Treating canine Cushing's syndrome: Current options and future prospects.

Naturally occurring hypercortisolism, also known as Cushing's syndrome, is a common endocrine disorder in dogs that can be caused by an adenocorticotrophic hormone (ACTH)-producing pituitary adenoma (pituitary-dependent hypercortisolism, PDH; 80-85% of cases), or by an adrenocortical tumor (ACT; 15-20% of cases). To determine the optimal treatment strategy, differentiating between these two main causes is essential. Good treatment options are surgical removal of the causal tumor, i.e. hypophysectomy for PDH and adrenalectomy for an ACT, or radiotherapy in cases with PDH. Because these options are not without risks, not widely available and not suitable for every patient, pharmacotherapy is often used. In cases with PDH, the steroidogenesis inhibitor trilostane is most often used. In cases with an ACT, either trilostane or the adrenocorticolytic drug mitotane can be used. Although mostly effective, both treatments have disadvantages. This review discusses the current treatment options for canine hypercortisolism, and considers their mechanism of action, efficacy, adverse effects, and effect on survival. In addition, developments in both adrenal-targeting and pituitary-targeting drugs that have the potential to become future treatment options are discussed, as a more selective and preferably also tumor-targeted approach could have many advantages for both PDH and ACTs.

Steroidogenic factor-1 inverse agonists as a treatment option for canine hypercortisolism: in vitro study.

Hypercortisolism is one of the most commonly diagnosed endocrinopathies in dogs, and new targeted medical treatment options are desirable. Steroidogenic factor-1 (SF-1), an orphan nuclear hormone receptor, is a key regulator of adrenal steroidogenesis, development, and growth. In pituitary-dependent hypercortisolism (PDH), high plasma ACTH concentrations increase the transcriptional activity of SF-1. In adrenal-dependent hypercortisolism, SF-1 expression is significantly greater in dogs with recurrence after adrenalectomy than in those without recurrence. Inhibition of SF-1 could therefore be an interesting treatment option in canine spontaneous hypercortisolism. We determined the effects of 3 SF-1 inverse agonists, compounds IsoQ A, #31, and #32, on cortisol production, on the messenger RNA (mRNA) expression of steroidogenic enzymes and SFs, and on cell viability, in primary adrenocortical cell cultures of 8 normal adrenal glands and of 3 cortisol-secreting adrenocortical tumors (ATs). To mimic PDH, the normal adrenocortical cell cultures were stimulated with ACTH. The results show that only compound #31 inhibited cortisol production and SF-1 target gene expression in non-ACTH-stimulated and ACTH-stimulated normal adrenocortical cells but did not affect cell viability. In the AT cell cultures, the effects of #31 on cortisol production and target gene expression were variable, possibly caused by a difference in the SF-1 mRNA expressions of the primary tumors. In conclusion, inhibition of SF-1 activity shows much promise as a future treatment for canine hypercortisolism.

In vitro and in vivo effects of kisspeptin antagonists p234, p271, p354, and p356 on GPR54 activation.

Kisspeptins (KPs) and their receptor (GPR54 or KiSS1R) play a key-role in regulation of the hypothalamic-pituitary-gonadal axis and are therefore interesting targets for therapeutic interventions in the field of reproductive endocrinology. As dogs show a rapid and robust LH response after the administration of KP10, they can serve as a good animal model for research concerning KP signaling. The aims of the present study were to test the antagonistic properties of KP analogs p234, p271, p354, and p356 in vitro, by determining the intracellular Ca2+ response of CHEM1 cells that stably express human GPR54, and to study the in vivo effects of these peptides on basal plasma LH concentration and the KP10-induced LH response in female dogs. Exposure of the CHEM1 cells to KP-10 resulted in a clear Ca2+ response. P234, p271, p354, and p356 did not prevent or lower the KP10-induced Ca2+ response. Moreover, the in vivo studies in the dogs showed that none of these supposed antagonists lowered the basal plasma LH concentration and none of the peptides lowered the KP10-induced LH response. In conclusion, p234, p271, p354, and p356 had no antagonistic effects in vitro nor any effect on basal and kisspeptin-stimulated plasma LH concentration in female dogs.

The Influence of Pituitary Size on Outcome After Transsphenoidal Hypophysectomy in a Large Cohort of Dogs with Pituitary-Dependent Hypercortisolism.

