Co-Culture Of Mouse Embryos: Effects Of Physiological Status And Mechanisms Of Action Of Somatic Cells

In general, the development of early mammal embryos is arrested or blocked when they are cultured in simple media. Although mouse and cattle embryos have been in vitro cultured successfully in defined media to blastocyst stage in recent years, co-culture is still necessary for embryos to overcome the developmental "block" in many species. In addition, embryo co-culture with somatic cells is a suitable model system for studying embryo-maternal interactions. Therefore, the study of influencing factors and crosstalk mechanism between embryos and somatic cells during co-culture has become indispensable in the interim. Firstly, to study the effect of species origin, degree of confluence and number of passages of somatic cells on in vitro development of mouse embryos, pronuclear embryos derived in vivo were co-cultured with mouse, goat or chick oviductal epithelial cells (OEC) at different days of confluence and different passages. Secondly, to specify the physiological features of different types of somatic cells, the cell cycle was determined by a flow cytometric analysis and apoptosis was examined by Annexin-V and PI staining in goat OEC at different days of confluence and passages. Finally, the molecular mechanisms of interaction between the co-cultured embryos and somatic cells were studied by renewing the conditioned medium at different time intervals, well-well tunnel and filter separation co-culture, and the concentration of hypoxanthine (HX) in the co-culture medium was analyzed with high-performance liquid chromatography. The result are summarized as follows:1. The proportion of mouse pronuclear embryos developing to blastocysts (61.7%, 57.2% and 56.2%, respectively) did not differ when co-cultured with primary mouse, goat and chick OEC at day 1 of confluence, suggesting that species of somatic cells had no effect on the in vitro development of mouse embryos.2. When mouse embryos were co-cultured with primary mouse, goat and chick OEC at different days of confluence, the proportion of embryos developing to blastocysts was significantly greater at day I of confluence than at other days of confluence. This result suggested that the degree of confluence of somatic cells had a remarkable effect on development of co-cultured embryos.3. When mouse embryos were co-cultured with mouse, goat and chick OEC at day 1 of confluence of different passages, the blastocyst rates were significantly higher when co-cultured with primary mouse, goat and chick OEC (51.1%; 54.3% and 57.2%, respectively) and with mouse and goat passage 3 OEC (57.1% and 52.4%, respectively) than cultured with OEC at other passages. This suggested that the number of passages of somatic cells had a remarkable effect on the development of co-cultured embryos.4. Flow cytometric analysis of cell cycles of goat OEC at different days of confluence and different passages showed that the percentage of S stage cells decreased but that of Gl+GO stage cells increased significantly with prolonged culture and passage. When apoptosis of goat OEC at different days of confluence and different passages was examined by Annexin- V and PI staining, it was found that the percentage of late apoptotic and necrotic cells increased with prolonged culture and passage. A better development of co-cultured embryos was achieved when the percentage of S stage cells increased but that of Gl+GO stage and late apoptotic + necrotic cells decreased significantly.5. To study whether the effect of co-culture needs direct embryo-to-cell contact, we renewed the conditioned medium at different time intervals and found that around 10% embryos co-cultured developed to blastocysts when the conditioning time was reduced to 3 h. When embryos were co-cultured by well-well tunnels and filter separation, the blastocyst rates increased significantly to 25% and 54%, respectively. These results suggested that the beneficial effect of co-culture did not necessarily require direct embryo-to-cell contact but rather it needed a continuous molecular exchange between somatic cells and embryos.6. The second cleavage of mouse embryos was blocked when 40uM HX was added to the CZB medium without co-culture. When mouse embryos were co-cultured with goat OEC at different days of confluence in CZB supplemented with 40 uM HX, the proportion of embryos developing to blastocysts was greater and the concentration of hypoxanthine in the conditioned medium was lower significantly at day 1 than at other days of confluence and than in CZB alone. The results indicated that somatic cells metabolize HX, alleviating its inhibitory effect on embryo development, but the ability of OEC to metabolize HX decreased with prolonged culture.