Response to kisspeptin and gonadotropin-releasing hormone agonist administration in Holstein-Friesian dairy heifers with positive or negative genetic merit for fertility traits.

Previous research has identified that Holstein-Friesian dairy heifers with positive (POS) genetic merit for fertility traits (FertBV) reach puberty earlier than heifers with negative (NEG) FertBV. The hypothalamus-pituitary-gonadal (HPG) axis is functional in heifers before the onset of puberty, with increased LH release evident as heifers progress toward puberty. We investigated the functionality of the HPG axis in peripubertal Holstein-Friesian dairy heifers with divergent POS or NEG FertBV, hypothesizing that the earlier puberty onset of POS heifers is associated with earlier activation of the HPG axis than in NEG heifers. In experiment 1, we tested the dose responsiveness of POS heifers to an intravenous injection of either kisspeptin [Kiss; 2, 4, or 8 µg/kg of body weight (BW); n = 3 per dose] or a GnRH agonist (buserelin; 5, 10, or 20 ng/kg of BW; n = 3 per dose). The use of these 2 agonists investigates the status of the HPG axis in both the hypothalamus (Kiss) and pituitary (buserelin) glands. Doses of 4 µg/kg BW of Kiss and 10 ng/kg BW of buserelin produced submaximal LH responses and were used in experiment 2, in which previously unused POS (n = 22) and NEG (n = 18) FertBV heifers were challenged with both agonists at 10 and 12 mo of age in a partial crossover design. Heifers were randomly allocated to treatment groups, balanced for age and BW. The LH response to buserelin was greater in POS heifers than NEG heifers at 10 mo of age, with no difference in response at 12 mo. The FSH response to buserelin and the LH and FSH responses to Kiss did not differ between the POS and NEG heifers at either age. These results indicate an association between divergent genetic merit for fertility and the LH release to buserelin at 10 mo of age, supporting the hypothesis that gonadotropin responsiveness to a GnRH agonist is more advanced in POS heifers than in NEG heifers.

Effect of gonadotropin-inhibitory hormone on luteinizing hormone secretion in humans.

BACKGROUND: Gonadotropin-inhibitory hormone (GnIH, human homologue of RFRP-3) suppresses gonadotropin secretion in animal models, but its effects have not been studied in the human. OBJECTIVE: We tested the hypotheses that exogenous GnIH inhibits LH secretion (i) in postmenopausal women and (ii) in men concurrently administered exogenous kisspeptin. DESIGN: Following in vitro and in vivo preclinical studies to functionally characterize the GnIH peptide, a dose-finding study (human GnIH: 1·5-150 µg/kg/h, iv for 3 h) was undertaken, and 50 µg/kg/h selected for further evaluation. Five postmenopausal women were administered 50 µg/kg/h iv infusion for 3 h or vehicle on two separate days. Four men were administered kisspeptin-10 (0·3 µg/kg iv bolus) with simultaneous infusion of GnIH (50 µg/kg/h, iv for 3 h) or vehicle. PARTICIPANTS: Healthy postmenopausal women (mean age 58 ± 2 years, LH: 30·8 ± 2·9 IU/l, FSH: 78·7 ± 6·4 IU/l, oestradiol: <50 pmol/l) and men (39·8 ± 2·1 years, mean total testosterone 12·1 ± 1·8 nmol/l, LH 2·2 ± 0·2 IU/l). PRIMARY OUTCOME: Change in area under curve (AUC) of LH during GnIHvs vehicle. RESULTS: During GnIH administration in postmenopausal women, LH secretion decreased (ΔAUC: -9·9 ± 1·8 IU/3 h) vs vehicle (ΔAUC: -0·5 ± 1·7 IU/3 h; P = 0·02). Kisspeptin-10-stimulated LH responses in men were not affected by GnIH co-administration (60-min AUC of LH 6·2 ± 0·8 IU/h with kisspeptin-10 alone, 6·3 ± 1·0 IU/h, kisspeptin-10 with GnIH, P = 0·72). Exogenous GnIH was well tolerated, with no adverse events reported. CONCLUSIONS: Gonadotropin-inhibitory hormone decreased LH secretion in postmenopausal women in this first-in-human study. Kisspeptin-stimulated LH secretion in men was not inhibited during concomitant administration of GnIH.

Expression of regulatory neuropeptides in the hypothalamus of red deer (Cervus elaphus) reveals anomalous relationships in the seasonal control of appetite and reproduction.

