Immunohistochemical localization of androgen and oestrogen receptors in canine hair follicles

Skin biopsies from seven different body sites were obtained from 21 dogs of different breeds (short, normal, long hair), which were presented for euthanasia. Commercially available polyclonal antibodies were used for the immunohistochemical detection of androgen and oestrogen receptors. Both receptors showed a similar distribution in canine skin, with specific intranuclear staining. In the epidermis, the percentage of androgen receptor (AR)-positive cells, but not that of oestrogen receptor (ER)-positive cells, was significantly higher in samples from the thorax and the flank. In the dermal papilla, the percentage of ER-positive (but not AR-positive) cells was significantly lower in biopsies from the flank. No significant difference was found for both receptors between the locations in the outer root sheath, among the three different hair types, between sex and between intact and castrated dogs.     

Androgens, progestagens and agonistic behaviour: a review

The relationship between androgens, progestagens and agonistic behaviour is reviewed. Most literature concerned the effects of hormones on aggression; little information was available on hormonal influences on fear. Difference in aggression levels between males and females may be explained by assuming the existence of a gender difference in motivation, which, among other factors, is controlled by androgen and progestagen levels in peripheral blood. Androgens and progestagens are metabolised mainly by 5 alpha-reductase in the target organs. In the brain, aromatisation of testosterone also plays a role. The metabolites of testosterone may exert the same organising and activating influence as testosterone on juvenile and adult brain tissue, respectively. In some animal species testosterone secretion appears to be influenced by social and environmental variables. Conversely, alterations in plasma androgen levels have been found to affect behaviour. Dominant and/or aggressive individuals tend to show higher plasma testosterone levels than submissive and/or less aggressive animals. Among other mechanisms, competitive inhibition of androgen action at a central level, by progestagens acting as antagonists of androgens, may be important.     

Testicular feminization syndrome in a mare.

Testicular feminization syndrome was diagnosed in a mare with aggressive, stallion like behavior and a history of infertility. She was found to have a high baseline testosterone concentration suggesting that testicular tissue was present, and ovarian-like structures examined by use of transrectal ultrasonography had the appearance typical of testicular tissue. Although her external female genitalia appeared normal, her vagina ended in a blind sac, and no cervix or uterus were identified. Surgery was performed, and structures removed from the abdominal cavity were determined to be hypoplastic testicles. Removal of the testicular tissue resulted in complete resolution of her aggressive behavior. Chromosomal evaluation revealed that the mare had 64X,Y (normal male) karyotype. Testicular feminization syndrome is a condition characterized by insensitivity of reproductive tissues to androgens during development because of an abnormality in androgen receptors. This androgen insensitivity results in development of normal external female genitalia, with high testosterone concentrations being released from developing testicles. Testicular feminization syndrome has not been commonly diagnosed in horses, but should be considered as a differential diagnosis for overly aggressive mares with a history of infertility.     

Neuro-imaging the serotonin 2A receptor as a valid biomarker for canine behavioural disorders

The serotonergic system is disturbed in different mood and affective disorders, with especially the serotonin (5-HT) 2A receptor involved in impulsive aggressiveness and anxiety. The aim of the study was to evaluate the involvement of the brain 5-HT 2A receptor in dogs with different behavioural disorders. Three groups of drug naive dogs were studied: 22 dogs showing impulsive aggressive behaviour, 22 showing normal behaviour, and 22 showing anxious behaviour. The serotonin 2A receptor was evaluated with Single Photon Emission Computed Tomography (SPECT) and the serotonin 2A receptor-selective radiopharmaceutical (123)I-R91150. A serotonin 2A receptor binding index (BI), proportional to the cortical receptor density, was calculated. A receiver operating characteristic (ROC) analysis was performed to determine cut-off values at which optimal sensitivity and specificity are achieved and to evaluate the general performance of the BI in reflecting the state of the dog, i.e., impulsive aggressive, normal or anxious. Significantly (P<0.0056) altered 5-HT 2A receptor binding indices were found in bilateral frontal, temporal and occipital cortical brain areas of the dogs behaving abnormally, with consistently increased BI in impulsive aggressive dogs and decreased BI in anxious dogs. These results provide clear evidence for a disturbed serotonergic balance in canine impulsive aggression and anxiety disorders. A right frontal cut-off value of ≥1.92 with 86.4% sensitivity and 2.3% (1-specificity) was obtained for the impulsive aggressive dogs. Differentiating the anxious dogs from the rest of the population was possible with a cut-off value of ≤1.73 with 86.4% sensitivity and 18.2% (1-specificity). We conclude that SPECT imaging with the radioligand (123)I-R91150 can be a helpful tool in evaluating the involvement of the serotonin 2A receptor in the complex mechanisms of impulsive aggressive and anxious behaviour. The 5HT-2A binding index of the right frontal cortex appears to be a valid biomarker in differentiating the studied canine behavioural disorders.     

