Development and DNA polymerase activities in cultured preimplantation mouse embryos: comparison with embryos developed in vivo.

Embryos from superovulated female mice that developed in vitro from the two-cell stage were compared with in vivo embryos with respect to yield of blastocytes, number and types of cells, morphology in histologic section, and DNA polymerase activities. Significantly more embryos developed into blastocytes in vitro (93%) than in vivo (18%). Inner cell mass (ICM) cells comprised approximately 30% of total cells in late morula/early blastocyst stage embryos developed either in vitro or in vivo. However, the in vitro embryos developed approximately half the number of total cells as in vivo embryos, did not develop endoderm, and did not develop abembryonic trophoblast cells with morphologic characteristics of late preimplantation in vivo embryos. DNA-dependent DNA polymerase activities in in vitro embryos decreased in correspondence with the decrease in cell number resulting in per cell levels comparable to in vivo embryos. In contrast, the poly (A).oligo(dT)-dependent DNA polymerase activity was the same in embryos developing either in vitro or in vivo, indicating different regulatory mechanisms for the two enzyme activities. A variety of nutrients and growth factors in the culture medium did not increase cell numbers or DNA polymerase activities in embryos cultured for 3 days; extending the culture an additional 24 hours resulted in a loss of ICM cells and decreases in both DNA polymerase activities. These results show that the retarded growth of embryos in vitro is equally distributed between ICM and trophoblast, is not reversed by culture conditions that include serum growth factors, and is not due to decreased cellular levels of DNA polymerase activities.

Histochemistry of otic capsule sclerotic lesions in Palmerston North autoimmune strain mice.

Otic capsule osteogenesis is a common finding in temporal bones from autoimmune disease individuals. However, the underlying cellular mechanisms are poorly understood. Therefore, to better understand this relationship of autoimmune disease and otic capsule pathology, inner ear sclerotic lesions of the Palmerston North autoimmune disease mouse were histochemically stained to identify their content and potential osteogenic processes. Lesions stained positive for calcium, amyloid, fibrinoid, and glycoproteins (PAS), but negative for collagen, calcium oxalate, reticular fibers and glycosaminoglycans (Alcian Blue). Amyloid and fibrinoid deposition are associated with other immune disease, which suggests these local processes may provide a protein substructure that calcifies in lesion progression. Similar cellular mechanisms may underlie certain types or phases of human autoimmune otic capsule disease.

Fluorescence of Trypan blue in frozen-dried embryos of the rat.

Freeze-drying and fluorescence microscopy techniques were combined to create a sensitive method for the visualization of the teratogenic dye, Trypan blue, in both protein-bound and free forms. In the development and initial application of this method, visceral yolk sacs of several gestational ages as well as normal appearing, 12-day embryos obtained from dye-injected rats were utilized. Observations on paraffinized sections of the yolk sac placentae demonstrated that only the protein-bound form of the dye exists in the yolk sac cavity whereas both forms of the dye exist in supranuclear regions of cells of the visceral endoderm. Paraffin sections of the normal appearing, 12-day embryos displayed the protein-bound form of dye within lumina of mid- and hind-gut, and both forms of dye in the primitive mucosa of mid- and hind-gut. The advantages of the method are derived not only from the use of fluorescence microscopy but also from the avoidance of solvents that are employed in more routine microtechniques.