Estrogenic Pastures: A Source of Endocrine Disruption in Sheep Reproduction.

Phytoestrogens can impact on reproductive health due to their structural similarity to estradiol. Initially identified in sheep consuming estrogenic pasture, phytoestrogens are known to influence reproductive capacity in numerous species. Estrogenic pastures continue to persist in sheep production systems, yet there has been little headway in our understanding of the underlying mechanisms that link phytoestrogens with compromised reproduction in sheep. Here we review the known and postulated actions of phytoestrogens on reproduction, with particular focus on competitive binding with nuclear and non-nuclear estrogen receptors, modifications to the epigenome, and the downstream impacts on normal physiological function. The review examines the evidence that phytoestrogens cause reproductive dysfunction in both the sexes, and that outcomes depend on the developmental period when an individual is exposed to phytoestrogen.

Expression of genes for Kisspeptin (KISS1), Neurokinin B (TAC3), Prodynorphin (PDYN), and gonadotropin inhibitory hormone (RFRP) across natural puberty in ewes.

Expression of particular genes in hypothami of ewes was measured across the natural pubertal transition by in situ hybridization. The ewes were allocated to three groups (n = 4); prepubertal, postpubertal and postpubertally gonadectomized (GDX). Prepubertal sheep were euthanized at 20 weeks of age and postpubertal animals at 32 weeks. GDX sheep were also euthanized at 32 weeks, 1 week after surgery. Expression of KISS1, TAC3, PDYN in the arcuate nucleus (ARC), RFRP in the dorsomedial hypothalamus and GNRH1 in the preoptic area was quantified on a cellular basis. KISS1R expression by GNRH1 cells was quantified by double-label in situ hybridization. Across puberty, detectable KISS1 cell number increased in the caudal ARC and whilst PDYN cell numbers were low, numbers increased in the rostral ARC. TAC3 expression did not change but RFRP expression/cell was reduced across puberty. There was no change across puberty in the number of GNRH1 cells that expressed the kisspeptin receptor (KISS1R). GDX shortly after puberty did not increase expression of any of the genes of interest. We conclude that KISS1 expression in the ARC increases during puberty in ewes and this may be a causative factor in the pubertal activation of the reproductive axis. A reduction in expression of RFRP may be a factor in the onset of puberty, removing negative tone on GNRH1 cells. The lack of changes in expression of genes following GDX suggest that the effects of gonadal hormones may differ in young and mature animals.

Patterns of preoptic-hypothalamic neuronal activation and LH secretion in female sheep following the introduction and withdrawal of novel males.

The neuroendocrine response of female sheep to a novel male involves neural activation in the hypothalamus. However, if males are removed, the gonadotrophic signal declines, so the neural activity is likely to change. We examined Fos-immunoreactive (IR) cells in hypothalamic tissues from seasonally anovulatory female sheep exposed to males for 2 or 6h, or for 2h followed by 4h isolation from males. Control females were killed in the absence of male exposure. Male introduction increased LH secretion in all females; male removal was associated with a reduction only in mean and basal LH concentrations. Females exposed to males for 2h had more Fos-IR cells in the arcuate nucleus (ARC), ventromedial nucleus of the hypothalamus (VMH) and organum vasculosum of the lamina terminalis (OVLT) than control females. Fos-IR cells in the preoptic area (POA) were only greater than in control females after 6h exposure to a male. Removal of males decreased the number of Fos-IR cells in the ARC, VMH and OVLT, but not in the POA. Thus, hypothalamic neural activation and LH secretion in female sheep are stimulated by males and decline after male removal. However, activation in the POA persists after removal and may explain the incomplete decline in the LH response.

Surgical Considerations for Vitamin D Deficiency in Foot and Ankle Surgery.

Vitamin D deficiency affects nearly one-sixth of the world's population and is common in patients undergoing foot and ankle surgery. Vitamin D is critical for calcium homeostasis and plays an important role in the maintenance of bone health. Patients undergoing foot and ankle procedures can be evaluated preoperatively with vitamin D level testing, and deficiencies can be addressed with either preoperative or postoperative supplementation. Current data suggest that patients with adequate vitamin D levels may have better outcomes, but the details are not yet clear. Vitamin D supplementation is well tolerated with rare side effects.

