The Ventral Premammillary Nucleus Links Fasting-Induced Changes in Leptin Levels and Coordinated Luteinizing Hormone Secretion

Physiological conditions of low leptin levels like those observed during negative energy balance are usually characterized by the suppression of luteinizing hormone (LH) secretion and fertility. Leptin administration restores LH levels and reproductive function. Leptin action on LH secretion is thought to be mediated by the brain. However, the neuronal population that mediates this effect is still undefined. The hypothalamic ventral premammillary nucleus (PMV) neurons express a dense concentration of leptin receptors and project to brain areas related to reproductive control. Therefore, we hypothesized that the PMV is well located to mediate leptin action on LH secretion. To test our hypothesis, we performed bilateral excitotoxic lesions of the PMV in adult female rats. PMV-lesioned animals displayed a clear disruption of the estrous cycle, remaining in anestrus for 15-20 d. After apparent recovery of cyclicity, animals perfused in the afternoon of proestrus showed decreased Fos immunoreactivity in the anteroventral periventricular nucleus and in gonadotropin releasing hormone neurons. PMV-lesioned animals also displayed decreased estrogen and LH secretion on proestrus. Lesions caused no changes in mean food intake and body weight up to 7 weeks after surgery. We further tested the ability of leptin to induce LH secretion in PMV-lesioned fasted animals. We found that complete lesions of the PMV precluded leptin stimulation of LH secretion on fasting. Our findings demonstrate that the PMV is a key site linking changing levels of leptin and coordinated control of reproduction.

Isolation and restraint stress results in differential activation of corticontrophin-releasing hormone and arginine vasopressin neurones in sheep

This study investigated sex differences in the stress-induced activation of neurons containing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin in the paraventricular nucleus (PVN) of gonadectomized male and female sheep. Groups (n=3) of both sexes were either subjected to 90 min isolation and restraint stress (stress group) or were not stressed. Blood samples were taken every 10 min for 90 min prior to and after stress to monitor cortisol levels in plasma. Brains were harvested after 90 min of stress. Stress caused elevation of plasma cortisol levels to a similar extent in both sexes. Double-labeling immunohistochemistry for Fos and either CRH, AVP or enkephalin was undertaken to quantify the numbers of neurons staining for CRH, AVP and enkephalin that also immunostained for Fos. Stress increased Fos immunostaining in all cell types. There was a greater proportion of CRH than AVP neurons activated in stressed animals. There were no sex differences in the activation of CRH and AVP neurons although females had a greater proportion of enkephalin cells staining for Fos than males in both control and stressed animals. There were no differences between control and stressed animals in the proportion of cells co-staining for CRH and AVP. We conclude that isolation and restraint stress activates neurons producing CRH, AVP and enkephalin in sheep and that CRH may play a greater role than AVP in regulating adrenocorticotrophic hormone secretion in response to this stressor in sheep. Finally, isolation and restraint stress does not influence co-localization of CRH and AVP in sheep.

Co-localization and distribution of corticotrophin-releasing hormone, arginine vasopressin and enkephalin in the paraventricular nucleus of sheep : a sex comparison

The paraventricular nucleus (PVN) is integral to regulation of the hypothalamo-pituitary-adrenal (HPA) axis and contains cells producing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin. We used immunohistochemistry to map these peptides and to resolve the extent of co-localization within PVN cells in intact and gonadectomized male and female sheep. Immunoreactive (ir) CRH, AVP and enkephalin cells were mapped in two rams and two ewes at 180 μm intervals throughout the rostro-caudal extent of the PVN. Similar distributions of AVP-ir cells occurred in both sexes whereas CRH-ir and enkephalin-ir cells extended more rostrally in rams. In groups (n=4) of intact and gonadectomized sheep of both sexes, co-localization and distribution of neuropeptides was influenced by sex and gonadectomy. Males had more AVP and CRH cells than females. Intact animals had more AVP cells than gonadectomized animals. There were no differences between groups in the number or percentage of cells that stained for both CRH and AVP or in the number of cells that stained for both CRH and enkephalin. Differences were observed in the percentage of enkephalin cells that contained CRH with males having a greater percentage of co-localized cells than did females. Differences were also observed in the number and percentage of cells that stained for both enkephalin and AVP; the number of cells that stained for both neuropeptides was greater in males than in females and greater in intact animals than in gonadectomized animals. Differences were observed in the percentage of AVP cells that contained enkephalin, and in the percentage of enkephalin cells that contained AVP with males having a greater percentage of co-localized cells than did females. We conclude that sex and gonadal status affect peptide distribution in the PVN of the sheep which may provide an anatomical basis for sex differences in HPA axis

