EFFECT OF AMINO-ACIDS AND ALPHA-AMANITIN ON THE DEVELOPMENT OF RABBITEMBRYOS IN MODIFIED PROTEIN-FREE KSOM WITH HEPES

Four experiments were conducted to test the effects of Eagle's nonesse ntial amino acids (NEAA) and essential amino acids (EAA), glycine, and the RNA polymerase inhibitor, alpha-amanitin, on the development of p reimplantation rabbit embryos in modified protein-free KSOM medium. Em bryos-were distributed randomly into different treatments and cultured in 5% O-2:5% CO2:90% N-2. 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 additi on of 1X NEAA and 0.5X EAA at 0, 12, 24, 36, and 48 hr of culture resu lted, 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 mu M) was added to the medium c ontaining Eagle's amino acids after 0, 12, 24, 36, and 48 hr of cultur e most embryos cleaved only once or twice after adding the alpha-amani tin. Without the inhibitor, 94% of the zygotes developed into blastocy sts. These results indicate that modified KSOM or KSOM plus glycine co uld not support rabbit embryo development past the morula stage, but t his block was overcome by adding Eagle's amino acids. An exogenous sou rce 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 divi sions, with no blastocyst development. (C) 1996 Wiley-Liss, Inc.