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Sex differences in the stress-induced activity of the hypothalamo-pituitary-adrenal axis in sheep appear to be dependent on the stressor encountered and occur irrespective of the presence of gonadal steroids. We tested the hypotheses that cortisol responses to exercise, endotoxin, wetting (experiment 1), and isolation/restraint (experiment 2) stress differ between gonadectomized male and female sheep. At weekly intervals (in experiment 1), we subjected gonadectomized rams and ewes (n = 6/group) to control conditions, to exercise stress, to iv injection of endotoxin, and to wetting stress. In a second experiment (experiment 2), we subjected gonadectomized rams and ewes (n = 5/group) to control conditions or to isolation/restraint stress. In both experiments, we measured plasma concentrations of cortisol before, during, and after stress at a frequency of at least 15 min with samples collected (from an indwelling jugular catheter) at a greater frequency around the time of the stressor. Cortisol responses to wetting (experiment 1) and isolation/restraint (experiment 2) stress were significantly higher in females compared with males but in response to exercise (experiment 1) and endotoxin (experiment 1) stress, there were no differences between the sexes. For some stressors, there are sex differences in sheep in the stress-induced activity of the hypothalamo-pituitary-adrenal axis that are independent of the presence of the sex steroids, but the existence of these sex differences and the direction of these sex differences differs, depending on the stressor imposed.

Kisspeptin is the product of the Kiss1 gene that regulates GnRH secretion. In sheep, Kiss1 mRNA-expressing cells are found in the preoptic area (POA) and arcuate nucleus (ARC), and expression is up-regulated in the caudal ARC during the periovulatory period. We hypothesized that kisspeptin neurons in the ARC are activated by estradiol-17beta prior to the preovulatory LH surge. Ovariectomized ewes were treated as follows: 1) estradiol-17beta implants (sc 2 wk) to cause tonic negative feedback; 2) vehicle (no estrogen negative or positive feedback); or 3) positive feedback/GnRH surge-inducing injection of estradiol-17beta (50 microg iv). For groups 2 and 3, brains were collected 1 h after treatment and kisspeptin/Fos immunoreactivity was examined. In the caudal and mid-ARC, the percentage of kisspeptin cells that were Fos immunoreactive increased after acute estradiol treatment (group 3) over that seen in the other two groups. Kisspeptin/Fos colocalization was also quantified in ewes during the luteal and late-follicular phase of the estrous cycle, showing a trend toward an increase in colocalization in the late-follicular phase. Kisspeptin/Fos colocalization was similar in the POA across groups in both experiments. Analysis of Kiss1 mRNA by in situ hybridization revealed an increase in expression during the late-follicular phase in the caudal ARC and POA. These data suggest kisspeptin neurons located in the caudal extent of the ARC are involved in generating the positive feedback preovulatory GnRH/LH surge in the ewe, but there may also be a role for Kiss1-expressing cells in the POA.

There is strong evidence that kisspeptin acts to regulate GnRH secretion, but whether there is also a component of action on the gonadotropes is not clear. Using quantitative RT-PCR, we found that G protein-coupled receptor-54 mRNA is expressed in ovine pituitary cell fractions enriched for gonadotropes as well as in somatotropes and lactotropes. To test whether kisspeptin acts directly on the pituitary gonadotropes, we first examined LH release from primary ovine pituitary cell cultures treated with kisspeptin. We found that kisspeptin treatment increased the concentration of LH in culture media by 80%, compared with control, but only in pituitary cultures from ewes during the follicular phase of the estrous cycle. After this, we determined whether kisspeptin acts on the pituitary gland in vivo. Using GnRH-replaced ovariectomized hypothalamo-pituitary-disconnected ewes, we were not able to achieve any effect of kisspeptin on LH under steady-state conditions or during the period of an estrogen-induced LH surge. Finally, we collected hypophysial portal blood samples from ovariectomized ewes and measured kisspeptin levels. Low but detectable amounts of kisspeptin were found in portal plasma, but levels were similar in ovariectomized ewes that were untreated or given estrogen to elicit an LH surge. Thus, although we observed an effect of kisspeptin on LH release in vitro in some situations, similar findings were not obtained in vivo. Moreover, the low concentrations of kisspeptin in hypophysial portal blood and the lack of any change during the period of an estrogen-induced GnRH/LH surge suggest that action on the pituitary gland is not of major consequence in terms of LH release.

We determined whether kisspeptin could be used to manipulate the gonadotropin axis and ovulation in sheep. First, a series of experiments was performed to determine the gonadotropic responses to different modes and doses of kisspeptin administration during the anestrous season using estradiol-treated ovariectomized ewes. We found that: 1) injections (iv) of doses as low as 6 nmol human C-terminal Kiss1 decapeptide elevate plasma LH and FSH levels, 2) murine C-terminal Kiss1 decapeptide was equipotent to human C-terminal Kiss1 decapeptide in terms of the release of LH or FSH, and 3) constant iv infusion of kisspeptin induced a sustained release of LH and FSH over a number of hours. During the breeding season and in progesterone-synchronized cyclical ewes, constant iv infusion of murine C-terminal Kiss1 decapeptide-10 (0.48 mumol/h over 8 h) was administered 30 h after withdrawal of a progesterone priming period, and surge responses in LH occurred within 2 h. Thus, the treatment synchronized preovulatory LH surges, whereas the surges in vehicle-infused controls were later and more widely dispersed. During the anestrous season, we conducted experiments to determine whether kisspeptin treatment could cause ovulation. Infusion (iv) of 12.4 nmol/h kisspeptin for either 30 or 48 h caused ovulation in more than 80% of kisspeptin-treated animals, whereas less than 20% of control animals ovulated. Our results indicate that systemic delivery of kisspeptin provides new strategies for the manipulation of the gonadotropin secretion and can cause ovulation in noncyclical females.

Unlabelled: This study examines the effect of long-term ICV administration of leptin in ewes. We found that central application significantly decreased osteoblast activity as measured by serum analysis as well as by histomorphometry, resulting in decreased trabecular bone volume. These data provide additional evidence that bone formation and therefore bone remodeling is at least in part centrally controlled., Introduction: Genetic studies in mice have identified leptin as a potent inhibitor of bone formation acting through the central nervous system and unraveled the central nature of bone mass control and its disorders. Although these studies have radically enhanced our understanding of skeletal physiology because they have established a hypothalamic regulation of bone remodeling through the sympathetic nervous system, controversy remains about the physiological relevance of these observations because leptin's effect on bone after intracerebroventricular (ICV) application has only been shown in mice. To address whether leptin has a role in regulating bone mass beyond rodents, we treated ewes with long-term ICV application of leptin and analyzed the bone phenotype after a treatment period of 3 months., Materials and Methods: Three groups of corriedale sheep were compared: (1) control entire (control), (2) ovariectomy (OVX) and ICV application of cerebrospinal fluid (CSF); and (iii) OVX and ICV application of leptin (leptin). Analysis included histomorphometric characterization of iliac crest, spine and femur by histology and biomechanical testing and measurement of bone turnover parameters in serum and urine., Results: Central application of leptin decreased bone formation by 70% and mineralizing surface (MS/BS, 39.4 +/- 3.3% versus 16.1 +/- 2.1%) significantly (p < 0.01). Whereas OVX increased osteoclast indices and urinary cross-lap excretion by two and three times, respectively, serum parameters of osteoblast activity were significantly reduced by ICV application of leptin (p < 0.01). Consequently, ewes treated with leptin were osteopenic (iliac crest BV/TV entire, 22.7 +/- 1.3%; CSF, 18.9 +/- 2.4%; leptin, 12.4 +/- 2.6%), whereas bone torsional failure load reflecting the cortex of the tibia was not yet changed after 3 months of treatment (p < 0.01)., Conclusions: Taken together, these data suggest that leptin controls bone formation after ICV application, leading to reduction of trabecular bone mass in sheep. Most importantly, however, they show that the central regulation of bone formation is not limited to rodents, but is also found in large animals, providing further evidence that bone remodeling in vertebrates is centrally controlled.

