ABSTRACT
PROBLEM: The type I interferon (IFN), IFN-tau (τ), is the primary embryonic signal for pregnancy maintenance in ruminants. This study determined the effects of heat shock upon IFN-τ (IFNT) gene expression by bovine blastocysts in vitro. METHOD OF STUDY: In vitro-produced blastocyst-stage embryos were exposed to 42°C for 4 hr, and mRNA for heat-shock protein 70 (HSP70) and IFNT quantified. RESULTS: Heat shock increased both HSP70 and IFNT expression. There was a significant correlation between HSP70 and IFNT transcript levels irrespective of whether a blastocyst had been exposed to heat shock or not. CONCLUSION: The increase in IFNT as a result of heat shock suggests that a proportion of the variation in IFNT expression observed in blastocyst-stage embryos is a response to stress.
Subject(s)
Blastocyst/metabolism , Gene Expression Regulation, Developmental , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Response , Interferon Type I/metabolism , Pregnancy Proteins/metabolism , Animals , Cattle , Female , HSP70 Heat-Shock Proteins/genetics , Interferon Type I/genetics , Pregnancy , Pregnancy Proteins/geneticsABSTRACT
To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.
