Halit Kanca

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.

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 β (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.

Vascular endothelial (VEGF) and epithelial growth factor (EGF) as well as platelet-activating factor (PAF) and receptors are expressed in the early pregnant canine uterus.

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.

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 and Activity of Matrix Metalloproteinases in the Uterus of Bitches After Spontaneous and Induced Abortion

Aim of this study was to determine the intrauterine activity of matrix metalloproteinases (MMP)-2 and -9 after cessation of the local effect of progesterone. For this purpose, pregnancy was terminated in 10 bitches at mid-gestation with the progesterone receptor antagonist aglepristone (10 mg/kg body weight, sc, Alizine®; Virbac, France) at two subsequent days (group IRA = induced resorption/abortion). The IRA group was divided into two subgroups (Group I, n = 5, days 25-35 of pregnancy; group II, n = 5, days 36-45). Five further bitches were introduced with beginning abortion (group SRA = spontaneous resorption/abortion). Seven healthy bitches between day 25 and 45 of gestation served as controls. After ovariohysterectomy at the end of abortion and between days 25 and 45 of gestation, respectively, the distribution and activity of collagenases were investigated by immunohistochemistry and gelatin zymography. At placental sites, MMP-2 activity in the endometrium was significantly lower in IRA groups than in the SRA group (33.7 ± 11.8% and 39.3 ± 5.4% vs 52.2 ± 10.2%, p < 0.05); however, MMP-2 expression was lowest in the control group (control: 21.4 ± 6.3%; p < 0.01) and similarly in the myometrium (controls: 13.1 ± 2.5%; p < 0.05). MMP-9 activity was also lower in the endometrium and myometrium of the control group in comparison to SRA and IRA groups (11.8 ± 3.2%; p < 0.01 and 28.4 ± 32.8%; p < 0.05). At interplacental sites, the amount of active collagenases in the myometrium was significantly lower in the control group. It is concluded that the blockade of the biological progesterone effect was associated with an increase in activity of both collagenases.

Expression of MHC-I and -II in Uterine Tissue from Early Pregnant Bitches

The aim of the study was to investigate the expression of major histocompatibility complex (MHC)-I and -II in uterine tissues from pregnant and non-pregnant bitches, taken at different time periods after mating. The pregnant bitches were ovariohysterectomized during the pre-implantation (group 1, n = 4), implantation (group 2, n = 7) and placentation stage (group 3, n = 7). Non-pregnant animals in diestrus served as controls (group 4, n = 7). The expression of MHC- I and -II in salpinx, apex, middle horn, corpus uteri and at implantation sites was investigated by immunohistochemistry as well as qualitative and quantitative RT-PCR; MHC-I mRNA was detected in all tissues and with quantitative RT-PCR, and no significant changes were detected until placentation. Immunohistologically, at the apex and corpus site, the average number of MHC-II positive cells increased from the pre-implantation to the post-implantation stage (apex: 1.54 +/- 1.21 to 3.82 +/- 2.93; corpus: 1.62 +/- 1.9 to 5.04 +/- 4.95; p < 0.05). The greatest numbers of MHC-II positive cells were observed at placentation sites (6.64 +/- 5.9). In parallel, a marked increase in the relative mRNA expression of MHC-II in uterine tissues was assessed from the pre-implantation to the placentation stage (relative to Glycerinaldehyd-3-phosphate-Dehydrogenase (GAPDH): 6.9 +/- 9.5, 8.4 +/- 5.8, p > 0.05). Immunohistologically, in the salpinx, significantly greater numbers of MHC-II positive cells were found in the tissues of pregnant animals than in the control group (p < 0.05). It is proposed that the increase in MHC-II is pregnancy-related, even though the impact on maintenance of canine pregnancy is still unclear.

Prostaglandin F receptor expression in intrauterine tissues of pregnant rats

In this investigation, we studied the expression and localization of rat prostaglandin F (FP) receptor in uterine tissues of rats on gestational Days 10, 15, 18, 20, 21, 21.5 and postpartal Days 1 and 3 using Western blotting analysis, real-time PCR, and immunohistochemistry. A high level of immunoreactivity was observed on gestational Days 20, 21, and 21.5 with the most significant signals found on Day 20. FP receptor protein was expressed starting on gestational Day 15, and a fluctuating unsteady increase was observed until delivery. Uterine FP receptor mRNA levels were low between Days 10 and 18 of gestation (p < 0.05). The transcript level increased significantly on Day 20 and peaked on Day 21.5 just before labor (p < 0.05). There was a positive correlation between FP receptor mRNA expression and serum estradiol levels (rs = 0.78; p < 0.01) along with serum estradiol/progesterone ratios (rs = 0.79; p < 0.01). In summary, we observed an increase FP receptor expression in rat uterus with advancing gestation, a marked elevation of expression at term, and a concominant decrease during the postpartum period. These findings indicate a role for uterine FP receptors in the mediation of uterine contractility at term.

