In the free calcium concentration of the egg cytoplasm sperm triggers a series of repetitive spikes at fertilization that are responsible for activation. The interest of this very short developmental period resides in three particular aspects. First of all, it is well established that the processes of oocyte activation co-ordinate a complex epigenetical chain of events resulting in the remodelling of the parental genomes. Secondly this initial biochemical activity of the egg cytoplasm has the unique property of ‘reprogramming’ a somatic nucleus when it is exposed to its environment. The third aspect resides in the fact this global and very complex molecular dynamics of the oocyte are triggered and paced by a repetitive Ca²⁺ signal that is comparatively simple and lasts a very short period of time.

While considerable knowledge on how sperm triggers calcium oscillations has recently been produced, still the extreme variability in signal kinetics between eggs, i.e. amplitude, number and frequency, usually observed after fertilization, makes it practically impossible to run meaningful experiments on the exact functions and biological impacts of signal parameters.

Since this calcium dynamic orchestrates the many changes through which a single fertilized egg cell turns into a complex organism, taking over the control of this process might give us the possibility of graduating and driving the early developmental events in order to establish correlations between early and late developmental events.

Several approaches can be used to stimulate or bypass the natural mechanisms that cause repetitive Ca²⁺ increase. Our initial approach uses electropermeabilisation to create a Ca²⁺ influx into the cell to either reproduce the natural signal on non-fertilized oocytes that are not capable of releasing Ca²⁺ from intracellular stores, or to stimulate the calcium-induced calcium release processes in fertilized eggs. This technique is simple and very common but it has a dramatic impact on the cell when the electrical current generated by the electrical pulse is too high. In fact, the eggs usually die after repetitive electrical stimulation. Since the impact depends on the external ionic concentration we have developed a microfluidic processor that assures us of fast washing before the electrical pulse to minimise the ionic conductivity of the media before the pulse and a very fast washing after the pulse to block the calcium influx by facilitating membrane healing. This microfluidic artifice associated with transient electropermeabilisation of the membrane gives us good control over the amplitude and frequency of the signal for an entire oocyte population, thus assuring standardization and reproducibility.

Using this technology, we have produced several pieces of information. Firstly, the calcium stimulus is the most efficient signal activating mammalian eggs when it is applied in a repetitive manner. Secondly, repetitive Ca²⁺ signals drive the onset and progression of a series of cellular and biochemical events that characterize fertilization, i.e. cortical granules exocytosis, cell cycle resumption with concomitant decrease in MPF and MAP kinases activities, recruitment of maternal mRNAs differently. The Ca²⁺ regimen appears to affect the methylation pattern of several DNA segments but the dynamics of early cleavage does not appear to be determined by either the frequency or the amplitude of the calcium signal. Thirdly, in the rabbit species, amplitude and temporal modulation of the Ca²⁺ signals during the early minutes of oocyte activation impacts the extent of developmental organization and differentiation at the post-implantation stages of parthenogenetic embryos.

On the basis of these results, artificial control of the Ca²⁺ dynamic during oocyte activation leads us to several important deductions. First of all, oocytes have the potential to decode even minute levels of perturbation during the process of activation. Secondly, some post-implantation developmental variations are caused by changes in the regime of calcium signalling. Thirdly, by assuring the fidelity of the Ca²⁺ response, new approaches are opened to determining the biochemical mechanisms driving the egg-to-embryo transition, thus making it possible to better understand hidden epigenetic regulation. In addition, such an approach improves developmental predictions in applied biotechnology.

By refining our technology we hope to be able to drive the calcium dynamic in fertilized eggs in order to establish a link between early and late developmental events until term to assist the process of fertilization.

This study was supported by funds from INRA JE #77 and from MRT 01H0228.