We investigated the effect of the presence and absence of lambs and suckling by lambs to attenuate activation of the hypothalamo-pituitary-adrenal (HPA) axis to isolation and restraint stress in lactating sheep. In experiment 1, blood samples were collected every 10 min from nonlactating (n = 5) and lactating (n = 5) ewes for 4 h before and during stress. In experiment 2, ewes (n = 6) were allocated to 1) nonlactating, 2) lactating with lambs absent, 3) lactating with lambs present but unable to suckle, and 4) lactating with lambs present and able to suckle. Blood samples were collected over 8 h with no stress (control day) and for 4 h before and 4 h during stress (stress day). In experiment 1, the mean (+/-SEM) cortisol concentrations increased significantly (P < 0.05) in nonlactating ewes during stress but did not change in lactating ewes. In experiment 2, cortisol did not vary on the control day or pretreatment of the stress day but increased (P < 0.05) during stress in all groups except lactating ewes with lambs present and able to suckle. The greatest cortisol response occurred in nonlactating ewes followed by lactating ewes with lambs absent and lactating ewes with lambs present but unable to suckle. During stress, the ACTH concentrations increased (P < 0.05) in nonlactating ewes and lactating ewes with lambs absent but not in lactating ewes with lambs present. We conclude that the activity of the HPA axis during isolation and restraint is reduced in lactating ewes and that the presence of lambs increases this level of attenuation.

Kisspeptin is a peptide that has been implicated in the regulation of GnRH cells in the brain. Immunohistochemical studies were undertaken to examine the distribution of kisspeptin-immunoreactive (IR) cells in the ovine diencephalon and determine the effect of ovariectomy in the ewe. We report that kisspeptin colocalizes to a high proportion of GnRH-IR cells in the preoptic area, which is a novel finding. A high level of colocalization of kisspeptin and GnRH was also seen in varicose neuronal fibers within the external, neurosecretory zone of the median eminence. Apart from the kisspeptin/GnRH cells, a population of single-labeling kisspeptin-IR cells was also observed in the preoptic area. Within the hypothalamus, kisspeptin-IR cells were found predominantly in the arcuate nucleus, and there was an increase in the number of immunohistochemically identified cell within this nucleus after ovariectomy. Kisspeptin-IR cells were also found in the periventricular nucleus of the hypothalamus, but the number observed was similar in gonad-intact and ovariectomized ewes. The colocalization of GnRH and kisspeptin within cells of the preoptic area and GnRH neurosecretory terminals of the median eminence suggests that the two peptides might be cosecreted into the hypophyseal portal blood to act on the pituitary gland. Effects of ovariectomy on the non-GnRH, Kisspeptin-IR cells of the hypothalamus suggest that kisspeptin production is negatively regulated by ovarian steroids.

The present study investigated photorefractoriness in the prolactin (PRL) axis in hypothalamopituitary-disconnected (HPD) sheep exposed to prolonged long days. In experiment 1, HPD Soay rams transferred from short (8L:16D) to long (16L:8D) days for 48 wk to induce a cycle of activation, decline (photorefractoriness), and reactivation in PRL secretion were treated chronically with bromocriptine (dopamine-receptor agonist) or vehicle from the onset of photorefractoriness. Bromocriptine (0.01-0.04 mg kg-1 day-1; 12-24 wk of long days) blocked PRL release and caused a rebound response after the treatment, but it had no effect on the long-term PRL cycle (posttreatment PRL minimum, mean +/- SEM, 35.3 +/- 0.6 and 37.0 +/- 0.4 wk for bromocriptine and control groups, respectively; not significant). In experiment 2, HPD rams were treated with sulpiride (dopamine-receptor antagonist) during photorefractoriness. Sulpiride (0.6 mg/kg twice daily; 22-30 wk of long days) induced a marginal increase in blood PRL concentrations, but again, it had no effect on the long-term PRL cycle (PRL minimum, 37.9 +/- 0.4 and 37.6 +/- 0.9 wk for sulpiride and control groups, respectively; not significant). The 24-h blood melatonin profile consistently reflected the long-day photoperiod throughout, and blood FSH concentrations were minimal, confirming the effectiveness of the HPD surgery. The results support the conclusion that photorefractoriness is regulated at the level of the pituitary gland independently of the PRL output signal.

Stress responses are thought to act within the hypothalamopituitary unit to impair the reproductive system, and the sites of action may differ between sexes. The effect of isolation and restraint stress on pituitary responsiveness to GnRH in sheep was investigated, with emphasis on possible sex differences. Experiments were conducted during the breeding season and the nonbreeding season. In both experiments, 125 ng of GnRH was injected i.v. every 2 h into hypothalamopituitary disconnected, gonadectomized rams and ewes on 3 experimental days, with each day divided into two periods. During the second period on Day 2, isolation and restraint stress was imposed for 5.5 h. Plasma concentrations of LH and cortisol were measured in samples of blood collected from the jugular vein. In the second experiment (nonbreeding season), plasma concentrations of epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylglycol were also measured. In both experiments, there was no effect of isolation and restraint stress on plasma concentrations of cortisol in either sex. During the breeding season, there was no effect of isolation and restraint stress on plasma concentrations of LH in either sex. During the nonbreeding season, the amplitude of the first LH pulse after the commencement of stress was significantly reduced (P < 0.05) in rams and ewes. In the second experiment, during stress there was a significant increase (P < 0.05) in plasma concentrations of epinephrine in rams and ewes and significantly higher (P < 0.05) basal concentrations of norepinephrine in ewes than in rams. These results suggest that in sheep stress reduces responsiveness of the pituitary gland to exogenous GnRH during the nonbreeding season but not during the breeding season, possibly because of mediators of the stress response other than those of the hypothalamus-pituitary-adrenal gland axis.

Thalidomide (Thd) is clinically useful in a number of conditions where its efficacy is probably related to its anti-TNF-alpha activity. More recently, Thd has also been shown to co-stimulate T cells and second generation co-stimulatory (IMiD trade mark ) analogues are currently being assessed in the treatment of cancer patients. However, in contrast to their known suppressive effects during inflammatory stimuli, the effects of Thd/IMiDs on TNF-alpha and TNF receptors (TNFRs) during T cell co-stimulation are not known. We sought to determine the effect of Thd, two clinically relevant IMiDs (CC-4047, ACTIMID trade mark and CC-5013, REVIMID trade mark ) and a non-stimulatory SelCID analogue (CC-3052) on TNF-alpha production and on the expression and shedding of TNFRs during co-stimulation. We found that co-stimulation of PBMC with Thd/IMiDs, but not CC-3052, prevented alphaCD3-induced T cell surface expression of TNFR2 and thereby reduced soluble TNFR2 (sTNFR2) levels. However, there was no effect on total (surface/intracellular) TNFR2 protein expression, suggesting inhibition of trafficking to the cell membrane. The extent of co-stimulation by Thd/IMiDs (assessed by CD69/CD25 expression and IL-2/sIL-2Ralpha production) was similar for CD4+ and CD8+ T lymphocytes and correlated with TNFR2 inhibition. Co-stimulation, but not the early inhibitory effect on TNFR2, was IL-2-dependent and led to increased TNF-alpha production by both CD4+ and CD8+ T lymphocytes. The clinical relevance of this observation was confirmed by the elevation of serum TNF-alpha during REVIMID trade mark treatment of patients with advanced cancer. Together, these results suggest a possible role for TNF-mediated events during co-stimulation and contrast with the TNF inhibitory effects of Thd and its analogues during inflammatory stimuli.

Analyses of FSH secretion suggest pulsatile, nonpulsatile, or compositely pulsatile and nonpulsatile release modes. This may reflect the reduced signal-to-noise ratio inherent in FSH pulse estimation procedures and/or immunological-biological assay inconsistencies. To address these issues, we sampled cavernous sinus and jugular venous blood concomitantly from ovariectomized sheep at either 5-min or 1-min intervals. Samples from the former were assayed by RIA, and those from the latter by RIA and bioassay. Waveform-independent peak detection revealed FSH pulses occurring at high frequency. Pulsatile FSH secretion accounted for 28% of total secretion. Approximate entropy analysis showed that FSH secretion was nearly random. There was synchronous release of LH and FSH, but most FSH secretion was not associated with LH release; 13% of discrete FSH and LH pulses were concordant. We infer that FSH secretion exhibits pulsatile and basal/nonpulsatile features, with high-entropy features. Linear and nonlinear statistical measures revealed joint sample-by-sample synchrony of FSH and LH release, indicating pattern coordination despite sparse synchrony of pulses. We postulate that pattern synchrony of FSH and LH release is effected at the level of the gonadotrope. Concordant FSH and LH pulses probably result from pulsatile GnRH input, but other mechanisms could account for independent FSH pulses.

