Expression of cyclooxygenase-2 in the canine lower urinary tract with regard to the effects of gonadal status and gender

As pituitary gonadotrophins can induce prostaglandin (PG) synthesis and receptors for LH and FSH are present in the canine lower urinary tract (LUT), the objectives of this study were to (i) investigate the expression of COX-2, a key rate-limiting enzyme in PG production, in the canine LUT and (ii) determine if COX-2 expression differs between gender, gonadal status (intact and gonadectomised) and LUT regions. Four regions (body and neck of the bladder as well as proximal and distal urethra) of the LUT were obtained from 20 clinically healthy dogs (5 intact males, 5 intact anoestrous females, 4 castrated males, 6 spayed females). In situ hybridization and immunohistochemistry were performed to determine the presence of COX-2 mRNA and protein, respectively. The mRNA and protein expression was semi-quantitatively assessed. The scoring system combined both the distribution and intensity of positive staining and was carried out separately on the three tissue layers (epithelium, sub-epithelial stroma and muscle) for each of four regions of the LUT. In comparison to intact dogs, lower expression (P < 0.001) of COX-2 and its mRNA in gonadectomised males and females was observed in all tissue layers of each region of the LUT except in the distal urethra where there was no difference in mRNA expression between gonadal statuses. Regardless of region and tissue layer, intact females expressed more (P < 0.05) COX-2 and its mRNA than intact males. However, in gonadectomised dogs, mRNA expression of COX-2 did not differ between genders; males had higher (P < 0.001) protein level of COX-2 compared to females. In conclusion, both COX-2 and its mRNA were expressed in the canine LUT and COX-2-regulated PG synthesis in the canine LUT may differ between gonadal statuses and genders. The lower expression of COX-2 in gonadectomised dogs may impair normal function of the LUT and probably implicated in the development of neutering-induced urinary incontinence in the dog.     

Effects of the dopamine agonist cabergoline on the pulsatile and TRH-induced secretion of prolactin, LH, and testosterone in male beagle dogs

In the present study, the pulsatile serum profiles of prolactin, LH and testosterone were investigated in eight clinically healthy fertile male beagles of one to six years of age. Serum hormone concentrations were determined in blood samples collected at 15 min intervals over a period of 6 h before (control) and six days before the end of a four weeks treatment with the dopamine agonist cabergoline (5 microg kg(-1) bodyweight/day). In addition, the effect of cabergoline administration was investigated on thyrotropin-releasing hormone (TRH)-induced changes in the serum concentrations of these hormones. In all eight dogs, the serum prolactin concentrations (mean 3.0 +/- 0.3 ng ml(-1)) were on a relatively constant level not showing any pulsatility, while the secretion patterns of LH and testosterone were characterised by several hormone pulses. Cabergoline administration caused a minor but significant reduction of the mean prolactin concentration (2.9 +/- 0.2 ng ml(-1), p < 0.05) and did not affect the secretion of LH (mean 4.6 +/- 1.3 ng ml(-1) versus 4.4 +/- 1.7 ng ml(-1)) or testosterone (2.5 +/- 0.9 ng ml(-1) versus 2.4 +/- 1.2 ng ml(-1)). Under control conditions, a significant prolactin release was induced by intravenous TRH administration (before TRH: 3.8 +/- 0.9 ng ml(-1), 20 min after TRH: 9.1 +/- 5.9 ng ml(-1)) demonstrating the role of TRH as potent prolactin releasing factor. This prolactin increase was almost completely suppressed under cabergoline medication (before TRH: 3.0 +/- 0.2 ng ml(-1), 20 min after TRH: 3.3 +/- 0.5 ng ml(-1)). The concentrations of LH and testosterone were not affected by TRH administration. The results of these studies suggest that dopamine agonists mainly affect suprabasal secretion of prolactin in the dog.     

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.     

A 'window of time' during which testosterone determines the opiatergic control of LH release in the adult male rat

Male rats castrated before puberty (when 26 days of age) showed a progressively decreasing susceptibility to the inhibitory effects of morphine (5 mg/kg) upon LH secretion for up to 28 days after gonadectomy (approximately 100%, 40% and 10% inhibition at 5, 12 and 28 days after castration), but thereafter morphine again caused approximately 50% reduction in serum LH values; the minimum inhibition found at 28 days after castration (age 54 days) occurred at the time at which male rats normally reach puberty. When rats were castrated at 59 days of age, morphine maximally suppressed serum LH concentrations (to less than 70%) 2 and 5 days after castration, but had no effect thereafter. In prepubertal castrates, testosterone replacement between Days 26 and 50 of life resulted in responses to morphine similar to those found in rats castrated after puberty, i.e. serum LH levels were not reduced. Morphine significantly reduced LH levels in prepubertal castrates given testosterone after 60 days of age. Treatment with morphine consistently elevated serum prolactin concentrations (greater than 100%) in castrated rats of all ages, regardless of the time elapsed after gonadectomy. These results indicate a transient fall in the inhibitory opioidergic tone upon LH secretion as the normal age of puberty approaches, that the ability of opiates to alter LH release in adulthood may depend upon testicular steroids secreted during the peripubertal period, and that the LH responses do not reflect general changes in the neuroendocrine response to opiates after castration since the prolactin response to morphine remains intact in rats castrated before and after puberty.     

