302 A MICROFLUIDIC DEVICE TO CONTROL Ca²⁺ SIGNALLING IN FERTILIZED AND NON FERTILIZED EGGS

Abstract

The process of egg activation has been the subject of much attention for many years. It has recently gained even more interest since nuclear transfer experiments have revealed the unique and extraordinary properties of the oocyte cytoplasm to cause massive reorganization of a somatic nucleus. The transduction mechanisms by which the incoming sperm triggers a series of intracellular Ca²⁺ oscillations is becoming clearer, but how activation impacts the process of remodelling is still unknown. One possibility to get a better view is to experimentally drive the dynamics of Ca²⁺ oscillations to monitor the changes in a sperm nucleus in maternal chromosomes or in a somatic nucleus during activation. For this purpose, we have used the modern technology of microfluidics in association with electropermeabilization of the cell membrane to drive the process of egg activation. We designed a microdevice that assures precise control of [Ca²⁺]I and makes it possible to slow down or accelerate the dynamics of Ca²⁺ signalling in fertilized and non-fertilized eggs during the process of activation. This device consists of a micro-chamber 400 μm wide, 0.5 mm high, and 25 mm long, making a volume of 5 μL. The lateral walls of the chamber are made of gold to subject the cells to an electric field pulse. The bottom surface has a slot 20 μm wide used to hold the oocytes by suction. The space above is left open to allow access with a mouth-pipette to lodge a maximum of a hundred eggs. At any moment eggs can be put in place or removed. This chamber is embedded in a disposable cartridge made for us by Micronics (www.micronics.net). A series of computer-driven pumps and micro-valves embedded in the cartridge makes it possible to multiplex very rapid washing with four reagents. The total reagent content of the chamber can be replaced in less than 1 s while eggs are maintained aligned. This washing process makes it possible to control the electropermeabilization technique while embryos are cultured in vitro. To evaluate the survival to term of fertilized eggs cultured and stimulated in this microsystem, 60 fertilized embryos were subjected to 24 electrical pulses (1.45 kV cm⁻¹) every 8 min for 3 hours in the absence of Ca²⁺ ions during the electrical pulse. Imaging recording showed that the ongoing Ca²⁺ signal dynamic triggered by fertilization is not altered by the treatment. Among the 60 treated embryos transferred to pseudopregnant recipients, 45 (75%) were capable of giving birth to viable young. Since this new microtechnology appears to be biologically safe, it becomes possible to automatically drive a great variety of signals onto and into the cell during in-vitro culture, to better evaluate how oocytes integrate signals during the process of activation, and to give a biological response at later stages of development. Moreover, this new microtechnology will provide standardization of the biological response after fertilization, ICSI or nuclear transfer experiments.