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

Document Type: Full paper (Original article)

Authors

1 Graduated from Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran

2 Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran;

3 Department of Anatomical Sciences, Afzalipour School of Medicine, University of Medical Sciences of Kerman, Kerman, Iran

4 DVM Student, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran

Abstract

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 (Ppercentage 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 (Pblastocyst 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%, Pcould 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.

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