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

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

Global transcriptomic analysis of the canine Corpus Luteum (CL) during the first half of diestrus and changes induced by in vivo inhibition of prostaglandin synthase 2 (PTGS2/COX2)

The canine luteal phase exhibits several peculiarities compared with other species. In early diestrus, the corpus luteum (CL) is, at least in part, independent of gonadotropins, and prostaglandins (PGs) appear to be among its main regulators. This was also observed with the inhibition in vivo of COX2, when also transcriptional capacity, vascularization and immune-related factors were affected. Here, we aimed to further investigate the potential effects of PGs withdrawal on the CL transcriptome by performing deep RNA sequencing (RNA-Seq). Samples from a previous in vivo study were used; bitches were treated for 5, 10, 20, or 30 days after ovulation with firocoxib (Previcox®), a PTGS2/COX2 inhibitor, or a placebo. Analysis of results was performed with SUSHI (framework from FGCZ) and with pathways and functional networks analyzers. Time-dependent effects were also investigated and used for quality control. More highly represented differentially expressed genes (DEGs, P < 0.01, FDR < 0.1) in the early CL (days 5 and 10) referred to proliferation and immune system, while in the mature CL (days 20 and 30) they were related with steroidogenesis. The absence of genes concomitantly affected by the treatment at all time-points suggested stage-dependency in the observed effects. Little effect was observed on days 5 and 10. Day 20 had the highest number of DEGs (n = 1,741), related with increased immune response. On day 30, DEGs found (n = 552) referred to decreased steroidogenesis and vascularization. Our results suggest the presence of strong compensatory effects in the early CL and multidirectional effects toward gonadotropin-dependency of the CL after COX2 inhibition

Canine decidualization <i>in vitro</i>: extracellular matrix modification, progesterone mediated effects and selective blocking of prostaglandin E2 receptors

Recently, we established an in vitro model with immortalized dog uterine stromal (DUS) cells for investigations into canine-specific decidualization. Their capability to decidualize was assessed with cAMP and prostaglandin (PG) E2. Here, we show that the effects of PGE2 are mediated through both of the cAMP-mediating PGE2 receptors (PTGER2/4). Their functional inhibition suppressed gene expression of PRLR and PGR in DUS cells. We also assessed the effects of cAMP and PGE2 on selected extracellular matrix components and CX43, and showed that cAMP, but not PGE2, increases COL4, extracellular matrix protein 1 (ECM1) and CX43 protein levels during in vitro decidualization, indicating a mesenchymal-epithelial decidual transformation in these cells. Thus, although PGE2 is involved in decidualization, it does not appear to regulate extracellular matrix. Further, the role of progesterone (P4) during in vitro decidualization was addressed. P4 upregulated PRLR and PGR in DUS cells, but these effects were not influenced by PGE2; both P4 and PGE2 hormones appeared to act independently. P4 did not affect IGF1 expression, which was upregulated by PGE2, however, it suppressed expression of IGF2, also in the presence of PGE2. Similarly, P4 did not affect PGE2 synthase (PTGES), but in the presence of PGE2 it increased PTGER2 levels and, regardless of the presence of PGE2, suppressed expression of PTGER4. Our results indicate a reciprocal regulatory loop between PGE2 and P4 during canine in vitro decidualization: whereas P4 may be involved in regulating PGE2-mediated decidualization by regulating the availability of its receptors, PGE2 regulates PGR levels in a manner dependent on PTGER2 and -4

Uterine and placental distribution of selected extracellular matrix (ECM) components in the dog

For many years, modifications of the uterine extracellular matrix (ECM) during gestation have not been considered as critical for successful canine (Canis lupus familiaris) pregnancy. However, previous reports indicated an effect of free-floating blastocysts on the composition of the uterine ECM. Here, the expression of selected genes involved in structural functions, cell-to-cell communication and inhibition of matrix metalloproteinases were targeted utilizing qPCR and immunohistochemistry. We found that canine free-floating embryos affect gene expression of FN1, ECM1 and TIMP4 This seems to be associated with modulation of trophoblast invasion, and proliferative and adhesive functions of the uterus. Although not modulated at the beginning of pregnancy, the decrease of structural ECM components (i.e. COL1, -3, -4 and LAMA2) from pre-implantation toward post-implantation at placentation sites appears to be associated with softening of the tissue in preparation for trophoblast invasion. The further decrease of these components at placentation sites at the time of prepartum luteolysis seems to be associated with preparation for the release of fetal membranes. Reflecting a high degree of communication, intercellular cell adhesion molecules are induced following placentation (Cx26) or increase gradually toward prepartum luteolysis (Cx43). The spatio-temporal expression of TIMPs suggests their active involvement in modulating fetal invasiveness, and together with ECM1, they appear to protect deeper endometrial structures from trophoblast invasion. With this, the dog appears to be an interesting model for investigating placental functions in other species, e.g. in humans in which Placenta accreta appears to share several similarities with canine subinvolution of placental sites (SIPS). In summary, the canine uterine ECM is only moderately modified in early pregnancy, but undergoes vigorous reorganization processes in the uterus and placenta following implantation

Global Transcriptomic Analysis of the Canine corpus luteum (CL) During the First Half of Diestrus and Changes Induced by in vivo Inhibition of Prostaglandin Synthase 2 (PTGS2/COX2)

The canine luteal phase exhibits several peculiarities compared with other species. In early diestrus, the corpus luteum (CL) is, at least in part, independent of gonadotropins, and prostaglandins (PGs) appear to be among its main regulators. This was also observed with the inhibition in vivo of COX2, when also transcriptional capacity, vascularization and immune-related factors were affected. Here, we aimed to further investigate the potential effects of PGs withdrawal on the CL transcriptome by performing deep RNA sequencing (RNA-Seq). Samples from a previous in vivo study were used; bitches were treated for 5, 10, 20, or 30 days after ovulation with firocoxib (Previcox®), a PTGS2/COX2 inhibitor, or a placebo. Analysis of results was performed with SUSHI (framework from FGCZ) and with pathways and functional networks analyzers. Time-dependent effects were also investigated and used for quality control. More highly represented differentially expressed genes (DEGs, P < 0.01, FDR < 0.1) in the early CL (days 5 and 10) referred to proliferation and immune system, while in the mature CL (days 20 and 30) they were related with steroidogenesis. The absence of genes concomitantly affected by the treatment at all time-points suggested stage-dependency in the observed effects. Little effect was observed on days 5 and 10. Day 20 had the highest number of DEGs (n = 1,741), related with increased immune response. On day 30, DEGs found (n = 552) referred to decreased steroidogenesis and vascularization. Our results suggest the presence of strong compensatory effects in the early CL and multidirectional effects toward gonadotropin-dependency of the CL after COX2 inhibition.ISSN:1664-239

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

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

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

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