BACKGROUND: Transsphenoidal hypophysectomy is one of the treatment strategies in the comprehensive management of dogs with pituitary-dependent hypercortisolism (PDH). OBJECTIVES: To describe the influence of pituitary size at time of pituitary gland surgery on long-term outcome. ANIMALS: Three-hundred-and-six dogs with PDH. METHODS: Survival and disease-free fractions were analyzed and related to pituitary size; dogs with and without recurrence were compared. RESULTS: Four weeks after surgery, 91% of dogs were alive and remission was confirmed in 92% of these dogs. The median survival time was 781 days, median disease-free interval was 951 days. Over time, 27% of dogs developed recurrence of hypercortisolism after a median period of 555 days. Dogs with recurrence had significantly higher pituitary height/brain area (P/B) ratio and pre-operative basal urinary corticoid-to-creatinine ratio (UCCR) than dogs without recurrence. Survival time and disease-free interval of dogs with enlarged pituitary glands was significantly shorter than that of dogs with a non-enlarged pituitary gland. Pituitary size at the time of surgery significantly increased over the 20-year period. Although larger tumors have a less favorable prognosis, outcome in larger tumors improved over time. CONCLUSIONS AND CLINICAL IMPORTANCE: Transsphenoidal hypophysectomy is an effective treatment for PDH in dogs, with an acceptable long-term outcome. Survival time and disease-free fractions are correlated negatively with pituitary gland size, making the P/B ratio an important pre-operative prognosticator. However, with increasing experience, and for large tumors, pituitary gland surgery remains an option to control the pituitary mass and hypercortisolism.

The effects of kisspeptin agonist canine KP-10 and kisspeptin antagonist p271 on plasma LH concentrations during different stages of the estrous cycle and anestrus in the bitch.

Kisspeptin (KP) plays a key role in the regulation of the hypothalamic-pituitary-gonadal axis via the release of GnRH. As normal KP signaling is essential for reproductive function, it could be an interesting new target for therapeutic interventions, e.g., nonsurgical contraception in dogs. The aims of the present study were to investigate the effect of KP-10 administration on plasma LH concentration in different stages of the reproductive cycle and to investigate the suitability of p271 as KP antagonist in the bitch. Two groups of six adult Beagle bitches were used. In one group, plasma LH concentration was determined before (40 and 0 minutes) and 10, 20, 40, and 60 minutes after the intravenous administration of 0.5-µg/kg body weight (BW) canine KP-10. In the other group, the bitches received a continuous intravenous infusion with p271 (50 µg/kg BW/h) for 3 hours, and 0.5-µg/kg BW canine KP-10 was administered intravenously 2 hours after the start of the p271 infusion. Their plasma LH concentration was determined before (-40 and 0 minutes) and 30, 60, 90, 120, 130, 140, 160, and 180 minutes after the start of the p271 infusion. In both groups, the experiments were performed during the follicular phase, the first and second half of the luteal phase, and during anestrus. Canine KP-10 induced an increase of plasma LH concentration during all estrous cycle stages and anestrus. There was no difference in LH response between the two groups. The lowest LH response was seen during the follicular phase and the highest response during anestrus. The area under the curve (AUC) for LH and LH increment in the follicular phase were lower than those in anestrus. The AUC LH and LH increment in the first half of the luteal phase were lower than those in the second half of the luteal phase and anestrus. The AUC LH and LH increment in the second half of the luteal phase were not different from those in anestrus. Continuous administration of the antagonist p271 did not alter basal plasma LH concentration and could not prevent or lower the LH response to KP-10 in any of the cycle stages and anestrus. It can be concluded that the LH response to KP-10 is dependent on estrous cycle stage and that peripheral administrated p271 cannot be used as KP antagonist in the dog. This provides new insight in reproductive endocrinology of the bitch, which is important when KP signaling is considered for therapeutic interventions, such as for estrus induction or nonsurgical contraception in the bitch.

The prognostic value of perioperative profiles of ACTH and cortisol for recurrence after transsphenoidal hypophysectomy in dogs with corticotroph adenomas.