The canine prostate cancer cell line CHP‐1 shows over‐expression of the co‐chaperone small glutamine‐rich tetratricopeptide repeat‐containing protein α

Although androgen therapy resistance and poor clinical outcomes are seen in most canine prostate cancer cases, there are only a few tools for analysing canine prostate cancer by using a cell biological approach. Therefore, to evaluate androgen‐independent neoplastic cell growth, a new canine prostate cancer cell line (CHP‐1) was established in this study. CHP‐1 over‐expressed the co‐chaperone small glutamine‐rich tetratricopeptide repeat‐containing protein α (SGTA), which is over‐expressed in human androgen‐independent prostate cancer. The CHP‐1 xenograft also showed SGTA over‐expression. Although CHP‐1 shows poor androgen receptor (AR) signalling upon dihydrotestosterone stimulation, forced expression of AR enabled evaluation of AR signalling. Taken together, these results suggest that CHP‐1 will be a useful model for investigating the pathogenesis of androgen‐dependent and androgen‐independent canine prostate cancer.     

Androgen receptor expression in normal, hyperplastic and neoplastic hepatoid glands in the dog

Neoplasms of the perianal glands are common in the dog, particularly in the male. The occurrence of these tumours appears to be hormone related and castration, without excision of the tumour, has sometimes resulted in regression of the tumour. The aim of this study was to investigate the expression of androgen receptors (AR) in normal, hyperplastic and neoplastic hepatoid glands in the dog. Thirty-one samples of canine hepatoid gland tissues were investigated. The lesions, classified according to WHO criteria, were comprised of 19 hyperplastic tissues, 10 benign lesions (2 hepatoid gland epithelioma and 8 hepatoid adenomas), and 19 carcinomas. Five samples from normal hepatoid glands were also investigated. The AR expression was evaluated by immunohistochemistry using a streptavidin-biotin peroxidase method. The immunoexpression was scored by two pathologists as the percentage of positive nuclei. The intensity of staining was also considered. AR expression was detected in all normal and abnormal glands. However, in hyperplastic tissues the percentage of positive nuclei was significantly higher than in normal tissue and especially in reserve basaloid cells. A similar increase in the percent of positive nuclei was also observed in hepatoid epitheliomas, while in hepatoid adenoma the percent of AR-immunolabelling was only slightly increased compared to normal tissue. In hepatoid carcinomas the percent of AR-positive cells was similar to that observed in benign tumours. The grade of differentiation of hepatoid carcinomas did not affect AR expression. These results demonstrate that increased AR expression is maintained throughout perianal gland cancer progression and that hepatoid gland carcinomas still express AR. Although further studies may be required to evaluate the hormonal background of these diseases, dogs bearing those carcinomas might benefit from castration or anti hormonal therapy.     

