Utero-placental expression and functional implications of HSD11B1 and HSD11B2 in canine pregnancy†.

Glucocorticoids modulate the feto-maternal interface during the induction of parturition. In the dog, the prepartum rise of cortisol in the maternal circulation appears to be erratic, and information about its contribution to the prepartum luteolytic cascade is scarce. However, the local placental upregulation of glucocorticoid receptor (GR/NR3C1) at term led to the hypothesis that species-specific regulatory mechanisms might apply to the involvement of cortisol in canine parturition. Therefore, here, we assessed the canine uterine/utero-placental spatio-temporal expression of hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1; reduces cortisone to cortisol), and -2 (HSD11B2; oxidizes cortisol to the inactive cortisone). Both enzymes were detectable throughout pregnancy. Their transcriptional levels were elevated following implantation, with a strong increase in HSD11B2 post-implantation (days 18-25 of pregnancy), and in HSD11B1 at mid-gestation (days 35-40) (P < 0.05). Interestingly, when compared pairwise, HSD11B2 transcripts were higher during post-implantation, whereas HSD11B1 dominated during mid-gestation and luteolysis (P < 0.05). A custom-made species-specific antibody generated against HSD11B2 confirmed its decreased expression at prepartum luteolysis. Moreover, in mid-pregnant dogs treated with aglepristone, HSD11B1 was significantly higher than -2 (P < 0.05). HSD11B2 (protein and transcript) was localized mostly in the syncytiotrophoblast, whereas HSD11B1 mRNA was mainly localized in cytotrophoblast cells. Finally, in a functional approach using placental microsomes, a reduced conversion capacity to deactivate cortisol into cortisone was observed during prepartum luteolysis, fitting well with the diminished HSD11B2 levels. In particular, the latter findings support the presence of local increased cortisol availability at term in the dog, contrasting with an enhanced inactivation of cortisol during early pregnancy.

Luteal expression of factors involved in the metabolism and sensitivity to oestrogens in the dog during pregnancy and in non-pregnant cycle.

The canine corpus luteum (CL) is the main source of reproductive steroids during dioestrus in the dog and remains active even in the absence of pregnancy (non-pregnant dioestrus, physiological pseudopregnancy). Whereas the biological effects of 17ß-oestradiol (E2) in the canine CL remain unclear, the transcriptional availability of oestrogen receptors, ESR1 and ESR2, as well as other modulators of local availability of E2, for example, HSD17B7 (converts oestrone into oestradiol), SULT1E1 (inactivates E2 binding capacity to its own receptors through sulphonation) and STS (reverts E2 sulphonation), were previously detected in the CL of non-pregnant bitches. The aim of the present work was to evaluate the mRNA amounts of these factors involved in luteal sensitivity and metabolism of E2 in the canine CL during the course of non-pregnant dioestrus (days 10, 20, 30, 40, 50 and 60 post-ovulation, n = 5/group) and at different stages of pregnancy (n = 4-6/group): pre-implantation (days 8-12), post-implantation (days 18-25), mid-gestation (days 35-40) and prepartum luteolysis. During pregnancy, the availability of ESR1, HSD17B7, SULT1E1 and STS decreased from mid-pregnancy to prepartum luteolysis. The main findings during non-pregnant dioestrus were as follows: increased ESR2:ESR1 ratio on days 40 and 50 after ovulation, decreasing during luteal regression (day 60); increased STS at day 30 when SULT1E1 levels decreased; increased availability of SULT1E1 transcripts during luteal regression; and decreased amounts of HSD17B7 mRNA in early dioestrus, increasing towards later stages. These results suggest that E2 signalling and biologically active local concentrations could diverge in response to time and pregnancy status of the bitch.

Determination of novel reference genes for improving gene expression data normalization in selected canine reproductive tissues - a multistudy analysis.

