32 EFFECTS OF HIGH HYDROSTATIC PRESSURE ON EXPRESSION PROFILES OF IN VITRO-PRODUCED, VITRIFIED BOVINE BLASTOCYSTS

Abstract

High hydrostatic pressure (HHP) has been used to enhance stress tolerance and to promote embryo survival before they are subjected to insulting procedures such as cryopreservation. However, the molecular mechanisms of the beneficial effects of HHP are poorly understood. Here in vitro-produced bovine blastocysts were treated with 40, 60, and 80 MPa of HHP for 1 h at either 25 or 37°C, followed by 3 different recovery periods (0, 1, and 2 h) after HHP before vitrification by the solid surface vitrification method (Dinnyes et al. 2000). The re-expansion rates after vitrification-warming were significantly (P < 0.05) higher in embryos treated with 40 or 60 MPa than controls, demonstrating that HHP promotes the in vitro developmental competence of vitrified bovine embryos. However, 80 MPa resulted in significantly reduced re-expansion rates, suggesting that this pressure started to be lethal to bovine blastocysts. In addition, no significant difference was found on re-expansion rates between 25 and 37°C; data were therefore combined for the 2 temperatures. Microarray analysis revealed a total of 399 differentially expressed transcripts, representing 254 unique genes, among different treatment groups. Gene ontology analysis revealed that HHP at 40 and 60 MPa promoted embryo competence through down-regulation of genes involved in cell death and apoptosis, and up-regulation of RNA processing, cellular growth, and proliferation. Moreover, gene expression was also changed by the length of the recovery time after HHP. The significantly over-represented groups are apoptosis and cell death in the 1-h group, and protein folding, response to unfolded protein, and cell cycle in the 2-h group. Although 80 MPa also up-regulated expression of genes for apoptosis, but it also significantly down-regulated genes for protein folding and cell cycle, which may explain why these embryos stopped developing. Taken together, these data suggest that HHP induces specific responses in vitrified bovine blastocysts and promotes their developmental competence through modest transcriptional reprogramming.