Development of vitrified-warmed mouse embryos co-cultured with polarized or non-polarized uterine epithelial cells using sequential culture media

Abstract

Purpose: To our knowledge, little is known about the effect of polarized and non-polarized uterine epithelial cells on cryopreserved embryo growth. This study was, therefore, set up to investigate the effect of these monolayers together with sequential culture media on vitrified-warmed mouse embryos in terms of blastocyst development, blastocyst quality, incidence of apoptosis and related genes expression.

Methods: Two cell vitrified-warmed mouse embryos were cultured in G-1ver3 medium to the eight-cell stage when they were randomly assigned to three treatment groups of no co-culture (control), non-polarized and polarized mouse uterine epithelial monolayer co-culture. The culture medium was G-2ver3 during the treatment phase. After 96 h on treatment, the significance of differences were evaluated by the one way analysis of variance for continuous data.

Results: In the polarized monolayer group, the hatched blastocyst formation and blastocyst quality improved significantly than other two groups (P < 0.05). Whereas the incidence of apoptosis and related gene expression such as Bax were higher in the blastocysts of control group (P < 0.05). The relative abundance of Bcl-2 mRNA to the beta-tubulin was similar for all treatments.

Conclusion: Co-culture system involving polarized uterine epithelial cells and sequential culture media is a promising method for the improvement of vitrified-warmed mouse embryo development.

Fig. 1

Transmission electron micrograph of mouse uterine epithelial cells in vitro. A Polarized epithelial cells, culture on ECM gel; B non-polarized epithelial cells, culture on plastic surface; the accumulation of dense (bold arrow) and clear (thin arrow) cytoplasmic vacuoles in the polarized epithelial cells cultured on ECM gel. Bar: A 750 nm, B 1 μm

Fig. 2

Relative abundance of Bax and Bcl-2 mRNA expression and Bax/Bcl-2 mRNA expression in blastocysts drived from vitrified-warmed mouse embryos and developed in sequential media alone or by co-culture with non-polarized or polarized mouse uterine epithelial cell monolayers. Control: culture in sequential media alone (G-1TM ver3 followed by G-2TM ver3 media); treatment 1: co-culture in sequential media with non-polarized monolayer; treatment 2: co-culture in sequential media with polarized monolayer. The relative abundance of each mRNA species is the ratio of the intensity of each band to the intensity of the corresponding β-tubulin. Columns within a variate with different letters are significantly different (ANOVA, P < 0.05)