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.

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.

[Plasma concentrations of folic acid, vitamin B12 and progesterone of cyclic bitches, bitches during pregnancy and induced abortion and bitches with pyometra]. / Plasmakonzentrationen von Folsäure, Vitamin B12 und Progesteron während des Sexualzyklus, Gravidität, Abortinduktion und Pyometra bei der Hündin.

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.

A screening for the occurrence of autoreactive antibodies in sera of pregnant and non-pregnant bitches.

Sera of healthy pregnant (group I, n = 11) and non-pregnant (group II, n = 11) bitches were screened for autoantibodies (AAb). In both groups, blood samples were drawn every fifth day between days 5 and 55 after mating. Serum was analysed via indirect immunofluorescence (IIF) with the Canine ANA HEp-2 Screening Kit. In all animals, anticytoplasmic AAb were detected. Utilizing primate-heart substrate slides AAb against contractile proteins of the cytoplasm could be observed. The predominating fluorescence pattern in pregnant animals resembled above all desmin, which was proven via Western blot. The sera were then pre-incubated with tropomyosin, actin, vimentin, vinculin and keratin solutions, and assessed on HEp-2 slides and on human and canine fibroblasts as well. The latter substrate was used to verify whether the detected Ab were in fact AAb. Utilizing tropomyosin, revealed elimination of the cytoplasmic fluorescences on all three substrates. It is therefore assumed, that in sera of healthy dogs, AAb against contractile structure proteins of the cytoplasm are present regularly. The majority of pregnant bitches presented with higher end-point titres (EPT), than to be found in non-pregnant dogs. AAb against desmin played the key role in those patterns. In addition, sera were screened for thyroid specific AAb, namely thyroglobulin, thyroid peroxidase (TPO), T3 and T4, and for AAb against insulin by ELISA or Western blot (TPO). Only in two of the pregnant bitches a weak positive reaction (1:100) for T3-AAb was detected.

The activity of matrix metalloproteinase-2 and -9 in serum of pregnant and non-pregnant bitches.

The aim of this study was to investigate, whether the activity of matrix metalloproteinase (MMP)-2 and -9 in the serum of pregnant and non-pregnant bitches differs significantly. For this purpose, 81 blood samples were taken from pregnant bitches at days 5-13, 15-21, 24-31, 34-40 and 41-50 after mating, and 51 samples from non-pregnant animals at corresponding times. Relative enzyme activity, calculated as the percentage of serum enzyme activity on enzyme activity in a control sample, was determined with a commercially available assay after activation of serum MMPs with p-aminophenylmercuric acetate (APMA). In addition, serum oestradiol (E(2)) and progesterone (P(4)) concentrations were measured with an enzymeimmunoassay (EIA). In the pregnant bitches, at days 5-13 and 15-21 after mating, the mean activity of both MMPs was significantly higher than in non-pregnant animals (28.5% vs 24.5% and 27.7% vs 22.6%; p < 0.01). Moreover, in the pregnant bitches, significant correlations were detected between the serum enzyme activity and the serum concentrations of E(2) (-0.900; p < 0.05) and P(4) (+0.667; p < 0.05).

[Comparison of various examination methods used in ovarian diagnostics in cattle]. / Vergleich verschiedener Untersuchungsmethoden bei der Ovardiagnostik des Rindes.

In this study diagnostic certainty of ultrasonography and rectal palpation concerning the detection of follicles and C.I. was compared by evaluation of the findings obtained with ultrasonography in waterbath and dissection of the ovaries after slaughter. Clinical examinations were performed on a total of 30 cows (transrectally and ultrasonographically, 5.0 mhz, linear) in slaughterhouse. In the laboratory ovaries were evaluated after slaughter both macroscopically and by ultrasonography in waterbath. Diagnostic reliabilities of these methods were compared. No difference between the methods was determined concerning the longitudinal measurements of corpora lutea (19.96 +/- 4.83 mm, 20.41 +/- 5.41 mm, 21.45 +/- 5.26 mm by ultrasonography, waterbath and macroscopy respectively). By means of determining the correct identification of corpora lutea, the error rate was 24.1% and 17.2% for rectal palpation and ultrasonography respectively. The comparison of rectal palpation and macroscopy showed that three small corpora lutea and two corpora lutea with small cavity were determined wrongly as small follicles and two corpora lutea were determined whereas they were not present actually. With ultrasonography four small C.I. could not be detected and one C.I. with cavity was wrongly determined as follicle. It was noticed that follicles bigger than 10 mm (F2 = 10-15 mm, F3 = 16-20 mm) could be determined more accurately by means of ultrasonography than by rectal palpation (with ultrasonography: F2 = 90.48%, F3 = 100.0%; with rectal palpation, F2 = 61.9%, F3 = 200.0%). The correlation of the findings of rectal palpation or ultrasonography and blood progesterone levels was 86.2% and 89.7% respectively. This accordance was 96.6% for progesterone levels and waterbath and macroscopic findings.

Use of exfoliative cytology for diagnosis of transmissible venereal tumour and controlling the recovery period in the bitch.

Transmissible venereal tumour (TVT) is a rare vaginal tumour that can be treated surgically or cryosurgically as well as by radiotherapy or chemotherapy. Vincristine has been found to be very effective for treating TVT. Since vaginal secretion or discharge may contain neoplastic cells, the cytological identification of TVT cells is possible. The present study was carried out in 12 bitches. Vaginal smears were obtained with cotton swab from the anterior vagina and TVT suspected structures. The smears were stained according to Papanicolaou and assessed by light microscopy. Additionally the general condition of the patients was evaluated by haematological and radiographic examinations. In bitches with TVT vincristine sulphate was administered intravenously at weekly intervals. The total treatment period was three to six weeks until no atypical cells were found in the smear. This was the case after an average of 3.2 +/- 1.3 applications. Tumour masses became smaller and by this non-visible from the rima vulva after 4.2 +/- 0.7 applications. During the treatment, two of the 12 bitches (16.7%) suffered from vomiting and diarrhoea while three (25%) showed neutropenia. Twelve months after completion of treatment, the bitches were examined again and vaginal smears were taken in order to control the recovery process or a possible recurrence of TVT. No atypical cells were found in any vaginal smear. By this exfoliative cytology has proved to be a safe and easy method for TVT diagnosis and for observing the recovery process.