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 cellsmonolayer on the development of mouse early embryos. One-cell mouse embryos were obtained from NMRImice 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-oocytecomplexes (COCs). To produce monolayer of cumulus and fibroblast cells, 1.0 × 105 cells/ml were platedinto culture dishes in 100 μl droplets. The collected mouse embryos were cultured randomly into six differentconditions, being supplemented (experiment, Exp) or not (control, Con) with 0.28 μg/ml of retinol acetatemethyl-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 wasAlpha Modification of Minimum Essential Medium Eagle (α-MEM) + 10% fetal bovine serum (FBS) with100 IU/ml penicillin and 100 μg/ml streptomycin. The proportions of embryos passing the two-cell blockwere significantly higher in the MEF (Con 3) group compared to the other treatment groups (Ppercentage of the two-cell passed embryos developing to the blastocyst stage was significantly higher in theco-culture groups than that of the culture medium alone (Pblastocyst stage for both groups of CC co-culture treatment (Con 2 and Exp 2) was identical but, adding RAinto the MEF co-culture (Exp 3) resulted significantly lower in vitro development than that of the Con 3group (29.2% vs. 57.7%, Pcould not affect the embryos passing the block and developing to the blastocyst stage, although the presenceof RA into the culture medium alone may improve passing the critical two-cell stage. Also, in vitro additionof RA to cells without receptors for retinol during long term co-culture may result early embryonic growthretardation.