PARENT SESSION
MINISYMPOSIUM X. Biopore Minisymposium. Egg Activation at Fertilization: Where It All Begins.
11:00 AM-12:30 PM
Grand Ballroom VI
Chair: Jaffe, Laurinda¹, ¹

(M31) DEVELOPMENTAL EFFECTS OF THE DYNAMICS OF CALCIUM SIGNALS TRIGGERING OOCYTE ACTIVATION IN MAMMALS.

Ozil, Jean-Pierre¹, ¹ Institut National de la Recherche Agronomique, Jouy en Josas, France

ABSTRACT- At fertilization, the mammalian sperm triggers a series of repetitive spikes in the free calcium concentration of the egg cytoplasm. These spikes are responsible for activating the egg. The process of oocyte activation is attracting increased attention because it is situated at the junction between fundamental research and applied biotechnology. The former focuses mainly on the field of epigenetic phenomena that impact the long-term developmental processes while the latter seeks new methodologies to assist activation. The interest of this very short developmental period resides in two particular aspects. First of all, it is well established that the activation processes coordinate a complex epigenetical chain of events resulting in the remodeling of the parental genome after fertilization. Also, 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 second aspect resides in the fact that the global and very complex molecular dynamics of the oocyte is triggered and paced by a repetitive Ca₂₊ signal that is comparatively simple and lasts a very short period of time in comparison to the total length of pregnancy. This contrast between the complex nature of epigenetical phenomena ensuring proper gene activation during the activation period and the simplicity of the driving signal that regulates overall cellular activity, offers an original and new experimental opportunity to investigate biological complexity through experimental simplicity. While considerable knowledge on how sperm triggers calcium oscillations has recently been produced, still the extreme variability in signal kinetics, 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 such signal parameters. To get around this problem, we have developed a microfluidic research device that makes it possible to control the process of oocyte activation in mammals without getting involved in natural mechanisms of transduction triggered by fertilization. This artificial procedure short-circuits the complex enzymatic processes engendered by the entry of the spermatozoon by using electropermeabilization of the membrane to modulate transmembrane Ca₂₊ influx. This artifice 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 progression of a series of cellular and biochemical events that characterize fertilization. The Ca₂₊ regimen affects 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, amplitude and temporal modulation of the Ca₂₊ signals during the early minutes impact the extent of developmental organisation and differentiation at the implantation stages. This artificial approach of oocyte activation is valuable for two main reasons. By assuring fidelity of the Ca₂₊ response, it opens new approaches in determining the biochemical mechanisms driving the egg to embryo transition. In addition, it improves developmental predictions in applied biotechnology. We therefore appear to be moving into an era of more rapid transfer of knowledge through the use of a common microfluidic interface. Through the advent of numerization we are learning to rely on computerized tools to carry out precise cellular intervention at the level of the single cell. Such intervention in the synchronization of biochemical events using Ca₂₊ signaling will lead to a better understanding of the chronology of biochemical processes that impact the developmental processes at later stages.

KEY WORDS: egg, activation, development, microfluidic