147 HYDROSTATIC PRESSURE STRESS TREATMENT OF MOUSE OOCYTES INFLUENCES PROTEIN SYNTHESIS AT THE 4-CELL STAGE

Abstract

Sublethal stress treatment [namely hydrostatic pressure (HP)] has been reported to improve the cryotolerance and developmental competence of mammalian gametes and embryos at cryopreservation, in vitro maturation, parthenogenetic activation and somatic cell NT (Pribenszky and Vajta 2011 Reprod. Fertil. Dev. 23, 48–55). In our previous study mouse oocytes were HP stress treated and then vitrified, warmed, fertilized by intracytoplasmic sperm injection (ICSI), cultured and transferred. Blastocyst rate, inner cell mass cell number and birth rate were significantly increased in the treated groups (Pribenszky et al. 2010 Fertil. Steril. 94, S32). In our present study we implemented gene expression microarray experiments to analyse the transcriptome of HP-treated eggs and 4-cell stage embryos developed from these oocytes. At first, 4 pools of 50 mouse oocytes were treated with 20 MPa for 60 min at 37°C in a programmable HP machine (Cryo-Innovation Inc., Budapest, Hungary). Second, mouse oocytes were treated with HP likewise, fertilized by ICSI and cultured to 4-cell stage. The controls were handled identically with no HP treatment. After ICSI, 78% of the treated and 82% of the control oocytes survived the procedure, of which 76 and 65% developed to 4-cell stage; furthermore 3–3 pools of 30 to 44 and 24 to 36 embryos were used for RNA isolation, respectively. The labelled cRNA target generated from 240 pg of total RNA was hybridized to 4 × 44 K whole mouse genome chips (Agilent Technologies, Palo Alto, CA, USA). All the microarray data have been deposited in the NCBI Gene Expression Omnibus database (accession GSE28443). The results were validated using RT-qPCR by analysing 9 genes per experiment. The GeneSpring GX 11 analysis of the normalized data showed no change in the global gene expression profile of the oocytes after the HP treatment; however, in the 4-cell embryo study, the analysis identified 676 significantly changed genes, confirmed by the similar expression of 8 genes by RT-qPCR. To characterise the affected pathways among these genes, we used the functional annotation tools of DAVID Bioinformatics Resources (Huang et al. 2009 Nat. Protoc. 4, 44–57). The cluster with the most outstanding gene ontology terms contained the ribosome, structural constituent of ribosome, ribonucleoprotein complex, translation and structural molecule activity (P < 0.001). Among the 2 directly ribosome-related categories, there were 35 and 40 down-regulated genes along with only 2-2 up-regulated genes. These results showed that principally the protein synthesis machinery is affected by the HP stress, with a robust down-regulation. In conclusion, this study suggests that ribosomal processes play a central role in the HP-treated and fertilized oocytes during pre-implantation development. Stress effects in the transcriptional processes are visible after the embryonic genome activation showing down-regulation in the most energy-consuming processes. How these processes support higher blastocyst rate, cell number and birth rate needs further elucidation.

Supported by OM-00069/2008, EGG_Care; EU FP7 (EpiHealth FP7-HEALTH-2011-278418).