Alan J. Tilbrook

Variation in Kisspeptin and RFamide-Related Peptide (RFRP) Expression and Terminal Connections to Gonadotropin-Releasing Hormone Neurons in the Brain: A Novel Medium for Seasonal Breeding in the Sheep

Reproductive activity in sheep is seasonal, being activated by short-day photoperiods and inhibited by long days. During the nonbreeding season, GnRH secretion is reduced by both steroid-independent and steroid-dependent (increased response to estradiol negative feedback) effects of photoperiod. Kisspeptin (also known as metastin) and gonadotropin-inhibitory hormone (GnIH, or RFRP) are two RFamide neuropeptides that appear critical in the regulation of the reproductive neuroendocrine axis. We hypothesized that expression of kisspeptin and/or RFRP underlies the seasonal change in GnRH secretion. We examined kisspeptin and RFRP (protein and mRNA) expression in the brains of ovariectomized (OVX) ewes treated with estradiol (OVX+E) during the nonbreeding and breeding seasons. In OVX+E ewes, greater expression of kisspeptin and Kiss1 mRNA in the arcuate nucleus and lesser expression of RFRP (protein) in the dorsomedial nucleus of the hypothalamus were concurrent with the breeding season. There was also a greater number of kisspeptin terminal contacts onto GnRH neurons and less RFRP-GnRH contacts during the breeding season (compared with the nonbreeding season) in OVX+E ewes. Comparison of OVX and OVX+E ewes in the breeding and nonbreeding season revealed a greater effect of steroid replacement on inhibition of kisspeptin protein and Kiss1 mRNA expression during the nonbreeding season. Overall, we propose that the two RFamide peptides, kisspeptin and RFRP, act in concert, with opposing effects, to regulate the activity of GnRH neurons across the seasons, leading to the annual change in fertility and the cyclical seasonal transition from nonbreeding to breeding season.

Central administration of leptin to ovariectomized ewes inhibits food intake without affecting the secretion of hormones from the pituitary gland: evidence for a dissociation of effects on appetite and neuroendocrine function.

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.

Potent Action of RFamide-Related Peptide-3 on Pituitary Gonadotropes Indicative of a Hypophysiotropic Role in the Negative Regulation of Gonadotropin Secretion

We identified a gene in the ovine hypothalamus encoding for RFamide-related peptide-3 (RFRP-3), and tested the hypothesis that this system produces a hypophysiotropic hormone that inhibits the function of pituitary gonadotropes. The RFRP-3 gene encodes for a peptide that appears identical to human RFRP-3 homolog. Using an antiserum raised against RFRP-3, cells were localized to the dorsomedial hypothalamic nucleus/paraventricular nucleus of the ovine brain and shown to project to the neurosecretory zone of the ovine median eminence, predicating a role for this peptide in the regulation of anterior pituitary gland function. Ovine RFRP-3 peptide was tested for biological activity in vitro and in vivo, and was shown to reduce LH and FSH secretion in a specific manner. RFRP-3 potently inhibited GnRH-stimulated mobilization of intracellular calcium in gonadotropes. These data indicate that RFRP-3 is a specific and potent mammalian gonadotropin-inhibiting hormone, and that it acts upon pituitary gonadotropes to reduce GnRH-stimulated gonadotropin secretion.

Effect of RF-Amide-Related Peptide-3 on Luteinizing Hormone and Follicle-Stimulating Hormone Synthesis and Secretion in Ovine Pituitary Gonadotropes

GnRH provides the primary stimulus for the reproductive axis, but original work also revealed the existence of a gonadotropin-inhibitory hormone (GnIH) in birds. In mammals, GnIH properties are displayed by a hypothalamic dodecapeptide, which is a member of the RF-amide family, namely RF-amide-related peptide (RFRP)-3. This peptide inhibits GnRH-stimulated gonadotropin secretion from ovine pituitary cells in culture, but it is not known whether there are effects on gonadotropin synthesis. The aim of the present study was to determine the effects of RFRP-3 on the expression of genes for beta-subunits of the gonadotropins in ovine pituitary cells from gonadectomized ewes and rams. Cells in primary culture were given GnRH or vehicle pulses every 8 h for 24 h with and without RFRP-3 treatment. GnRH stimulated LH and FSH secretion, which was reduced by RFRP-3. Quantitative real-time PCR revealed increased expression of LHbeta and FSHbeta subunit genes after GnRH treatment and a specific reduction in expression after RFRP-3 treatment. There was no effect on the expression of GH, proopiomelanocortin, or prolactin genes. Western blotting showed that GnRH stimulated phosphorylation of ERK (phospho-ERK-1/2), and this effect was abolished by RFRP-3. We conclude that RFRP-3 acts on the pituitary gonadotropes to inhibit synthesis of the gonadotropins, and this effect may be mediated by a reduction in the GnRH-stimulated second messenger phospho-ERK-1/2.

