Conduct, Oversight, and Ethical Considerations of Clinical Trials in Companion Animals with Cancer: Report of a Workshop on Best Practice Recommendations.

Development of effective and safe treatments for companion animals with cancer requires the collaboration of numerous animal health professionals and the full engagement of animal owners. Establishing 'Best Practice Recommendations' for clinical trials in veterinary oncology represents an important step toward meeting the goal of rigorous clinical trial design and conduct that is required to establish valid evidence. Likewise, optimizing patient welfare and owner education and advocacy is crucial to meet the unique ethical obligations to both owners and animals enrolled in these clinical trials and to ensure trust in the team conducting the research. To date, 'Best Practice Recommendations' for clinical trial conduct have not been reported for veterinary oncology. This document summarizes the consensus of a workshop held in November, 2014 to identify relevant ethical principles and to ensure responsible conduct of clinical research in companion animals with cancer. It is intended as a working document that will be updated as advances in science and ethical considerations require. To the extent possible, existing guidelines for the conduct and oversight of clinical trials in humans have been adapted for veterinary trials to avoid duplicative effort and to facilitate integration of clinical trials such that translational research with benefits for both companion animals and humans are encouraged.

Best practice in the conduct of key nonclinical cardiovascular assessments in drug development: current recommendations from the Safety Pharmacology Society.

A cardiovascular safety pharmacology assessment is routinely conducted prior to first administration of a new chemical entity or biopharmaceutical to man. These assessments are used to inform clinicians of potential effects in those initial clinical studies. They may also indicate more subtle effects having more relevance for longer term patient treatment studies such as a potential effect in a Thorough QT (TQT) study or a small persistent increase in blood pressure. Many pharmaceutical companies use the nonclinical studies for early decision making to avoid the clinical development of any compound likely to have a positive signal in a TQT study. These latter purposes generally require more sensitive assay systems and a confidence in their translation to man. At present it is often unclear whether any given study meets the standard required to convincingly detect these subtle effects. The Safety Pharmacology Society (SPS) brought together a group of over 50 experts to discuss best practices for dog and monkey cardiovascular assessments in safety pharmacology and toxicology studies in order to build overall confidence in the ability of a study to test a given hypothesis. It is clearly impossible to dictate a very specific standard practice for assays which are conducted globally in very different facilities using different equipment. However it was clear that a framework could be described to improve comparison and interpretation. Recommendations can be summarized on the basis of three key criteria: 1) know your study population quantitatively and qualitatively, 2) know how well your current study matches the historical data and 3) support your conclusions on the basis of the specific study's determined ability to detect change.

Nitration and increased alpha-synuclein expression associated with dopaminergic neurodegeneration in equine pituitary pars intermedia dysfunction.

Equine pituitary pars intermedia dysfunction (PPID) is a spontaneously occurring progressive disease affecting aged horses and ponies. The pathogenesis of PPID is poorly understood, but the available evidence supports a loss of dopaminergic inhibition of the melanotropes of the pars intermedia. Horses with PPID have increased plasma concentrations of pars intermedia pro-opiomelanocortin-derived peptides that decrease in response to dopamine or dopamine agonist administration. Dopamine and dopamine metabolite concentrations are decreased in the pars intermedia of affected horses compared to age-matched control horses. Horses with disease that are treated with the dopamine agonist pergolide show improvement in clinical signs and normalisation of diagnostic test results. In the present study, immunohistochemical evaluation of pituitary and hypothalamic tissue demonstrated reduced tyrosine hydroxylase immunoreactivity in affected horses compared to age-matched and young controls, supporting the role of dopaminergic neurodegeneration in PPID. In addition, immunohistochemical evaluation revealed an increase in the oxidative stress marker, 3-nitrotyrosine and in nerve terminal protein, alpha-synuclein that colocalised in the pars intermedia of horses with disease. These findings suggest a role for nitration of overexpressed alpha-synuclein in the pathogenesis of neurodegeneration in PPID.

Overexpression of HER2/neu in solid tumours: an immunohistochemical survey.

