Canine decidualization in vitro: 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.

Gene expression profiling of the canine placenta during normal and antigestagen-induced luteolysis.

The domestic dog is the only domestic animal species that does not produce steroids in the placenta and instead relies on luteal steroids throughout pregnancy. Nevertheless, the canine placenta is highly responsive to steroids, and withdrawal of progesterone (P4) affects the feto-maternal unit, initializing the parturition cascade. Similar effects can be observed during antigestagen-induced abortion. Here, aiming to provide new insights into mechanisms involved in the termination of canine pregnancy, next generation sequencing (NGS, RNA-seq) was applied. Placental transcriptomes derived from natural prepartum and antigestagen-induced abortions were analyzed and compared with fully developed mid-gestation placentas. The contrast "prepartum luteolysis over mid-gestation" revealed 1973 differentially expressed genes (DEG). Terms associated with apoptosis, impairment of vascular function and activation of signaling of several cytokines (e.g., IL-8, IL-3, TGF-ß) were overrepresented at natural luteolysis. When compared with mid-term, antigestagen treatment revealed 135 highly regulated DEG that were involved in the induced luteolysis and showed similar associations with functional terms and expression patterns as during natural luteolysis. The contrast "antigestagen-induced luteolysis over prepartum luteolysis" revealed that, although similar changes occur in both conditions, they are more pronounced during natural prepartum. Among P4-regulated DEG were those related to immune system and cortisol metabolism. It appears that, besides inducing placental PGF2α output, both natural and induced P4 withdrawal is associated with disruption of the feto-maternal interface, leading to impaired vascular functions, apoptosis and controlled modulation of the immune response. The time-related maturation of the feto-maternal interface needs to be considered because it may be clinically relevant.

Luteal ANGPT-TIE system during selected stages of pregnancy, and normal and antigestagen-induced luteolysis in the dog.

Abstract: Rapid establishment of a vascular network is essential for normal functionality of the corpus luteum (CL). The early luteal phase is associated with increased expression of the VEGF system in canine CL. Acting in synchrony with angiopoietins (ANGPTs), VEGF system plays major roles in stabilization of blood vessels. However, the expression of the ANGPT system has not yet been investigated in the dog. Therefore, here, we investigated the luteal expression of ANGPT1, -2, and of their receptors TIE1 and -2, in pregnant dogs at selected time points during pregnancy and at normal and antigestagen-induced luteolysis. Additionally, luteal cells from early CL were incubated with PGE2 and its effects on the ANGPT system were assessed. Whereas the luteal ANGPT1 was stable until mid-gestation, TIE1 was elevated post-implantation, their expression decreased toward prepartum luteolysis. The ANGPT2- and TIE2-mRNA did not vary during pregnancy. The ANGPT2/ANGPT1 ratio was elevated during prepartum luteolysis. PGE2 increased ANGPT2, but suppressed ANGPT1 levels. None of the ANGPT-system members was affected by antigestagen treatment in mid-pregnancy. Localization of ANGPT1 was predominantly found in the tunica intima and media of vessels and ANGPT2 stained strongly in luteal cells. Both ANGPTs were localized in macrophages. TIE1 stained in the vascular tunica media, in luteal cells and macrophages, whereas TIE2 was colocalized with ANGPT1 in vascular components. In conclusion, high expression of ANGPT1 during the increased presence of VEGFA in early canine CL implies its contribution to vascular network development. The upregulation of the ANGPT2/ANGPT1 ratio during prepartum luteolysis indicates involvement of the ANGPT system in PGF2α-mediated vascular destabilization.

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.

Morphological Characterization of Basally Located Uninucleate Trophoblast Cells as Precursors of Bovine Binucleate Trophoblast Giant Cells.

