RESUMEN
Recombinant human Insulin-like growth factor-I (hIGF-1) was administered to one ovary of prepubertal and postpubertal cattle to determine its effects on (1) oocyte developmental competence, (2) the expression pattern of six developmentally important genes (GLUT3, GLUT8, AKT1, BCL-XL, BAD, and BAX), and (3) its relationship with apoptosis (female Holstein-Friesian). Oocytes were retrieved from 7- to 10-mo-old prepubertal dairy calves (preP), 11- to 18-mo-old postpubertal heifers (postP), and cows via ultrasound-guided follicular aspiration. Immature oocytes were matured in vitro then fertilized and cultured up to the blastocyst stage. Apoptosis was determined by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) in 8-d blastocysts. Similar low blastocyst yields were observed in the IGF-1-treated preP group (11.2+/-2.4%), the control preP group (10.4+/-3.0%), and in the IGF-1 postP group (10.9+/-2.3%). These were lower (PAsunto(s)
Apoptosis/fisiología
, Blastocisto/fisiología
, Bovinos/embriología
, Factor I del Crecimiento Similar a la Insulina/administración & dosificación
, Oocitos/crecimiento & desarrollo
, Maduración Sexual/genética
, Animales
, Blastocisto/química
, Blastocisto/citología
, Desarrollo Embrionario/fisiología
, Femenino
, Fertilización In Vitro/veterinaria
, Expresión Génica/fisiología
, Proteínas Facilitadoras del Transporte de la Glucosa/genética
, Etiquetado Corte-Fin in Situ
, Oocitos/química
, Oocitos/efectos de los fármacos
, Ovario/efectos de los fármacos
, Proteínas Proto-Oncogénicas c-akt/genética
, ARN Mensajero/análisis
, Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
, Maduración Sexual/fisiología
, Proteína X Asociada a bcl-2/genética
, Proteína Letal Asociada a bcl/genética
, Proteína bcl-X/genética
RESUMEN
The glycophenotyping of mammalian cells with plant lectins maps aspects of the glycomic profile and disease-associated alterations. A salient step toward delineating their functional dimension is the detection of endogenous lectins. They can translate sugar-encoded changes into cellular responses. Among them, the members of the lectin family of galectins are emerging regulators of cell adhesion, migration and proliferation. Focusing on galectins-1, -3 and -7, we addressed the issue whether their expression is regulated during wound healing in porcine skin as model. A conspicuous upregulation is detected for galectin-1 in the dermis and a neoexpression in the epidermis, where an increased level of galectin-7 was also found. Applying biotinylated tissue lectins as probes, the signal intensities for accessible binding sites decreased, intimating an interaction of the cell lectin with reactive sites. In contrast, galectin-3 parameters remained rather constant. Of note, epidermal cells in culture also showed an increase in expression/presence of galectin-1, measured on the levels of mRNA and protein, in this case by Western blotting and quantitative immunocytochemistry. Used as matrix, galectin-1 conferred resistance to trypsin treatment to attached human keratinocytes and reduced migration into scratch-wound areas in vitro. This report thus presents new information on endogenous lectins in wound healing and differential regulation among the three tested cases.
Asunto(s)
Movimiento Celular , Galectinas/metabolismo , Queratinocitos/metabolismo , Piel/metabolismo , Cicatrización de Heridas , Animales , Sitios de Unión , Biotinilación , Western Blotting , Adhesión Celular , Células Cultivadas , Galectina 1/metabolismo , Galectina 3/metabolismo , Galectinas/genética , Humanos , Inmunohistoquímica , Queratinocitos/patología , ARN Mensajero/metabolismo , Piel/lesiones , Piel/patología , Porcinos , Porcinos Enanos , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Bovine embryos can be generated by in vitro fertilization or somatic nuclear transfer; however, these differ from their in vivo counterparts in many aspects and exhibit a higher proportion of developmental abnormalities. Here, we determined for the first time the transcriptomes of bovine metaphase II oocytes and all stages of preimplantation embryos developing in vivo up to the blastocyst using the Affymetrix GeneChip Bovine Genome Array which examines approximately 23,000 transcripts. The data show that bovine oocytes and embryos transcribed a significantly higher number of genes than somatic cells. Several hundred genes were transcribed well before the 8-cell stage, at which the major activation of the bovine genome expression occurs. Importantly, stage-specific expression patterns in 2-cell, 4-cell, and 8-cell stages, and in morulae and blastocysts, were detected, indicating dynamic changes in the embryonic transcriptome and in groups of transiently active genes. Pathway analysis revealed >120 biochemical pathways that are operative in early preimplantation bovine development. Significant differences were observed between the mRNA expression profiles of in vivo and in vitro matured oocytes, highlighting the need to include in vivo derived oocytes/embryos in studies evaluating assisted reproductive techniques. This study provides the first comprehensive analysis of gene expression and transcriptome dynamics of in vivo developing bovine embryos and will serve as a basis for improving assisted reproductive technology.
