 200 genes differentially expressed between the AI and IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category "RNA processing" was over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on the ability to transcribe maternal RNA stores. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI with IVD embryos.


Assuntos
Técnicas de Cultura Embrionária , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Fertilização in vitro , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Bovinos , Epigênese Genética/genética , Cromossomo X/genética
13.
Reprod Fertil Dev ; 21(1): 60-6, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19152746

RESUMO

Studies on cloned pigs are scant compared with those in mice and cattle. Expression profiles of cloned pig embryos on full-term cloned pigs are even more limited owing to the limited availability of DNA microarray technology in the pig. We have conducted expression profile comparisons between pigs from somatic cell nuclear transfer and pigs from conventional breeding at birth and 1 month of age. Differentially expressed genes that are subjected to DNA methylation were also examined for their DNA methylation status. These data will be presented in the 2009 Annual Meeting of the International Embryo Transfer Society in San Diego. In the present review, we focus on summarising existing findings on epigenetic and other changes in cloned embryo, cloned pigs and their offspring by conventional breeding.


Assuntos
Clonagem de Organismos , Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento/genética , Sus scrofa , Animais , Perfilação da Expressão Gênica , Impressão Genômica/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Telômero/genética
14.
Reprod Fertil Dev ; 21(1): 37-44, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19152744

RESUMO

Somatic cells can be reprogrammed to a totipotent state through nuclear transfer or cloning, because it has been demonstrated that the oocyte has the ability to reprogramme an adult nucleus into an embryonic state that can initiate the development of a new organism. Therapeutic cloning, whereby nuclear transfer is used to derive patient-specific embryonic stem cells, embraces an entire new opportunity for regenerative medicine. However, a key obstacle for human therapeutic cloning is that the source of fresh human oocytes is extremely limited. In the present review, we propose prospective sources of human oocytes by using oocyte cryopreservation, such as an oocyte bank and immature oocytes. We also address some potential issues associated with nuclear transfer when using cryopreserved oocytes. In the future, if the efficacy and efficiency of cryopreserved oocytes are comparable to those of fresh oocytes in human therapeutic cloning, the use of cryopreserved oocytes would be invaluable and generate a great impact to regenerative medicine.


Assuntos
Criopreservação/métodos , Técnicas de Transferência Nuclear , Oócitos/citologia , Medicina Regenerativa/métodos , Animais , Humanos , Medicina Regenerativa/tendências
15.
Reprod Fertil Dev ; 21(1): 22-30, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19152742

RESUMO

Microarray technology enables the interrogation of thousands of genes at one time and therefore a systems level of analysis. Recent advances in the amplification of RNA, genome sequencing and annotation, and the lower cost of developing microarrays or purchasing them commercially, have facilitated the analysis of single preimplantation embryos. The present review discusses the components of embryonic expression profiling and examines current research that has used microarrays to study the effects of in vitro production and nuclear transfer.


Assuntos
Fertilização in vitro/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos
16.
Theriogenology ; 140: 109-116, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31473493

RESUMO

The study was designed to determine the impact of magnesium (Mg2+) on bovine embryo development. We found that two commercially available sources of bovine serum albumin (BSA) and fetal bovine serum (FBS) contained different amounts of Mg2+ residue: 4 ppm in ICPbio BSA, 114 ppm in Sigma BSA, and 44 ppm in FBS. When CR1 was used as basal medium, PVA and ICPbio BSA produced the lowest blastocyst yield (2.2-2.3%), whereas Sigma BSA increased blastocyst yield to 18.9% (P < 0.05). Supplementation of 1.4 mM MgCl2 into the medium increased the blastocyst rate in the ICPbio BSA group (29.4%) but not in the PVA group (5.4%; P < 0.05) to a level comparable to that of the FBS group (33.7%; P > 0.05). We next found that increasing concentrations of MgCl2 in the culture medium (ICPbio BSA) elevated blastocyst rate from 2.6% (0 mM), 38.4% (0.35 mM) to 50.2% (1.4 mM; P < 0.05), further maintained at 44.9% (2.1 mM) and 43.4% (2.8 mM) (P > 0.05). However, blastocyst rate was reduced to 31.4% (4.2 mM) and 29.4% (5.6 mM) when MgCl2 supplement was increased (P < 0.05). Comparable blastocyst development was achieved in both ICPbio BSA (30.0-33.1%) and Sigma BSA (37.4-38.7%) groups when 1.4 mM Mg2+ was supplemented regardless of its source (MgCl2 vs. MgSO4; P > 0.05). In embryo transfer experiments, higher rates of pregnancy (54.3 vs. 41.5%) and calving (44.3 vs. 32.5%) were achieved in the CR1-Mg2+-supplemented BSA group compared with the FBS group with co-culture, respectively (P < 0.05). These results demonstrate that Mg2+ is a key ion that promotes competent blastocyst and term development. Therefore, a simple and efficient defined medium (CR1-Mg2+-BSA) can successfully replace complex serum and somatic cell co-culture.