Effect of naloxone-precipitated morphine withdrawal on c-fos expression in rat corticotropin-releasing hormone neurons in the paraventricular hypothalamus and extended amygdala

Morphine withdrawal is characterized by physical symptoms and a negative affective state. The 41 amino acid polypeptide corticotropin-releasing hormone (CRH) is hypothesized to mediate, in part, both the negative affective state and the physical withdrawal syndrome. Here, by means of dual-immunohistochemical methodology, we examined the co-expression of the c-Fos protein and CRH following naloxone-precipitated morphine withdrawal. Rats were treated with slow-release morphine 50 mg/kg (subcutaneous, s.c.) or vehicle every 48 h for 5 days, then withdrawn with naloxone 5 mg/kg (s.c.) or saline 48 h after the final morphine injection. Two hours after withdrawal rats were perfused transcardially and their brains were removed and processed for immunohistochemistry. We found that naloxone-precipitated withdrawal of morphine-dependent rats increased c-Fos immunoreactivity (IR) in CRH positive neurons in the paraventricular hypothalamus. Withdrawal of morphine-dependent rats also increased c-Fos-IR in the central amygdala and bed nucleus of the stria terminalis, however these were in CRH negative neurons.

Effects of postbreeding gonadotropin treatments on conception rates of lactating dairy cows subjected to timed artificial insemination or embryo transfer in a tropical environment

The objective of experiment 1 was to evaluate the effects of treatments with human chorionic gonadotropin (hCG) or GnRH 7 d after induced ovulation on reproductive performance of lactating dairy cows submitted to timed artificial insemination (TAI) or timed embryo transfer (TET). A total of 834 potential breedings were used from 661 lactating Holstein cows (37.3 +/- 0.3 kg of milk/d). Cows had ovulation synchronized and were assigned randomly to receive TAI on d 0 or TET on d 7. Within each group, cows were assigned randomly to receive on d 7 no additional treatment (control; n(TAI) = 156; n(TET) = 126), a 100 mu g i.m. injection of GnRH (n(TAI) = 155; n(TET) = 124), or a 2,500 TU i.m. injection of hCG (ITA = 151; n(TET) = 122). Postbreeding treatment affected the percentages of pregnant cows at TET on d 28 (control: 38.1%; GnRH: 52.4%; hCG: 45.1%) and on d 60 (control: 32.5%; GnRH: 41.1%; hCG: 38.5%), but postbreeding treatment did not affect percentages of pregnant cows at TAT on d 28 (control: 30.1%; GnRH: 32.2%; hCG: 32.4%) or on d 60 (control: 25.6%; GnRH: 27.1%; hCG: 29.8%). The objective of experiment 2 was to evaluate the effect of a treatment with GnRH 7 d after TET on reproductive performance of lactating dairy cows that received a previous GnRH treatment at TET. A total of 285 potential breedings were used from 257 lactating Holstein cows (35.1 +/- 0.8 kg of milk/d). Cows had ovulation synchronized and were assigned for TET on d 7. Immediately after TET, all cows were treated with a 100 mu g i.m. injection of GnRH. on d 14, cows were assigned randomly to receive (G7-14; n = 147) or not (G7; n = 138) an additional injection of GnRH. Pregnancy diagnosis were performed on d 28 and 60. The additional treatment with GnRH on d 14 did not affect the percentages of pregnant cows on d 28 (G7: 48.5%; G7-14: 42.9%) or on d 60 (G7: 39.8%; G7-14: 37.4%). In conclusion, treatment with GnRH or hCG 7 d after induced ovulation increased conception rates in lactating dairy cows submitted to TET, but not in cows submitted to TAI. Moreover, treatment with GnRH 7 d after TET did not enhance reproductive performance of lactating dairy cows that received a previous GnRH treatment at TET.