Asunto(s)
Desarrollo Embrionario/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Animales , Bovinos , Femenino , Genoma , Familia de Multigenes , Análisis de Secuencia por Matrices de Oligonucleótidos , Oocitos/metabolismo , Transcripción GenéticaRESUMEN
Early embryogenesis depends on a tightly choreographed succession of gene expression patterns which define normal development. Fertilization and the first zygotic cleavage involve major changes to paternal and maternal chromatin and translation of maternal RNAs which have been sequestered in the oocyte during oogenesis. At a critical species-specific point known as the major onset of embryonic expression, there is a dramatic increase in expression from the new diploid genome. The advent of array technology has, for the first time, made possible to determine the transcriptional profile of all approximately 20,000 mammalian genes during embryogenesis, although the small amount of mRNA in a single embryo necessitates either pooling large numbers of embryos or a global amplification procedure to give sufficient labeled RNA for analysis. Following array hybridization, various bioinformatic tools must be employed to determine the expression level for each gene, often based on multiple oligonucleotide probes and complex background estimation protocols. The grouped analysis of clusters of genes which represent specific biological pathways provides the key to understanding embryonic development, embryonic stem cell proliferation and the reprogramming of gene expression after somatic cloning. Arrays are being developed to address specific biological questions related to embryonic development including DNA methylation and microRNA expression. Array technology in its various facets is an important diagnostic tool for the early detection of developmental aberrations; for improving the safety of assisted reproduction technologies for man; and for improving the efficiency of producing cloned and/or transgenic farm animals. This review discusses current approaches and limitations of DNA microarray technology with emphasis on bovine embryos.
Asunto(s)
Embrión de Mamíferos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Desarrollo Embrionario/genética , Perfilación de la Expresión Génica/veterinaria , Humanos , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos/tendencias , Análisis de Secuencia por Matrices de Oligonucleótidos/veterinaria , ARN Mensajero/análisisRESUMEN
The goal of this study was to establish and validate a protocol for preparing bovine cardiomyocytes from slaughterhouse material for nuclear transfer experiments. The cardiomyocyte was selected because it is a terminally differentiated cell and strongly expresses a unique subset of genes which can be monitored during the reprogramming period. A total of 39 trials were conducted, and an optimized protocol was developed yielding individual contractile cardiomyocytes from 3-5-month-old bovine fetuses The basic protocol involves stabilization of bovine heart tissue for transportation from the slaughterhouse to the laboratory by perfusion with Custodiol. This was followed by an enzymatic dissociation with collagenase in calcium-free medium and yielded individual contractile rod-shaped cardiomyocytes. Subsequent addition of Ca2+ caused the cardiomyocytes to round up which was an essential pre-condition for drawing them into glass transfer pipettes for delivery into the perivitelline space and for efficient electrofusion with cytoplasts derived from in vitro matured bovine oocytes. The use of cardiomyocytes maintained at 37 degrees C in nuclear transfer, resulted in a significantly reduced proportion of blastocysts compared to adult fibroblasts (14.0% versus 32.7%). Storage of cardiomyocytes at 4 degrees C prior to nuclear transfer was not compatible with blastocyst development. It is expected that this system will be valuable for investigating the reprogramming of gene expression which occurs after somatic cell nuclear transfer.
