Applying a novel kinomics approach to study decidualization and the effects of antigestagens using a canine model†.

Maternal decidual cells are crucial for the maintenance of canine pregnancy as they are the only cells expressing the nuclear progesterone (P4) receptor (PGR) in the placenta. Interfering with P4/PGR signaling adversely affects decidual cells and terminates pregnancy. Although immortalized dog uterine stromal (DUS) cells can be decidualized in vitro using cAMP, the involvement of cAMP-dependent kinases in canine decidualization had not been investigated. Therefore, the present project investigated changes in the kinome of DUS cells following in vitro decidualization, using the serine/threonine kinase (STK) PamChip assay (PamGene). Decidualization led to a predicted activation of 85 STKs in DUS cells, including protein kinase (PK) A, PKC, extracellular signal-regulated kinase (ERK)1/2 and other mitogen-activated protein kinases (MAPKs), calcium/calmodulin-dependent protein kinases (CAMKs), and Akt1/2. In addition, blocking PGR with type 2 antigestagens (aglepristone or mifepristone) decreased the activity of virtually all kinases modulated by decidualization. The underlying transcriptional effects were inferred from comparison with available transcriptomic data on antigestagen-mediated effects in DUS cells. In targeted studies, interfering with PKA or MAPK kinase (MEK)1/2 resulted in downregulation of important decidualization markers (e.g., insulin-like growth factor 1 (IGF1), prostaglandin E2 synthase (PTGES), prolactin receptor (PRLR), PGR, and prostaglandin-endoperoxide synthase 2 (PTGS2/COX2)). Conversely, blocking of PKC decreased the mRNA availability of IGF1, PGR, and PTGS2, but not of PTGES and PRLR. Moreover, suppressing PKA decreased the phosphorylation of the transcription factors cJUN and CREB, whereas blocking of PKC affected only cJUN. This first kinomics analysis to target decidualization showed an increased activity of a wide range of STKs, which could be hindered by disrupting P4/PGR signaling. Decidualization appears to be regulated in a kinase-dependent manner, with PKA and PKC evoking different effects.

Membrane-bound progesterone receptors in the canine uterus and placenta; possible targets in the maintenance of pregnancy.

To date, the biological functions of P4 within the canine placenta have been attributed to maternal stroma-derived decidual cells as the only placental cells expressing the nuclear P4 receptor (PGR). However, P4 can also exert its effects via membrane-bound receptors. To test the hypothesis that membrane-bound P4 receptors are involved in regulating placental function in the dog, the expression of mPRα, -ß, -γ, PGRMC1 and -2 was investigated in the uterine and placental compartments derived from different stages of pregnancy and from prepartum luteolysis. Further, to assess the PGR signaling-mediated effects upon membrane P4 receptors in canine decidual cells, in vitro decidualized dog uterine stromal (DUS) cells were treated with type II antigestagens (aglepristone or mifepristone). The expression of all membrane P4 receptors was detectable in reproductive tissues and in DUS cells. The main findings indicate their distinguishable placental spatio-temporal distribution; PGRMC2 was predominantly found in decidual cells, PGRMC1 was strong in maternal endothelial compartments, and syncytiotrophoblast showed abundant levels of mPRα and mPRß. In vitro decidualization was associated with increased expression of PGRMC1 and -2, while their protein levels were diminished by antigestagen treatment. The involvement of membrane-bound P4 signaling in the regulation of canine placental function is implied, with P4 effects being directly exerted through maternal and fetal cellular compartments. The indirect effects of PGR might involve the modulation of membrane-bound receptors availability in decidual cells, implying a self-regulatory loop of P4 in regulating the availability of its own receptors in the canine placenta.

Endometrial extracellular matrix components do not change over the course of embryonic diapause and reactivation in the roe deer (Capreolus capreolus).