Interleukin-12 (IL-12) is a key immunomodulatory cytokine produced by antigen-presenting cells that promotes cellular immunity and enables the generation of protective immunity against intracellular pathogens and tumours. Therefore, modulation of IL-12 activity is a primary immunotherapeutic goal. However, little is known about its regulation. Signalling via p38 MAPK has been implicated in the control of inflammatory responses and is therefore a potential therapeutic target. We have used the highly selective p38 MAPK inhibitor (SB203580) to examine the effect of this pathway on the production of IL-12. Surprisingly, we found that SB203580 strongly up-regulated LPS induced IL-12p40 at the protein (intracellular and secreted) and mRNA levels in PBMC cultures. The effect on IL-12 was apparent using both T cell-independent and T cell-dependent stimuli but not in unstimulated cultures, indicating that activation signals are required. Furthermore, the production of IFN-gamma by T cells is crucial as production was not increased in LPS-stimulated, purified adherent monocytes/macrophages without the addition of exogenous IFN-gamma. These results provide evidence that p38 MAPK has an unexpected suppressive effect on IL-12p40 gene transcription, and suggests interplay between p38 MAPK- and IFN-gamma -mediated signals in the regulation of IL-12 production by monocytes/macrophages. Furthermore, the importance of IL-12 as a key immunoregulatory cytokine suggests that the clinical application of pyrinidyl imidazole inhibitors, such as SB203580, may need to be reassessed.

Whilst there have been many studies in various species examining the effects of leptin on food intake, there is a paucity of data comparing responsiveness in the two sexes. We have, therefore, addressed this issue in sheep. Because this species shows seasonal variation in voluntary food intake (VFI), we also considered the possibility that there might be seasonal variation in the responsivity to leptin. Centrally administered leptin was relatively ineffective as a satiety factor in either sex during AUTUMN: In Spring, leptin had a profound inhibitory effect on VFI in the females, but only a slight effect in males. These data indicate that responsiveness to leptin depends on sex and also on season in animals that are substantially affected by photoperiod.

This minireview considers the state of knowledge regarding the interactions of testicular hormones to regulate the secretion and actions of GnRH in males, with special focus on research conducted in rams and male rhesus monkeys. In these two species, LH secretion is under the negative feedback regulation of testicular steroids that act predominantly within the central nervous system to suppress GnRH secretion. The extent to which these actions of testicular steroids result from the direct actions of testosterone or its primary metabolites, estradiol or dihydrotestosterone, is unclear. Because GnRH neurons do not contain steroid receptors, the testicular steroids must influence GnRH neurons via afferent neurons, which are largely undefined. The feedback regulation of FSH is controlled by inhibin acting directly at the pituitary gland. In male rhesus monkeys, the feedback regulation of FSH secretion is accounted for totally by the physiologically relevant form of inhibin, which appears to be inhibin B. In rams, the feedback regulation of FSH secretion involves the actions of inhibin and testosterone and interactions between these hormones, but the physiologically relevant form of inhibin has not been determined. The mechanisms of action for inhibin are not known.

This experiment determined if the degree of stimulation of the pituitary gland by GnRH affects the suppressive actions of inhibin and testosterone on gonadotropin secretion in rams. Two groups (n = 5) of castrated adult rams underwent hypothalamopituitary disconnection and were given two i.v. injections of vehicle or 0.64 microg/kg of recombinant human inhibin A (rh-inhibin) 6 h apart when treated with i.m. injections of oil and testosterone propionate every 12 h for at least 7 days. Each treatment was administered when the rams were infused i.v. with 125 ng of GnRH every 4 h (i.e., slow-pulse frequency) and 125 ng of GnRH every hour (i.e., fast-pulse frequency). The FSH concentrations and LH pulse amplitude were lower and the LH concentrations higher during the fast GnRH pulse frequency. The GnRH pulse frequency did not influence the ability of rh-inhibin and testosterone to suppress FSH secretion. Testosterone did not affect LH secretion. Following rh-inhibin treatment, LH pulse amplitude decreased at the slow, but not at the fast, GnRH pulse frequency, and LH concentrations decreased at both GnRH pulse frequencies. We conclude that the degree of stimulation of the pituitary by GnRH does not influence the ability of inhibin or testosterone to suppress FSH secretion in rams. Inhibin may be capable of suppressing LH secretion under conditions of low GnRH.

We have used in situ hybridization to compare the distributions of estrogen receptor alpha (ERalpha) and ERbeta messenger RNA (mRNA)-containing cells in the preoptic area and hypothalamus of ewes and rams. Perfusion-fixed brain tissue was collected from luteal phase ewes and intact rams (n = 4) during the breeding season. Matched pairs of sections were hybridized with sheep-specific, 35S-labeled riboprobes, and semiquantitative image analysis was performed on emulsion-dipped slides. A number of sex differences were observed, with females having a greater density of labeled cells than males (P < 0.001) and a greater number of silver grains per cell (P < 0.01) in the ventromedial nucleus for both ER subtypes. In addition, in the retrochiasmatic area, males had a greater (P < 0.05) cell density for ERalpha mRNA-containing cells than females, whereas in the paraventricular nucleus, females had a greater density (P < 0.05) of ERalpha mRNA-containing cells than males. There was a trend (P = 0.068) in the arcuate nucleus for males to have a greater number of silver grains per cell labeled for ERalpha mRNA. In both sexes, there was considerable overlap in the distributions of ERalpha and ERbeta mRNA-containing cells, but the density of labeled cells within each nucleus differed in a number of instances. Nuclei that contained a higher (P < 0.001) density of ERalpha than ERbeta mRNA-containing cells included the preoptic area, bed nucleus of the stria terminalis, and ventromedial nucleus, whereas the subfornical organ (P < 0.001), paraventricular nucleus (males only, P < 0.05), and retrochiasmatic nucleus (females only, P < 0.05) had a greater density of ERalpha than ERbeta mRNA-containing cells. The anterior hypothalamic area and supraoptic nucleus had similar densities of cells containing both ER subtypes. The lateral septum and arcuate nucleus contained only ERalpha, whereas only ERbeta mRNA-containing cells were seen in the zona incerta. The sex differences in the populations of ER mRNA-containing cells in the ventromedial and arcuate nuclei may explain in part the sex differences in the neuroendocrine and behavioral responses to localized estrogen treatment in these nuclei. Within sexes, the differences between the distributions of ERalpha and ERbeta mRNA-containing cells may reflect differential regulation of the actions of estrogen in the sheep hypothalamus. Low levels of ERbeta mRNA in the preoptic area and ventromedial and arcuate nuclei, regions known to be important for the regulation of reproduction, suggest that ERbeta may not be involved in these functions.

The application of molecular techniques to the characterization of caliciviruses has resulted in an extensive database of sequence information. This information has led to the identification of 4 distinct genera. The human enteric caliciviruses have been assigned to 2 of these genera. This division is reflected not only in sequence diversity but in a fundamental difference in genome organization. Complete genome sequences are now available for 5 enteric caliciviruses and demonstrate that human and animal enteric caliciviruses are phylogenetically closely related. Currently, there is no cell culture system for the human viruses; therefore, studies have relied on heterologous expression and in vitro systems. These studies have shown that in both human and animal viruses the viral nonstructural proteins are produced from a polyprotein precursor that is cleaved by a single viral protease. The purpose of this article is to provide an overview of the current knowledge of genome structure and gene expression in the enteric caliciviruses.

The International Committee on Taxonomy of Viruses (ICTV) has recently approved several proposals submitted by the present Caliciviridae Study Group. These proposals include the division of the family into 4 new genera designated Lagovirus, Vesivirus, "Norwalk-like viruses (NLVs), and "Sapporo-like viruses (SLVs); the latter 2 genera were assigned temporary names until acceptable names can be determined by the scientific community. The genera have been further divided into the following species: Feline calicivirus and Vesicular exanthema of swine virus (genus Vesivirus), Rabbit hemorrhagic disease virus and European brown hare syndrome virus (genus Lagovirus), Norwalk virus (genus NLV), and Sapporo virus (genus SLV). In addition, the ICTV approved a proposal to remove the hepatitis E virus from the Caliciviridae into an "unassigned classification status.