Binucleate trophoblast giant cells (TGCs) are one characteristic feature of the ruminant placenta. In cows, the frequency of TGCs remains constant for most of the duration of pregnancy. As TGCs are depleted by their fusion with uterine epithelial cells, they need to be constantly formed. It is still unclear whether they develop from stem cells within the trophectoderm or whether they can arise from any uninucleate trophoblast cell (UTC). Within the latter, generally accepted theory, a basally located uninucleate cell (BUC) without contact to the feto-maternal interface would represent a transient cell between a UTC and a TGC. So far, no evidence for the existence of such transient cells or for the presence of stem cells has been shown. The aim of the present study is to morphologically characterize the early stages of TGC development. Placentomal tissue of 6 pregnant cows from different gestational stages (gestational days 51-214) was examined for BUCs, UTCs, and TGCs either in serial sections (light and transmission electron microscopy, TEM, n = 3), in single sections (TEM, n = 2), or by serial block face-scanning electron microscopy (n = 1). These investigations revealed the occurrence of BUCs, as well as young TGCs showing contact with the basement membrane (BM), but without apical contact to the feto-maternal interface. The study morphologically defines these 2 cell types as early stages of TGC development and shows that binucleation of TGCs can precede detachment from the BM.

Expression of insulin-like growth factor 1 and its receptor in preovulatory follicles and in the corpus luteum in the bitch.

In the bitch, ovarian follicular and corpus luteum (CL) development and function are regulated by gonadotropins as well as local factors, the role of which is especially important during the early CL phase of relative gonadotrophic independence. We assumed that insulin-like growth factor 1 (IGF1) has a paracrine/autocrine regulatory role in ovarian follicular and luteal function in the dog. To address our hypothesis, we studied gene and protein expression of IGF1 and its receptor (IGF1R) in preovulatory follicles and in the CL of pregnant and non-pregnant dogs, and following antigestagen (aglepristone, progesterone receptor blocker) treatment in mid-gestation. Ovaries in the follicular phase were collected from five bitches. CL were collected on pregnancy Days 8-12 (pre-implantation), 18-25 (post-implantation), 35-40 (mid-gestation), at prepartum luteolysis, and 24 h and 72 h after aglepristone treatment in mid-gestation (n = 3-5 per group). From non-pregnant bitches, CL were collected on Days 5, 15, 25, 35, 45, 65 after ovulation (n = 4-5 per group). Semi-quantitative real-time (TaqMan) PCR and immunohistochemistry were applied. IGF1 immunostaining in preovulatory follicles seemed stronger in theca interna than granulosa cells. IGF1R signals appeared more intense in granulosa cells at the apical part of mural folds. In pregnant dogs, luteal IGF1 mRNA expression decreased significantly from pre-implantation to prepartum luteolysis, while IGF1R expression increased at prepartum luteolysis. Aglepristone treatment in mid-gestation had no effect on IGF1 and IGF1R mRNA levels. In non-pregnant bitches, highest IGF1 mRNA concentrations were found in the early CL and decreased by Days 45 and 65, while IGF1R expression did not change. In the CL of pregnant bitches, signals for IGF1 and IGF1R in luteal cells were strongest at pre- and post-implantation and weakest at prepartum luteolysis. IGF1 and IGF1R immunostaining was also detected in macrophages and in blood vessels. In conclusion, IGF1 may have a paracrine or autocrine role in granulosa and theca interna cells in preovulatory follicles. As IGF1 was highest represented in early luteal stages in pregnant and non-pregnant bitches, this may support a role for IGF1 in steroid synthesis, angiogenesis and cell proliferation as well as in immune function in the early canine CL. The unaffected mRNA levels after aglepristone treatment may support that IGF1 is not directly regulated by local progesterone in an auto- or paracrine manner.

Transcriptome analysis reveals differences in mechanisms regulating cessation of luteal function in pregnant and non-pregnant dogs.

