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Tetraploid mouse embryos usually cease to develop early after implantation, though they can develop to blastocysts. To characterize the failure of development in detail, tetraploid mouse embryos at the preimplantation period were examined as to both their morphology and number of cells. The tetraploid embryos were produced by 12 hr treatment with cytochalasin B (CB) at the 2-cell stage of backcross of (C57BL/6 x C3H/He) F1 x C3H/He. The tetraploid embryos in the preimplantation period exhibited compaction at 72 hr after hCG injection and blastocyst formation at 96 hr, as well as diploid embryos, but the number of cells composing the embryos was significantly smaller than that in the diploid embryos. At the term 60-96 hr after hCG injection, mean cell cycles were 14.03 hr in the tetraploid embryos, but 12.02 hr in the diploid. When tetraploid embryos were transferred into the oviducts of pseudopregnant recipients immediately after CB treatment, the number of cells in tetraploid blastocysts was increased compared with the embryos cultured in vitro, though the number did not reach that of diploid embryos. These results suggested that compaction and blastocyst formation in preimplantation development of tetraploid embryos depended on the time after hCG injection, irrespective of the number of cells or the length of the cell cycle. The lengthening of the cell cycle in tetraploid embryos may be one of the causes of failure in postimplantation development.

Preimplantation mouse embryos were incubated in vitro with mouse recombinant gamma-interferon (IFN). The effect of the gamma-IFN on major histocompatibility complex (MHC) class I antigen expression was tested using an ELISA procedure. It was found that there is a doubling of Db antigens and a tripling of Qa-2 antigens on C57BL/6 mouse embryos cultured from the 8-cell stage for 24 h in the presence of 10(5) units/ml gamma-IFN. The effect of gamma-IFN on the rate of preimplantation embryonic development was tested by culturing 2-cell embryos for 48 h and 8-cell embryos for 24 h in the presence of varying concentrations of gamma-IFN up to 10(6) units/ml. Two methods were used to assess the cell number per embryo after the culture period: incorporation of [3H]thymidine into DNA, and direct counting of nuclei in fixed and stained embryos. Both methods showed that treatment with gamma-IFN increases the rate of development of preimplantation mouse embryos. Since rate of preimplantation embryonic development is genetically controlled by the Ped gene, it is suggested that gamma-IFN has a direct effect on the Ped gene phenotype of preimplantation mouse embryos.

A two-step polymerase chain reaction (PCR) assay was used to determine the sex of mouse preimplantation embryos obtained from oocytes fertilized and cultured in vitro, to investigate the differences in the developmental rates of mouse embryos according to the sex. All the in vitro developed embryos could be analyzed by this method. When the embryos were classified according to the time of morula to blastocyst transition as fast-intermediate- and slow-growing embryos, a significantly high percentage (78.0%) of the fast-developing embryos were identified as males; while a significantly lower percentage (42.5%) of slow-developing embryos were identified as males. The intermediate-developing embryos presented a sex ratio not significantly different from the total (57.5%). The deviation of sex ratio was further confirmed by embryo transfer experiment, where fast- and slow-developing embryos gave 76.2% and 25.7% male fetuses, respectively. We concluded that male mouse embryos fertilized and cultured in vitro develop faster than female embryos.

We have confirmed the faster growth of male preimplantation mouse embryos. We have also studied the transcription of Y chromosomal genes postulated to have a role in sex determination, using the highly sensitive technique of reverse-transcription polymerase chain reaction at these early stages. We find that two sex-determining region genes, Sry and Zfy, are transcribed during mouse preimplantation development, while the Zfy homologs Zfx and Zfa and a sex-determining region gene originally called A1s9 (now called Ube1y-1) are not. We also show that the anti-Müllerian hormone gene, which contains a Sry consensus binding element in its 5' promoter region, is not transcribed at this time. Developmental curves show that Sry and Zfy are expressed commencing at the two-cell stage. These results suggest that mammalian sex determination starts prior to gonad differentiation.

Mouse cells of developing embryos at the 2-4 cell, morula and blastocyst stages, were bombarded by high velocity tungsten microprojectiles. About 70% of developing embryos survived the bombardment. The general embryo structure did not change as a result of the bombardment. Penetration of the tungsten microparticles into the embryo cell nuclei was found at all stages being investigated, and tungsten particle localization on mitotic chromosomes was demonstrated. The total DNA of the mice born from the bombarded embryos was analyzed by dot-blot hybridization and PCR with post-hybridization. The most important results were obtained in experiments with blastocysts. In three cases of blastocyst bombardment, the presence of transferred plasmid DNA (pSV3-neo) was revealed. Transfected cells were shown to be located in the fetal membrane as well as in the embryo. The bombardment of mouse culture cells resulted in their transfection and the production of G418-resistant clones.

The genetic control of the rate of preimplantation development was studied in the mouse embryos. The number of cells in the embryo and the percentage of embryos at the blastocyst stage were determined on the 3.5 day of pregnancy. The experiments were carried out with CBA, A/He, C57Bl/Mib mice and mice homozygous by the mutant genes white (Miwh), fidget (fi) and ocular retardation (or), congenic with the inbred C57Bl/Mib mice. Contrasting differences were found between C57Bl/Mib and fi/fi mice. The rate of development of the morphologically normal C57Bl/Mib and fi/fi and F1 embryo was shown to depend on the maternal genotype, rather than on the paternal one. The effect of maternal genotype of the rate of preimplantation development was related to differences in the time of beginning of the cleavage. The rate of cleavage is similar for the C57Bl/Mib, fi/fi and F1 embryos.

A study was made of the duration of early developmental stages in F1CBA/C57BL mouse hybrid embryos. It was found that the longest were zygota and 2-cell stages. During cleavage the developmental stages became about 2 times shorter. The 4-cell stage was the shortest. The duration of the cleavage stages in the test mouse embryos was different from that in mouse embryos of other strains reported in the literature. These findings suggest that the genetic features of mice belonging to different strains exert an effect on the duration of cleavage stages.