Evaluation of mouse preimplantation embryos exposed to oxidative stress cultured with insulin-like growth factor I and II, epidermal growth factor, insulin, transferrin and selenium.
Blastocyst culture requires strictly defined culture media to sustain its viability and quality. Although blastocyst media are commercially available, they do not meet all the needs and research focused on blastocyst-promoting agents is on the way. The aims of the study were to evaluate the significance of insulin-like growth factors I (IGF-I) and II (IGF-II); epidermal growth factor (EGF) and a mixture of insulin, transferrin and selenium (ITS) on the development of embryos exposed to oxidative stress. C3B6F1 mice were stimulated with 5 IU of pregnant mare serum gonadotropin following by administration of 5 IU of equine chorionic gonadotropin and mating with DBA males. The mice were killed 40 h after eCG injection by cervical dislocation and then the 2 cell embryos were flushed out from the fallopian tubes. To evaluate whether the growth factors may compensate the unfavorable--oxidative milieu created by hydrogen peroxide (H2O2), the embryos were transferred to 1/ control medium, 2/ control medium+0.1 mM (H2O2) or 3/ control medium+H2O2 enriched with 10(-7) g/ml of IGF-I, IGF-II, EGF or a mixture of insulin (5x10(-6) g/ml), transferrin (5 x10(-6) g/ml) and selenium (5x10(-9) g/ml; ITS). Embryos were evaluated 96-144 hours following eCG injection. In the study the dynamics of embryo development and blastocyst cell numbers (including inner cell mass) were assessed. The morphological evaluation comprised viability and apoptosis (TUNEL). In oxidative stress setting, IGF-I, IGF-II, EGF and ITS minimized the negative influence of H2O2, and embryos developed faster than in control conditions. Blastocysts cultured with hydrogen peroxide and growth factors or ITS displayed normal morphology and had more cells--also within the inner cell mass--than those treated only with H2O2. The positive TUNEL reactions were sporadically observed in embryos cultured with hydrogen peroxide supplemented with growth factors. IGF-I, IGF-II, EGF and ITS have a positive effect on pre-implantation embryo development in detrimental culture conditions of oxidative stress.
Kurzawa
Rafał
R
Clinic for Reproduction and Gynecology, Pomeranian Academy of Medicine, Szczecin, Poland. kurzawa@sci.pam.szczecin.pl
Głabowski
Wojciech
W
Baczkowski
Tomasz
T
Brelik
Paweł
P
D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D016923:Cell Death; D003470:Culture Media; D046508:Culture Techniques; D047108:Embryonic Development; D005314:Embryonic and Fetal Development / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005260:Female; D020287:In Situ Nick-End Labeling; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008811:Mice, Inbred DBA; D018384:Oxidative Stress; D011247:Pregnancy; D012643:Selenium / Q000494:pharmacology; D013194:Staining and Labeling; D013997:Time Factors; D014168:Transferrin / Q000494:pharmacology
D003470:Culture Media; D007328:Insulin; D014168:Transferrin; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D012643:Selenium