Arcuate nucleus kisspeptin response to increased nutrition in rams.

Rams respond to acute nutritional supplementation by increasing the frequency of gonadotrophin-releasing hormone (GnRH) pulses. Kisspeptin neurons may mediate the effect of environmental cues on GnRH secretion, so we tested whether the ram response to nutrition involves activation of kisspeptin neurons in the arcuate nucleus (ARC), namely kisspeptin, neurokin B, dynorphin (KNDy) neurons. Rams were given extra lupin grain with their normal ration. Blood was sampled before feeding, and continued until animals were killed for collection of brain tissue at 2 or 11h after supplementation. In supplemented rams, LH pulse frequency increased after feeding, whereas control animals showed no change. Within the caudal ARC, there were more kisspeptin neurons in supplemented rams than in controls and a higher proportion of kisspeptin cells coexpressed Fos, regardless of the time the rams were killed. There were more Fos cells in the mid-ARC and mid-dorsomedial hypothalamus of the supplemented compared with control rams. No effect of nutrition was found on kisspeptin expression in the rostral or mid-ARC, or on GnRH expression in the preoptic area. Kisspeptin neurons in the caudal ARC appear to mediate the increase in GnRH and LH production due to acute nutritional supplementation, supporting the hypothesised role of the KNDy neurons as the pulse generator for GnRH.

The localization of kisspeptin and kisspeptin receptor in the canine ovary during different stages of the reproductive cycle.

Kisspeptin is a neuropeptide involved in the hypothalamic regulation of reproduction in many species. Recent studies have revealed kisspeptin within the ovaries of rats, Siberian hamsters and humans, indicating a local role in reproduction. However, the role of kisspeptin in the ovary is poorly understood in the bitch. This study investigated the presence and location of kisspeptin protein (KISS1) and kisspeptin receptors (KISS1R) in the canine ovary during different stages of the reproductive cycle (pre-pubertal, anoestrus and cycling) by means of immunohistochemical staining. Ovaries from 24 bitches presented at local veterinary clinics for routine ovariohysterectomy were collected and grouped based on reproductive stage (pre-pubertal, anoestrus and cycling (proestrus, oestrus and dioestrus)). The presence or absence of immunoreactive KISS1 and KISS1R was recorded without any quantification of the levels of expression within cells. Immunoreactive KISS1 was found in the oocytes during all stages of the oestrous cycle, in the granulosa cells during all stages except anoestrus and in the corpus luteum (CL) during dioestrus. KISS1 was absent in the ovaries of pre-pubescent bitches. Immunoreactive KISS1R were consistently found in the oocytes, primordial follicles, the granulosa cells and CL in cycling bitches. The finding of KISS1R in the granulosa cells is suggestive that kisspeptin and progesterone may be linked as this pattern of staining is seen in animals that show preovulatory luteinisation of follicles during oestrus, KISS1R were also observed in the ovaries of pre-pubescent and anoestrous bitches, suggesting a possible role of kisspeptin in oocyte proliferation, development and maturation of granulosa cells, and progesterone production. This study provides a starting point for the establishment of a canine model for kisspeptin regulation within the ovary.

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.

Evaluation of variable new antigen receptors (vNARs) as a novel cathepsin S (CTSS) targeting strategy.

Aberrant activity of the cysteine protease Cathepsin S (CTSS) has been implicated across a wide range of pathologies. Notably in cancer, CTSS has been shown to promote tumour progression, primarily through facilitating invasion and migration of tumour cells and augmenting angiogenesis. Whilst an attractive therapeutic target, more efficacious CTSS inhibitors are required. Here, we investigated the potential application of Variable New Antigen Receptors (vNARs) as a novel inhibitory strategy. A panel of potential vNAR binders were identified following a phage display panning process against human recombinant proCTSS. These were subsequently expressed, purified and binding affinity confirmed by ELISA and SPR based approaches. Selected lead clones were taken forward and were shown to inhibit CTSS activity in recombinant enzyme activity assays. Further assessment demonstrated that our lead clones functioned by a novel inhibitory mechanism, by preventing the activation of proCTSS to the mature enzyme. Moreover, using an intrabody approach, we exhibited the ability to express these clones intracellularly and inhibit CTSS activity whilst lead clones were also noted to impede cell invasion in a tumour cell invasion assay. Collectively, these findings illustrate a novel mechanistic approach for inhibiting CTSS activity, with anti-CTSS vNAR clones possessing therapeutic potential in combating deleterious CTSS activity. Furthermore, this study exemplifies the potential of vNARs in targeting intracellular proteins, opening a range of previously "undruggable" targets for biologic-based therapy.

