Calcitonin impairs the anabolic effect of PTH in young rats and stimulates expression of sclerostin by osteocytes.

The therapeutic goal of increasing bone mass by co-treatment of parathyroid hormone (PTH) and an osteoclast inhibitor has been complicated by the undefined contribution of osteoclasts to the anabolic activity of PTH. To determine whether active osteoclasts are required at the time of PTH administration, we administered a low dose of the transient osteoclast inhibitor salmon calcitonin (sCT) to young rats receiving an anabolic PTH regimen. Co-administration of sCT significantly blunted the anabolic effect of PTH as measured by peripheral quantitative computer tomography (pQCT) and histomorphometry in the femur and tibia, respectively. To determine gene targets of sCT, we carried out quantitative real time PCR and microarray analysis of metaphyseal samples 1.5, 4 and 6.5h after administration of a single injection of PTH, sCT or PTH+sCT. Known targets of PTH action, IL-6, ephrinB2 and RANKL, were not modified by co-administration with sCT. Surprisingly, at all time points, we noted a significant upregulation of sclerostin mRNA by sCT treatment, as well as down-regulation of two other osteocyte gene products, MEPE and DMP1. Immunohistochemistry confirmed that sCT administration increased the percentage of osteocytes expressing sclerostin, suggesting a mechanism by which sCT reduced the anabolic effect of PTH. Neither mRNA for CT receptor (Calcr) nor labeled CT binding could be detected in sclerostin-enriched cells differentiated from primary calvarial osteoblasts. In contrast, osteocytes freshly isolated from calvariae expressed a high level of Calcr mRNA. Furthermore immunohistochemistry revealed co-localization of CT receptor (CTR) and sclerostin in some osteocytes in calvarial sections. Taken together these data indicate that co-treatment with sCT can blunt the anabolic effect of PTH and this may involve direct stimulation of sclerostin production by osteocytes. These data directly implicate calcitonin as a negative regulator of bone formation through a previously unsuspected mechanism.

Evaluation of hot saline solution and restriction endonuclease techniques in cytogenetic studies of Cycloneda sanguinea L. (Coccinellidae).

The goal of the present study was to determine if simple methods, especially hot saline solution (HSS) and MspI and HaeIII restriction endonucleases, which do not require special equipments, may be helpful in studies of genetic variability in the lady beetle, Cycloneda sanguinea. The HSS method extracted the heterochromatin region, suggesting that it is composed mostly of DNA rich in A-T base pairs. However, the X and y chromosomes were resistant to HSS banding. These bands facilitated the identification of each chromosome. In this study, we used the restriction endonucleases with different G-C base target sequences: MspI C/GGC and HaeIII GG/CC. The use of restriction enzyme MspI did not show an effect on the autosomal chromosomes. On the other hand, the sex pair showed a pale staining, to help in the recognition of these chromosomes. HaeIII produced characteristic bands which were identified all along the chromosomes, facilitating the identification of each chromosome. Based on these results, we can consider the heterochromatin being heterogeneous. The findings obtained here, using different chromosomal banding techniques, may be useful in the identification of intraspecific chomosome variability, specifically in Coccinellidae (Coleoptera) chromosomes, even without special equipment.

Isolation and restraint stress results in differential activation of corticotrophin-releasing hormone and arginine vasopressin neurons in sheep.

This study investigated sex differences in the stress-induced activation of neurons containing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin in the paraventricular nucleus (PVN) of gonadectomized male and female sheep. Groups (n=3) of both sexes were either subjected to 90 min isolation and restraint stress (stress group) or were not stressed. Blood samples were taken every 10 min for 90 min prior to and after stress to monitor cortisol levels in plasma. Brains were harvested after 90 min of stress. Stress caused elevation of plasma cortisol levels to a similar extent in both sexes. Double-labeling immunohistochemistry for Fos and either CRH, AVP or enkephalin was undertaken to quantify the numbers of neurons staining for CRH, AVP and enkephalin that also immunostained for Fos. Stress increased Fos immunostaining in all cell types. There was a greater proportion of CRH than AVP neurons activated in stressed animals. There were no sex differences in the activation of CRH and AVP neurons although females had a greater proportion of enkephalin cells staining for Fos than males in both control and stressed animals. There were no differences between control and stressed animals in the proportion of cells co-staining for CRH and AVP. We conclude that isolation and restraint stress activates neurons producing CRH, AVP and enkephalin in sheep and that CRH may play a greater role than AVP in regulating adrenocorticotrophic hormone secretion in response to this stressor in sheep. Finally, isolation and restraint stress does not influence co-localization of CRH and AVP in sheep.