Leptin is a secreted product of the adipocytes that regulates a variety of functions. The presence of the leptin receptor (LR) has been demonstrated in the endocrine and neuroendocrine tissue, but only limited information is available regarding cell-specific expression in the anterior pituitary gland. We have used double-label immunofluorescence histochemistry to study the distribution of LR-like immunoreactivity (LR-ir) in the corticotropes, somatotropes, and gonadotropes of the ovine anterior pituitary. LR-ir was found in 34% of cells in the pars distalis and 94% of the cells in the pars tuberalis. In the pars distalis, LR-ir was present in 27% of corticotropes, 69% of somatotropes, and 29% of gonadotropes. In contrast, 90% of the gonadotropes in the pars tuberalis were immunopositive for LR. There was no alteration in the number of gonadotropes containing LR-ir during the various phases of the estrous cycle (n = 3/group) in the pars distalis (luteal phase, 36%; follicular phase, 32%; and estrous phase, 32%). In conclusion, we show that, in the pars distalis, LR-ir is expressed to a greater extent in the somatotropes than in the gonadotropes or corticotropes. This is in accordance with the documented effects of leptin on pituitary GH secretion. The differential expression of LR-ir between the gonadotropes of the pars distalis and pars tuberalis probably reflects the different phenotypes of the cells in these two regions. Lower levels of LR-ir expression in gonadotropes and corticotropes of the pars distalis may suggest that leptin does not substantially influence these particular cells, at least in this species.

We have developed a ruminant model to study long-term alterations in adiposity on the expression of appetite-regulating peptides in the hypothalamus. In this model endocrine and metabolic status are fully defined as well as body composition. The current study sought to define the effects of altered adiposity on the expression of genes for neuropeptide Y (NPY), POMC, enkephalin (ENK), and melanin-concentrating hormone (MCH). Ovariectomized ewes with high (60 +/- 1 kg) (FAT) or low (37 +/- 3 kg) body weights (THIN) were blood sampled every 10 min for 8 h to determine metabolic and endocrine status. The animals were then killed and the brains perfused for in situ hybridization. Body composition analysis was performed on the carcass using dual energy x-ray absorptiometry; this indicated that the FAT animals were 36 +/- 1% fat, whereas the THIN animals were 15 +/- 2% fat. The LH interpulse interval was lower and mean GH concentrations were higher in the THIN animals; cortisol and TSH levels were not different between the two groups but free T4 and free T3 levels were lower; the FT3:FT4 ratio was higher in THIN ewes. Levels of insulin, lactate, and nonesterified fatty acids were lower in the THIN group, and plasma glucose and urea concentrations were similar in THIN and FAT animals. Levels of gene expression of NPY and MCH were higher in THIN ewes. POMC expression was similar in the two groups. In the THIN animals, ENK expression was lower in the paraventricular and ventromedial nuclei but higher in the periventricular region. In conclusion, we have shown that alterations in adiposity influence the expression of appetite-regulating peptides in the absence of ovarian steroids. The appetite stimulators, NPY and MCH, appear to be involved in the metabolic response to altered adiposity, whereas ENK in the periventricular region may be linked to the secretion of GH and possibly LH. Our results suggest that altered expression of appetite- regulating peptides can be linked with the endocrine and metabolic adaptations that occur with long-term changes in adiposity.

Hypothalamo-pituitary disconnected Soay rams were exposed to two photoperiodic treatments: 1) constant long days (16L:8D) for 48 wk after pretreatment under short days (LD group), and 2) constant short days (8L:16D) for 48 wk after pretreatment under long days (SD group). In the LD group, plasma prolactin (PRL) concentrations increased from 0 to 8 wk (maximum: 143.3 +/- 8.4 microg/l; 8.8 +/- 1. 2 wk), decreased from 9 to 34 wk (minimum: 15.6 +/- 1.6 microg/l; 34. 5 +/- 1.5 wk), and finally increased again under the constant conditions, with a similar cyclical pattern for all individuals. In the SD group, PRL concentrations showed an inverse pattern (minimum: 8.6 +/- 2.6 microg/l; 17.1 +/- 2.0 wk; maximum: 46.4 +/- 5.5 microg/l; 30.2 +/- 3.2 wk), with more variability. Plasma concentrations of FSH were basal in both groups. The duration of the daily nocturnal melatonin peak (measured at 10, 24, and 44 wk) remained close to 8 h under long days (high-fidelity melatonin signal) but decreased significantly (13.8 h to 9.3 h) under short days (low-fidelity melatonin signal). The results support the conclusion that the melatonin signal encoding photoperiod acts within the pituitary gland to induce both acute (inductive) and chronic (refractory) effects photoperiod on PRL secretion.

We have studied the effect of leptin on food intake and neuroendocrine function in ovariectomized ewes. Groups (n = 5) received intracerebroventricular infusions of either vehicle or leptin (20 microg/h) for 3 days and were blood sampled over 6 h on days -1, 2, and for 3 h on day 3 relative to the onset of the infusion. The animals were then killed to measure hypothalamic neuropeptide Y expression by in situ hybridization. Plasma samples were assayed for metabolic parameters and pituitary hormones. Food intake was reduced by leptin, but did not change in controls. Leptin treatment elevated plasma lactate and nonesterified fatty acids, but did not affect glucose or insulin levels, indicating a state of negative energy balance that was met by the mobilization of body stores. Pulse analysis showed that the secretion of LH and GH was not affected by leptin treatment, nor were the mean plasma concentrations of FSH, PRL, or cortisol. Expression of messenger RNA for neuropeptide Y in the arcuate nucleus was reduced by the infusion of leptin, primarily due to reduced expression per cell rather than a reduction in the number of cells observed. Thus, the action of leptin to inhibit food intake is dissociated from neuroendocrine function. These results suggest that the metabolic effects of leptin are mediated via neuronal systems that possess leptin receptors rather than via endocrine effects.

The effects of neonatal immunization against GnRH were studied in sheep after they had reached adulthood (3-4 yr) and the antibody titers had fallen to undetectable levels. The immunized animals had small gonads, and the females did not have large follicles (>3 mm) or corpora lutea in their ovaries. Compared with controls, the immunized animals had low or nondetectable levels of LH and FSH in peripheral plasma, and the immunized animals generally failed to respond to a single i.v. GnRH challenge. After ovariectomy, the control ewes, but not the immunized ewes, showed an elevation in plasma LH and FSH levels. The sampling of hypophysial portal blood, with a newly described method, showed that the secretion of GnRH was reduced in the immunized animals, but the amount of GnRH in the median eminence was similar in the control and immunized ewes. The pituitary content of LH and FSH was reduced in the immunized ewes as was messenger RNA for the gonadotropin subunits and the GnRH receptor. These data indicate that neonatal immunization does not affect the synthesis of GnRH in adulthood but reduces the secretion of GnRH, causing long-term sterility in these animals.

Hypothalamo-pituitary disconnected (HPD) and control Soay rams were treated chronically for 48 wk with s.c., continuous-release implants of melatonin while under long days (16L:8D). The implants produced continuously elevated blood concentrations of melatonin 2-3 times higher than the normal nocturnal maximum. The long-term treatment induced a biphasic effect on prolactin secretion in both the HPD and control rams, with a marked decrease in the blood prolactin concentrations for 10 wk followed by a gradual increase. The introduction of a second melatonin implant after 20 wk failed to affect prolactin secretion. The treatment with melatonin also caused a dynamic effect on FSH secretion, but this occurred in the control rams only. Blood concentrations of FSH in the HPD rams were very low throughout, but there were minor changes in testicular diameter that were correlated with variations in prolactin. Overall, the results support the conclusion that 1) melatonin acts primarily in the pituitary gland to affect prolactin secretion, and partial refractoriness develops at this level for control of prolactin; and 2) melatonin acts most probably in the hypothalamus to affect gonadotropin secretion, and refractoriness develops at the level of the neural tissue regulating GnRH release for control of gonadotropins.