Stress and Reproduction: Central Mechanisms and Sex Differences in Non-rodent Species

Despite extensive research, the mechanisms by which stress affects reproduction are unknown. Activation of stress systems could potentially influence reproduction at any level of the hypothalamo-pituitary gonadal axis. Nonetheless, the predominant impact is on the secretion of gonadotrophin releasing hormone (GnRH) from the brain and the secretion of the gonadotrophins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), from the gonadotrophs of the anterior pituitary gland. When stress is prolonged, it is likely that secretion of the gonadotrophins will be suppressed but the effects of acute stress or repeated acute stress are not clear. Different stressors activate different pathways for varying durations, and the actions of stress vary with sex and are influenced by the predominance of particular sex steroids in the circulation. The mechanisms by which stress influences reproduction are likely to involve complex interactions between a number of central and peripheral pathways and may be different in males and females. To understand these mechanisms, it is important to determine the stress pathways that are activated by particular stressors and to establish how these pathways affect the secretion and actions of GnRH. Furthermore, there is a need to know how stress influences the feedback actions of gonadal steroids and inhibin.

Negative Feedback Regulation of the Secretion and Actions of Gonadotropin-Releasing Hormone in Males

Abstract 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.

The glucocorticoid contribution to obesity.

Obesity is fast becoming the scourge of our time. It is one of the biggest causes of death and disease in the industrialized world, and affects as many as 32% of adults and 17% of children in the USA, considered one of the worlds fattest nations. It can also cost countries billions of dollars per annum in direct and indirect care, latest estimates putting the USA bill for obesity-related costs at

The effects of handling by humans at calving and during milking on the behaviour and milk cortisol concentrations of primiparous dairy cows

147 billion in 2008. It is becoming clear that the pathophysiology of obesity is vastly more complicated than the simple equation of energy in minus energy out. A combination of genetics, sex, perinatal environment and life-style factors can influence diet and energy metabolism. In this regard, psychological stress can have significant long-term impact upon the propensity to gain and maintain weight. In this review, we will discuss the ability of psychological stress and ultimately glucocorticoids (GCs) to alter appetite regulation and metabolism. We will specifically focus on (i) GC regulation of appetite and adiposity, (ii) the apparent sexual dimorphism in stress effects on obesity and (iii) the ability of early life stress to programme obesity in the long term.

Neuroendocrine mechanisms of innate states of attenuated responsiveness of the hypothalamo-pituitary adrenal axis to stress

This study examined the effects of human presence at the time of calving of primiparous cows, and the behaviour of the stockperson during milking on the behaviour of the cows during milking and in response to an experimenter in a standard 5-min test (human test). Cows which were not handled around the time of their first calving (n=7) displayed a higher (P<0.05) number of flinch, step and kick (FSK) responses during the first 20 weeks of lactation than cows which were handled (n=7); most of these FSK responses occurred in the close presence of the stockperson. It is of interest that although the two treatments did not differ in the amount of extra assistance given by the stockperson, the number of FSK responses of cows was strongly (P<0.01) and positively correlated with the amount of extra assistance given by the stockperson in the form of attaching a kick-bar, replacing dislodged cups or steadying the cow by holding the tail or leg. Cows in the no handling treatment were also slower (P<0.05) to enter areas within 1 and 2 m of the experimenter in the human test conducted in the sixth week of lactation and had higher (P<0.05) mean cortisol concentrations in their milk than cows in the handling treatment. It is proposed that the lower FSK response of cows that received human contact at the time of their calving was a result of these cows being less fearful of humans. Although the two stockpersons differed significantly (P<0.01) in the proportion of physical interactions which were negative (aversive) in nature that they directed towards cows, the stockpersons had no significant influence on the behaviour of the cows during milking. However, the number of physical interactions that the cow received that were of a positive nature was significantly (P<0.05) correlated with the total number of FSK responses displayed in the first 2 weeks of lactation. It appears that stockpersons recognize those cows with a high FSK response and attempt to quieten them with the use of interactions that are positive in nature.

Long-term alterations in adiposity affect the expression of melanin-concentrating hormone and enkephalin but not proopiomelanocortin in the hypothalamus of ovariectomized ewes.

Neuroendocrine responses to stress vary between sexes and reproductive states and are influenced by the type of stressor. Stress responses are attenuated in some physiological states, such as lactation and conditions of low visceral adipose tissue. Moreover, some individuals within a species characteristically display reduced stress responses. The neuroendocrine mechanisms for stress hyporesponsiveness are likely to include reduced synthesis and secretion of corticotropin releasing hormone (CRH) and arginine vasopressin (AVP) from the hypothalamus as a result of enhanced glucocorticoid negative feedback and/or reduced noradrenergic stimulatory input from the brain stem. A major limitation of research to date is the lack of direct measures of CRH and AVP secretion. Attenuated stress responsiveness is also commonly associated with reduced pituitary responsiveness to CRH and AVP. The possible roles of inhibitory central inputs to CRH and AVP neurons and of oxytocin and prolactin in attenuating the HPA axis responses to stress are unknown.