BACKGROUND: In the domestic dog, corpora lutea (CL) are the only source of progesterone (P4), both in pregnant and non-pregnant cycles because there is no placental steroidogenesis. The absence of an endogenous luteolysin in absence of pregnancy results in long-lasting physiological pseudopregnancy, strongly contrasting with the acute luteolysis observed prepartum. The underlying biological mechanisms and the involvement of P4 signalling remain, however, not fully understood. Therefore, here, next-generation sequencing (RNA-Seq) was performed on CL from the late luteal phase and compared with normally luteolyzing CL collected at the prepartum P4 decrease. RESULTS: The contrast "luteal regression over luteolysis" yielded 1595 differentially expressed genes (DEG). The CL in late luteal regression were predominantly associated with functional terms linked to extracellular matrix (p = 5.52e-05). Other terms related to transcriptional activity (p = 2.45e-04), and steroid hormone signalling (p = 2.29e-04), which were more highly represented in late regression than during luteolysis. The prepartum luteolysis was associated with immune inflammatory responses (p = 2.87e-14), including acute-phase reaction (p = 4.10e-06). Immune system-related events were also more highly represented in CL derived from normal luteolysis (p = 7.02e-04), compared with those from dogs in which luteolysis was induced with an antigestagen (1480 DEG in total). Additionally, the withdrawal of P4 at mid-gestation resulted in 92 DEG; over-represented terms enriched in antigestagen-treated dogs were related to the inflammatory response (p = 0.005) or response to IL1 (p = 7.29e-05). Terms related to proliferation, e.g., centrosome organization (p = 0.002) and steroid metabolic processes (p = 0.001), prevailed at mid-gestation. Thereby, our results revealed the nature of luteotropic effects of P4 within canine CL. It appears that, even though they result in diminished steroidogenic output, the effect of antigestagens is more related to the withdrawal of P4 support than to the PGF2alpha-related inflammatory reaction observed at physiological parturition. CONCLUSIONS: We report the differential gene expression associated with maintenance and cessation of luteal function in pregnant and non-pregnant dogs. Based on the differentially expressed genes, we indicate functional pathways and gene networks that are potentially involved in the underlying endocrine and molecular mechanisms. This study establishes future research directions that may be helpful in understanding some of the clinical conditions, such as luteal insufficiency, associated with negative pregnancy outcome in dogs.

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.

Functional implications of the utero-placental relaxin (RLN) system in the dog throughout pregnancy and at term.

Relaxin (RLN) is a key hormone of pregnancy in mammals best known for its involvement in connective tissue remodeling. In the domestic dog, placental RLN is the only known endocrine marker of pregnancy. However, knowledge is sparse regarding the spatio-temporal expression of RLN and its receptors (RXFP1 and RXFP2) in the canine uterus and placenta. Here, their expression was investigated in the pre-implantation uterus and utero-placental compartments (UtPl) at selected time points during gestation: post-implantation, mid-gestation, and at normal and antigestagen-induced luteolysis/abortion. Immunohistochemistry with newly generated, canine-specific antisera, in situ hybridization and semi-quantitative PCR were applied. In compartmentalization studies, placental and endometrial RLN increased continuously toward prepartum. The placental RXFP1 was time-related and highest during post-implantation and decreased together with RXFP2 at prepartum luteolysis. The endometrial levels of both receptors did not vary greatly, but myometrial RXFP2 decreased from mid-gestation to prepartum luteolysis. Antigestagen treatment resulted in suppression of RLN in UtPl and decreased RXFP1 and RXFP2 in the uterus. The placental RLN was localized mainly in the cytotrophoblast. Additionally, RXFP1 stained strongly in placental endothelial cells while RXFP2 was found mainly in maternal decidual cells. Uterine staining for all targets was found in epithelial cellular constituents and in myometrium. Finally, besides its endocrine functions, RLN seems to be involved in auto-/paracrine regulation of utero-placental functions in dogs in a time-dependent manner. New insights into feto-maternal communication was provided, in particular regarding the localization of RXFP2 in the maternal decidual cells, implying functional roles of RLN during the decidualization process.

Centrosome Clustering in the Development of Bovine Binucleate Trophoblast Giant Cells.