BACKGROUND: Real time RT-PCR (qPCR) is a useful and powerful tool for quantitative measurement of gene expression. The proper choice of internal standards such as reference genes is crucial for correct data evaluation. In female dogs, as in other species, the reproductive tract is continuously undergoing hormonal and cycle stage-dependent morphological changes, which are associated with altered gene expression. However, there have been few attempts published so far targeted to the dog aimed at determining optimal reference genes for the reproductive organs. Most of these approaches relied on genes previously described in other species. Large-scale transcriptome-based experiments are promising tools for defining potential candidate reference genes, but were never considered in this context in canine research. RESULTS: Here, using available microarray and RNA-seq datasets derived from reproductive organs (corpus luteum, placenta, healthy and diseased uteri) of dogs, we have performed multistudy analysis to identify the most stably expressed genes for expression studies, in each tissue separately and collectively for different tissues. The stability of newly identified reference genes (EIF4H, KDELR2, KDM4A and PTK2) has been determined and ranked relative to previously used reference genes, i.e., GAPDH, ß-actin and cyclophillin A/PPIA, using RefFinder and NormFinder algorithms. Finally, expression of selected target genes (luteal IL-1b and MHCII, placental COX2 and VEGFA, and uterine IGF2 and LHR) was re-evaluated and normalized. All proposed candidate reference genes were more stable, ranked higher and introduced less variation than previously used genes. CONCLUSIONS: Based on our analyses, we recommend applying KDM4A and PTK2 for normalization of gene expression in the canine CL and placenta. The inclusion of a third reference gene, EIF4H, is suggested for healthy uteri. With this, the interpretation of qPCR data will be more reliable, allowing better understanding of canine reproductive physiology.

First detection of heat shock protein 60 and 70 in the serum of early pregnant bitches.

Heat shock proteins (HSPs) belong to a group of cellular stress proteins. Heat shock protein 10 immunoregulates and promotes growth during early gestation in humans, while HSP70 is considered to regulate autophagy and apoptosis during pregnancy and parturition. Both HSPs are detectable in the serum and placentas of early pregnant women and considered to contribute to the establishment of pregnancy. Within this pilot study we aimed (1) to assess whether HSPs 10, 60 and 70 are measurable in the serum of healthy early pregnant and non-pregnant bitches, and (2) to explore whether measurable differences between groups indicate pregnancy. Blood was collected from 31 bitches on days 7, 14 and 21 after mating. At 21 days post mating, all bitches were examined for pregnancy by ultrasonography; 23 were pregnant, and the eight non-pregnant bitches served as controls. Pregnant bitches had normal parturitions and gave birth to healthy puppies. The serum concentrations of HSPs 10, 60 and 70 were measured by electrophoresis and western blot. Serum HSP10 was not detectable. Average serum HSP70 concentration was significantly (d7, P = 0.030; d14, P = 0.023; d21, P = 0.030) lower in pregnant animals at all days investigated, while serum HSP60 was significantly lower at day 21 of gestation (P = 0.024) when compared to the controls. HSP 60 and HSP70 concentrations correlated positively (d7, r = +0.386, P = 0.021; d14, r = 0.450, P = 0.008; d21, r = +0.472, P = 0.006). We conclude that in pregnant bitches, serum concentrations of HSP60 and HSP70 are significantly decreased between days 7 and 21 of gestation, in comparison to non-pregnant bitches in early dioestrus, raising the question about intrauterine functions during the peri-implantation period.

Uterine and placental distribution of selected extracellular matrix (ECM) components in the dog.

For many years, modifications of the uterine extracellular matrix (ECM) during gestation have not been considered as critical for successful canine (Canis lupus familiaris) pregnancy. However, previous reports indicated an effect of free-floating blastocysts on the composition of the uterine ECM. Here, the expression of selected genes involved in structural functions, cell-to-cell communication and inhibition of matrix metalloproteinases were targeted utilizing qPCR and immunohistochemistry. We found that canine free-floating embryos affect gene expression of FN1, ECM1 and TIMP4 This seems to be associated with modulation of trophoblast invasion, and proliferative and adhesive functions of the uterus. Although not modulated at the beginning of pregnancy, the decrease of structural ECM components (i.e. COL1, -3, -4 and LAMA2) from pre-implantation toward post-implantation at placentation sites appears to be associated with softening of the tissue in preparation for trophoblast invasion. The further decrease of these components at placentation sites at the time of prepartum luteolysis seems to be associated with preparation for the release of fetal membranes. Reflecting a high degree of communication, intercellular cell adhesion molecules are induced following placentation (Cx26) or increase gradually toward prepartum luteolysis (Cx43). The spatio-temporal expression of TIMPs suggests their active involvement in modulating fetal invasiveness, and together with ECM1, they appear to protect deeper endometrial structures from trophoblast invasion. With this, the dog appears to be an interesting model for investigating placental functions in other species, e.g. in humans in which Placenta accreta appears to share several similarities with canine subinvolution of placental sites (SIPS). In summary, the canine uterine ECM is only moderately modified in early pregnancy, but undergoes vigorous reorganization processes in the uterus and placenta following implantation.

