Ali Reha Ağaoğlu

Time related changes in luteal prostaglandin synthesis and steroidogenic capacity during pregnancy, normal and antiprogestin induced luteolysis in the bitch

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.

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.

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 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.

Uterine responses to early pre-attachment embryos in the domestic dog and comparisons with other domestic animal species

Abstract In the dog, there is no luteolysis in the absence of pregnancy. Thus, this species lacks any antiluteolytic endocrine signal as found in other species that modulate uterine function during the critical period of pregnancy establishment. Nevertheless, in the dog an embryo-maternal communication must occur in order to prevent rejection of embryos. Based on this hypothesis, we performed microarray analysis of canine uterine samples collected during pre-attachment phase (days 10-12) and in corresponding non-pregnant controls, in order to elucidate the embryo attachment signal. An additional goal was to identify differences in uterine responses to pre-attachment embryos between dogs and other mammalian species exhibiting different reproductive patterns with regard to luteolysis, implantation, and preparation for placentation. Therefore, the canine microarray data were compared with gene sets from pigs, cattle, horses, and humans. We found 412 genes differentially regulated between the two experimental groups. The functional terms most strongly enriched in response to pre-attachment embryos related to extracellular matrix function and remodeling, and to immune and inflammatory responses. Several candidate genes were validated by semi-quantitative PCR. When compared with other species, best matches were found with human and equine counterparts. Especially for the pig, the majority of overlapping genes showed opposite expression patterns. Interestingly, 1926 genes did not pair with any of the other gene sets. Using a microarray approach, we report the uterine changes in the dog driven by the presence of embryos and compare these results with datasets from other mammalian species, finding common-, contrary-, and exclusively canine-regulated genes. Summary Sentence Pre-implantation embryos invoke functional changes in the canine uterus related to ongoing structural remodeling and immunological modulation; comparisons with different mammals reveal similarities and differences in maternal pregnancy recognition.

Tarantula cubensis extract alters the degree of apoptosis and mitosis in canine mammary adenocarcinomas

In the present study, 13 clinical cases of canine mammary adenocarcinoma were evaluated in order to understand the effect of Tarantula cubensis extract (TCE) on tumor tissue. Punch biopsies were taken from the tumors before treatment with TCE. Subcutaneous injections of TCE were administered three times at weekly intervals (3 mL per dog). Between days 7 and 10 after the third injection, the tumor masses were extirpated by complete unilateral mastectomy. Pre- and post-treatment tumor tissues were immunohistochemically assessed. The expression of B-cell lymphoma 2 (Bcl-2) was found to be higher in pre-treatment compared to post-treatment tissues (p < 0.01) whereas Ki-67 expression was lower in post-treatment tissues (p < 0.01). No significant differences in fibroblast growth factor or vascular endothelial growth factor expression were observed between pre- and post-treatment tissues (p > 0.05). The apoptotic index was determined to be low before treatment and increased during treatment. These results suggest that TCE may be effective for controlling the local growth of canine mammary adenocarcinoma by regulating apoptosis.

The Effects of Separate and Combined Use of PGF2α and GnRH Hormones and the Addition of Βeta-Carotene on Fertility Parameters in Dairy Cows with Ovarian Cysts