The Impact of Music Therapy in the Postoperative Setting on Overall Patient Satisfaction Using Validated Outcome Scores.

PURPOSE: Previous studies have examined music therapy (MT) as a potential modality to relieve negative postoperative symptoms such as pain. This randomized control trial examined the use of MT on patient satisfaction in the postanesthesia care unit. DESIGN: Fifty patients undergoing outpatient orthopaedic surgery were enrolled and randomized into two groups, those receiving MT postoperatively and a control group who did not. METHODS: After hospital discharge, subjects were assessed with two validated outcome measurements for overall patient satisfaction, the visual analog satisfaction scale and the Patient Judgment of Hospital Quality survey. FINDINGS: The results showed no statistically significant differences between the MT and control group on the Patient Judgment of Hospital Quality survey (MT = 3.42, standard therapy = 3.41, P = .94) and the visual analog satisfaction scale (MT = 91.20, standard therapy = 91.65, P = .88). CONCLUSIONS: MT given in the postoperative setting has no impact on overall patient satisfaction.

Ontogeny of clock and KiSS-1 metastasis-suppressor (Kiss1) gene expression in the prepubertal mouse hypothalamus.

Kisspeptin is crucial for the generation of the circadian-gated preovulatory gonadotrophin-releasing hormone (GnRH)-LH surge in female rodents, with expression in the anteroventral periventricular nucleus (AVPV) peaking in the late afternoon of pro-oestrus. Given kisspeptin expression is established before puberty, the aim of the present study was to investigate kisspeptin and clock gene rhythms during the neonatal period. Anterior and posterior hypothalami were collected from C57BL/6J mice on Postnatal Days (P) 5, 15 and 25, at six time points across 24h, for analysis of gene expression by reverse transcription-quantitative polymerase chain reaction. Expression of aryl hydrocarbon receptor nuclear translocator-like gene (Bmal1) and nuclear receptor subfamily 1, group D, member 2 (Rev-erbα) in the anterior hypothalamus (containing the suprachiasmatic nucleus) was not rhythmic at P5 or P15, but Bmal1 expression exhibited rhythmicity in P25 females, whereas Rev-erbα expression was rhythmic in P25 males. KiSS-1 metastasis-suppressor (Kiss1) expression did not exhibit time-of-day variation in the anterior (containing the AVPV) or posterior (containing the arcuate nucleus) hypothalami in female and male mice at P5, P15 or P25. The data indicate that the kisspeptin circadian peak in expression observed in the AVPV of pro-oestrous females does not manifest at P5, P15 or P25, likely due to inadequate oestrogenic stimuli, as well as incomplete development of clock gene rhythmicity before puberty.

Unaltered Hypothalamic Metabolic Gene Expression in Kiss1r Knockout Mice Despite Obesity and Reduced Energy Expenditure.

Kisspeptin controls reproduction by stimulating gonadotrophin-releasing hormone neurones via its receptor Kiss1r. Kiss1r is also expressed other brain areas and in peripheral tissues, suggesting additional nonreproductive roles. We recently determined that Kiss1r knockout (KO) mice develop an obese and diabetic phenotype. In the present study, we investigated whether Kiss1r KOs develop this metabolic phenotype as a result of alterations in the expression of metabolic genes involved in the appetite regulating system of the hypothalamus, including neuropeptide Y (Npy) and pro-opiomelanocortin (Pomc), as well as leptin receptor (Lepr), ghrelin receptor (Ghsr), and melanocortin receptors 3 and 4 (Mc3r, Mc4r). Body weights, leptin levels and hypothalamic gene expression were measured in both gonad-intact and gonadectomised (GNX) mice at 8 and 20 weeks of age that had received either normal chow or a high-fat diet. We detected significant increases in Pomc expression in gonad-intact Kiss1r KO mice at 8 and 20 weeks, although there were no alterations in the other metabolic-related genes. However, the Pomc increases appeared to reflect genotype differences in circulating sex steroids, because GNX wild-type and Kiss1r KO mice exhibited similar Pomc levels, along with similar Npy levels. The altered Pomc gene expression in gonad-intact Kiss1r KO mice is consistent with previous reports of reduced food intake in these mice and may serve to increase the anorexigenic drive, perhaps compensating for the obese state. However, the surprising overall lack of changes in any of the hypothalamic metabolic genes in GNX KO mice suggests that the aetiology of obesity in the absence of kisspeptin signalling may reflect peripheral rather than central metabolic impairments.

