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.