Background: Ovarian cysts are commonly observed pathologies, which interfere with normal cyclic activity and adversely affect fertility in cows. Beta-carotene is effective in the reduction of reproductive problems by inducing the natural defence mechanisms of the body. There are several methods that can be used for the treatment of ovarian cysts. The separate and combined use of GnRH and PGF2α commonly uses in the treatment of ovarian cysts. Therefore, in the presented study the effects of Beta-carotene (βC) addition for the treatment of ovarian cysts either with GnRH solely or GnRH and PGF2α in combination on the fertility parameters of dairy cows were investigated. Materials, Methods & Results: Seventy-six Holstein Friesian cows having ovarian cysts diagnosed by ultrasonography (USG) were divided into three groups. Cows in Group I (GI, n = 27), were injected with GnRH (Buserelin acetate, 5 mL, im), PGF2α (Tiaprost-trometamol, 5 mL, im) and βC (20 mL/cow, into 4 regions by im route). In Group II (GII, n = 25) GnRH (Buserelin acetate, 5 mL, im) and PGF2α (Tiaprost-trometamol, 5mL, im) were administrated while GnRH (Buserelin acetate, 5 mL, im) solely in Group III (GIII, n = 24). Cysts were monitored via USG, and blood samples were collected on the on day of treatment (day 0) and on the 7th and 14th days following the administrations. Cows shoving oestrous were inseminated and pregnancy diagnoses were performed on the 40th day following insemination. Treatment results showed that there were statistically no significant differences between GI and GII (P > 0.05). Only numerical difference obtained in time from therapy to pregnancy and overall pregnancy index (P > 0.05). Overall pregnancy rate (85 %), first service pregnancy rates (40 %) and overall pregnancy index (2.11) in GI were found significantly higher than GIII (53.3 %; 20 %; 4.12) [P 0.05). It was found that βC administrations significantly increased βC levels in GI than GII and GIII on the 7th and 14th days (P < 0.05). Discussion: One of the most common problems encountered in modern dairy production is the development of ovarian cysts. Treatments for ovarian cyst are numerous and variable, and have changed considerably over the years. In the present study, GnRH and PGF2α were administered together as a combination, and as a result of this combined use, higher percentages were obtained for both pregnancy rate and fertility parameters in GI and GII, in comparison to the group administered with GnRH alone (GIII). Better outcome from combination therapy (GnRH and PGF2α) may be due to the fact that luteal thickening in the walls of cysts was determined by ultrasonography, but P4 values were not identified immediately and the treatments were not categorized in accord with these values. No statistically significant differences were determined between GI and GII concerning the fertility parameters investigated, however numerical and proportional differences were observed. βC levels were significantly higher on day 7 and 14 after treatment in GI which were administered βC additionally to the treatment protocol for ovarian cysts. This statistical difference suggests that administration of βC in combination therapy is also effective in the treatment of ovarian cysts. In conclusion, it was determined that high pregnancy rates were obtained by the combined treatment of ovarian cysts (GnRH + PGF2α) and number of inseminations per conception were at desired limits. Better percentile and numerical fertility parameters were achieved in the group, which additionally received βC, high numbers of infertility cases, βC supplementation could be a viable option for treatment.

Gene expression profiles of some cytokines, growth factors, receptors, and enzymes (GM-CSF, IFNγ, MMP-2, IGF-II, EGF, TGF-β, IGF-IIR) during pregnancy in the cat uterus

Early pregnancy is one of the most critical periods of pregnancy, and many factors such as cytokines, enzymes, and members of the immune system have to cooperate in a balanced way. In the present study, the gene expression profiles of factors associated with pregnancy such as EGF, transforming growth factor beta, granulocyte-macrophage colony-stimulating factor, interferon gamma, insulin-like growth factor 2, insulin-like growth factor 2 receptor, and matrix metalloproteinase 2 were analyzed in uterine tissues of female cats. The cats were assigned to five groups: G1 (embryo positive, n = 7; 7th day after mating), G2 (after implantation, n = 7; 20th day after mating), G3 (midgestation, n = 7; 24-25th day after mating), G4 (late gestation, n = 7; 30-45th day after mating), G5 (oocyte group, n = 7; 7th day after estrus). Tissue samples from the uterus and placenta were collected after ovariohysterectomy. Relative messenger RNA levels were determined by real-time polymerase chain reaction. All the factors examined were detected in all tissue samples. In the course of pregnancy, significantly higher expression of EGF and matrix metalloproteinase 2 in G2 than in G1 was observed (P < 0.05). Insulin-like growth factor 2 expression was higher in all groups than in G1 (P < 0.05). Upregulation of EGF during implantation was detected. The expression of interferon gamma was significantly higher in G3 than in G1 (P < 0.05). Transforming growth factor beta and granulocyte-macrophage colony-stimulating factor were constantly expressed in all groups. In conclusion, the expressions of these factors in feline uterine tissue at different stages of pregnancy might indicate that these factors play roles in the development of pregnancy such as trophoblast invasion, vascularization, implantation, and placentation.