Kisspeptin-10 and the G protein-coupled receptor 54 are differentially expressed in the canine pregnant uterus and trophoblast cells.

Uterine tissue was collected from bitches after ovariohysterectomy at different times after ovulation. Samples were assigned to four groups: metestrous non-pregnant, day 10-12, n = 4; pre-implantation, day 10-12, n = 9; post-implantation, day 18-25, n = 13; mid-gestation, day 30-40, n = 7. RT-qPCR detection was performed for kiss1 and the G protein-coupled receptor 54 (GPR54, specific receptor for kisspeptin). In addition, immunohistochemistry was performed for detection of kisspeptin-10 (KP-10), GPR54, as well as pan-cytokeratin and vimentin. The latter two were included to differentiate the different placental cell types. The percentage of positive stained cells was evaluated, and an immunoreactivity score (IRS) was obtained by multiplying the labelling intensity score (0-3) with the percentage of immunolabelled cells (range: 0-300). In non-pregnant and pre-implantation tissues, gene expression was highly variable for kiss1 and GPR54. Expression of GPR54 was higher before embryo adhesion than during post-implantation and mid-gestation (p < .05), whereas there was no difference found between groups for kiss1. Except during the pre-implantation period, KP-10 expression was higher in the non-pregnant uterus compared to all gestational periods investigated, indicating a pregnancy-related downregulation. In the pre-implantation period, KP-10 was present in larger vessels only, whereas the presence of GPR54 in vessels was found in all samples, with most labelling in the post-implantation period. KP-10 was present in superficial uterine glands, GPR54 in superficial and deep uterine glands of the post-implantation uterus. In myocytes, the highest staining for KP-10 was seen in the non-pregnant uterus, whereas the highest staining for GPR54 was seen in post-implantation and mid-gestation. Syncytiotrophoblast cells stained for both KP-10 and GPR54 in post-implantation and mid-gestation, with maximum intensity for GPR54 in the latter. We conclude that KP-10 and GPR54 are expressed in the canine uterus and trophoblast cells. However, during pregnancy, expression of both proteins seems to be differentially regulated.

GnRH and its receptor (GnRH-R) are expressed in the canine placenta and uterus.

In reproductive tissues, GnRH participates in the regulation of cell growth and proliferation by direct binding to the GnRH-R, which is essential for embryo implantation. However, there is no study on the expression and cellular localization of GnRH and GnRH-R in the canine uterus and placenta. Therefore, bitches were ovariohysterectomized 10 to 12 days after mating (vaginal cytology and progesterone measurement), the uteri were flushed, and if embryos were detectable, bitches were allocated to the embryo positive group (E-pos.; preimplantation, n = 5). Other bitches were operated at later stages and, dependent on the gestational age, either allotted to the post-implantation group (Day 18-25 after mating, n = 9), or the mid-gestation group (Day 30-40 after mating, n = 3). Dogs negative in embryo flushing served as controls (E-neg.; controls, n = 5). Samples of the entire uterine wall were taken from the middle of the horn in E-neg. and E-pos. groups, and from placental and interplacental uterine sites in post-implantation and mid-gestation groups. GnRH-R expression was localized at the mRNA and protein levels by immunohistochemistry and in situ hybridization. The expression of GnRH and GnRH-R mRNA was assessed by semiquantitative polymerase chain reaction. Additionally, both GnRH and GnRH-R mRNA were expressed in all tissues examined until mid-gestation. Relative expression of GnRH was higher than that of GnRH-R (P < 0.05). During the post-implantation stage, GnRH-R expression was significantly higher in uteroplacental than in interplacental tissues. In the uterus, GnRH-R stained strongly in the surface and glandular epithelial cells, and seemed to be weaker in myometrium and stroma. Placental signals were predominantly localized in fetal trophoblast cells and to a lesser extent in maternal decidual cells. These findings suggest a local regulatory function of GnRH during early canine pregnancy.

The effects of aglepristone alone and in combination with cloprostenol on hormonal values during termination of mid-term pregnancy in bitches.