Binucleate trophoblast giant cells (BNC) are the characteristic feature of the ruminant placenta. During their development, BNC pass through 2 acytokinetic mitoses and become binucleate with 2 tetraploid nuclei. In this study, we investigate the number and location of centrosomes in bovine BNC. Centrosomes typically consist of 2 centrioles surrounded by electron-dense pericentriolar material. Duplication of centrosomes is tightly linked to the cell cycle, which ensures that the number of centrosomes remains constant in proliferating diploid cells. Alterations of the cell cycle, which affect the number of chromosome sets, also affect the number of centrosomes. In this study, we use placentomal tissue from pregnant cows (gestational days 80-230) for immunohistochemical staining of γ-tubulin (n = 3) and transmission electron microscopy (n = 3). We show that mature BNC have 4 centrosomes with 8 centrioles, clustered in the angle between the 2 cell nuclei. During the second acytokinetic mitosis, the centrosomes must be clustered to form the poles of a bipolar spindle. In rare cases, centrosome clustering fails and tripolar mitosis leads to the formation of trinucleate "BNC". Generally, centrosome clustering occurs in polyploid tumor cells, which have an increased number of centrioles, but it is absent in proliferating diploid cells. Thus, inhibition of centrosome clustering in tumor cells is a novel promising strategy for cancer treatment. BNC are a cell population in which centrosome clustering occurs as part of the normal life history. Thus, they might be a good model for the study of the molecular mechanisms of centrosome clustering.

Uterine and placental expression of HPGD in cows during pregnancy and release of fetal membranes.

15-Hydroxyprostaglandin dehydrogenase (HPGD) plays a key role in prostaglandins (PGs) catabolism. Its expression and activity appear to be regulated by progesterone (P4). We investigated the HPGD mRNA-expression and protein localization in placentomes and interplacental uterine sites throughout gestation (Study I), and after fetal membranes retention (RFM) compared with normally delivered fetal membranes (DFM) (Study II). Furthermore, we analyzed the influence of aglepristone (AP), dexamethasone (GC) or cloprostenol (CP), on HPGD expression in bovine placentomes (Study III). Tissues from late gestation (D272) and at normal term (NT) served as controls. HPGD was highest in all sites at the beginning of pregnancy and at (NT). Following induced parturition HPGD was lower after (AP) and (GC) compared with (NT), and was similar in RFM and DFM. Placentomes stained primarily in fetal compartments; interplacentomal signals were observed in endometrial glandular and luminal epithelium. Results indicate that HPGD may play a role during establishment and termination of gestation.

Cellular localization, expression and functional implications of the utero-placental endothelin system during maintenance and termination of canine gestation.

Utero-placental (Ut-Pl) angiogenesis and blood flow are fundamental for successful outcome of pregnancy. They are controlled by numerous vasodilator and vasoconstrictor systems such as endothelins (EDNs) and the renin angiotensin system. Dogs possess an invasive type of placentation, classified as endotheliochorial. Despite increasing knowledge regarding canine Ut-Pl function, little information exists on uterine and placental vascular activity during initiation, maintenance and termination of pregnancy in this species. The current study investigated expression of EDNs and their receptors (EDNRA and EDNRB) in the pre-implantation uterus and Ut-Pl compartments during gestation and at normal parturition, as well as in mid-pregnant dogs treated with the antigestagen aglepristone. The Ut-Pl mRNA expression of EDN1 and EDNRA was constant until mid-gestation and increased significantly during prepartum luteolysis. In contrast, EDN2 was highest pre-implantation and decreased following placentation, remaining low thereafter. Expression of the EDN-activating enzyme ECE1 and mRNA of EDNRB increased towards mid-gestation and was further elevated at prepartum luteolysis. Antigestagen treatment resulted in increased levels of EDN1 and EDNRA. At the cellular level, the uterine expression of EDN1, ECE1 and EDNRB was found predominantly in the endometrial surface and glandular epithelial cells; uterine signals for EDNRA were weak. In Ut-Pl all targets were mainly localized in the placenta fetalis, with syncytiotrophoblast staining stronger for ECE1 and EDNRB. In contrast, EDNRA stained strongly at the base of the placental labyrinth. Expression and localization of EDNs (EDN1, -2), EDN receptors and ECE1 in the placenta fetalis suggests their involvement in the trophoblast invasion and proliferation.

Elevated utero/placental GR/NR3C1 is not required for the induction of parturition in the dog.