Neuro-imaging the serotonin 2A receptor as a valid biomarker for canine behavioural disorders

The serotonergic system is disturbed in different mood and affective disorders, with especially the serotonin (5-HT) 2A receptor involved in impulsive aggressiveness and anxiety. The aim of the study was to evaluate the involvement of the brain 5-HT 2A receptor in dogs with different behavioural disorders.Three groups of drug naive dogs were studied: 22 dogs showing impulsive aggressive behaviour, 22 showing normal behaviour, and 22 showing anxious behaviour. The serotonin 2A receptor was evaluated with Single Photon Emission Computed Tomography (SPECT) and the serotonin 2A receptor-selective radiopharmaceutical ¹²³I-R91150. A serotonin 2A receptor binding index (BI), proportional to the cortical receptor density, was calculated. A receiver operating characteristic (ROC) analysis was performed to determine cut-off values at which optimal sensitivity and specificity are achieved and to evaluate the general performance of the BI in reflecting the state of the dog, i.e., impulsive aggressive, normal or anxious.Significantly (P < 0.0056) altered 5-HT 2A receptor binding indices were found in bilateral frontal, temporal and occipital cortical brain areas of the dogs behaving abnormally, with consistently increased BI in impulsive aggressive dogs and decreased BI in anxious dogs. These results provide clear evidence for a disturbed serotonergic balance in canine impulsive aggression and anxiety disorders. A right frontal cut-off value of ⩾1.92 with 86.4% sensitivity and 2.3% (1-specificity) was obtained for the impulsive aggressive dogs. Differentiating the anxious dogs from the rest of the population was possible with a cut-off value of ⩽1.73 with 86.4% sensitivity and 18.2% (1-specificity).We conclude that SPECT imaging with the radioligand ¹²³I-R91150 can be a helpful tool in evaluating the involvement of the serotonin 2A receptor in the complex mechanisms of impulsive aggressive and anxious behaviour. The 5HT-2A binding index of the right frontal cortex appears to be a valid biomarker in differentiating the studied canine behavioural disorders.     

Aggressive dogs are characterized by low omega-3 polyunsaturated fatty acid status

Canine aggressive behaviour is one of the most common problems being reported by dog owners. However, the biochemical basis of this phenomenon remains unclear. In humans, alterations in omega-3 plasma polyunsatured fatty acids and elevated omega6/omega-3 ratio have been linked to behavioural alterations, including aggression. Thus far, however, the relationship between plasma polyunsatured fatty acid status and aggression has not been investigated in the dog. In the present study we sought to investigate whether polyunsatured fatty acid status could be altered in plasma of pathologically aggressive Canis familiaris. Eighteen adult male German Shepherd dogs, aged 4.9 ± 0.9 years, showing no clinical signs but aggression, were investigated. Eighteen healthy male dogs, aged 4.8 ± 0.7 years, with a negative history of behavioural and neurological disorders served as controls. Baseline fasting plasma polyunsatured fatty acid composition was determined by gas chromatography. Compared to normal dogs, aggressive dogs showed lower docosahexaenoic acid (22:6 n-3) concentrations and a higher omega6/omega-3 ratio. In addition, they showed reduced cholesterol and bilirubin concentrations compared to their normally behaving counterparts. Altogether, our results suggest that low omega-3 fatty acids may adversely impact behaviour in dogs, resulting in greater propensity to aggression. However, given the cross-sectional design of our study, we cannot claim any causal relationship between the presence of alterations in fatty acid status and canine aggressiveness. Whether omega-3 fatty acids supplementation may be useful to reduce aggressive behaviour in the dog deserves further investigation.     

Serum testosterone and estradiol 17-beta concentrations in 15 dogs with perineal hernia

Serum testosterone and estradiol 17-beta concentrations, and serum testosterone-to-estradiol ratio were evaluated in 15 dogs (greater than or equal to 5 years old) with perineal hernia (9 sexually intact males and 6 castrated males) and in 9 clinically normal sexually intact male dogs greater than or equal to 5 years old. There was no significant difference in serum testosterone-to-estradiol ratio between sexually intact male dogs with perineal hernia and clinically normal sexually intact male dogs. In castrated dogs with perineal hernia, serum testosterone concentration and testosterone-to-estradiol ratio were significantly (P less than 0.05) lower, compared with those values in sexually intact dogs with perineal hernia and in clinically normal sexually intact male dogs. There was no significant difference in serum estradiol 17-beta concentration among sexually intact male dogs with perineal hernia, castrated dogs with perineal hernia, and clinically normal sexually intact male dogs. Serum testosterone and estradiol 17-beta concentrations in dogs with perineal hernia did not differ from those values in clinically normal male dogs of the same age. Castration cannot be recommended for the treatment of perineal hernia unless a castration-responsive contributing factor such as prostatomegaly is identified, unless the pelvic diaphragm of dogs with perineal hernia has high sensitivity to normal or low serum testosterone and estradiol 17-beta concentrations, or unless there is documentation that other androgens and/or estrogens are involved.     