It has previously been shown in the ewe that luteinizing hormone (LH)-containing secretory granules become polarized to the side of the gonadotrope nearest to a vascular sinusoid during the preovulatory period. We have used laser scanning confocal microscopy to monitor the migration of LH and follicle stimulating hormone (FSH)-containing secretory granules in the gonadotrope of the ewe. Ovariectomized (OVX) ewes (n = 4/group) were given either 50 micrograms of estradiol benzoate (EB) or oil and were killed 16 h later for collection of the pituitary glands for immunohistochemistry and confocal microscopy. Pituitary sections were simultaneously immunolabelled for LH and FSH and were visualized on the same sections using fluorescent markers. All cells that contained LH also contained FSH and vice versa. The results showed that LH-containing granules were uniformly distributed throughout the cytoplasm in 83% of gonadotropes from controls, while in the remaining 17% the granules were predominantly located adjacent to the plasma membrane. Following EB treatment, LH-containing secretory granules were distributed around the plasma membrane in 84% of immunopositive gonadotropes; FSH remained uniformly distributed throughout the cytoplasm. We conclude that estrogen causes the movement of LH-containing granules to the plasma membrane in gonadotropes but does not influence the distribution of FSH-containing granules. Rather than being polarized to one side of the cell, the LH-containing granules were distributed around the periphery of the cell.

An immunogenic loop within the diphtheria toxin has been deleted from the B-subunit by a modification of the inverse polymerase chain reaction (IPCR) and replaced by a unique restriction endonuclease site. An oligonucleotide encoding an identified epitope sequence from the major outer membrane protein of Neisseria meningitidis of similar size and structure to that deleted has been introduced into the restriction site. Expression of the resulting chimeric B-subunit from Escherichia coli yielded a protein that was recognised by a panel of antibodies specific for the meningococcal epitope. Initial immunisation data suggest that this protein could elicit an antibody response against both diphtheria toxin and meningococcal proteins.

A reduction in endogenous opioid inhibition (disinhibition) of GnRH secretion is thought to be permissive for the preovulatory GnRH/LH surge. There are no published studies of the effects of highly specific opioid receptor agonists on the LH surge in any species, and the relative importance of the opioid receptor subtypes mu, delta and kappa in the mechanism of disinhibition is unknown. In sheep, attempts to block the LH surge with opiates have been largely unsuccessful, and there is little evidence for reduced opioid inhibition during the GnRH/LH surge. The opioid receptor subtypes regulating PRL secretion in sheep are also unknown. Conscious, ovariectomized ewes with permanent third ventricular cannulae were injected with estradiol benzoate (EB) 50 micrograms or oil im (t = 0 h). In this model, EB elicits a time-delayed surge in LH secretion after 13-18 h. Jugular venous blood was sampled at half hourly intervals between-2 and 0 h and 10 and 26 h. From 12-20 h, infusions were made into the third ventricle of either the highly specific mu-agonist DAGO (10, 20 or 40 nmol/h), the delta-agonist DPDPE (40 nmol/h), the kappa-agonist U50488 (40 nmol/h) or saline (vehicle). In oil-treated animals (n = 4-6), DAGO infusion at 20 and 40 nmol/h reduced plasma LH whereas DPDPE or U50488 had no effect. In EB-treated animals (n = 6), DAGO (40 nmol/h) delayed the LH surge (mean +/- SEM time to surge onset 21.4 +/- 0.3 h vs. 14.0 +/- 0.4 h in controls, P < 0.0001). DAGO at 10 nmol/h did not alter surge onset and at 20 nmol/h had variable effects. DPDPE or U50488 did not affect LH surge timing or amplitude. All doses of DAGO increased plasma PRL, whereas DPDPE and U50488 had no effect. We conclude that, in ovariectomized ewes, activation of opioid mu-receptors, but not delta- or kappa-receptors, inhibits GnRH secretion, can block the estrogen-induced GnRH/LH surge and increases PRL secretion. The results are consistent with the disinhibition hypothesis.

The objective in this study was to characterize direct effects of the LHRH agonist, deslorelin, on anterior pituitary gland function in male cattle in the absence of gonadal feedback. Castrated bulls (steers), 30 mo old, were allocated to four groups: group 1, control, no treatment (n = 8); group 2, five deslorelin implants (approximately 250 micrograms total deslorelin/day) for 42 days (n = 8); group 3, control+ LHRH (50 micrograms i.m.) at weekly intervals (n = 3); group 4, five deslorelin implants+LHRH as for group 3 (n = 3). Plasma LH was similar (p > 0.05) for steers in groups 1 and 2 on Day 0 and lower (p < 0.05) for steers in group 2 on Day 4, and continued to decrease to Day 41 (group 1, 1.71 +/- 0.20 ng/ml [mean +/- SEM]; group 2, 0.38 +/- 0.03 ng/ml [p < 0.001]). Mean plasma concentrations of FSH were similar (p > 0.05) for steers in groups 1 and 2 on Day 0 and lower (p < 0.05) for steers in group 2 on Day 7, and declined to Day 41 (group 1, 43.5 +/- 3.9 ng/ml; group 2, 17.5 +/- 1.5 ng/ml [p < 0.001]). Steers in group 3 showed increases in plasma LH after injection of LHRH on all occasions, while steers in group 4 did not show increases in plasma LH from Day 14 onward. Mean relative pituitary contents (arbitrary units) of LH beta- and FSH beta-subunit mRNAs were reduced on Day 42 in steers treated with deslorelin (LH beta: groups 1 and 3, 1.56 +/- 0.27; groups 2 and 4, 0.08 +/- 0.01 [p < 0.001]; FSH beta: groups 1 and 3, 1.01 +/- 0.08; groups 2 and 4, 0.34 +/- 0.07 [p < 0.001]). However, alpha-subunit mRNA was similar for control steers and steers treated with deslorelin (groups 1 and 3, 1.00 +/- 0.11; groups 2 and 4, 0.86 +/- 0.12 [p > 0.1]). Pituitary content of LH, but not FSH, was reduced in steers treated with deslorelin. In summary, steers treated with deslorelin showed desensitization to natural LHRH, and this was associated with reduced pituitary contents of LH and FSH beta-subunit mRNAs, a reduction in pituitary content of LH, and decreases in plasma concentrations of LH and FSH. This demonstrated, for the first time, a direct action of LHRH agonist on LH and FSH beta-subunit gene expression in cattle, independent of gonadal feedback. Also, there was a differential effect of treatment with deslorelin on gonadotropin alpha- and beta-subunit mRNA contents in the anterior pituitary.

We have tested the hypothesis that treatment of castrated rams (wethers) with human recombinant follistatin-288 (FS-288) suppresses plasma concentrations of FSH but has no effect on plasma concentrations of LH. Wethers were given an i.v. injection of vehicle or 50 micrograms FS-288 followed by a 12-h i.v. infusion of vehicle or 800 micrograms FS-288, respectively. This dose and treatment regimen was identical to that used in a previous study in which wethers were treated with vehicle or human recombinant inhibin A (hr-inhibin). Human recombinant follistatin significantly (p < 0.05) suppressed plasma concentrations of FSH, but vehicle had no effect. The maximal suppression of FSH occurred 12-15 h after the start of treatment, at which time the plasma concentrations were reduced by 20.6%. The reduction in plasma concentrations of FSH caused by FS-288 was about 2.6-fold less than that observed after the same treatment with hr-inhibin. FS-288 had no effect on plasma concentrations of LH; this result was similar to the findings we obtained with hr-inhibin. These data suggest that follistatin is a less potent negative regulator of FSH secretion than inhibin and that follistatin is not a significant regulator of the secretion of LH in rams.

Photoaffinity cross-linking methods presented here demonstrate a 55-kilodalton (kDa) GH-releasing factor (GRF) receptor in ovine pituitary membranes and in cell lines expressing the cloned human pituitary receptor complementary DNA. Covalent cross-linking of photoprobe to this high affinity site is strongly competed by 1 nM GRF. Competition shows strong specificity for GRF over related peptides. Reduced cross-linking in the presence of guanosine 5'-O-(3-thiotriphosphate) suggests that this is a G-protein-coupled receptor. Detection of cross-linking to this receptor required detergent extraction to reduce high nonspecific binding of GRF photoprobe. Partial deglycosylation of the cross-linked receptor with neuraminidase caused a shift in apparent size to 52 kDa. Complete deglycosylation with N-glycosidase caused a shift to 45 kDa, demonstrating that this receptor is an N-linked glycoprotein and agreeing with the protein size and single glycosylation site predicted from the cloned complementary DNA sequence. These sizes differ from those found in previous reports which used chemical cross-linking to identify GRF receptor. This photoaffinity cross-linking method will facilitate studies of receptor function and tissue distribution. Photoaffinity cross-linking can also be used to map regions of the receptor molecule and bound GRF that are in close proximity.