Asunto(s)
Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Técnicas de Transferencia Nuclear , Animales , Bovinos , Diferenciación Celular , Separación Celular/métodos , Separación Celular/veterinaria , Clonación de Organismos/métodos , Clonación de Organismos/veterinaria , Criopreservación , Corazón Fetal/citología , Corazón Fetal/metabolismo , Citometría de Flujo , Expresión Génica , Técnicas In VitroRESUMEN
Different alleles of the human and ovine prion protein gene correlate with a varying susceptibility to transmissible spongiform encephalopathies. However, the pathogenic implications of specific polymorphisms in the bovine prion protein gene (PRNP) are only poorly understood. Previous studies on the bovine PRNP gene investigated common European and North American cattle breeds. As a consequence of decades of intensive breeding for specific traits, these modern breeds represent only a small fraction of the bovine gene pool. In this study, we analysed PRNP polymorphisms in the native Brazilian Caracu breed, which developed in geographical isolation since the 16th century. A total of 10 single nucleotide polymorphisms (SNPs) were discovered in the coding region of the Caracu PRNP gene. Eight of the SNPs occurred at high frequencies in Caracu cattle (variant allele frequencies = 0.10-0.76), but were absent or only rarely observed in European and North American breeds. One of the Caracu SNPs was associated with an amino acid exchange from serine to asparagine (f = 0.17). This SNP was not detected in Holstein-Friesian, Simmental and German Gelbvieh and was only rarely detected in beef cattle (f = 0.01). We found 17 haplotypes for PRNP in the Caracu breed.
Asunto(s)
Bovinos/genética , Polimorfismo de Nucleótido Simple , Priones/genética , Animales , Brasil , Frecuencia de los Genes , Haplotipos , Mutación Missense , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ProteínaRESUMEN
Until recently, pronuclear microinjection of deoxyribonucleic acid (DNA) was the standard method for producing transgenic animals. This technique is now being replaced by more efficient protocols based on somatic nucleartransferthat also permit targeted genetic modifications. Lentiviral vectors and small interfering ribonucleic acid technology are also becoming important tools for transgenesis. Transgenic farm animals are important in human medicine as sources of biologically active proteins, as donors in xenotransplantation, and for research in cell and gene therapy. Typical agricultural applications include improved carcass composition, lactational performance and wool production, as well as enhanced disease resistance and reduced environmental impact. Product safety can be ensured by standardisation of procedures and monitored by polymerase chain reaction and array technology. As sequence information and genomic maps of farm animals are refined, it becomes increasingly practical to remove or modify individual genes. This approach to animal breeding will be instrumental in meeting global challenges in agricultural production in the future.
Asunto(s)
Animales Domésticos , Animales Modificados Genéticamente , Biotecnología/tendencias , Seguridad de Productos para el Consumidor , Animales , Bovinos/genética , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen/veterinaria , Cabras/genética , Humanos , Inmunidad Innata/genética , Ovinos/genética , Porcinos/genética , Trasplante HeterólogoRESUMEN
Until recently, transgenic rabbits were produced exclusively by pronuclear microinjection which results in additive random insertional transgenesis; however, progress in somatic cell cloning based on nuclear transfer will soon make it possible to produce rabbits with modifications to specific genes by the combination of homologous recombination and subsequent prescreening of nuclear donor cells. Transgenic rabbits have been found to be excellent animal models for inherited and acquired human diseases including hypertrophic cardiomyopathy, perturbed lipoprotein metabolism and atherosclerosis. Transgenic rabbits have also proved to be suitable bioreactors for the production of recombinant protein both on an experimental and a commercial scale. This review summarizes recent research based on the transgenic rabbit model.
Asunto(s)
Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Conejos/genética , Animales , Femenino , Técnicas de Transferencia de Gen , Hormonas/genética , Hormonas/metabolismo , Glándulas Mamarias Animales/metabolismo , Péptidos/genética , Péptidos/metabolismoRESUMEN
Human clotting factor VIII is probably the largest protein to be expressed to date in the mammary gland of a transgenic animal, and it requires extensive posttranslational modification to achieve full biological activity. The mammary gland specific construct mWAP-hFVIII-MT-I was injected into the pronuclei of rabbit zygotes, and three transgenic offspring were obtained. Founder 385 showed germ-line transmission of a single integrated copy, and a homozygous line was established from this animal. The rhFVIII was transcribed and translated exclusively in the mammary gland. The activity of rhFVIII in the rabbit milk ranged from 5 to 8% of that found in normal human plasma. Results indicate the suitability of the transgenic rabbit mammary gland for rhFVIII production.