Diurnal regulation of hypothalamic kisspeptin is disrupted during mouse pregnancy.

Kisspeptin, the neuropeptide product of the Kiss1 gene, is critical in driving the hypothalamic-pituitary-gonadal (HPG) axis. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (Arc) of the hypothalamus mediate differential effects, with the Arc regulating negative feedback of sex steroids and the AVPV regulating positive feedback, vital for the preovulatory surge and gated under circadian control. We aimed to characterize hypothalamic Kiss1 and Kiss1r mRNA expression in nonpregnant and pregnant mice, and investigate potential circadian regulation. Anterior and posterior hypothalami were collected from C57BL/6J mice at diestrus, proestrus, and days 6, 10, 14, and 18 of pregnancy, at six time points across 24h, for real-time PCR analysis of gene expression. Analysis confirmed that Kiss1 mRNA expression in the AVPV increased at ZT13 during proestrus, with a luteinizing hormone surge observed thereafter. No diurnal regulation was seen at diestrus or at any stage of pregnancy. Anterior hypothalamic Avp mRNA expression exhibited no diurnal variation, but Avpr1a peaked at 12:00h during proestrus, possibly reflecting the circadian input from the suprachiasmatic nucleus to AVPV Kiss1 neurons. Rfrp (Npvf) expression in the posterior hypothalamus did not demonstrate diurnal variation at any stage. Clock genes Bmal1 and Rev-erbα were strongly diurnal, but there was little change between diestrus/proestrus and pregnancy. Our data indicate the absence of the circadian input to Kiss1 in pregnancy, despite high gestational estradiol levels and normal clock gene expression, and may suggest a disruption of a kisspeptin-specific diurnal rhythm that operates in the nonpregnant state.

Pregnancy-induced adaptations of the central circadian clock and maternal glucocorticoids.

Maternal physiological adaptations, such as changes to the hypothalamic-pituitary-adrenal (HPA) axis, are central to pregnancy success. Circadian variation of the HPA axis is dependent on clock gene rhythms in the hypothalamus, but it is not known whether pregnancy-induced changes in maternal glucocorticoid levels are mediated via this central clock. We hypothesized that hypothalamic expression of clock genes changes across mouse pregnancy and this is linked to altered HPA activity. The anterior hypothalamus and maternal plasma were collected from C57Bl/6J mice prior to pregnancy and on days 6, 10, 14 and 18 of gestation (term=d19), across a 24-h period (0800, 1200, 1600, 2000, 0000, 0400 h). Hypothalamic expression of clock genes and Crh was determined by qPCR, plasma ACTH concentration measured by Milliplex assay and plasma corticosterone concentration by LC-MS/MS. Expression of all clock genes varied markedly across gestation, most notably at mid-gestation when levels of each gene were elevated. The pregnancy-induced increase in maternal corticosterone levels (by up to 14-fold on day 14) was not accompanied by a parallel shift in plasma ACTH (28% lower on day 14 compared with non-pregnant levels). Moreover, while circadian rhythmicity in corticosterone was maintained up to day 14 of gestation, this was effectively lost by day 18. Overall, our data show that the central circadian clock undergoes marked adaptations throughout mouse pregnancy, changes that are likely to contribute to maternal physiological adaptations. Importantly, however, neither hypothalamic clock genes nor plasma ACTH levels appear to drive the marked increase in maternal corticosterone after mid-gestation.

Prevalence of vitamin D deficiency in patients with foot and ankle injuries.