Assuntos
Bovinos/embriologia , Desenvolvimento Embrionário/efeitos dos fármacos , Magnésio/farmacologia , Animais , Técnicas de Cultura Embrionária/veterinária , Magnésio/fisiologia
17.
Physiol Genomics ; 33(1): 65-77, 2008 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-18089771

RESUMO

Transcription profiling of placentomes derived from somatic cell nuclear transfer (SCNT, n = 20), in vitro fertilization (IVF, n = 9), and artificial insemination (AI, n = 9) at or near term development was performed to better understand why SCNT and IVF often result in placental defects, hydrops, and large offspring syndrome (LOS). Multivariate analysis of variance was used to distinguish the effects of SCNT, IVF, and AI on gene expression, taking into account the effects of parturition (term or preterm), sex of fetus, breed of dam, breed of fetus, and pathological finding in the offspring (hydrops, normal, or other abnormalities). Differential expression of 20 physiologically important genes was confirmed with quantitative PCR. The largest effect on placentome gene expression was attributable to whether placentas were collected at term or preterm (i.e., whether the collection was because of disease or to obtain stage-matched controls) followed by placentome source (AI, IVF, or SCNT). Gene expression in SCNT placentomes was dramatically different from AI (n = 336 genes; 276 >2-fold) and from IVF (n = 733 genes; 162 >2-fold) placentomes. Functional analysis of differentially expressed genes (DEG) showed that IVF has significant effects on genes associated with cellular metabolism. In contrast, DEG associated with SCNT are involved in multiple pathways, including cell cycle, cell death, and gene expression. Many DEG were shared between the gene lists for IVF and SCNT comparisons, suggesting that common pathways are affected by the embryo culture methods used for IVF and SCNT. However, the many unique gene functions and pathways affected by SCNT suggest that cloned fetuses may be starved and accumulating toxic wastes due to placental insufficiency caused by reprogramming errors. Many of these genes are candidates for hydrops and LOS.


Assuntos
Bovinos/genética , Clonagem de Organismos , Perfilação da Expressão Gênica , Técnicas de Transferência Nuclear , Placenta/metabolismo , Animais , Células Cultivadas , Análise por Conglomerados , Embrião de Mamíferos , Feminino , Fertilização in vitro , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Gravidez
18.
Anim Biotechnol ; 19(1): 22-7, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18228173

RESUMO

The genes insulin-like growth factor 2 (IGF2) and H19 express paternally and maternally, respectively, in humans, mice, sheep, and cattle. Additionally, IGF2 has been shown to be regulated by at least four promoters in a tissue- or development-specific manner. In the domestic pigs, the promoter- and tissue-specific imprinting pattern of IGF2 has not been well characterized, nor is the imprinting pattern of H19. In the present study, we identified two polymorphisms in each of IGF2 (exons 2 and 9) and H19 (exons 1 and 5) and determined the imprinting status of these two genes in 13 organs / tissues of week-old pigs. IGF2 P1 transcript is bi-allelically expressed (not imprinted) in all major organs studied, while the majority of IGF2 transcripts are expressed from promoters 2-4 and are imprinted. H19 is exclusively expressed from the maternal allele in all major organs, concurrent with observations in other species.


Assuntos
Impressão Genômica , Fator de Crescimento Insulin-Like II/genética , RNA não Traduzido/genética , Suínos/genética , Animais , Genoma , Masculino , Polimorfismo Genético , RNA Longo não Codificante , Sêmen/citologia , Sêmen/fisiologia , Pele/citologia
19.
20.
Biol Reprod ; 76(2): 232-40, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17108336

RESUMO

Premature chromosome condensation (PCC) was believed to promote nuclear reprogramming and to facilitate cloning by somatic cell nuclear transfer (NT) in mammalian species. However, it is still uncertain whether PCC is necessary for the successful reprogramming of an introduced donor nucleus in cattle. In the present study, fused NT embryos were subjected to immediate activation (IA, simultaneous fusion and activation), delayed activation (DA, activation applied 4 h postfusion), and IA with aged oocytes (IAA, activation at the same oocyte age as group DA). The morphologic changes, such as nuclear swelling, the occurrence of PCC, and microtubule/aster formation, were analyzed in detail by laser-scanning confocal microscopy. When embryos were subjected to IA in both IA and IAA groups, the introduced nucleus gradually became swollen, and a pronuclear-like structure formed within the oocyte, but PCC was not observed. In contrast, delaying embryo activation resulted in 46.5%-91.2% of NT embryos exhibiting PCC. This PCC was observed beginning at 4 h postcell fusion and was shown as one, two, or multiple chromosomal complexes. Subsequently, a diversity of pronuclear-like structures existed in NT embryos, characterized as single, double, and multiple nuclei. In the oocytes exhibiting PCC, the assembled spindle structure was observed to be an interactive mass, closely associated with condensed chromosomes, but no aster had formed. Regardless of whether they were subjected to IA, IAA, or DA treatments, if the oocytes contained pronuclear-like structures, either one or two asters were observed in proximity to the nuclei. A significantly higher rate of development to blastocysts was achieved in embryos that were immediately activated (IA, 59.1%; IAA, 40.7%) than in those for which activation was delayed (14.2%). The development rate was higher in group IA than in group IAA, but it was not significant (P = 0.089). Following embryo transfer, there was no statistically significant difference in the pregnancy rates (Day 70) between two of the groups (group IA, 11.7%, n = 94 vs. group DA, 12.3%, n = 130; P > 0.05) or live term development (group IA, 4.3% vs. group DA, 4.6%; P > 0.05). Our study has demonstrated that the IA of bovine NT embryos results in embryos with increased competence for preimplantational development. Moreover, PCC was shown to be unnecessary for the reprogramming of a transplanted somatic genome in a cattle oocyte.


Assuntos
Bovinos , Reprogramação Celular , Cromossomos/fisiologia , Técnicas de Transferência Nuclear , Animais , Núcleo Celular/fisiologia , Clonagem de Organismos , Desenvolvimento Embrionário , Feminino , Técnicas In Vitro , Microscopia Confocal , Microtúbulos/ultraestrutura , Oócitos/fisiologia , Oócitos/ultraestrutura , Gravidez , Taxa de Gravidez
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