Red deer are seasonal with respect to reproduction and food intake, so we tested the hypothesis that their brains would show seasonal changes in numbers of cells containing hypothalamic neuropeptides that regulate these functions. We examined the brains of male and female deer in non-breeding and breeding seasons to quantify the production of kisspeptin, gonadotropin inhibitory hormone (GnIH), neuropeptide Y (NPY) and γ-melanocyte stimulating hormone (γ-MSH - an index of pro-opiomelanocortin production), using immunohistochemistry. These neuropeptides are likely to be involved in the regulation of reproductive function and appetite. During the annual breeding season there were more cells producing kisspeptin in the arcuate nucleus of the hypothalamus than during the non-breeding season in males and females whereas there was no seasonal difference in the expression of GnIH. There were more cells producing the appetite stimulating peptide, NPY, in the arcuate/median eminence regions of the hypothalamus of females during the non-breeding season whereas the levels of an appetite suppressing peptide, γ-MSH, were highest in the breeding season. Male deer brains exhibited the converse, with NPY cell numbers highest in the breeding season and γ-MSH levels highest in the non-breeding season. These results support a role for kisspeptin as an important stimulatory regulator of seasonal breeding in deer, as in other species, but suggest a lack of involvement of GnIH in the seasonality of reproduction in deer. In the case of appetite regulation, the pattern exhibited by females for NPY and γ-MSH was as expected for the breeding and non-breeding seasons, based on previous studies of these peptides in sheep and the seasonal cycle of appetite reported for various species of deer. An inverse result in male deer most probably reflects the response of appetite regulating cells to negative energy balance during the mating season. Differences between the sexes in the seasonal changes in appetite regulating peptide cells of the hypothalamus present an interesting model for future studies.

IGFBP-2 inhibits adipogenesis and lipogenesis in human visceral, but not subcutaneous, adipocytes.

BACKGROUND/OBJECTIVE: IGF-binding protein (IGFBP)-2 is the principal IGFBP produced by white adipocytes during adipogenesis, and circulating levels are reduced in obesity. Overexpression of IGFBP-2 in transgenic mice prevents obesity, but depot-specific effects of IGFBP-2 on adipo/lipogenesis are unknown. The present study aimed to investigate whether IGFBP-2 affects adipo/lipogenesis in a depot-specific manner and explore potential mechanisms. METHODS: Following adipocyte characterisation, IGFBP-2 levels were measured from human subcutaneous and visceral preadipocytes, and IGFBP-2 dose-responses were then undertaken with exogenous IGFBP-2 in an in vitro IGF-I-free system to examine adipo/lipogenesis. Following this, both types of adipocytes were transfected with human siRNA IGFBP-2 to assess auto-/para-/intra-crine effects, with and without additional add-back IGFBP-2. To elucidate the potential mechanisms, visceral preadipocytes were treated with either wild-type or Heparin Binding Domain (HBD)-mutant IGFBP-2 (which is unable to bind to cell-surface components), and experiments were also undertaken using Echistatin (an integrin receptor blocker). Outcomes included gene expression profiles, protein levels and phosphorylation and lipid staining. RESULTS: Human visceral adipocytes produced significantly more IGFBP-2 than subcutaneous adipocytes. Subsequent dose-responses to IGFBP-2 demonstrated significant reductions in adipo/lipogenesis in visceral, but not subcutaneous, adipocytes in response to increasing IGFBP-2. Silencing IGFBP-2 resulted in exaggerated adipo/lipogenesis in visceral, but not subcutaneous, adipocytes, an effect completely inhibited by add-back IGFBP-2. These effects occurred in the absence of changes in IGF-I levels. HBD-mutant IGFBP-2 had reduced effects compared with wild-type IGFBP-2. Wild-type IGFBP-2 increased phosphorylation of focal adhesion kinase (FAK) and decreased phosphatase and tensin homolog (PTEN) levels, suggestive of integrin-mediated signalling. Blockade of this signalling, using Echistatin, completely negated the effects of IGFBP-2 on visceral adipo/lipogenesis. CONCLUSION: IGFBP-2 inhibits both adipogenesis and lipogenesis in visceral, but not subcutaneous, adipocytes. This depot-specific impairment appears to be independent of IGF-I and involves cell-surface association of IGFBP-2 and activation of integrin signalling pathways.

