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Contents 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. [ABSTRACT FROM AUTHOR]

Contents The aim of this study was to investigate the course of expression of platelet-activating factor (PAF), PAF-receptor (PAF-R), epidermal growth factor (EGF), EGF-R, vascular endothelial growth factor (VEGF), VEGF-R1 and VEGF-R2 in uterine tissue during canine pregnancy. For this purpose, 20 bitches were ovariohysterectomized at days 10-12 (n = 10), 18-25 (n = 5) and 28-45 (n = 5) days after mating, respectively. The pre-implantation group was proven pregnant by embryo flushing of the uterus after the operation, the others by sonography. Five embryo negative, that is, non-pregnant, bitches in diestrus (day 10-12) served as controls. Tissue samples from the uterus (placentation sites and horn width, respectively) were excised and snap-frozen in liquid nitrogen after embedding in Tissue Tec®. Extraction of mRNA for RT-PCR was performed with Tri-Reagent. In the embryos, mRNA from all factors except VEGF was detected. In the course of pregnancy, significantly higher expression of PAF and PAFR as well as VEGF and VEGFR2 during the pre-implantation stage than in all other stages and a strong upregulation of EGF during implantation were characteristic. The course of EGF was in diametrical opposition to the course of the receptor. These results point towards an increased demand for VEGF, EGF and PAF during the earliest stages of canine pregnancy. [ABSTRACT FROM AUTHOR]

The goal of this study was to compare three different medications for the induction of abortion in bitches between day 22 and 40 after mismating, to reduce the dosage and frequency of PGF2alpha applications and to investigate the effects of additional intravaginal applications of misoprostol, a PGE1 analog. For this purpose, 22 bitches of different breeds were assigned to three groups: group 1 (n = 6) received cabergoline (Galastop, 10 microg/kg daily, po) until abortion was complete (first application = day 0) and one administration of alfaprostol (Gabbrostim, 10 microg/kg at day 3, s. c.); group 2 (n = 9) was administered cabergoline (10 microg/kg daily, po) and alfaprostol (10 microg/kg at days 0 and 3, s. c.); and group 3 (n = 7) received the same combination as group 2 in addition to misoprostol (Cytotec, 400 microg for bitches with < or = 20 kg bw, 600 microg for bitches with > 20 kg bw, every other day, intravaginally). Abortion occurred in 83.3%, 77.7% and 100% of cases in group 1, 2 and 3, respectively (p > 0.05). Cervical dilatation time was shortest in group 3 (3.1 +/- 0.9 days vs. group 1:5.4 +/- 2.8 and group 2: 5.5 +/- 2.5; p < 0.05). Minor side effects like transient anorexia, weakness, mild vomiting and diarrhea or low grade hair loss were found in 30.3%, 54.3% and 54.0% of cases in group 1, 2 and 3, respectively. We conclude that a combination of oral cabergoline (10 microg/kg) and alfaprostol (10 microg/kg) is an effective method for inducing termination of pregnancy in bitches. However, a combination of oral cabergoline (10 microg/kg), alfaprostol (10 microg/kg) and administration of additional intravaginal misoprostol may be even more effective.

Expression of cyclooxygenase 2 (COX2, now known as PTGS2), prostaglandin E2 synthase (PTGES, PGES), and prostaglandin F2alpha synthase (PGFS), of the respective receptors PTGFR (FP), PTGER2 (EP2), and PTGER4 (EP4) and of the progesterone receptor (PGR, PR) was assessed by real-time PCR, immunohistochemistry (IHC), or in situ hybridization (ISH) in utero/placental tissue samples collected from three to five bitches on days 8-12 (pre-implantation), 18-25 (post-implantation), and 35-40 (mid-gestation) of pregnancy and during the prepartal luteolysis. Additionally, ten mid-pregnant bitches were treated with the antiprogestin aglepristone (10 mg/kg bw (2x/24 h)); ovariohysterectomy was 24 and 72 h after the second treatment. Plasma progesterone and 15-ketodihydro-PGF2alpha (PGFM) concentrations were determined by RIA. Expression of the PGR was highest before implantation and primarily located to the endometrium; expression in the placenta was restricted to the decidual cells. PTGS2 was constantly low expressed until mid-gestation; a strong upregulation occurred at prepartal luteolysis concomitant with an increase in PGFM. PGFS was upregulated after implantation and significantly elevated through early and mid-gestation. PTGES showed a gradual increase and a strong prepartal upregulation. PTGFR, PTGER2, and PTGER4 were downregulated after implantation; a gradual upregulation of PTGFR and PTGER2 occurred towards parturition. ISH and IHC co-localized PGFS, PTGFR, PTGES, and PTGS2 in the trophoblast and endometrium. The changes following application of aglepristone were in the same direction as those observed from mid-gestation to prepartal luteolysis. These data suggest that the prepartal increase of PGF2alpha results from a strong upregulation of PTGS2 in the fetal trophoblast with the withdrawal of progesterone having a signalling function and the decidual cells playing a key role in the underlying cell-to-cell crosstalk.