Lesion Explorer: a comprehensive segmentation and parcellation package to obtain regional volumetrics for subcortical hyperintensities and intracranial tissue.

Subcortical hyperintensities (SH) are a commonly observed phenomenon on MRI of the aging brain (Kertesz et al., 1988). Conflicting behavioral, cognitive and pathological associations reported in the literature underline the need to develop an intracranial volumetric analysis technique to elucidate pathophysiological origins of SH in Alzheimer's disease (AD), vascular cognitive impairment (VCI) and normal aging (De Leeuw et al., 2001; Mayer and Kier, 1991; Pantoni and Garcia, 1997; Sachdev et al., 2008). The challenge is to develop processing tools that effectively and reliably quantify subcortical small vessel disease in the context of brain tissue compartments. Segmentation and brain region parcellation should account for SH subtypes which are often classified as: periventricular (pvSH) and deep white (dwSH), incidental white matter disease or lacunar infarcts and Virchow-Robin spaces. Lesion Explorer (LE) was developed as the final component of a comprehensive volumetric segmentation and parcellation image processing stream built upon previously published methods (Dade et al., 2004; Kovacevic et al., 2002). Inter-rater and inter-method reliability was accomplished both globally and regionally. Volumetric analysis showed high inter-rater reliability both globally (ICC=.99) and regionally (ICC=.98). Pixel-wise spatial congruence was also high (SI=.97). Whole brain pvSH volumes yielded high inter-rater reliability (ICC=.99). Volumetric analysis against an alternative kNN segmentation revealed high inter-method reliability (ICC=.97). Comparison with visual rating scales showed high significant correlations (ARWMC: r=.86; CHIPS: r=.87). The pipeline yields a comprehensive and reliable individualized volumetric profile for subcortical vasculopathy that includes regionalized (26 brain regions) measures for: GM, WM, sCSF, vCSF, lacunar and non-lacunar pvSH and dwSH.

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.

Visual rating versus volumetry to detect frontotemporal dementia.

BACKGROUND/AIMS: Automated, volumetrically defined atrophy in the left anterior cingulate (LAC) and anterior temporal regions (LAT) on MRI can be used to distinguish most patients with frontotemporal dementia (FTD) from controls. FTD and Alzheimer's disease (AD) can differ in the degree of anterior temporal atrophy. We explored whether clinicians can visually detect this atrophy pattern and whether they can use it to classify the 2 groups of dementia patients with the same accuracy. METHODS: Four neurologists rated atrophy in the LAC and LAT regions on MRI slices from 21 FTD, 21 controls, and 14 AD participants. Inter-rater reliability and diagnostic accuracy were assessed. RESULTS: All 4 raters agreed on the presence of clinically significant atrophy, and their atrophy scoring correlated with the volumes, but without translation into high inter-rater diagnostic agreement. CONCLUSIONS: Volumetric analyses are difficult to translate into routine clinical practice.

Diffusion tensor imaging abnormalities in depressed multiple sclerosis patients.

Depression is common in patients with multiple sclerosis, but to date no studies have explored diffusion tensor imaging indices associated with mood change. This study aimed to determine cerebral correlates of depression in multiple sclerosis patients using diffusion tensor imaging. Sixty-two subjects with multiple sclerosis were assessed for depression with the Beck Depression Inventory (BDI-II). All subjects underwent magnetic resonance imaging. Whole brain and regional volumes were calculated for lesions (hyper/hypointense) and normal-appearing white and grey matter. Fractional anisotropy and mean diffusivity were calculated for each brain region. Magnetic resonance imaging comparisons were undertaken between depressed (Beck Depression Inventory > or = 19) and non-depressed subjects. Depressed subjects (n = 30) had a higher hypointense lesion volume in the right medial inferior frontal region, a smaller normal-appearing white matter volume in the left superior frontal region, and lower fractional anisotropy and higher mean diffusivity in the left anterior temporal normal-appearing white matter and normal-appearing grey matter regions, respectively. Depressed subjects also had higher mean diffusivity in right inferior frontal hyperintense lesions. Magnetic resonance imaging variables contributed to 43% of the depression variance. We conclude that the presence of more marked diffusion tensor imaging abnormalities in the normal-appearing white matter and normal-appearing grey matter of depressed subjects highlights the importance of more subtle measures of structural brain change in the pathogenesis of depression.

Sexual health of adults working in pornographic films.

We report the frequency of sexually transmitted infections (STIs) diagnosed in performers in the adult pornographic film industry. Over a 13 month period, 445 STI screens were performed in 115 patients, 56 women and 59 men. All reported unprotected sex during filming. Seventy-five percent (86) had at least one sexual partner outside work, and 90% used condoms inconsistently with them. Women worked exclusively with women (23%), men only (38%) or both genders (39%). Almost all men (97%) worked exclusively heterosexually. Thirty-eight percent (44/115) were diagnosed with 77 STIs, including non-specific urethritis (51), gonorrhoea (10), chlamydia (6) and genital warts (6). Gonorrhoea was found exclusively at the pharynx in three heterosexual men. There were no cases of HIV, syphilis, hepatitis B or hepatitis C. Monthly screening and certification is a working requirement for this population but STIs are common in an industry where unprotected sex is the norm.

Multisubstrate adduct inhibitors: drug design and biological tools.

In drug discovery, different methods exist to create new inhibitors possessing satisfactory biological activity. The multisubstrate adduct inhibitor (MAI) approach is one of these methods, which consists of a covalent combination between analogs of the substrate and the cofactor or of the multiple substrates used by the target enzyme. Adopted as the first line of investigation for many enzymes, this method has brought insights into the enzymatic mechanism, structure, and inhibitory requirements. In this review, the MAI approach, applied to different classes of enzyme, is reported from the point of view of biological activity.

Alum Activates the Bovine NLRP3 Inflammasome.

There has been a move away from vaccines composed of whole or inactivated antigens toward subunit-based vaccines, which although safe, provide less immunological protection. As a result, the use of adjuvants to enhance and direct adaptive immune responses has become the focus of much targeted bovine vaccine research. However, the mechanisms by which adjuvants work to enhance immunological protection in many cases remains unclear, although this knowledge is critical to the rational design of effective next generation vaccines. This study aimed to investigate the mechanisms by which alum, a commonly used adjuvant in bovine vaccines, enhances IL-1ß secretion in bovine peripheral blood mononuclear cells (PBMCs). Unlike the case with human PBMCs, alum promoted IL-1ß secretion in a subset of bovine PBMCs without priming with a toll-like receptor agonist. This suggests that PBMCs from some cattle are primed to produce this potent inflammatory cytokine and western blotting confirmed the presence of preexisting pro-IL-1ß in PBMCs from a subset of 8-month-old cattle. To address the mechanism underlying alum-induced IL-1ß secretion, specific inhibitors identified that alum mediates lysosomal disruption which subsequently activates the assembly of an NLRP3, ASC, caspase-1, and potentially caspase-8 containing complex. These components form an inflammasome, which mediates alum-induced IL-1ß secretion in bovine PBMCs. Given the demonstrated role of the NLRP3 inflammasome in regulating adaptive immunity in murine systems, these results will inform further targeted research into the potential of inflammasome activation for rational vaccine design in cattle.

Atmospheric changes from solar eclipses.

This article reviews atmospheric changes associated with 44 solar eclipses, beginning with the first quantitative results available, from 1834 (earlier qualitative accounts also exist). Eclipse meteorology attracted relatively few publications until the total solar eclipse of 16 February 1980, with the 11 August 1999 eclipse producing the most papers. Eclipses passing over populated areas such as Europe, China and India now regularly attract scientific attention, whereas atmospheric measurements of eclipses at remote locations remain rare. Many measurements and models have been used to exploit the uniquely predictable solar forcing provided by an eclipse. In this paper, we compile the available publications and review a subset of them chosen on the basis of importance and novelty. Beyond the obvious reduction in incoming solar radiation, atmospheric cooling from eclipses can induce dynamical changes. Observations and meteorological modelling provide evidence for the generation of a local eclipse circulation that may be the origin of the 'eclipse wind'. Gravity waves set up by the eclipse can, in principle, be detected as atmospheric pressure fluctuations, though theoretical predictions are limited, and many of the data are inconclusive. Eclipse events providing important early insights into the ionization of the upper atmosphere are also briefly reviewed.This article is part of the themed issue 'Atmospheric effects of solar eclipses stimulated by the 2015 UK eclipse'.

Using the ionospheric response to the solar eclipse on 20 March 2015 to detect spatial structure in the solar corona.

The total solar eclipse that occurred over the Arctic region on 20 March 2015 was seen as a partial eclipse over much of Europe. Observations of this eclipse were used to investigate the high time resolution (1 min) decay and recovery of the Earth's ionospheric E-region above the ionospheric monitoring station in Chilton, UK. At the altitude of this region (100 km), the maximum phase of the eclipse was 88.88% obscuration of the photosphere occurring at 9:29:41.5 UT. In comparison, the ionospheric response revealed a maximum obscuration of 66% (leaving a fraction, Φ, of uneclipsed radiation of 34±4%) occurring at 9:29 UT. The eclipse was re-created using data from the Solar Dynamics Observatory to estimate the fraction of radiation incident on the Earth's atmosphere throughout the eclipse from nine different emission wavelengths in the extreme ultraviolet (EUV) and X-ray spectrum. These emissions, having varying spatial distributions, were each obscured differently during the eclipse. Those wavelengths associated with coronal emissions (94, 211 and 335 Å) most closely reproduced the time varying fraction of unobscured radiation observed in the ionosphere. These results could enable historic ionospheric eclipse measurements to be interpreted in terms of the distribution of EUV and X-ray emissions on the solar disc.This article is part of the themed issue 'Atmospheric effects of solar eclipses stimulated by the 2015 UK eclipse'.

Functional characterisation of a TLR accessory protein, UNC93B1, in Atlantic salmon (Salmo salar).

Toll-like receptors (TLRs) are indispensable components of the innate immune system, which recognise conserved pathogen associated molecular patterns (PAMPs) and induce a series of defensive immune responses to protect the host. Biosynthesis, localisation and activation of TLRs are dependent on TLR accessory proteins. In this study, we identified the accessory protein, UNC93B1, from Atlantic salmon (Salmo salar) whole-genome shotgun (WGS) contigs aided by the conserved gene synteny of genes flanking UNC93B1 in fish, birds and mammals. Phylogenetic analysis showed that salmon UNC93B1 grouped with other vertebrate UNC93B1 molecules, and had highest amino acid identity and similarity to zebrafish UNC93B1. The salmon UNC93B1 gene organisation was also similar in structure to mammalian UNC93B1. Our gene expression studies revealed that salmon UNC93B1 was more highly expressed in spleen, liver and gill tissues but was expressed at a lower level in head kidney tissue in post-smolts relative to parr. Moreover, salmon UNC93B1 mRNA transcripts were up-regulated in vivo in spleen tissue from polyI:C treated salmon and in vitro in polyI:C or IFNγ stimulated Salmon Head Kidney-1 (SHK-1) cells. Initial studies into the functional role of salmon UNC93B1 in fish TLR signalling found that both wild type salmon UNC93B1 and a molecule with a site-directed mutation (H424R) co-immunoprecipitated with salmon TLR19, TLR20a and TLR20d. Overall, these data illustrate the potential importance of UNC93B1 as an accessory protein in fish TLR signalling.

Inhibition of luteinizing hormone secretion in ovariectomized ewes during the breeding season by gamma-aminobutyric acid (GABA) is mediated by GABA-A receptors, but not GABA-B receptors.

We have investigated the effects of agonists and antagonists to gamma-aminobutyric acid-A (GABA-A) and GABA-B receptors microinjected (1 microliters) into the preoptic area (POA) on LH in ovariectomized (OVX) ewes with or without estrogen (E) treatment during the breeding season. Guide tubes were placed into the preoptic area of OVX ewes using lateral ventriculograms for localization of the target area. Doses of 10 micrograms of the GABA agonists muscimol (GABA-A) and baclofen (GABA-B) or the GABA antagonists bicuculline (GABA-A) and phaclofen (GABA-B) were injected into the POA of tame conscious animals. Jugular venous blood was collected at 10-min intervals for 3 h, the injection of GABA drug or vehicle was given, and samples were collected for a further 3 h. The plasma samples were assayed for LH. On completion of the experiments, the brains were sectioned to locate the site of injection. In the first year, 17 ewes were used, of which 16 had correct guide tube placement. In OVX sheep, both muscimol and bicuculline injections caused suppression of plasma LH concentrations, with a cessation of pulses in many instances. When OVX sheep were treated with 1.0-cm Silastic implants (sc) of E for at least 7 days, there was a decrease in LH interpulse interval. In these sheep, muscimol and bicuculline injections had effects on plasma LH concentrations similar to those in OVX sheep. In addition, bicuculline was injected into the POA of OVX ewes treated with 2.0-cm E implants. Despite E treatment resulting in reduced plasma LH levels, due to an increase in the interpulse interval, bicuculline injection further suppressed plasma LH levels. Neither baclofen nor phaclofen injection had any effect on plasma LH secretion in either OVX ewes or OVX ewes given 1.0-cm E implants. In the second year, eight sheep were used, all of which had correct guide tube placement. These sheep were treated with 2.0-cm E implants and injected with phaclofen. Phaclofen had no effect on LH secretion. These results suggest that GnRH secretion is regulated by GA-BAergic neurons at the level of the GnRH cell bodies in the POA and that during the breeding season, this is effected by GABA-A and not GABA-B receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence that changes in the function of the subtypes of the receptors for gamma-amino butyric acid may be involved in the seasonal changes in the negative-feedback effects of estrogen on gonadotropin-releasing hormone secretion and plasma luteinizing hormone levels in the ewe.

We have investigated the effects of gamma-amino butyric acid (GABA) agonists and antagonists to GABAA and GABAB receptors microinjected (1 microliter) into the medial preoptic area (MPOA) on LH secretion in ovariectomized (OVX) ewes with or without estrogen (E) treatment, during the nonbreeding season. Guide tubes (19 gauge) were placed into the MPOA of OVX ewes and injections of 1 microgram or 10 micrograms of the GABAA agonist muscimol or the GABAA antagonist bicuculline, and 10 micrograms of the GABAB agonist baclofen or the GABAB antagonist phaclofen were made into conscious animals. Jugular venous blood was collected at 10-min intervals for 3 h, the injection of GABA drug or vehicle was given and samples collected for a further 3 h. The plasma samples were assayed for LH. On completion of the experiments the brains were sectioned to locate the sites of injection. Thirty-four ewes were used, of which 30 had correct guide tube placement. In OVX sheep, both muscimol and bicuculline injection caused suppression of plasma LH concentrations, with a cessation of pulsatile release in many instances. Injection with baclofen or phaclofen into these sheep had no effect on LH secretion. When OVX sheep were treated with 0.5-cm Silastic implants (sc) of E for at least 7 days, there was a variable response to muscimol and bicuculline injection, depending on the degree of suppression of LH secretion by E. When E had little effect on plasma LH levels, muscimol and bicuculline both suppressed LH secretion in a similar manner to that observed in OVX ewes. When E treatment fully suppressed plasma LH levels, muscimol and bicuculline both stimulated LH secretion. This stimulation often took the form of a sustained, nonpulsatile secretion of LH. Baclofen injection into OVX ewes treated with E increased mean plasma LH levels through an increase in pulse amplitude, although there was only an effect on the amplitude of the first pulse after injection. Conversely, phaclofen injection in OVX ewes treated with E resulted in a reduction in LH pulse amplitude. These results provide evidence for a seasonal shift in the regulation of GnRH secretion by GABAergic neurons in the MPOA of the ewe. In contrast to the breeding season, where there was no effect of GABAB ligands, these appear to function in non-breeding season, this may be part of the mechanism for the seasonal shift in the negative-feedback effect of E on LH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Differential regulation of luteinizing hormone release by gamma-aminobutyric acid receptor subtypes in the arcuate-ventromedial region of the castrated ram.

We investigated the effects on LH secretion of infusing gamma-aminobutyric acid (GABA) agonists muscimol and baclofen (GABAA and GABAB receptor agonists, respectively) into either the medial preoptic area (mPOA) or the arcuate-ventromedial region (ARC-VMR) of the hypothalamus of castrated rams during the nonbreeding season. Bilateral microdialysis of artificial cerebrospinal fluid for 4 h followed by treatment with artificial cerebrospinal fluid, baclofen (1 mM), or muscimol (1 mM in the ARC-VMR, 250 microM in the mPOA) for 4 h was carried out on three separate occasions in random order. Simultaneously, jugular venous blood was collected at 10-min intervals, and plasma later was assayed for LH. The estimated dose of baclofen delivered to each unilateral microdialysis site was 7.9 micrograms; for muscimol, it was 1.1 micrograms for the mPOA and 4.5 micrograms for the ARC-VMR. In the mPOA, baclofen had no detectable effect, whereas muscimol had a delayed suppressive effect on mean LH (P < 0.01). In the ARC-VMR muscimol rapidly reduced mean LH (P < 0.01). In contrast, baclofen increased mean LH (P = 0.01) and LH pulse amplitude (P = 0.05) without altering the LH interpulse interval (P > 0.10). These results support the involvement of GABAA receptors in the mPOA in regulating LH secretory patterns. More importantly, both GABAA and GABA(B) receptors in the ARC-VMR appear to differentially modulate LH and, presumably, GnRH release. Whether GABA acts directly on the GnRH neuron or indirectly through other neural systems remains to be determined.

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.

Neonatal immunization against gonadotropin-releasing hormone (GnRH) results in diminished GnRH secretion in adulthood.

The effects of neonatal immunization against GnRH were studied in sheep after they had reached adulthood (3-4 yr) and the antibody titers had fallen to undetectable levels. The immunized animals had small gonads, and the females did not have large follicles (>3 mm) or corpora lutea in their ovaries. Compared with controls, the immunized animals had low or nondetectable levels of LH and FSH in peripheral plasma, and the immunized animals generally failed to respond to a single i.v. GnRH challenge. After ovariectomy, the control ewes, but not the immunized ewes, showed an elevation in plasma LH and FSH levels. The sampling of hypophysial portal blood, with a newly described method, showed that the secretion of GnRH was reduced in the immunized animals, but the amount of GnRH in the median eminence was similar in the control and immunized ewes. The pituitary content of LH and FSH was reduced in the immunized ewes as was messenger RNA for the gonadotropin subunits and the GnRH receptor. These data indicate that neonatal immunization does not affect the synthesis of GnRH in adulthood but reduces the secretion of GnRH, causing long-term sterility in these animals.