The modification of the endometrial extracellular matrix (ECM) is a crucial step for embryo implantation in many mammalian species. The embryo of the European roe deer (Capreolus capreolus) displays a 4-5 months long temporary reduction of developmental pace termed embryonic diapause. A reduction of epithelial cell height during diapause has previously been described. Co-occurring ECM modifications may contribute to the changes of the intra-uterine milieu during reactivation at which the embryo regains developmental velocity. We assessed the localization of five ECM proteins (collagen I and IV, fibronectin, laminin, and extracellular matrix protein 1) using immunohistochemistry in animals with early, late, and post-diapause (elongating) embryos. While our results confirmed the reduction of epithelial height during diapause, we only detected marginal differences in localization and staining intensities of the selected ECM proteins. Major ECM remodelling events in the roe deer endometrium are thus likely to occur only at implantation.

Ovarian and uterine changes during the oestrous cycle in female dogs.

CONTEXT: An accurate staging of sexual cycle is essential for the optimum timing of medical interventions. AIMS: Here, an updated insight into clinical, endocrinological and vagino-cytological parameters, and their correlation with histomorphology of ovarian and uterine tissue samples is presented. METHODS: Samples from 39 dogs were collected at various stages of the oestrous cycle: pro-oestrus (n =8), oestrus (n =12), dioestrus (n =9) (luteal phase) and anoestrus (n =10), according to clinical observations. Final allocation of samples was done after histomorphological evaluation of all tissues. Peripheral oestradiol-17ß (E2) and progesterone (P4) concentrations were measured, P4 by both chemiluminescent immunoassay (CLIA) and radioimmunoassay (RIA). KEY RESULTS: Differences were observed between determination of the stage of the oestrous cycle, either by clinical, endocrinological or histomorphological evaluation. Individuals considered to be in clinical and endocrinological oestrus, had entered the luteal phase according to histomorphology. P4 concentrations measured by two different assays differed, underlying the importance to understand that absolute P4 concentrations may deviate depending on the used assay. Comparison of E2 and P4 concentrations is suggested to be useful when defining the transition from early follicular phase to the time of ovulation. CONCLUSIONS AND IMPLICATIONS: Based on parallel histomorphological observations, combined with clinical and endocrinological findings on the same individuals, the present study emphasises that an accurate classification of the stage of the cycle in female dogs based solely on clinical and endocrinological assessments can be difficult. The histomorphological findings presented herein provide new insights into the transitional phases between the different stages of the oestrous cycle in the dog.

Transcriptomic profiling of canine decidualization and effects of antigestagens on decidualized dog uterine stromal cells.

Maternal-stroma derived decidual cells, the only cell population in the canine placenta expressing the nuclear progesterone (P4) receptor (PGR), are crucial for the maintenance of canine pregnancy. Decreased circulating progesterone (P4) levels, or blockage of PGR function with antigestagens, terminate canine pregnancy. As an in vitro model for canine decidualization, dog uterine stromal (DUS) cells can be decidualized in vitro with cAMP. The antigestagens aglepristone and mifepristone ablate the expression of decidualization markers in DUS cells (e.g., PGR, PRLR, IGF1 or PTGES). Here, the transcriptome profile of DUS cells was investigated to acquire deeper insights into decidualization-associated changes. Additionally, effects mediated by antigestagens (competitive PGR blockers) in decidualized cells were assessed. Decidualization led to the upregulation of 1841 differentially expressed genes (DEGs, P and FDR < 0.01) involved in cellular proliferation and adhesion, mesenchymal-epithelial transition, extracellular matrix organization, and vaso- and immunomodulation. The 1475 DEGs downregulated after decidualization were mostly associated with apoptosis and cell migration. In decidualized DUS cells, aglepristone modulated 1400 DEGs and mifepristone 1558 DEGs. Interestingly, around half of the identified DEGs were modulated by only one of the antigestagens. In all cases, however, PGR-blockage was mainly associated with an inversion of several decidualization-induced effects. Comparison between antigestagen-mediated effects and transcriptional changes in the canine placenta at term allowed the identification of 191 DEGs associated with diminished cell proliferation and adhesion, and vascular and immune modulation. This study emphasizes the importance of P4/PGR signaling for decidual cell function, providing new insights into the maintenance of canine pregnancy.