Distribution and sequence of gonadotropin-inhibitory hormone and its potential role as a molecular link between feeding and reproductive systems in the Pekin duck (Anas platyrhynchos domestica).

The reproductive status of adult Pekin drakes is very sensitive to nutritional status. Thus, the purpose of this study was to increase our understanding of the neurobiology underlying the depressive effect of fasting on the secretion of reproductive hormones. It was hypothesized that this effect was mediated by gonadotropin-inhibitory hormone (GnIH). Networks of GnIH fibers were present throughout the diencephalon, and cell bodies were present primarily, in the hypothalamic paraventricular nucleus (PVN). The duck GnIH gene was cloned and sequenced and found to encode GnIH and two GnIH-related peptides (GnIH-RP1, GnIH-RP2) which have a similar identity to those found in other avian species. Intracerebroventricular injection of GnIH, but not of GnIH-RP1, depressed plasma LH and stimulated feeding. Fasting for 48h depressed plasma LH and induced fos expression in about half the population of GnIH-ir neurons. These data suggest that GnIH neurons are mediators between feeding and reproductive systems in Pekin drakes.

Inhibition of compensatory renal growth by the N-terminus of a sheep-derived peptide.

The N-terminal sequence of a novel sheep-derived peptide with growth inhibitory activity has been obtained. The N-terminal fragment was chemically synthesised and designated EPL001. The kidney was chosen as the first mammalian system in which to study EPL001 since kidney growth can be accurately quantified following a surgical reduction in renal mass. Cell proliferation was measured in mouse collecting duct kidney (MCDK) cells stimulated with insulin-like growth factor I (IGF-I). Compensatory renal growth (CRG) was induced in Wistar rats and either EPL001 or an EPL001 antibody delivered by continuous renal tissue infusion. Mouse monoclonal antibodies to EPL001 were generated for immunoneutralisation, rabbit polyclonal antibodies were generated for immunohistochemistry. EPL001 had no apparent effect on IGF-I stimulated cell proliferation in MCDK cells in vitro, yet provoked a dose-dependent inhibition of CRG in vivo. An EPL001 antibody potentiated CRG, in the absence of exogenous EPL001, consistent with an inhibitory role in kidney growth for an endogenous peptide containing the EPL001 sequence. Tubular staining for epitopes to the EPL001 sequence was detected in normal human kidney sections and enhanced in renal cell carcinoma. Results support the presence of growth inhibitory activity in the N-terminus of a sheep-derived peptide with evidence for both its presence and endogenous activity in the kidney. Attempts to further characterise its structure and activity are ongoing.

Characterization of the projections to the hypothalamic paraventricular and periventricular nuclei in the female sheep brain, using retrograde tracing and immunohistochemistry.

The paraventricular nucleus (PVN) and the periventricular nucleus (Pe) are important neuroendocrine centers, but the neuronal input to these regions is poorly defined in nonrodent species. We utilized the retrograde transport of injected tracers to determine the neural input to these two nuclei in the ovine brain. Adult Corriedale ewes were studied following FluoroGold injection into either the PVN (n = 5) or the Pe (n = 3). Both the PVN and the Pe were found to receive neuronal input from a number of hypothalamic nuclei. Projections to the PVN from the lateral hypothalamic area were from neurons that produce melanin-concentrating hormone or orexins and a subset of those from the arcuate nucleus were immunopositive for neuropeptide Y and gamma-melanocyte stimulating hormone. This pathway was verified by staining of terminals in the PVN. Input to the PVN from the brain stem was seen to originate from the catecholaminergic and serotoninergic neurons. The projections to the PVN and Pe from hypothalamic and brain stem regions in the sheep brain are generally similar to those in the rat, with some minor differences. These studies highlight the differences in the afferent input to these two closely related nuclei in the ovine brain.

Estradiol enables cortisol to act directly upon the pituitary to suppress pituitary responsiveness to GnRH in sheep.

