Charged Amino Acids in the Transmembrane Helix Strongly Affect the Enzyme Activity of Aromatase.

Estrogens play critical roles in embryonic development, gonadal sex differentiation, behavior, and reproduction in vertebrates and in several human cancers. Estrogens are synthesized from testosterone and androstenedione by the endoplasmic reticulum membrane-bound P450 aromatase/cytochrome P450 oxidoreductase complex (CYP19/CPR). Here, we report the characterization of novel mammalian CYP19 isoforms encoded by CYP19 gene copies. These CYP19 isoforms are all defined by a combination of mutations in the N-terminal transmembrane helix (E42K, D43N) and in helix C of the catalytic domain (P146T, F147Y). The mutant CYP19 isoforms show increased androgen conversion due to the KN transmembrane helix. In addition, the TY substitutions in helix C result in a substrate preference for androstenedione. Our structural models suggest that CYP19 mutants may interact differently with the membrane (affecting substrate uptake) and with CPR (affecting electron transfer), providing structural clues for the catalytic differences.

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.

[Initiation and endocrine control of parturition in domestic mammals - Part 1]. / Initiierung und endokrine Kontrolle der Geburt bei Haussäugetieren ­ Teil 1.

Endocrine regulation of parturition is based on an intense exchange of signals between the fetus, placenta and mother. Apart from sheep, our knowledge of the endocrine control of parturition is still very incomplete. However, current observations suggest significant differences between the species. For the maintenance of pregnancy, progesterone (P4) is the crucial superordinate regulatory factor, although in some species, such as the horse, functions of P4 are at least partially fulfilled by other progestogens. In general, prepartum P4 withdrawal is considered a prerequisite for the onset of physiological birth. In species with exclusive (dog) or predominant (e. g., cattle, goat, pig) luteal P4 at the end of gestation, luteolysis is the crucial event. In sheep, where P4 is of placental origin prior to parturition, the prepartum P4 decline is due to a switch in placental steroid metabolism. The mechanism of prepartum progestogen withdrawal in the mare is still largely unclear. In sheep, initiation of parturition proceeds from maturation of the fetal hypothalamic-pituitary-adrenal (HPA) axis, which leads to a steep prepartum rise in fetal cortisol concentrations stimulating the collapse of placental P4 production. In cattle, fetal cortisol probably triggers luteolysis via stimulation of placental prostaglandin secretion. In several other domestic mammalian species, there is also evidence that the initiation of parturition proceeds from maturation of the fetal HPA axis. However, the functional relationships between fetal cortisol and prepartum P4 withdrawal are largely unknown in nonruminant species.

[Induction and endocrine control of parturition in domestic mammals - Part 2 - Species-specific aspects and their relevance to the applicability of birth induction procedures]. / Initiierung und endokrine Kontrolle der Geburt bei Haussäugetieren ­ Teil 2.

The endocrine regulation of birth is based on an intensive exchange of signals between fetus, placenta and mother. Apart from sheep, our knowledge of the underlying processes is still very incomplete. However, current observations suggest substantial species differences. Of critical importance for the onset of the final steps of the signaling cascade leading to active labor is "prepartum progesterone withdrawal," which is based on luteolysis (e. g., cattle, goat, buffalo, camelids, pig) or a breakdown in placental progestogen production (sheep, horse), depending on the relevant progestogen source in late pregnancy. Knowledge of birth-associated regulatory processes allows species-specific regulatory mechanisms to be mimicked for drug-based induction of labor. Furthermore, species-independent mechanisms such as the inhibition of progesterone receptors are available. In addition to efficacy, other aspects such as tolerability for dams and offspring as well as drug regulations must be taken into account when selecting active ingredients under practical conditions.

Effect of uterine torsion intrapartum on concentrations of placental estrogens and progesterone in cattle.

