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Egg activation in mammals is caused by cytosolic Ca2+oscillations that are essential for development. However, despite increasing knowledge about signal transduction mechanisms, the functional linkage between frequency number, amplitude and duration of the Ca2+signal and the kinetics of pronucleus formation has not yet been defined. While a wide range of Ca2+signal parameters are efficient in causing egg activation, the basic rules governing how the egg integrates these signalling events are not yet clear. Thus, in the perspective of better understanding how the egg processes Ca2+signalling events, the objective of this study was to determine experimentally whether the efficiency of egg activation and the subsequent early developmental stages rely on Ca2+signalling summation. Non-fertilized, but freshly ovulated mouse eggs, were subjected to a series of repetitive Ca2+influxes of various patterns modulated by a non-invasive membrane electropermeabilization method. Using a combination of two suboptimal treatments we have shown that mouse eggs can sum up the effects caused by various patterns of intracellular Ca2+concentrations transient during the period of egg activation. In addition, overloading the intracellular milieu by repetitive Ca2+influxes did not seem to inhibit the process of activation. The kinetics of pronuclear formation among a population of eggs treated in the same conditions became accelerated when the total dose of Ca2+signal ‘experienced’ by the eggs was increased. The results suggested that summation of the biological effects of all Ca2+signals constitutes an important mode of Ca2+signal integration.

Ca(2+) oscillations and signaling represent a basic mechanism for controlling many cellular events. Activation of mouse eggs entrains a temporal series of Ca(2+)-dependent events that include cortical granule exocytosis, cell cycle resumption with concomitant decreases in MPF and MAP kinase activities, and recruitment of maternal mRNAs. The outcome is a switch in cellular differentiation, i.e., the conversion of the egg into the zygote. By activating mouse eggs with experimentally controlled and precisely defined Ca(2+) transients, we demonstrate that each of these events is initiated by a different number of Ca(2+) transients, while their completion requires a greater number of Ca(2+) transients than for their initiation. This combination of differential responses to the number of Ca(2+) transients provides strong evidence that a single Ca(2+) transient-driven signaling system can initiate and drive a cell into a new developmental pathway, as well as can account for the temporal sequence of cellular changes associated with early development.

Although the dynamics of oscillations of cytosolic Ca2+ concentration ([Ca2+]cyt) play important roles in early mammalian development, the impact of the duration when [Ca2+]cyt is elevated is not known. To determine the sensitivity of fertilization-associated responses [i.e., cortical granule exocytosis, resumption of the cell cycle, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, recruitment of maternal mRNAs] and developmental competence of the parthenotes to the duration of a [Ca2+]cyt transient, unfertilized mouse eggs were subjected to a prolonged [Ca2+]cyt change for 15, 25, or 50 min by means of repetitive Ca2+ electropermeabilization at 2-min intervals. The initiation and completion of fertilization-associated responses are correlated with the duration of time in which the [Ca2+]cyt is elevated, with the exception that autonomous CaMKII activity is down-regulated with prolonged elevated [Ca2+]cyt. Activated eggs from 25- or 50-min treatments readily develop to the blastocyst stage with no sign of apoptosis or necrosis and some implant. Ca2+ influx into unfertilized eggs causes neither Ca2+ release from intracellular stores nor rapid removal of cytosolic Ca2+. Thus, the total Ca2+ signal input appears to be an important regulatory parameter that ensures completion of fertilization-associated events and oocytes have a surprising degree of tolerance for a prolonged change in [Ca2+]cyt.

Postfertilization manipulation of mammalian embryos results in various developmental alterations. To determine whether the manipulation of the Ca2+ regime causing oocyte activation is a valuable experimental means in helping understand the biological process by which embryos integrate signals from outside and later regulate gene expression, we linked Ca2+ signal parameters i.e. amplitude, number and frequency, with the efficiency and quality of postimplantation development. Freshly ovulated rabbit oocytes were subjected to repetitive and modulated Ca2+ influx. The results provide three major pieces of information. Firstly, the Ca2+ stimulus is the most efficient signal activating mammalian eggs when it is applied in a repetitive manner, the amplitude being the crucial factor. Secondly, the dynamics of early cleavage does not appear to be determined by either the frequency or the amplitude of modulation of the Ca2+ signal that activates the oocyte. Thirdly, amplitude and temporal modulation of the Ca2+ signal in the early minutes influences the developmental performance and the morphology of the rabbit parthenogenetic conceptus at day 11.5 of pregnancy. The results demonstrate the importance of epigenetic events during postfertilization as well as the possible uses of Ca2+ modulation in studying long term developmental effects.

In immunoblotting analysis of fully grown oocyte proteins separated by sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis, four polypeptides reacted specifically with cytokeratin antibodies: Mr 66 000, IEP 5.6; Mr 64 000, IEP 5.4; Mr 59 000, IEP 5.3; Mr 55 000, IEP 5.2. These proteins remained insoluble after extraction in high salt buffer and Triton X-100. In oocytes isolated from small antral follicles, only two polypeptides of Mr 66 000 and Mr 55 000 were detected. Immunofluorescence microscopy revealed a bright granular staining throughout the oocyte with an accumulation of granules in the perinuclear and cortical regions. Using electron microscopy and immunogold staining after treatment with cytokeratin antibody, gold particles were found on discrete amorphous material distributed throughout the cell. In the subcortical region of the oocyte, dense aggregates, whose diameters ranged from 3 to 8 μm, were also covered with gold particles. From these results it appears that cytokeratin-like proteins are present in sheep oocytes in a non-fibrillar form.

The distribution of actin was studied during early events of sheep fertilization by fluorescence microscopy after staining with 7-nitrobenz-2-oxal-1.3 diazole (NBD)-phallacidin and anti-actin antibody and by electron microscopy after heavy meromyosin labelling. Unfertilized and fertilized eggs exhibited a continuous band of fluorescence with both NBD-phallacidin and anti-actin antibody. Unlike in mice, no high concentration of actin overlying the spindle was detected in ovulated sheep oocytes. At the site of sperm head incorporation, the fertilization cone developed above the decondensing male chromatin and was underlined by a submembranous area rich in microfilaments. A similar actin network was observed in the cortex of the second polar body. Cytochalasin D was used to investigate the role of actin during the fertilization process. This drug did not prevent sperm fusion and incorporation but inhibited polar body abstriction and fertilization cone development and retarded sperm tail incorporation. Moreover, in the presence of the drug, the anchorage of the metaphase II spindle at the surface of the egg was destroyed. The role of microfilaments in these early events is discussed.