Asunto(s)
Animales Modificados Genéticamente/metabolismo , Factor VIII/genética , Glándulas Mamarias Animales/metabolismo , Leche/metabolismo , Animales , Coagulación Sanguínea/fisiología , Factor VIII/metabolismo , Femenino , Humanos , Lactancia , ConejosRESUMEN
Bovine in vitro-produced (IVP) and nuclear transfer (NT)-derived embryos differ from their in vivo-developed counterparts in a number of characteristics. A preeminent observation is the occurrence of the large offspring syndrome, which is correlated with considerable embryonic fetal and postnatal losses. We summarize here results from our studies in which we compared gene expression patterns from IVP and NT-derived embryos with those from their IVP counterparts. Numerous aberrations were found in IVP and NT-derived embryos, including a complete lack of expression, an induced expression, or a significant up- or downregulation of a specific gene. These alterations may affect a number of physiological functions and are considered as a kind of stress response of the embryos to deficient environmental conditions. We hypothesize that the alterations are caused by epigenetic modifications, primarily by changes in the methylation patterns. Unravelling these epigenetic modifications is promising to reveal the underlying mechanisms of the large offspring syndrome.
Asunto(s)
Regulación hacia Abajo , Transferencia de Embrión , Desarrollo Embrionario y Fetal/genética , Técnicas de Transferencia Nuclear , Regulación hacia Arriba , Animales , Blastocisto/metabolismo , Bovinos , Núcleo Celular/patología , Medios de Cultivo/farmacología , Compensación de Dosificación (Genética) , Femenino , Fertilización In Vitro/métodos , Masculino , ARN Mensajero/metabolismo , Factores Sexuales , Factores de Tiempo , Cromosoma XRESUMEN
The aim of this study was to determine the relative abundance of mRNAs for the insulin-like growth factor I (IGF-I) and IGF-II ligands, and for the IGF receptors (IGF-IR and IGF-IIR) in in vitro preimplantation bovine embryos from the oocyte to the hatched blastocyst stage using two different culture systems: TCM-199 supplemented with oestrous cow serum, or synthetic oviduct fluid supplemented with polyvinyl alcohol. Development to the two- to four-cell stage and blastocyst stage was significantly higher (P < or = 0.05) in embryos cultured in TCM supplemented with oestrous cow serum than in those cultured in synthetic oviduct fluid supplemented with polyvinyl alcohol (61 and 25% versus 55 and 17%, respectively). A semi-quantitative RT-PCR assay did not detect IGF-I transcripts at any stage of preimplantation bovine development, including the hatched blastocyst stage. In both culture systems, IGF-IR, IGF-II and IGF-IIR were expressed throughout preimplantation development up to the hatched blastocyst stage in a varying pattern. The expression patterns of IGF-IR, IGF-II and IGF-IIR in embryos generated in the two culture systems were not significantly different, except at the expanded blastocyst stage, at which significantly higher amounts of IGF-IIR were observed in the TCM system than in the synthetic oviduct fluid system. These results indicate that transcripts of IGF-IR and IGF-IIR follow the standard pattern in which maternal stores of mRNA in the oocyte are slowly depleted up to the 16-cell stage and then re-established at the onset of embryonic expression of these genes. The lack of detectable IGF-I transcripts in the bovine embryo indicates a predominantly paracrine mode of action. The bovine embryo is capable of producing IGF-II, IGF-IIR and IGF-IR in large amounts, particularly after hatching, which may be important for the formation of the filamentous conceptus. Results indicate an autocrine mechanism for IGF-II and modulation of IGF family expression by culture conditions.
Asunto(s)
Blastocisto/metabolismo , Fertilización In Vitro/métodos , Factor II del Crecimiento Similar a la Insulina/genética , ARN Mensajero/metabolismo , Receptores de Somatomedina/genética , Análisis de Varianza , Animales , Bovinos , Medios de Cultivo , Cartilla de ADN , Factor I del Crecimiento Similar a la Insulina/análisis , Oogénesis , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
POU transcription factors are involved in transcriptional regulation during early embryonic development and cell differentiation. Oct-4, a member of this family, has been shown to be under strict regulation during murine development. The expression of Oct-4 correlates with the undifferentiated cell phenotype of the mouse preimplantation embryo. In this study, expression of a gene construct consisting of selected parts of the region upstream from the murine Oct-4 gene as promoter/enhancer, enhanced green fluorescent protein (EGFP) as reporter and the five exons of the murine Oct-4 gene (GOF18-delta PE EGFP) was evaluated in murine, porcine, and bovine preimplantation embryos. For comparison, expression of the endogenous Oct-4 gene was also analyzed in all three species by immunocytochemistry. The transgene construct was microinjected into zygotes cultured in vitro to various developmental stages. The EGFP fluorescence was visualized in developing embryos by excitation with blue light at different days following microinjection and showed similar expression patterns in all three species. Most embryos displayed a mosaic pattern of transgene expression. The EGFP fluorescence was not restricted to the inner cell mass (ICM) but was also seen in trophoblastic cells. An affinity-purified polyclonal antibody specific to Oct-4 was used for immunocytochemical analysis of in vivo- and in vitro-derived bovine and porcine blastocysts and also of in vivo-derived murine blastocysts. In the in vivo-derived murine embryos, Oct-4 protein was detectable in the ICM but not the trophectoderm, whereas in porcine and bovine blastocysts, derived in vivo or in vitro, Oct-4 protein was detected in both the ICM and the trophectoderm. Thus, in the two large animal species, Oct-4 expression from the endogenous gene was clearly not restricted to the pluripotent cells of the early embryo. These results show that Oct-4 regulation differs between these species and that the presence of Oct-4 protein may not be sufficient for selection of undifferentiated cell lines in domestic animals.