Pre-pubertal treatment with a GnRH agonist in bitches-Effect on the uterus and hormone receptor expression.

Aim of the study was to examine the effect of deslorelin on uterine tissues of eleven pre-pubertal bitches aged 4.2 ± 0.6 m. Implants containing placebo (sodium chloride 0.9%; n = 4, G I), 4.7 mg (n = 3, GII) or 9.4 mg (n = 4, GIII) deslorelin acetate (Suprelorin® ; Virbac, France), were administered subcutaneously. Signs of oestrus, vaginal cytology, serum progesterone (P4) and estradiol-17ß (E2) concentrations were monitored until the occurrence of oestrus. Bitches were ovariohysterectomized and sections from the uterine tissue were subjected to immunohistochemistry (IHC) for detection of GnRH receptor (R), Kisspeptin (KP)10, Kisspeptin receptor (GPR54), androgen receptor (AR), oestrogen receptor (ER) α,ß, and progesterone receptor (PR). Tissue sections were scored semi-quantitatively using an immunoreactivity score (IRS) ranging from 0 to 300 (3). Since some animals were ovariohysterectomized before puberty (n = 1 from GII and n = 2 from GIII), and some in metestrus (all controls and 2 from GII and GIII each), results from these animals were separately evaluated and compared to the controls. Results: No abnormalities were seen in uterine tissues. Kisspeptin 10 expression was low in all cell types, highest IRS were seen in the vascular endothelial cells. The GPR54 was mainly detected in the luminal epithelial cells, superficial and deep uterine glands. The expression of GPR54 and ERα,ß was especially high in bitches operated prepubertally. No difference was observed between the controls and experimental bitches operated in their first metestrus. The PR and ERα,ß were exclusively expressed in superficial and deep uterine glands and luminal surface epithelial cells. The AR and GnRH-R expression was negative in all cells of all groups. We conclude that application of 4.7 or 9.4 mg deslorelin at the age of 4 months did not cause uterine disturbances. GPR54 expression might be influenced by pre-pubertal deslorelin treatment or the changings related to approaching puberty; the latter is supposed in case of ERα,ß.

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

In the dog, there is no luteolysis in the absence of pregnancy. Thus, this species lacks any anti-luteolytic 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.

Functional implications of the utero-placental relaxin (RLN) system in the dog throughout pregnancy and at term.

Relaxin (RLN) is a key hormone of pregnancy in mammals best known for its involvement in connective tissue remodeling. In the domestic dog, placental RLN is the only known endocrine marker of pregnancy. However, knowledge is sparse regarding the spatio-temporal expression of RLN and its receptors (RXFP1 and RXFP2) in the canine uterus and placenta. Here, their expression was investigated in the pre-implantation uterus and utero-placental compartments (UtPl) at selected time points during gestation: post-implantation, mid-gestation, and at normal and antigestagen-induced luteolysis/abortion. Immunohistochemistry with newly generated, canine-specific antisera, in situ hybridization and semi-quantitative PCR were applied. In compartmentalization studies, placental and endometrial RLN increased continuously toward prepartum. The placental RXFP1 was time-related and highest during post-implantation and decreased together with RXFP2 at prepartum luteolysis. The endometrial levels of both receptors did not vary greatly, but myometrial RXFP2 decreased from mid-gestation to prepartum luteolysis. Antigestagen treatment resulted in suppression of RLN in UtPl and decreased RXFP1 and RXFP2 in the uterus. The placental RLN was localized mainly in the cytotrophoblast. Additionally, RXFP1 stained strongly in placental endothelial cells while RXFP2 was found mainly in maternal decidual cells. Uterine staining for all targets was found in epithelial cellular constituents and in myometrium. Finally, besides its endocrine functions, RLN seems to be involved in auto-/paracrine regulation of utero-placental functions in dogs in a time-dependent manner. New insights into feto-maternal communication was provided, in particular regarding the localization of RXFP2 in the maternal decidual cells, implying functional roles of RLN during the decidualization process.

Membrane-bound progesterone receptors in the canine uterus and placenta; possible targets in the maintenance of pregnancy.

