Jean-Pierre Ozil

Repetitive calcium stimuli drive meiotic resumption and pronuclear development during mouse oocyte activation

Freshly ovulated (12 hr post hCG) F1 (C57BL/6 x CBA) hybrid mouse oocytes were parthenogenetically activated by repetitive elevation of Ca2+ induced by carefully controlled electrical pulses. Different patterns of stimulation were employed to examine the role of repetitive calcium changes on meiotic resumption and pronuclear development. In the first series of experiments oocytes received 33 electrical pulses of 1.8 kV/cm delivered every 4 min. The pulse duration decreased according to a negative exponential equation from a 900-microseconds first pulse to give a total pulse duration of 18.721 msec. The strength of calcium stimuli was varied by changing the concentration of CaCl2 in the medium. Ninety-eight percent of the oocytes stimulated with 12 microM calcium extruded the second polar body by the end of treatment and 92% completed pronuclear formation between 3.5 and 8 hr after the first pulse. For higher or lower Ca2+ concentrations the proportion of oocytes developing pronuclei decreased; the timing of pronuclear formation was retarded and the majority of oocytes failed to form a pronucleus after extrusion of the second polar body. In the second series of experiments, the strength of the calcium stimuli was modulated by changing the duration of the 33 electrical pulses given in the presence of 12 microM calcium. By increasing the total pulse duration to 33.958 msec, 100% of the oocytes activated and completed pronuclear formation between 3 and 5 hr after the first electric pulse. Stimulation protocols of lower total pulse duration (less than 18.721 msec) gave rise to high rates of partial activation (up to 95%). Examination of these partially activated oocytes showed metaphases with haploid sets of chromatids characteristic of third meiotic metaphase arrest. The results indicate that repetitive calcium stimuli can regulate the rate and extent of meiotic resumption and the time course of pronuclear formation during mouse oocyte activation. They suggest that meiotic resumption in mammalian oocytes is regulated by the amplitude and frequency of cytosolic calcium oscillations induced by the activating stimulus.

De novo formation of centrioles in parthenogenetically activated, diploidized rabbit embryos

Summary— In rabbit oocytes activated parthenogenetically by repetitive electric pulses, centrioles develop de novo in blastocysts. Centrioles were not observed in earlier stages of development, not until the blastocoele is formed. Up to the morula stage (between 8–32 cells), a filamentous, electron‐dense material develops and aggregates with a small vesicle fraction within the well developed Golgi apparatus. A spherical to ovoid electron dense mass forms, which is comparable to the deuterosome or to the blepharoplast. The quantity of the electron dense material enlarges and it seems to give rise to the centriole “generating complex”. Centrioles arise in all three differentiated cell types of the blastocysts, the mural and polar trophoblasts and the embryonal cell mass at the same time. Some of the forming centrioles in parthenotes have a co‐linear arrangement, as in control blastocysts. It is not yet known whether the co‐linearly arranged centrioles represent a maturation phase, prior to the formation of the usual diplosome, with centrioles oriented perpendicularly to each other. Nor is it known whether the forming centrioles are functioning as the polar organizer of the mitotic spindle or if they can perform any other centriolar function.

Role of calcium oscillations in mammalian egg activation: experimental approach.

Biological rhythms are everywhere; the pulsatility of intracellular signals appears to maximise the cellular processes better than constant signaling. The aim of this paper is, firstly, to review the cellular mechanisms that modulate calcium oscillator activity during fertilisation and, secondly, to describe recent results we have obtained by artificially imposing rhythmical calcium stimulation on fertilised rabbit eggs during in vitro culture. The key finding in these experiments is that the egg appears to be sensitive to repetitive signalling during a period that goes far beyond the time of meiosis reinitiation. When delivered at the proper rhythm transient signalling can optimise developmental processes.