Asunto(s)
Blastocisto/metabolismo , Proteínas de Unión al ADN/biosíntesis , Factores de Transcripción/biosíntesis , Animales , Bovinos , Proteínas de Unión al ADN/genética , Femenino , Colorantes Fluorescentes , Genes Reporteros/genética , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Técnicas In Vitro , Proteínas Luminiscentes , Ratones , Ratones Endogámicos , Microinyecciones , Microscopía Confocal , Factor 3 de Transcripción de Unión a Octámeros , Embarazo , Especificidad de la Especie , Porcinos , Factores de Transcripción/genética , Cigoto/metabolismoRESUMEN
The success of somatic nuclear transfer critically depends on the cell cycle stage of the donor nucleus and the recipient cytoplast. In this study we tested serum deprivation as well as two reversible cell cycle inhibitors, aphidicolin and butyrolactone I, for their ability to synchronize porcine fetal fibroblasts at either G0 stage or G1/S or G2/M transition. The synchronization efficiency of the various protocols was determined by fluorescence-activated cell sorting (FACS), cell proliferation assays, and semiquantitative multiplex reverse transcription-polymerase chain reaction detection of the cell cycle-regulated porcine Polo-like kinase mRNA (Plk-p). FACS measurements revealed that 66.6-73.3% of the porcine fetal fibroblasts were in G0/G1 stage (2C DNA content) in serum-supplemented medium. Short periods of 24-72 h of serum deprivation significantly increased the proportion of cells at G0/G1 phase to 77.9-80.2%, and mitotic activity had already terminated after 48 h. Prolonged culture in serum-deprived medium induced massive DNA fragmentation. Aphidicolin treatment led to an accumulation of 81.9 +/- 4.9% of cells at the G1/S transition. Butyrolactone I arrested 81.0 +/- 5.8% of the cells at the end of G1 stage and 37.0 +/- 6.8% at the G2/M transition. The effects of both chemical inhibitors were fully reversible, and their removal led to a rapid progression in the cell cycle. The measurement of Plk-p expression allowed discrimination between the presumptive G0 phase induced by serum deprivation and the G1/S transition arrest achieved by chemical inhibitors. These data indicate that porcine fetal fibroblasts can be effectively synchronized at various cell cycle stages without compromising their proliferation capacity.