To date, the biological functions of P4 within the canine placenta have been attributed to maternal stroma-derived decidual cells as the only placental cells expressing the nuclear P4 receptor (PGR). However, P4 can also exert its effects via membrane-bound receptors. To test the hypothesis that membrane-bound P4 receptors are involved in regulating placental function in the dog, the expression of mPRα, -ß, -γ, PGRMC1 and -2 was investigated in the uterine and placental compartments derived from different stages of pregnancy and from prepartum luteolysis. Further, to assess the PGR signaling-mediated effects upon membrane P4 receptors in canine decidual cells, in vitro decidualized dog uterine stromal (DUS) cells were treated with type II antigestagens (aglepristone or mifepristone). The expression of all membrane P4 receptors was detectable in reproductive tissues and in DUS cells. The main findings indicate their distinguishable placental spatio-temporal distribution; PGRMC2 was predominantly found in decidual cells, PGRMC1 was strong in maternal endothelial compartments, and syncytiotrophoblast showed abundant levels of mPRα and mPRß. In vitro decidualization was associated with increased expression of PGRMC1 and -2, while their protein levels were diminished by antigestagen treatment. The involvement of membrane-bound P4 signaling in the regulation of canine placental function is implied, with P4 effects being directly exerted through maternal and fetal cellular compartments. The indirect effects of PGR might involve the modulation of membrane-bound receptors availability in decidual cells, implying a self-regulatory loop of P4 in regulating the availability of its own receptors in the canine placenta.

Monitoring of canine pregnancy by considering Anti-Mullerian hormone, C-reactive protein, progesterone and complete blood count in pregnant and non-pregnant dogs.

In this study, we measured the serum concentration of anti-Mullerian hormone (AMH), C-reactive protein (CRP), progesterone (P4), and the complete blood count (CBC) in pregnant and non-pregnant bitches. The aim was to investigate the suitability of these parameters for monitoring canine pregnancy. Blood samples were collected from all bitches introduced for timed mating on the day of first mating (>5 ng/ml). The first blood sample after mating was obtained on day 12 post-copulation. The dogs whose pregnancy was confirmed on days 25 and 35, were allotted to the pregnancy positive group (G+) and those that were not pregnant were grouped as pregnancy negative (G). Ultrasonography (US) was performed on days 25, 35, 45 and 55 in pregnant (N = 13) and non-pregnant (N = 7) animals; The sonographic examinations in non-pregnant bitches were continued up to day 63, and in pregnant bitches they were also carried out one day after parturition (D+1). Blood samples were taken in parallel with these periods. Furthermore, the pregnant bitches were classified as G1A (1-2 puppies), G1B (3-4 puppies), and G1C (5-11 puppies) based on the number of puppies, and G1X (10 kg), G1Y (10-20 kg), and G1Z (>20 kg) based on their body weight. No significant difference was found between G+ and G-with regard to AMH, except on day 45, when AMH was higher in G+ (P < 0.01). On the other hand, the CRP values in the G+ exceeded those in the G-group on day 25 (38.26 vs. 15.66 mg/L, P < 0.05), on day 35 (32.54 vs. 15.97 mg/L, P < 0.05) and on day one after parturition (36.24 vs. 10.10 mg/L, P < 0.01). When puppy number was considered, it was discovered that CRP values significantly increased with puppy number on days 12 and 45 (G1A vs. G1B day 12: 4.13 vs 15.84 mg/L, P < 0.05; day 45: 12.40 vs. 25.76 mg/L, P < 0.001), and on day 35 (G1B vs. G1C: 24.18 vs. 38.87 mg/L, P < 0.01). With regards to AMH, this was only detectable on day 12 (G1A vs. G1B: 0.56 vs. 1.13 ng/mL, P < 0.05). When the body weight of the pregnant bitches was considered, bitches <10 kg had significantly higher AMH values than bitches bitch >20 kg on days 12 and 25 (day 12: 1.20 vs. 0.21 ng/mL, P < 0.01; day 25: 0.91 vs. 0.21 ng/mL, P < 0.05). This was not found in the case of CRP. The white blood cells (WBC) and the granulocytes (GRAN) were found to be higher in the G+ group (P < 0.01) on day 55, while the hematocrit (HCT) was significantly lower on day 45 (P < 0.05) and day 55 (P < 0.01). The increased GRAN was still detectable one day after parturition (P < 0.05). In conclusion, measurement of the AMH and CRP concentrations may contribute to determination of gestation stage and monitoring of the course of pregnancy; values are related to maternal body weight and number of puppies; however, AMH did not change over the course of a normal pregnancy. Sonography, the increase in CRP and complete blood count values may be beneficial for monitoring canine pregnancy. More studies are necessary to prove these findings.