OBJECTIVE: The current study investigates how uterine torsion influences placental oestrogens and progesterone blood concentrations in intrapartum cows. Our research tests the hypothesis that intrapartum uterine torsion impairs the ability of the placenta to synthesize steroids and may also suppress the release of synthesized steroids into the maternal circulation. METHODS: The study included a total number of 37 intrapartum dairy cows of various breeds and ages. These animals were transported to our clinic by their owners. Furthermore, general and obstetrical examinations of all these animals were performed in our clinic. The uterine torsion (UT) group consisted of 20 animals. The presence of UT was verified during clinical general examinations by vaginal and transrectal examination. The comparison (C) group included 17 animals whose birth was undisturbed or could be terminated with moderate obstetrical assistance. The clinical examination of group C animals showed no problems with their general health and genital organs. Blood samples were collected immediately after the initial obstetrical examination from 37 cows for radioimmunological measurement of estradiol-17ß (E2), free total estrogen (FTE), conjugated total estrogen (CTE), and progesterone (P4). RESULTS: In terms of P4, there was no statistical difference between the two groups. For all estrogen parameters, however, concentrations were significantly lower in the UT group than in the C group. In the correlation analysis, there was a significant correlation between the P4 and the FTE in the C group. Furthermore, the positive correlation between all estrogen parameters in the UT group was significant. In group C, significant positive correlations were found apart from the correlation between E2 and CTE. CONCLUSIONS: The results are consistent with the hypothesis and suggest that in UT animals processes dependent on estrogens or other placental hormones may be impaired during the peri- or postpartum period.

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.

Epididymectomy as a novel surgical procedure; application in the domestic cat.

Feline overpopulation raises issues concerning health, ecology, economy, and ethics. Procedures to limit overpopulation should carefully address animal welfare, efficiency, costs, and feasibility. Vasectomy in unowned cats is suggested as preferable to standard neutering as it maintains male sexual behaviour which may induce ovulation and pseudopregnancy in intact females and may prevent immigration of other males. Vasectomy is not performed routinely because it is fastidious, time consuming and requires more material than standard neutering. We describe epididymectomy as an alternative. In a first experiment, we analysed semen, testosterone, behaviour and pain in six experimental cats before and after epididymectomy, and after castration two months later. Excised tissues were analysed histologically. Testosterone concentrations did not differ significantly between intact and epididymectomised animals but were significantly different after castration. Sexual behaviour and testicular spermatogenesis persisted after epididymectomy, but with a marked drop in the semen count after 7 days. The Glasgow pain scores did not differ significantly after epididymectomy and castration. In a subsequent experiment, 20 privately owned cats were epididymectomised and castrated immediately afterwards, to analyse the learning curve and perioperative complications. The time required for an epididymectomy was significantly shorter than for castration. The study confirms that epididymectomy is quicker and less invasive than castration, it is associated with minimal risks and post-operative pain while easy to learn and inexpensive. Further field studies are required to test its efficiency for feline feral population control or in other species such as in bears, lions or deer, where infertility is required and castration not wanted.

Ultrastructural and Immunohistochemical Characterization of Maternal Myofibroblasts in the Bovine Placenta around Parturition.

Myofibroblasts are contractile cells that exhibit features of both fibroblasts and smooth muscle cells. In the synepitheliochorial placenta of the cow myofibroblasts are found in the maternal stroma. However, a deeper understanding of the structure and function of the stromal myofibroblasts in the developed bovine placenta is still missing. Thus, immunohistochemical and ultrastructural analyses in bovine term placentomes, compared to non-pregnant caruncle samples, were conducted. To investigate functional aspects, contractility of placentomal caruncle slices was assessed in an in vitro contraction assay. Additionally, a three-dimensional reconstruction of a bovine placental myofibroblast was created. Immunofluorescent staining revealed a characteristic pattern, including cytoplasmic expression of α-smooth muscle actin, strong perinuclear signal for the intermediate filament vimentin and nuclear progesterone receptor staining. Ultrastructurally, stress fibers, extended cisternae of the rough endoplasmic reticulum and perinuclear intermediate filaments were observed. Moreover, in vitro stimulation with angiotensin-II, but not with prostaglandin F2α, induced contraction of placental caruncle tissue. Altogether, these results indicate that progesterone-responsive myofibroblasts represent a mesenchymal phenotype that is involved in the contractile properties of bovine placental stroma. Therefore, the present findings suggest a potential involvement of myofibroblasts in post-partum events of cattle, i.e., expulsion of fetal membranes and uterine involution.

Ovarian and uterine changes during the oestrous cycle in female dogs.