BACKGROUND: Vitamin D deficiency has been identified as one of the most common causes of fragility fractures and poor fracture healing. Although rates of vitamin D deficiency have been delineated in various orthopaedic populations, little is known about the prevalence of vitamin D deficiency in patients with foot and ankle disorders. The goal of this study was to identify the prevalence of vitamin D deficiency in patients with a low energy fracture of the foot or ankle. METHODS: Over a 6-month period, a serum 25-OH vitamin D level was obtained from consecutive patients with a low energy ankle fracture, fifth metatarsal base fracture, or stress fracture of the foot or ankle. For comparative purposes, vitamin D levels in patients with an ankle sprain and no fracture were also examined. RESULTS: The study cohort included 75 patients, of which 21 had an ankle fracture, 23 had a fifth metatarsal base fracture, and 31 had a stress fracture. The mean age was 52 (range, 16-80) years. Thirty-five of the fracture patients (47%) had an insufficient vitamin D level (below the recommended level of 30 ng/mL), and 10 of the patients (13%) had a level that was deficient (< 20 ng/mL). Vitamin D levels were significantly lower in those with a fracture than in those with an ankle sprain (P = .02). In the fracture cohort, the factors significantly associated with vitamin D insufficiency in the multivariate analysis were smoking (P = .03), obesity (P = .003), and other medical risk factors for vitamin D deficiency (P = .03). CONCLUSION: Hypovitaminosis D was common among patients with a foot or ankle injury seen at our institution. Patients with a low energy fracture of the foot or ankle were at particular risk for low vitamin D, especially if they smoked, were obese, or had other medical risk factors. Given that supplementation with vitamin D (± calcium) has been shown to reduce the risk of fragility fractures and improve fracture healing, monitoring of 25-OH vitamin D and supplementation should be considered in patients with fractures. LEVEL OF EVIDENCE: Level III, prospective case control.

Characterizing the neuroendocrine and ovarian defects of androgen receptor-knockout female mice.

Homozygous androgen receptor (AR)-knockout (ARKO) female mice are subfertile due to both intra- and extraovarian (neuroendocrine) defects as defined by ovary transplantation. Using ARKO mice, this study set out to reveal the precise AR-regulated pathways required for optimal androgen-regulated ovulation and fertility. ARKO females exhibit deficient neuroendocrine negative feedback, with a reduced serum luteinizing hormone (LH) response to ovariectomy (OVX) (P < 0.01). Positive feedback is also altered as intact ARKO females, at late proestrus, exhibit an often mistimed endogenous ovulatory LH surge. Furthermore, at late proestrus, intact ARKO females display diminished preovulatory serum estradiol (E2; P < 0.01) and LH (P < 0.05) surge levels and reduced Kiss1 mRNA expression in the anteroventral periventricular nucleus (P < 0.01) compared with controls. However, this reduced ovulatory LH response in intact ARKO females can be rescued by OVX and E2 priming or treatment with endogenous GnRH. These findings reveal that AR regulates the negative feedback response to E2, E2-positive feedback is compromised in ARKO mice, and AR-regulated negative and positive steroidal feedback pathways impact on intrahypothalamic control of the kisspeptin/GnRH/LH cascade. In addition, intraovarian AR-regulated pathways controlling antral to preovulatory follicle dynamics are disrupted because adult ARKO ovaries collected at proestrus have small antral follicles with reduced oocyte/follicle diameter ratios (P < 0.01) and increased proportions of unhealthy large antral follicles (P < 0.05) compared with controls. As a consequence of aberrant follicular growth patterns, proestrus ARKO ovaries also exhibit fewer preovulatory follicle (P < 0.05) and corpora lutea numbers (P < 0.01). However, embryo development to the blastocyst stage is unchanged in ARKO females, and hence, the subfertility is a consequence of reduced ovulations and not altered embryo quality. These findings reveal that the AR has a functional role in neuroendocrine regulation and timing of the ovulatory LH surge as well as antral/preovulatory follicle development.

An eGFP-expressing subpopulation of growth hormone secretagogue receptor cells are distinct from kisspeptin, tyrosine hydroxylase, and RFamide-related peptide neurons in mice.