The use of a 4.7 mg deslorelin slow release implant in male dogs in the field.

OBJECTIVE: Slow-release GnRH agonist implants (SRI) are used for reversible medical downregulation of testicular function in male dogs as an alternative to surgery. The 4.7 mg deslorelin SRI should reduce testosterone after 6-8 weeks and induce castration-like effects for 6 months (mon). However, some individual variation is described in the field in regard to onset and duration of effect. For this reason, we aimed to study the effects of the 4.7 mg deslorelin SRI in a larger cohort. MATERIAL AND METHODS: In total 50 intact, healthy male dogs (12-48 months, mon; 9-40 kg) were treated with a 4.7 mg deslorelin SRI into the umbilical area (TG, n=45) or served as untreated controls (CG, n=5). CG dogs were surgically castrated after measurement of testicular dimensions and blood sampling for testosterone. In TG, SRIs remained for 5 mon in place and subsequently 3-7 male dogs were surgically castrated at removal (week, W 0) or 1, 2, 3, 4, 5, 6, 7, 8 or 10 weeks later. Examination parameters were testicular dimensions (before treatment, at 4, 8, 12 W, 5 mon, weekly until castration), testosterone (before treatment, at 8 W, 5 mon, castration) and testicular histology (castration). RESULTS: Whereas examination parameters did not differ between CG and TG before treatment, testicular volume and testosterone was significantly reduced at all time points during treatment. In all but 3 (8 W) and 2 male dogs (5 mon) testosterone was basal during treatment before removal, whereas the parameters were significantly reduced compared to pre-treatment in the respective dogs. After implant removal, testosterone and testicular volumes increased. However, different to earlier studies, the "restart" was more variable with individual basal testosterone until W7, but also physiological testosterone concentrations in W2. Similarly, histological testicular findings at castration were quite variable: besides an arrest on spermatogonia and spermatocytes, elongated spermatids with normal spermatogenesis were found in individual dogs. CONCLUSION: Our study confirms the efficacy of the deslorelin SRI, but also individual variation especially regarding reversibility of effects on endocrine and germinative testicular function. CLINICAL RELEVANCE: Deslorelin SRIs offer a suitable alternative to surgical castration with individual variation to be considered when used in clinical practice.

Prepubertal Use of Long-Term GnRH Agonists in Dogs: Current Knowledge and Recommendations.

The search for an alternative approach of estrus control (induction or suppression) in dogs is an important issue and the use of slow GnRH agonist-releasing implants has been the subject of frequent research in recent years. Studies to date demonstrate that the short- and long-term effects of deslorelin implants applicated at different time points of the prepubertal period are similar to those of adult dogs; however, there are important differences. The age of the prepubertal bitch and the dosage appear to be the main determinants of the response to deslorelin, as well as the individual metabolism of the bitch. Recent studies reported that the deslorelin-mediated long-term delay of puberty does not have negative carry-over effects on subsequent ovarian functionality, serum steroid hormone concentrations, uterine health, and fertility; however, more molecular studies are needed to determine the effects of application time of GnRH agonists on hormone concentrations and peripheral receptor expression. Furthermore, the long-term effects of delay of puberty with deslorelin on joint health, tumor development, the immune system, and social behavior deserve further investigations.

Computerized histogram analysis of the canine placenta during normal pregnancy.