AIMS: Using a standardized immunohistochemical assay we have evaluated 575 primary neoplasms of different histogenesis to determine the incidence of HER2 overexpression in some of the most common categories of human solid neoplasms. This study addresses the variable incidence of HER2 overexpression previously published for some tumour types. METHODS AND RESULTS: The immunohistochemical staining was performed on paraffin sections of surgical specimens and a well-defined scoring system based upon numbers of HER2 receptors expressed on the cell surface was applied. Overexpression of HER2 as defined as a HER2 score of equal or greater than 2 was seen in breast cancer (22%), pulmonary adenocarcinoma (28%), colorectal adenocarcinomas (17%), pulmonary squamous (11%) and gastric adenocarcinomas (11%). As expected, the proportion of cases with a HER2 score of 3 was highest in breast cancer. Contrary to published results prostate and pancreas adenocarcinomas showed a very low incidence of HER2 overexpression. CONCLUSIONS: Overexpression of HER2 is detected immunohistochemically in a proportion of epithelial neoplasms of diverse histogenesis in addition to ductal breast cancer. The standardized format of the assay will allow comparative analyses of studies performed at different institutions.

Tissue disposition and pharmacokinetics of recombinant human nerve growth factor after acute and chronic subcutaneous administration in monkeys.

In this study, we have characterized the metabolism, tissue disposition, excretion routes, and plasma pharmacokinetics of recombinant human nerve growth factor after single and multiple s.c. administration in male cynomolgus monkeys. Unlabeled nerve growth factor (NGF; 2 mg/kg) was administered three times a week for 4 weeks and a full pharmacokinetic profile was obtained for doses 1 and 12. For the tissue distribution studies, 0.8 microg/kg of trace (125)I-labeled recombinant human nerve growth factor was dosed. Histological analysis of emulsion-microautoradiography indicated that specific (125)I-NGF labeling was confined to sections of nerves most frequently localized adjacent to large vessels in sections of kidney, spleen, liver, and salivary gland. A small percentage of large neurons within the sympathetic ganglia were intensely labeled, as well as large neurons within the dorsal root ganglia. We found an increased disposition of (125)I-NGF in parts of the peripheral nervous system (including sympathetic ganglia) from 8 to 24 h postdose. In contrast, radioactivity in most non-neuronal tissues declined. This suggests specific uptake in these target tissues known to express specific receptors for NGF. We also identified changes in pharmacokinetic parameters after single versus chronic s. c. administration. These studies demonstrated that s.c. administration of NGF at 0.8 microg/kg doses in monkeys is capable of accessing and localizing in the target tissues.

Hypertension and endothelial dysfunction in apolipoprotein E knockout mice.

Mice lacking ApoE (Apoe(-/-)) develop initially hypercholesterolemia and lastly atherosclerosis. This study examined hemodynamics and endothelial function in 6-week-old Apoe(-/-) mice with hypercholesterolemia only, 7.5-months-old Apoe(-/-) mice with both hypercholesterolemia and atherosclerosis, and age matched controls. One day after implantation of catheters into the carotid artery, arterial pressure was measured in conscious, unrestrained mice. Compared with the respective controls, there was a significant increase in arterial pressure and the ratio of left ventricular weight to body weight in 7.5-month-old Apoe(-/-) mice but not in 6-week-old Apoe(-/-) mice. Histopathological analysis demonstrated significant renal artery disease in the form of extensive atheromatous plaques only in 7.5-month-old Apoe(-/-) mice, whereas no atherosclerotic lesions were found in 6-week-old Apoe(-/-) mice. For evaluation of endothelial function, a laser Doppler perfusion imager with a computer-controlled optical scanner was used to measure cutaneous blood perfusion on the dorsal side of one hind paw before and after topical application of mustard oil, which is known to induce nitric oxide-mediated vasodilation. The mustard oil treatment elicited a substantial increase in blood perfusion (P<0.01), which was similar between 6-week-old Apoe(-/-) mice and controls but significantly blunted in 7.5-month-old Apoe(-/-) mice versus control mice, suggesting nitric oxide-mediated vasodilation is diminished in 7.5-month-old Apoe(-/-) mice but not in 6-week-old Apoe(-/-) mice. In contrast, the increase in blood perfusion induced by topical administration of cilostazol, which induces vasodilation via cyclic adenosine monophosphate, was not different between 7.5-month-old Apoe(-/-) mice and controls. Thus hypertension and endothelial dysfunction observed in 7.5-month-old Apoe(-/-) mice may be due mainly to atherosclerosis.

A mouse model of human familial hypercholesterolemia: markedly elevated low density lipoprotein cholesterol levels and severe atherosclerosis on a low-fat chow diet.