BACKGROUND: Transsphenoidal hypophysectomy is an effective treatment for dogs with pituitary-dependent hypercortisolism (PDH). However, long-term recurrence of hypercortisolism is a well-recognized problem, indicating the need for reliable prognostic indicators. OBJECTIVES: The aim of this study was to evaluate the prognostic value of perioperative plasma ACTH and cortisol concentrations for identifying recurrence of hypercortisolism after transsphenoidal hypophysectomy. ANIMALS: A total of 112 dogs with PDH that underwent transsphenoidal hypophysectomy met the inclusion criteria of the study. METHODS: Hormone concentrations were measured preoperatively and 1-5 hours after surgery. Both absolute hormone concentrations and postoperative concentrations normalized to preoperative concentrations were included in analyses. The prognostic value of hormone concentrations was studied with Cox's proportional hazard analysis. RESULTS: Median follow-up and disease-free period were 1096 days and 896 days, respectively. Twenty-eight percent of patients had recurrence, with a median disease-free period of 588 days. Both absolute and normalized postoperative cortisol concentrations were significantly higher in dogs with recurrence than in dogs without recurrence. High ACTH 5 hours after surgery, high cortisol 1 and 4 hours after surgery, high normalized ACTH 3 hours after surgery, high normalized cortisol 4 hours after surgery and the random slope of cortisol were associated with a shorter disease-free period. CONCLUSIONS AND CLINICAL IMPORTANCE: Individual perioperative hormone curves provide valuable information about the risk of recurrence after hypophysectomy. However, because no single cutoff point could be identified, combination with other variables, such as the pituitary height/brain area (P/B) ratio, is still needed to obtain a good estimate of the risk for recurrence of hypercortisolism after hypophysectomy.

Insulin-like growth factor--phosphatidylinositol 3 kinase signaling in canine cortisol-secreting adrenocortical tumors.

BACKGROUND: Hypercortisolism is a common endocrine disorder in dogs, caused by a cortisol-secreting adrenocortical tumor (AT) in approximately 15% of cases. In adrenocortical carcinomas of humans, activation of the phosphatidylinositol 3 kinase (PI3K) signaling pathway by insulin-like growth factor (IGF) signaling represents a promising therapeutic target. OBJECTIVES: To investigate the involvement of PI3K signaling in the pathogenesis of ATs in dogs and to identify pathway components that may hold promise as future therapeutic targets or as prognostic markers. ANIMALS: Analyses were performed on 36 canine cortisol-secreting ATs (11 adenomas and 25 carcinomas) and 15 normal adrenal glands of dogs. METHODS: mRNA expression analysis was performed for PI3K target genes, PI3K inhibitor phosphatase and tensin homolog (PTEN), IGFs, IGF receptors, IGF binding proteins and epidermal growth factor receptors. Mutation analysis was performed on genes encoding PTEN and PI3K catalytic subunit (PIK3CA). RESULTS: Target gene expression indicated PI3K activation in carcinomas, but not in adenomas. No amino acid-changing mutations were detected in PTEN or PIK3CA and no significant alterations in IGF-II or IGFR1 expression were detected. In carcinomas, ERBB2 expression tended to be higher than in normal adrenal glands, and higher expression of inhibitor of differentiation 1 and 2 (ID1 and ID2) was detected in carcinomas with recurrence within 2.5 years after adrenalectomy. CONCLUSIONS AND CLINICAL IMPORTANCE: Based on these results, ERBB2 might be a promising therapeutic target in ATs in dogs, whereas ID1 and 2 might be valuable as prognostic markers and therapeutic targets.

Clinical, pathologic, and immunohistochemical prognostic factors in dogs with thyroid carcinoma.

BACKGROUND: Prognostic markers for dogs with thyroid tumors are limited. HYPOTHESIS/OBJECTIVES: To identify clinical, pathologic, and immunohistochemical prognostic factors for dogs with thyroid tumors. ANIMALS: Seventy dogs with thyroid neoplasia. METHODS: Retrospective study. Dogs with thyroid neoplasia were included when follow-up information and formalin-fixed paraffin-embedded tumor samples were available. Immunohistochemistry (IHC) was performed for thyroglobulin, calcitonin, Ki-67, and E-cadherin. Correlation of tumor variables (diameter, volume, localization, scintigraphic uptake, thyroid function, IHC) with local invasiveness and metastatic disease was performed on all tumor samples. Forty-four dogs treated by thyroidectomy were included in a survival analysis. RESULTS: Fifty dogs (71%) had differentiated follicular cell thyroid carcinoma (dFTC) and 20 (29%) had medullary thyroid carcinoma (MTC). At diagnosis, tumor diameter (P = .007; P = .038), tumor volume (P = .020), tumor fixation (P = .002), ectopic location (P = .002), follicular cell origin (P = .044), and Ki-67 (P = .038) were positively associated with local invasiveness; tumor diameter (P = .002), tumor volume (P = .023), and bilateral location (P = .012) were positively associated with presence of distant metastases. Forty-four dogs (28 dFTC, 16 MTC; stage I-III) underwent thyroidectomy. Outcome was comparable between dogs with dFTC and MTC. Macroscopic (P = .007) and histologic (P = .046) vascular invasion were independent negative predictors for disease-free survival. Although time to presentation, histologic vascular invasion and Ki-67 were negatively associated with time to metastases, and time to presentation was negatively associated with time to recurrence, no independent predictors were found. E-cadherin expression was not associated with outcome. CONCLUSIONS AND CLINICAL IMPORTANCE: Prognostic factors have been identified that provide relevant information for owners and clinicians.