Transcriptional regulation of HIF1α-mediated STAR expression in murine KK1 granulosa cell line involves cJUN, CREB and CBP-dependent pathways.

Gonadal function is connected to hypoxia, with hypoxia-inducible factor (HIF) 1α, as a component of HIF1-complexes, regulating cellular adaptation to hypoxic conditions. In the ovary, it regulates follicular maturation, ovulation and luteal development. At the cellular level, HIF1-complexes coordinate the expression of steroidogenic acute regulatory protein (STAR), and thereby ovarian steroidogenesis. The functionality of STAR is associated with the cAMP/PKA-dependent pathways. In vitro, HIF1α is required for basal and cAMP-induced STAR expression, under ambient and reduced oxygen (O2) tension. Lowering O2 increases the responsiveness of the Star promoter towards cAMP and PKA mediates activation/phosphorylation (P) of several transcriptional factors, including cJUN and cAMP response element-binding protein (CREB), whose functionality is linked to HIF1 through utilization of CREB-binding protein (CBP). Since the mechanisms underlying HIF1α-dependent expression of STAR remain unknown, we investigated the involvement of HIF1α in CREB-, cJUN- and CBP-mediated expression of STAR using a well-characterized steroidogenic model, murine KK1 granulosa cells; ambient and lowered (10%) O2 were applied. Our main findings were that while functional suppression of the α-subunit of HIF1 lowered STAR/P-STAR and steroidogenic output from granulosa cells, surprisingly the levels of P-CREB and its transcriptional activity were strongly induced. However, its association with the Star promoter was decreased, indicating dissociation of P-CREB from the promoter. Further, suppression of HIF1 activity ultimately diminished the expression of cJUN/P-cJUN and CBP. Finally, the study suggests that HIF1-complex: (1) regulates cJUN expression in granulosa cells, (2) is involved in regulating the recruitment of P-CREB to the Star promoter in (3) a mechanism which possibly involves the HIF1-dependent regulation of CBP expression.

Insulin induces steroidogenesis in canine luteal cells via PI3K-MEK-MAPK.

Glucose uptake increases in canine luteal cells under insulin treatment. We hypothesize that insulin also increases luteal cell steroidogenesis. Dogs underwent elective ovariohysterectomy from days 10-60 post ovulation and their corpora lutea (CL) and blood samples were collected. Deep RNA sequencing determined differentially expressed genes in CL; those related to insulin signaling and steroidogenesis were validated in vivo by qPCR and their respective proteins by Western blotting and immunofluorescence. Next, luteal cell cultures were stimulated with insulin with or without inhibition of MAPK14, MAP2K1 and PI3K. Studied proteins except P450 aromatase showed the same expression pattern of coding genes in vivo. The expression of HSD3B and CYP19A1 was higher in insulin-treated cells (P < 0.005). Following respective pathway blockades, the culture medium had decreased concentrations of progesterone (P4) and 17b-estradiol (E2) (P < 0.01). Our results indicate that insulin increases HSD3B and CYP19A1 expression via MAPK and PI3K, and contributes to the regulation of P4 and E2 production in canine luteal cells.

The involvement of hypoxia-inducible factor 1α (HIF1α)-stabilising factors in steroidogenic acute regulatory (STAR) protein-dependent steroidogenesis in murine KK1 granulosa cells in vitro.

