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.

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

“…It needs to be emphasized that, in agreement with our previous findings (Graubner et al 2017a), and in contrast to other species including humans and rodents, the above described functional changes in the canine uterus driven by the presence of embryos, take place without any morphologically visible alterations of uterine structures. The deep structural remodeling processes of the canine uterus start following implantation and placentation and are associated with increased expression of COLs (COL1, -3, and -4), ECM1 and FN1 in the uterine wall (i.e., inter-Pl).…”

“…Expression of the following proteins was assessed: COL1, COL3, COL4, ECM1, FN1, LAMA2 and aSMA. Freefloating embryos were previously reported to increase the uterine expression of LAMA2 (Graubner et al 2017a). Here, the uterine adjustments during early In the non-pregnant group, TIMP2 was weakly detectable in the deep glands (B).…”

“…The present study explored the spatiotemporal expression of ECM components selected from our preceding microarray analysis (Graubner et al 2017a), and known for their involvement in modulating uterine and placental functions in other animals, in the canine uterus and placenta throughout pregnancy. Quantitative assessment was done by qPCR, and cellular localization was investigated qualitatively by IHC.…”

“…COL1, COL3, COL4, Cx26 and -43 were not affected at the beginning of early pregnancy (P > 0.05, not shown). Effects of the presence of embryos on LAMA2 and TIMP2 mRNA expression in the pre-implantation uterus were investigated previously (Graubner et al 2017a) and were, therefore, excluded from the present study. Next, the uterine pre-implantation stage was compared to inter-Pl sites and to the Ut-Pl compartments from different stages of pregnancy (i.e., post-implantation, mid-gestation and prepartum luteolysis).…”

“…Their response to a decidual stimulus (i.e.-increased secretory and proliferative activity) provides evidence that also in dogs uterine ECM modulation during decidualization occurs (Kautz et al 2015). Furthermore, functional genomics studies imply that in vivo canine free-floating embryos transmit signals to the uterus and discretely, i.e., without clearly visible morphological changes, affect uterine ECM composition at the early stage of pregnancy (Graubner et al 2017a). Indicating the beginning of canine decidualization, these embryo-derived signals control the expression of uterine biochemical markers of decidualization (Kautz et al 2014).…”