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.

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.

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.

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.

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.

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.

The role of hypoxia and HIF1α in the regulation of STAR-mediated steroidogenesis in granulosa cells.

The adaptive responses to hypoxia are mediated by hypoxia-inducible factor 1 alpha (HIF1α). Its role, however, in regulating steroidogenesis remains poorly understood. We examined the role of hypoxia and HIF1α in regulating steroid acute regulatory protein (STAR) expression and steroidogenesis in immortalized (KK1) mouse granulosa cells under progressively lowering O2 concentrations (20%, 15%, 10%, 5%, 1%). Basal and dbcAMP-stimulated progesterone synthesis was decreased under severe hypoxia (1% and 5% O2). The partial hypoxia revealed opposing effects, with a significant increase in steroidogenic response at 10% O2 in dbcAMP-treated cells: Star-promoter activity, mRNA and protein expression were increased. The hypoxia-stimulated STAR expression was PKA-dependent. Binding of HIF1α to the Star-promoter was potentiated under partial hypoxia. Inhibition of the transcriptional activity or expression of HIF1α suppressed STAR-expression. HIF1α appears to be a positive regulator of basal and stimulated STAR-expression, which under partial hypoxia is capable of increasing the steroidogenic capacity of granulosa cells.

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.

In vivo investigations on luteotropic activity of prostaglandins during early diestrus in nonpregnant bitches.

The aim of this study was to test for the postulated luteotropic effect of prostaglandin E2 during early diestrus in the dog in an in vivo study. This study was performed on 30 bitches which were randomly assigned to a treatment group (TG) and a control group. Starting on the day of ovulation (Day 0), dogs of the TG were treated for 5, 10, 20, or 30 days with 10 mg firocoxib/kg body weight per day (Previcox, a selective PTGS2 inhibitor) and ovariohysterectomized for collection of corpora lutea on the last day of treatment. Similarly, dogs of the control group were ovariohysterectomized on Days 0, 5, 10, 20, and 30. Blood samples for progesterone measurement were collected every second day; additionally, the area of luteal cell nuclei and the expression of 3ß-hydroxysteroid-dehydrogenase at the mRNA and the protein levels were assessed. Mean P4 concentrations were lower in TGs; however, a significant difference was only observed on Day 10. This observation is in line with the finding that treatment with firocoxib reduced expression of 3ß-hydroxysteroid-dehydrogenase mRNA and protein (P < 0.05) and the area of luteal cell nuclei (P < 0.05). The results of this study further point to the postulated luteotropic function of prostaglandin E2.

LPS-challenged TNFα production, prostaglandin secretion, and TNFα/TNFRs expression in the endometrium of domestic cats in estrus or diestrus, and in cats with pyometra or receiving medroxyprogesterone acetate.

Progesterone (P4) derivatives which are commonly used to block the cyclicity of domestic cats disturb the endocrine balance in the endometrium. The aims of this study were (i) to examine whether lipopolysaccharide (LPS) is responsible for enhancement of tumor necrosis factor-α (TNFα) secretion by the feline endometrial epithelial and stromal cells in vitro, (ii) to know whether immunolocalization of TNFα/TNFR1 and TNFR2 differs in cats at estrus or diestrus, receiving medroxyprogesterone acetate and suffering from pyometra, and (iii) to determine if TNFα-challenged prostaglandin secretion is stopped by prostaglandin synthases inhibitors. A total of 37 domestic adult cats in estrus or diestrus, receiving octane medroxyprogesterone or having clinical symptoms of pyometra, were enrolled in this study. The results obtained showed a distinct increase in LPS-challenged TNFα secretion in endometrial epithelial, but not stromal cells. TNFα augmented PG secretion was blocked by phospholipase A2 (PLA2) and cyclooxygeanase-2 (COX-2), but not by mitogen-activated protein kinase (MAPK) inhibitor. TNFα/TNFR1 and 2 protein expressions were limited mostly to the surface and glandular epithelium. TNFα/TNFRs protein was upregulated in the inflammatory uterus and hence may be involved in development of pathologic changes in the endometrial glands in cats receiving exogenous P4 as a hormonal contraceptive.

Placental origin of prostaglandin F2α in the domestic cat.

In the present study, the question was addressed whether the feline placenta can synthesize prostaglandin F2α (PGF2α). The PGFS protein was elevated, particularly at 2.5-3 weeks of pregnancy compared to 7-8 (P < 0.05) and 8.5-9 weeks (P < 0.001). Transcripts for PGFS were significantly upregulated at 2.5-3 weeks of pregnancy and then gradually declined towards the end of gestation (P < 0.001). Transcripts for PTGS2 were only upregulated in placentas from queens close to term (P < 0.001) compared with earlier phases. Staining of PTGS2 showed distinct positive signals in placentas obtained during the last week before labor, particularly in the strongly invading trophoblast surrounding blood vessels, and also in decidual cells. Shortly after implantation, signals for PGFS were localized in the trophoblast cells. Near term, PGFS staining was seen mainly in decidual cells. Both placental PGF2α and plasma PGFM were elevated towards the end of pregnancy (P < 0.001) compared with earlier weeks of pregnancy. The content of PGF2α in extracted placenta mirrored the PGFM level in plasma of pregnant females. During late gestation there is a significant increase in PGFM levels in maternal blood and of PGF2α levels in placental tissue concomitant with an upregulation of placental PTGS2.