We have investigated the effects of gamma-amino butyric acid (GABA) agonists and antagonists to GABAA and GABAB receptors microinjected (1 microliter) into the medial preoptic area (MPOA) on LH secretion in ovariectomized (OVX) ewes with or without estrogen (E) treatment, during the nonbreeding season. Guide tubes (19 gauge) were placed into the MPOA of OVX ewes and injections of 1 microgram or 10 micrograms of the GABAA agonist muscimol or the GABAA antagonist bicuculline, and 10 micrograms of the GABAB agonist baclofen or the GABAB antagonist phaclofen were made into conscious animals. Jugular venous blood was collected at 10-min intervals for 3 h, the injection of GABA drug or vehicle was given and samples collected for a further 3 h. The plasma samples were assayed for LH. On completion of the experiments the brains were sectioned to locate the sites of injection. Thirty-four ewes were used, of which 30 had correct guide tube placement. In OVX sheep, both muscimol and bicuculline injection caused suppression of plasma LH concentrations, with a cessation of pulsatile release in many instances. Injection with baclofen or phaclofen into these sheep had no effect on LH secretion. When OVX sheep were treated with 0.5-cm Silastic implants (sc) of E for at least 7 days, there was a variable response to muscimol and bicuculline injection, depending on the degree of suppression of LH secretion by E. When E had little effect on plasma LH levels, muscimol and bicuculline both suppressed LH secretion in a similar manner to that observed in OVX ewes. When E treatment fully suppressed plasma LH levels, muscimol and bicuculline both stimulated LH secretion. This stimulation often took the form of a sustained, nonpulsatile secretion of LH. Baclofen injection into OVX ewes treated with E increased mean plasma LH levels through an increase in pulse amplitude, although there was only an effect on the amplitude of the first pulse after injection. Conversely, phaclofen injection in OVX ewes treated with E resulted in a reduction in LH pulse amplitude. These results provide evidence for a seasonal shift in the regulation of GnRH secretion by GABAergic neurons in the MPOA of the ewe. In contrast to the breeding season, where there was no effect of GABAB ligands, these appear to function in non-breeding season, this may be part of the mechanism for the seasonal shift in the negative-feedback effect of E on LH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Oestrogen can act on the brain to exert negative and positive feedback effects on the secretion of gonadotrophin releasing hormone (GnRH), but this cannot be effected through the GnRH cells themselves because they have no oestrogen receptors. We have used intraventricular injection of agonists and antagonists of various transmitters to determine which systems are involved in the control of GnRH by steroid hormones. We have also developed a model for the injection of drugs directly into the medial pre-optic area of the sheep brain to study the action on the GnRH cell bodies. Opioid systems seem to be involved in the mediation of the feedback effects of progesterone, but not oestrogen. Noradrenaline appears to be involved in the negative feedback regulation of GnRH secretion during the anoestrous period. Thus, under the strong negative influence of oestrogen at this time GnRH/luteinizing hormone (LH) secretion is reduced but can be restored by the preoptic micro-injection of noradrenaline. Gamma-amino-butyric acid (GABA) agonists and antagonists inhibit LH secretion when injected into the medial pre-optic region of the hypothalamus of ovariectomized ewes with or without oestrogen treatment. We have no evidence that GABA is involved in the negative feedback regulation of GnRH, since neither agonists nor antagonists will reverse the effects of steroid feedback. There appears to be a shift in the GABA receptor subtype function with season; in the breeding season only GABAA ligands are effective, whereas during the non-breeding season both GABAA and GABAB type ligands will affect LH secretion. This suggests that the seasonal shift in responsiveness to the negative feedback effects of oestrogen involves a shift in the function of GABA receptor subtypes.

This study tested the hypothesis that inhibin is a major negative feedback regulator of FSH secretion but has minimal effects on LH secretion in rams. In experiment 1, castrated rams (wethers) were given either vehicle or human recombinant inhibin A (hr-inhibin) as three s.c. or three i.v. 50-micrograms injections 6 h apart or as one 50-micrograms i.v. injection followed by 100-micrograms infusion over 12 h. Human recombinant inhibin suppressed plasma FSH while the vehicle had no effect. The greatest suppression in plasma FSH was achieved following i.v. administration of hr-inhibin given either by repeated injection or by infusion. In experiment 2, wethers were given vehicle or a 50-micrograms i.v. injection followed by 800-micrograms infusion of hr-inhibin over 12 h. Infusion of hr-inhibin suppressed plasma FSH with a maximal suppression of 53.3% occurring between 15 and 24 h after the start of treatment. During this period, the plasma concentrations of FSH and inhibin were in the range of values for intact rams. Human recombinant inhibin did not influence plasma LH in either experiment. This study demonstrated that physiological treatment with inhibin, in the absence of testosterone, has the capacity to suppress plasma concentrations of FSH in wethers to the levels found in intact rams.

There have been a number of studies of GnRH secretion into the hypophysial portal blood at the time of the estrogen-induced LH surge, but the exact pattern of secretion at the onset of the positive feedback event remains a point of some dispute. In the present study, GnRH concentrations in portal plasma and jugular venous LH concentrations were measured in samples taken at 2.5-min intervals from five ovariectomized control ewes and seven ovariectomized ewes that were treated with 50 micrograms estradiol benzoate (im) to cause a LH surge. The frequency of GnRH pulses was calculated using a modification of previous criteria because of the frequent sampling. It was found that GnRH pulse frequency increased during the LH surge, being greatest during the "leading edge" of the surge. Perhaps more importantly, the data show that variable patterns of GnRH secretion occur at the start of the LH surge and during the surge, suggesting that no single pattern can be identified as an initiation signal of the positive feedback event. These data are consistent with other results that have been obtained in this laboratory in cyclic animals.

We have investigated the effects of agonists and antagonists to gamma-aminobutyric acid-A (GABA-A) and GABA-B receptors microinjected (1 microliters) into the preoptic area (POA) on LH in ovariectomized (OVX) ewes with or without estrogen (E) treatment during the breeding season. Guide tubes were placed into the preoptic area of OVX ewes using lateral ventriculograms for localization of the target area. Doses of 10 micrograms of the GABA agonists muscimol (GABA-A) and baclofen (GABA-B) or the GABA antagonists bicuculline (GABA-A) and phaclofen (GABA-B) were injected into the POA of tame conscious animals. Jugular venous blood was collected at 10-min intervals for 3 h, the injection of GABA drug or vehicle was given, and samples were collected for a further 3 h. The plasma samples were assayed for LH. On completion of the experiments, the brains were sectioned to locate the site of injection. In the first year, 17 ewes were used, of which 16 had correct guide tube placement. In OVX sheep, both muscimol and bicuculline injections caused suppression of plasma LH concentrations, with a cessation of pulses in many instances. When OVX sheep were treated with 1.0-cm Silastic implants (sc) of E for at least 7 days, there was a decrease in LH interpulse interval. In these sheep, muscimol and bicuculline injections had effects on plasma LH concentrations similar to those in OVX sheep. In addition, bicuculline was injected into the POA of OVX ewes treated with 2.0-cm E implants. Despite E treatment resulting in reduced plasma LH levels, due to an increase in the interpulse interval, bicuculline injection further suppressed plasma LH levels. Neither baclofen nor phaclofen injection had any effect on plasma LH secretion in either OVX ewes or OVX ewes given 1.0-cm E implants. In the second year, eight sheep were used, all of which had correct guide tube placement. These sheep were treated with 2.0-cm E implants and injected with phaclofen. Phaclofen had no effect on LH secretion. These results suggest that GnRH secretion is regulated by GA-BAergic neurons at the level of the GnRH cell bodies in the POA and that during the breeding season, this is effected by GABA-A and not GABA-B receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct amplification of the major outer membrane protein (MOMP) gene by polymerase chain reaction (PCR) was used to identify Chlamydia trachomatis in eye swabs from clinically active cases of endemic trachoma in a Gambian village. Chlamydial DNA was detected in 51% of 96 subjects with clinically active disease and in 5% of 37 clinically negative individuals. The PCR detection was combined with typing, using nested primers to variable sequences (VS) 1, 2, and 4 of the MOMP genes to distinguish between trachoma genotypes A, B, and C, respectively. Genotypes A and B were detected in the village, with some individuals harboring both genotypes within the same eye. DNA sequencing revealed strain variants of both genotypes. Typing of genotype and strain variants is now in progress to study trachoma transmission within the village.