Estrogen and androgen receptor expression in relation to steroid concentrations in the adult boar epididymis

The steroid hormone regulation of the epididymis in a high estrogen producing animal like the boar is not currently understood. To test the hypothesis that the boar epididymis is an estrogen and androgen responsive tissue, the presence of estrogen and androgen receptors, in conjunction with steroid hormone concentrations were investigated in the boar epididymis. Epididymal (caput, corpus, cauda) and testicular samples of boars (1–2.5 years; n = 5) were collected for immunolocalization of estrogen receptor alpha (ER), estrogen receptor beta (ERβ) and androgen receptor (AR). Concentrations of testosterone, estradiol and estrogen conjugates (EC) in the tissue were also determined. AR and ERβ were localized in the principal and basal cells of all three epididymal regions. ER was localized in the principal cells of the caput, some cells of the corpus and was not present in the cauda. Testosterone (p < 0.0001), estradiol (p < 0.0001) and EC (p < 0.005) were significantly lower in the epididymis compared with the testis. The epididymal regions were not significantly different from each other for testosterone (p > 0.15) or estradiol (p > 0.09). EC were significantly higher in the corpus than either the caput (p = 0.003) or cauda (p = 0.002). These results suggest that the boar epididymis is responsive to both estrogens and androgens and that both steroid hormones are important for proper epididymal function. Since testosterone and estradiol concentrations are similar throughout the epididymis, regional differences in steroid hormone regulation are likely due to differences in receptor expression.     

Role of monocyte recruitment in hemangiosarcoma metastasis in dogs

Canine hemangiosarcoma (HSA) is a highly malignant tumour associated with short survival times because of early and widespread metastasis. In humans and rodents, monocytes play key roles in promoting tumour metastasis through stimulating tumour cell extravasation, seeding, growth and angiogenesis. Therefore, we investigated the potential association between monocyte infiltration and tumour metastasis in HSA and other common canine tumours. Immunohistochemistry was used to quantify CD18⁺ monocytes within metastases. We found that HSA metastases had significantly greater numbers of CD18⁺ monocytes compared with metastases from other tumour types. HSA cells were the highest producers of the monocyte chemokine CCL2, and stimulated canine monocyte migration in a CCL2 dependent manner. These results are consistent with the hypothesis that overexpression of CCL2 and recruitment of large numbers of monocytes may explain in part the aggressive metastatic nature of canine HSA. Thus, therapies designed to block monocyte recruitment may be an effective adjuvant strategy for suppressing HSA metastasis in dogs.     

Androgen receptor CAG repeat polymorphisms in canine prostate cancer

Background: Relatively shorter lengths of the polymorphic polyglutamine repeat‐1 of the androgen receptor (AR) have been associated with an increased risk of prostate cancer (PC) in humans. In the dog, there are 2 polymorphic CAG repeat (CAGr) regions. Objective: To investigate the relationship of CAGr length of the canine AR‐gene and the development of PC. Animals: Thirty‐two dogs with PC and 172 control dogs were used. Methods: DNA was extracted from blood. Both CAG repeats were amplified by polymerase chain reaction (PCR) and PCR products were sequenced. Results: In dogs with PC, CAG‐1 repeat length was shorter (P= .001) by an increased proportion of 10 repeats (P= .011) and no 12 repeats (P= .0017) than in the control dogs. No significant changes were found in CAG‐3 length distribution. CAG‐1 and CAG‐3 polymorphisms proved not to be in linkage disequilibrium. Breed difference in allelic distribution was found in the control group. Of the prostate‐disease sensitive breeds, a high percentage (64.5%) of the shortest haplotype 10/11 was found in the Doberman, whereas Beagles and German Pointers had higher haplotype 12/11 (47.1 and 50%). Bernese Mountain dogs and Bouvier dogs both shared a high percentage of 11 CAG‐1 repeats and 13 CAG‐3 repeats. Differences in (combined) allelic distributions among breeds were not significant. Conclusions and Clinical Importance: In this preliminary study, short CAG‐1 repeats in the AR‐gene were associated with an increased risk of developing canine PC. Although breed‐specific differences in allelic distribution of CAG‐1 and CAG‐3 repeats were found, these could not be related to PC risk.     