Asunto(s)
Ciclo Celular/fisiología , Feto/fisiología , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Animales , Afidicolina/farmacología , Bromodesoxiuridina/farmacología , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/fisiología , Inhibidores Enzimáticos/farmacología , Feto/citología , Fibroblastos/fisiología , Citometría de Flujo , Etiquetado Corte-Fin in Situ , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Transcripción Genética/genética , Transcripción Genética/fisiología , Quinasa Tipo Polo 1RESUMEN
By targeting the expression of sequences encoding non-milk proteins to the mammary gland of transgenic farm animals, the organ could serve as a 'bioreactor' for producing pharmacologically active proteins on a large scale. Here we report the generation of transgenic sheep bearing a fusion gene construct with the human blood clotting factor VIII (hFVIII) cDNA under the transcriptional control of a 2.2 kb fragment of the mammary gland specific promoter of the ovine beta-Lactoglobulin (beta-Lac) gene. Six founder animals were generated bearing a hFVIII cDNA construct with the introns of the murine metallothionein (MtI) gene (beta-Lac/hFVIII-MtI). Founders transmitted the transgene in a Mendelian fashion and two transgenic lines were generated. Ten out of 12 transgenic F1-females expressed rhFVIII mRNA in exfoliated mammary epithelial cells isolated from the milk. But only in transgenic F1 ewes 4010 and 603 hFVIII clotting activity estimated at 4-6 ng/ml was detected in defatted milk. Furthermore, the presence of rhFVIII-protein in ovine milk was demonstrated by a specific band at approximately 190 kD following immunoprecipitation and immunoblotting. Transgenic founder 395 expressed rhFVIII mRNA in biopsied mammary gland tissue, in exfoliated mammary cells as well as ectopically in brain, heart, spleen, kidney and salivary gland, suggesting that the employed beta-Lac promoter fragment lacks essential sequences for directing expression exclusively to the mammary gland. A rhFVIII standard preparation (rhFVIIIstd) was rapidly sequestered in a saturable fashion in ovine milk, thus rendering it largely inaccessible to immunoprecipitation although its biological activity was retained. Recovery of hFVIIIstd was dependent on milk donor, storage temperature and dilution of milk sample.
Asunto(s)
Animales Modificados Genéticamente/genética , Factor VIII/genética , Glándulas Mamarias Animales/metabolismo , Ovinos/genética , Animales , Animales Modificados Genéticamente/metabolismo , Factor VIII/biosíntesis , Factor VIII/aislamiento & purificación , Femenino , Humanos , Immunoblotting , Lactoglobulinas/biosíntesis , Lactoglobulinas/genética , Ratones , Leche/química , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ovinos/metabolismoRESUMEN
In preimplantation bovine embryos, the relative abundance of various developmentally important gene transcripts was determined by a semi-quantitative RT-PCR assay to analyze the effects of two medium supplements, serum or polyvinyl alcohol (PVA). Development to morula, blastocyst, and hatched blastocyst stages was higher (P < or = 0.05) in medium supplemented with serum than in medium supplemented with PVA. Connexin43 mRNA expression virtually disappeared from the 8-16 cell stage onward, but reappeared in the hatched blastocyst in serum-supplemented medium, whereas it was detected in PVA-derived embryos throughout development. No differences were found for plakophilin mRNA between both culture groups. Desmocollin II mRNA showed a sharp increase at the blastocyst stage in both groups with a higher transcription level in PVA-generated embryos. A significant difference in desmocollin III transcripts was detectable at the 8-16-cell stage between serum- and PVA-derived embryos. Transcripts for desmoglein 1 and desmocollin I were not detected at any preimplantation stage, irrespective of medium supplementation. The relative abundance of glucose-transporter-1 mRNA was significantly increased at the 8-16-cell stage in embryos produced in medium supplemented with PVA, but not serum. Heat shock protein and poly(A)polymerase mRNA were continuously expressed during preimplantation development in both culture groups. Although poly(A)polymerase mRNA was significantly elevated in PVA- over serum-derived embryos, heat shock protein mRNA expression was significantly enhanced in serum-generated embryos over PVA-derived embryos. Interferon tau mRNA showed a significant increase at the hatched blastocyst stage only in PVA-supplemented medium. These data suggest that alterations in mRNA expression are associated with culture environment. Timing and magnitude of the alterations varied among the different transcripts and were significantly affected by the presence of exogenous protein in a stage-specific manner, predominantly at critical developmental time points.
