Embedment procedures were modified from Dykstra. Compact morulae were removed from fixative and washed three times for a total of 15 min in 0.1 M PBS. A solution of low melting point agarose (4% w:v) was prepared in 0.2 M PBS, and embryos were individually transferred into the liquid agarose at 37°C. The agarose was cooled to 25°C and allowed to harden. Agarose-embedded embryos were then removed as individual 3-mm3 blocks with a razor blade. Agarose blocks were postfixed in 2% osmium tetroxide in 0.2 M PBS for 45 min, washed three times in deionized water for a total of 15 min, subjected to an alcohol dehydration series, and individually embedded into Spurr’s resin. Resin blocks were solidified at 80°C for at least 8 h. Ultrathin sections (80 nm) from each compact morula were collected onto copper grids and poststained with methanolic uranyl acetate and lead citrate. Sections were visualized on a transmission electron microscope, and 7 to 9 random micrographs were taken to represent each embryo. Micrographs were printed at a final magnification of X6720. An average of 37 534 ± 773 ^m2 (mean ± SEM) was analyzed for each compact morula.
General procedures. The volume density of cellular components was determined utilizing the point-count method. Briefly, a transparent grid consisting of 576 fine and 64 coarse test points was laid over each micrograph. The number of test points falling on an individual structure was recorded, as was the total number of test points available on the test grid. The volume occupied by each component was equivalent to the number of points falling on that structure divided by the total number of test points available on the test grid. This figure was then expressed as a percentage. Within an individual embryo, the volume density, or proportional volume, was computed based on the combined total counts from all micrographs for that embryo.
Mitochondria. Three types of mitochondria were analyzed: 1) mature mitochondria, containing well-developed and evenly stacked cristae; 2) immature mitochondria, having poorly developed, peripheral cristae or a hooded appearance; and 3) vacuolated mitochondria, containing a membrane-bound vesicle. The volume density of total mitochondria was calculated based on the sum of the densities for the three individual mitochondrial types.
Other cellular components. The volume densities of lipid droplets, vacuoles, inclusion bodies, apoptotic bodies, intercellular space, debris between cells, nuclei, and cytoplasm, as well as the cytoplasmic-to-nuclear ratio, were determined.
The volume densities for all cellular and intercellular components were analyzed by General Linear Models procedures of the Statistical Analysis System. When a significant F-statistic was found, treatment differences were identified using Duncan’s multiple range test. Effects of treatment were considered statistically different at P < 0.05. All data are reported as least-squares means ± SEM.