Effect of amino acids and alpha-amanitin on the development of rabbit embryos in modified protein-free KSOM with HEPES.

Abstract

Four experiments were conducted to test the effects of Eagle's non-essential amino acids (NEAA) and essential amino acids (EAA), glycine, and the RNA polymerase inhibitor, alpha-amanitin, on the development of preimplantation rabbit embryos in modified protein-free KSOM medium. Embryos were distributed randomly into different treatments and cultured in 5% O2:5% CO2:90% N2. In experiment 1, 100% of the embryos became blastocysts in the medium with Eagle's 1X NEAA and 0.5X EAA, but 100% stopped development at the morula stage in KSOM without amino acids. These morulae failed to develop further when transferred to amino acid supplemented medium after 72 hr of culture. Glycine alone in modified KSOM (experiment 2) was ineffective in supporting development of 8-16-cell stage embryos past the morula stage. In experiment 3, the addition of 1X NEAA and 0.5X EAA at 0, 12, 24, 36, and 48 hr of culture resulted, respectively, in 57, 65, 65, 44, and 14% blastocysts on Day 3 (P < 0.05) and 86, 77, 77, 78, and 69% on Day 5 (P > 0.05). Omission of Eagle's amino acids until 48 hr clearly delayed embryo development. In experiment 4, when alpha-amanitin (20 microM) was added to the medium containing Eagle's amino acids after 0, 12, 24, 36, and 48 hr of culture most embryos cleaved only once or twice after adding the alpha-amanitin. Without the inhibitor, 94% of the zygotes developed into blastocysts. These results indicate that modified KSOM or KSOM plus glycine could not support rabbit embryo development past the morula stage, but this block was overcome by adding Eagle's amino acids. An exogenous source of amino acids was not critical for embryo development during the first 24 hr of culture, but was required after that for development to equal controls. Addition of alpha-amanitin at multiple pre-blastocyst stages limited further embryo development to one or two cleavage divisions, with no blastocyst development.