In mammals, the dynamic of Ca2+ oscillations induced by fertilization causes the remodeling of the parental chromosomes and activates the developmental processes. The objective of the present study was to evaluate whether the modulation of the regime of Ca2+ oscillations during the process of egg activation is capable of inducing specific chromatin modification. The sensitivity of chromatin modification during oocyte activation was evaluated by comparison of the methylation profile of Igf2r, the maternal allele of which is methylated, and H19, the maternal allele of which is unmethylated. Freshly ovulated mouse oocytes were parthenogenetically activated by a series of repetitive Ca2+ ion influxes induced by electropermeabilization of the plasma membrane in a microfluidic processor. The first treatment (T1) consisted of 24 electrical pulses (1.45kVcm−1) given every 8min for 3h in the presence of 100μM of Ca2+. The treated oocytes were almost all activated, 98% (328/338), and formed pronuclei 3 or 4h after the first pulse. The second treatment (T2) was made up of 16 electrical pulses of lower amplitude (1.12kVcm−1), given every 8 min for 2h under the same conditions. The rate of egg activation dropped to 29%, (86/298), and the time course of pronucleus formation was completed 7h after the first pulse. The remaining oocytes, 71% (212/298), extruded the second polar body, but their chromatin did not undergo full decondensation and then did not proceed further to pronucleus formation. The methylation profiles of the two imprinted genes were analyzed by using the bisulfite sequencing method. Oocytes that formed pronuclei after being subjected to either the T1 or the T2 treatment displayed methylation profiles of the two alleles similar to those of the non-treated oocytes. The Igf2r-DMR was found fully methylated on all 13 copies examined, and the H19 DMR was found mostly unmethylated (13 out of 19 copies; 6 were partially methylated). Thus, when parthenogenetic activation results in formation of a pronucleus the methylation profile of these two genes is maintained. In contrast, those from oocytes that responded by PB extrusion showed the following: fully unmethylated Igf2r-DMR copies (9/9) and partially methylated H19-DMR copies (4/11). These results reveal that rapid de novo methylating and demethylating activity are present when cells are subjected to partial activation. Thus, when the biological response of the oocyte is incomplete, that is to say, when the chromatin remains partially decondensed after suboptimal activation, both active demethylation and de novo methylation can be remarkably dynamic. While the molecular mechanism needs to be clarified, these results reveal that modulation of the process of egg activation might give new opportunities to better understand the epigenetic chromatin changes caused by fertilization.
133SUBOPTIMAL OOCYTE ACTIVATION CAUSES METHYLATION CHANGE IN TWO
IMPRINTED GENES IN THE MOUSE