Evaluation of hot saline solution and restriction endonuclease techniques in cytogenetic studies of Cycloneda sanguinea L. (Coccinellidae)

The goal of the present study was to determine if simple methods, especially hot saline solution (HSS) and MspI and HaeIII restriction endonucleases, which do not require special equipments, may be helpful in studies of genetic variability in the lady beetle, Cycloneda sanguinea. The HSS method extracted the heterochromatin region, suggesting that it is composed mostly of DNA rich in A-T base pairs. However, the X and y chromosomes were resistant to HSS banding. These bands facilitated the identification of each chromosome. In this study, we used the restriction endonucleases with different G-C base target sequences: MspI C/GGC and HaeIII GG/CC. The use of restriction enzyme MspI did not show an effect on the autosomal chromosomes. On the other hand, the sex pair showed a pale staining, to help in the recognition of these chromosomes. HaeIII produced characteristic bands which were identified all along the chromosomes, facilitating the identification of each chromosome. Based on these results, we can consider the heterochromatin being heterogeneous. The findings obtained here, using different chromosomal banding techniques, may be useful in the identification of intraspecific chomosome variability, specifically in Coccinellidae (Coleoptera) chromosomes, even without special equipment.

Elevated KiSS-1 expression in the arcuate nucleus prior to the cyclic preovulatory gonadotrophin-releasing hormone/lutenising hormone surge in the ewe suggests a stimulatory role for kisspeptin in oestrogen-positive feedback.

Kisspeptins are encoded by the gene KiSS-1 and regulate gonadotrophin-releasing hormone (GnRH) and gonadotrophin secretion in various species, including humans. Here, we quantify gene expression of KiSS-1 in the arcuate nucleus (ARC) across the ovine oestrous cycle and demonstrate an increase in the caudal division of the ARC during the preovulatory period. These data strongly suggest that kisspeptins are involved in the generation of the preovulatory GnRH and luteinising hormone surge.

Colocalization of kisspeptin and gonadotropin-releasing hormone in the ovine brain.

Kisspeptin is a peptide that has been implicated in the regulation of GnRH cells in the brain. Immunohistochemical studies were undertaken to examine the distribution of kisspeptin-immunoreactive (IR) cells in the ovine diencephalon and determine the effect of ovariectomy in the ewe. We report that kisspeptin colocalizes to a high proportion of GnRH-IR cells in the preoptic area, which is a novel finding. A high level of colocalization of kisspeptin and GnRH was also seen in varicose neuronal fibers within the external, neurosecretory zone of the median eminence. Apart from the kisspeptin/GnRH cells, a population of single-labeling kisspeptin-IR cells was also observed in the preoptic area. Within the hypothalamus, kisspeptin-IR cells were found predominantly in the arcuate nucleus, and there was an increase in the number of immunohistochemically identified cell within this nucleus after ovariectomy. Kisspeptin-IR cells were also found in the periventricular nucleus of the hypothalamus, but the number observed was similar in gonad-intact and ovariectomized ewes. The colocalization of GnRH and kisspeptin within cells of the preoptic area and GnRH neurosecretory terminals of the median eminence suggests that the two peptides might be cosecreted into the hypophyseal portal blood to act on the pituitary gland. Effects of ovariectomy on the non-GnRH, Kisspeptin-IR cells of the hypothalamus suggest that kisspeptin production is negatively regulated by ovarian steroids.

Evidence that projections from the bed nucleus of the stria terminalis and from the lateral and medial regions of the preoptic area provide input to gonadotropin releasing hormone (GNRH) neurons in the female sheep brain.

