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Sugars such as trehalose are effectively used by various organisms as protective agents to undergo anhydrobiosis and cryobiosis. The objective of this study was first to establish a method for quantitative delivery of trehalose as a model sugar into oocytes, and then to evaluate its effect on development of mouse zygotes. To this end, a quantitative microinjection technique was developed using volumetric response of microdroplets suspended in dimethylpolysilaxene. To verify accuracy of this technique, both microdroplets and oocytes were microinjected with fluorophore-labeled dextran. Thereafter, injection volumes were calculated from fluorescence intensity, and volumetric responses of both microdroplets and oocytes. Comparison of calculated injection volumes revealed that this technique reflects microinjection into oocytes with pL-accuracy. The next series of experiments focused on toxicity of injection buffers (i.e., 10 mM Tris and 15 mM Hepes) and trehalose. Microinjection of Hepes and Tris buffer in the presence of 0.1 M trehalose resulted in blastocyst rates of 86 and 72%, respectively, without a significant difference when compared to controls (86%). In subsequent experiments, Hepes was used as the injection buffer, and embryonic development of zygotes was studied as a function of intracellular trehalose concentrations. Microinjection of trehalose up to 0.15 M resulted in development to blastocyst stage similar to controls (85 and 87%, respectively) while the blastocyst rate was significantly decreased (43%) in the presence of 0.20 M intracellular trehalose. When transferred to foster mothers, trehalose-injected zygotes (0.1 M) implanted and developed to day 16 fetuses similar to controls, healthy pups were born. The findings of this study suggest that trehalose at effective intracellular concentrations does not impair development of mouse zygotes. [Copyright &y& Elsevier]

Nkx2.5, an evolutionarily conserved homeodomain containing transcription factor, is one of the earliest cardiogenic markers. Although its expression continues through adulthood, its function in adult cardiomyocytes is not well understood. To examine the effect of Nkx2.5 in terminal differentiated postnatal cardiomyocytes, we generated transgenic mice expressing either wild-type Nkx2.5 (TG-wild), a putative transcriptionally active mutant (carboxyl-terminus deletion mutant: TG-ΔC) or a DNA non-binding point mutant of Nkx2.5 (TG-I183P) under α-myosin heavy chain promoter. Most TG-wild and TG-ΔC mice died before 4 months of age with heart failure associated with conduction abnormalities. Cardiomyocytes expressing wild-type Nkx2.5 or a putative transcriptionally active mutant (ΔC) had dramatically reduced expression of connexin 43 and changed sarcomere structure. Wild-type Nkx2.5 adenovirus-infected adult cardiomyocytes demonstrated connexin 43 downregulation as early as 16 h after infection, indicating that connexin 43 downregulation is due to Nkx2.5 overexpression but not due to heart failure phenotype in vivo. These studies indicate that overexpression of Nkx2.5 in terminally differentiated cardiomyocytes dramatically alters cardiac cell structure and function. [Copyright &y& Elsevier]