Asunto(s)

Puntos de Acupuntura , Anatomía Comparada , Meridianos , Animales , Biología , Humanos , Coloración y Etiquetado

RESUMEN

The temporal progressive increase of interferon tau (IFNτ) secretion from the bovine trophoblast is a major embryonic signal of establishing pregnancy. Here, we cultured and isolated bovine trophoblast cells (BTs) from IVM/IVF oocytes and in vitro produced blastocysts, used them, for the first time, as donor cells for nuclear transfer and compared them with adult fibroblasts (AFs) as donor cells. BTs were reprogrammed in enucleated oocytes to blastocysts with similar efficiency to AFs (14.5% and 15.6% respectively, P≤0.05). The levels of IFNτ, CDX2 and OCT4 expression in IVF-, BT- and AF-derived blastocysts were analyzed using reverse transcription polymerase chain reaction and reverse transcription quantitative polymerase chain reaction (RT-PCR and RT-qPCR). IVF-produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid, expanded and hatching blastocysts. RT-PCR and RT-qPCR studies showed that IFNτ expression was higher in BT-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and BT-derived blastocysts showed a progressive increase in IFNτ expression as blastocyst development advanced when it compared with AF-derived blastocysts. OCT4 was inversely related with IFNτ expression, while CDX2 was found to be directly related with IFNτ temporal expression. Persistence of high expression of IFNτ and CDX2 was found to be higher in BT-derived embryos than in IVF- or AF-derived embryos. In conclusion, using BTs expressing IFNτ as donor cells for bovine NT could be a useful tool for understanding the IFNτ genetics and epigenetics.

Asunto(s)

Reprogramación Celular , Ectogénesis , Implantación del Embrión , Regulación del Desarrollo de la Expresión Génica , Oocitos/metabolismo , Trofoblastos/metabolismo , Animales , Blastocisto/citología , Blastocisto/metabolismo , Bovinos , Células Cultivadas , Femenino , Fertilización In Vitro/veterinaria , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Técnicas de Transferencia Nuclear/veterinaria , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Oocitos/citología , Embarazo , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , ARN Mensajero/metabolismo , Trofoblastos/citología

RESUMEN

We have previously shown that the in vitro embryonic development and the yield of viable calves were increased by using a two-step chemically defined medium for post-fertilization culture of bovine embryos. In this study, we explored the embryonic development and the temporal behavioral interaction of the genes involved in IFNτ gene expression and how they behave in an orchestrated manner to increase the developmental competence of IVF produced embryos by culturing in the chemically defined medium. Behavior of genes included ETS2, CDX2, GATA2, GATA3, OCT4 and NANOG was analyzed in early bovine IVF produced embryos, (from compact morulae to the blastocyst hatching stages), by semi- and relative quantitative PCR and compared between two in vitro culture (IVC) systems, two-step chemically defined medium and modified synthetic oviductal fluid (mSOF) containing 8 mg/mL, BSA. Early embryonic development was found to be better in two-step chemically defined culture system than that of mSOF as indicated by the increment of blastocyst yield, 33.1% in two-step culture system vs 18.8% in mSOF medium, and the blastocyst hatching, 52.3% in two-step culture system vs 33.5% in mSOF medium. Relative quantitative gene expression showed harmonic behavior in the two-step culture system rather than the culture in mSOF, IFNτ showed even increase throughout the embryonic development in the two-step culture medium while it decreased with blastocyst hatching in mSOF culture condition. Temporal dominance of OCT4 over all the transcription factors was found in regulation of IFNτ expression (the major factor of expression regulation but in inverse manner). However, ETS2, CDX2, GATA2 and GATA3 are potent IFNτ stimulator in cumulative manner but in case of OCT4 decrement. CDX2 directly related with IFNτ, but still under OCT4 dominance and also regulated by the subservient of OCT4 which is NANOG. In conclusion, this study confirmed our previous results about the usefulness of using the two-step chemically defined culture medium for increasing the developmental competence of IVF produced embryos and elucidated the dominance of OCT4 over the other genes implicated in regulation of IFNτ expression.

Asunto(s)

Medios de Cultivo/farmacología , Técnicas de Cultivo de Embriones , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Animales , Bovinos , Femenino , Fertilización In Vitro , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Masculino , Oocitos/citología , Oocitos/crecimiento & desarrollo , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermatozoides/citología

RESUMEN

The effect of pulse current (PC) on Ni-W alloy electrodeposition was investigated to fill the trenches with aspect ratio 1:2 for MEMS applications. Rapid deposition of top side in trench produced a void in electrodeposits with direct current (DC). Morphology of Ni-W electrodeposition inside trench was strongly influenced by the applied current density and the current off-time (t(off)). Enhanced filling phenomena were observed with increasing current off-time because of decreasing concentration gradient between top and bottom in trench. The complete Ni-W filling of trench was achieved with a current density of 200 mA/cm2 and a current on- and off-time of 100 ms and 300 ms, respectively. Amorphous structure was revealed in both DC and PC electrodeposits and the concentration of W in Ni-W electrodeposits for complete filling of trench was about 24% in atomic ratio.