This study was designed to assess endocrine changes associated with termination of mid-term pregnancy after use of two different protocols. For this purpose we compared the effects of aglepristone (AGL) alone and in combination with cloprostenol (CLO) on serum concentrations of progesterone (P4), estradiol (E2) and relaxin (RLN) measured at short-term intervals during the abortion period in bitches. Fourteen pregnant bitches between day 25 and 32 of gestation were used in the study. In the AGL group (n=7), aglepristone was administered solely (10mg/kg body weight (BW), subcutaneously, once daily on two consecutive days) whereas in the AGL-CLO group (n=7), aglepristone (dosage as in AGL group) and cloprostenol (1µg/kg BW, subcutaneously, same with aglepristone) were combined. All pregnancies were successfully terminated 5.2±1.6 days after initiation of treatments, which was significant in both groups (P>0.05). At the time of the start of abortion (SA) and the end of abortion (EA), the mean P4 concentrations were 26.6±7.3 and 12.0±6.4ng/ml in AGL group, and 2.7±0.7 and 0.9±0.1ng/ml, in AGL-CLO group, respectively (P<0.01). Serum E2 concentrations were significantly higher (P<0.05) in AGL group at 42, 48, 54h and SA after initiation of treatment. In the AGL-CLO group, serum RLN concentrations did not significantly change from the initiation of treatment to EA (P>0.05). However, markedly higher RLN concentrations (P<0.05) were observed in the AGL group at 48h (1.5±0.7ng/ml) and at SA (1.6±0.5ng/ml). The results of the present study indicate that changes in the hormonal concentrations affect the mechanism of abortion in different ways. Further in depth studies investigating changes in the expression of hormone receptors inside the ovary, endometrium and placenta might be helpful to our understanding of the endocrinological differences observed in this study.

Expression of genes involved in the embryo-maternal interaction in the early-pregnant canine uterus.

Although there is no acute luteolytic mechanism in the absence of pregnancy in the bitch, a precise and well-timed embryo-maternal interaction seems to be required for the initiation and maintenance of gestation. As only limited information is available about these processes in dogs, in this study, the uterine expression of possible decidualization markers was investigated during the pre-implantation stage (days 10-12) of pregnancy and in the corresponding nonpregnant controls. In addition, the expression of selected genes associated with blastocyst development and/or implantation was investigated in embryos flushed from the uteri of bitches used for this study (unhatched and hatched blastocysts). There was an upregulated expression of prolactin receptor (PRLR) and IGF2 observed pre-implantation. The expression of PRL and of IGF1 was unaffected, and neither was the expression of progesterone- or estrogen receptor ß (ESR2). In contrast, (ESR1) levels were elevated during early pregnancy. Prostaglandin (PG)-system revealed upregulated expression of PGE2-synthase and its receptors, PTGER2 and PTGER4, and of the PG-transporter. Elevated levels of AKR1C3 mRNA, but not the protein itself, were noted. Expression of prostaglandin-endoperoxide synthase 2 (PTGS2) remained unaffected. Most of the transcripts were predominantly localized to the uterine epithelial cells, myometrium and, to a lesser extent, to the uterine stroma. PGES (PTGES) mRNA was abundantly expressed in both groups of embryos and appeared higher in the hatched ones. The expression level of IGF2 mRNA appeared higher than that of IGF1 mRNA in hatched embryos. In unhatched embryos IGF1, IGF2, and PTGS2 mRNA levels were below the detection limit.

Uterine progesterone receptor and leukaemia inhibitory factor mRNA expression in canine pregnancy.

The study investigated the expression of genes for progesterone receptor (PR) and for the cytokine leukaemia inhibitory factor (LIF) in the uterine tube and uterine horn tissues from pregnant and non-pregnant bitches. The aim was to study whether a relation existed between the likely biological effectiveness of progesterone (P(4)) and the change in the uterine expression of LIF mRNA during pregnancy, as has been described in primates. For this purpose, 20 pregnant bitches were ovariohysterectomized after being allotted to three groups according to gestational age (pre-implantation: days 10 to 12, n = 7; peri-implantation: days 18 to 25, n = 7; post-placentation: days 28 to 45, n = 7). Tissue samples were obtained from the uterine tubes, one uterine horn (including placentation sites and interplacental sites in bitches that had already implanted) and the corpus uteri, stored at -80 degrees C, and then analysed by qualitative and quantitative PCR for PR and LIF mRNA expression. From the pre-implantation to the placentation stage, a decrease in the relative expression of PR mRNA in uterine tissue was obvious and significant when expressed relative to beta-actin (11.2 +/- 6.8 vs 2.7 +/- 1.9; p < 0.05). However, over the same period, the relative expression of LIF mRNA increased (10.1 +/- 16.1 vs 50.0 +/- 32.3; p < 0.05). In addition, PR mRNA went from being detectable to no longer detectable in the uterine tube, and no longer detectable in interplacental-site uterine tissue. We conclude that LIF is important for the establishment of canine pregnancy; that decreased uterine PR mRNA expression may contribute to the increase in uterine LIF mRNA; and, that the ability of the embryo to preserve PR mRNA expression at implantation and placentation sites while expression is lost in the remainder of the uterus represent an effect important to the establishment and maintenance of pregnancy. We additionally propose that canine embryo secretory proteins have a regulatory effect on both PR and LIF before as well as at and after implantation.

Cytokines, growth factors and prostaglandin synthesis in the uterus of pregnant and non-pregnant bitches: the features of placental sites.