Mutations in the low density lipoprotein (LDL) receptor gene cause familial hypercholesterolemia, a human disease characterized by premature atherosclerosis and markedly elevated plasma levels of LDL cholesterol and apolipoprotein (apo) B100. In contrast, mice deficient for the LDL receptor (Ldlr-/-) have only mildly elevated LDL cholesterol levels and little atherosclerosis. This difference results from extensive editing of the hepatic apoB mRNA in the mouse, which limits apoB100 synthesis in favor of apoB48 synthesis. We have generated Ldlr-/- mice that cannot edit the apoB mRNA and therefore synthesize exclusively apoB100. These mice had markedly elevated LDL cholesterol and apoB100 levels and developed extensive atherosclerosis on a chow diet. This authentic model of human familial hypercholesterolemia will provide a new tool for studying atherosclerosis.

DNAse treatment does not improve the survival of lupus prone (NZB x NZW)F1 mice.

OBJECTIVE: To examine the efficacy of deoxyribonuclease I (DNAse) therapy in the (NZB x NZW)F1 murine model of lupus. METHODS: Lupus-prone female (NZB x NZW)F1 mice were treated daily with 0-15 microg/g of recombinant DNAse for 1-6 months. Parameters including anti-DNA autoantibody production, activation of cytokine secreting cells, kidney function and longevity were monitored. RESULTS: DNAse treatment selectively reduced the number of B cells secreting anti-dsDNA antibodies for approximately one month. However, neither short-term nor long-term treatment altered cytokine production, delayed the onset or reduced the severity of glomerulonephritis, or prolonged survival. CONCLUSION: DNAse treatment initiated before, during, or after the onset of murine lupus did not improve clinical outcome.

Genetic absence of gamma-interferon delays but does not prevent diabetes in NOD mice.

Cytokines, particularly interferons, may participate in the development of type I diabetes. This involvement could be from direct cytotoxic actions of the interferons on the pancreatic beta-cells or from an indirect influence on the number, activity, or type of inflammatory cells that invade the islets in type I diabetes. To examine directly the role of interferon (IFN)-gamma in a mouse model of type I diabetes, we have introduced an inactivating mutation in the IFN-gamma gene (ifg) into NOD mice. The genetic absence of IFN-gamma does not prevent either insulitis or diabetes in the NOD mice, but it does increase the time to onset. Although it might have been predicted that the absence of IFN-gamma in these mice would lead to an increase in expression of Th2 T-helper cell-related cytokines, we found instead a profound decrease in the expression of two of the characteristic Th2 cytokines, interleukin (IL)-4 and IL-10. We also demonstrate that the splenocytes taken from IFN-gamma-deficient diabetic mice are fully capable of transferring diabetes to naive recipients.

Neurotrophin-3 reverses experimental cisplatin-induced peripheral sensory neuropathy.

Cisplatin, a widely used chemotherapeutic agent, induces a sensory neuropathy with selective loss of vibration sense and proprioception. Here we demonstrate that neurotrophin-3 (NT-3), a member of the nerve growth factor family of neurotrophic factors, restored to normal levels the reduced H-reflex-related sensory nerve conduction velocity induced by cisplatin in rats. NT-3 treatment corrected an abnormal cytoplasmic distribution of neurofilament protein in large sensory neurons in dorsal root ganglia and the reduction in the numbers of myelinated fibers in sural nerves caused by cisplatin. The NT-3-dependent reversal of cisplatin neurotoxicity thus suggests the possible use of NT-3 in the treatment of peripheral sensory neuropathy.

Selective modulation of the expression of L-selectin ligands by an immune response.