The endocrine mechanisms that lead to initiation of parturition in dogs are still not fully understood. The prepartum luteolysis is associated with increased prostaglandin (PG) F2α secretion; however, there is no pregnancy- or parturition-related increase in estrogens. Moreover, unlike in other mammalian species, in the dog, increased peripartum levels of cortisol measured sporadically in maternal peripheral blood are not mandatory for normal parturition. Nevertheless, auto/paracrine effects of cortisol at the placental feto-maternal level cannot be excluded. Therefore, the aim of this study was to investigate the expression and localization of glucocorticoid receptor (GR/NR3C1) in canine utero/placental (Ut/Pl) units and uterine interplacental sites at selected time points during pregnancy (pre-implantation, post-implantation and mid-gestation), and at normal and antigestagen-induced parturition. The Ut/Pl expression of GR/NR3C1 did not change significantly from pre-implantation until mid-gestation; however, it was strongly induced during the prepartum luteolysis. Within the interplacental samples, expression of GR/NR3C1-mRNA was greater post-implantation than pre-implantation and did not change afterward, i.e. toward mid-gestation. Compartmentalization studies within the Ut/Pl units, involving placenta, endometrium and myometrium separately, performed at the prepartum luteolysis revealed the highest GR/NR3C1-mRNA levels in placenta compared with endometrium and myometrium. Interestingly, in antigestagen-treated mid-pregnancy dogs, Ut/Pl and interplacental GR/NR3C1-mRNA expression remained unaffected. At the cellular level, placental GR/NR3C1 was clearly detectable in placenta fetalis, i.e. in trophoblast cells. In conclusion, increased expression of GR/NR3C1 during normal parturition, but not following antigestagen-treatment, suggest that it is not required for initiating the signaling cascade of PG synthesis leading to the induction of parturition in the dog.

Expression and functional implications of luteal endothelins in pregnant and non-pregnant dogs.

Luteal development is regulated by many locally produced mediators, e.g., prostaglandins and angiogenic factors. However, the role and function of vasoactive factors in the canine corpus luteum (CL) remain largely unknown. Consequently, expression of the endothelin (ET) receptors-A and -B (ETA and ETB, revealing vasoconstriction and vasodilator properties respectively), the ET-converting enzyme (ECE1) and ET1, -2 and -3 were investigated in CL from non-pregnant dogs (days 5, 15, 25, 35, 45 and 65 post-ovulation), and at selected stages of pregnancy (pre-implantation, post-implantation, mid-gestation), and during normal and antigestagen-induced prepartum luteolysis/abortion. The interrelationship between PGE2 and the ET system was investigated in PGE2-treated canine primary lutein cells from early CL. ET1 did not change significantly over time; ET2, ECE1 and ETB were elevated in early CL and were downregulated towards the mid/late-luteal phase. The prepartum increase of ET2 was significant. ET3 increased gradually, and was highest in late CL and/or at prepartum luteolysis. ETA remained constant until the late CL phase and increased only during prepartum luteolysis. ET1 was localized to the luteal cells, and ET2, ET3 and ETA to vascular endothelium. ECE1 and ETB were detected at both locations. Except for upregulated ET1 and lack of effect on ET2, antigestagen applied to mid-pregnant dogs evoked similar changes to those observed during normal luteolysis. PGE2 upregulated ETB in treated cells; ETA and ET1 remained unaffected, and ET2 decreased. A modulatory role of the ETs in canine CL, possibly in association with other factors (e.g., PGE2 and progesterone receptor), is strongly indicated.

Quantitative morphological changes in the interplacentomal wall of the gravid uterine horn of cattle during pregnancy.