Epididymis of Viscacha (Lagostomus maximus maximus): A Morphological Comparative Study in Relation to Sexual Maturity

The morphological variations and the androgen receptor (AR) expression were studied in viscacha epididymis in relation to sexual maturity. The animals were divided into immature, pre‐pubertal and adult, according to their corporal weight and testicular histology. The epididymides were studied by light microscopy, immunohistochemistry for AR and morphometric analysis. In pre‐pubertal and adult animals, four well‐differentiated segments (initial, caput, corpus and cauda) were observed, while in immature animals, three segments were identified (initial‐caput segment, corpus and cauda). In each segment, the structural parameters and the relative cell distribution were different between the groups. The serum testosterone levels of pre‐pubertal and adults showed a very significant increase related to sexual maturity. The AR expression in epithelial and fibromuscular stromal cells was different between the groups. In conclusion, the present work demonstrates that the morphological characteristics of the viscacha epididymis vary while sexual maturity is reached, the development of initial and caput is subsequent to corpus and cauda development and the androgens might play an important role during this process.     

Comparison of thyroid analytes in dogs aggressive to familiar people and in non-aggressive dogs

A cross-sectional study was performed in order to examine the association between canine aggression to familiar people and serum concentrations of total thyroxine (TT4), free thyroxine (fT4), thyroxine autoantibodies (T4AA), total triiodothyronine (TT3), free triiodothyronine (fT3), triiodothyronine autoantibodies (T3AA), thyroid stimulating hormone (TSH), and thyroglobulin autoantibodies (TgAA). The subjects were 31 dogs historically aggressive to familiar people and 31 dogs with no history of aggression. Behavioral evaluation and physical examination were completed for each dog in addition to a complete blood count, serum chemistry panel, TT4, fT4 by equilibrium dialysis, TT3, fT3, TgAA, T3AA, and T4AA. Significant differences were found between the two groups with respect to only T4AA, which was increased in the aggressive group, but the concentrations for both groups were within the normal reference range. There were no differences between the two groups in the thyroid analytes most commonly measured by veterinary practitioners evaluating thyroid function in dogs. The results of this study revealed no significant difference between aggressive and non-aggressive dogs in the thyroid concentrations most commonly used to diagnose canine hypothyroidism.     

Estrogen-dependent induction of cyclooxygenase-2 in the canine prostate in vivo

Cyclooxygenase (COX)-2 is involved in several physiologic and pathologic processes. COX-2 is overexpressed in human and canine prostate cancer, but little is known about COX-2 inducers in the prostate. Our objective was to investigate the effect of sex steroid hormones on COX-2 expression in the canine prostate in vivo. COX-2 expression was evaluated by immunohistochemistry in intact and castrated dogs treated with exogenous androgen or estrogen. Results showed that no COX-2 staining was observed in prostates of untreated or androgen-treated castrated or intact dogs. However, treatment of intact and castrated dogs with estrogen resulted in squamous metaplasia with intense COX-2 expression observed in both squamous epithelial cells and in cells of acini without metaplasia. This is the first report to demonstrate the induction of COX-2 by estrogen in the prostate in vivo.     

