I. Kucukaslan

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.

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.

The intravaginal application of misoprostol improves induction of abortion with aglepristone

The aim of the present study was to compare the clinical and endocrinological effects of four different treatments for the induction of abortion in bitches. For this purpose, 28 pregnant bitches between days 25 and 35 of gestation, were randomly assigned to four groups. In group I (n = 7), only aglepristone (AGL, 10mg/kg bw, two injections 24 h apart, s.c.) was administered. In group II (n = 7), AGL (as in group I), cabergolin (CAB, 5 μg/kg, daily p.o., until completion of abortion) and misoprostol (MIS, 200 μg for bitches with ≤ 20 kg bw, 400 μg for bitches with > 20 kg bw, daily intravaginally, until completion of abortion) were administered. In group III (n = 7), AGL (as in group I) and MIS (as in group II) were administered. In group IV (n = 7) AGL, (as in group I) and cloprostenol (CLO, 1μg/kg bw, s.c., two injections 24 h apart with the AGL injections) were combined. In all groups, bitches were examined daily, clinically and ultrasonographically to monitor resorptions/abortions. To measure serum progesterone (P4) and total estrogen (TE) concentrations, blood samples were collected in all groups immediately after the first AGL administration and every other day until completion of abortion. No statistical differences were found between groups concerning the duration until completion of abortion following treatment (n.s.); however, in Group III, 6 d after the start of treatment all pregnancies were terminated whereas in Group I, II and IV, only 57.1% (4/7), 85.7 % (6/7) and 42.8 % (3/7) of pregnancies were terminated. In the latter groups, all pregnancies were terminated between days 8 and 10 after the start of treatment. In Group IV, P4 concentrations on days two and one before the beginning of abortion and the day the abortion started was significantly lower than in the other groups (P < 0.01). No statistical differences were found between groups for TE concentrations (P > 0.05). In Groups I, II and III, no severe side effects occurred. Severe vomiting after each treatment and until the end of abortion was observed in Group IV only. In conclusion, only when a combination of AGL and MIS was used abortion was completed within 6 d in all bitches whereas the additional use of CAB did not improve the treatment.

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.

Immunolocalization of vascular endothelial growth factor, its receptors (flt1/fms, flk1/KDR, flt4) and vascular endothelial growth inhibitor in the bitch uterus during the sexual cycle

Angiogenesis is regulated by proangiogenic and antiangiogenic factors. Vascular endothelial growth factor (VEGF) is a prime proangiogenic regulator, whereas vascular endothelial growth inhibitor (VEGI) is a specific antiangiogenic cytokine. To clarify temporal changes in the localization of pro-angiogenic and anti-angiogenic factors in the uterus of normal bitches during the proestrus, estrus, diestrus and anestrus phases of the estrous cycle, the expressions of VEGF and its receptors (flt1/fms, flk1/KDR and flt4) and their correlation with VEGI were analyzed using immunohistochemistry. Uteruses were collected after ovariohysterectomy. Immunohistochemical staining was evaluated semi-quantitatively by an immunohistochemical total score consisting of the sum of the intensity and proportional scores. The results in the bitch uterus demonstrated that positive immunohistochemical staining was found exclusively in the cytoplasm and apical membrane of luminal and glandular epithelial, stromal and smooth muscle cells and nuclear staining was observed in the flt1/fms, flk4 and VEGI during proestrous and estrous. Semi-quantitative analyses revealed that the total score for VEGF in the glandular epithelial cells was significantly higher than that of luminal, endometrial stromal and myometrial smooth muscle cells during proestrous (p<0.05). The total score for flk1/KDR and flt4 in the glandular epithelium was also significantly higher than that of endometrial stromal cells during proestrous, whilst the total score for flt1/fms in the glandular epithelium was significantly higher than that of endometrial stromal cells during anestrus (p<0.05). We conclude that, in the bitch uterus, cyclic changes may be precisely regulated by the combined functions of VEGF family members, angiogenic VEGF and VEGF receptors, and the angiogenesis inhibitor VEGI.

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.

The expression of epidermal growth factor receptors and their ligands (epidermal growth factor, neuregulin, amphiregulin) in the bitch uterus during the estrus cycle

In order to study the possible role of EGFR receptors in the bitch reproductive process, we have analyzed the expression pattern and localization of EGFR receptors and some of their ligands epidermal growth factor (EGF), neuregulin (NRG), amphiregulin (AREG), in the uterus during the estrus cycle using immunohistochemistry. The immunostaining for receptors and ligands of EGFR/ligand system was confined to membrane and cytoplasm of the target cells. Variations were observed, not only at the different stages of the estrous cycle, but also in the different tissue compartments of the uterus. However, it was detected that the immunostainings for NRG and AREG in the different cells do not show important differences at stages of the estrus cycle. In the luminal epithelium, strong immunostaining for ErbB1/HER1, ErbB2/HER2, ErbB4/HER4 and EGF was found at estrus. In the glandular epithelium, strong immunostaining for ErbB4/HER4 was observed at diestrus, while strong immunostaining for EGF was detected in both of estrus and diestrus. ErbB3/HER3 immunoreactivity in the stromal cells was higher at diestrus and anestrus, while ErbB4/HER4 immunoreactivity was lower at anestrus. In the myometrium, the highest levels of immunoreactivity of ErbB2/HER2 were found at estrus, while ErbB3/HER3 immunoreactivity was higher at anestrus. EGF immunoreactivity was lower at anestrus compared to other stage of cycle. Altered EGFR/ligand system expression during the estrus cycle suggests this growth factor system is a potent regulator of proliferation and differentiation events during preparation for implantation of bitch uterus.

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.

Quantitative Software Analysis of Ultrasonographic Textures in Experimental Testicular Torsion

AIM Ultrasonography (US) has high diagnostic value in testicular torsion but is vulnerable to several potential errors, especially in the early period. Echotexture (ETX) analysis software provides a numerical expression of B-mode images and allows quantitative evaluation of blood flow due to ischemic damage using power Doppler US (PDUS) analysis. Our aim in this study was to determine the diagnostic value and effective parameters of EXT analysis software in the early period of torsion using B-mode and PDUS images. MATERIALS AND METHODS In this study, eight rats were used. Following anesthesia, the right testis was rotated to a 1080-degree counterclockwise position whereas the left testis was left in place to have a control group. B-mode and PDUS images of both sides were recorded with a portable US device immediately (0 hour) and 1 and 2 hours after torsion. The B-mode images were analyzed in terms of gradient, homogeneity, and contrast using the BS200pro software (BAB Digital Imaging System 2007, Ankara, Turkey). Intensity (I)-red and area (A)-red values were measured on PDUS images with the Pixelflux (Version 1.0, Chameleon-Software, Leipzig, Germany). The data were evaluated by the Mann-Whitney U and Wilcoxon tests. RESULTS Data from B-mode US image EXT analysis showed no significant difference between the right and left testicles in 0 to 2 hours (p > 0.05). The values obtained from PDUS analysis (I-red and A-red) significantly decreased at the testicular torsion side at the end of the second hour (p < 0.05). I-red and A-red values at 0 to 1 hour of torsion indicated similar blood flow alterations (p > 0.05) whereas the flow was significantly lower at 2 hours (p < 0.05). CONCLUSION In experimental testicular torsion, ischemic changes can be detected by PDUS power/angio mode using blood flow alterations as early as the second hour. Tissue damage cannot be evaluated within the first 2 hours of torsion with B-mode ETX analysis.