Asunto(s)
Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Alcohol Polivinílico , Albúmina Sérica Bovina , Animales , Bovinos , Medios de Cultivo , Desarrollo Embrionario/fisiología , Desarrollo Embrionario y Fetal , Femenino , Embarazo , ARN MensajeroRESUMEN
This study investigated the effects of a semi-defined culture system on the temporal pattern of expression of RNA from genes involved in compaction and cavitation: gap junction protein connexin43 (Cx43), desmosomal glycoproteins desmoglein 1 (Dg 1), desmocollins I, II and III (Dc I, Dc II, Dc III), desmosomal protein plakophilin (Plako); metabolism glucosetransporter-1 (Glut-1); RNA processing poly(A)polymerase (PolyA); heat shock protein 70.1 (HSP); and trophoblastic function trophoblast protein (TP) in bovine oocytes and embryos generated in vitro using TCM199 supplemented with BSA as the culture medium. Morulae and blastocysts derived in vivo were collected from superovulated heifers and also used for this study. Poly(A)+ RNA was extracted from pools of 20-50 oocytes or embryos, analysed by reverse transcription-polymerase chain reaction and the amplified fragments were verified by sequencing. Assays were repeated at least three times for each developmental stage and provided consistent results in all replicates. In bovine embryos produced in vitro, mRNA encoding Cx43 was detectable up to the morula stage, whereas blastocysts and hatched blastocysts did not express this gene. No transcripts were found for Dg 1 and Dc I throughout the tested preimplantation stages. Dc II and Dc III transcripts were found from 2-4-cell embryos up to the hatched blastocyst stage. mRNA encoding Plako was detected in immature and mature oocytes and zygotes, while no transcripts were seen in 2-4-cell and 8-16-cell embryos. The gene was expressed again from the morulae to the hatched blastocyst stage. Oocytes and bovine embryos produced in vitro showed transcripts for Glut-1, PolyA and HSP throughout preimplantation development up to the hatched blastocyst stage. The gene encoding TP was transcribed only in blastocysts and hatched blastocysts. Morulae and blastocysts produced in vivo showed the same expression as their in vitro counterparts, with one exception: the in vivo embryos transcribed Cx43. The results of this study reveal for the first time the transcriptional pattern of a set of 'marker' genes involved in various processes in early bovine embryonic development. Transferable morulae and blastocysts produced in vitro expressed most genes similar to their in vivo counterparts. These data contribute to the molecular characterization of this widely used in vitro culture system for bovine embryos and provide a major advance towards production of 'physiologically normal' embryos.
Asunto(s)
Blastocisto/fisiología , Bovinos/fisiología , Técnicas de Cultivo/métodos , Genes , ARN Mensajero/metabolismo , Transcripción Genética , Animales , Conexina 43/genética , Medios de Cultivo , Proteínas del Citoesqueleto/genética , Desmocolinas , Desmogleína 1 , Desmogleínas , Desmoplaquinas , Proteínas HSP70 de Choque Térmico/genética , Interferón Tipo I/genética , Proteínas de Transporte de Monosacáridos/genética , Mórula/fisiología , Placofilinas , Reacción en Cadena de la Polimerasa , Polinucleotido Adenililtransferasa/genética , Proteínas Gestacionales/genética , Proteínas/genética , Albúmina Sérica BovinaRESUMEN
We have developed a protocol for fast, nonradioactive, mRNA differential display reverse transcription PCR (DDRT-PCR) based on a commercial automated sequencer with RNA isolated from pig granulosa cells. We sought to discover conditions that would minimize the problem of using relatively small primers labeled with large infrared dye molecule, IR41, required for the sequencer. Extended IR41-labeled primers IR41-AAGC-T11-A, IE41-AAGC-T11-C and IR41-AAGC-T11-G gave more consistent differential display patterns than shorter anchored primers (IR41-T11A, IR41-T11C and IR41-T11G) without the additional (AAGC) cloning site. The optimal concentration of the extended labeled (downstream) primers was 20 pmol when 13-mer arbitrary (upstream) primers were used at a concentration of 4 pmol. Background smear and the intensity of amplified bands was significantly improved by changing from conventional Taq DNA polymerase to AmpliTaq Gold polymerase, which permits an improved "hot start" for the reaction. Running time (during which a digitized gel image is recorded) for a 26-cm polyacrylamide gel was 4 h, enabling us to analyze 90 reactions in an 8-h day. This protocol offers a rapid and reliable nonradioactive method for comparing gene expression patterns for various research or diagnostic purposes.
