[In vitro development of murine embryos using different types of microinjections]. / Razvitie myshinykh émbrionov in vitro pri razlichnykh variantakh mikroin''ektsii.

We studied the effects of different types of microinjections, such as the mechanical damage to cytoplasmic and nuclear membranes of the zygote and the injection of various gene-engineering constructs or buffer solutions into the cytoplasm or the pronucleus, on the preimplantation of murine embryos (CBA x x C57BL)F1. The survival rate of the embryos was estimated by their capacity to develop in vitro to the blastocyst or hatched blastocyst stages. Puncture of the cytoplasm using a microneedle and injection of buff or foreign DNA did not affect the zygotes capacity for further in vitro development. But, the puncture of the pronucleus and microinjection of gene-engineering constructs or buffer into it reliably decreased the survival rate of embryos, as compared to the control. The differences were found in the capacity of murine zygotes for in vitro development after injection with gene-engineering constructs.

[Investigation of the effect of various buffer solutions used for microinjection of gene-engineering constructs on preimplantation development of mouse embryos]. / Izuchenie vliianiia razlichnykh rastvorov, ispol'zuemykh pri mikroin''ektsii genno-inzhenernykh konstruktsii, na doimplantatsionnoe razvitie myshinykh émbrionov.

We studied the effects of some buffer solutions used for microinjection in mammalian zygotes on preimplantation development of (CBA x C57BL)F1 mouse embryos in vitro. The rate of embryo survival was estimated according to their capacity to develop to the stages of blastocyst and blastocyst hatched from zona pellucida. The results obtained suggested a reduced rate of survival of zygotes to the blastocyst stage after the injection into a pronucleus of the buffers Tris-HCl with EDTA, Tris-HCl with MgCl2 and NaCl, and medium M2 (p < 0.05) and to the stage of blastocyst hatched from zone pellucida after injection of a Dulbecco solution, as compared to the control. No differences were found in the survival rate of zygotes injected with different buffer solutions.

[The biopsy of preimplantation embryos obtained from donor pigs with follicle-stimulating hormone-induced superovulation]. / Biopsiia doimplantatsionnykh émbrionov, poluchennykh ot svinei-donorov s vyzvannoi follikulostimuliruiushchim gormonom superovuliatsiei.

We have examined possible use of the follicle-stimulating hormone (FSH) for the induction of superovulation in pigs and studied the effect of biopsy of preimplantation pig embryos on their survival in vitro. Superovulation was induced by injecting FSH twice daily over a period of four days for a total dose of 25 units. per animal. Pigs receiving pregnant mare serum gonadotropin (PMSG) according to the standard scheme served as the control. The experiments demonstrated that FSH produces significantly better estrus figures as compared with PMSG (100% and 60%, respectively; p < 0.05). The mean number of ovulations per donor was also better in the FSH group, i.e., 36.5 as compared with 17.3 in the control group (p < 0.05). The mean number of embryos obtained from one donor was higher in FSH treated animals as compared with the animals that received PMSG (29.2 vs. 19.3), however, this difference was not statistically significant. Biopsy of preimplantation pig embryos was conducted by a "manual" technique without a micromanipulator; middle blastocysts collected at days 5-6 after insemination were the most convenient for this operation. Embryos at these developmental stages contained a well developed inner cell mass, which allows separation of trophoectodermal cells only, thus minimizing damage to the embryo. The number of cells in dissected fragments did not depend on the stage of development, and even smallest fragments (4 blastomeres) were sufficient for genome analysis with the aid of PCR. The survival of embryos in vitro after biopsy practically did not differ from that of control intact embryos. Thus, we demonstrated the effective use of FSH for the induction of superovulation in pigs; we also determined the developmental stages which are most convenient for conducting biopsies and developed a technique for preimplantation biopsy, which allows genome analysis of embryos without any decrease of their survival in vitro.

[The preservation of mammalian ova and embryos at positive near-zero temperatures. II. The general patterns and mechanisms of hypothermic exposure]. / Khranenie iaitsekletok i émbrionov mlekopitaiushchikh pri polozhitel'nykh okolonulevykh temperaturakh. II. Obshchie zakonomernosti i mekhanizmy gipotermicheskogo vozdeistviia.

General patterns of the sensitivity of embryos to hypothermia are described, such as its species-specific dependence, its relationship with the embryonic stage, etc. Data are provided on differences in the cold resistance of the inner cell mass and trophectoderm cells, as well as that of the nucleus and cytoplasm. The mechanisms underlying the effects of cold on mammalian eggs and embryos are considered, and ways of improving the method of hypothermic storage are discussed. The general technological principles of the storage of mammalian embryos at positive near-zero temperatures are formulated for the first time. Advantages and drawbacks of the method and its possible practical application are discussed.

[The development of mouse embryos in vitro in a protein-free medium depending on the numbers of embryos in a microvolume of the medium]. / Razvitie myshinykh émbrionov in virto v srede bez belka v zavisimosti ot kolichestva zarodyshei v mikroob''eme sredy.

We studied the development of mouse embryos in vitro depending on the number of embryos in a given microvolume of the Ham's F-10 medium without protein or with the addition of serum. The absence of serum from the culture medium did not affect the development of two-cell embryos cultivate in groups of 5-6 (about 90% embryos developed until the stage of blastocyst and over 50% left zona pellucida), but the development of single embryos in the protein-free medium proceeded significantly worse. Single two-cell embryos cultivated in the serum-containing medium developed similar to embryos cultivated in groups. At the same time, no significant differences in development was found between eight-cell embryos cultivated individually or in groups of up to 10 embryos in the Ham's F-10 medium, either in the presence of serum or without it (about 95% embryos developed until the stage of blastocyst and over 70% left zona pellucida). The increase in the number of cultivated embryos over 10 had the adverse effect on development of either two-cell or eight-cell embryos. The attachment of blastocysts to the substrate after leaving zona pellucida and growth of trophectoderm was observed only in the presence of serum. These results suggest that interaction between preimplantation embryos in culture can probably be mediated by factor(s) released by embryos into the medium. Serum appears to contain such factor(s). Sensitivity of embryos to the factor(s) apparently depends on the stage of development.