Abstract:
Parthenogenetic embryos obtained by electroactivation of mature oocytes have been used as models in developmental studies. The correct gene expression in early cleavage embryos is essential to sustain embryo development. The precise regulation of genes involved in pluripotency (Oct-4, Sox-2, and Nanog) is crucial to the formation of inner cell mass and trophoblast cells. Failure to do so can contribute to impaired development. We hypothesized that porcine embryos produced by fertilization in vitro and parthenogensis would possess a similar pattern of expression of Oct-4, Nanog, and Sox-2 during cleavage development. The objective of this study was to determine the developmental expression pattern of these three transcription factors in porcine oocytes and cleavage-stage embryos produced by either fertilization or parthenogenesis. Messenger RNAwas isolated from pools of 40-150 germinal vesicle (GV)- and MII-arrested oocytes and pools of 2-cell (2c), 4-cell (4c), 8-cell (8c), and blastocyst-stage embryos produced by in vitro fertilization (IVF) or electroactivation. Quantitative real-time PCR was performed following cDNA synthesis. Transcripts for Oct-4, Nanog, Sox-2, andYWHAG (housekeeping gene control) were amplified in duplicate across three to five experimental replicates. Transcripts were quantified using the comparative CT method using YWHAG as internal control and GV stage as normalizing stage. Fold activation and repression were analyzed with ANOVA and Tukey's post-hoc test. Our results show that porcine embryos produced by either IVF or electroactivation possess a similar pattern of pluripotent gene expression during cleavage-stage development. Oct-4 was found to be present in high abundance in the 2-cell parthenogenetic embryos and then repressed at the 8-cell stage (10-fold; P < 0.05, 2c v. 8c). In IVF embryos, Oct-4 was found in significantly higher amount at the 2-cell stage (35-fold; P < 0.05, 2c v. GV). Nanog transcripts were present at low levels from the GV oocyte until the 4-cell stage in both IVF and parthenogenetic embryos and then upregulated 10 000-fold at the 4-cell stage (P < 0.0001, GV v. 4c); at the blastocyst stage, Nanog transcript levels were similar to the levels found in the GV stage oocytes. Sox-2 transcripts were lower in MII oocytes and were significantly upregulated in 8-cell-stage embryos produced by either IVF or electroactivation (9- and 20-fold; P < 0.01, P < 0.0001, MII v. 8c, respectively). In addition, Sox-2 transcripts were significantly higher in parthenogenetic blastocysts compared to IVF-derived blastocysts (P < 0.05). This work demonstrates that cleavage-stage porcine embryos, produced by either electroactivation or IVF, undergo a similar pattern of activation of key regulatory genes; however, the activation method can have an influence on the transcript abundance of specific genes at defined stages.