Prostaglandin E2 functions as a luteotrophic factor in the dog.

The luteal phase in dogs is governed by many poorly understood regulatory mechanisms. Functioning of the corpus luteum (CL) is unaffected by hysterectomy. Recently, the role of prostaglandins in regulating canine CL function was addressed suggesting a luteotrophic effect of prostaglandin E2 (PGE2) during the early luteal phase. However, compelling functional evidence was lacking. The potential of PGE2 to stimulate steroidogenesis was tested in canine primary luteal cells isolated from developing CL of non-pregnant dogs. In addition, the luteal expression of prostaglandin transporter (PGT) and steroidogenic acute regulatory protein (STAR) was demonstrated and characterized in CL from non-pregnant bitches during the course of dioestrus as well as from pregnant animals during the pre-implantation, post-implantation and mid-gestation periods of pregnancy and during luteolysis; the luteal expression of PGE2 receptors (EP2 and EP4) has been investigated at the protein level throughout pregnancy. Our findings show that PGE2 is an activator of STAR expression in canine luteal cells from early luteal phase, significantly up-regulating STAR promoter activity and protein expression resulting in increased steroidogenesis. The 3ßHSD (HSD3B2) and P450scc (CYP11A1) expression remained unaffected by PGE2 treatment. The expression of PGT was confirmed in CL during both pregnancy and dioestrus and generally localized to the luteal cells. After initial up-regulation during the earlier stages of the CL phase, its expression declined towards the luteal regression. Together with the demonstration of EP2 and EP4 throughout pregnancy, and the decline in EP2 at prepartum, our findings further support our hypothesis that intra-luteal PGE2 may play an important role in regulating progesterone secretion in the canine CL.

Steroidogenic capacity of the placenta as a supplemental source of progesterone during pregnancy in domestic cats.

BACKGROUND: Until recently, the corpus luteum (CL) was considered to be the main source of progesterone (P4) during pregnancy in the domestic cat (Felis catus). However, other possible sources of P4 have not been ruled out. Although feline placental homogenates were found to be capable of synthesizing P4, expression of the respective steroidogenic enzymes has not been investigated at the molecular level. Therefore, in the present study, expression of the two major factors involved in the synthesis of P4 - 3beta-hydroxysteroid dehydrogenase (3betaHSD) and steroidogenic acute regulatory protein (StAR) - was investigated in the feline CL and placenta during the course of pseudopregnancy and pregnancy. METHODS: The mRNA levels of StAR and 3betaHSD were determined using Real Time PCR and their localizations were determined by immunohistochemistry. Placental P4 concentrations, after ethyl extraction, were measured by EIA. RESULTS: Luteal 3betaHSD and StAR mRNA levels were strongly time-dependent, peaking during mid-pregnancy. The placental 3betaHSD mRNA level was significantly upregulated towards the end of pregnancy. In the CL, 3betaHSD and StAR protein were localized in the luteal cells whereas in the placenta they were localized to the maternal decidual cells. Placental P4 concentrations were low in early pregnant queens, but increased along with gestational age. CONCLUSIONS: These results confirm that the placenta is an additional source of P4 in pregnant queens and can thereby be considered as an important endocrine organ supporting feline pregnancy.

Expression of prolactin receptors in normal canine mammary tissue, canine mammary adenomas and mammary adenocarcinomas.

BACKGROUND: Mammary tumors represent the most common neoplastic disease in female dogs. Recently, the promoting role of prolactin (PRL) in the development of human breast carcinoma has been shown. Possible proliferative, anti-apoptotic, migratory and angiogenic effects of PRL on human mammary cancer cells in vitro and in vivo were suggested. The effects of PRL are mediated by its receptor, and alterations in receptor expression are likely to play a role in tumor development. Currently, not much data is available about prolactin receptor (PRLR) expression in canine mammary tumors. To set the basis for investigations on the role of PRL in mammary tumorigenesis in this species, prolactin receptor expression was evaluated by semi-quantitative real time PCR and immunohistochemistry on 10 formalin-fixed, paraffin-embedded samples each of canine non-neoplastic mammary tissue, mammary adenomas and adenocarcinomas. RESULTS: The highest PRLR expression levels were found in normal mammary tissue, while adenomas, and to an even higher degree adenocarcinomas, showed a significant decrease in prolactin receptor expression. Compared to normal tissue, PRLR mRNA was reduced 2.4 fold (p = 0.0261) in adenomas and 4.8 fold (p = 0.008) in adenocarcinomas. PRLR mRNA expression was significantly lower in malignant than in benign lesions (p = 0.0165). Immunohistochemistry demonstrated PRLR expression in all three tissue types with signals mostly limited to epithelial cells. CONCLUSIONS: Malignant transformation of mammary tissue was associated with a decline in prolactin receptor expression. Further studies are warranted to address the functional significance of this finding.