Upregulation of the PI3K/Akt pathway in the tumorigenesis of canine thyroid carcinoma.

BACKGROUND: Information on the genetic events leading to thyroid cancer in dogs is lacking. HYPOTHESIS/OBJECTIVES: Upregulation of the PI3K/Akt pathway has an important role in the tumorigenesis of thyroid carcinoma in dogs. ANIMALS: Fifty-nine dogs with thyroid carcinoma and 10 healthy controls. METHODS: Quantitative RT-PCR was performed for VEGFR-1, VEGFR-2, EGFR, PIK3CA, PIK3CB, PDPK1, PTEN, AKT1, AKT2, COX-2, and CALCA. Mutation analysis was performed for known hotspots of RAS (N, K, H), PIK3CA, BRAF, RET, and for the entire coding region of PTEN. RESULTS: Forty-three dogs (73%) had follicular cell thyroid carcinoma (FTC) and 16 dogs (27%) had medullary thyroid carcinoma (MTC). The relative mRNA expressions of VEGFR-1 (P < .001), VEGFR-2 (P = .002), PDPK1 (P < .001), AKT1 (P = .009), and AKT2 (P < .001) were increased in FTC, and those of EGFR (P < .001), VEGFR-1 (P = .036), and PIK3CA (P = .019) were increased in MTC when compared to normal thyroid glands. Mutation analysis of K-RAS identified 2 activating missense mutations, which also have been described in thyroid cancer of humans. A G12R substitution was present in 1 FTC and an E63K substitution was present in 1 MTC. No functional mutations were found in the sequenced regions of H-RAS, N-RAS, PIK3CA, BRAF, RET, and PTEN. CONCLUSIONS AND CLINICAL IMPORTANCE: The increased expression of several genes associated with PI3K/Akt signaling suggests the involvement of this pathway in the pathogenesis of thyroid carcinoma in dogs, warranting further research on pathway activation and gene amplification. The mutations most frequently associated with thyroid cancer in humans are rare in dogs.

Expression of steroidogenic factor 1 in canine cortisol-secreting adrenocortical tumors and normal adrenals.

We report on a screening for the relative messenger RNA (mRNA) and protein expression of steroidogenic factor 1 (SF-1) in normal canine adrenals (n = 10) and cortisol-secreting adrenocortical tumors (11 adenomas and 26 carcinomas). The relative mRNA expression of SF-1 was determined by quantitative real-time polymerase chain reaction analysis and revealed no differences between normal adrenals, adenomas, and carcinomas. Immunohistochemistry demonstrated SF-1 protein expression in a nuclear pattern throughout the normal adrenal cortex and a predominantly nuclear staining pattern in adrenocortical tumors. Of the 15 dogs available for follow up, 7 dogs developed hypercortisolism within 2.5 yr after adrenalectomy, with metastatic disease in 6 dogs and adrenocortical tumor regrowth in 1 dog. The relative SF-1 mRNA expression in dogs with early recurrence was greater (2.46-fold, P = 0.020) than in dogs in remission for at least 2.5 yr after adrenalectomy. In conclusion, we demonstrated the presence of SF-1 expression in normal canine adrenals and adrenocortical tumors. The high SF-1 mRNA expression in carcinomas with early recurrence might indicate its value as a prognostic marker, as well as its potential for therapeutic development.

Immunohistochemical expression of potential therapeutic targets in canine thyroid carcinoma.