BACKGROUND: The adhesion molecule L-selectin is expressed on the cell surface of lymphocytes and mediates their migration from the bloodstream into lymph nodes. L-selectin is able to recognize four glycoprotein ligands, three of which--Sgp50, Sgp90, and Sgp200--are sulphated, bind specifically to L-selectin and are synthesized by the high endothelial venules of the peripheral and mesenteric lymph nodes. One of these three sulphated L-selectin ligands, Sgp90, has been shown to be identical to the known surface marker CD34 and is expressed on the cell surface of endothelial cells. The cDNA encoding Sgp50 has been cloned, and its product, which has been designated GlyCAM-1, is secreted. The third ligand, Sgp200, is both secreted and cell-associated. We have investigated how the expression of these sulphated glycoproteins is regulated during an immune response. RESULTS: Here we demonstrated that, during a primary immune response, the expression and secretion of both GlyCAM-1 and Sgp200 are reduced, recovering to normal levels 7-10 days after antigen stimulation. In contrast, the expression of cell-associated CD34 and Sgp200 is relatively unaffected. These results may account for the modest decreases in the binding of an L-selectin-IgG fusion protein to high endothelial venules of inflamed peripheral lymph nodes that have been observed after antigen exposure. In vivo experiments show that, following the decrease in the levels of secreted GlyCAM-1 and Sgp200, migration of lymphocytes from the blood stream into lymph nodes remains L-selectin-dependent, but more lymphocytes home to antigen-primed than unprimed peripheral lymph nodes. CONCLUSIONS: We suggest that the secreted forms of the L-selectin ligands GlyCAM-1 and Sgp200 act as modulators of cell adhesion, and that cell-associated CD34 and Sgp200 are the ligands that mediate the initial loose binding of lymphocytes to high endothelial venules.

Global vascular expression of murine CD34, a sialomucin-like endothelial ligand for L-selectin.

Extravasation of leukocytes into organized lymphoid tissues and into sites of inflammation is critical to immune surveillance. Leukocyte migration to peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and Peyer's patches (PP) depends on L-selectin, which recognizes carbohydrate-bearing, sialomucin-like endothelial cell surface glycoproteins. Two of these ligands have been identified at the molecular level. One is the potentially soluble mucin, GlyCAM 1, which is almost exclusively produced by high endothelial venules (HEV) of PLN and MLN. The second HEV ligand for L-selectin is the membrane-bound sialomucin CD34. Historically, this molecule has been successfully used to purify human pluripotent bone marrow stem cells, and limited data suggest that human CD34 is present on the vascular endothelium of several organs. Here we describe a comprehensive analysis of the vascular expression of CD34 in murine tissues using a highly specific antimurine CD34 polyclonal antibody. CD34 was detected on vessels in all organs examined and was expressed during pancreatic and skin inflammatory episodes. A subset of HEV-like vessels in the inflamed pancreas of nonobese diabetic (NOD) mice are positive for both CD34 and GlyCAM 1, and bind to an L-selectin/immunoglobulin G (IgG) chimeric probe. Finally, we found that CD34 is present on vessels of deafferentiated PLN, despite the fact that these vessels are no longer able to interact with L-selectin or support lymphocyte binding in vitro or trafficking in vivo. Our data suggest that the regulation of posttranslational carbohydrate modifications of CD34 is critical in determining its capability to act as an L-selectin ligand. Based on its ubiquitous expression, we propose that an appropriately glycosylated form of vascular CD34 may act as a ligand for L-selectin-mediated leukocyte trafficking to both lymphoid and nonlymphoid sites.

Islet expression of interferon-alpha precedes diabetes in both the BB rat and streptozotocin-treated mice.

The mechanism(s) leading to beta cell dysfunction in type I diabetes has not been defined. We have investigated whether islet expression of IFN alpha could be a cause of the lesions that are hallmarks of type I diabetes. Streptozotocin induces the expression of interferon-alpha by pancreatic islets prior to the diabetes induced by streptozotocin. Increased IFN alpha, induced by poly I/C or expressed from a transgene will exacerbate the diabetogenic effects of streptozotocin. In another rodent model of type I diabetes (the BB rat), islet expression of IFN alpha precedes lymphocytic infiltration and diabetes. As in the streptozotocin model, in the BB rats poly I/C will induce islet expression of IFN alpha and accelerate the onset of diabetes. These results are consistent with the hypothesis that islet expression of IFN alpha participates in causing type I diabetes.

Diagnostic testing for pituitary pars intermedia dysfunction in horses.

