93 EXPOSURE TIME IS CRITICAL IN BLASTOCYSTS ADAPTATIONS TO BRANCH-CHAIN AMINO-ACID DEPLETION

Abstract

Decreased branch-chain amino-acids (BCAA) availability during the preimplantation period has been linked with adverse phenotypes (e.g. high blood pressure) in mouse offspring born to females subjected to protein undernutrition. This study aimed to determine the effects of decreasing (50%, L-bcaa) or omitting (0%, 0-bcaa) BCAA during in vitro culture on preimplantation embryo development and blastocyst outgrowth formation. The control medium (100%, N-bcaa) consisted of KSOM supplemented with serum insulin (1 ng mL–1) and uterine luminal fluid amino-acid concentrations [including BCAA: valine (0.46 mM), isoleucine (0.21 mM), and leucine (0.32 mM)] found in MF1 mice fed with normal levels of protein (18%). Groups of 2-cell (n = 194 per treatment) and 8-cell (n = 180 per treatment) embryos from nonsuperovulated mice were cultured for 66 and 44 h, respectively. Resultant late blastocysts were either subjected to differential cell staining (10 replicates) or individually cultured in N-bcaa medium for a further 96 h for outgrowth formation (6 replicates). Treatment groups were compared within 2-cell or 8-cell embryos by nonparametric tests (Kruskal-Wallis, Mann–Whitney, chi-square, or Fisher exact test). Blastocyst formation was not affected by BCAA depletion in the 2-cell group. However, in the 8-cell group, more (P = 0.016) blastocysts were hatching (27.7%) and less (P = 0.010) at the late blastocyst stage (60.5%) when BCAA were omitted compared with N-bcaa (17.2% hatching; 73.3% late blastocysts). Decreasing BCAA did not affect development to late blastocysts or hatching (68.8 and 23.3%). Blastocysts derived from 2-cell embryos cultured in L-bcaa (n = 69) had less total and trophectoderm cells than did those cultured in N-bcaa (n = 72; total cells: 63.75 ± 1.47 v. 68.28 ± 1.48; P = 0.026; trophectoderm: 53.74 ± 1.41 v. 58.06 ± 1.44; P = 0.025). Omitting BCAA (n = 71) did not affect total (64.7 ± 1.43) or trophectoderm (55.14 ± 1.49) cell number in 2-cell-derived blastocysts. Cell numbers in blastocysts derived from 8-cell embryos were not altered by BCAA treatment. Similarly, cell numbers of the inner cell mass were not affected by BCAA treatment in either embryo category. At 48 h after plating for outgrowth formation under N-bcaa conditions, hatching and attachment were not affected by prior BCAA depletion. Active spreading was slightly advanced after L-bcaa treatment in 2-cell outgrowths compared to N-bcaa (52.1 v. 26.9%; P = 0.086), whereas 8-cell outgrowths were not affected by BCAA treatment. These results suggest that blastocyst phenotype and trophectoderm cell proliferation before implantation are sensitive to BCAA depletion and that initial blastocyst activation might be promoted in embryos sensing a lack of BCAA during the preimplantation period. These adaptations depend on exposure time and duration of BCAA depletion, thus potentially contributing to triggering specific developmental consequences. Funded by BBSRC BB/1001840/1.