Effect of zinc administration on thyrotropin releasing hormone-stimulated prolactinemia in healthy men

Previous in vitro studies have demonstrated zinc (Zn++) inhibition of basal and of potassium (K+) or thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion, in a selective, reversible, and dose-dependent manner. Thus, Zn++ may regulate physiologically pituitary PRL secretion. Furthermore, studies with patients with uremia, cirrhosis or prolactinoma, have shown the coexistence of hypozincemia and hyperprolactinemia and zinc supplementation did not correct hyperprolactinemia in these patients. In normal individuals Zn++ administration produced controversial results on PRL secretion. Here, we investigated whether zinc administration affects TRH-stimulated PRL in healthy men. We found that Zn++ administration does not change the TRH-stimulated PRL. Therefore, in normal conditions, Zn++ does not inhibit TRH-stimulated prolactinemia. In addition, we found that acute increases of blood PRL and TRH do not alter blood Zn++ levels.

Paracoccidioidomycosis in the region of Botucatu (state of São Paulo, Brazil). Evaluation of serum thyroxine (T4) and triiodothyronine (T3) levels and of the response to thyrotropin releasing hormone (TRH)

T4, T3 and TSH serum levels were measured in 25 patients with paracoccidioidomycosis. Thyroid T3 reserves were measured on the basis of the increase in T3 (ΔT3) 2 h after intravenous injection of 200 μg TRH, and pituitary TSH reserves were measured on the basis of TSH increase (ΔTSH) 20 min after the same injection. Twenty healthy volunteers with no history of thyroid disease were used as controls. When the two groups were compared, the following results were obtained: (a) there was no significant difference in mean T4, T3, ΔTSH between groups; (b) reduced T3 levels were detected more frequently in patients with paracoccidioidomycosis, especially among those with the acute form of the disease or with the severely disseminated chronic form. The results suggest the occurrence of a reduction in peripheral conversion of T4 to T3, but do not indicate the occurrence of hypothyroidism in any of its forms (thyroid, pituitary or hypothalamic). © 1988 Kluwer Academic Publishers.

Effects on performance and carcass and meat quality attributes following immunocastration with the gonadotropin releasing factor vaccine bopriva or surgical castration of bos indicus bulls raised on pasture in brazil

Bos indicus bulls 20. months of age grazed on pasture in Minas Gerais, Brazil either received 2 doses of the GnRF vaccine Bopriva at d0 and d91 (group IC, n. =. 144) or were surgically castrated on d91 (group SC, n. =. 144). Slaughter on d280, was 27. weeks after castration. Adverse safety issues in 8% of group SC bulls following surgery contrasted with 0% in group IC bulls. At d105 testosterone levels were suppressed to similar levels in both groups. Importantly, group IC bulls had higher live weight, hot carcass weight, ADG (P<. 0.005) and dressing percentage (P<. 0.0001) compared to group SC animals. There were no negative effects on carcass or meat quality traits, thus immunocastration was concluded to offer a safe and effective method that provides production gains, and improves animal welfare in Bos indicus beef bulls without impacting meat and carcass quality. © 2013 Elsevier Ltd.

Effects of a synthetic gonadotrophin-releasing hormone agonist, leuprolide, on rut-associated events in male red deer