Ghrelin acts on the growth hormone secretagogue receptor (GHSR) in the brain to elicit changes in physiological functions. It is associated with the neural control of appetite and metabolism, however central ghrelin also affects fertility. Central ghrelin injection in rats suppresses luteinizing hormone (LH) concentrations and pulse frequency. Although ghrelin suppresses LH and regulates kisspeptin mRNA in the anteroventral periventricular/periventricular nucleus (AVPV/PeN), there is no neuroanatomical evidence linking GHSR neural circuits to kisspeptin neurons. In this study, we first determined coexpression of GHSR and GnRH neurons using a GHSR-eGFP reporter mouse line. Using dual-label immunohistochemistry, we saw no coexpression. GHSR-eGFP expressing cells were present in the AVPV/PeN and over 90% of these expressed estrogen receptor-α (ERα). Despite this, we observed no evidence of GHSR-eGFP/kisspeptin coexpressing neurons in the AVPV/PeN. To further examine the phenotype of GHSR-eGFP cells in the AVPV/PeN, we determined coexpression with tyrosine hydroxylase (TH) and showed virtually no coexpression in the AVPV/PeN (<2%). We also observed no coexpression of GHSR-eGFP and RFamide-related peptide-3 (RFRP3) neurons in the dorsomedial hypothalamic nucleus. Importantly, we observed that approximately half of the GHSR-eGFP cells in the AVPV coexpressed Ghsr mRNA (as determined by in situ hybridization) so these data should be interpreted accordingly. Although ghrelin influences the hypothalamic reproductive axis, our data using a GHSR-eGFP reporter suggests ghrelin regulates neurons expressing ERα but does not directly act on GnRH, kisspeptin, TH, or RFRP3 neurons, as little or no GHSR-eGFP coexpression was observed.

Preweaning over- and underfeeding alters onset of puberty in the rat without affecting kisspeptin.

The perinatal nutritional environment can permanently influence body weight, potentially leading to changes in puberty onset and reproductive function. We hypothesized that perinatal under- or overfeeding would alter puberty onset and influence concentrations of a neuropeptide crucial for successful puberty, kisspeptin. We manipulated Wistar rat litter sizes to derive small (SL), control (CL), and large (LL) litters containing 4, 12, and 20 rat pups respectively. This manipulation results in an overweight phenotype in SL rats and a lean phenotype in LL that persists throughout life. To investigate whether successful puberty onset is affected by neonatal under- or overfeeding, we examined indices of growth and development, including the onset of puberty, as well as the central expression of Kiss1 mRNA in these pups. Male LL rats reached puberty later than those from CL. These males also had reduced plasma testosterone and elevated 17beta-estradiol concentrations at puberty. The age at puberty onset was not affected in SL males despite accelerated growth. In females, puberty onset was not significantly delayed by having a lean phenotype, and steroid hormones were not affected. The age at onset was, however, younger in the SL females. Kiss1 mRNA in the hypothalamus was not affected by neonatal nutrition either at puberty or 7 days later. Our findings show early life underfeeding in males and overfeeding in females significantly affects puberty onset, altering steroid hormone concentrations in males, but this is not related to changes in hypothalamic kisspeptin.

Seasonal variation in the gonadotropin-releasing hormone response to kisspeptin in sheep: possible kisspeptin regulation of the kisspeptin receptor.

