Among domestic animal species, the reproductive biology of the dog belongs to the most peculiar. This includes the conceptus-maternal communication and endocrine mechanisms involved in maintenance of pregnancy. Dogs fully depend on luteal progesterone (P4) throughout pregnancy, with similar steroid secretion patterns in pregnant and non-pregnant bitches until prepartum luteolysis. Thus, dogs lack the classical recognition of pregnancy. The luteal P4 is the most important hormone regulating the onset and maintenance of pregnancy in previously estrogenized bitches. Although the canine uterus is exposed to high P4 levels, decidualization is not spontaneous but induced by the presence of embryos. Following implantation, decidualization continues, associated with development of the invasive endotheliochorial placenta, leading to establishment of maternal decidual cells expressing the nuclear P4 receptor (PGR). Consequently, although not producing steroids, the canine placenta remains highly sensitive to circulating ovarian steroids. The placental conceptus-maternal communication is responsible for the maintenance of pregnancy, with functional withdrawal of PGR evoking a luteolytic cascade with prepartum PGF2α release. The fetal trophoblast is the major source of prepartum placental prostaglandins. This conceptus-maternal communication is unique to the dog and has clinical implications. Due to luteal steroids, there is no prepartum estradiol increase. Elevated cortisol levels are observed irregularly. This emphasizes the unique character of canine reproductive physiology and the challenges in transferring translational research to the dog. Further research is needed for better understanding of canine reproduction and improving clinical protocols, including the latest results obtained from applying modern laboratory technologies such as the transcriptomic approach.

Prostaglandins (PGs) are important regulators of the early corpus luteum (CL) in the dog. Whereas, initially, CL is gonadotropin independent, in the second half of its lifespan, hypophyseal support is required. The transition period is marked by decreased availability of PGs, in particular of PGE2. We previously reported that inhibition of COX2/PTGS2 in vivo suppressed luteal production of PGE2, lowered circulating progesterone and negatively affected luteal development. Therefore, bitches were treated with a COX2-specific blocker, firocoxib, for 5, 10, 20 and 30 days after ovulation, leading to suppression of the steroidogenic machinery. Control groups received a placebo for the same periods. Considering the wide range of possible modulatory roles of PGs shown in different organ systems, this follow-up project aimed to understand further possible PG-mediated effects in early canine CL. Thirty-four (34) factors related predominantly to vascularization and immune response were screened (mRNAs and proteins) on samples from the above described in vivo study. Most of the effects were observed during the transitional period (days 20 and 30). The inhibition of COX2 diminished the expression of angiopoietin family members ANGPT1, -2, Tie1 and -2 receptors. The expression of endothelin (ET)-1 was increased. Concerning the immune system, increased expression of the pro-inflammatory cytokines, IL1β, IL6 and IL12a, and elevated expression levels of CD4, was observed. Cumulatively, besides its involvement in regulating steroidogenesis, our results indicate a broader role of PGs in the canine CL, including modulation of angiogenesis, vascular stabilization and local immunomodulation. Possible cross-species translational effects are strongly implied.

Feto-maternal communication in the dog involves the differentiation of stromal cells into decidual cells. As the only placental cells expressing the nuclear progesterone (P4) receptor (PGR), decidual cells play crucial roles in the maintenance and termination of pregnancy. Accordingly, to investigate possible PGR-mediated mechanisms in canine decidual cells, in vitro decidualized dog uterine stromal (DUS) cells were treated with functional PGR-blockers, mifepristone and aglepristone. Effects on decidualization markers, epithelial and mesenchymal factors, and markers of cellular viability were assessed. Decidualization increased the expression of PTGES, PGR, IGF1, and PRLR, along with ECM1, COL4 and CX43, but downregulated IGF2. DUS cells retained their mesenchymal character, and the expression of COL4 indicated the mesenchymal-epithelial transformation. Antigestagen treatment decreased the availability of PTGES, PRLR, IGF1 and PGR. Furthermore, antigestagens decreased the mRNA and protein expression of CX43, and transcriptional levels of ECM1 and COL4. Additionally, antigestagens increased levels of activated-CASP3 (a proapoptotic factor), associated with lowered levels of PCNA (a proliferation marker). These data reveal important aspects of the functional involvement of PGR in canine decidual cells, regarding the expression of decidualization markers and acquisition of epithelial-like characteristics. Some of these mechanisms may be crucial for the maintenance and/or termination of canine pregnancy.

The canine corpus luteum (CL) is the main source of reproductive steroids during dioestrus in the dog and remains active even in the absence of pregnancy (non‐pregnant dioestrus, physiological pseudopregnancy). Whereas the biological effects of 17β‐oestradiol (E2) in the canine CL remain unclear, the transcriptional availability of oestrogen receptors, ESR1 and ESR2, as well as other modulators of local availability of E2, for example, HSD17B7 (converts oestrone into oestradiol), SULT1E1 (inactivates E2 binding capacity to its own receptors through sulphonation) and STS (reverts E2 sulphonation), were previously detected in the CL of non‐pregnant bitches. The aim of the present work was to evaluate the mRNA amounts of these factors involved in luteal sensitivity and metabolism of E2 in the canine CL during the course of non‐pregnant dioestrus (days 10, 20, 30, 40, 50 and 60 post‐ovulation, n = 5/group) and at different stages of pregnancy (n = 4‐6/group): pre‐implantation (days 8–12), post‐implantation (days 18–25), mid‐gestation (days 35–40) and prepartum luteolysis. During pregnancy, the availability of ESR1, HSD17B7, SULT1E1 and STS decreased from mid‐pregnancy to prepartum luteolysis. The main findings during non‐pregnant dioestrus were as follows: increased ESR2:ESR1 ratio on days 40 and 50 after ovulation, decreasing during luteal regression (day 60); increased STS at day 30 when SULT1E1 levels decreased; increased availability of SULT1E1 transcripts during luteal regression; and decreased amounts of HSD17B7 mRNA in early dioestrus, increasing towards later stages. These results suggest that E2 signalling and biologically active local concentrations could diverge in response to time and pregnancy status of the bitch.

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.