How Does High Osmolarity Induce the 2-cell Block in Pre-Implantation Mouse Embryos?

Mouse embryos develop optimally from the 1-cell to blastocyst stage in media such as KSOM that have osmolarity as low as 250mOsM, much lower than the in vivo osmolarity in the oviduct (~300-310mOsM). Previous work by our laboratory and others showed that, when any of several amino acids or derivatives are present, embryos are rescued and can develop from fertilized eggs at normal oviductal osmolarity in vitro. One of these, glycine rescues embryo development at normal in vivo osmolarity, because it is required for cell volume regulation in 1-cell embryos. We wished to determine the mechanism by which embryos stop developing and become blocked at the 2-cell stage at 310mOsM in the absence of glycine. Our first hypothesis was that increased osmolarity will induce apoptosis. We cultured 1-cell embryos in culture medium with osmolarity of 250mOsM and 310mOsM. We then assessed mouse embryos for several apoptotic processes on day2 (48hr post-hCG; 2-cell stage) and day3 (72hr post-hCG; normally 4-cell to 8-cell stage in vitro) after culture at each osmolarity. TUNEL labeling was used to detect DNA fragmentation, Cytochrome C immunolocalization to detect whether Cytochrome C was released from mitochondria, and Annexin V binding to live embryos to determine whether phospshatidylserine (PS) translocated to the external cell surface. TUNEL-positive nuclei were never detected in embryos on either day 2 or 3, including in the embryos at 310mOsM, which still remained at the 2-cell stage on day 3. After continued culture, TUNEL-positive nuclei were first detected at the blastocyst stage, primarily in the inner cell mass, indicating that DNA fragmentation does not normally appear in embryos cultured under our conditions until the blastocyst stage. Immunolocalization of Cytochrome C revealed a punctuate pattern in 2- and 4-cell embryos, which was not different in the embryos cultured at 310mOsM that were still at the 2-cell stage on day 3. In contrast, Cytochrome C staining was substantially decreased in 2-cell embryos treated with H2O2 (1mM), previously shown to induce Cytochrome C release from mitochondria. Finally, no 2-cell embryos on either day were positive for Annexin V binding, again contrasting with the clear Annexin V binding by H2O2-exposed embryos. Therefore, the failure of embryos to develop past the 2-cell stage at 310mOsM does not appear to be due to apoptosis.Our second hypothesis was that high osmolarity will prevent zygotic genome activation (ZGA) that normally occurs at the 2 cell stage. We chose H47, Eif1a and Zscan4d genes as ZGA markers, since H47 and Eif1a exhibit among the largest increases in expression at ZGA, and Zscan4d is transiently expressed during ZGA. Using quantitative real-time RT-PCR, we found the expected patterns of expression in in vivo- and in vitro-derived 1-cell and 2-cell embryos. All three markers were expressed in embryos cultured at 310mOsM at the same time as control embryos at 250mOsM and embryos cultured at 310mOsM with glycine, indicating that at least some components of ZGA still occurred in embryos that would fail to develop further. These results indicate that increased osmolarity does not induce apoptosis nor block ZGA in 2-cell mouse embryos. Therefore, the developmental block likely occurs nearer to the end of the second cell cycle, possible at the G2-M border. Research supported by a China-Canada Joint (NSFC-CIHR) Health Research grant. (poster)

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