Uterine tissue from pregnant bitches was investigated by qualitative RT-PCR for the gene expression of local factors potentially important for the implantation of canine embryos. For this purpose, 10 bitches identified as being at the time of implantation or early placentation by means of ultrasonography before ovariohysterectomy (days 20-35, n = 10) provided tissues for comparison to tissue collected in a previous study and identified as early pregnant (n = 10) or non-pregnant (n = 4) by embryo flushing after ovariohysterectomy (days 10-12 after mating; Schäfer-Somi et al. 2008). Uterine tissue was excised from the middle of the left horn from early pregnant and non-pregnant animals, including from interplacental and placentation sites. The following genes were investigated: CD-4, -8; cyclooxygenase (COX)-1, -2; granulocyte macrophage-colony stimulating factor (GM-CSF); hepatocyte growth factor (HGF); insulin-like growth factor (IGF)-1, -2; transforming growth factor (TGF) and tumour necrosis factor (TNF)-alpha; interferon (IFN)-gamma; interleukin (IL)-1beta, -2, -4, -6, -8, -10, -12; leukaemia inhibitory factor (LIF) and leptin. Gene expression for CD-8, COX-1, TGF-beta, HGF, IGF-1, IL-2, -4,-10, IFN-gamma and LIF were detected in the pre-implantation uterus, and all except IL-2 and -10 were still detectable during the implantation and placentation stage. During implantation, mRNA for IGF-2 and GM-CSF were additionally detected. The dioestrous uterus differed from the pregnant uterus because of the absence of CD-8, IL-4 and IFN-gamma and the expression of CD-4, TNF-alpha and IL-6. The results suggest that IL-4, IFN-gamma, CD-8, GM-CSF and IGF-2 are regulated in a pregnancy-specific manner and that GM-CSF and IGF-2 probably have growth supporting and immune modulating functions during implantation of the canine embryo.

Induction of abortion with aglepristone significantly changed the expression of progesterone and estrogen receptors in canine endometrial stromal cells.

In the present study, resorption/abortion was induced between days 25 and 45 of gestation with aglepristone (group IRA, n=10). The aim was to observe the change in the distribution of progesterone (PR) and estrogen receptors (ER), in comparison to a group of spontaneous resorptions/abortions (group SRA, n=5), and a further group of normal healthy pregnant animals, ovariohysterectomized between days 25 and 45 of gestation (controls, n=7). The receptors were assessed by means of immunohistochemistry (IHC) and RT-PCR, at the placental and interplacental sites of the uterine horn as well as in the corpus uteri. Significant differences were observed between the controls on one side and the groups of resorption/abortion on the other side. The total scores of the progesterone receptors (TPR) in the placental and interplacental part of the uterine horn, was significantly lower in the endometrial stromal cells (ESC) of the control group than in those of the SRA- and IRA-group, respectively (placenta: 5.8 vs. 6.5 and 6.7, p<0.01; interplacental sites: 5.6 vs. 6.6 and 6.6, p<0.05). In contrary, the total scores of the estrogen receptors (TER) at interplacental sites and the corpus uteri, respectively, was significantly higher in the myometrial smooth muscle cells (MSMC) and the ESC (p<0.05) of the controls. We therefore conclude, that the here observed differences between groups point to an up-regulation of TPR- and a down-regulation of TER-scores in endometrial stromal cells at different uterine sites during resorption/abortion, which indicates a special role of these cells.

Expression of genes in the canine pre-implantation uterus and embryo: implications for an active role of the embryo before and during invasion.

The aim of the present study was to assess genes expressed in maternal uterine tissue and pre-implantation embryos which are presumably involved in maternal recognition and establishment of canine pregnancy. For this purpose, 10 pregnant bitches were ovariohysterectomized between days 10 and 12 after mating. Four non-pregnant bitches served as controls. Early pregnancy was verified by flushing the uterine horns with PBS solution. The collected embryos (n = 60) were stored deep-frozen (-80 degrees C). Uterine tissue was excised, snaps frozen in liquid nitrogen and homogenized using TRI Reagent. All embryos from one litter were thawed together and also homogenized in TRI Reagent. RT-PCR was performed to prove mRNA expression of progesterone receptor, key enzymes of the prostaglandin synthesis pathway, selected growth factors, cytokines, immune cell receptors, major histocompatibility complex (MHC) and matrix-metalloproteinases (MMP). Only pregnant uteri revealed the presence of mRNA for interferon (IFN)-gamma, IL-4 and CD-8, which resembles the milieu in humans and other mammalians. Similarly, in day 10 embryos, mRNA for transforming growth factor-beta, insulin-like growth factor-1,-2, hepatocyte growth factor, leukaemia inhibitor factor, tumour necrosis factor-alpha, interleukin-1beta,-6,-8, cyclooxygenase-2, CD4(+) cells, and MMP-2 and -9 were detected, but not MHC-I or -II. We therefore suppose that the canine embryo, like its human counterpart, actively initiates measures to prevent attacks from the maternal immune system to prepare its own adhesion, nidation, growth and further development.