190 EXPRESSION OF GENES ASSOCIATED WITH WINGLESS-RELATED MOUSE MAMMARY TUMOUR VIRUS SIGNALING IN THE PRE-IMPLANTATION BOVINE EMBRYO

Abstract

Wingless-related mouse mammary tumour virus (WNT) signalling participates in early embryonic development to maintain pluripotency, controls cell–cell communication, and modulates cell polarization and migration. To gain an understanding of the regulation of WNT signalling during embryonic development, expression patterns of a variety of molecules involved in WNT signal transduction were evaluated. Specific genes were DKK1, an endogenous inhibitor of canonical WNT signalling, the WNT co-receptors LRP5 and LRP6, WNT-responsive transcription factors, LEF1 and TCF7, and two repressors of WNT-regulated genes, the bovine orthologue of GROUCHO (LOC505120) and AES. Embryos were produced in vitro from oocytes obtained from ovaries collected at a local abattoir. Following oocyte maturation, fertilization was performed with sperm pooled from three randomly selected bulls; a different pool of bulls was used for each replicate. Groups of 30 matured oocytes or embryos at the 2-cell [28–32 h post-insemination (hpi)], 3–4 cell (44–48 hpi), 5–8 cell (50–55 hpi), 9–16 cell (72–75 hpi), morula (120–123 hpi), and blastocyst (168–171 hpi) stages were collected. The zona pellucida was removed with proteinase, RNA was purified, cDNA synthesised using random hexamer primers and real-time qPCR performed. Data analysed were ΔC_T values, which were calculated by subtracting the C_T value of the geometric mean of the three housekeeping genes (GAPDH, YWHAZ, and SDHA) from the C_T value of the sample. The relative transcript abundance was calculated as the 2^ΔCT. Data were analysed by least-squares ANOVA using the Proc GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). A total of 5 replicates were analysed for each developmental stage. Results show significant effects of stage of development for each gene that ranged from P = 0.004 for LRP5 to P ≤ 0.0001 for AES, DKK1, LEF, LOC505120, LRP6, and TCF7. In all cases, expression declined as development advanced. Except for AES, lowest expression occurred at the blastocyst stage. Lowest expression for AES was at the morula stage; expression remained low at the blastocyst stage. For two genes, DKK1 and LEF1, there was no detectable expression at the blastocyst stage. The timing of decline in expression varied between genes, first occurring at the 9–16-cell stage (AES, LEF1, and LOC505120) or morula stage (DKK1, LRP5, LRP6, or TCF7). For DKK1, LEF1, and LRP6, there was also a slight increase in expression from the oocyte to two-cell stage. Results suggest that canonical WNT signalling is reduced at the morula and blastocyst stages relative to earlier stages in development.

Research was supported by USDA-NIFA 2011-67015-30688.