Distribution of the androgen receptor in the diencephalon and the pituitary gland in goats: Co-localisation with corticotrophin releasing hormone, arginine vasopressin and corticotrophs

Previously it has been shown that androgen suppresses transportation-induced increases in plasma adrenocorticotropic hormone (ACTH), possibly by suppressing the secretion of corticotrophin releasing hormone (CRH) or arginine vasopressin (AVP) from the hypothalamus, or secretion of ACTH from the pituitary gland. The aim of the present study was to examine androgen target sites in the caprine diencephalon and pituitary gland using immunohistochemical methods. The androgen receptor (AR) was expressed strongly in the bed nucleus of the stria terminalis, the medial preoptic area, the arcuate nucleus, the ventromedial hypothalamic nucleus and the suprachiasmatic nucleus in the diencephalon. Between 8% and 11% of CRH and AVP neurons in the paraventricular hypothalamic nucleus (PVN) expressed AR. In the pituitary gland, 7.1% of corticotrophs expressed AR. The results are consistent with the proposal that androgen acts directly and indirectly on CRH and/or AVP neurons in the PVN. The possibility of a direct action of androgen on the corticotrophs in the pituitary gland was also considered.     

Canine storage disease characterized by hereditary progressive neurogenic muscular atrophy: breeding experiments and clinical manifestation

Progressive neurogenic muscular atrophy due to storage of a compound lipid in the lower motor neurons was diagnosed in 3 English Pointers that were littermates. Using 2 clinically normal littermates of these 3 affected dogs and 2 clinically normal dogs of the 2nd litter from the parents of the original 3 affected dogs as the initial breeding stock, a breeding experiment was performed, resulting in a breeding line of 26 dogs, 4 of which had the disease and 6 of which died before 3 months of age. Results indicated that the disease may have an autosomal recessive mode of inheritance. The clinical manifestation and electrophysiologic findings indicated lower motor neuron involvement in the affected dogs produced by breeding consistent with findings in the original 3 affected dogs. Upper motor neurons or the sensory system was not involved. The disease appeared to be distinct from other canine storage diseases previously reported.     

Clinicopathological study of canine transmissible venereal tumour in leishmaniotic dogs

Introduction : Canine transmissible venereal tumour is occasionally observed in leishmaniotic dogs, and Leishmania amastigotes can be harboured in canine transmissible venereal tumour cells. Objectives : The aim of this paper was to investigate the clinicopathological significance of the association of both diseases. Methods : Nineteen dogs affected by canine transmissible venereal tumour and canine leishmaniasis were studied retrospectively. Results : In these dogs, the tumour manifested a large size and often aggressive behaviour (42%) and no predictive sign of spontaneous regression was observed. Sporadic Leishmania amastigotes were found within the canine transmissible venereal tumour in three cases, probably transported by infected macrophages often infiltrating the tumour. A high Leishmania parasitisation of canine transmissible venereal tumour was observed in two other cases and verified by immunohistochemistry. Clinical Significance : Canine transmissible venereal tumour is a tumour of the dog able to harbour a large number of Leishmania parasites. Alternatively, the systemic disease (canine leishmaniasis) may lower the immune defence against malignancy (canine transmissible venereal tumour).     

Androgens promote the acquisition of maturation competence in bovine oocytes

Recent studies in mice suggest that androgens are important for normal follicle development. However, there have been few reports concerning the action of androgens in the growth of oocytes from large animals. The purpose of this study was to determine the roles of androgens in bovine oocyte growth in vitro. Oocyte-granulosa cell complexes (OGCs) collected from 0.4−0.7 mm early antral follicles were cultured for 14 days with 17β-estradiol (E₂) and a non-aromatizable androgen, dihydrotestosterone (DHT). We also examined the ability of an androgen receptor (AR) inhibitor, hydroxyflutamide, to antagonize the effect of androgens on the oocytes. During growth culture, the OGC structures collapsed in the medium with DHT alone, while in the presence of E₂, the OGC structures were maintained. In the medium with both androgens and E₂, the mean diameter of oocytes was increased from 95 μm to around 120 μm, larger than those grown with E₂ alone (115 μm). Also in the maturation culture, oocytes grown with androgens (A₄ or DHT) and E₂ showed higher percentages of metaphase II oocytes (63% or 69%, respectively) than those grown with E₂ alone (32%). Moreover, these maturation rates were decreased by hydroxyflutamide in a dose-dependent manner. Immunostaining showed that ARs were expressed in oocytes and granulosa cells in early antral follicles, and the nuclei of granulosa cells showed intense AR expression. In conclusion, although E₂ supports the OGC structure, additional androgens promote oocyte growth and their acquisition of meiotic competence via AR during in vitro growth culture.