Kisspeptin signaling in the hypothalamus appears critical for the onset of puberty and driving the reproductive axis. In sheep, reproduction is seasonal, being activated by short days and inhibited by long days. During the non-breeding (anestrous) season, gonadotropin-releasing hormone (GnRH) and gonadotropin secretion is reduced, as is the expression of Kiss1 mRNA in the brain. Conversely, the luteinizing hormone response to kisspeptin during this time is greater. To determine whether the GnRH response to kisspeptin is increased during anestrus, we utilized hypophysial portal blood sampling. In anestrus ewes, the GnRH and LH responses to kisspeptin were greater compared to the breeding season (luteal phase). To ascertain whether this difference reflects a change in Kiss1r, we measured its expression on GnRH neurons using in situ hybridization. The level of Kiss1r was greater during the non-breeding season compared to the breeding season. To further examine the mechanism underlying this change in Kiss1r, we examined Kiss1r/GnRH expression in ovariectomized ewes (controlling for sex steroids) during the breeding and non-breeding seasons, and also ovariectomized non-breeding season ewes with or without estradiol replacement. In both experiments, Kiss1r expression on GnRH neurons was unchanged. Finally, we examined the effect of kisspeptin treatment on Kiss1r. Kiss1r expression on GnRH neurons was reduced by kisspeptin infusion. These studies indicate the kisspeptin response is indeed greater during the non-breeding season and this may be due in part to increased Kiss1r expression on GnRH neurons. We also show that kisspeptin may regulate the expression of its own receptor.

Gonadotropin-inhibitory hormone is a hypothalamic peptide that provides a molecular switch between reproduction and feeding.

OBJECTIVE: Gonadotropin-inhibitory hormone (GnIH)-3 is a neuropeptide that plays a major role in the regulation of reproduction and feeding in mammals. MATERIALS AND METHODS: We measured endocrine and behavioural parameters of reproduction in sheep, and sexual behaviour in sheep, mice and cynomolgus monkeys. In addition, GnIH gene expression (in situ hybridization) was examined in ewes, and effects of GnIH-3 on food intake and energy expenditure were measured in various species. GnIH-3 was infused (i.v.) into ewes after an i.m. injection of estradiol benzoate to determine whether the peptide blocks the surge in luteinizing hormone (LH) secretion. RESULTS: GnIH gene expression was reduced in the preovulatory period in ewes. Infusion (i.v.) of GnIH-3 blocked the estrogen-induced LH surge (in ewes). Intracerebroventricular infusion had no effect on female or male sexual behaviour in each of the three species, but increased food intake. There were no effects on energy expenditure in sheep or rats. GnIH increased fos protein (immunohistochemistry) was seen in orexigenic neurons (in sheep and rats), but also in anorexigenic neurons (in sheep). CONCLUSIONS: GnIH-3 reduces reproductive hormone levels and increases food intake in mammals without reducing energy expenditure. There is minimal effect on reproductive behaviour. The dual effect on reproduction and feeding suggests that GnIH-3 provides a molecular switch between these two functions. Blockade of the positive feedback effect of estrogen with parenteral infusion indicates that this peptide may have utility as a blocker of reproductive function in mammals.

Hypothalamic expression of KISS1 and gonadotropin inhibitory hormone genes during the menstrual cycle of a non-human primate.

Kisspeptin, the product of the KISS1 gene, stimulates gonadotropin-releasing hormone (GnRH) secretion; gonadotropin inhibitory hormone (GnIH), encoded by the RF-amide-related peptide (RFRP) or NPVF gene, inhibits the reproductive axis. In sheep, kisspeptin neurons are found in the lateral preoptic area (POA) and the arcuate nucleus (ARC) and may be important for initiating the preovulatory GnRH/luteinizing hormone (LH) surge. GnIH cells are located in the ovine dorsomedial hypothalamic nucleus (DMN) and paraventricular nucleus (PVN), with similar distribution in the primate. KISS1 cells are found in the primate POA and ARC, but the function that kisspeptin and GnIH play in primates has not been elucidated. We examined KISS1 and NPVF mRNA throughout the menstrual cycle of a female primate, rhesus macaque (Macaca mulatta), using in situ hybridization. KISS1-expressing cells were found in the POA and ARC, and NPVF-expressing cells were located in the PVN/DMN. KISS1 expression in the caudal ARC and POA was higher in the late follicular phase of the cycle (just before the GnRH/LH surge) than in the luteal phase. NPVF expression was also higher in the late follicular phase. We ascertained whether kisspeptin and/or GnIH cells project to GnRH neurons in the primate. Close appositions of kisspeptin and GnIH fibers were found on GnRH neurons, with no change across the menstrual cycle. These data suggest a role for kisspeptin in the stimulation of GnRH cells before the preovulatory GnRH/LH surge in non-human primates. The role of GnIH is less clear, with paradoxical up-regulation of gene expression in the late follicular phase of the menstrual cycle.