Thyroid function and vitamin A deficiency.

Rats, when vitamin A deficient, had increased plasma T₃, T₄ and free thyroxine indexes. Pituitary TSH and hypothalamic TRH content were increased in vitamin A deficient animals compared to pair-fed controls. The plasma TSH response to TRH was normal in the vitamin A deficient rats. Basal prolactin, LH and FSH levels did not differ significantly in the two groups. Both groups had significant increases in LH and FSH after LRH. Vitamin A deficiency produces biochemical hyperthyroidism. Our data are consistent with an abnormality in thyroid hormone feedback on the hypothalamic pituitary axis.     

The effect of dexamethasone on TSH and prolactin secretion after TRH stimulation

Serum thyrotropin (TSH) and prolactin levels were measured after intravenous administration of 400 μg of synthetic thyrotropin-releasing hormone (TRH) in 13 normal subjects and six hypothyroid patients before and after three days of administration of dexamethasone 2 mg per day. In the normal subjects dexamethasone suppressed baseline serum levels and secretion of TSH after TRH stimulation. On the other hand, it had no effect on the hypothyroid patients. In the control group dexamethasone also suppressed baseline serum levels but not secretion of prolactin after TRH stimulation. Dexamethasone had no effect on prolactin levels in the hypothyroid group. It is concluded that in normal patients short-term administration of dexamethasone has an inhibitory effect on TSH secretion at the pituitary level. As for prolactin, our results could indicate that TRH is a more potent stimulator of prolactin secretion than of TSH secretion, or that TSH and prolactin pituitary thresholds for TRH are different.     

Opioid regulation of luteinising hormone and prolactin release in the horse—identical or independent endocrine pathways?

In order to determine if endogenous opioids regulate luteinising hormone (LH) and prolactin secretion via a common, gonadotropin-releasing hormone (GnRH) dependent pathway in the horse, effects of the opioid antagonist naloxone (300 mg) and the GnRH agonist buserelin (20 μg) on prolactin and LH secretion were investigated in stallions (n = 22), long-term castrated geldings (n = 15) and non-lactating mares during the luteal phase of the oestrous cycle (n = 16). Blood samples for determination of LH and prolactin concentrations were withdrawn at 15 min intervals for 120 min. After 60 min of sampling, animals were treated with either naloxone, buserelin or saline. In stallions, naloxone significantly increased LH as well as prolactin release (P < 0.05), indicating an opioid inhibition of both hormones, whereas in mares, naloxone stimulated only LH secretion (P < 0.05). No changes in plasma LH or prolactin concentrations after injection of naloxone were found in geldings. In all animal groups, buserelin induced a significant release of LH (P < 0.05) without affecting prolactin. We conclude that endogenous opioids inhibit LH and prolactin release in the horse but the regulation of these two hormones involves independent opioid pathways. These are activated differentially in stallions, geldings and mares. The opioid regulation of prolactin secretion is not mediated via GnRH.     

Effect of bombesin on basal and stimulated secretion of some pituitary hormones in humans

The effect of bombesin (5 ng/kg/min X 2.5 h) on basal pituitary secretion as well as on the response to thyrotropin releasing hormone (TRH; 200 micrograms) plus luteinizing hormone releasing hormone (LHRH; 100 micrograms) was studied in healthy male volunteers. The peptide did not change the basal level of growth hormone (GH), prolactin, thyroid-stimulating hormone (TSH), luteinizing hormone (LH) and follicle-stimulating hormone (FSH). On the contrary, the pituitary response to releasing hormones was modified by bombesin administration. When compared with control (saline) values, prolactin and TSH levels after TRH were lower during bombesin infusion, whereas LH and FSH levels after LHRH were higher. Thus bombesin affects in man, as in experimental animals, the secretion of some pituitary hormones.     

Sex differences in acute luteinizing hormone responses to gonadectomy remain after progesterone antagonist and dopamine agonist treatment.