As a component of hypoxia-inducible factor1 (HIF1)-complexes, HIF1α regulates the expression of steroidogenic acute regulatory (STAR) protein in granulosa cells. However, severe hypoxia or exaggeratedly expressed HIF1α have detrimental effects. HIF1α is regulated by factor inhibiting HIF (FIH), prolyl hydroxylases (PHD1, 2, 3) and von Hippel-Lindau (VHL) suppressor protein. In this study, the expression of FIH, PHD1, 2, 3 and VHL was investigated in murine ovaries and immortalised KK1 granulosa cells. We found FIH, VHL and PHD2 transcripts predominantly in growing tertiary follicles. Functional aspects were assessed in KK1 cells exposed to decreasing O2 (20%, 10%, 1%), by determining HIF1α, FIH, VHL, PHD1-3 and STAR expression. The main findings indicated gradually increasing PHD2 under lowered O2. Functional blocking of PHDs revealed biphasic effects on STAR expression; concomitantly with increasing HIF1α, STAR expression, which was initially induced, decreased significantly when HIF1α was strongly stabilised. Finally, PHD2 in particular might act as a specific regulator of HIF1α and, thereby, of STAR availability in granulosa cells.

Anti-Müllerian hormone, testosterone, and insulin-like peptide 3 as biomarkers of Sertoli and Leydig cell function during deslorelin-induced testicular downregulation in the dog.

The role of anti-Müllerian hormone (AMH) and insulin-like peptide 3 (INSL3) in male infertility is not fully understood. We used the downregulated testis as a model of gonadotropin-dependent infertility. Serum testosterone and AMH concentrations were studied in five adult male Beagles implanted (day 0) with 4.7 mg deslorelin (Suprelorin®, Virbac) (DES group). Testicular expression of LH receptor (LHR) and androgen receptor (AR), AMH, type 2 AMH receptor (AMHR2), INSL3 and its receptor (RXFP2) was evaluated 112 days (16 weeks) after deslorelin treatment by qPCR and immunohistochemistry, and compared to untreated adult (CON, n = 6) and prepubertal (PRE, n = 8) dogs. Serum testosterone concentration decreased significantly by the onset of aspermia on study day 14 (four dogs) or day 21 (one dog), and was baseline on day 105 (week 15). In contrast, serum AMH started to increase only after the onset of aspermia and reached the maximum detectable concentration of the assay by day 49-105 in individual dogs. Testicular LHR gene expression in DES was lower than in CON and PRE (P < 0.0001), while AR gene expression in DES was similar to CON and significantly higher than PRE (P < 0.0001). Testicular AMH expression in DES was intermediate compared to the lowest mRNA levels found in CON and the highest in PRE (P ≤ 0.006). AMHR2 gene expression was similar between groups. AMH protein was detected in Sertoli cells only, while AMHR2 immunoreactivity was principally detected in Leydig cells which appeared to be increased in DES. INSL3 and RXFP2 gene expression was significantly downregulated in the DES testis along with noticeably weak Leydig cell immunosignals compared to CON. In conclusion, deslorelin treatment caused testicular LH insensitivity without affecting androgen sensitivity, and de-differentiation of Sertoli and Leydig cells. In DES, upregulation of the AMH-AMHR2 feed-back loop and downregulation of the INSL3-RXFP2 feed-forward loop are paracrine-autocrine mechanisms that may additionally regulate testosterone production independent of gonadotropins. Our results support AMH and INSL3 as unique biomarkers and paracrine-autocrine regulators of testis function involved in the intimate interplay between Sertoli and Leydig cells.

Macrophages in bovine term placenta: An ultrastructural and molecular study.

