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The pronuclear and chromosomal changes which occur prior to and during the first cleavage division were studied in 216 mouse zygotes. Shortly before prophase the pronuclei are next to one another in the center of the cells. Contact or fusion between opposing pronuclear surfaces does not occur. The development of chromosomes at prophase takes place prior to the dismantling of the pronuclear envelopes. In the mouse, thus, parental pronuclei enter prophase in an independent fashion. At the time the pronuclear membranes break down, the pronuclei are invaded by microtubules which originate from the adjacent ooplasm in close association with clusters of small vesicles. In metaphase, the microtubules become organized into a spindle, the poles of which lack centrioles and are associated with groups of vesicles similar to those seen at the site of origin of the microtubules in prophase. At late metaphase, the chromosomes line on the equatorial region of the spindle and exhibit morphological characteristics suggesting that longitudinal splitting into chromatids has occurred. At anaphase, these separate and move toward the opposite poles of the spindle where they clump at telophase forming the nuclear complements of the two developing blastomeres. This stage is characterized also by the reconstitution of nucleoli and of nuclear membranes, and by advanced cytokinesis.

The effect of polymorphonuclear leukocytes (PMNLs) on the development of pre-implantation mouse embryos cultured from the two-cell stage to blastocyst was investigated. PMNLs (1–2 × 106) blocked development of most embryos beyond the four- or eight-cell stage. The embryotoxic activity declined with decreasing PMNL concentration. The activity of intact PMNLs or equal numbers of PMNLs that were homogenized or lyophilized following homogenization was similar. However, acidification of PMNL homogenates prior to lyophilization appeared to increase their activity, while homogenization of PMNLs under hypotonic conditions appeared to decrease their activity. The embryotoxic activity was not destroyed by storage of whole PMNLs for 1 week at 4 C.

The effect of estrogen on uptake and incorporation of amino acids was investigated in mouse embryos cultured in vitrofrom the 2-cell to hatched blastocyst stage. Embryos at various stages of preimplantation development were incubated with 14C-amino acids and different concentrations of 17β-estradiol, prepared for determination of amino acid uptake or incorporation into peptides and counted in a scintillation counter. Estradiol did not stimulate uptake or incorporation of 14C-valine into morulae. There was a slight but evident increase in both uptake and incorporation in early blastocysts. When expanded blastocysts were incubated with 14C-valine and 17β-estradiol, uptake and incorporation were significantly [p< 0.05] increased over control values at 10−10, 10−9, and 10−5Mestradiol. At 10−8, 10−7, and 10−6Mcounts were equivalent to or lower than counts in embryos incubated in 14C-valine alone. The same pattern of estrogen stimulation was observed when these embryos were labeled with a mixture of 14C-valine, lysine, and aspartic acid. In hatched blastocysts, estradiol had no effect on valine incorporation but greatly stimulated its uptake. These results demonstrate that estrogen can stimulate uptake and incorporation of amino acids in preimplantation embryos by acting directly on the blastocyst, and they suggest that the mouse embryo may develop an estrogen-sensitive mechanism just prior to implantation.