BACKGROUND: Thyroid carcinoma is a common endocrine tumor in the dog. Local invasive growth frequently precludes surgical excision and, in up to 38% of dogs, the tumor has already metastasized by the time of diagnosis. Therefore, it is important to investigate new treatment modalities that may be useful for the large number of dogs with inoperable tumors or metastatic disease. HYPOTHESIS/OBJECTIVES: To investigate the immunohistochemical expression of potential therapeutic targets in canine thyroid tumors. ANIMALS: 74 dogs with thyroid neoplasia. METHODS: Immunohistochemistry was performed for thyroglobulin, calcitonin, vascular endothelial growth factor (VEGF), p53, cycloxygenase-2 (cox-2), and P-glycoprotein (P-gp). RESULTS: Fifty-four (73%) tumors were classified as follicular cell thyroid carcinomas (FTCs) and 20 (27%) as medullary thyroid carcinomas (MTCs). Eighty percent of FTCs and all MTCs had a high percentage (76-100%) of neoplastic cells immunopositive for VEGF. Thirteen percent of FTCs and 50% of MTCs expressed cox-2. Seven percent of FTCs and 70% of MTCs expressed P-gp. No tumor was immunopositive for p53 expression. Expression of VEGF (P = .034), cox-2 (P = .013), and P-gp (P < .001) was significantly higher in MTCs compared to FTCs. CONCLUSIONS AND CLINICAL IMPORTANCE: VEGF is a potential therapeutic target in both FTC and MTC in dogs. Cox-2 and P-gp may be useful molecular targets in canine MTC.

Expression of angiogenesis-related genes in canine cortisol-secreting adrenocortical tumors.

The aim of this study was to evaluate the expression of angiogenesis-related genes in canine cortisol-secreting adrenocortical tumors (ATs). Quantitative RT-PCR analysis revealed mRNA encoding for vascular endothelial growth factor, vascular endothelial growth factor receptors 1 and 2, angiopoietin 1 and 2 (ANGPT1 and ANGPT2), the splice variant ANGPT2443, the ANGPT-receptor Tie2, and basic fibroblast growth factor in 38 canine cortisol-secreting ATs (26 carcinomas and 12 adenomas) and 15 normal adrenals. The relative expression of both ANGPT2 and ANGPT2443 was higher in adenomas (P = 0.020 for ANGPT2 and P = 0.002 for ANGPT2443) and carcinomas (P = 0.003 for ANGPT2 and P < 0.001 for ANGPT2443) compared with normal adrenals, and this enhanced expression was also detected with Western blot analysis. Immunohistochemistry indicated expression of ANGPT2 protein in AT cells and in vascular endothelial cells of carcinomas, whereas Tie2 was mainly present in the tumor vascular endothelial cells. The ANGPT2-to-ANGTPT1 ratio, a marker for a proangiogenic state, was higher in both adenomas (P = 0.020) and carcinomas (P = 0.043). With the use of the human H295R cortisol-producing adrenocortical carcinoma cell line, we were able to demonstrate that the ANGPT2 expression was stimulated by cyclic adenosine monophosphate and progesterone but not by cortisol. In conclusion, canine cortisol-secreting ATs have enhanced ANGPT2 expression with a concomitant shift toward a proangiogenic state. On the basis of this information, treatment modalities may be developed that interfere with ANGPT2 expression, including inhibition of the cyclic adenosine monophosphate/protein kinase A pathway, or of the effect of ANGPT2, by using specific ANGPT2 inhibitors.

Activating mutations of GNAS in canine cortisol-secreting adrenocortical tumors.

BACKGROUND: Cushing's syndrome or hypercortisolism is a common endocrinopathy in dogs. In approximately 15% of cases, the disorder is caused by adrenocorticotropin (ACTH)-independent hypersecretion of cortisol by an adrenocortical tumor (AT). Without other explanation, the cortisol hypersecretion has been referred to as autonomous. OBJECTIVES: To investigate whether ACTH-independent hypersecretion of cortisol may be associated with aberrant activation of the melanocortin 2 receptor (MC2R)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway. ANIMALS: All analyses were performed on 44 cortisol-secreting ATs (14 adenomas and 30 carcinomas) derived from dogs diagnosed with ACTH-independent hypercortisolism. METHODS: Mutation analysis was performed of genes encoding the stimulatory G protein alpha subunit (GNAS), MC2R, and PKA regulatory subunit 1A (PRKAR1A) in all ATs. RESULTS: Approximately one-third of all ATs harbored an activating mutation of GNAS. Missense mutations, known to result in constitutive activation, were present in codon 201 in 11 ATs, in codon 203 (1 AT), and in codon 227 (3 ATs). No functional mutations were found in MC2R and PRKAR1A. CONCLUSIONS AND CLINICAL IMPORTANCE: Activation of cAMP signaling is a frequent event in canine cortisol-secreting ATs and may play a crucial role in both ACTH-independent cortisol production and tumor formation. To the best of our knowledge, this is the first report of potentially causative mutations in canine cortisol-secreting ATs.