This study examined the effectiveness of leuprolide, a gonadotrophin-releasing hormone agonist, in suppressing rut-associated events in farmed male red deer. In mid-January (~6 weeks before the rut period in the southern hemisphere) adult red deer (Cervus elaphus scoticus) stags that had been allocated to three groups (n = 10 per group) received leuprolide, administered subcutaneously in a 90-day release formulation, at zero (control), low (22.5 mg) or high (45 mg) doses. Following treatment with leuprolide there was evidence of suppression of mean plasma luteinising hormone concentration that was significant (P < 0.05) at 9 weeks. Mean plasma testosterone concentration of all three groups rose following treatment, then declined prematurely in the low- and high-dose leuprolide-treated groups, so that it was significantly (P < 0.05) suppressed (0.66 ± 0.29 and 2.0 ± 0.88 ng mL–1, low and high dose respectively) in early April when the peak value (9.0 ± 1.94 ng mL–1) was recorded from control stags. A reduction in mean liveweight occurred in all three groups through February–April and this did not differ among treatments. However, a corresponding reduction in mean body condition score was greater in the control stags (P < 0.05). There was little effect of leuprolide treatment on aggressive behaviours, but it lowered roaring frequency in the latter period of the rut. The results indicate that this gonadotrophin-releasing hormone agonist has potential for application in the deer farming industry to suppress undesirable effects of the rut.

GH-Releasing Hormone Receptor Gene: A Novel Splice-Disrupting Mutation and Study of Founder Effects

Background: Mutations in GH-releasing hormone receptor gene (GHRHR) are emerging as the most common cause of autosomal recessive isolated GH deficiency (IGHD). Objective: To search for GHRHR mutations in patients with familial or sporadic IGHD and to investigate founder effects in recurring mutations. Methods: The coding region of GHRHR was entirely amplified and sequenced from DNA of 18 patients with IGHD (16 unrelated) with topic posterior pituitary lobe on MRI. Haplotypes containing promoter SNPs and microsatellites flanking GHRHR were analyzed in patients with c.57+1G>A (IVS1+1G>A) mutation of our previously published kindred and also a Brazilian patient and 2 previously reported Japanese sisters with c. 1146G>A (p.E382E) mutation. Results: A novel homozygous intronic GHRHR c.752-1G>A (IVS7-1G>A) mutation, predicting loss of the constitutive splice acceptor site, was identified in two siblings with IGHD. A compound heterozygous c.[57+1G>A];[1146G>A] and a heterozygous c.527C>T (p.A176V) were found in two sporadic cases. Haplotype analysis provided evidence for a founder effect for the c.57+1G>A mutation and independent recurrence for the c.1146G>A mutation. Conclusion: We report a novel splice-disrupting mutation in GHRHR in 2 siblings and provide evidence that all c.57+1G>A (IVS1+1G>A) mutant chromosomes have the same haplotype ancestor, indicating the occurrence of a founder effect in Brazilian patients with IGHD. Copyright (C) 2012 S. Karger AG, Basel

The consequences of growth hormone-releasing hormone receptor haploinsufficiency for bone quality and insulin resistance

Objective Growth hormone (GH)/insulin-like growth factor (IGF) axis and insulin are key determinants of bone remodelling. Homozygous mutations in the GH-releasing hormone receptor (GHRHR) gene (GHRHR) are a frequent cause of genetic isolated GH deficiency (IGHD). Heterozygosity for GHRHR mutation causes changes in body composition and possibly an increase in insulin sensitivity, but its effects on bone quality are still unknown. The objective of this study was to assess the bone quality and metabolism and its correlation with insulin sensitivity in subjects heterozygous for a null mutation in the GHRHR. Patients and methods A cross-sectional study was performed on 76 normal subjects (68.4% females) (N/N) and 64 individuals (64.1% females) heterozygous for a mutation in the GHRHR (MUT/N). Anthropometric features, quantitative ultrasound (QUS) of the heel, bone markers [osteocalcin (OC) and CrossLaps], IGF-I, glucose and insulin were measured, and homeostasis model assessment of insulin resistance (HOMAIR) was calculated. Results There were no differences in age or height between the two groups, but weight (P = 0.007) and BMI (P = 0.001) were lower in MUT/N. There were no differences in serum levels of IGF-I, glucose, T-score or absolute values of stiffness and OC, but insulin (P = 0.01), HOMAIR (P = 0.01) and CrossLaps (P = 0.01) were lower in MUT/N. There was no correlation between OC and glucose, OC and HOMAIR in the 140 individuals as a whole or in the separate MUT/N or N/N groups. Conclusions This study suggests that one allele mutation in the GHRHR gene has a greater impact on energy metabolism than on bone quality.