[Deoxyribonucleic acid and protein synthesis in delayed mouse blastocysts in vitro]

Developmental ability, DNA and protein synthesis were examined in mouse blastocysts which were delayed by proteinase inhibitors (nitrophenol-p-guanidino benzoate (NPGB) & soybean trypsin inhibitor. Cell division is depressed in the culture which contains proteinase inhibitors: Delayed blastocysts showed no increase in number after 5 days of culture. Mitotic figures were absent 120 hours after culture. On the other hand, most of the blastocysts showed a substantial increase in number in the first few days following the onset of delay. The DNA synthesis of nuclei in delayed blastocysts was measured by autoradiography incorporating [3H]-TdR. Although the incorporation rate was 54 +/- 8 cells/embryo (83 +/- 7%) on the first day of culture, it was decreased to 1/3 (36 +/- 5, 24 +/- 1%) on the 10th day of culture. Protein synthesis was measured with the incorporation rate of [35S]-methionine. The incorporation rate of delayed blastocysts declined sharply on the 5th day of culture (1.6 +/- 0.4 pmol/embryo/hr.) in contrast to that of normal implanted blastocysts (2.5 +/- 0.3 pmol/embryo/hr.). Qualitative changes in synthesized protein were estimated by SDS-PAGE. About 110 of the autoradiographic bands on 1-D gel were observed in the 4th day blastocysts, and actin is the major protein synthesized, located between 50,000 and 55,000 daltons. No differences in these bands were seen between normal and delayed blastocysts. However, two-dimensional gel showed that the qualitative patterns of protein synthesis of delayed embryos are significantly different from those of normal implanted blastocysts. In delayed blastocysts, trophoblast-specific spots varied versatile. Above all, protein synthesis of trophoblast in delay is selectively suppressed by proteinase inhibitors, but not ICM. Therefore, delayed blastocysts are not always metabolically dormant.