“The role of L-proline in pre-implantation mouse embryo development in vitro”
Amino acids are known to play important roles in pre-implantation embryo development, including regulation of cell volume and metabolism. Inclusion of L-glutamine, glycine and betaine in embryo culture medium has been shown to improve development in vitro by acting as organic osmolytes, thereby regulating cell volume. The purpose of the present study was to determine the role of L-proline in pre-implantation mouse embryo development in vitro. In order to ensure the osmotic effect of L-proline, the osmolality of culture medium was prepared in different ranges (258, 270, 296 and 336 mOsm/kg). One-cell embryos were cultured in amino acid free modified human tubal fluid medium and potassium simplex optimization medium, at low density (1embryo/100μl) and high density (1embryo/μl). Amino acids (L-glutamine, L-proline, D-proline, glycine, L-alanine, betaine) were added to the culture medium and development of the embryos was scored every 24h until day 6 of pregnancy (blastocyst stage). Blastocysts were fixed, stained with DAPI and imaged on a confocal microscope. Cell numbers in each blastocyst were then counted. At low density, with 270−336mOsm/kg L-proline increased development at day 6 (p<0.001) to the blastocyst stage, and also increased the proportion of blastocysts that were hatching at day 6 (p<0.01) compared to embryos cultured in the absence of L-proline. However, there was no change in the number of cells in the embryos that reached the blastocyst stage. In contrast, at high density, L-proline had no effect on development compared to the absence of L-proline. L-proline improved development and blastocyst expansion despite when embryos were cultured in upon and sub-optimal (hyper osmolality and low density) conditions.