In this study, the aim was to evaluate digitalized images obtained via B-mode sonography (USG) during different stages of pregnancy and to assess features of the canine placenta, typical for the different gestational periods. For this purpose, ultrasound pictures were digitalized and a computerized histogram analysis performed. Finally, 151 USG images were obtained from 37 healthy, pregnant dogs of different breeds, aged 4.4 ± 1.8 years, between days 17 and 62 of gestation, and 755 Regions of Interest (ROI) within these images were evaluated. Five ROI per picture were situated in the ventral and dorsal uterus/placenta (early stages) or exclusively the placenta (later stages). The data were grouped into GI: gestational day 17-25, GII: gestational day 26-40, GIII: gestational day 41-50 and GIV: gestational day 51-62. The mean grey value (MGV), standard deviation of grayscale value (StdDGV), minimum grey value (MinGV), maximum grey value (MaxGV), Modal/Mode grey value (ModGV), median grey value (MedGV), homogeneity (Angular Second Moment, ASM), contrast (CONT), correlation (COR), regional homogeneity (Inverse Difference Moment, IDM) and the entropy (ENT) parameters in the Region of Interest (ROI) were calculated. In the course of pregnancy, the grey value parameters, CONT and the local homogeneity (IDM) changed characteristically. The MGV increased between days 17 and 40 (p < 0.0001) and decreased towards late gestation (GIV, p < 0.0001). In parallel, the ModGV, MedGV and MaxGV values changed significantly (p < 0.0001). Similarly, CONT value significantly increased between implantation (GI) and later gestational periods, together with the increasing grayscale variation, probably indicating the massive increase in vascularisation during mid-gestation. This is supported by the fact that local homogeneity (IDM) was highest during implantation and decreased towards late pregnancy (p < 0.01). There was no difference between groups in terms of ASM, COR and ENT (p > 0.05). We conclude that some of the parameters evaluated by computational analysis increased or decreased with the progression of pregnancy, and correlated with the gestational period of bitches. Further studies will reveal whether this technique can contribute to early diagnosis of placental alterations.

Expression of Anti-Müllerian Hormone and Its Type 2 Receptor in the Ovary of Pregnant and Cyclic Domestic Cats.

To evaluate the expression of AMH and its receptor AMHRII, ovaries of 33 p cats were investigated by western blot and immunohistochemistry. After ovariohysterectomy, the cats were grouped according to pregnancy stages and ovarian/placental endocrine activity: group I (n = 3, 24−29 days), II (n = 8, 32−40 days), III (n = 4, 41−46 days), IV (n = 6, 53−61 days) and according to cycle stages: V (n = 6, interestrus) and VI (n = 6, estrus). Serum progesterone- and AMH-concentration was measured. Follicle numbers did not differ between groups. The number of corpora lutea was higher in pregnant cats than in the non-pregnant cats. Serum AMH concentration was at maximum between day 30 and 50 of gestation, and was higher than in non-pregnant cats, then decreased towards term (p < 0.05). In the ovaries, AMH immunopositivity was observed in granulosa cells of secondary and antral follicles, and in interstitial cells of corpora lutea; highest percentage of immunopositive areas was detected in group III (p < 0.05). A positive correlation between the number of corpora lutea and the positive AMH signals in ovarian tissue was determined (r2 = 0.832, p < 0.05); however, only during mid-gestation (group II). Expression of AMHRII was in close co-localization with AMH and strong in the interstitial cells surrounding follicles undergoing atresia. AMHRII expression did not differ between pregnant groups but was higher compared to estrus cats (p ˂ 0.05). We conclude that AMH and AMHRII expression in the feline ovary is comparable to other species. The high serum AMH concentration and ovarian AMHRII expression between day 30 and 50 of gestation are probably related to ovarian activity and follicular atresia.

The Efficacy of a 3ß-Hydroxysteroid Dehydrogenase Inhibitor for the Termination of Mid-Term Pregnancies in Dogs.

Progesterone (P4) is the only hormone needed to maintain pregnancy in dogs. Therefore, a competitive inhibitor of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) could be a safe and effective option to terminate pregnancy by inhibiting P4 synthesis. To address this hypothesis, we investigated the efficacy of trilostane (TRL), a competitive inhibitor of 3ß-HSD, in terminating pregnancy in dogs. Twenty-one dogs between days 30 and 38 of pregnancy were randomly assigned to one of two treatment groups (trilostane (TRL) and aglepristone (AGL)) and an untreated control (CON) group (n = 7 dogs each). Fetal heart rates (FHRs) (measured at 12 h intervals) and serum P4 concentrations (measured at 6 h intervals) were evaluated. The pregnancy termination rates were 0% and 100% in the TRL and AGL groups, respectively. The decrease in the FHR in the TRL and AGL groups was significantly lower than that observed in the CON group. There was a marked decrease in P4 concentrations in the TRL group 6, 54, and 102 h after the initiation of treatment. The luteal expression of StAR appeared to be weaker in the AGL group than the CON group. In conclusion, although a treatment-induced decrease was observed in plasma P4 concentrations, a seven-day TRL treatment alone was not effective in terminating pregnancies. Further studies are needed on the effects of the prolonged administration of TRL with varying doses and frequencies for the termination of mid-term pregnancy in dogs.