We have shown that cortisol infusion reduced the luteinizing hormone (LH) response to fixed hourly GnRH injections in ovariectomized ewes treated with estradiol during the non-breeding season (pituitary-clamp model). In contrast, cortisol did not affect the response to 2 hourly invariant GnRH injections in hypothalamo-pituitary disconnected ovariectomized ewes during the breeding season. To understand the differing results in these animal models and to determine if cortisol can act directly at the pituitary to suppress responsiveness to GnRH, we investigated the importance of the frequency of GnRH stimulus, the presence of estradiol and stage of the circannual breeding season. In experiment 1, during the non-breeding season, ovariectomized ewes were treated with estradiol, and pulsatile LH secretion was restored with i.v. GnRH injections either hourly or 2 hourly in the presence or absence of exogenous cortisol. Experiments 2 and 3 were conducted in hypothalamo-pituitary disconnected ovariectomized ewes in which GnRH was injected i.v. every 2 h. Experiment 2 was conducted during the non-breeding season and saline or cortisol was infused for 30 h in a cross-over design. Experiment 3 was conducted during the non-breeding and breeding seasons and saline or cortisol was infused for 30 h in the absence and presence of estradiol using a cross-over design. Samples were taken from all animals to measure plasma LH. LH pulse amplitude was reduced by cortisol in the pituitary clamp model with no difference between the hourly and 2-hourly GnRH pulse mode. In the absence of estradiol, there was no effect of cortisol on LH pulse amplitude in GnRH-replaced ovariectomized hypothalamo-pituitary disconnected ewes in either season. The LH pulse amplitude was reduced in both seasons in experiment 3 when cortisol was infused during estradiol treatment. We conclude that the ability of cortisol to reduce LH secretion does not depend upon the frequency of GnRH stimulus and that estradiol enables cortisol to act directly on the pituitary of ovariectomized hypothalamo-pituitary disconnected ewes to suppress the responsiveness to GnRH; this effect occurs in the breeding and non-breeding seasons.

Kisspeptin is present in ovine hypophysial portal blood but does not increase during the preovulatory luteinizing hormone surge: evidence that gonadotropes are not direct targets of kisspeptin in vivo.

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.

Psychosocial stress suppresses attractivity, proceptivity and pulsatile LH secretion in the ewe.

Various stressors suppress pulsatile secretion of luteinizing hormone (LH) in ewes and cortisol has been shown to be a mediator of this effect under various conditions. In contrast, little is known about the impact of stress and cortisol on sexual behavior in the ewe. Therefore, we tested the hypothesis that both psychosocial stress and stress-like levels of cortisol will reduce the level of attractivity, proceptivity and receptivity in addition to suppressing LH secretion in the ewe. In Experiment 1, a layered stress paradigm of psychosocial stress was used, consisting of isolation for 4 h with the addition of restraint, blindfold and noise of a barking dog (predator stress) at hourly intervals. This stress paradigm reduced LH pulse amplitude in ovariectomized ewes. In Experiment 2, ovariectomized ewes were artificially induced into estrus with progesterone and estradiol benzoate treatment and the layered stress paradigm was applied. LH was measured and sexual behavior was assessed using T-mazes and mating tests. Stress reduced pulsatile LH secretion, and also reduced attractivity and proceptivity of ewes but had no effect on receptivity. In Experiment 3, ewes artificially induced into estrus were infused with cortisol for 30 h. Cortisol elevated circulating plasma concentrations of cortisol, delayed the onset of estrus and resulted in increased circling behavior of ewes (i.e. moderate avoidance) during estrus and increased investigation and courtship from rams. There was no effect of cortisol on attractivity, proceptivity or receptivity during estrus. We conclude that psychosocial stress inhibits LH secretion, the ability of ewes to attract rams (attractivity) and the motivation of ewes to seek rams and initiate mating (proceptivity), but cortisol is unlikely to be the principal mediator of these effects.

Neural connectivity in the mediobasal hypothalamus of the sheep brain.

The ventromedial nucleus of the hypothalamus (VMN) and the arcuate nucleus (ARC) are two centres regulating energy balance and food intake, but inter-connectivity of these nuclei is not well defined in non-rodent species. In this study, we performed retrograde tracing and immunohistochemistry in the ovine brain with ewes receiving FluoroGold (FG) injections into either ARC or VMN for the mapping of retrogradely labelled cells. Strong reciprocal connections were found between the two regions. The distribution of the FG labelled neurons in other regions of the hypothalamus and brain stem was also mapped. Some of the cells projecting from ARC to VMN were immunopositive for neuropeptide Y, galanin, adrenocorticotropin (marker of pro-opiomelanocortin cells) or tyrosine hydroxylase (marker of dopaminergic cells). Melanin-concentrating hormone and orexin neurons in the lateral hypothalamic area were also found to provide input to the VMN and ARC. This observed interconnectivity between regions important for metabolic regulation and other neuroendocrine functions presumably allows coordinated functions. Input to both the ARC and VMN from other brain regions, such as brain stem cell groups, provides a further level of regulation. These data provide a substrate upon which further understanding of appetite regulation and neuroendocrine function can be derived in this species.