Endometrial echotexture parameters in Turkish Saanen Goats (Akkeci) during oestrus and early pregnancy.

This experiment was conducted to evaluate endometrial echotexture in oestrus and early pregnancy and its association with ovarian hormones and foetal count in goats. Akkeci goats (Saanen×Kilis crossbreed, n=40) were randomly divided into two groups. Ten does (NAT) were mated on natural oestrus and 30 does (SYN) were subjected to synchronisation-prior to mating. The uterus was scanned on the days of sponge insertion (d -14), sponge removal (d -2) and mating (d 0) as well as 17 (d 17) and 30 (d 30) days after mating. Mean gray level (MGL), homogeneity (HOM) and contrast (CON) values were calculated. Blood samples were collected on days ultrasonography was performed. Data were analyzed by Chi-square, ANOVA, regression tests. HOM value reached the highest level on the mating day and then continuously decreased (P<0.0001). Overall, HOM values were greater for SYN does than for NAT does after mating. CON values were virtually stable during the experimental period. MGL value fluctuated during the breeding period (P<0.03) at a similar fashion in NAT and SYN does. Foetal count was not correlated with plasma hormones and echotexture parameters. Plasma progesterone concentration was correlated with echotexture parameters (r=-0.28 for HOM; r=0.29 for CON; r=0.25 for MGL; P<0.05 for all) during post-mating. In conclusion, echotexture parameters changed during the breeding period, in association with plasma progesterone concentration. Future studies should test if the echotextural changes during embryonic fixation days can be used as a marker for early detection of pregnancy in does.

Expression of iNOS, COX-2 and VEGF in canine mammary tumours and non-neoplastic mammary glands: Association with clinicopathological features and tumour grade

The aim of the present study was to investigate the relationship between the expression of iNOS, COX-2 and VEGF mRNA levels and malignancy degree in canine malignant mammary tumours. Thirty-five bitches presented with the complaint of mammary masses, aged 6-10 years and representing different breeds, were used. The expressions of iNOS, COX-2 and VEGF mRNA levels were significantly higher in both benign and malignant tumours than in the adjacent nonneoplastic mammary glands (P < 0.05). The iNOS, COX-2 and VEGF mRNA expression levels of grade 2 tumours were higher than those of grade 1 tumours; however, the highest expression levels were detected in grade 3 tumours. Thus, increased iNOS, COX-2 and VEGF gene mRNA levels were found to be related with the histological grade of malignancy in dogs with mammary tumours.

Is apoptosis a regulatory mechanism during early canine pregnancy

Fas is a membrane-bound protein which upon activation causes programmed cell death. Fas ligand (FasL) binds Fas on target cells. Both these factors are known to regulate apoptosis at implantation in different species and thus might be involved in the regulation of implantation in dogs. The aim of the study was to assess the expression of Fas and FasL in canine uterine tissue throughout pregnancy as well as in pre-implantation embryos using RT-PCR and RT-qPCR. Uterine tissues was collected from of 21 healthy pregnant bitches (group I: days 10-12, n = 5; group II: days 18-25, n = 6; group III: days 28-45, n = 6) and from 4 non-pregnant bitches (controls: days 10-12). Pregnancy stage was determined by days after mating, that is, 2-3 days after ovulation as determined by vaginal cytology and progesterone measurement. After ovariohysterectomy, uteri from group I bitches were flushed with PBS and the embryos washed and stored frozen at -80°. Tissues from the other groups were taken from the implantation and placentation sites, respectively, covered with Tissue Tek(®) and frozen at -80°. Extraction of RNA was performed with Trizol Reagent and RT-qPCR using SYBR green probes. In pre-implantation embryos, only FasL but not Fas could be detected. In all tissues from pregnant and non-pregnant bitches, both parameters were detectable. Before implantation (group I) expression of FasL resembled that of non-pregnant bitches in early dioestrus and decreased significantly during implantation and thereafter (p < 0.05). Expression of Fas did not change significantly until day 45. The relative expression of Fas exceeded that of FasL at each stage investigated, which is comparable to observations of other species; however, high standard deviations indicate high individual differences. These preliminary results point towards a regulatory function of the Fas/FasL system during early canine pregnancy.