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

Effects of sex, DKK1 and the interaction on expression of genes involved in blastocyst differentiation and pluripotency.^a

<p>^a Units are log2 of fluorescent intensity.</p><p>Effects of sex, DKK1 and the interaction on expression of genes involved in blastocyst differentiation and pluripotency.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133587#t002fn001" target="_blank">^a</a></p

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 sex and DKK1 on AMOT expression in Day 6 morula (Experiment 2).

<p>Data are shown as ΔC_t data (top panel) and as fold-change relative to female controls (bottom panel). Data are least-squares means ± SEM (top) or were calculated from the least-squares means of ΔC_t (bottom). RNA abundance was affected by sex (P = 0.0527) and DKK1 ((P = 0.0222) but not by the interaction (P = 0.5791).</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

Differences in gene expression between female and male embryos is predictive that cell survival is enhanced in females.

<p>Genes upregulated in females are in red while genes upregulated in males are in green. Cellular and molecular functions predicted to be activated are in orange while functions predicted to be inhibited in blue. Orange arrows represents predicted activation of the function, blue arrows represent predicted inhibition, yellow arrows indicates that the relationship is inconsistent with the prediction while gray lines represent that the effect is not predicted. Note <i>EXO5</i> is identified as DEM1 in the microarray database.</p

Comparison of the fold-change in gene expression due to sex, DKK1 and DKK1 x sex as determined by microarray and by qPCR. ^a

<p>^a Abbreviation: fem—female.</p><p>Comparison of the fold-change in gene expression due to sex, DKK1 and DKK1 x sex as determined by microarray and by qPCR. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133587#t003fn001" target="_blank">^a</a></p