BACKGROUND: The interplacentomal wall of the gravid uterine horn in cattle is the subject of reports dealing mainly with specific aspects of early pregnancy or the peripartal period. Only a very limited number of early and descriptive studies includes the whole period of pregnancy. Thus, there is a gap concerning quantitative morphological data of the uterine wall during pregnancy. We hypothesized that the specific requirements of pregnancy are reflected by significant and characteristic morphologic changes. METHODS: Interplacentomal segments of the fetus-bearing horn of the uterus of 47 cows were collected at slaughter, assessed quantitatively by light microscopy, grouped into trimesters (trim), and data were analyzed statistically. RESULTS: During pregnancy there were significant increases (p<0.05) in the measured parameters: heights of the endometrial surface epithelium (31 increased to 46 and 46 µm, in the 1st, 2nd and 3rd trim, respectively), glandular epithelium (19.6 to 22.4 and 25.4 µm, respectively), diameters of glands (94 to 166 to 239 µm, respectively) and glandular lumina (56 to 122 to 188 µm, respectively). Volume density of the glandular epithelium did not change, while that of glandular lumina increased significantly (8 to 26 to 40% in the 1st, 2nd and 3rd trim, respectively) and of endometrial stroma decreased with ongoing pregnancy (67 to 46 to 37%; p<0.05). Diameters of myometrial smooth muscle cells (MSMC) (9.7 to 12.4 and 12.9 µm, respectively, for the 1st, 2nd and 3rd trim; p<0.05), and the volume fraction of myometrial stroma increased (6 to 10 to 13%; p<0.05), while decreases were observed in MSMC nuclear volume density (4.4 and 4.0 to 2.4%; p<0.05). The fraction of MSMC cytoplasm (89 to 85%) and the nucleus:cytoplasm ratio (0.05 to 0.03%) both decreased for the 1st vs. 3rd trim, respectively (p<0.05). CONCLUSIONS: These results indicate that the interplacentomal wall of the gravid uterine horn is subjected to significant morphological changes during pregnancy, underlining the importance of endometrial surface epithelium and of gland hypertrophy for nourishment of the conceptus, of increased myometrial extracellular matrix for uterine tensile strength and of myometrial smooth muscle hypertrophy for expulsion of the fetus at term.

Leptin in the canine uterus and placenta: possible implications in pregnancy.

BACKGROUND: Leptin (Lep) is known for its involvement in the regulation of reproductive functions. It is important for uterine receptivity, implantation, placental growth and maternal energy homeostasis in several species, but Lep's function in the pregnant dog has not been investigated. METHODS: Pregnant bitches were ovariohysterectomized at pre-implantation, post-implantation, mid-gestation and prepartum luteolysis. Two additional groups were treated with aglepristone in mid-gestation, and ovariohysterectomized 24 or 72 h later. Lep and leptin receptor (LepR) gene expression was detected by semi-quantitative real-time PCR in pre-implantation and inter-placental uterine sections (Ut) and in utero-placental compartments (Ut/Pl). Immunohistochemistry and in situ hybridization (ISH) were performed for Lep and LepR protein and mRNA localization. Parametric one-way ANOVA, paired t-test and Wilcoxon signed-rank test were used for statistical analysis. RESULTS: In the Ut/Pl, Lep expression was higher at post-implantation and prepartum luteolysis than at mid-gestation, while in the Ut, Lep mRNA levels did not change during pregnancy. LepR expression in the Ut/Pl was up-regulated at prepartum luteolysis compared to the earlier stages. In the Ut, highest LepR mRNA was found at pre- and post-implantation. LepR expression was down-regulated in the Ut/Pl compared to the Ut at post-implantation and at mid-gestation. Aglepristone treatment resulted in a decrease of Lep mRNA levels from 24 to 72 h in the Ut without concomitant changes in the Ut/Pl or in LepR levels. Lep and LepR immunoreactivities were strong in the luminal and glandular epithelium in the Ut with abundant LepR signals in the subepithelial stroma. In the Ut/Pl, fetal trophoblasts stained stronger for Lep and LepR than decidual cells, and signals for both proteins were also detected in the glandular chambers. The myometrium, blood vessel media, and sporadically also the endothelium stained for Lep and LepR. ISH showed similar signal distribution in the Ut and Ut/Pl. CONCLUSIONS: Lep and LepR are differentially expressed in the canine uterus and placenta during pregnancy, and their presence in various cell types indicates paracrine/autocrine roles. The Lep signaling system may be one of the pathways involved in feto-maternal cross-talk, implantation and maintenance of pregnancy, and may have a regulatory role around parturition.

In vitro decidualisation of canine uterine stromal cells.

