Canonical WNT signaling regulates development of bovine embryos to the blastocyst stage

Objectives were to evaluate the role of canonical WNT signaling in development of the preimplantation embryo. Signaling was activated with 2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) and inhibited with Dickkopf-related protein 1 (DKK1). Treatment of bovine embryos with AMBMP at day 5 after insemination decreased development to the blastocyst stage at day 7 and reduced numbers of trophectoderm and inner cell mass cells. At high concentrations, AMBMP caused disorganization of the inner cell mass. DKK1 blocked actions of AMBMP but did not affect development in the absence of AMBMP. Examination of gene expression in day 6 morulae by microarray revealed expression of 16 WNT genes and other genes involved in WNT signaling; differences in relative expression were confirmed by PCR for 7 genes. In conclusion, the preimplantation embryo possesses a functional WNT signaling system and activation of the canonical pathway can inhibit embryonic development

Influence of Sex on Basal and Dickkopf-1 Regulated Gene Expression in the Bovine Morula

<div><p>Sex affects function of the developing mammalian embryo as early as the preimplantation period. There were two goals of the current objective. The first was to determine the degree and nature of differences in gene expression between female and male embryos in the cow at the morula stage of development. The second objective was to determine whether DKK1, a molecule known to alter differentiation of the blastocyst, would affect gene expression differently for female and male morulae. In Experiment 1, female and male embryos were treated with DKK1 at Day 5 after insemination. Morulae were harvested 24 h after treatment, pooled in groups of 20 for microarray analysis and RNA subjected to analysis of gene expression by microarray hybridization. There were 662 differentially expressed genes between females and males and 128 of these genes had a fold change ≥ 1.5 between the two sexes. Of the genes upregulated in females, 49.5% were located in the X chromosome. Functional analysis predicted that cell survival was greater in female embryos. Experiment 2 involved a similar design except that transcripts for 12 genes previously reported to be affected by sex, DKK1 or the interaction were quantified by quantitative polymerase chain reaction. Expression of all genes tested that were affected by sex in experiment 1 was affected in a similar manner in Experiment 2. In contrast, effects of DKK1 on gene expression were largely not repeatable in Experiment 2. The exception was for the Hippo signaling gene <i>AMOT</i>, which was inhibited by DKK1. In Experiment 3, embryos produced by fertilization with unsorted sperm were treated with DKK1 at Day 5 and abundance of transcripts for <i>CDX2</i>, <i>GATA6</i>, and <i>NANOG</i> determined at Days 5, 6 and 7 after insemination. There was no effect of DKK1 on expression of any of the three genes. In conclusion, female and male bovine embryos have a different pattern of gene expression as early as the morula stage, and this is due to a large extent to expression of genes in the X chromosomes in females. Differential gene expression between female and male embryos is likely the basis for increased resistance to cell death signals in female embryos and disparity in responses of female and male embryos to changes in the maternal environment.</p></div

Number of differentially expressed genes due to DKK1, sex and the interaction.

<p>Number of differentially expressed genes due to DKK1, sex and the interaction.</p

Effect of day after insemination on expression of <i>CDX2</i>, <i>NANOG</i> and <i>GATA6</i> (Experiment 3).

<p>Data are shown as ΔC_t data (left panel) and as fold-change relative to Day 5 controls (right panel). Data are least-squares means ± SEM (left) or were calculated from the least-squares means of ΔC_t (right). Embryos were morulae (Days 5 and 6) or blastocysts (Day 7). Day of insemination is considered Day 0. Blue bars represent control and red bars represent DKK1. Day effects are indicated by superscripts. Days with different superscripts differ (P<0.05). There were no effects of DKK1 or day x DKK1.</p