Sex steroid control of hypothalamic Kiss1 expression in sheep and rodents: comparative aspects.

In recent years, the Kiss1 gene has been cast into the reproductive spotlight. In the short period since the discovered link between kisspeptins, the encoded peptides of Kiss1, and fertility, these peptides are now known to be critical for the neuroendocrine control of reproduction. Kisspeptin producing cells in the hypothalamus are poised to become the 'missing link' in the sex steroid feedback control of GnRH secretion. These cells contain all the necessary components to relay information of the sex steroid environment to GnRH neurons, which possess the kisspeptin receptor, GPR54. Sex steroids regulate Kiss1 mRNA, and kisspeptin expression in the hypothalamus, in a manner consistent with both negative and positive feedback control of GnRH. The precise nature of sex steroid effects, in particular those of estrogen, on Kiss1 expression have been extensively studied in the female rodent and ewe. In the arcuate nucleus (ARC) of both species, kisspeptin cells appear to forward signals pertinent to negative feedback regulation of GnRH, although in the ewe it appears this population of Kiss1 cell is also responsible for positive feedback regulation of GnRH at the time of the preovulatory GnRH/LH surge. In rodents, these positive feedback signals appear to be mediated by kisspeptin cells exclusively within the anteroventral periventricular nucleus (AVPV). There are no Kiss1 cells in the ovine AVPV, but there is a population in the preoptic area. The role these preoptic area cells play in the sex steroid feedback regulation of GnRH secretion, if any, is yet to be revealed.

Kisspeptin and seasonality in sheep.

Sheep are seasonal breeders, experiencing a period of reproductive quiescence during spring and early summer. During the non-breeding period, kisspeptin expression in the arcuate nucleus is markedly reduced. This strongly suggests that the mechanisms that control seasonal changes in reproductive function involve kisspeptin neurons. Kisspeptin cells appear to regulate GnRH neurons and transmit sex-steroid feedback to the reproductive axis. Since the non-breeding season is characterized by increased negative feedback of estrogen on GnRH secretion, the kisspeptin neurons seem to be fundamentally involved in the determination of breeding state. The reduction in kisspeptin neuronal function during the non-breeding season can be corrected by infusion of kisspeptin, which causes ovulation in seasonally acyclic females.

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.

KiSS-1 messenger ribonucleic acid expression in the hypothalamus of the ewe is regulated by sex steroids and season.

The KiSS-1 gene encodes a family of peptides called kisspeptins, which are endogenous ligands for the G protein-coupled receptor GPR54. Kisspeptin function appears to be critical for GnRH secretion and the initiation of puberty. To test the hypothesis that steroid hormones regulate KiSS-1 mRNA expression in the ewe, we examined the brains of ovary-intact (luteal phase) and ovariectomized (OVX) ewes, as well as OVX ewes that received estradiol (E) or progesterone (P) replacement. KiSS-1 mRNA-expressing cells were predominantly located in the arcuate nucleus (ARC). Here, expression was increased after OVX but returned to the level of gonad-intact animals with E treatment. Treatment with P partially restored KiSS-1 expression toward gonad-intact levels. Double-label immunohistochemistry revealed that approximately 86% of kisspeptin-immunoreactive cells in the ARC are also P-receptor positive. Finally, we tested the hypothesis that KiSS-1 mRNA is lower during anestrus, due to a non-steroid-dependent seasonal effect. In OVX ewes, expression in the ARC was lower at the time of year corresponding to anestrus. We conclude that KiSS-1 expression in the ARC of the ewe brain is negatively regulated by chronic levels of E and P, suggesting that both steroids may exert negative feedback control on GnRH secretion through altered kisspeptin signaling. Furthermore, a seasonal alteration in KiSS-1 expression in the ARC of OVX ewes strongly suggests that kisspeptin is fundamentally involved in the control of seasonal changes in reproductive function.