IGF-1/IGFBP-1 increases blastocyst formation and total blastocyst cell number in mouse embryo culture and facilitates the establishment of a stem-cell line.
BACKGROUND
Apoptosis occurs frequently for blastocysts cultured in vitro, where conditions are suboptimal to those found in the natural environment. Insulin-like growth factor-1 (IGF-1) plays an important role in preventing apoptosis in the early development of the embryo, as well as in the progressive regulation of organ development. We hypothesize that IGF-1 and its dephosphorylated binding protein (IGFBP-1) may be able to improve embryo culture with an associated reduced cell death, and that the resultant increase in the total cell number of the embryo could increase the chances of establishing an embryonic stem-cell line.


RESULTS
In vivo fertilized zygotes were cultured in medium containing supplementary IGF-1, or IGFBP-1/IGF-1. The stages of the resultant embryos were evaluated at noon on day five post-hCG injection. The extent of apoptosis and necrosis was evaluated using Annexin V and propidium iodine staining under fluorescent microscopy. The establishment of embryonic stem-cell lines was performed using the hatching blastocysts that were cultured in the presence of IGF-1 or IGFBP-1/IGF-1. The results show that the rate of blastocyst formation in a tissue-culture system in the presence of IGF-1 was 88.7% and IGFBP-1/IGF-1 it was 94.6%, respectively, and that it was significantly greater than the figure for the control group (81.9%). IGFBP-1/IGF-1 also resulted in a higher hatching rate than was the case for the control group (68.8% vs. 48.6% respectively). IGF-1 also increased the number of Annexin V-free and propidium iodine-free blastocysts in culture (86.8% vs. 75.9% respectively). Total cell number of blastocyst in culture was increased by 18.9% for those examples cultured with dephosphorylated IGFBP-1/IGF-1. For subsequent stem-cell culture, the chances of the successful establishment of a stem-cell line was increased for the IGF-1 and IGFBP-1/IGF-1 groups (IGF-1 vs. IGFBP-1/IGF-1 vs. control: 45.8% vs. 59.6% vs. 27.3% respectively).


CONCLUSIONS
IGF-1 or dephosphorylated IGFBP-1/IGF-1 supplement does result in an anti-apoptotic effect for early embryo development in culture, with a subsequent increased total cell number resulting from cell culture. The effect is beneficial for the later establishment of a stem-cell line.
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001755:Blastocyst / Q000187:drug effects* / Q000378:metabolism*; D002452:Cell Count; D002460:Cell Line; D046508:Culture Techniques; D004624:Embryo Transfer; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects* / Q000473:pathology; D005314:Embryonic and Fetal Development / Q000187:drug effects; D005260:Female; D005307:Fertilization in Vitro; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000378:metabolism / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D009336:Necrosis; D010766:Phosphorylation; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects*; D015053:Zygote / Q000187:drug effects
D018970:Insulin-Like Growth Factor Binding Protein 1; D007334:Insulin-Like Growth Factor I