Retention of foetal membranes (RFM) is a major reproductive disorder in dairy cows. An appropriate immune response is important for a physiological expulsion of the foetal membranes at parturition. Our study aims to provide a deeper insight into characteristics of foetal and maternal macrophages in bovine term placenta. We used transmission electron microscopy (TEM), immunohistochemistry and semi-quantitative RT-PCR to provide a deeper insight into characteristics of foetal and maternal macrophages in bovine term placenta. Semi-quantitative RT-PCR was used to define macrophage polarization in foetal and maternal compartments of normal term placenta. Gene expression of factors involved in M1 polarization [interferon regulatory factor-5 (IRF5), interleukin (IL)-12A, IL12B] and in M2 polarization (IL10) were studied. Ultrastructurally, foetal macrophages showed an irregular shape and large vacuoles, whereas the maternal macrophages were spindle shaped. By immunohistochemistry, macrophages were identified by a strong staining with the lysosomal marker Lysosome-associated membrane glycoprotein 1 (LAMP-1), while myofibroblast in the maternal stroma was positive for alpha-smooth muscle actin. We used the LAMP-1 marker to compare the density of foetal stromal macrophages in placentas of cows with RFM and in controls, but no statistically significant difference was observed. RT-PCR showed a higher expression of all studied genes in the maternal compartment of the placenta and generally a higher expression of M1-, compared to M2-associated genes. Our results indicated that at parturition placental macrophages predominantly show the pro-inflammatory M1 polarization. The higher expression of all the target genes in the maternal compartment may denote that maternal macrophages in bovine term placenta are more frequent than foetal macrophages.

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.

Factors affecting the fate of the canine corpus luteum: Potential contributors to pregnancy and non-pregnancy.

The fate of the canine corpus luteum (CL) differs from that of other domestic species: beyond the extended luteal regression observed in both pregnant and non-pregnant cycles, active luteolysis is observed only in pregnant dogs. Luteal regression in the absence of pregnancy lacks a luteolytic trigger. The CL lifespan during pregnancy is around 60 days, as long as that of the cyclic CL. Although they are already available in the first half of diestrus, LH and especially prolactin (PRL) play a decisive luteotropic role from approximately day 25 post-ovulation onwards. Nevertheless, many locally-produced factors are orchestrated to ensure a fully functional CL, which in the bitch produces progesterone (P4), 17b-estradiol, and other local regulators. Recently, insulin has been described as another luteotropic factor in this species, able to increase glucose uptake in luteal cells and contribute to steroid biosynthesis. The locally-produced PGE2 is also a potent luteotropic factor in the first half of diestrus, promoting STAR expression, as are also proliferating, vasoactive- and immunomodulatory factors. These, in turn, all contribute to the formation and maintenance of the canine CL. Meanwhile PGF2a, produced by the utero-placental compartment, participates actively in triggering pre-partum luteolysis. Cytokines play different roles, either contributing as luteotropic or as acute inflammation molecules. So far, the one clinically most efficient mechanism of interrupting a pregnancy in the dog is to block P4 receptors, using an antigestagen (e.g., aglepristone) in the second half of diestrus. To enhance the chances of pregnancy, however, several luteotropic factors could be used.

Endometrial luminal epithelial cells sense embryo elongation in the roe deer independent of interferon-tau†.

Numerous intrauterine changes take place across species during embryo development. Following fertilization in July/August, the European roe deer (Capreolus capreolus) embryo undergoes diapause until embryonic elongation in December/January. Embryonic elongation prior to implantation is a common feature among ungulates. Unlike many other ruminants, the roe deer embryo does not secrete interferon-tau (IFNτ). This provides the unique opportunity to unravel IFNτ-independent signaling pathways associated with maternal recognition of pregnancy (MRP). This study aimed at identifying the cell-type-specific endometrial gene expression changes associated with the MRP at the time of embryo elongation that are independent of IFNτ in roe deer. The messenger RNA (mRNA) expression of genes known to be involved in embryo-maternal communication in cattle, pig, sheep, and mice was analyzed in laser capture microdissected (LMD) endometrial luminal, glandular epithelial, as well as stromal cells. The mRNA transcript abundances of the estrogen (ESR1), progesterone receptor (PGR), and IFNτ-stimulated genes were lower in the luminal epithelium in the presence of an elongated embryo compared to diapause. Retinol Binding Protein-4 (RBP4), a key factor involved in placentation, was more abundant in the luminal epithelium in the presence of an elongated embryo. The progesterone receptor localization was visualized by immunohistochemistry, showing an absence in the luminal epithelium and an overall lower abundance with time and thus prolonged progesterone exposure. Our data show a developmental stage-specific mRNA expression pattern in the luminal epithelium, indicating that these cells sense the presence of an elongated embryo in an IFNτ-independent manner.

