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• W2023302785 abstract "Objective: Sugars such as trehalose are effectively used by various organisms as protective agents to undergo anhydrobiosis and cryobiosis. Furthermore, it has recently been shown that small amounts of intracellular trehalose protect different mammalian cells including human and mouse oocytes against freezing-associated stresses. The objective of this study was first to establish a method for quantitative delivery of sugars into oocytes, and then to evaluate their effect on static osmometric behavior and development of mouse eggs. Design: In vitro comparative study. Materials/Methods: Microinjection was accomplished by applying a regulated pressure using a pneumatic device for a given pulse duration. To quantitatively introduce sugars into eggs, the injection pipettes were precalibrated using a system based on volumetric response of medium microdroplets suspended in dimethylpolysilaxene. To test accuracy of this technique, both microdroplets and eggs were microinjected with a fluorophore-labeled dextran. Next, microinjection volumes were calculated from fluorescence intensity, as well as from volumetric responses of microdroplets and eggs. To study osmometric behavior, eggs were injected with 0.1 M of glucose, trehalose, and stachyose and exposed osmolalities ranging from 400 to 1300 mOsm/kg. Next series of experiments focused on toxicity of injection buffers (i.e., 10 mM Tris and 15 mM Hepes) and trehalose. For the culture of eggs microinjected with 0.1, 0.15, and 0.20 M trehalose, a bicarbonate-buffered medium was formulated with osmolalities of 320, 340, and 360 mOsm, respectively. Results: The egg volume in the presence of sugars was found to be linear function of the reciprocal of the extracellular osmolality. Comparison of calculated injection volumes revealed that volumetric response of the microdroplets reflects microinjection into oocytes with pL-accuracy. Microinjection of Hepes and Tris buffer in the presence of 0.1 M trehalose resulted in blastocyts rates of 86% and 72%, respectively, without significant difference when compared to controls (86%). In subsequent experiments, Hepes was used as the injection buffer, and embryonic development of pronuclear eggs 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%) after microinjection of 0.20 M trehalose. When transferred to foster mother, similar proportion of control and trehalose-injected eggs implanted (77% and 86%, respectively) and developed to Day 16 fetuses (54% and 57%, respectively). Moreover, trehalose-injected eggs developed to term resulting in birth of healthy pups. Conclusions: Mouse eggs in the presence of intracellular sugars behave as ideal osmometers. Quantitative microinjection of trehalose suggests that cryoprotective concentrations of intracellular trehalose do not impair development of mouse eggs. Supported by: This study was funded by grateful patients." @default.
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• W2023302785 date "2002-09-01" @default.
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• W2023302785 title "Quantitative microinjection of sugars into mouse eggs and their effect on static osmometric behavior and development" @default.
• W2023302785 doi "https://doi.org/10.1016/s0015-0282(02)03592-6" @default.
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