Evaluation of the oral fludrocortisone suppression test for diagnosing primary hyperaldosteronism in cats.

BACKGROUND: Primary hyperaldosteronism (PHA) in cats is suggested by clinical signs and an elevated plasma aldosterone-to-renin ratio (ARR), but a test to confirm the diagnosis is lacking. HYPOTHESIS: Fludrocortisone does not suppress urinary aldosterone excretion in cats with PHA, but does so in cats with arterial hypertension because of other causes. ANIMALS: Nineteen client-owned cats with arterial hypertension because of PHA (n = 9) or other causes (n = 10). METHODS: Prospective clinical study. The urinary aldosterone-to-creatinine ratio (UACR) was determined in morning urine before, during, and after 4 days of oral fludrocortisone administration in a dose of 0.05 mg/kg q12h. Arterial blood pressure and plasma potassium concentration were measured before and after fludrocortisone administration. RESULTS: A basal UACR above 46.5 × 10(-9), the upper limit of the reference range, was found in 3 cats with PHA. All PHA cats had basal UACRs >7.5 × 10(-9). In all non-PHA cats with a basal UACR >7.5 × 10(-9), fludrocortisone administration induced >50% suppression. In contrast, fludrocortisone administration resulted in <50% suppression in 6 of the 9 PHA cats. Neither basal UACR, nor UACR after suppression testing, correlated with the etiology of PHA (adenoma, adenocarcinoma, or suspected bilateral hyperplasia of the zona glomerulosa). Fludrocortisone induced hypokalemia in 7 cats, but did not induce or exacerbate arterial hypertension. CONCLUSIONS AND CLINICAL IMPORTANCE: Measuring the UACR before and after 4 days of administering fludrocortisone is a practical method of confirming most cases of PHA in cats, and of substantiating the absence of PHA in cats having an ARR within the reference range.

Disorders of sexual development and associated changes in the pituitary-gonadal axis in dogs.

Normal sexual differentiation depends on completion of chromosomal sex determination, gonadal differentiation, and development of the phenotypic sex. An irregularity in any of these three steps can lead to a disorder in sexual development (DSD). We examined nine dogs with DSD by abdominal ultrasonography, laparotomy, histologic examination of the gonads, and reproductive tract, cytogenetic analysis, and mRNA expression of the SRY gene. We also determined the plasma concentrations of luteinizing hormone (LH), estradiol-17ß, and testosterone before and after administration of gonadotropin-releasing hormone (GnRH) and compared these results with those obtained in anestrous bitches and male control dogs. The gonads of three dogs with DSD contained both testicular and ovarian tissue, while in the other six only testicular tissue was found. Each of the dogs had a uterus. Based on gynecologic examination, cytogenetic analysis, and the histology of the gonads, seven of the nine dogs appeared to be XX sex reversals. Three of these were XX true hermaphrodites and four were XX males; the other two dogs had incomplete XY gonadal dysgenesis. All seven XX sex-reversed dogs were found to be negative for the SRY gene by polymerase chain reaction. The basal plasma luteinizing hormone (LH) concentration was significantly higher in dogs with DSD than in anestrous bitches but not significantly different from that in male dogs. The basal plasma LH concentration increased significantly after GnRH administration in all dogs with DSD. The basal plasma estradiol concentration was significantly higher in dogs with DSD than in anestrous bitches but not significantly different from that in male dogs. The basal plasma testosterone concentration was lower in dogs with DSD than in male dogs. In all dogs with DSD both the basal and GnRH-induced plasma testosterone concentrations were above the upper limit of their respective ranges in the anestrous bitches. In conclusion, the secretion of LH and estradiol in these dogs with DSD, all of which had testicular tissue in their gonads, was similar to that in male control dogs. These results indicate that the basal and/or GnRH-stimulated plasma testosterone concentration might be used to detect the presence of testicular tissue in dogs with DSD.

