297 A METHOD TO DRIVE CALCIUM SIGNALLING DYNAMICS IN FERTILIZED MOUSE EGGS

Abstract

Following fertilization, eggs exhibit a series of repetitive increases in intracellular calcium that activate development. The developmental impact of the long-lasting series of Ca²⁺ signals is still a subject of controversy. Although several studies using parthenogenetically activated eggs suggest that Ca²⁺ dynamics affect post-implantation development, artificial stimulation of Ca²⁺ signaling after ICSI in bovine eggs shows that development still remains poor in comparison to fertilized eggs. Such divergence between parthenogenetic studies and those aimed at stimulating ICSI eggs makes it impossible to draw any conclusions regarding the function of Ca²⁺ signaling for two reasons. First, non-fertilized eggs do not release Ca²⁺ from intracellular stores and their development is compromised due to the absence of paternally-derived chromosomes. Second, because ICSI eggs are excitable, Ca²⁺ stimulation generates additional Ca²⁺ oscillations that might compromise their development. Moreover, in both cases, Ca²⁺ signaling is not physiological. To understand better the function of Ca²⁺ signaling at fertilization, we developed a new approach based on micro fluidic technology that makes it possible to drive Ca²⁺ signal dynamics of fertilized eggs with no apparent deleterious effects. This method relies on the fact that the properties of the IP3 receptor (IP3R) calcium channel are changed after fertilization, and IP3 and Ca²⁺ act as co-agonists to cause Ca²⁺-induced Ca²⁺ release (CICR) from intracellular stores. Because Ca²⁺ has both an inhibiting and a stimulating function, we exploited these opposing properties. First, we inhibited Ca²⁺ release by external washing with Ca²⁺-free medium;; this extra cellular washing decreases cytosolic [Ca²⁺]I, and facilitates dissociation of Ca²⁺ ions from the IP3R that in turn decreases the probability of IP3R channel opening. Second, once the IP3R is inhibited, a simple injection of Ca²⁺ ions by electropermeabilization triggers channel opening and induces Ca²⁺ release. Then, by just varying the time interval and the number of the electrical pulses, it is possible to drive the dynamics of the CICR process that initiates development. Intracellular Ca²⁺ imaging demonstrated that fertilized eggs subjected to 24 electrical pulses (1.45 kV cm⁻¹) every 8 min for 3 h in the microfluidic processor responded by exhibiting 24 induced-Ca²⁺ transients that are caused by calcium release from intracellular stores. All auto-regenerative responses between pulses were inhibited. Among 60 treated embryos transferred to pseudo-pregnant recipients, 40 (67%) developed to term, with birth of live offspring, thus demonstrating that this new methodology does not compromise development. Because the eggs are fertilized, it now becomes possible to study the function of Ca²⁺ signaling during egg activation and to evaluate its developmental impact, if any, in association with genomic approaches.