Stimulation of growth hormone by kisspeptin antagonists in ewes.

Kisspeptin signalling is indispensable for fertility, stimulating gonadotropin-releasing hormone (GnRH) secretion and mediating gonadal steroid feedback on GnRH neurons. Moreover, kisspeptin neurons have been implicated in other non-reproductive neuroendocrine roles. Kisspeptin appears to also regulate growth hormone secretion but much of the data appear contradictory. We sought to clarify a potential role of kisspeptin in growth hormone (GH) regulation by examining the effect of kisspeptin antagonists on GH secretion in ewes under various physiological conditions. Our data show clear and robust increases in GH secretion following lateral ventricle or third ventricle infusion of kisspeptin antagonists p-234 and p-271 in either ovariectomized or anestrous ewes. Central infusion of kisspeptin-10 had no effect on GH secretion. To determine the level at which kisspeptin may influence GH secretion, we examined expression of the cognate kisspeptin receptor, GPR54, in pituitary cells and showed by immunocytochemistry that the majority of somatotropes express GPR54 while expression was largely negative in other pituitary cells. Overall, we have demonstrated that blocking kisspeptin signalling by antagonists stimulates GH secretion in ewes and that this is likely mediated by inhibiting endogenous kisspeptin activation of GPR54 expressed on somatotropes. The findings suggest that endogenous kisspeptin inhibits GH secretion through GPR54 expressed on somatotropes.

Kisspeptin synchronizes preovulatory surges in cyclical ewes and causes ovulation in seasonally acyclic ewes.

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.

Measurement of growth hormone-releasing hormone and somatostatin in hypothalamic-portal plasma of unanesthetized sheep. Spontaneous secretion and response to insulin-induced hypoglycemia.

To elucidate the role of growth hormone (GH)-releasing hormone (GRH) and somatostatin (SRIH) in the regulation of the growth hormone (GH) secretory pattern, we collected portal blood from five unanesthetized ovariectomized ewes for repeated measurements of GRH and SRIH simultaneous with those of peripheral GH. Hormones were measured at 10-min intervals for 5.5 h and their interrelationships analyzed. Mean portal GRH was 20.4 +/- 6.7 (SD) pg/ml and the estimated overall secretion rate was 13 pg/min. GRH secretion was pulsatile with peaks of 25-40 pg/ml and a mean pulse interval of 71 min. Mean portal SRIH was 72 +/- 33 pg/ml and the estimated overall secretion rate was 32 pg/min. SRIH secretion was also pulsatile with peaks of 65-160 pg/ml and a mean pulse interval of 54 min. The GH pulse interval was 62 min. A significant association was present between GRH and GH secretory peaks though not between GRH and SRIH or SRIH and GH. Insulin hypoglycemia resulted in a rapid and brief stimulation of SRIH secretion followed by a decline in GH levels. No effect was observed on GRH secretion until 90 min, when a slight increase occurred. The results suggest (a) the presence of an independent neural rhythmicity of GRH and SRIH secretion with a primary role of GRH in determining pulsatile GRH secretion, and (b) that the inhibitory effects of insulin hypoglycemia on GH in this species are attributable to a combination of enhanced SRIH secretion and possibly other factors, though without significant inhibition of GRH.

Studies of the secretion of corticotropin-releasing factor and arginine vasopressin into the hypophysial-portal circulation of the conscious sheep. II. The central noradrenergic and neuropeptide Y pathways cause immediate and prolonged hypothalamic-pituitary-adrenal activation. Potential involvement in the pseudo-Cushing's syndrome of endogenous depression and anorexia nervosa.