CONTEXT: An accurate staging of sexual cycle is essential for the optimum timing of medical interventions. AIMS: Here, an updated insight into clinical, endocrinological and vagino-cytological parameters, and their correlation with histomorphology of ovarian and uterine tissue samples is presented. METHODS: Samples from 39 dogs were collected at various stages of the oestrous cycle: pro-oestrus (n =8), oestrus (n =12), dioestrus (n =9) (luteal phase) and anoestrus (n =10), according to clinical observations. Final allocation of samples was done after histomorphological evaluation of all tissues. Peripheral oestradiol-17ß (E2) and progesterone (P4) concentrations were measured, P4 by both chemiluminescent immunoassay (CLIA) and radioimmunoassay (RIA). KEY RESULTS: Differences were observed between determination of the stage of the oestrous cycle, either by clinical, endocrinological or histomorphological evaluation. Individuals considered to be in clinical and endocrinological oestrus, had entered the luteal phase according to histomorphology. P4 concentrations measured by two different assays differed, underlying the importance to understand that absolute P4 concentrations may deviate depending on the used assay. Comparison of E2 and P4 concentrations is suggested to be useful when defining the transition from early follicular phase to the time of ovulation. CONCLUSIONS AND IMPLICATIONS: Based on parallel histomorphological observations, combined with clinical and endocrinological findings on the same individuals, the present study emphasises that an accurate classification of the stage of the cycle in female dogs based solely on clinical and endocrinological assessments can be difficult. The histomorphological findings presented herein provide new insights into the transitional phases between the different stages of the oestrous cycle in the dog.

Stress Assessment of Wild Boar (Sus scrofa) in Corral-Style Traps Using Serum Cortisol Levels.

Capture of wild boar in corral-style traps with subsequent culling is increasingly used for population management. The method is debated due to animal welfare concerns making welfare studies in traps necessary. While previous studies focused on behaviour and injuries, this study dealt with the physiological aspect. Cortisol levels in wild boar caught in corral-style traps (50-90 qm2, n = 138) were compared with those killed during single (n = 37) and driven hunts (n = 90). Collected sera were purified by solid phase extraction (SPE) and analysed via radioimmunoassay. Cortisol levels in blood samples were stable under cooled (4-7 °C) conditions for a storage time of up to 87 h before centrifugation. Cortisol levels were significantly higher in wild boar killed in corral-style traps than during driven hunts and single hunts. Wild boar caught in groups of five or more showed lower cortisol levels than single animals or in smaller groups. Therefore, time span inside the trap and of culling should be reduced to a minimum, and capturing groups of animals should be preferred to reduce stress. For animal welfare assessment of wild boar live-trapping, additional data from behavioural analyses and pathological examinations must be integrated.

Regulation of Porcine Oviduct Epithelium Functions via Progesterone and Estradiol Is Influenced by Cortisol.

Preimplantation maternal stress, characterized by elevated glucocorticoids (GCs), has been linked to reproductive failures caused by impaired oviduct functionality, which is known to be predominantly regulated by the sex steroids, progesterone (P4) and (17)estradiol (E2). Although steroid receptors share analogous structures and binding preferences, the interaction between GCs and E2/P4 in the oviduct has attracted little attention. Using an air-liquid interface culture model, porcine oviduct epithelial cells were stimulated with single (cortisol, E2, P4) or hormone mixtures (cortisol/E2, cortisol/P4) for 12 hours and 72 hours. Cultures were subsequently assessed for epithelial morphometry, bioelectrical properties, and gene expression responses (steroid hormone signaling, oviductal function, immune response, and apoptosis). Results confirmed the suppressive role of P4 in regulating oviduct epithelium characteristics, which was partially opposed by E2. Besides increasing the ratio of ciliated cells, cortisol antagonized the effect of P4 on epithelial polarity and modified sex steroid-induced changes in transepithelial electrical properties. Both sex steroids affected the glucocorticoid receptor expression, while cortisol downregulated the expression of progesterone receptor. The overall gene expression pattern suggests that sex steroid dominates the cotreatment, but cortisol contributes by altering the gene responses to sex steroids. We conclude that besides its individual action, maternal cortisol interplays with sex steroids at phenotypic and molecular levels in the oviduct epithelium, thereby influencing the microenvironment of gametes and early embryos.

What Happens in Male Dogs after Treatment with a 4.7 mg Deslorelin Implant? II. Recovery of Testicular Function after Implant Removal.