The arcuate nucleus/ventromedial hypothalamic nucleus (ARC/VMH) region is thought to relay estrogen feedback signals to gonadotropin-releasing hormone (GnRH) cells in the sheep brain. This region sends major projections to the lateral preoptic area (lPOA), ventral bed nucleus of the stria terminals (vBnST) and the ventro-caudal division of the median preoptic nucleus (vcMePON) with little direct input to GnRH cell bodies, suggesting interneuronal relay to GnRH neurons. The brain stem also provides input to the POA. The present study aimed to identify possible relay circuits in the POA and BnST to GnRH neurons. Biotinylated dextran amine (BDA) was injected into lPOA (n=6), vBnST (n=2), vcMePON (n=3) and periventricular nucleus (PeriV; n=1) of ewes for anterograde tracing. GnRH immunoreactive (IR) perikarya appearing to receive input from BDA-containing varicosities were identified by fluorescence microscopy, with further analysis by confocal microscopy. When BDA was injected into rostral and caudal regions of lPOA (n=3), no tracer-filled varicose fibers were found in contact with GnRH-IR perikarya. Injections into the center of the lPOA (n=3) indicated direct projections to GnRH-IR cells. Injections into the vBnST, vcMePON and PeriV indicated that cells of these regions also provide input to GnRH cells. BDA-containing varicosities found in the MPOA were immunoreactive for NPY or were GABAergic or glutamatergic when the tracer was injected into vBnST and lPOA, but not when injections were placed in the vcMePON. With injection into the PeriV, tracer-filled varicosities in the MPOA were not immunoreactive for somatostatin or enkephalin. Injection of FluoroGold into ventral POA retrogradely labeled cells in the above mentioned areas, but few were also immunoreactive for estrogen receptor-alpha. Thus, cells of the vBnST, lPOA, vcMePON and PeriV project to GnRH neurons. These cells may provide an interneuronal route to GnRH neurons from the ARC/VMH, the brain stem and other regions of the brain.

A RAPD marker associated with B chromosomes in Partamona helleri (Hymenoptera, Apidae).

The hymenopteran Partamona helleri is found in southwestern Brazil in the Mata Atlântica from the north of the state of Santa Catarina until the south of Bahia. This work shows that P. helleri can carry up to four B chromosomes per individual. In order to obtain more information about P. helleri B chromosomes, the RAPD technique was used to detect DNA fragments associated with these chromosomes. The results showed that the RAPD technique is useful to detect specific sequences associated with B chromosomes. One RAPD marker was identified, cloned and used as probe in a DNA blot analysis. This RAPD marker hybridized with sequences present only in individuals containing B chromosomes.

Imitate to integrate: reviewing the pathway for B chromosome integration in Trypoxylon (Trypargilum) albitarse (Hymenoptera, Sphecidae).

B chromosomes are genomic "intruders" normally characterized by their total dispensability counteracted by a variety of drive mechanisms, which assures their presence regardless of their harmful effects on the host genome. From an evolutionary standpoint, the relationship between standard (A) and B chromosomes can go through different pathways, from an everlasting arms race to a cordial B integration. Examples underlying the first situation are fairly common; B integration, however, has been more a theoretical than a practical possibility. The B chromosome in the haplodiploid solitary wasp Trypoxylon albitarse is probably the first example of a "mimetic" B, which is being integrated into the A genome by limiting itself to one B per haploid genome, the same dosage as the A chromosomes. Here we review some of the findings underlying this hypothesis and discuss the T. albitarse B strategy as a possible mechanism for B chromosome integration as a regular member of the chromosome complement in haplodiploid organisms.

Dinoponera lucida Emery (Formicidae: Ponerinae): the highest number of chromosomes known in Hymenoptera.

We report the remarkable karyotype of Dinoponera lucida, a Brazilian endemic ponerine ant. Its chromosome number is 2n=106, most of the chromosomes are acrocentric and of very small size, and the karyotype formula is 88A+18M. A chromosome pair of the AM(t) type is reported. This is the largest number of chromosomes reported for the Hymenoptera order until now.

Neuropeptide Y (NPY) delays the oestrogen-induced luteinizing hormone (LH) surge in the ovariectomized ewe: further evidence that NPY has a predominant negative effect on LH secretion in the ewe.

Studies in rats suggest that neuropeptide Y (NPY) plays a stimulatory role in the generation of the preovulatory luteinizing hormone (LH) surge, via the Y1 receptor. We have investigated this issue using the oestradiol benzoate (EB)-treated ovariectomized (OVX) ewe which is a model for the preovulatory LH surge. A Y1 receptor antagonist (BIBO3304) was infused (25 microg/h) into the third cerebral ventricle (III-V) from 2 h before EB injection for 24 h, and had no effect on the ensuing LH surge. Using in situ hybridization, we then examined expression of NPY mRNA in the arcuate nucleus during the luteal, follicular and oestrous phases of the oestrous cycle, and found that levels were greatest during the luteal phase. Thus, reduced NPY synthesis might be an integral factor in the events leading to the cyclic preovulatory LH surge. This was tested by infusion of NPY (25 microg/h) into the III-V (as above). The NPY infusion delayed the LH surge until the infusion was ceased. High levels of NPY expression during the luteal phase of the oestrous cycle may be caused by progesterone. Thus, we determined whether NPY cells possess progesterone receptors (PR) and whether progesterone treatment up-regulates NPY mRNA expression in the arcuate nucleus. Immunohistochemistry for NPY and PR was performed in OVX, oestrogen-treated ewes, but no NPY cells of the arcuate nucleus were seen to colocalize PR. In situ hybridization for NPY was performed in OVX and OVX ewes treated with progesterone. There was no significant effect of progesterone treatment on NPY mRNA expression in the arcuate nucleus. We conclude that chronically elevated levels of NPY block the preovulatory surge of gonadotropin-releasing hormone/LH secretion in sheep, but high levels of NPY mRNA expression in the luteal phase of the oestrous cycle cannot be explained by an action of progesterone.