Pituitary pars intermedia dysfunction is a slowly progressive disorder that afflicts most breeds of horses. Because it shares features with human Cushing disease, it has been referred to as equine Cushing disease. A variety of tests of pituitary-adrenocortical function were performed on horses with evidence of pituitary pars intermediate dysfunction, and results were compared with those in healthy control horses. Diurnal variations in plasma cortisol concentration were not statistically different between control horses and those with pituitary pars intermedia dysfunction. An ACTH stimulation (1 U of natural ACTH gel/kg of body weight, IM) test or a combined dexamethasone suppression test (10 mg, IM) and ACTH stimulation (100 mg of synthetic ACTH, IV) test also failed to distinguish horses with pituitary pars intermedia dysfunction from control horses. A significant (P < 0.001) dose-related suppression of cortisol concentration in response to increasing doses (5, 10, 20, and 40 micrograms/kg) of dexamethasone was observed in control horses but not in those with pituitary pars intermedia dysfunction. On the basis of plasma cortisol concentration, the dexamethasone suppression test, using 40 micrograms/kg, whether initiated at 5 PM with sample collection at 15 (8 AM) and 19 (12 PM) hours after dexamethasone administration, or initiated at 12 AM with sample collection at 8 (8 AM), 12 (12 PM), 16 (4 PM), 20 (8 PM), and 24 (12 AM) hours after dexamethasone administration, reliably distinguished between control horses and those with pituitary pars intermedia dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of gonadotropin-releasing hormone-stimulated luteinizing hormone release in cultured male equine anterior pituitary cells by gonadal steroids.

The objective of the present study was to determine whether the testicular steroids, i.e., testosterone (T), dihydrotestosterone (DHT), estradiol (E2), estrone (E1), and estrone sulfate (E1SO4), play a physiological role in regulating LH release in the male horse by direct actions at the anterior pituitary gland. Enzymatically dispersed anterior pituitary cells from stallions (n = 4) or geldings (n = 3) were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse medium. To determine the effects of the steroids on the LH response to GnRH, the cells were incubated for 24 h in fresh media with or without 10(-10) M E2 or 10(-8) M T or DHT followed by a 4-h incubation +/- GnRH (10(-11) to 10(-7) M). Media and cells were analyzed for LH by RIA. In the stallion, GnRH increased LH release (p < 0.001) in a dose-dependent manner (ED50 GnRH = 4.5 x 10(-9) M), and this response was unaltered by T or DHT but greatly enhanced by E2 (p < 0.001). E2 lowered the ED50 for GnRH to 5 x 10(-10) M and increased the maximum LH response to GnRH by 350%. The LH release in response to a constant dose of 1 nM GnRH was unaltered by varying doses of T, DHT, or E1SO4 (10(-11) to 10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Increased serum growth hormone concentration in feline hypertrophic cardiomyopathy.

Serum growth hormone concentration was measured by radioimmunoassay in 31 cats with hypertrophic cardiomyopathy, 38 normal cats, and 35 cats with other cardiac disease. Cats with hypertrophic cardiomyopathy had a significantly increased serum growth hormone concentration when compared with normal cats and cats with other cardiac disease. The serum growth hormone concentration in cats with hypertrophic cardiomyopathy was less than that previously reported in cats with growth hormone secreting pituitary tumors. Pituitary tumors were not identified in eight of the cats with hypertrophic cardiomyopathy examined at necropsy. An increased serum growth hormone concentration may be measured in cats with hypertrophic cardiomyopathy but it is unclear if the increased serum growth hormone concentration is a cause or effect of hypertrophic cardiomyopathy.

Pituitary tumor size, neurologic signs, and relation to endocrine test results in dogs with pituitary-dependent hyperadrenocorticism: 43 cases (1980-1990).

Pituitary neoplasm was identified in 43 dogs with pituitary-dependent hyperadrenocorticism via necropsy (n = 33), diagnostic imaging with computerized tomography or magnetic resonance imaging (n = 5), or diagnostic imaging and necropsy (n = 5). All dogs had clinical signs and clinicopathologic test results typical of hyperadrenocorticism. Thirty-seven dogs had grossly visible pituitary tumors, and 6 dogs had microscopic pituitary tumors. Fifteen dogs had developed neurologic signs typical of those resulting from an enlarging pituitary mass. Twenty-three dogs had pituitary tumors greater than or equal to 1 cm in diameter. Provocative testing of the pituitary-adrenocortical axis was performed on all dogs. Dogs with grossly visible pituitary tumors and dogs with neurologic signs had significantly (P less than 0.05) higher mean plasma endogenous ACTH concentrations, compared with values from dogs with microscopic tumors and dogs without neurologic signs, respectively. Dogs with grossly visible pituitary tumors and dogs with tumors greater than or equal to 1 cm in diameter had significantly (P less than 0.05) lower adrenocortical responsiveness to exogenous ACTH, compared with dogs with microscopic pituitary tumors and dogs with tumors less than 1 cm in diameter, respectively. Despite these differences, there was overlap between test results among dogs. On the basis of endocrine test results, it would appear difficult to distinguish dogs with pituitary-dependent hyperadrenocorticism and large pituitary tumors from those with pituitary-dependent hyperadrenocorticism and microscopic pituitary tumors prior to onset of neurologic signs.

