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.

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.

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.

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.

Hypothalamus as an endocrine organ.

The endocrine hypothalamus constitutes those cells which project to the median eminence and secrete neurohormones into the hypophysial portal blood to act on cells of the anterior pituitary gland. The entire endocrine system is controlled by these peptides. In turn, the hypothalamic neuroendocrine cells are regulated by feedback signals from the endocrine glands and other circulating factors. The neuroendocrine cells are found in specific regions of the hypothalamus and are regulated by afferents from higher brain centers. Integrated function is clearly complex and the networks between and amongst the neuroendocrine cells allows fine control to achieve homeostasis. The entry of hormones and other factors into the brain, either via the cerebrospinal fluid or through fenestrated capillaries (in the basal hypothalamus) is important because it influences the extent to which feedback regulation may be imposed. Recent evidence of the passage of factors from the pars tuberalis and the median eminence casts a new layer in our understanding of neuroendocrine regulation. The function of neuroendocrine cells and the means by which pulsatile secretion is achieved is best understood for the close relationship between gonadotropin releasing hormone and luteinizing hormone, which is reviewed in detail. The secretion of other neurohormones is less rigid, so the relationship between hypothalamic secretion and the relevant pituitary hormones is more complex.

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.

Dietary antioxidants at supranutritional doses improve oxidative status and reduce the negative effects of heat stress in sheep.

The present study was undertaken to investigate the impact of heat (thermal) stress and dietary antioxidant supplementation on the oxidative and physiological status of sheep. Twenty-four Merino × Poll Dorset crossbred ewes were housed in 1 of 2 climatic chambers (thermoneutral or heat stress) and offered either a control (10 IU vitamin E/kg DM and 0.24 mg Se/kg DM) or high antioxidant (100 IU vitamin E/kg DM and 1.20 mg Se/kg DM) diet. The sheep were exposed to 2 thermal (temperature) treatments (thermoneutral [TN]: 18-21°C and 26-30% relative humidity; and heat stress [HS]: 28-40°C and 40-50% relative humidity) for 2 wk in a single reversal design. After 1 wk of dietary treatment, animals in 1 chamber were subjected to HS for 1 wk, with the temperature being increased to 40°C between 0900 and 1700 h and then maintained at 28°C overnight. Those sheep in the TN group were maintained at 18 to 21°C. Physiological parameters were recorded 4 times a day (0900, 1300, 1700, and 2100 h) and blood samples were collected on d 1 and 7 of heat treatment. Plasma samples and red blood cell lysates were assayed for oxidative stress biomarkers. The thermal treatments were then reversed and the above measures repeated. All measured physiological parameters were elevated (P < 0.001) by thermal treatment. Respiration rate was lower during HS in sheep supplemented with antioxidants as indicated by a diet × temperature × time interaction (P = 0.010). There was 13% decline (P = 0.014) in feed intake of the unsupplemented animals during HS whereas the same was maintained in sheep supplemented with high doses of antioxidants. Plasma reactive oxygen metabolites concentrations were reduced (114 vs. 85 units/dL; P < 0.005) while biological antioxidant potential tended to be increased (3,688 vs. 3,985 µmol/L; P = 0.070) in heat stressed sheep supplemented with antioxidants. The oxidative stress index was 30% lower (P < 0.001) in supplemented sheep (2.16 ± 0.06 arbitrary units) during HS than in unsupplemented sheep (3.12 ± 0.08 arbitrary units). Plasma advanced oxidation protein products tended (P = 0.070) to decrease in antioxidant supplemented heat stressed sheep as compared to their unsupplemented counterparts. It was concluded that heat stress negatively affects the oxidative status of sheep along with the physiological responses and some of these affects can be ameliorated through dietary antioxidants supplementation at supranutritional concentrations.

Kisspeptin cells in the ovine arcuate nucleus express prolactin receptor but not melatonin receptor.