The production of alpha-amidated peptide hormones from their glycine-extended precursors is catalyzed by the specific enzyme peptidylglycine alpha-amidating monooxygenase (PAM). In the present study, the distribution and subcellular localization of PAM activity in the sheep brain was examined and compared with known sites of amidated peptide synthesis and release. Of the brain regions assayed, the preoptic anterior and medial basal areas of the hypothalamus contained the greatest concentration of amidating activity. Lower concentrations (greater than 3-fold less) were found in the anterior and neurointermediate pituitary, median eminence, cerebral cortex, hippocampus, pons-medulla, and brainstem. Very low amounts of activity were present in the cerebellum and pineal gland. In most tissues tested, PAM activity was 40-75% higher in the membrane-associated fraction than in the soluble fraction. In the hypothalamus, affinity constants were identical for both membrane-associated and soluble fractions, and ranged from 12.3-13.3 microM. Maximal velocity was higher in the membrane fraction (4.7-4.8 pmol/microgram.h) than in the soluble fraction (2.6-2.9 pmol/microgram/h). Levels of amidating activity in hypophysial-portal and jugular plasma were similar and were 20- to 25-fold lower than in hypothalamic extracts. Insulin-induced hypoglycemia did not significantly alter PAM levels in portal or peripheral plasma, suggesting that amidating activity is not released during this stress. These results indicate that the hypothalamus is the richest source of amidating activity in the sheep brain, and suggest that amidation of neurohypophysial and hypothalamic releasing peptides may occur before axonal transport, given the much lower levels in median eminence, neurointermediate pituitary, and portal plasma.

This study aimed to delineate the hypothalamic and/or pituitary actions of testosterone and its primary metabolites 5 alpha-dihydrotestosterone and estradiol (E) in adult castrated rams (wethers) during the breeding season. In Exp 1, wethers were treated for a week with twice daily injections (im) of peanut oil, 8, 16 or 32 mg/day testosterone propionate (TP) or dihydrotestosterone benzoate (DHTB) or an sc silastic implant containing 1 or 3 cm E. TP decreased plasma LH concentrations, increased (P less than 0.05) LH interpulse interval, did not have consistent effects on LH pulse amplitude, and had minimal effects on plasma FSH concentrations. DHTB decreased LH and FSH concentrations and increased (P less than 0.05) LH interpulse interval. E reduced (P less than 0.05) plasma LH and FSH concentrations and increased LH interpulse interval but had no effects on LH pulse amplitude. In Exp 2, hypothalamo-pituitary disconnected wethers given 125 ng GnRH every 2 h, were treated with either peanut oil, 32 mg/day TP or DHTB or 3 cm E. None of the treatments affected plasma LH or FSH concentrations or LH pulse amplitude. Exp 3 investigated the effects on GnRH of treatment of wethers either with peanut oil or TP. TP reduced GnRH concentrations (P less than 0.05) and pulse amplitude (P less than 0.01) and increased interpulse interval (P less than 0.05). These data provide evidence that, during the breeding season, the principal site of negative feedback of testicular steroids in the ram is the hypothalamus, resulting in decreased GnRH secretion; feedback effects at the pituitary are minimal.

Previous studies have shown that the circulating half-life (t 1/2) of serum FSH in ewes after hypophysectomy (HPX) increased 10-fold after ovariectomy (OVEX). The basis for this difference was examined in this study by determining the circulating half-life of serum FSH and LH in HPX ewes after administration of pituitary extracts and gonadotropin isoform fractions. High-speed supernatants of pituitaries from gonadal-intact and OVEX ewes were fractionated by electrofocusing in sucrose gradients and based on the pI distribution of FSH and LH divided into four pools, pH 4.3-4.8, 4.8-5.55, 5.8-6.7, and 6.7-10. These extracts were administered by iv bolus injection to HPX gonadal-intact ewes and blood samples collected between 15-1000 min later. The clearance pattern for both serum FSH and LH was heterogenous, indicative of a major rapid and a minor slow dissociating component. A significant (P less than 0.05) difference in circulating half-lives (rapid component) was observed between pituitary extracts from intact and OVEX ewes for FSH (t 1/2 = 32.8 +/- 8.6 min vs. 89.9 +/- 32.3 min) but not LH (31.3 +/- 9.2 min vs. 39.3 +/- 6.1 min, respectively), whereas no significant difference was observed between the corresponding FSH or LH isoform preparations. To establish if the difference in circulating half-lives obtained after HPX and bolus iv injection was due to mode of delivery, an extract of pituitaries from OVEX ewes was infused for 12 h into HPX sheep and the t 1/2 values determined after cessation of treatment and compared to those after a bolus injection. The clearance of both FSH and LH from plasma after infusion was significantly prolonged than after a bolus injection. It is concluded that the difference in circulating half-lives of FSH between pituitary extracts from intact and OVEX ewes after bolus administration is due to a difference in pituitary FSH composition. However, the prolonged clearance with infusion compared to bolus administration suggests that extrapituitary factors are also responsible.

These studies were undertaken to test the hypothesis that stimulation of the central noradrenergic and adrenergic pathways activates the hypothalamic-pituitary-adrenal axis in vivo in the conscious sheep. Blood samples were taken at 10-min intervals over 4 h to establish the baseline state, and then each animal received an intracerebroventricular (icv) injection of NaCl (control animals) or catecholamine [norepinephrine (NE) or epinephrine (EPI)]. A more frequent rate of venous sampling was used for the 30-min period after the icv injection, after which time the 10-min rate of blood sampling was continued for another 3.5 h. NaCl (n = 4) caused no change in pituitary-adrenal secretion. In contrast, 10 micrograms NE (n = 4) caused acute 1.9- and 3.2-fold increases in mean plasma ACTH and cortisol levels over the 1 h period post injection, and 1.6- and 2.3-fold increments in their concentrations over the 4 h postinjection period. Although 10 micrograms EPI (n = 4) did not elevate mean plasma ACTH, it produced significant 1.7- and 1.5-fold increases in plasma cortisol during the 1- and 4-h periods post injection. However, when 100 micrograms EPI was injected (n = 4), acute 9.5- and 5.5-fold increases in plasma ACTH and cortisol were seen over the 1 h period post injection, and 6.1- and 4.2-fold increments in their plasma concentration were noted during the entire post-injection period. To determine the predominant site of action of the catecholamines, we also examined the ability of NE and EPI to release ACTH from cultured ovine anterior pituitary cells. NE and EPI (10(-9)-10(-6) M) stimulated the release of ACTH in a dose-dependent manner, but with maximal increments only 1.5-fold greater than the basal secretion. NE and EPI also increased the maximal ACTH response to CRF, but did not alter the maximal ACTH release induced by arginine vasopressin.(ABSTRACT TRUNCATED AT 400 WORDS)

These studies characterized the secretion of GH-releasing factor (GRF) and somatostatin (SRIF) into the hypophysial portal circulation in ewes after long term restricted feeding. In addition, we examined the temporal relationship between the concentrations of these two hypothalamic peptides in portal blood and the concentration of GH in jugular blood. Six sheep were fed 1000 g hay/day (normal feeding) and 6 sheep were fed 400-600 g hay/day (restricted feeding). This resulted in a wt loss of 35% in restricted animals compared with 6% in control animals after 20 weeks. Fluctuations in portal levels of GRF indicated a pulsatile pattern of secretion with approximately 60% of pulses coincident with, or immediately preceding, a GH pulse. Similarly, 65% of GH pulses were associated with GRF pulses. Restricted feeding increased (P less than 0.01) mean ( +/- SEM) plasma GH levels (9.8 +/- 1.4 vs. 2.9 +/- 0.6 ng/ml) and mean GH pulse amplitude (7.9 +/- 1.8 vs. 2.8 +/- 0.3 ng/ml) but did not affect mean GH pulse frequency (6.0 +/- 1.1 vs. 5.7 +/- 1.1 pulses/8 h). The level of feeding had no effect on mean portal concentration of GRF (restricted: 5.5 +/- 0.8, normal: 6.6 +/- 1.4 pg/ml), GRF pulse amplitude (14.7 +/- 2.3 vs. 13.5 +/- 0.7 pg/ml), or GRF pulse frequency (5.3 +/- 1.1 vs. 6.7 +/- 0.9 pulses/8 h). Portal concentrations of SRIF in sheep on a restricted diet were half (P less than 0.01) those of sheep fed a normal diet (10.2 +/- 2.3 vs. 19.6 +/- 1.6 pg/ml). Pulses of SRIF were not significantly associated with changes in GH or GRF concentrations. These data indicate a functional role for hypothalamic GRF in initiating GH pulses. Furthermore, the increase in GH secretion in underfed sheep was most probably due to a decrease in the release of SRIF into hypophysial portal blood. Restricted feeding had no affect on GRF secretion, but because of the reduced exposure of the pituitary gland to SRIF, it is possible that responsiveness to GRF is enhanced.

