Mechanisms of cell number regulation in the peri-implantation mouse blastocyst.

Low viability of manipulated or in vitro cultured is caused primarily by the reduced cell number in the implanting blastocysts. In order to investigate the effect of implantation delay on embryo viability and cell number, mouse blastocysts were transferred into oviducts of day 0 pseudopregnant females. This type of transfer improved embryo survival rates, indicating that embryos retarded by in vitro culture restored their viability during 3 days of delayed implantation. Our results showed that even in the cases when the initial cell count was as low as 28.2 +/- 0.7 cells per blastocyst (vs 60.5 +/- 1.4 cells in the control blastocysts, developed in vivo), implantation delay increased this number to 107.2 +/- 3.5 cells (control blastocysts had at this stage on average 111.0 +/- 3.7 cells). Half-blastocysts, developed from the single blastomeres of the 2-cell embryos or from experimentally produced tetraploids, had around 50 cells after 3 days of implantation delay. This indicates that the start of blastocyst dormancy is triggered during the eighth cell cycle and independent of the absolute cell number or the number cytokineses. Implantation-delayed blastocysts, developed from the half-embryos with the doubled volume of cytoplasm, had on average 70.5 +/- 2.4 cells, suggesting that embryo fall into quiescence is also dependent upon the attainment of a definite nucleo-cytoplasmic ratio. We conclude that blastocyst readiness for implantation is determined by two factors: number of cell cycles and nucleo-cytoplasmic ratio.

[The first and second polar bodies in mouse oogenesis]. / Izuchenie pervogo i vtorogo poliarnykh telets v oogeneze myshi.

Using light and electron microscopy, we studied several characteristics of the interphase nuclei and mitotic chromosomes in embryonic cells Cyclops kolensis (Lill) before and after chromatin diminution. We detected the reduction of one of the two nucleoli in somatic cells resulting from chromatin diminution. However, the ratio of the total diameter of the nucleoli to the diameter of the nucleus or to the diameter of the cell remains unchanged. Granules of the chromatin to be eliminated become visible in somatic cells of the C. kolensis embryos at the interphase of the fourth cleavage division. The structure of interphase nuclei and mitotic chromosomes of somatic cells of C. kolensis embryos is different before and after chromatin diminution. Possible mechanisms underlying these differences are discussed.

Preimplantation development of manipulated mouse zygotes fused with the second polar bodies: a cytogenetic study.

Immediately after fertilization one chromatid of each maternally-derived chromosome is extruded into the second polar body (2PB). We tested the ability of these "extra" chromosomes to support preimplantation development. Micromanipulation and electrofusion techniques were used to fuse 2PBs with diploid, haploid, or enucleated mouse zygotes. Androgenetic haploids, intact embryos, and digynic triploids served as the controls for the reconstructed embryos. Androgenetic haploid zygotes developed to the blastocyst stage only when fused with the 2PBs. This result demonstrates that even when extruded into the 2PB, chromosomes retain their ability to support normal preimplantation development. However, 2PB fusion with diploid zygote impaired preimplantation development. Normal development of experimentally produced digynic triploids (zygotes with one extra maternal pronucleus) indicated that developmental arrests, caused by the 2PB fusion, were not the result of triploidy or micromanipulation procedures. Cytogenetic studies showed that developmental failures of the reconstructed embryos were caused by premature chromosome condensation of the polar body chromosomes. This result indicates that 2PB must be removed from the zygotes' perivitelline space during animal cloning experiments. In addition, we showed that 2PB fusion with enucleated zygote is a reliable method for 2PB karyotyping and may be used in the studies of mammalian meiosis.

The role of the nucleocytoplasmic ratio in development regulation of the early mouse embryo.

The hypothesis suggesting that the blastocoele is able to form only at a definite nucleocytoplasmic ratio was tested. We compared the development of preimplantation mouse embryos under different conditions. The results demonstrated that the start of cavitation is not dependent on the number of cell divisions. Thus, a definite nucleocytoplasmic ratio is not required for blastocoele formation to start. Our studies on embryos with microsurgically altered cytoplasm content provided evidence for the following biological clock mechanism: a change in the cell program of morphogenesis needs definite concentration of the products of a previous genetic program.