Asunto(s)
Células de la Granulosa/metabolismo , ARN Mensajero/análisis , Animales , Femenino , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , PorcinosRESUMEN
In this study we have examined the presence of mRNA encoding connexin 43 (Cx43) in bovine embryos derived in vivo and in vitro. Cumulus-oocyte complexes, immature and matured oocytes liberated from cumulus cells, zygotes, 2-4-cell and 8-16-cell embryos, morulae, blastocysts and hatched blastocysts were produced in vitro from ovaries obtained from an abattoir using TCM 199 supplemented with hormones and 10% oestrous cow serum for maturation. Cumulus-oocyte complexes matured for 24 h were exposed to bull spermatozoa for 19 h and then cultured in TCM 199 supplemented with 10% oestrous cow serum to the desired developmental stage. Morulae and blastocysts derived in vivo were collected from superovulated donor cows. Total RNA was extracted from pools of 60-200 bovine oocytes or embryos using a modified phenol-chloroform extraction method and analysed by reverse transcriptase polymerase chain reaction. Before reverse transcription, aliquots of DNase-digested embryonic RNA were tested by polymerase chain reaction using bovine-specific primers to control for residual genomic DNA contamination. DNA-free, total RNA was reverse transcribed after preincubation with the Cx43 specific 3'primer. The resultant cDNA was amplified by polymerase chain reaction using Cx43 specific primers that define a 516 bp fragment of Cx43. The reverse transcriptase polymerase chain reaction product was verified by restriction enzyme analysis with Alu I and sequencing. Assays were repeated at least twice for each developmental stage and provided identical results between replicates. Cx43 transcripts were detected in bovine morulae and blastocysts grown in vivo. In contrast, whereas the early in vitro stages from cumulus-oocyte complexes to morulae expressed Cx43, blastocysts and hatched blastocysts did not have detectable concentrations of mRNA from this gene. Restriction enzyme cutting revealed three fragments of the predicted size (139, 177, 200 bp). The amplified product showed 100% identity with the published bovine genomic DNA sequence. Under our in vitro conditions the Cx43 gene either had never been activated, which would require that the maternal transcript was stable through early development, or embryonic gene expression that had been active was then terminated prematurely. The differences in transcription between bovine embryos derived in vivo or in vitro indicate that culture conditions affect gene expression. This affords a tool for the further optimization of in vitro production systems for bovine embryos and contributes towards physiological characterization by definition of transcription phenotype of bovine embryos produced in vitro.
Asunto(s)
Blastocisto/fisiología , Bovinos/metabolismo , Conexina 43/genética , Animales , Fertilización In Vitro , Expresión Génica , Mórula/fisiología , Oocitos/fisiología , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisisRESUMEN
Molecular biological methods developed for basic research have been increasingly employed in animal production. For example, individual animals can be identified by their unique DNA sequences in DNA fingerprints and their genetic relationships can be established by more detailed sequence comparisons. It is also possible to diagnose gene defects and to determine the sex of preimplantation embryos. This information can then be used as the basis for an efficient breeding program. Methods for the analysis of RNA yield information about genetic or environmental factors which influence the expression of specific genes for example genes of economic value in milk production or genes which are important for preimplantation embryo development. This information will enable further improvements in embryo transfer and associated biotechniques which promise to improve the rate of genetic improvement (NIEMANN and MEINECKE, 1993). In the following section, we will briefly describe these molecular biological methods and their applications in animal breeding using examples from current research in our research group.
Asunto(s)
Cruzamiento/métodos , Biología Molecular , Animales , Northern Blotting/veterinaria , Southern Blotting/veterinaria , ADN/análisis , Dermatoglifia del ADN/veterinaria , Hibridación in Situ/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , ARN/análisisRESUMEN
A group of radiation-induced perinatal-lethal deletions that include the albino (c) locus on mouse chromosome 7 causes failure of expression of various hepatocyte-specific genes when homozygous (S. Gluecksohn-Waelsch, 1979, Cell 16:225-237). The transcription of such genes could be controlled in trans by a regulatory gene(s) located within the proximal region of the C14CoS deletion. To identify this potential regulatory gene, a microclone library was established from microdissected D and E bands of chromosome 7. Three nonoverlapping microclones (E305, E336B, and E453B) hybridizing with wildtype but not with C14CoS/C14CoS DNA were isolated. E336B represents a single-copy DNA fragment, whereas E305 and E453B hybridized with 3 and 10 EcoRI DNA restriction fragments, respectively. All fragments map exclusively within the deletion. The microclones hybridized to DNA of viable C6H/C14CoS deletion heterozygotes but not to DNA of homozygotes for the lethal mutation c10R75M, which belongs to the same complementation group as c14CoS. DNA of viable homozygous mutant C62DSD, which carries a deletion breakpoint proximal to that of c6H, hybridized only with E453B. This microclone identified 6 EcoRI restriction fragments in C62DSD/C62DSD DNA. The results demonstrate that of the isolated microclones, E453B identifies a locus (D7RT453B) that maps closest to the hsdr-1 (hepatocyte-specific developmental regulation) locus (S. Gluecksohn-Waelsch, 1989, Mouse Newslett. 83: 149), which maps between the proximal breakpoints of deletions c10R75M and c62DSD.