Luteal and placental function in the bitch: spatio-temporal changes in prolactin receptor (PRLr) expression at dioestrus, pregnancy and normal and induced parturition.

BACKGROUND: Endocrine mechanisms governing canine reproductive function remain still obscure. Progesterone (P4) of luteal origin is required for maintenance of pregnancy. Corpora lutea (CL) are gonadotrop-independent during the first third of dioestrus; afterwards prolactin (PRL) is the primary luteotropic factor. Interestingly, the increasing PRL levels are accompanied by decreasing P4 concentrations, thus luteal regression/luteolysis occurs in spite of an increased availability of gonadotropic support. PRL acts through its receptor (PRLr), the expression of which has not yet been thoroughly investigated at the molecular and cellular level in the dog. METHODS: The expression of PRLr was assessed in CL of non-pregnant dogs during the course of dioestrus (days 5, 15, 25, 35, 45, 65 post ovulation; p.o.) as well as in CL, the utero/placental compartments (Ut/Pl) and interplacental free polar zones (interplacental sites) from pregnant dogs during the pre-implantation, post-implantation and mid-gestation period of pregnancy and during the normal and antigestagen-induced luteolysis. Expression of PRLr was tested by Real Time PCR, immunohistochemistry and in situ hybridization. RESULTS: In non-pregnant CL the PRLr expression was significantly upregulated at day 15 p.o. and decreased significantly afterwards, towards the end of dioestrus. CL of pregnancy showed elevated PRLr expression until mid gestation while prepartal downregulation was observed. Interestingly, placental but not interplacental expression of PRLr was strongly time-related; a significant upregulation was observed towards mid-gestation. Within the CL PRLr was localized to the luteal cells; in the Ut/Pl it was localized to the fetal trophoblast and epithelial cells of glandular chambers. Moreover, in mid-pregnant animals treated with an antigestagen, both the luteal and placental, but not the uterine PRLr were significantly downregulated. CONCLUSIONS: The data presented suggest that the luteal provision of P4 in both pregnant and non-pregnant dogs may be regulated at the PRLr level. Furthermore, a role of PRL not only in maintaining the canine CL function but also in regulating the placental function is strongly suggested. A possible functional interrelationship between luteal P4 and placental and luteal PRLr expression also with respect to the prepartal luteolysis is implied.

Changes in antioxidant enzyme activities and metabolic parameters in dairy cows during different reproductive periods.

Our study aimed to monitor the alterations in antioxidant enzyme activities and the metabolic profile parameters and their relationships in dairy cows during different reproductive stages (gestational-, dry-, pre- and post-partum). The collection of blood samples from thirty healthy pregnant Holstein cows took place at a commercial dairy farm, between September 2015 and June 2016. The cows covered eight different reproductive stages (4-6 weeks before the dry period (D4-6w)), at the beginning of the dry period (D0; on day 210 of gestation), the first month of the dry period (D1m), antepartum day 8 (APd8), postpartum (PP) day 3 (PPd3), PP day 8 (PPd8), PP between 3rd-4th weeks (PP3-4w) and PP between 80 and 90 days (PP80-90d). The activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) were analysed to monitor and evaluate the antioxidant system. Concentrations of beta-hydroxybutyrate (BHBA), non-esterified fatty acids (NEFA), albumin, glucose, total bilirubin, cholesterol, calcium (Ca) and the enzyme activities of aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), gamma-glutamyl transferase (GGT) were also determined as metabolic profile parameters. GPx activities increased in D1m (P < 0.05) and peaked during the PP3d and PP8d periods (P < 0.0001), with values decreasing again at the PP3-4w period. The most significant change in SOD activities was detected in the PP3-4w with a significant decrease (P < 0.05). BHBA and NEFA concentrations increased significantly during the PPd3 and PPd8 periods (P < 0.05), with BHBA decreased to low values in the PP3-4w (P < 0.05). Significant positive correlations were found between BHBA, NEFA, total bilirubin and GPx (0.784, 0.874 and 0.871; P < 0.05, P < 0.01 and P < 0.01 respectively). A significant negative correlation between calcium and cholesterol and GPx was found (-0.857, P < 0.01; and -0.681, P < 0.05, respectively). As a result, a relationship was determined between blood antioxidant enzymes and metabolic parameters at different periods. High GPx and SOD activities, especially in the early period before and after parturition, is thought to be related to the adaptation of the animals to this process. The lack of adaptation abilities of animals during these periods causes many problems seen during early postpartum. Therefore, considering GPx and SOD activity measurements in pre- and postpartum periods contributes to better management of these periods and the prevention of complications.