BACKGROUND: The uterine response to the presence of embryos is poorly understood in the domestic dog (Canis familiaris). The intimate embryo-maternal cross-talk, which begins following the hatching of blastocysts and embryo attachment leads to strong structural and functional remodelling of the uterus. A part of this process is decidualisation, comprising morphological and biochemical changes that result in formation of maternal stroma-derived decidual cells. These are an integral part of the canine placenta materna, which together with the maternal vascular endothelium are the only cells of the canine endotheliochorial placenta able to resist trophoblast invasion. These cells are also the only ones within the canine placenta expressing the progesterone receptor (PGR). Understanding the decidualisation process thus appears essential for understanding canine reproductive physiology. METHODS: Here, we investigated the capability of canine uterine stromal cells to decidualise in vitro, thereby serving as a canine model of decidualisation. A dbcAMP-mediated approach was chosen during a time course of 24 - 72 h. Tissue material from six (n = 6) healthy, dioestric bitches was used (approximately 2 weeks after ovulation). Cells were characterized by differential staining, nearly 100 % of which were vimentin-positive. Scanning and transmission electron microscope analyses were applied, and morphological changes were recorded with a live cell imaging microscope. Expression of several decidualisation markers was investigated. RESULTS: The in vitro cultured stromal cells acquired characteristics of decidual cells when incubated with 0.5 mM dbcAMP for 72 h. Their shape changed from elongated to rounded, while ultrastructural analysis revealed higher numbers of mitochondria and secretory follicles, and an increased proliferation rate. Elevated expression levels of IGF1, IGF2, PRLR and ERα were observed in decidualised cells; PRL and ERß remained mostly below the detection limit, while PGR remained unaffected. The expression of smooth muscle α actin (αSMA), another decidualisation marker, was strongly induced. Among prostaglandin system members, levels of COX2 (PTGS2) and of PGE2-synthase (PTGES) were upregulated. Expression of the PGE2 receptors, PTGER2 and PTGER4, was clearly detectable. CONCLUSION: An in vitro decidualisation model with canine uterine stromal cells was successfully established, allowing future, more detailed studies to be undertaken on the underlying molecular and endocrine mechanisms of canine decidualisation.

Endocrine control of canine mammary neoplasms: serum reproductive hormone levels and tissue expression of steroid hormone, prolactin and growth hormone receptors.

BACKGROUND: Neoplasms of the mammary gland are among the most common diseases in female domestic dogs (Canis familiaris). It is assumed that reproductive hormones influence tumorigenesis in this species, although the precise role of the endocrine milieu and reproductive state is subject to continuing discussion. In line with this, a recent systematic review of available data on the development of mammary neoplasms revealed weak evidence for risk reduction after neutering and an effect of age at neutering. Investigation of several hormone receptors has revealed decreased expression of estrogen receptor-alpha (ERα, ESR1), progesterone (P4) receptor (PGR), prolactin (PRL) receptor (PRLR) and growth hormone receptor (GHR) associated with neoplastic differentiation of mammary tissues. In other studies, increased levels of estrogens, progesterone and prolactin were found in serum and/or tissue homogenates of dogs with malignant neoplasms. However, the association between these entities within one animal population was never previously examined. Therefore, this study investigated the association between circulating serum concentrations of estradiol-17ß, progesterone and prolactin, and gene expression of ERα (ESR1), ERß (ESR2), PGR, PRLR, PRL and GHR, with respect to reproductive state (spayed vs. intact) and cycle stage (anestrus vs. diestrus). Additionally, the expression of E-cadherin (CDH-1) was evaluated as a possible indicator of metastatic potential. RESULTS: For all receptors, the lowest gene expression was found in malignant tumors compared to normal tissues of affected dogs. Steroid levels were not influenced by their corresponding receptor expression in mammary neoplasms, but increased PRL levels were negatively associated with low PRLR gene expression in malignant tumors. The expression of CDH-1 was influenced by tumor malignancy and cycle stage, i.e., the highest gene expression was found in benign mammary tumors in diestrous dogs compared to normal and malignant mammary tissues of anestrous and spayed dogs. CONCLUSIONS: Herein, it has been confirmed that transformation towards malignant neoplasms is associated with significant reduction of gene expression of particular hormone receptors. Only PRLR in malignant tumors seems to be influenced by circulating PRL levels. In dogs, CDH-1 can be used as a prognostic factor; its expression, however, in benign tumors is influenced by cycle stage.

