Uterine responses to early pre-attachment embryos in the domestic dog and comparisons with other domestic animal species

Abstract

In the dog, there is no luteolysis in the absence of pregnancy. Thus, this species lacks any anti-luteolytic endocrine signal as found in other species that modulate uterine function during the critical period of pregnancy establishment. Nevertheless, in the dog an embryo-maternal communication must occur in order to prevent rejection of embryos. Based on this hypothesis, we performed microarray analysis of canine uterine samples collected during pre-attachment phase (days 10-12) and in corresponding non-pregnant controls, in order to elucidate the embryo attachment signal. An additional goal was to identify differences in uterine responses to pre-attachment embryos between dogs and other mammalian species exhibiting different reproductive patterns with regard to luteolysis, implantation, and preparation for placentation. Therefore, the canine microarray data were compared with gene sets from pigs, cattle, horses, and humans. We found 412 genes differentially regulated between the two experimental groups. The functional terms most strongly enriched in response to pre-attachment embryos related to extracellular matrix function and remodeling, and to immune and inflammatory responses. Several candidate genes were validated by semi-quantitative PCR. When compared with other species, best matches were found with human and equine counterparts. Especially for the pig, the majority of overlapping genes showed opposite expression patterns. Interestingly, 1926 genes did not pair with any of the other gene sets. Using a microarray approach, we report the uterine changes in the dog driven by the presence of embryos and compare these results with datasets from other mammalian species, finding common-, contrary-, and exclusively canine-regulated genes.

Keywords: dog (Canis lupus familiaris); early pregnancy; embryo-maternal communication.

Figure 1.

Heatmap showing the microarray analysis of DEG in early pregnant (P1-4) canine uterus at days 10-12, and corresponding non-pregnant controls (C1-3). In total, 412 genes (FDR: 10%) were differentially expressed between the two groups. A total of 314 genes were up-regulated and 98 down-regulated. The main functional terms determined by DAVID analysis are presented for the identification of those functional terms affected by the presence of free-floating embryos in the canine pre-implantation uterus. The full list of DEG (FDR: 10%) is provided in Supplemental File 2.

Figure 2.

Cytoscape analysis of functional networks over-represented in canine uterus exposed to pre-implantation free-floating embryos. As input, DEG (FDR: 10%) were used (see Supplemental File 2). The redundant and noninformative terms were removed, and the resulting network was manually rearranged. For each network, the size of the node implies the number of genes, while the color intensity denotes the level of enrichment (see legend to illustration). Functional networks more highly represented in early pregnant canine uterus refer predominantly to ECM, immune response, and angiogenesis.

Figure 3.

IPA of canine DEG (early pregnancy vs. non-pregnant). (A) The canonical pathways analysis with the output gene sets ranked according to log (P-value). The overlapping of gene sets and the trend of the status (up- or down-regulated) are indicated. (B) The main functional terms identified by IPA were determined based on the identified canonical pathways.

Figure 4.

GSEA. The entire set of genes detected in canine uterus was ranked according to their expression levels (a score calculated from the log2 fold change and the P-value; see Methods). The ranked list was compared to sets of up-regulated (UP-regulated) genes (A-C) or down-regulated (DOWN-regulated) genes (D-F) from bovine (Bos taurus/BTA) uterus at day 15 of pregnancy (A and D), swine (Sus scrofa/SSC) uterus at day 12 of pregnancy (B and E) and horse (Equus caballus/ECA) uterus at day 16 of pregnancy (C and F). Enrichment scores and quantitatively strongest overlapping with the canine gene set are indicated. For details see the text. The list of overlapping genes is provided as Supplemental File 3.

Figure 5.

Venn diagrams showing the intersection between genes differentially expressed in the presence of pre-implantation embryos in different domestic animal species are presented using the canine gene set as reference. (A) Overlapping of the top 2000 up-regulated (2000 best positive scores) canine genes with up-regulated genes in other species; (B) comparison of the top 700 down-regulated (700 best negative scores) canine genes with genes down-regulated in other species; (C) cumulative analysis of the canine genes with genes up- and down-regulated in other species. Hsa WOI = Homo sapiens during the window of implantation (WOI), Bta = Bos taurus (days 15–16 of pregnancy), Ssc = Sus scrofa (day 12), and Eca = Equus caballus (days 12 and 16). The list of overlapping genes is provided as Supplemental File 4.

Figure 6.

Expression of selected target genes as determined by real-time (TaqMan) RT-PCR. AIF1 = allograft inflammatory factor 1, CXCL16 = chemokine ligand 16, CXCR7 = chemokine receptor 7, LXR = liver X receptor, CXCR6 = chemokine receptor 6, PTGDR = prostaglandin D2 receptor, LAMA2 = laminin alpha 2, IDO1 = idolamin 2,3-dioxygenase 1, TIMP2 = tissue inhibitor of matrix metalloproteinase-2, PAPPA2 = pappalysin2. An unpaired, two-tailed Student t-test was applied. Bars with different asterisks differ at P = 0.001 (PTGDR), P = 0.002 (AIF1, LXR), P = 0.009 (CXCL16), P = 0.01 (LAMA2, TIMP2), P = 0.02 (CXCR7, CXCR6, IDO1), P = 0.04 (PAPPA2). Numerical data are presented as geometric means Xg ± geometric standard deviation (SD).