In male rats, LH pulse frequency and amplitude increase dramatically by 24 h after gonadectomy; in females they increase only slightly by this time. Mean FSH levels increase significantly in both sexes by 24 h after gonadectomy. The objectives of the present studies were to compare pulsatile LH, FSH, and prolactin (PRL) secretion in intact versus gonadectomized and in male versus female rats, and to determine whether the acute postovariectomy lag in LH rise is due to a lingering effect of the higher PRL and/or progesterone (P) levels seen in intact females. LH pulse amplitude, frequency, and mean levels increased significantly by 24 h after gonadectomy in both sexes, but the increases were greater in the males. FSH mean levels, but not pulse amplitude or frequency, increased similarly in both sexes by 24 h after gonadectomy. PRL did not change with gonadectomy. Treatment with CB-154 (a dopamine agonist), with or without RU486 (a P antagonist), 1 h before gonadectomy significantly suppressed pulsatile PRL secretion 1 day later in both sexes. There was no effect of either treatment on LH secretion. We have demonstrated that there is a sex difference in LH, but not FSH or PRL, pulsatility at 24 h after gonadectomy, and that female rats' higher PRL and P levels do not account for their slow rate of LH rise after ovariectomy.     

Prolactin suppresses GnRH but not TSH secretion

BACKGROUND/AIMS: In animal models, prolactin increases tuberoinfundibular dopamine turnover, which has been demonstrated to suppress both hypothalamic GnRH and pituitary TSH secretion. To test the hypothesis that prolactin suppresses GnRH and TSH secretion in women, as preliminary evidence that a short-feedback dopamine loop also operates in the human, the effect of hyperprolactinemia on GnRH and TSH secretion was examined. METHODS: Subjects (n=6) underwent blood sampling every 10 min in the follicular phase of a control cycle and during a 12-hour recombinant human prolactin (r-hPRL) infusion preceded by 7 days of twice-daily subcutaneous r-hPRL injections. LH and TSH pulse patterns and menstrual cycle parameters were measured. RESULTS: During the 7 days of r-hPRL administration, baseline prolactin increased from 16.0+/-3.0 to 101.6+/-11.6 microg/l, with a further increase to 253.7+/-27.7 microg/l during the 12-hour infusion. LH pulse frequency decreased (8.7+/-1.0 to 6.0+/-1.0 pulses/12 h; p<0.05) with r-hPRL administration, but there were no changes in LH pulse amplitude or mean LH levels. There were also no changes in TSH pulse frequency, mean or peak TSH. The decreased LH pulse frequency did not affect estradiol, inhibin A or B concentrations, or menstrual cycle length. CONCLUSION: These studies demonstrate that hyperprolactinemia suppresses pulsatile LH secretion but not TSH secretion and suggest that GnRH secretion is sensitive to hyperprolactinemia, but that TSH secretion is not. These data further suggest that the degree of GnRH disruption after 7 days of hyperprolactinemia is insufficient to disrupt menstrual cyclicity.     

Reversible downregulation of endocrine and germinative testicular function (hormonal castration) in the dog with the GnRH-Agonist Azagly-Nafarelin as a removable implant “Gonazon”; a preclinical trial

Downregulation of anterior pituitary GnRH-receptors by application of a slow release GnRH-implant offers an effective and reversible alternative to surgical castration of the male dog.Aim of the present study was to test the efficacy and the underlying mechanisms of a new non-biodegradable controlled-release device implant (Gonazon^®, Intervet, containing 18.5 mg of the GnRH-agonist Azagly-Nafarelin). Eight male beagle dogs were implanted s.c. at the para-umbilical region. In four dogs implant removal was after 180 days (group 1), in the other four dogs after 365 days (group 2). Eleven weeks after implantation availability of LH was reduced (p < 0.0001) by 70%. After an initial increase lasting for about 4 days, testosterone (T) and estradiol (E2) concentrations decreased (p < 0.0001) to basal levels within 17.5 ± 8.4 days. Size of testes was decreased by about 82% after 17 weeks, size of prostate by about 46% after 5 weeks (p < 0.0001). Five to 7 weeks after implantation all dogs were aspermic.Testosterone and estradiol concentrations, together with testicular and prostatic size remained suppressed in all dogs in group 1 and one dog of group 2 until implant removal. The other three dogs of group 2 escaped from down-regulation between 223 and 324 days.Effects on the availability of LH, T, E2 and on testicular and prostatic size were fully reversible after implant removal or escape from down-regulation. In six dogs semen quality was back to pre-treatment values after about 29 weeks, however, one dog developed oligozoospermia while another one stayed azoospermic, probably due to an obstruction within the epididymal duct.     