Utero-Placental Immune Milieu during Normal and Aglepristone-Induced Parturition in the Dog.

Maternal immunotolerance is required for the maintenance of pregnancy, in sharp contrast with the uterine pro-inflammatory activity observed during parturition in several species. Correspondingly, in the dog, increased immune signaling at term has been suggested, but a deeper understanding of the uterine immune milieu is still missing. Thus, the availability of 30 immune-related factors was assessed in utero-placental samples collected during post-implantation (days 18-25 of pregnancy) and mid-gestation (days 35-40) stages, and at the time of prepartum luteolysis. Gene expression and/or protein localization studies were employed. Samples collected from antigestagen (aglepristone)-treated dogs were further analyzed. Progression of pregnancy was associated with the downregulation of IL1ß and upregulation of IL10 (p < 0.05) at mid-gestation. When compared with mid-gestation, a higher availability of several factors was observed at term (e.g., CD206, CD4, TLR4). However, in contrast with natural parturition, MHCII, CD25, CCR7, TNFα, IDO1 and AIF1 were upregulated after aglepristone treatment (p < 0.05), but not TNFR1 or CCL13 (p > 0.05). Altogether, these results show an increased immune activity during canine parturition, involving, i.a., M2 macrophages, Treg and Th cells, with strong support for progesterone-mediated immunomodulation. Furthermore, differences between term and induced parturition/abortion could relate to differences in placental maturation towards parturition and/or functional traits of antigestagens.

Expression of the insulin-like growth factor (IGF) gene family in feline uterus during pregnancy.

Members of the IGF gene family participate in cell differentiation and proliferation during pregnancy. We used 35 cats assigned to experimental groups (G) based on pregnancy stages: G1, pre-implantation; G2, implantation; G3, early pregnancy; G4, mid-pregnancy; G5, nonpregnant. Quantitative polymerase chain reaction (qPCR) and immunohistochemistry were used to analyze the expression of the IGF gene family. During pregnancy, expression of IGF-1 gene was significantly greater at implantation sites in the G1 and G2 groups than at placentation sites in G3 and G4 groups. IGF-2 expression was greater in the G2, G3 and G4 groups than in G1. Expression of the IGF-1R gene was significantly greater at placental sites in G3 than in G1 and G4. IGF-2R genes were expressed in all groups. Insulin-like growth factor binding proteins (IGFBPs) were expressed at intensities that depended on the stage of pregnancy; they were detected in different cell types and at different sites in the uterus. We found that members of the IGF gene family were expressed differentially in the endometrium during pregnancy. Our findings suggest that the IGF family may be a regulatory factor for pregnancy in cats.

The Effect of Comfort- and Hot-Period on the Blood Flow of Corpus Luteum (CL) in Cows Treated by an OvSynch Protocol.

The values of luteal blood flow (LBF), total corpus luteum (CL) area (TAR), and progesterone (P4), during and after OvSynch (OvS) protocol in comfort (CP; n = 40) and hot periods (HP; n = 40) were compared. We investigated how low and high P4 values obtained before the application affected the parameters above during CP and HP periods. Blood samples were collected before the OvS application on day 0 (OVSd0), day 9 (OeG), and day 18 (9th day after OeG: OvSd9). The P4 (ng/mL) values of the animals exhibiting dominant follicles were between 0.12-0.82 in HC and 0.1-0.88 in CP (P4-2: 4.36-4.38 and P4-3: ≥7.36 ng/mL). The LBF values were measured on days 7 (OvSd7) and 9 (OvSd9) after the OeG. The P4 mean values at day 0 (OvSd0) were classified as low (P4-1), medium (P4-2), and high (P4-3). The LBF and the TAR values in the P4-2 and P4-3 on OeG day 9 were higher than in HP (p < 0.05; 0.001), but there was no significant difference in the P4-1. In conclusion, when the OvS program was initiated with low P4 values, no difference was observed between HP and CP in terms of LBF values; however, when the program was started with high P4 values, there were significant increases in LBF and TAR values in the CP compared to the HP.