Progesterone receptor isoforms in the mammary gland of cats and dogs.

Progesterone exerts its effect by binding to specific progesterone receptors (PR) within the cell. In dogs and cats, no data are available on PR isoforms as found in other species. We therefore investigated the sequence of the PR gene and encoded protein in dogs and cats, the expression of PR isoforms in mammary tissue using Western blots and the presence of PR in mammary tissue using immunohistochemistry. Comparison of the amino acid sequence of the canine and feline PR with human PR revealed major differences in the PR-B-specific upstream segment (BUS). However, the essential activation function 3 (AF3) domain was intact in the cat but mutated in the dog. The DNA and ligand-binding domains were highly similar among the species. In cats with fibroadenomatous hyperplasia (FAH), high expression of PR mRNA together with growth hormone (GH), GH receptor (GHR) and IGF-I mRNA was found in comparison with feline mammary carcinomas. Immunohistochemical analysis showed strong nuclear as well as cytoplasmic staining for PR in FAH. Western blot analysis revealed expression of the PR-A and PR-B isoforms in the feline mammary gland. In canine mammary tissue, the most abundant PR staining was found in proliferative zones of the mammary gland. Western blot analyses showed mainly staining for PR-A with lower PR-B staining. It is concluded that in dogs and cats both PR isoforms are expressed. The role of mutations found in the canine PR-B is discussed.

Effects of gonadotropin-releasing hormone administration on the pituitary-gonadal axis in male and female dogs before and after gonadectomy.

GnRH-stimulation tests were performed in 14 female and 14 male client-owned dogs of several breeds, before and 4 to 5 mo after gonadectomy. The aim of the study was to obtain more insight into the pituitary-gonadal axis in intact and neutered dogs and to establish reference values. Basal plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations were increased significantly after gonadectomy in both bitches and male dogs. In both males and females ranges of the basal plasma FSH concentrations, before and after gonadectomy, did not overlap as opposed to the overlap in ranges of the basal plasma LH concentrations. Before gonadectomy basal plasma LH concentrations were lower and basal plasma FSH concentrations were higher in bitches than in male dogs. After gonadectomy these basal values did not differ significantly. GnRH administration before gonadectomy resulted in an increase in plasma LH and FSH concentrations in both genders. GnRH administration after gonadectomy produced an increase only in plasma LH concentrations in both genders, and a just significant increase in plasma FSH in castrated male dogs. GnRH administration before gonadectomy resulted in a significant increase in plasma testosterone concentration in both genders. In males ranges of basal and GnRH-stimulated plasma testosterone concentrations before and after gonadectomy did not overlap. Basal plasma estradiol concentrations were significantly higher in intact males than in castrated males and their ranges did not overlap. The basal estradiol concentrations in bitches before and after ovariectomy were not significantly different. At 120 min after GnRH administration, ranges of plasma estradiol concentration of intact and ovariectomized bitches no longer overlapped. In conclusion, basal plasma FSH concentration appears to be more reliable than basal plasma LH concentration for verification of neuter status in both male and female dogs. The basal plasma testosterone concentration appears to be reliable for verification of neuter status in male dogs. The plasma estradiol concentration at 120 min after GnRH administration can be used to discriminate between bitches with and without functional ovarian tissue.

Effects of the 3ß-hydroxysteroid dehydrogenase inhibitor trilostane on luteal progesterone production in the dog.