Gonadotropin, prolactin and thyrotropin pituitary secretion after exogenous dehydroepiandrosterone-sulfate administration in normal women.

The effect of exogenous dehydroepiandrosterone-sulfate (DHAS) on luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL) and thyroid-stimulating hormone (TSH) pituitary secretion was studied in 8 normal women during the early follicular phase. The plasma levels of these hormones were evaluated after gonadotropin-releasing hormone (GnRH)/thyrotropin-releasing hormone (TRH) stimulation performed after placebo or after 30 mg DHAS i.v. administration. The half-life of DHAS was also calculated on two subjects; two main components of decay were detected with half-times of 0.73-1.08 and 23.1-28.8 h. The results show an adequate response of all hormones to GnRH or TRH tests which was not significantly modified, in the case of LH, FSH and PRL, when performed in the presence of high levels of DHAS. However, the TSH response to TRH was significantly less suppressed (p less than 0.05) (39%) after DHAS administration than during repeated TRH stimulation without DHAS (51%). The data support the hypothesis that DHAS does not affect LH, FSH and PRL secretion, while TSH seemed to be partially influenced.     

Gonadectomy negatively impacts social behavior of adolescent male primates

Social behavior changes dramatically during primate adolescence. However, the extent to which testosterone and other gonadal hormones are necessary for adolescent social behavioral development is unknown. In this study, we determined that gonadectomy significantly impairs social dominance in naturalistic settings and changes reactions to social stimuli in experimental settings. Rhesus macaques were castrated (n =  6) or sham operated (n = 6) at age 2.4 years, group-housed for 2 years, and ethograms were collected weekly. During adolescence the gonadally intact monkeys displayed a decrease in subordinate behaviors and an increase in dominant behaviors, which ultimately related to a rise in social status and rank in the dominance hierarchy. We measured monkey's reactions to emotional faces (fear, threat, neutral) of conspecifics of three ages (adult, peer, infant). Intact monkeys were faster to retrieve a treat in front of a threatening or infant face, while castrated monkeys did not show a differential response to different emotional faces or ages. No group difference in reaction to an innate fear-eliciting object (snake) was found. Approach and proximity responses to familiar vs unfamiliar conspecifics were tested, and intact monkeys spent more time proximal to a novel conspecific as compared to castrates who tended to spend more time with a familiar conspecific. No group differences in time spent with novel or familiar objects were found. Thus, gonadectomy resulted in the emergence of significantly different responses to social stimuli, but not non-social stimuli. Our work suggests that intact gonads, which are needed to produce adolescent increases in circulating testosterone, impact social behavior during adolescences in primates.     

Prolactin and LH release in response to LH-RH and TRH in ewes during dioestrus, pregnancy and post partum

With advancing pregnancy in the ewe there was a marked decline in plasma LH concentrations and pituitary LH-RH responsiveness (integrated LH release) and a marked increase in plasma prolactin values and pituitary TRH responsiveness (integrated prolactin release). In lactating ewes plasma LH levels and pituitary LH-RH responsiveness had returned to values found in the luteal phase of the normal cycle by 21 days post partum, whereas at 42 days post partum prolactin levels were still high. No interaction between TRH and LH-RH on prolactin and LH release in dioestrous ewes was detected. In non-pregnant ewes plasma prolactin levels were significantly higher in June than in January but TRH responsiveness was similar. It is concluded that, in sheep, pituitary LH secretion recovers more rapidly from the chronic negative feedback effect of oestrogens and progesterone in pregnancy than prolactin secretion recovers from the chronic positive feedback effects of oestrogens. This finding may be a contributory factor in the resistance to resumption of breeding activity.     

Plasma concentrations of testosterone and LH in the male dog

Blood samples were withdrawn every 20 min from 3 conscious intact and 2 castrated mature males during non-consecutive periods of 12 h during the light and dark phases of the lighting schedule (intact dogs) and of 11 h during the light period (castrated dogs). In the intact dogs testosterone concentrations ranged from 0.4 to 6.0 ng/ml over the 24-h period. LH concentrations varied from 0.2 to 12.0 ng/ml. In all animals, LH peaks were clearly followed, after about 50 min, by corresponding testosterone peaks, but no diurnal rhythm could be established. LH concentrations in the castrated dogs were high (9.8 +/- 2.7 (s.e.m.) ng/ml), and still showed an episodic pattern in spite of the undetectable plasma testosterone levels.     

Cyclosporine A inhibits the secretion of certain anterior pituitary hormones in patients with nephrotic syndrome.

