Apoptosis In Mouse EmbryosCo-Cultured With Polarized Or Non-Polarized Uterine Epithelial CellsUsing Sequential Culture Media



Introduction: This study investigated the effects of the in vitro co-culture of mouse embryos with non-polarized or polarized uterine epithelial cells, using sequential culture media, on their development to blastocysts, blastocyst quality (blastocyst diameter and cell number) ,the onset and frequency of apoptosis as well as morphological changes that confirm to the general criteria of apoptosis by using a terminal deoxy nucleaotidyl transferasemediated dUDP nike-end labeling(TUNEL) assay and expression of apoptotic related gene including Bcl-2 and Bax using RT-PCR.
Materials and Methods: There were three treatments, all of which used sequential culture media. The treatments were no coculture (control), non-polarized or polarized epithelial cell monolayer co-culture in 24-well tissue culture plates. Mouse uterine epithelial cells were isolated enzymatically and were seeded either on the surface of the culture plate (non-polarized monolayer) or on a Millipore filter insert coated with extra-cellular matrix extract (polarized monolayer) that was then placed in the culture plate. Two-cell mouse embryos were cultured in G-1TMver3 medium to the 8-cell stage when they were randomly assigned to the treatments. The culture medium was G-2TMver3 during the treatment phase of the study. Significances of differences were evaluated by the one way analysis of variance for continuous data.
Results: The epithelial cells cultured on Millipore filters became polarized and their morphology compared favorably with those cultured on the surface of the culture plate and in vivo uterine epithelial cells. After 96 h on the treatments, the polarized monolayer had supported the development of significantly more hatched blastocysts (80.0%; p