Interference with the pregnancy-maintaining influence of progesterone is the basis of most methods for termination of unwanted pregnancy in dogs. The currently available methods are based on induction of luteolysis or blocking of the progesterone receptor. Inhibition of progesterone synthesis using a competitive inhibitor of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) could be another strategy to terminate unwanted pregnancies. In this study we investigated the effects of the 3ß-HSD inhibitor trilostane on corpus luteum function in non-pregnant bitches. Trilostane was administered orally for seven consecutive days in either the pituitary-independent part of the luteal phase (PIP, start of treatment on D11 after ovulation, n = 6) or the pituitary-dependent part (PDP, start of treatment on D31 after ovulation, n = 6), in an oral dose of about 4.5 mg/kg bw, twice daily. Results were compared with those obtained in control bitches (n = 6). ACTH stimulation tests were performed to assess adrenocortical reserve capacity. Trilostane caused no apparent side effects and ACTH stimulation tests revealed good suppression of cortisol secretion. Trilostane also caused a significant decrease in plasma progesterone concentration. When it was stopped during PIP, progesterone secretion was completely restored and there was no difference in the length of the luteal phase between those dogs and control dogs (99 days, range 70-138 d and 99 d, range 60-112 d, respectively). When trilostane was stopped during PDP there was no post-treatment recovery of progesterone secretion and although the luteal phase tended to be shorter (66 d, range 41-101 d) the difference was not significant (P = 0.09). Plasma prolactin concentration did not increase after the trilostane-induced decrease in plasma progesterone. The interoestrous interval in dogs treated during PIP (234 d, range 175-269 d) or PDP (198 d, range 120-287 d) was not significantly shorter than the control interval (247 d, range 176-313 d). In conclusion, trilostane treatment was effective in decreasing plasma progesterone concentration in bitches during the luteal phase, but the dose regimen used in this study produced less clear-cut inhibition of ovarian steroidogenesis than have other strategies to decrease plasma progesterone concentration. Further studies are warranted to determine whether trilostane can be used to terminate unwanted pregnancy in the bitch without inducing adrenocortical insufficiency.

The pituitary-ovarian axis in dogs with remnant ovarian tissue.

It can be difficult to confirm the presence of remnant ovarian tissue (ROT) in bitches that are presumed to be ovariohysterectomised. A GnRH stimulation test can be used to distinguish ovariectomised bitches from those in anoestrus, but it is uncertain whether the GnRH-induced changes in plasma LH and oestradiol concentrations that occur in intact bitches also occur in ROT-bitches. We report here eighteen ROT-bitches and compare the results of GnRH stimulation tests with those of six ovariectomised and six bitches in anoestrus. The basal (n = 17) and/or GnRH-stimulated (n = 18) plasma oestradiol concentration was above the detection limit of the assay, i.e., < 7 pmol/l, in all ROT-bitches but below the detection limit in all ovariectomised bitches. Basal plasma LH concentration was significantly higher in ROT-bitches (4.1 ± 0.7 µg/L) than those in anoestrus (0.64 ± 0.04 µg/L), and significantly lower than in ovariectomised bitches (20.2 ± 3.6 µg/L). Basal plasma LH concentration was relatively high in bitches in which there was a long interval between ovariectomy and appearance of oestrus. GnRH administration resulted in a significant increase in plasma LH and oestradiol concentrations in ROT-bitches. The GnRH-induced increase and subsequent decline in plasma LH concentration were significantly less in ROT-bitches than in either ovariectomised bitches or those in anoestrus. The GnRH-induced increase in plasma oestradiol concentration was significantly smaller in ROT-bitches than in those in anoestrus. In conclusion, the results of this study demonstrate that in dogs ROT is associated with noticeable changes in the pituitary-ovarian axis and suggest that a GnRH stimulation test may be used to distinguish between completely ovariectomised bitches and those with ROT.

Expression of the ACTH receptor, steroidogenic acute regulatory protein, and steroidogenic enzymes in canine cortisol-secreting adrenocortical tumors.

Studies of human adrenocortical tumors (ATs) causing Cushing's syndrome suggest that hypersecretion of cortisol is caused by altered expression of steroidogenic enzymes and that steroidogenesis can only be maintained when there is expression of the ACTH receptor (ACTH-R). Here we report the screening for the mRNA expression of the ACTH-R, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase, 21-hydroxylase (all in 38 cortisol-secreting ATs), 17α-hydroxylase, and 11ß-hydroxylase (both in 28 cortisol-secreting ATs). Real-time PCR (RT-PCR) was applied in all samples and was compared with that in normal canine adrenal glands. Messenger-RNA encoding StAR, steroidogenic enzymes, and ACTH-R were present in both normal adrenal glands and cortisol-secreting ATs. The amounts of mRNA encoding StAR and enzymes of the steroidogenic cluster needed for cortisol production did not differ significantly between either adenomas or carcinomas and normal adrenal glands. The amount of mRNA encoding ACTH-R was significantly lower in carcinomas than in normal adrenal glands (P = 0.008). In conclusion, RT-PCR analysis revealed no overexpression of StAR and steroidogenic enzymes in canine cortisol-secreting ATs. Significant downregulation of ACTH-R in carcinomas might be associated with the malignant character of the AT.