In the sheep, in contrast to the rat, arginine vasopressin (AVP) is a more potent stimulus to ACTH secretion from the anterior pituitary (AP) than CRF. To further explore this difference, we have compared [3H]AVP and [125I]-[Nle21 Tyr32]ovine CRF binding in membranes prepared from rat and sheep AP. Between species, no difference in affinity of binding was found for either ligand. In contrast, the concentration of AVP receptors in sheep AP was twice that in rat, whereas that of CRF receptors was only one tenth. AVP receptor concentration in sheep AP was not altered by chronic (10 day) dexamethasone administration, but fell to 60% of control after chronic (60 day) hypothalamo-pituitary-disconnection. The increased level of AVP receptors and the much lower level of CRF receptors in sheep compared with rat may thus provide an explanation for our previous findings of increased sensitivity to AVP and a very poor response to CRF in stimulating ACTH release from the sheep AP. In addition the finding that AVP receptor numbers are reduced in the hypothalamo-pituitary-disconnected sheep suggests that hypothalamic factors may play a role in regulating AVP receptor concentration in the ovine AP gland.

Studies were performed to determine whether the isolated ovine anterior and intermediate pituitary might rhythmically secrete three POMC peptides, ACTH, ir-beta-endorphin (ir-beta-EP), and ir-alpha-melanocyte stimulating hormone (ir-alpha-MSH) in vivo. When blood was taken at 10-min intervals from four ewes with hypothalamo-pituitary-disconnection (HPD), a distinct POMC-peptide and cortisol ultradian rhythm was noted. A comparison of the four HPD ewes with five nonstressed hypothalamopituitary-intact (HPI) ewes revealed that the mean plasma levels of the three POMC-peptides and cortisol were increased, the mean ACTH and ir-alpha-MSH pulse amplitudes were increased, and the mean ir-beta-EP and ir-alpha-MSH interpulse intervals were decreased. When four HPI ewes were subjected to a mild stress, plasma POMC-peptide and cortisol levels increased significantly when compared with the five unstressed HPI animals. In addition, the ACTH and cortisol pulse amplitudes increased and the ir-beta-EP and ir-alpha-MSH interpulse intervals decreased. Although plasma ACTH levels in the stressed HPI and HPD ewes were comparable, mean plasma cortisol levels were 2-fold greater in the stressed HPI animals. To determine whether the ACTH hypersecretion in the HPD ewe might reflect a net reduction in hypothalamic inhibitory influence over ACTH secretion, we examined the effects of dopamine (DA), somatostatin (SS-14), and rat atrial natriuretic peptide [rANF(1-28)] on the secretion of ACTH from cultured ovine anterior pituitary cells. DA and SS-14 did not exert a discernible effect on basal, CRF-, or arginine vasopressin (AVP)-stimulated ACTH secretion. Although basal ACTH secretion was unaffected by rANF(1-28) (10(-12)-10(-8) M), a significant inhibition of CRF- and AVP-stimulated ACTH release was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Ovariectomized ewes were treated with 100 IU insulin, iv, which caused reductions in blood sugar and plasma LH concentrations. The effect was prevented by the infusion (iv) of glucose, suggesting that neuroglycopenia and not a direct action of insulin was the cause of reduced LH secretion. An iv infusion of naloxone (40 mg/h for 2 h), which commenced 25 min before the insulin injection, blocked the inhibitory effect of insulin on LH secretion, but it did not prevent the decrease in plasma glucose concentrations. In this treatment group and in a group treated only with naloxone, the opioid antagonist significantly stimulated LH secretion. To determine whether CRF might be involved in the insulin-induced decrease in LH secretion, 50 micrograms CRF were injected into ovariectomized sheep. Despite producing very high circulating concentrations of CRF within 2 min of injection and the stimulation of cortisol secretion during most of the 4-h posttreatment period, plasma LH levels were not affected. In addition, the intracerebroventricular administration of 10 micrograms CRF or 10 micrograms CRF plus 10 micrograms arginine vasopressin (AVP) did not affect LH secretion. These observations suggest that insulin-induced hypoglycemia decreased LH secretion by neuroglycopenia. This may involve an opioidergic mechanism, but does not involve activation of the hypothalamo-pituitary-adrenocortical axis.

The effects of long term restricted feeding on the synthesis, storage, and release of GH, LH, FSH, and PRL were examined in adult ovariectomized ewes. Two groups of six ewes were fed a diet of either 1000 g/day (normal feeding) or 400-600 g/day (restricted feeding) hay for 20 weeks. Restricted feeding increased mean plasma GH concentrations and the amplitude of GH pulses, but did not affect GH pulse frequency. In contrast, mean plasma LH and FSH concentrations and LH pulse frequency were decreased by restricted feeding. Mean plasma PRL concentrations were unaffected by treatment. The levels of mRNA for GH in pituitary cytosol were increased by restricted feeding, but no changes were seen in mRNA levels of alpha-subunit, LH beta, FSH beta, or PRL. The pituitary contents of hormones measured did not change with the level of feeding. In conclusion, these data show that long term restricted feeding affects anterior pituitary function in adult ewes, presumably reflecting alterations in the secretion of hypothalamic releasing and inhibiting factors.

A heterologous RIA for ovine inhibin was developed which was sufficiently sensitive and specific to describe the peripheral concentrations of immunoreactive inhibin (iINH) during the estrous cycle of the ewe and to examine the effects of cautery of ovarian follicles on concentrations of iINH in ovarian and jugular venous plasma. Parallel logit-log dose-response lines were observed among ovine follicular fluid, ewe plasma, and pure native ovine (31 kDa) and bovine (31 kDa) inhibin. iINH could not be detected in ovariectomized ewe plasma, and there was no apparent cross-reactivity with a variety of structurally related and unrelated hormones and peptides, except a monomeric form of the alpha-subunit of INH, iINH in follicular fluid was 10(4)-fold higher than that in ovarian venous plasma, which was 3-fold higher than that in peripheral plasma. Cautery of the follicles resulted in a 35% reduction in iINH and an 81% reduction in estrogen concentrations in the ovarian vein within 10 min. During the estrous cycle, iINH and FSH were inversely related in samples taken over 30 h in the luteal phase (r = -0.69; P less than 0.001) and in the pre- and postovulatory phases (r = -0.45; P less than 0.001). iINH and LH were not related in the luteal phase, but were weakly positively correlated in the follicular phase (r = 0.31; P less than 0.01). iINH and estrogen concentrations in the follicular phase were also weakly correlated (r = 0.30; P less than 0.001). Furthermore, iINH concentrations rose in the follicular phase and decreased within 3-6 h of the preovulatory surges of LH and FSH, reaching a nadir around the time of the second rise in FSH 24-48 h later. It is concluded that 1) large antral follicles are a major source of peripheral iINH during the ovine estrous cycle; 2) iINH levels increase in the follicular phase with the growth of the dominant follicle and may be inhibited by the preovulatory surge of gonadotropin; 3) the fall in inhibin after the LH surge may be responsible for the second rise in FSH; and 4) the inverse relationship between FSH and iINH is consistent with the hypothesis that inhibin is involved in the feedback regulation of FSH.