Although deslorelin slow-release implants are widely used in the clinic, detailed published information about the recovery of testosterone concentrations (T), semen quality, and testicular and prostatic volume (TV, PV) after treatment is still missing. This article aims to characterize changes during restart after a five-months treatment and subsequent implant removal. Seven male Beagle dogs were treated with deslorelin (treatment group, TG), and three saline-treated dogs served as controls (CG). Deslorelin implants were removed after five months (D ex), followed by detailed andrological examinations for TV, PV, semen collection, and blood sampling for T-analysis with/without GnRH/hCG stimulation tests. TV, PV, and T increased rapidly after D ex in TG, not differing from CG from D91 (TV), D49 (PV), and D14 (T). The first sperm-containing ejaculates were collected between D49 and 70, whereas the samples were normospermic between D84 and 133. A T increase (>0.1 ng/mL) subsequent to the GnRH/hCG stimulation test was observed from D28/29 onwards, respectively. Histological assessment of testicular tissue at the end of the observational period (D149 after implant removal) revealed normal spermatogenesis. Our data confirm that the restart of endocrine and germinative testicular function is highly variable, but nevertheless, all of the effects induced were reversible.

What Happens in Male Dogs after Treatment with a 4.7 mg Deslorelin Implant? I. Flare up and Downregulation.

Although registered since 2007, knowledge about changes in testosterone concentrations (T), testicular and prostatic volumes (TV, PV) and semen quality, as well as the time point of infertility following treatment with a 4.7 mg deslorelin (DES) slow-release implant, is limited. Therefore, seven sexually mature male dogs were treated with DES (TG); three male dogs treated with saline served as controls (CG). The study assessed local tolerance, TV, PV, semen parameters and T subsequent to GnRH/hCG stimulation in regular intervals. Local tolerance was good. In TG, T was increased right after treatment, but decreased four hours afterwards. Subsequently, TV, PV, semen quality and T decreased over time in TG, but not CG. T was basal (≤0.1 ng/mL) from D28 onwards. Response to GnRH/hCG stimulation was variable, with two TG dogs having increased T post-stimulation on all study days independent of pre-treatment concentrations. A(zoo)spermia in TG was observed from D35-D77 in all seven dogs. Whereas treatment was still effective in six TG dogs five months after implant insertion, it was fully reversed in one dog in terms of T and spermatozoa on the last examination. These results indicate high variation in individual dogs, necessary to consider when advising dog owners.

Serum immunoglobulin G concentration after plasma transfusion in neonatal foals with hypogammaglobulinemia in various health status.

Due to the time-limited intestinal uptake of colostral immunoglobulins, the suggested treatment of hypogammaglobulinemia in new-born foals is usually plasma transfusion. The aims of this study were twofold: firstly, to investigate the course of serum IgG concentration after plasma transfusion in newborn foals; and secondly, to determine the amount of transfusion required for a significant increase in serum IgG concentration. For this purpose, the IgG concentration was measured in 23 foals at three different points in time: before transfusion, 1 hour after transfusion, and 24 hours after transfusion. There was an increase in IgG concentration in the blood of 18 foals (78.3%). In five foals (21.7%), no increase in serum IgG concentration were detected after plasma transfusion. Transfusion of 1 mg IgG caused an average increase in IgG level of 0.03 mg/dl (0.001-0.268 mg/dl) 1 hour after transfusion. After 24 hours, the same amount of IgG caused a larger increase of 0.05 mg/dl (0.002-0.537 mg/dl). None of the foals demonstrated adverse reactions to the plasma transfusion. These values provide a guidance how much IgG is needed to increase serum IgG concentration to a desired level. However, this study has shown that there is a high variability in serum IgG concentration after plasma transfusion. Which highlights the necessity for monitoring IgG concentration following transfusion.

Biomarkers for Non-Invasive Stratification of Coronary Artery Disease and Prognostic Impact on Long-Term Survival in Patients with Stable Coronary Heart Disease.

