In mammals fertilization triggers a series of Ca2+ oscillations that not only are essential for events of egg activation but also stimulate oxidative phosphorylation. Little is known, however, about the relationship between quantitative changes in egg metabolism and specific long-term effects in offspring. This study assessed whether post-natal growth is modulated by early transient changes in NAD(P)H and FAD2+ in zygotes. We report that experimentally manipulating the redox potential of fertilized eggs during the pronuclear (PN) stage affects post-natal body weight. Exogenous pyruvate induces NAD(P)H oxidation and stimulates mitochondrial activity with resulting offspring that are persistently and significantly smaller than controls. Exogenous lactate stimulates NAD+ reduction and impairs mitochondrial activity, and produces offspring that are smaller than controls at weaning but catch up after weaning. Cytosolic alkalization increases NAD(P)+ reduction and offspring of normal birth-weight become significantly and persistently larger than controls. These results constitute the first report that post-natal growth rate is ultimately linked to modulation of NAD(P)H and FAD2+ concentration as early as the PN stage.

Alteration of the postnatal phenotype has sparked great concern about the developmental impact of culture media used at fertilization. However, the mechanisms and compounds involved are yet to be determined. Here, we used the Ca responses from mouse eggs fertilized by ICSI as a dynamic and quantitative marker to understand the role of compounds in egg functioning and establish possible correlations with adult phenotypes. We computed 134 Ca responses from the first to the last oscillation in media with specific formulations. Analyses demonstrate that eggs generated two times as many Ca oscillations in KSOM as in M16 media (18.8 ± 7.0 vs 9.2 ± 2.5). Moreover, the time increment of the delay between two consecutive oscillations, named TIbO, is the most sensitive coefficient characterizing the mechanism that paces Ca oscillations once the egg has been fertilized. Neither doubling external free Ca nor dispermic fertilization increased significantly the total number of Ca oscillations. In contrast, removing Mg from the M16 boosted Ca oscillations to 54.0 ± 35.2. Hence, [Mg]/[Ca] appears to determine the number, duration and frequency of the Ca oscillations. These changes were correlated with long-term effects. The rate of female's growth was impacted with the 'KSOM' females having only half the fat deposit of 'M16' females. Moreover, adult animals issued from M16 had significantly smaller brain weight vs 'KSOM' and 'control' animals. TIbO is a new Ca coefficient that gauges the very early functional impact of culture media. It offers the possibility of establishing correlations with postnatal consequences according to IVF medium formulation.Free French abstract: A French translation of this abstract is freely available at http://www.reproduction-online.org/content/154/5/675/suppl/DC2.

Following fertilization, eggs exhibit a series of repetitive increases in intracellular calcium that activate development. The developmental impact of the long-lasting series of Ca 2+ signals is still a subject of controversy. Although several studies using parthenogenetically activated eggs suggest that Ca 2+ dynamics affect post-implantation development, artificial stimulation of Ca 2+ signaling after ICSI in bovine eggs shows that development still remains poor in comparison to fertilized eggs. Such divergence between parthenogenetic studies and those aimed at stimulating ICSI eggs makes it impossible to draw any conclusions regarding the function of Ca 2+ signaling for two reasons. First, non-fertilized eggs do not release Ca 2+ from intracellular stores and their development is compromised due to the absence of paternally-derived chromosomes. Second, because ICSI eggs are excitable, Ca 2+ stimulation generates additional Ca 2+ oscillations that might compromise their development. Moreover, in both cases, Ca 2+ signaling is not physiological. To understand better the function of Ca 2+ signaling at fertilization, we developed a new approach based on micro fluidic technology that makes it possible to drive Ca 2+ signal dynamics of fertilized eggs with no apparent deleterious effects. This method relies on the fact that the properties of the IP3 receptor (IP3R) calcium channel are changed after fertilization, and IP3 and Ca 2+ act as co-agonists to cause Ca 2+ -induced Ca 2+ release (CICR) from intracellular stores. Because Ca 2+ has both an inhibiting and a stimulating function, we exploited these opposing properties. First, we inhibited Ca 2+ release by external washing with Ca 2+ -free medium; this extra cellular washing decreases cytosolic [Ca 2+ ]I, and facilitates dissociation of Ca 2+ ions from the IP3R that in turn decreases the probability of IP3R channel opening. Second, once the IP3R is inhibited, a simple injection of Ca 2+ ions by electropermeabilization triggers channel opening and induces Ca 2+ release. Then, by just varying the time interval and the number of the electrical pulses, it is possible to drive the dynamics of the CICR process that initiates development. Intracellular Ca 2+ imaging demonstrated that fertilized eggs subjected to 24 electrical pulses (1.45 kV cm −1 ) every 8 min for 3 h in the microfluidic processor responded by exhibiting 24 induced-Ca 2+ transients that are caused by calcium release from intracellular stores. All auto-regenerative responses between pulses were inhibited. Among 60 treated embryos transferred to pseudo-pregnant recipients, 40 (67%) developed to term, with birth of live offspring, thus demonstrating that this new methodology does not compromise development. Because the eggs are fertilized, it now becomes possible to study the function of Ca 2+ signaling during egg activation and to evaluate its developmental impact, if any, in association with genomic approaches.