299 OOCYTE ACTIVATION IS THE RESULT OF CALCIUM SIGNAL SUMMATION

Abstract

Egg activation in mammals is caused by a series of cytosolic Ca²⁺ oscillations that are essential for development. However, the specific functional significance of the number or the frequency of the Ca²⁺ signals is not clear. The objective of this study was to determine whether the efficiency of egg activation relies on the frequency or on the number of signals. Given the repetitive nature of the signal, it is nearly impossible to discriminate the specific effect of the frequency from the number because increasing the time interval between signals directly reduces their number in a given time unit and vice versa. To bypass such difficulty, we subjected non-fertilized mouse oocytes, which are not capable of releasing Ca²⁺ from intracellular stores, to a coherent series of four treatments. Repetitive intracellular Ca²⁺ signals were introduced into freshly ovulated mouse oocytes by electropermeabilisation of Ca²⁺ ions by means of a microfluidic processor . The first regime (T1) was a monotonic [Ca²⁺]i increase lasting about 10 min and provoked by 4 consecutive electrical pulses (1.45 kV cm⁻¹) given at 2-min intervals. This large calcium signal gave a poor activation rate: 6% (4/67). The second regime (T2) was made up of six lower pulses (1.12 kV cm⁻¹) given at 30-min intervals. The rate of egg activation reached 38% (75/195). The third treatment (T3) consisted in subjecting oocytes consecutively to the T1 and the T2 treatments. In that case, the egg activation reached 88% (121/138). The remarkable increase in egg activation efficiency can be attributed to the increase in the total [Ca²⁺]i load, which is the only change in T3. Thus, it appears that the large monotonic increase (T1) potentiates the response as revealed by the periodic stimulation delivered by T2. It therefore seems that the process downstream of the signal is incremental in nature, and entrance in interphase of the cell cycle is reached when the summation of individual signals reaches a sufficient level. We next evaluated whether the putative summation effect makes it possible to regularly get 100% of egg activation by increasing the cytosolic Ca²⁺load. Oocytes were subjected to a last treatment (T4) that consisted of T1 and then 24 pulses (1.45 kV cm⁻¹) given every 8 min. In that case 100% of the oocytes were activated (80/80). Thus, it appears that all oocytes are subject to egg activation if they receive a sufficient amount of calcium signal. Moreover, the frequency does not seem to play a crucial role for entrance into interphase because reducing the time interval from 30 min (T3) to 8 min (T4) resulted in high activation rates in both cases (88% and 100%, respectively). The question remains as to whether the distribution of the Ca²⁺ signal throughout the process of egg activation might affect some epigenetic modifications that impact developmental processes.