The development of mouse early embryos in vitro in fibroblasts and cumulus cells co-cultures supplemented with retinoic acid

This study was designed to examine the effects of retinoic acid adding to cumulus and/or fibroblast cells monolayer on the development of mouse early embryos. One-cell mouse embryos were obtained from NMRI mice after superovulation by an intraperitoneal injection of 5 IU equine chorionic gonadotrophin (eCG) followed 48 hrs later by 5 IU human chorionic gonadotrophin (hCG). Mouse embryonic fibroblasts (MEF) were obtained from mouse fetuses and cumulus cells (CC) were prepared from mouse cumulus-oocyte complexes (COCs). To produce monolayer of cumulus and fibroblast cells, 1.0 × 105 cells/ml were plated into culture dishes in 100 µl droplets. The collected mouse embryos were cultured randomly into six different conditions, being supplemented (experiment, Exp) or not (control, Con) with 0.28 µg/ml of retinol acetate methyl-β-cyclodextrin (RA) for 96 hrs at 37°C in 5% CO2 in air, including: (1) culture media only (Con 1); (2) culture media plus RA (Exp 1); (3) co-culture with CC (Con 2); (4) co-culture with CC plus RA (Exp 2); (5) co-culture with MEF (Con 3) and (6) co-culture with MEF plus RA (Exp 3). The culture medium was Alpha Modification of Minimum Essential Medium Eagle (α-MEM) + 10% fetal bovine serum (FBS) with 100 IU/ml penicillin and 100 µg/ml streptomycin. The proportions of embryos passing the two-cell block were significantly higher in the MEF (Con 3) group compared to the other treatment groups (P<0.05). The percentage of the two-cell passed embryos developing to the blastocyst stage was significantly higher in the co-culture groups than that of the culture medium alone (P<0.05). After 96 hrs in culture, the rate of blastocyst stage for both groups of CC co-culture treatment (Con 2 and Exp 2) was identical but, adding RA into the MEF co-culture (Exp 3) resulted significantly lower in vitro development than that of the Con 3 group (29.2% vs. 57.7%, P<0.05). These results suggest that supplementation of co-culture groups with RA could not affect the embryos passing the block and developing to the blastocyst stage, although the presence of RA into the culture medium alone may improve passing the critical two-cell stage. Also, in vitro addition of RA to cells without receptors for retinol during long term co-culture may result early embryonic growth retardation.