Stereological study of mouse uterine and in vitro grown blastocysts: cell numbers and volumes

Mouse blastocysts were studied to determine if there were differences in cell number and volumes between those that were 1) derived from the uterus prior to implantation on the afternoon of day 4 of pregnancy and 2) those that were cultured for 72 hr from two-cell-stage embryos. Blastocysts were fixed, embedded in resin, and serially sectioned at 1.5 or 2 microns. Photographic prints of alternate sections were used to count the numbers of inner cell mass (ICM) and trophectoderm cells. Cavalieri's direct estimator was applied to the same prints to estimate the volume of the whole blastocyst. Point counting was used to determine the volumes of the ICM, trophectoderm, and zona pellucida. The number of cells and size of the ICM were similar between the two groups of blastocysts, although it was found that the ICM of uterine embryos that did not have a zona pellucida were smaller than the ICM of those that did. There were twice as many trophectoderm cells in the blastocysts that were cultured from two-cell embryos, and these cells were also found to be larger. Furthermore, the volume of the zona pellucida was less in the uterine blastocysts. This study indicates that, while trophectoderm proliferation is enhanced in vitro, the ICM is more constant and thus may be self-regulating and independent of the growth conditions of the blastocysts as a whole. This study also suggests partial zona lysis occurs in utero and occurs either at a reduced rate or not at all in vitro.