In nonpregnant and pregnant dogs the corpora lutea (CL) are the only source of progesterone (P4) which shows an almost identical secretion pattern until the rapid decrease of P4 prior to parturition. For the nonpregnant dog clear evidence has been obtained that physiological luteal regression is devoid of a functional role of the PGF2alpha-system and seems to depend on the provision of StAR. Yet in pregnant dogs the rapid prepartal luteal regression, coinciding with an increase of PGF2alpha, may be indicative for different regulatory mechanisms. To assess this situation and by applying semi-quantitative Real Time (Taq Man) RT-PCR, expression patterns were determined for the following factors in CL of pregnant and prepartal dogs and of mid-pregnant dogs treated with the antiprogestin Aglepristone: cyclooxygenase 2 (Cox2), prostaglandin E2 synthase (PGES), prostaglandin F2alpha synthase (PGFS), its receptors (EP2, EP4 an FP), the steroidogenic acute regulatory protein (StAR), 3beta-hydroxysteroid-dehydrogenase (3betaHSD) and the progesterone receptor (PR). Peripheral plasma P4 concentrations were determined by RIA. CL were collected via ovariohysterectomy from pregnant bitches (n=3-5) on days 8-12 (Group 1, pre-implantation period), days 18-25 (Group 2, post-implantation period), days 35-40 (Group 3, mid-gestation period) and during the prepartal progesterone decline (Group 4). Additionally, CL were obtained from groups of 5 mid-pregnant dogs (days 40-45) 24h, respectively 72h after the second treatment with Aglepristone. Expression of Cox2 and PGES was highest during the pre-implantation period, that of PGFS and FP during the post-implantation period. EP4 and EP2 revealed a constant expression pattern throughout pregnancy with a prepartal upregulation of EP2. 3betaHSD and StAR decreased significantly from the pre-implatation period to prepartal luteolysis, it was matched by the course of P4 concentrations. Expression of the PR was higher during mid-gestation and prepartal luteolysis than in the two preceding periods. After application of Aglepristone the overall mRNA-expression resembled the situation during prepartal luteolysis except for EP2, which remained unchanged. These data suggest that - as in the nonpregnant bitch - also in the pregnant bitch luteal production of prostaglandins is associated with luteal support rather than luteolysis. On the other hand induction of luteolysis by the PR blocker Aglepristone points to a role of luteal P4 as an autocrine factor in a positive loop feedback system controlling the availability of P4, StAR and 3betaHSD.

The aim of the present study was to investigate the effects of two medications on two subsequent abortions and plasma hormone concentrations of dogs. For this purpose, two groups of bitches (n=5 each), received the antiprogesterone aglepristone (Alizine) at 10mg/kg body weight on two subsequent days around day 30 after mating. In group II, the antiprolactin cabergoline (Galastop) was additionally administered po at 5 microg/kg body weight until the start of abortion. The plasma concentrations of relaxin, progesterone (P4) and estradiol-17beta (E2) were measured before, during and after each abortion. During the next cycle after the abortion, the same bitches were mated again and in pregnant animals, induction of abortion was performed as before. During the third cycle, pregnant bitches were allowed to whelp. Termination of first pregnancy occurred significantly earlier after the combined treatment (6.8 versus 10.6 days, p<0.05). In both groups and during both abortions, relaxin varied between individuals; however, there was a continuous decrease after the abortions and no significant differences between groups (p>0.05). In one bitch with high relaxin concentrations before treatment (11.6 ng/ml), a cystic endometrial hyperplasia was diagnosed. In the aglepristone only group, P4 concentrations increased significantly after the first application (p<0.05), then decreased continuously until day 45 after the beginning of abortion. In the combined group, there was a continuous decrease until day 45 (p>0.05). At this time, P4 concentrations between 0.47 and 84.9 nmol/l were measured in both groups. The level of E2 over time was not influenced by any medication. We therefore note that the two medications mainly influenced plasma concentrations of P4 in different ways, probably due to specific treatment-hormone interactions. However, all measurements fell within the range considered normal.

The aim of this study was to investigate the plasma concentrations of folic acid, vitamin B12 and progesterone at different stages of the sexual cycle and pregnancy, during induced abortion and in bitches with pyometra. Bitches (n = 97) were assigned to groups as follows: a) oestrous cycle (n = 42) b) pregnancy (n = 25) c) induction of abortion (n = 10 and d) pyometra (n = 20). Oestrous cycle stages were determined by vaginal inspection and cytology. Pregnancies were estimated by ultrasound (5.0 Mhz; linear transducer; Schimadzu) at days 15-25, 35-45 and 46-63 of pregnancy. Treatments for the induction of abortion were started between days 25 and 35 after mating (5 microg/kg cabergoline daily, Galastop; 5-10 microg/kg Alfaprostol every other day, Gabbrostim). Diagnosis of pyometra was confirmed by ultrasound and vaginoscopy. Folic acid and vitamin B12 concentrations did not differ among different stages of the oestrous cycle. The mean concentration of folic acid during early pregnancy (days 15-25) exceeded levels of later stages (days 46-63): 9.4 +/- 3.7 microg/ml and 4.7 +/- 1.8 microg/ml, respectively (p < 0.01). A positive correlation between folic acid and vitamin B12 was determined in pregnant dogs ( r = 0.925; p < 0.02). Before the induction of abortion, the concentration of folic acid was 9.6 +/- 5.2 microg/ml; during abortion it decreased to 5.0 +/- 3.2 microg/ml (p < 0.01). A significant correlation (r = 0.925; p < 0.02) between progesterone and folic acid was obtained in bitches with abortion. The mean concentration of folic acid in bitches with pyometra significantly differed from that of bitches at different stages of the oestrous cycle (p < 0.05). The mean concentration of folic acid was significantly lower in metoestrous bitches when compared to bitches with pyometra (p < 0.05). The decrease of serum concentrations of folic acid during pregnancy and induced abortion show that fetal growth and abortion caused higher consumption of folic acid. Concerning bitches did not show any deficiency symptoms, which is why it can be concluded that this decrease is physiological.