Understanding gene expression patterns in response to altered environmental conditions at different time points of the preimplantation period would improve our knowledge on regulation of embryonic development. Here we aimed to examine the effect of alternative in vivo and in vitro culture conditions at the time of major embryonic genome activation (EGA) on the development and transcriptome profile of bovine blastocysts. Four different blastocyst groups were produced under alternative in vivo and in vitro culture conditions before or after major EGA. Completely in vitro- and in vivo-produced blastocysts were used as controls. We compared gene expression patterns between each blastocyst group and in vivo blastocyst control group using EmbryoGENE's bovine microarray. The data showed that changing culture conditions from in vivo to in vitro or vice versa, either before or after the time of major EGA, had no effect on the developmental rates; however, in vitro conditions during that time critically influenced the transcriptome of the blastocysts produced. The source of oocyte had a critical effect on developmental rates and the ability of the embryo to react to changing culture conditions. Ontological classification highlighted a marked contrast in expression patterns for lipid metabolism and oxidative stress response between blastocysts generated in vivo versus in vitro, with opposite trends. Molecular mechanisms and pathways that are influenced by altered culture conditions during EGA were defined. These results will help in the development of new strategies to modify culture conditions at this critical stage to enhance the development of competent blastocysts.

Summary: MicroRNAs (miRNAs) are small endogenous molecules that are involved in a diverse of cellular process. However, little is known about their abundance in bovine oocytes and their surrounding cumulus cells during oocyte development. To elucidate this situation, we investigated the relative expression pattern of sets of miRNAs between bovine oocyte and the surrounding cumulus cells duringin vitromaturation using miRNA polymerase chain reaction (PCR) array. Results revealed that a total of 47 and 51 miRNAs were highly abundant in immature and matured oocytes, respectively, compared with their surrounding cumulus cells. Furthermore, expression analysis of six miRNAs enriched in oocyte miR-205, miR-150, miR-122, miR-96, miR-146a and miR-146b-5p at different maturation times showed a dramatic decrease in abundance from 0 h to 22 h of maturation. The expression of the same miRNAs in preimplantation stage embryos was found to be highly abundant in early stages of embryo development and decreased after the 8-cell stage to the blastocyst stage following a typical maternal transcript profile. Similar results were obtained by localization of miR-205 in preimplantation stage embryos, in which signals were higher up to the 4-cell stage and reduced thereafter. miR-205 and miR-210 were localizedin situin ovarian follicles and revealed a spatio-temporal expression during follicular development. Interestingly, the presence or absence of oocytes or cumulus cells during maturation was found to affect the expression of miRNAs in each of the two cell types. Hence, our results showed the presence of distinct sets of miRNAs in oocytes or cumulus cells and the presence of their dynamic degradation during bovine oocyte maturation.

Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and chromosome segregation in IVP blastocyst groups. Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively. Therefore, this finding indicated that suboptimal culture condition during preimplantation embryo development induced changes in the DNA methylation landscape of the resulting blastocysts in a stage dependent manner and the altered DNA methylation pattern was only partly explained the observed aberrant gene expression patterns of the blastocysts.

This study was conducted to investigate the gene expression profile of in vivo-derived bovine embryo biopsies based on pregnancy outcomes after transferring to recipients. For this, biopsies of 30–40% embryos were taken from grade I blastocysts (International Embryo Transfer Society Manual) and the remaining 60–70% of the intact embryos were transferred to recipients. Frozen biopsies were pooled into three distinct groups based on the pregnancy outcome after transferring the corresponding parts, namely those resulting in no pregnancy (NP), pregnancy loss (PL), and calf delivery (CD). Array analysis revealed a total of 41 and 43 genes to be differentially expressed between biopsies derived from blastocysts resulting in NP versus CD and PL versus CD respectively. Genes regulating placental development and embryo maternal interaction (PLAC8) were found to be upregulated in embryo biopsies that ended up with CD. Embryo biopsies that failed to induce pregnancy were enriched with mitochondrial transcripts (Fl405) and stress-related genes (HSPD1). Overall, gene expression profiles of blastocysts resulting in NP and CD shared similar expression profiles with respect to genes playing significant roles in preimplantation development of embryo. Finally, comparing the transcript signatures of in vivo- and in vitro-derived embryos with developmental competence to term revealed a similarity in the relative abundance of 18 genes. Therefore, we were able to present a genetic signature associated with term developmental competence independent of the environmental origin of the transferred blastocysts.