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.

Prostaglandin-mediated effects in early canine corpus luteum: In vivo effects on vascular and immune factors.

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, IL1ß, 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.

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.

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.

Luteal and hypophyseal expression of the canine relaxin (RLN) system during pregnancy: Implications for luteotropic function.

By acting through its receptors (RXFP1, RXFP2), relaxin (RLN) exerts species-specific effects during pregnancy; possible luteotropic effects through stimulation of prolactin (PRL) release have been suggested. In the domestic dog (Canis lupus familiaris) serum PRL increases in pregnant bitches shortly after RLN appears in the circulation, and a possible functional relationship between the RLN and the PRL systems in regulating progesterone secretion has been implied. Therefore, here (Study 1) the luteal expression and localization of the RLN system was investigated by immunohistochemistry using custom-made antibodies and semi-quantitative PCR, at selected time points during gestation: pre-implantation (d. 8-12), post-implantation (d. 18-25), mid-gestation (d. 35-40) and at normal and antigestagen-induced luteolysis. Further, (Study 2) hypophyseal expression of the RLN system and its spatial association with PRL was assessed. Luteal expression of RLN, but not of its receptors, was time-dependent: it increased significantly following implantation towards mid-gestation and decreased at prepartum. Antigestagen treatment resulted in downregulation of RLN and RXFP2. Whereas RLN was localized in steroidogenic cells, RXFP1 and RXFP2 also stained strongly in macrophages and vascular endothelial cells. The RLN system was detected in the canine adenohypophysis and was co-localized with PRL in hypophyseal lactotrophs. The intraluteal RLN seems to be involved in regulating the canine corpus luteum (CL) in a time-dependent manner. The presence of RLN family members in the adenohypophysis implies their possible involvement in regulating the availability of PRL and other pituitary hormones.

Cells expressing CD4, CD8, MHCII and endoglin in the canine corpus luteum of pregnancy, and prepartum activation of the luteal TNFα system.

In the dog, knowledge about involvement of the immune system in controlling luteal function is restricted to observations showing a time-dependent invasion of immune cells into the corpus luteum (CL) of non-pregnant bitches. Therefore, this study investigated the presence of CD4-, CD8-, MHCII- and endoglin-expressing cells in CL collected throughout pregnancy from pre-implantation until prepartum luteolysis. Immunohistochemistry and semi-quantitative RT-PCR were applied. The time-dependent expression of CD4, CD8 and endoglin was more strongly related to formation of the CL, whereas MHCII was induced during luteolysis. Next, the luteal expression of TNFα and its receptors, TNFR1 and TNFR2, was analyzed in non-pregnant dogs between days 5-65 after ovulation and during pregnancy. Moreover, the effects of progesterone withdrawal were investigated in mid-pregnant dogs treated with an antigestagen aglepristone. The TNFα system was induced in the early CL of non-pregnant dogs. In pregnant dogs, expression of TNFα did not vary much, contrasting with increased expression of both receptors in the post-implantation period and significantly decreased expression at mid-gestation; prepartum luteolysis was characterized by increased TNFR2 expression. Apart from the downregulated expression of TNFR1, the changes observed following antigestagen treatment resembled those observed during normal prepartum luteolysis. A modulatory function of the TNFα system during formation of the canine CL is suggested, possibly related to the strong accompanying vascularization and luteal infiltration with activated macrophages. Contrasting with the slow luteal regression in non-pregnant dogs, in pregnant animals the upregulation of TNFR2 expression during prepartum luteolysis implies functional involvement of the TNFα system during that time.

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.