Development of mouse embryos co-cultured with polarized or non-polarized uterine epithelial cells using sequential culture media.
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), apoptosis, Bcl-2 and Bax gene expression. There were three treatments, all of which used sequential culture media. The treatments were no co-culture (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-1 ver3 medium to the eight-cell stage when they were randomly assigned to the treatments. The culture medium was G-2 ver3 during the treatment phase of the study. Significances of differences were evaluated by the one-way analysis of variance for continuous data. 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<0.05) than the non-polarized monolayer (63.4%) or the control (61.4%) culture treatments. Co-culture resulted in the production of blastocysts with significantly more cells (non-polarized monolayer 56.7+/-2.1, polarized monolayer 61.9+/-2.1) than the control culture (42.8+/-2.6; P<0.05) but the diameter and shape of the blastocysts were not significantly different. The proportion of blastocysts with apoptotic blastomere was higher for the control culture (94.4%) than for the non-polarized (68.2%) or polarized (66.7%) co-culture systems (P<0.05). Moreover, the apoptotic index was significantly higher in control blastocysts (5.6+/-0.9; P<0.05) than in non-polarized (1.7+/-0.3) or polarized (1.5+/-0.3) co-culture. In the control, Bax mRNA was strongly expressed when compared to co-culture treatments (P<0.05), whereas, the relative abundance of Bcl-2 mRNA to the beta-tubulin was lower than co-culture treatments (P<0.05). It is concluded that a co-culture system involving polarized uterine epithelial cells and sequential culture media is a promising method of producing mouse embryos.
Azadbakht
Mehri
M
Department of Anatomy, Tarbiat Modarres University, P.O. Box 14115-111, Tehran, Iran.
Valojerdi
Mojtaba Rezazadeh
MR
Mowla
Sayed Javad
SJ
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D003470:Culture Media / Q000737:chemistry*; D046149:Embryo Culture Techniques / Q000379:methods*; D004622:Embryo, Mammalian / Q000502:physiology*; D004847:Epithelial Cells / Q000502:physiology / Q000648:ultrastructure*; D005260:Female; D005786:Gene Expression Regulation; D051379:Mice; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014599:Uterus / Q000166:cytology*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
D003470:Culture Media; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein