Characterizing the relationship between gonadotropin releasing hormone (GnRH), kisspeptin, and RFamide related peptide 3 (RFRP-3) neurons in the equine hypothalamus across the estrous cycle and in the anovulatory seasons.

To understand better the role that kisspeptin plays in regulating seasonal and estrous cycle changes in the mare, this study investigated the number, location and interactions between GnRH, kisspeptin and RFRP-3 neurons in the equine hypothalamus. Hypothalami were collected from mares during the non-breeding season, vernal transition and various stages of the breeding season. Fluorescent immunohistochemistry was used to label the neuropeptides of interest. GnRH cells were observed primarily in the arcuate nucleus (ARC), while very few labeled cells were identified in the pre-optic area (POA). Kisspeptin cells were identified primarily in the ARC, with a small number of cells observed dorsal to the ARC, surrounding the third ventricle (3V). The mean number of kisspeptin cells varied between animals and typically showed no pattern associated with season or stage of estrous cycle, but a seasonal difference was identified in the ARC population. Small numbers of RFRP-3 cells were observed in the ARC, ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH). The mean number of RFRP-3 cells appeared higher in pre-ovulatory animals compared to all other stages. The percentage of GnRH cell bodies with kisspeptin appositions did not change with season or stage of estrous cycle. The percentage of kisspeptin cells receiving inputs from RFRP-3 fibers did not vary with season or stage of estrous cycle. These interactions suggest the possibility of the presence of an ultra-short loop feedback system between these three peptides. The changes in RFRP-3 neurons suggest the possibility of a role in the regulation of reproduction in the horse, but it is unlikely to be as a gonadotropin inhibitory factor.

The role of Cathepsin S as a marker of prognosis and predictor of chemotherapy benefit in adjuvant CRC: a pilot study.

BACKGROUND: The aim of this pilot retrospective study was to investigate the immunohistochemical expression of Cathepsin S (CatS) in three cohorts of colorectal cancer (CRC) patients (n=560). METHODS: Prevalence and association with histopathological variables were assessed across all cohorts. Association with clinical outcomes was investigated in the Northern Ireland Adjuvant Chemotherapy Trial cohort (n=211), where stage II/III CRC patients were randomised between surgery-alone or surgery with adjuvant fluorouracil/folinic acid (FU/FA) treatment. RESULTS: Greater than 95% of tumours had detectable CatS expression with significantly increased staining in tumours compared with matched normal colon (P>0.001). Increasing CatS was associated with reduced recurrence-free survival (RFS; P=0.03) among patients treated with surgery alone. Adjuvant FU/FA significantly improved RFS (hazard ratio (HR), 0.33; 95% CI, 0.12-0.89) and overall survival (OS; HR, 0.25; 95% CI, 0.08-0.81) among 36 patients with high CatS. Treatment did not benefit the 66 patients with low CatS, with a RFS HR of 1.34 (95% CI, 0.60-3.19) and OS HR of 1.33 (95% CI, 0.56-3.15). Interaction between CatS and treatment status was significant for RFS (P=0.02) and OS (P=0.04) in a multivariate model adjusted for known prognostic markers. CONCLUSION: These results signify that CatS may be an important prognostic biomarker and predictive of response to adjuvant FU/FA in CRC.

Ovarian steroids modulate neuroendocrine dysfunction in polycystic ovary syndrome.

OBJECTIVE: Neuroendocrine dysfunction in polycystic ovary syndrome (PCOS) was addressed by studying the steroid hormone changes in women with PCOS with either high or normal LH levels leading to inferences regarding the primacy of elevated LH in the pathophysiology of PCOS. METHODS: A cross-sectional study was designed in an academic clinical facility involving 234 women with PCOS. Patients were divided into two groups based on an LH/FSH ratio < or >1 and hormonal and metabolic studies were performed in both groups. Factors were determined by binomial logistic regression that predicted group membership of these women. RESULTS: Higher follicular phase estradiol (E2) and androstenedione (A4) levels as well as greater insulin sensitivity were the only factors that predicted the presence of neuroendocrine dysfunction with elevated A4 being necessary for neuroendocrine dysfunction. CONCLUSIONS: It was concluded that uncoupling of hypothalamic E2 inhibition by elevated ovarian A4 associated with E2 related sensitization of pituitary LH leads to neuroendocrine dysfunction in PCOS.

Sex differences in the distribution and abundance of androgen receptor mRNA-containing cells in the preoptic area and hypothalamus of the ram and ewe.

Rams and ewes show a negative-feedback response to peripheral treatment with testosterone, with both sexes having a similar degree of suppression in luteinizing hormone (LH) secretion during the breeding season. At least part of the action of testosterone to suppress gonadotropin-releasing hormone/LH secretion is exerted via interaction with an androgen receptor. The distribution of androgen receptor-containing cells in the hypothalamus has been described for the ram, but similar studies have not been performed in the ewe. In the present study, we tested the hypothesis that levels of androgen receptor mRNA expression in the preoptic area and hypothalamus would be similar in rams and ewes. Perfusion-fixed brain tissue was obtained from adult Romney Marsh ewes (luteal phase) and rams during the breeding season (n = 4/sex). Androgen receptor mRNA expression was quantified in hypothalamic sections by in situ hybridization using an (35)S-labelled riboprobe and image analysis. Hybridizing cells were found in the medial preoptic area, bed nucleus of the stria terminalis, anterior hypothalamic area, ventromedial nucleus, arcuate nucleus and premamillary nucleus. The level of androgen receptor mRNA expression was higher in rams than ewes in the rostral preoptic area, caudal preoptic area and rostral portion of the bed nucleus of the stria terminalis, with no sex difference in other regions. The preoptic area and bed nucleus of the stria terminalis are important for reproductive behaviour and the sex differences in androgen receptor mRNA expression at these levels may relate to this. The high level of androgen receptor mRNA expression in the basal hypothalamus, with no sex difference, is consistent with the role of this region in the regulation of gonadotropin secretion.

Progesterone and testosterone in combination act in the hypothalamus of castrated rams to regulate the secretion of LH.

We tested the hypotheses that progesterone enhances the negative feedback actions of testosterone in rams and that this occurs through actions at the hypothalamus. In the first part of this study, blood samples were collected every 10 min for 12 h before and after 7 days of treatment (i.m.) of castrated Romney Marsh rams (n=5 per group) with vehicle, progesterone (4 mg/12 h), testosterone (4 mg/12 h) or a combination of progesterone (4 mg/12 h) and testosterone (4 mg/12 h). In the second part of this study the brains of four gonad-intact Romney Marsh rams were collected, the hypothalamus was sectioned and in situ hybridisation of mRNA for progesterone receptors conducted. After 7 days of treatment with vehicle or progesterone or testosterone alone, there were no changes in the secretion of LH. In contrast, treatment with a combination of progesterone and testosterone resulted in a significant (P<0.01, repeated measures ANOVA) decrease in mean plasma concentrations of LH, the number of LH pulses per hour and the pre-LH pulse nadir and a significant (P<0.01) increase in the inter-LH pulse interval. We found cells containing mRNA for progesterone receptors throughout the hypothalamus, including the preoptic area (where most GnRH neurons are located in sheep), the periventricular, ventromedial and arcuate nuclei and the bed nucleus of the stria terminalis. This study shows that progesterone is capable of acting centrally with testosterone to suppress the secretion of LH in castrated rams and that cells containing mRNA for progesterone receptors are located in the hypothalamus of rams in the vicinity of GnRH neurons.

The distribution of cells containing estrogen receptor-alpha (ERalpha) and ERbeta messenger ribonucleic acid in the preoptic area and hypothalamus of the sheep: comparison of males and females.

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

Gonadal steroid receptors in the regulation of GnRH secretion in farm animals.

The sites of action and mechanisms by which gonadal steroids regulate gonadotrophin-releasing hormone (GnRH) in domestic animals remain largely unknown. This review summarises information gained from sheep regarding the distribution of the gonadal steroid receptors in the brain, the neurochemical identity and the projections of these steroid receptor-containing neurones. The cells in the hypothalamus that contain each of the gonadal steroid receptors (oestrogen receptor alpha (ERalpha), oestrogen receptor beta (ERbeta), progesterone receptor (PR) and androgen receptor (AR)) show a remarkably similar distribution, although the PR and AR-containing cells are less widespread than oestrogen receptors (ERs). There is considerable overlap in the distribution of ERalpha- and ERbeta-containing cells but also some unique sites for each subtype. This suggests differential regulation of the actions of oestrogen. There appears to be little sexual dimorphism in the distribution of the gonadal steroid receptors in the hypothalamus, with the notable exception of the ventromedial nucleus where females appear to have greater numbers of both ERalpha- and ERbeta-containing cells. Neuronal tracing studies have identified projections of some of the ERalpha-containing cells to sites that may allow interaction with the GnRH system. The receptor mapping, neuronal tracing and microimplantation studies suggest that the ventromedial nucleus is likely to be a key hypothalamic nucleus in the steroid regulation of GnRH secretion in sheep.

Hypothalamic sites of action for testosterone, dihydrotestosterone, and estrogen in the regulation of luteinizing hormone secretion in male sheep.

Testosterone (T) inhibits LH secretion partly by acting at unknown sites within the brain to inhibit GnRH secretion. We tested the hypothesis that the preoptic area (POA) and arcuate-ventromedial region (ARC/VMR), areas rich in androgen and estrogen (E) receptors, are neural sites at which T and the T metabolites, dihydrotestosterone (DHT) and estrogen (E), act to suppress LH secretion. Bilateral guide cannulae were surgically implanted into either the POA or ARC/VMR of castrated male sheep. Experiments were conducted under a long day photoperiod to maximize the inhibitory effect of the steroids. In Exp 1, all sheep (n = 6/site) sequentially received bilateral implants of cholesterol (CHOL), T, or E at each site. Jugular blood samples were taken at 10-min intervals for 4 h both immediately before implant insertion and 5 days later. In Exp 2, all sheep (n = 6/site) sequentially received bilateral implants of CHOL, DHT, or E at each site according to a latin square design. Blood samples were taken before and 7 days after implant insertion. In Exp 3, which followed the same design as Exp 2, implants of E, T, or DHT were placed only in the ARC/VMR. In the final experiment, the effects of T and CHOL implants in the ARC/VMR were compared. Neither T, DHT, nor CHOL implants at either site affected LH secretion. In contrast, E treatment in the ARC/VMR suppressed mean plasma LH levels (P < 0.01), primarily due to an increase in interpulse interval (P < 0.01). Estrogen implants in the POA caused a small, but nonsignificant (P > 0.05), decrease in mean LH levels in the first experiment and an increase in LH interpulse interval (P < 0.05) in the second experiment. These results suggest that the ARC/VMR and possibly the POA are sites at which E acts to reduce GnRH secretion in male sheep.

Enhancing patient outcomes through an understanding of intercultural medicine: guidelines for the practitioner.

As cultural and ethnic diversity increase within American society, physicians face new challenges in recognizing patients' culturally defined expectations about medical care and the cultural/ethnic dictates that influence physician-patient interactions. Patients present to practitioners with many mores related to concepts of disease and illness, intergenerational communication, decision-making authority, and gender roles. In addition, many cultural groups follow folk medicine traditions, and an increasing number of Americans seek treatment by practitioners of alternative therapies before seeking traditional western medical attention. To facilitate patient assessments, enhance compliance with health care instructions, and thus achieve the best possible medical outcomes and levels of satisfaction, practitioners must acknowledge and respect the cultural differences patients bring to medical care environments.

Influence of testosterone on LHRH release, LHRH mRNA and proopiomelanocortin mRNA in male sheep.

The mechanism whereby testosterone (T) reduces pulsatile LHRH and LH release is unknown. We tested the hypothesis that hypothalamic levels of LHRH mRNA decrease and proopiomelanocortin (POMC) mRNA increase coincident with reduced LHRH release induced by either long-term or short-term T treatment in male sheep. Experiment 1 examined the effect of long-term T exposure on LHRH and LH release and LHRH and POMC mRNA levels. Yearling Suffolk rams were castrated and assigned to one of four treatments: 1) castrated (n = 4); 2) castrated, portal cannula (n = 5); 3) castrated+T (n = 4) and 4) castrated+T, portal cannula (n = 4). T-treated males received ten 10-cm silastic T-implants immediately after castration. Surgical placement of devices for collecting hypophyseal-portal blood occurred 2 to 3 months after castration. Seven to 10 days after surgery, blood samples were collected at 10-min intervals for 8 h from portal cannulated males or for 5 h from non-cannulated males to assess pulsatile LHRH and/or LH release. Immediately after blood sample collection, hypothalamic tissue was collected for in situ measurement of LHRH or POMC mRNA. T-treatment decreased (P < 0.01) mean LHRH and LH and decreased (P < 0.01) LHRH and LH pulse frequency. T did not significantly affect (P > 0.10) silver grain area per LHRH neuron, but decreased (P < 0.01) silver grain area per POMC neuron. Portal cannulation tended to decrease (P = 0.057) silver grain area per LHRH neuron without significantly affecting (P > 0.10) LHRH cell numbers while reducing (P < 0.01) silver grain area per POMC neuron and POMC cell numbers. A second experiment examined the effect of 72 h of T-infusion on LHRH and POMC mRNA levels. Castrated yearling males were assigned to receive either vehicle (n = 4) or T (768 ug/kg/day; n = 4). Blood samples were collected at 10 min intervals for 4 h prior to and during the final 4 h of infusion. Infusion of T decreased (P < 0.01) mean LH and LH pulse frequency. T did not significantly affect (P > 0.10) silver grain area per LHRH neuron or LHRH cell numbers. T reduced (P < 0.01) silver grain area per POMC neuron without affecting (P > 0.10) POMC cell number. We reject our hypothesis and conclude that reduced LHRH or heightened POMC gene expression are not mechanisms whereby T reduces pulsatile LHRH release in male sheep.

The role of neuropeptide Y (NPY) in the control of LH secretion in the ewe with respect to season, NPY receptor subtype and the site of action in the hypothalamus.

Neuropeptide Y1-36 (NPY1-36) acts through Y1 and Y2 receptors while the C-terminal NPY fragments NPY18-36 and N-acetyl[Leu28,31]pNPY24-36 act only through the Y2 receptor. We have investigated the effects of intracerebroventricular (i.c.v.) administration of NPY1-36, NPY18-36 and N-acetyl[Leu28,31]pNPY24-36 on LH secretion in the ovariectomised (OVX) ewe. These peptides were administered into a lateral ventricle (LV) or the third ventricle (3V) of OVX ewes during the non-breeding and breeding seasons. Microinjections of NPY were also made into the preoptic area (POA) during both seasons to investigate the effects of NPY at the level of the GnRH cell bodies. Tamed sheep were fitted with 19 gauge guide tubes into the LV, 3V or the septo-preoptic area (POA). Jugular venous blood samples were taken every 10 min for 3 h. Sheep were then given NPY1-36 (10 micrograms), NPY18-36 (100 micrograms) or saline vehicle into the LV; N-acetyl[Leu28,31]pNPY24-36 (100 micrograms), NPY1-36 (10 micrograms or 100 micrograms), NPY18-36 (10 micrograms or 100 micrograms) or saline vehicle into the 3V, or NPY1-36 (1 microgram, 5 micrograms, 10 micrograms) into the POA. Blood sampling continued for a further 3 h. LH was measured in plasma by radioimmunoassay. LV or 3V injection of 10 micrograms NPY1-36 caused a small but significant (P < 0.025) increase in the interval from the last pre-injection pulse of LH to the first post-injection LH pulse during the breeding season. Other LH pulse parameters were not significantly affected. NPY18-36 did not produce any significant change in LH pulsatility when injected into the LV, and neither peptide had any effect on plasma prolactin or GH levels. There was a significant (P < 0.01) reduction in LH pulse frequency after 3V injection of 10 micrograms and 100 micrograms NPY and 100 micrograms NPY18-36. Pulse amplitude was reduced by 3V administration of the Y2 agonist, N-acetyl[Leu28-31]pNPY24-36 and 100 micrograms NPY18-36. When the amplitude of the first post-injection LH pulse was analysed, 10 micrograms NPY also had a significant (P < 0.05) suppressive effect. During the non-breeding season, 100 micrograms NPY1-36 (but not 10 micrograms) decreased (P < 0.01) LH pulse frequency. LH pulse amplitude was significantly (P < 0.01) decreased by 100 micrograms NPY18-36. Doses of 10 micrograms NPY1-36 and 100 micrograms NPY18-36 had greater inhibitory effects on pulse frequency during the breeding season but the suppressive effect of 100 micrograms NPY was similar between seasons. Microinjections of NPY into the POA decreased (P < 0.01) average plasma LH levels during the non-breeding season at a dose of 10 micrograms but did not significantly affect pulse frequency or amplitude. We conclude that a substantial component of the inhibitory action of NPY on LH secretion in the absence of steroids is mediated by the Y2 receptor. This inhibition is probably exerted by way of a presynaptic action on GnRH terminals in the median eminence as NPY does not modulate the frequency or amplitude of LH pulses at the level of the GnRH cell bodies in the POA.

Adrenergic regulation of growth hormone secretion in the ewe.

This study examined the role of the adrenergic system in the regulation of growth hormone (GH) secretion in sheep. Intravenous infusion of noradrenaline (0.5 microgram/kg per min for 2 hr) totally suppressed plasma GH concentrations. Concomitant treatment of animals with the beta-adrenergic antagonist propranolol completely blocked the noradrenaline-induced suppression of GH. In contrast, intravenous injection of the centrally acting alpha 2-agonist clonidine (2 micrograms/kg) elicited a release of GH. To further investigate the central adrenergic regulation of GH secretion 10 micrograms of noradrenaline or adrenaline was microinjected (1 microliter) directly into the preoptic area of the hypothalamus of ovariectomized ewes. When the time of injection coincided with a GH trough period, both noradrenaline and adrenaline caused an increase in plasma GH concentrations, whereas if the injection coincided with an endogenous pulse of GH no additional GH response was obtained. In conclusion, these results provide evidence for the involvement of the adrenergic system in the regulation of GH secretion in sheep. Centrally, adrenergic pathways exert a stimulatory effect on GH release via an alpha 2-adrenergic system, whereas peripherally adrenergic pathways exert an inhibitory effect via beta-adrenergic mediated mechanisms. Furthermore, adrenergic stimulation of the preoptic area may inhibit somatostatin activity and directly facilitate a GH pulse. Alternatively, adrenergic innervation of the preoptic area may influence neurons (somatostatin or other) that project to the arcuate nucleus and stimulate the release of GH-releasing factor.