Melatonin is secreted at night by the pineal gland and governs the reproductive system in seasonal breeders, such as sheep. The mechanism by which melatonin regulates reproduction is not known. The circannual rhythmicity of other factors, including prolactin, is also regulated by photoperiod via changes in melatonin secretion. In sheep, plasma prolactin levels are higher in the nonbreeding season than the breeding season. Kisspeptin, synthesised by neurones in the ovine arcuate nucleus (ARC) and preoptic area, is a key regulator of reproduction through stimulation of gonadotrophin-releasing hormone secretion and its expression in the ARC is reduced during the nonbreeding season. We hypothesised that kisspeptin expression is directly, or indirectly, regulated by melatonin and/or prolactin. We first examined the expression of melatonin receptor (MTNR1A) in kisspeptin (Kiss1 mRNA) neurones in the ARC of ovariectomised (OVX) sheep using double-label in situ hybridisation. MTNR1A mRNA was not expressed by kisspeptin neurones, whereas strong expression was detected in the pars tuberalis. We then examined the expression of the long-form prolactin receptor (PRLR-L) in ARC kisspeptin neurones. In OVX ewes, approximately 60% of kisspeptin neurones expressed PRLR-L mRNA at similar levels in the breeding and nonbreeding seasons. We then aimed to determine whether prolactin treatment during the breeding season regulates kisspeptin expression in the ARC. Continuous central infusion of prolactin (20 µg/h for 7 days) in oestradiol-treated OVX sheep did not alter Kiss1 mRNA expression or luteinising hormone secretion, although it induced substantial phosphorylated signal transducer and activator of transcription 5-immunoreactive nuclei staining in the mediobasal hypothalamus. We conclude that the seasonal change in kisspeptin neurones cannot be regulated directly by melatonin, although it may be a result of changes in prolactin levels. Despite this, kisspeptin expression was unchanged after exogenous prolactin treatment in breeding season ewes.

The role of kisspeptin and gonadotropin inhibitory hormone (GnIH) in the seasonality of reproduction in sheep.

Sheep are seasonal breeders and reproductive status is controlled by photoperiod. Recent recognition of the significant role for kisspeptin and gonadotropin inhibitory hormone (GnIH) in the regulation of gonadotropin releasing hormone (GnRH) cells has provided a new perspective in the seasonal regulation of reproductive activity. Virtually all kisspeptin cells express estrogen receptors and kisspeptin is a potent stimulator of GnRH secretion. Thus, kisspeptin cells provide a conduit by which changes in estrogen feedback effects may be exerted upon GnRH cells. Changes in the activity of kisspeptin cells with season indicate a major role in the seasonal changes in reproductive activity in the ewe. GnIH is an inhibitor of reproductive function and there is mounting evidence that changing activity of this system is also an important determinant of reproductive status. Reciprocal changes in kisspeptin and GnIH activity explain seasonal changes in the function of GnRH cells.

Estradiol-17beta-responsive A1 and A2 noradrenergic cells of the brain stem project to the bed nucleus of the stria terminalis in the ewe brain: a possible route for regulation of gonadotropin releasing hormone cells.

We have studied brain stem cells in the ewe brain that project to the bed nucleus of the stria terminalis (BNST) and determined if these cells are activated by estradiol-17beta. This would predicate an indirect role in the estradiol-17beta regulation of gonadotropin releasing hormone (GnRH) cells, since these receive input from the BNST. Ovariectomized ewes received 50 mug estradiol-17beta benzoate (i.m.) 1 h prior to brain collection, so that activated cells could be identified by Fos immunohistochemistry. Retrograde tracer (FluoroGold; FG), was injected into the three divisions of the BNST and labeled cells were mapped to the A1 and A2 regions and the parabrachial nucleus (PBN) of the brain stem. With FG injection into the dorsal and lateral BNST, all FG-containing cells in the caudal A1 and 45% of those in A2 stained for dopamine-beta-hydroxylase (DBH), indicating noradrenergic type. No FG-labelled cells in the PBN were DBH-positive. In A1 and A2 respectively, 42% and 46% of FG-labelled cells were Fos-positive, with no double-labeling in cells of the PBN. In ewes receiving FG injections into the ventral BNST, estrogen receptor (ER)alpha-immunoreactive nuclei were found in 82% of A1-FG labeled and 38% of A2-FG labeled cells. No FG-labelled cells of the PBN were ERalpha-positive. Anterograde tracing from A1 with microruby injection identified projections to the PBN, BNST and preoptic area (POA). Thus, A1 and A2 noradrenergic neurons project to the BNST in the ewe brain, express ERalpha and are activated by estradiol-17beta. These noradrenergic, estrogen-responsive cells may provide indirect input to GnRH cells, via the BNST.

Kisspeptin neurons in the ovine arcuate nucleus and preoptic area are involved in the preovulatory luteinizing hormone surge.

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.

Projections of RFamide-related peptide-3 neurones in the ovine hypothalamus, with special reference to regions regulating energy balance and reproduction.

RFamide-related peptide-3 (RFRP-3) is a neuropeptide produced in cells of the paraventricular nucleus and dorsomedial nucleus of the ovine hypothalamus. In the present study, we show that these cells project to cells in regions of the hypothalamus involved in energy balance and reproduction. A retrograde tracer (FluoroGold) was injected into either the arcuate nucleus, the lateral hypothalamic area or the ventromedial nucleus. The distribution and number of retrogradely-labelled RFRP-3 neurones was determined. RFRP-3 neurones projected to the lateral hypothalamic area and, to a lesser degree, to the ventromedial nucleus and the arcuate nucleus. Double-label immunohistochemistry was employed to identify cells receiving putative RFRP-3 input to cells in these target regions. RFRP-3 cells were seen to project to neuropeptide Y and pro-opiomelanocortin neurones in the arcuate nucleus, orexin and melanin-concentrating hormone neurones in the lateral hypothalamic area, as well as orexin cells in the dorsomedial nucleus and corticotrophin-releasing hormone and oxytocin cells in the paraventricular nucleus. Neurones expressing gonadotrophin-releasing hormone in the preoptic area were also seen to receive input from RFRP-3 projections. We conclude that RFRP-3 neurones project to hypothalamic regions and cells involved in regulation of energy balance and reproduction in the ovine brain.

Cortisol disrupts the ability of estradiol-17beta to induce the LH surge in ovariectomized ewes.

Stress disrupts the preovulatory luteinizing hormone (LH) surge in females, but the mechanisms are unknown. We tested the hypothesis that cortisol compromises the ability of estrogen to induce a preovulatory-like LH surge in ovariectomized ewes in both the breeding and nonbreeding season. Luteinizing hormone surges were induced in ovariectomized ewes by treatment with progesterone followed by a surge-inducing estradiol-17beta (E2) stimulus using a crossover design. The experiment was replicated in the breeding and nonbreeding seasons. Cortisol reduced the incidence of LH surges irrespective of season. Cortisol increased the latency from E2 stimulus to the onset of the surge in the breeding season only and suppressed the LH surge amplitude during both seasons (P<0.01). We conclude that cortisol can interfere with the LH surge in several ways: delay, blunt, and in extreme cases prevent the E2-induced LH surge. Furthermore, the effect of cortisol to delay the E2-induced LH surge is more pronounced in the breeding season. These results show that cortisol disrupts the positive feedback effect of E2 to trigger an LH surge and suggest the involvement of multiple mechanisms.

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.

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.

Adipose tissue hormones and the regulation of food intake.

Over the past decade, adipose tissue has been shown to produce numerous factors that act as hormones. Many of these act on the brain to regulate energy balance via dual effects on food intake and energy expenditure. These include well-characterised hormones such as leptin, oestrogen and glucocorticoids and novel factors such as adiponectin and resistin. This review provides a perspective on the role of these factors as lipostats.

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.

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.

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.

Kisspeptin expression in the brain: catalyst for the initiation of puberty.

In 2003, two independent groups of researchers discovered almost simultaneously that inactivating mutations of the G protein coupled receptor, GPR54, cause hypogonadotropic hypogonadism in mice and men. Since this discovery, kisspeptins, the natural ligands for GPR54, have been thrust into the reproductive neuroendocrine spotlight, as major regulators of GnRH function. Kisspeptins are the peptide products of the KiSS-1 gene, and potently stimulate gonadotrophin secretion when administered either centrally or peripherally. Expression of KiSS-1 has been localised to specific regions of the hypothalamus in many species and is regulated by gonadal steroids and across the estrous cycle. It appears that kisspeptin transmits steroid feedback signals to GnRH cells, especially the positive feedback effect of estrogen that causes the preovulatory GnRH/LH surge. Importantly, kisspeptin function appears to be fundamental to the initiation of puberty.