N-acetylation and C-terminal proteolysis of beta-endorphin in the anterior lobe of the horse pituitary.

beta-Endorphin is post-translationally processed to both N-acetylated and C-terminally shortened derivatives in the anterior lobe of the horse pituitary, a processing pattern qualitatively different from that of the rat and virtually every other mammalian species. Thus, separation of the molecular forms of beta-endorphin using gel filtration and ion exchange chromatography showed that the horse anterior lobe primarily contains beta-endorphin-1-31 and N-acetyl-beta-endorphin-1-27 along with smaller amounts of beta-lipotropin, beta-endorphin-1-27, and N-acetyl-beta-endorphin-1-31 and -1-26, in contrast to the rat anterior lobe, which contains approximately equal amounts of beta-lipotropin and beta-endorphin-1-31. Immunohistochemical experiments using an antiserum which specifically recognizes N-acetylated beta-endorphin peptides confirmed that N-acetyl-beta-endorphin immunoreactivity is present in the anterior lobe of the horse, but not the rat. The intermediate lobe of both species primarily synthesizes N-acetylated, C-terminally shortened beta-endorphin peptides, and while distinct species differences do occur, they were relatively minor, consisting of quantitative differences in the relative proportion of each peptide. These results are consistent with earlier reports that beta-endorphin processing in the rat pituitary is tissue specific; the anterior and intermediate lobes produce entirely different sets of beta-endorphin peptides. In the equine pituitary, however, both pituitary lobes produce the same multiple beta-endorphin forms, possessing both opioid and nonopioid properties, although their relative amounts differ.

Gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) secretion from perifused equine pituitaries.

In vitro responsiveness of the horse anterior pituitary (AP) gonadotropes to single and multiple GnRH challenges was examined. The pituitaries were collected from reproductively sound mares in estrus (n = 5) and diestrus (n = 5). Uniform 0.5 mm AP slices were subdivided using a 3 mm biopsy punch and then bisected for use in the perifusion chamber. Four bisected sections per chamber were perifused at 0.5 ml/min at 37 C for 560 min in Medium 199 saturated with 95% 0(2)/5% CO2. Ten minute fractions were collected after an initial 2 hr equilibration period. Four different treatment regimes of GnRH (10(-10) M) were evaluated: (A) three consecutive 10 min GnRH pulses separated by 80 and 100 min, respectively; (B) a single 120 min GnRH infusion; (C) a 10 min GnRH pulse followed 80 min later by a 120 min GnRH infusion and (D) two 10 min GnRH pulses separated by 60 min followed 80 min later by a 120 min GnRH infusion. Estimated total pituitary LH content was higher in estrous than diestrus mares (p less than 0.05). The total amount of LH released in response to GnRH tended to be greater in estrus than diestrus (p less than 0.1), whereas the percentage of LH released in estrus and diestrus was similar. An increase in the area under the LH response curve was noted with each successive 10 min pulse of GnRH during both estrus and diestrus (p less than 0.05), demonstrating a self-priming effect of GnRH. In addition, a significant increase in the peak LH amplitude (p less than 0.05) and the slope to peak amplitude (p less than 0.05) were observed for the 120 min GnRH pulse in regime C and D indicating that prior exposure to short-term pulses of GnRH increased the acute LH secretory response. These results suggest that in the cycling mare (1) the responsiveness of the pituitary (amount of LH released as percent of total LH) is similar in both estrus and diestrus, however, the magnitude of the LH response (total microgram amount of LH released) differs with the stage of the estrous cycle, being highest in estrus, and appears to be related, in part, to pituitary LH content and (2) GnRH self-priming occurs independently of the stage of the estrous cycle. Furthermore, we have demonstrated that the pulsatile mode of GnRH can act directly on the anterior pituitary to dictate the pulsatile release pattern of LH in the cycling mare.