Studies were performed to determine the effects of intracerebroventricular norepinephrine (NE) or neuropeptide Y (NPY) on the ovine hypothalamic-pituitary-adrenal (HPA) axis. NE (50 micrograms) increased mean hypophysial-portal corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) levels (1 h, 1.3- and 2.9-fold; 4 h, 2.2- and 5.7-fold) and caused acute and sustained increases in mean plasma ACTH and cortisol. NPY (50 microgram) also increased mean CRF and AVP levels (1 h, 1.4- and 4.2-fold; 4 h, 1.1- and 1.9-fold), increased pituitary-adrenal activity at 1 h, and caused ACTH hypersecretion at 4 h. When added to cultured ovine anterior pituitary cells, NPY neither increased basal ACTH release nor augmented CRF- or AVP-induced ACTH release. We conclude that: (a) activation of either the central noradrenergic or NPY pathways causes an acute and sustained stimulation of the ovine HPA axis; (b) such activation increases the AVP/CRF ratio, suggesting a dominant role for AVP in the ovine stress response; and (c) the central noradrenergic or NPY systems may cause sustained HPA activation by attenuating or disrupting the glucocorticoid negative feedback on those brain areas concerned with regulation of the HPA axis. The possible roles of the central noradrenergic and NPY systems in the etiology of the hypercortisolemia of endogenous depression and anorexia nervosa are discussed.

Isolation and restraint stress results in differential activation of corticotrophin-releasing hormone and arginine vasopressin neurons 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.

Changes in insulin, glucose and ketone bodies, but not leptin or body fat content precede restoration of luteinising hormone secretion in ewes.

The reproductive system, including pulsatile luteinising hormone (LH) secretion, is inhibited by deficits in energy availability and restored by energy surfeits. Plasma LH, insulin, leptin, ghrelin, glucose, ketone body, and nonesterified fatty acid concentrations were measured in ovariectomised, food-restricted ewes before and after return to ad libitum feeding to determine the factors that change in time to account for the restoration of pulsatile LH secretion. At 07.00 h, blood was sampled every 10 min for 5 h from ovariectomised, hypogonadotrophic, chronically food-restricted and ad libitum-fed ewes (Fed). At 12.00 h, four of the food-restricted sheep were given ad libitum access to food (Re-Fed), while three ewes continued to be food restricted (Restricted). Sampling continued for 5 h and resumed again on the mornings of days 2, 4, and 9. A pulse of LH was seen within 1 h of re-feeding in all Re-Fed ewes, and interpulse interval (IPI) was significantly shorter in Re-Fed compared to Restricted ewes and longer than in Fed ewes during the period after re-feeding. Re-Fed LH IPI was not restored to that of Fed ewes until sometime between days 4 and 9. The first pulse occurred within minutes, whereas restoration of IPI occurred after 4-8 days. Prior to the initial LH pulses seen in Re-Fed ewes, plasma ketone bodies first fell and then rose to levels significantly above those in Restricted ewes. Significant changes in circulating insulin, ghrelin, glucose, and total ketone body concentrations, daily food intake and lean body mass preceded restoration of Re-Fed LH IPI some time between days 4 and 9, but there were no significant changes in adiposity or circulating leptin concentrations, consistent with the hypothesis that LH pulses are reinitiated by changes in the availability of oxidisable metabolic fuels and possibly insulin, but not leptin concentrations.

Alteration in the relationship between tanycytes and gonadotrophin-releasing hormone neurosecretory terminals following long-term metabolic manipulation in the sheep.

The activity of the hypothalamic-pituitary gonadal axis is influenced by energy reserves, such that an increase or a decrease in adiposity may perturb the secretion and action of gonadotrophin-releasing hormone (GnRH). This is considered to be a result of the signalling of hormones such as leptin, which act upon neuronal systems controlling GnRH secretion. Other work shows plasticity in the relationship between tanycytes and GnRH neurosecretory terminals in the median eminence across the oestrous cycle and we hypothesised that a similar plasticity may occur with altered metabolic status. We studied Lean, Normal and Fat ovariectomised ewes, which displayed differences in gonadotrophin status, and investigated the relationship between tanycytes and GnRH neuroterminals. Under both Lean and Fat conditions, an altered anatomical arrangement between these two elements was observed in the vicinity of the blood vessels of the primary plexus of the hypophysial portal blood system. These data suggest that such plasticity is an important determinant of the rate of secretion of GnRH in animals of differing metabolic status and that this also contributes to the relative hypogonadotrophic condition prevailing with metabolic extremes.

Activation of the hypothalamo-pituitary-adrenal axis by isolation and restraint stress during lactation in ewes: effect of the presence of the lamb and suckling.

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.

Colocalization of kisspeptin and gonadotropin-releasing hormone in the ovine brain.

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.