Growth factors protect in vitro cultured embryos from the consequences of oxidative stress

Abstract

The aim of the study was to evaluate the effect of insulin-like growth factors (IGF1 and IGF2), stem cell factor (SCF) and epidermal growth factor (EGF) on the development of embryos exposed to oxidative stress. C3B6F1 female mice were stimulated with 5 IU of pregnant mare serum gonadotropin and 5 IU of equine chorionic gonadotropin (eCG). Two-cell embryos were flushed out from the fallopian tubes 40 h after eCG administration and mating with DBA males. In each experiment embryos were divided into three groups and cultured in (1) control medium, (2) control medium with 0.1 mM hydrogen peroxide and (3) control medium with hydrogen peroxide and separately with IGF1, IGF2, SCF or EGF in concentrations of 1 ng/ml, 10 ng/ml and 100 ng/ml. Under phase-contrast microscopy, 8-cell and compacted embryos, and early, expanded, hatched and outgrown blastocysts were counted at 24 h. The total blastocyst (TB) and inner cell mass (ICM) cell numbers were established by differential staining. Blastocyst cell viability was examined under fluorescence microscopy. To detect apoptosis, TUNEL was performed and visualized under a laser scanning confocal microscope. Hydrogen peroxide decreased embryo growth, blastocyst rates, blastocyst cell viability as well as TB and ICM counts. The TUNEL reaction revealed significantly more apoptotic cells in oxidative stress conditions. Tested factors revealed a varying extent of protective activity against oxidative stress caused by hydrogen peroxide. In media containing hydrogen peroxide and one of the four tested factors (IGF1, IGF2, SCF or EGF) the embryos developed faster than in media with hydrogen peroxide alone. IGF1, IGF2 and EGF increased both TB and (or) ICM counts in embryos exposed to hydrogen peroxide. All tested factors reduced the number of apoptotic cells (TUNEL) in embryos exposed to hydrogen peroxide.