Expression and localization of vascular endothelial growth factor A (VEGFA) and its two receptors (VEGFR1/FLT1 and VEGFR2/FLK1/KDR) in the canine corpus luteum and utero-placental compartments during pregnancy and at normal and induced parturition.

VEGFA is one of the most potent known inducers of angiogenesis. However, the function of angiogenic factors in the canine corpus luteum (CL) of pregnancy and in the pregnant uterus and placenta has not yet been elucidated. Therefore, here we investigated the expression and localization of VEGFA and its receptors (VEGFR1/FLT1 and VEGFR2/FLK1/KDR) in the canine CL and utero-placental compartments (ut-pl) throughout pregnancy until prepartum luteolysis. Antigestagen-mediated effects on expression of VEGF system in ut-pl were elucidated in mid-pregnant dogs. While displaying high individual variation, the luteal VEGFA was elevated during pre-implantation and post-implantation, followed by a decrease during mid-gestation, which was more pronounced at the mRNA level, and showed constant expression afterwards. Within the uterus, it increased following implantation and during mid-gestation in ut-pl compartments, but was downregulated at prepartum luteolysis. Luteal VEGFR1 expression resembled that of VEGFA; VEGFR2 remained unaffected throughout pregnancy. In ut-pl compartments, both receptors increased gradually towards mid-gestation; a prepartum decrease was observed for VEGFR1. Antigestagen-treatment resulted in decreased expression of ut-pl VEGFR1. In the CL, VEGFA stained in luteal cells. Uterine signals of VEGFA and its two receptors were observed in epithelial and vascular compartments, and in myometrium. In placental labyrinth, additionally, trophoblast stained positively. Luteal VEGFR1 was localized to the luteal cells and tunica media of blood vessels, whereas VEGFR2 stained only in capillary endothelial cells. The upregulation of luteal and the ut-pl VEGF system during early gestational stages supports the increased vascularization rate during this time. The diminishing effects of the prepartum endocrine milieu on VEGFA function seem to be more pronounced in the ut-pl units.

Canine placental prostaglandin E2 synthase: expression, localization, and biological functions in providing substrates for prepartum PGF2alpha synthesis.

The prepartum output of PGF2alpha in the bitch is associated with increased placental PGE2-synthase (PTGES) mRNA levels. Contrasting with this is a decreased expression of PGF2alpha-synthase (PGFS/AKR1C3) in uteroplacental compartments during prepartum luteolysis, suggesting an involvement of alternative synthetic pathways in PGF2alpha synthesis, for example, conversion of PGE2 to PGF2alpha. However, because the expression and possible functions of the respective PTGES proteins remained unknown, no further conclusion could be drawn. Therefore, a canine-specific PTGES antibody was generated and used to investigate the expression, cellular localization, and biochemical activities of canine uteroplacental PTGES throughout pregnancy and at prepartum luteolysis. Additionally, the biochemical activities of these tissues involved in the conversion of PGE2 to PGF2alpha were investigated. The endometrial PTGES was localized in the uterine surface epithelium at preimplantation and in superficial and deep uterine glands, endothelial cells, and myometrium throughout pregnancy and at parturition. Placental signals were mostly in the trophoblast. The biochemical properties of recombinant PTGES protein were confirmed. Additionally, expression of two PGE2-receptors, PTGER2/EP2 and PTGER4/EP4, revealed their decreasing expression during luteolysis. In contrast, the uteroplacental expression of prostaglandin transporter (PGT) was strongly elevated prior to parturition. These localization patterns resembled that of PTGES. The increased expression of PTGES and PGT at parturition, together with the accompanying decreased levels of PGE2-receptors and the capability of canine uterine and placental homogenates to take part in the conversion of PGE2 to PGF2alpha, as found in this study, suggest that PGE2 could be used locally as a substrate for prepartum PGF2alpha synthesis in the dog.