Improved embryo development with decreased apoptosis in blastomeres after the treatment of cloned bovine embryos with beta-mercaptoethanol and hemoglobin.
In preliminary experiments, the treatment of donor somatic cells with beta-mercaptoethanol (ME) or hemoglobin (Hb) improved in vitro-development of bovine cloned embryos. This study was subsequently evaluated whether the exposure to Hb and/or ME during in vitro-maturation or embryo culture could further promote the development of embryos cloned with ME-treated donor cells. A prospective, randomized study was conducted and, embryo development, cell number, and apoptosis in blastocysts were monitored. A significant (P < 0.05) effect was found after the combined treatment of cloned embryos with Hb (1 microg/ml) and ME (10 microM); the development of morulae (53 vs. 35%) was greatly improved, which resulted in enhanced blastocyst formation (38%). However, cell number and apoptosis in blastocysts were predominantly affected by ME rather than Hb; a significant increase in total cell number of blastomeres (142-154 vs. 123 cells/embryo), inner cell mass (ICM) (39-41 vs. 27), and trophectoderm (TE) (103-114 vs. 98), and the ratio of ICM to TE cell number (0.26-0.27 vs. 0.22) was found. Also, the apoptosis index indicating the ratio of apoptotic cell to normal blastomere number was greatly reduced after ME treatments (0.85 vs. 0.056-0.069). When embryos cloned with ME-treated cells were cultured in Hb + ME-containing medium, any of the treatments to recipient oocytes before enucleation did not further promote the development. In conclusion, combined treatment of cloned embryos with Hb + ME not only improved in vitro-development but also decreased blastomere apoptosis. The use of ME-treated donor cells and the culture of cloned embryos in Hb + ME-containing medium yielded the optimal results for promoting the production of blastocysts with improved quality.
Park
Eul Soon
ES
School of Agricultural Biotechnology, Seoul National University, Seoul, Korea.
Hwang
Woo Suk
WS
Kang
Sung Keun
SK
Lee
Byeong Chun
BC
Han
Jae Yong
JY
Lim
Jeong Mook
JM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects; D001757:Blastomeres / Q000166:cytology / Q000187:drug effects; D002417:Cattle; D019976:Cloning, Organism; D047108:Embryonic Development / Q000187:drug effects*; D005260:Female; D006454:Hemoglobins / Q000494:pharmacology*; D066298:In Vitro Techniques; D008623:Mercaptoethanol / Q000494:pharmacology*; D053652:Nuclear Transfer Techniques
D006454:Hemoglobins; D008623:Mercaptoethanol