Development of observation system to investigate both intracellular calcium concentration and mechanical stimuli to mammalian embryos

Abstract

Using an air-actuating device, we investigated the cellular response to mechanical stimuli (MS) in mouse blastocysts. Both MS and intracellular calcium concentration ([Ca2+]i) were quantified based on time-resolved confocal microscopy images in the polydimethylsiloxane (PDMS) microfluidic channels by deforming a 0.1-mm membrane. [Ca2+]i was measured in a stained mouse embryo with Fluo-4 AM using confocal fluorescence microscopy. We captured a z-series stack of sections encompassing the entire embryo. When translocation velocities of the embryo and shear stress were 40 µm/s and 0.01 dyne/cm2, respectively, a 10% increase in the sum of fluorescent intensities (FI) was observed. When blastocysts were compressed, FI also increased in response to the applied MS. Compressive force estimated from the shape of the blastocysts was approximately 0.5–2.0 µN according to a force deformation curve for the mouse embryo. The average FI and sum of FIs increased by a factor of 1.1–1.2 times compared with those observed before MS. The increase in the sum of FI indicated that enhancement of [Ca2+]i would be induced by these MS.