To clarify the effects of cyclosporine A (CsA) on the secretion of serum thyrotropin (TSH), prolactin (PRL), luteinizing hormone (LH) and follicular stimulating hormone (FSH), we performed TRH and LH-RH testing in 4 patients with the nephrotic syndrome before and after the administration of CsA, 6 mg/kg/day for 4 to 12 weeks. Prior to CsA all patients responded normally to TRH with respect to TSH and PRL secretion. Two patients showed normal response of LH and FSH to LH-RH stimulation while the response in 2 other patients, who were both menopausal, was exaggerated. By the third or fourth week of CsA administration the basal and peak TSH and PRL values declined significantly in all patients in response to TRH stimulation while those of LH and FSH showed only a modest decrease in response to LH-RH stimulation. Two to 4 weeks after the cessation of CsA the response of TSH, PRL and FSH returned to the pretreatment level. These observations suggest that: 1) CsA exerts an inhibitory effect on the secretion of at least TSH and PRL in humans, and 2) the effect of CsA on the pituitary may be partially reversible after the cessation of the therapy.     

Serum concentrations of reproductive hormones after administration of various anesthetics to immature and young adult male rats

Immature and young adult male rats were either castrated or unoperated. One of seven anesthetic agents (Rompun, Bio-Tal, Thiopental, pentobarbital, ketamine, halothane, or ether) was administered. When the animals were clearly anesthetized, they were decapitated. Control rats were decapitated without anesthesia. Serum concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, testosterone, and androstenedione were determined by radioimmunoassay. None of the anesthetics was clearly suitable for study of all these hormones. Most would be suitable for acute LH studies. Ketamine and halothane appeared inappropriate for FSH studies in immature rats. Pentobarbital, Rompun, and ether caused increases in serum prolactin. Most of the agents appeared to cause a reduction in serum testosterone in intact rats but an increase in castrated animals, suggesting an inhibition of testicular androgen secretion and a stimulation of adrenal androgen secretion.     

Thyrotropin Releasing Hormone (TRH) in goldfish (Carassius auratus): role in the regulation of feeding and locomotor behaviors and interactions with the orexin system and cocaine- and amphetamine regulated transcript (CART)

TRH is a peptide produced by the hypothalamus which major function in mammals is the regulation of TSH secretion by the pituitary. In fish, TRH does not appear to affect TSH secretion, suggesting that it might regulate other functions. In this study, we assessed the effects of central (intracerebroventricular, icv) injections of TRH on feeding and locomotor behavior in goldfish. TRH at 10 and 100 ng/g, but not 1 ng/g, significantly increased feeding and locomotor behaviors, as indicated by an increase in food intake and in the number of total feeding acts as compared to saline-injected fish. In order to assess possible interactions between TRH and other appetite regulators, we examined the effects of icv injections of TRH on the hypothalamic expression of orexin, orexin receptor and CART. The mRNA expression levels of all three peptides were significantly increased in fish injected with TRH at 100 ng/g as compared to saline-injected fish. Fasting increased TRH, orexin, and orexin receptor hypothalamic mRNA levels and decreased CART hypothalamic mRNA levels. Our results suggest that TRH is involved in the regulation of feeding/locomotor activity in goldfish and that this action is associated with a stimulation of both the orexin and CART systems.     

The hypothalamic-pituitary axis in diabetes mellitus

The hormonal response to LHRH and TRH was evaluated in three groups of male diaetics. Five patients were receiving therapy with the hypoglycemic agent glibenclamide, five were on NPH insulin and five were on dietary therapy alone. When compared to controls, the latter two groups had intact gonadotropin responses to LHRH. Despite normal basal gonadotropin levels, however, the group receiving glibenclamide therapy showed significantly exaggerated LH and FSH responses to LHRH. Both basal PRL and TSH levels, as well as the responses to TRH were normal in all three groups. These results indicate that LH, FSH, TSH and PRL secretion is intact in uncomplicated diabetes mellitus. The exaggerated LH and FSH responses to LHRH in the glibenclamide treated subjects are probably related to primary gonadal involvement; alternatively, there may be augmented pituitary gonadotropin secretion in this group.     

The effects of thyrotropin releasing hormone (TRH) on the gastrointestinal tract.

Thyrotropin-releasing hormone (TRH) immunoreactivity is distributed throughout the gastrointestinal tract and the pancreas. We have studied the effect of TRH on several gastrointestinal functions in intact, unanesthetized dogs. Intravenous TRH stimulated gastric action potentials (p<0.01) and transiently inhibited tetragastrin-stimulated gastric acid secretion (p<0.05). TRH had no effect on basal or secretin-stimulated pancreatic exocrine secretion. TRH did not alter water absorption in dogs with Thiry-Vella loops constructed from proximal jejunum.