Knowledge about cardiac and inflammatory biomarkers in patients with stable coronary artery disease (CAD) is limited. To address this, we analyzed 3072 patients (36% female) with a median follow-up of 10 years in the Leipzig LIFE Heart Study with suspected CAD with coronary angiography. Selected biomarkers included troponin T (hsTNT), N-terminal pro B-type natriuretic peptide (NT-proBNP), copeptin, C-reactive protein (hsCRP), and interleukin-6 (IL-6). Patients were stratified by CAD severity: CAD0 (no sclerosis), CAD1 (non-obstructive, i.e., stenosis < 50%), and CAD2 (≥one stenosis ≥ 50%). Group comparison (GC) included GC1: CAD0 + 1 vs. CAD2; GC2: CAD0 vs. CAD1 + 2. CAD0, CAD1, and CAD2 were apparent in 1271, 631, and 1170 patients, respectively. Adjusted for classical risk factors, hs-cTnT, NT-proBNP, and IL-6 differed significantly in both GC and hsCRP only in GC2. After multivariate analysis, hs-cTnT, NT-proBNP, and IL-6 remained significant in GC1. In GC2, hs-cTnT (p < 0.001) and copeptin (p = 0.014) reached significance. Ten-year survival in groups CAD0, CAD1, and CAD2 was 88.3%, 77.3%, and 72.4%. Incorporation of hs-cTnT, NT-proBNP, copeptin, and IL-6 improved risk prediction (p < 0.001). The studied cardiac and inflammatory biomarkers enable fast and precise non-invasive identification of mortality risk in CAD patients, allowing the tailoring of primary and secondary CAD prevention.

Comparison of Different Methods to Determine the Absorption of Colostral IgG in Newborn Foals.

The timely diagnosis of abnormalities in the passive transfer of colostral immunoglobulins is important for the health and development of newborn foals. This study investigated three different methods for measuring immunoglobulin G concentration in neonatal foals. Comparison of a commercial SNAP assay, total protein concentration determination, and total globulin calculation by subtracting the albumin fraction from total protein as an indirect parameter was performed on a quantitative ELISA, which served as a reference method. The study included 119 samples from 148 foals between the age of 1 and 6 days. A blood concentration of 800 mg/dL was considered to indicate adequate absorption of immunoglobulins, and a concentration of less than 400 mg/dL was considered to be hypogammaglobulinemia. The sensitivity of the SNAP test was 64.5% and specificity was 94.7% for diagnosing sufficient absorption of immunoglobulin G at a value of 800 mg/dL. A value of 54 g/L was found to be most appropriate for the use of total protein and provided a sensitivity of 67.3% and specificity of 84.2%. For total globulins, the most appropriate value was 27 g/L, which yielded a sensitivity of 74.5% and specificity of 81.6%. At values under 400 mg/dL, the sensitivity of the SNAP test was 89.4% and the specificity was 83.0%. Here, the most suitable value for the total protein was 51 g/L. This provides a sensitivity of 65.2% and a specificity of 76.8%. The most suitable concentration for the use of total globulin was determined to be 24 g/L, which provided a sensitivity of 75.8% and a specificity of 78.1%. The study and its results show that the SNAP test, the TP, and the TP-A method perform similarly well compared to the ELISA in determining IgG concentration of ≥800 mg/dL. Based on the 95% confidence intervals, however, the Snap test and the TP-A method appear to perform similarly well but better than the TP approach for IgG concentrations <400 mg/dL.

Chronic Immune-Mediated Orchitis Is the Major Cause of Acquired Non-obstructive Azoospermia in Dogs.

Azoospermia, the lack of spermatozoa in the ejaculate, is the most common finding in infertile but otherwise healthy male dogs and represents an increasing reproductive health issue in men, too. The diagnosis can be further classified as non-obstructive azoospermia and obstructive azoospermia due to an obstruction of the deferent ducts. Although non-obstructive azoospermia comprises more than half of azoospermic cases in men and is a common cause of infertility in the male dog, knowledge of the underlying etiology and pathophysiology is still strongly limited, and much uncertainty exists about the true incidence and possible treatment options. Therefore, this study aims to investigate and characterize infertile canine patients in detail by combining results of andrological examinations (clinical parameters, semen analysis, bacterial examination of semen, and Brucella canis serology), endocrine analysis (luteinizing hormone, testosterone, estradiol-17ß, and thyroid function), analysis of the alkaline phosphatase in seminal plasma, and histological assessment of testicular biopsies of 10 azoospermic dogs. Our results not only verify non-obstructive etiology for 9/10 cases of canine azoospermia but also further identified significant histopathological changes of the testicular tissue with severely disrupted spermatogenesis, including fibrotic remodeling, vacuolization, Sertoli-cell-only syndrome, tubular shadows, and an increase of the interstitial and vascular area. In addition, three dogs showed local and six dogs generalized immune-cell infiltration, indicating chronic immune-mediated orchitis. Only in one case (no. 1) that no immune cells were found, and obstructive azoospermia was suspected due to low alkaline phosphatase activity. Furthermore, the detection of anti-thyroideal antibodies in two dogs indicates an autoimmune thyroid disease and a correlation between the occurrence of thyroidal disorders and azoospermia. Our results confirm previous findings and contribute additional evidence suggesting that chronic immune-mediated orchitis is the major cause of infertility in dogs. Further studies should focus on uncovering underlying inflammatory processes behind spermatogenic failure in these cases and identify possible treatment options to (re-)initialize spermatogenesis.

Comparison of three progesterone quantification methods using blood samples drawn from bitches during the periovulatory phase.

Background and Aim: Measuring blood progesterone (P4) concentration has become an essential diagnostic tool in small animal reproductive medicine. Methods enabling precise and rapid on-site measurements are in high demand, especially for the optimization of breeding management in bitches. This study aimed to compare two commercial on-site methods (Speed™ P4, Virbac [M1] and mini VIDAS®, bioMérieux [M2]) and a well-established radioimmunoassay (RIA), which was used as a reference method. Materials and Methods: Comparative measurements were performed on 52 blood serum samples collected from 45 clinically healthy bitches of different breeds. The dogs had been presented to determine the estrus cycle stage and predict the time of ovulation. Each sample was divided into three aliquots. In aliquot 1, P4 was measured immediately applying M2. Aliquots 2 and 3 were stored at -20°C until analysis was performed using RIA and M1. The consistency of the three methods was investigated by pairwise linear regression analyses. Results: In RIA, the P4 concentrations ranged between 1.1 and 25.4 ng/mL. Regression analyses revealed highly significant (p<0.0001) positive correlations between the three methods applied (M1 vs. RIA: R=0.94; M2 vs. RIA: R=0.98; and M1 vs. M2: R=0.91). Conclusion: The results show that the two commercial on-site methods tested exhibit approximately equal, high consistency with the radioimmunological reference method and can, therefore, be used beneficially in a clinical setting. However, biological interpretation of data must be performed in a method-specific manner.

Transcriptional regulation of HIF1α-mediated STAR expression in murine KK1 granulosa cell line involves cJUN, CREB and CBP-dependent pathways.

Gonadal function is connected to hypoxia, with hypoxia-inducible factor (HIF) 1α, as a component of HIF1-complexes, regulating cellular adaptation to hypoxic conditions. In the ovary, it regulates follicular maturation, ovulation and luteal development. At the cellular level, HIF1-complexes coordinate the expression of steroidogenic acute regulatory protein (STAR), and thereby ovarian steroidogenesis. The functionality of STAR is associated with the cAMP/PKA-dependent pathways. In vitro, HIF1α is required for basal and cAMP-induced STAR expression, under ambient and reduced oxygen (O2) tension. Lowering O2 increases the responsiveness of the Star promoter towards cAMP and PKA mediates activation/phosphorylation (P) of several transcriptional factors, including cJUN and cAMP response element-binding protein (CREB), whose functionality is linked to HIF1 through utilization of CREB-binding protein (CBP). Since the mechanisms underlying HIF1α-dependent expression of STAR remain unknown, we investigated the involvement of HIF1α in CREB-, cJUN- and CBP-mediated expression of STAR using a well-characterized steroidogenic model, murine KK1 granulosa cells; ambient and lowered (10%) O2 were applied. Our main findings were that while functional suppression of the α-subunit of HIF1 lowered STAR/P-STAR and steroidogenic output from granulosa cells, surprisingly the levels of P-CREB and its transcriptional activity were strongly induced. However, its association with the Star promoter was decreased, indicating dissociation of P-CREB from the promoter. Further, suppression of HIF1 activity ultimately diminished the expression of cJUN/P-cJUN and CBP. Finally, the study suggests that HIF1-complex: (1) regulates cJUN expression in granulosa cells, (2) is involved in regulating the recruitment of P-CREB to the Star promoter in (3) a mechanism which possibly involves the HIF1-dependent regulation of CBP expression.