Longitudinal differentiation in Melipona mandacaia (Hymenoptera, Meliponini) chromosomes.

Melipona mandacaia is a stingless bee endemic to northeast Brasil. We describe the M. mandacaia karyotype using C-banding technique. fluorochrome staining and treatment with restriction enzymes and discuss the position of this species in the context of the phylogeny of the genus. Melipona mandacaia has 2n = 18 (14 SM + 2 M + 2 A). Heterochromatin was detected in the pericentromeric region of pairs 1, 2 and 8 and in the form of small blocks in the remaining pairs. Staining with base-specific fluorochromes showed that this heterochromatin was rich AT (QM and DAPI), except in the region corresponding to the NOR which was rich GC (CMA3) and was cleaved by the HaeIII enzyme. Melipona mandacaia is a member of Group I Melipona. Treatment with DraI/Giemsa discloses a larger number of bands than treatment with DraI/QM. Pre-cleavage with DraI gave rise to a larger number of bands following QM staining; a circumstance evidently due to a removal of the DNA-protein complex that prevented the association of the fluorochrome with AT-rich DNA. The results highlight the complex nature of heterochromatin.

Seasonal changes in the inputs to gonadotropin-releasing hormone neurones in the ewe brain: an assessment by conventional fluorescence and confocal microscopy.

The seasonal pattern of breeding in sheep offers an opportunity to examine plasticity of neuronal inputs to gonadotropin-releasing hormone (GnRH) neurones. We used conventional fluorescence microscopy and confocal microscopy to compare the extent of input to GnRH neurones from various neuropeptide/neurotransmitter systems in ewes during the breeding and anestrous seasons. Using double-labelling immunohistochemistry, we counted close appositions between GnRH cells and varicosities that were immunoreactive for either glutamic acid decarboxylase (GAD; for gamma-amino butyric acid-GABA-neurones), dopamine beta hydroxylase (DBH; for noradrenergic neurones), vesicular glutamate transporter-1 (VGluT-1, for glutamatergic neurones), neuropeptide Y (NPY) and tyrosine hydroxylase (TH; for dopaminergic/noradrenergic neurones). The percentage of GnRH cells displaying close appositions to GABA-ergic varicosities was higher (P < 0.02) in anestrus than in the breeding season. The percentage of GnRH cells receiving input from varicosities that were positive for TH, DBH and VGluT-1 was similar in both seasons. Approximately 26-49% of GnRH neurones were seen to receive inputs from NPY, TH, GABAergic or noradrenergic neurones, while a larger number of GnRH cells (72-75%) received input from glutamatergic neurones. Conventional microscopy consistently overestimated the number of close contacts on GnRH neurones compared to confocal microscopy. For TH-immunoreactive varicosities in the preoptic area, only 16-35% were also immunoreactive for DBH, suggesting that the remainder are dopaminergic. Approximately half of the noradrenergic inputs in the preoptic area were also immunoreactive for NPY. In conclusion, we present numerical data on the consensus between light and confocal microscopy and the level of input of various neuronal systems to GnRH cells; the data indicate a seasonal change in the GABAergic input to GnRH neurones.

Long-term alteration in bodyweight and food restriction does not affect the gene expression of either preproorexin or prodynorphin in the sheep.

Various hypothalamic neuropeptides are involved in central regulation of food intake and expression of genes encoding these peptides changes with alterations in the bodyweight/metabolic status/nutritional status. Orexin(s) and dynorphin have been implicated in the regulation of appetite and neuroendocrine systems, but the function of these peptides is not well understood. We have employed in situ hybridization to examine the effects of long-term alterations in the bodyweight on expression of mRNA for preproorexin and prodynorphin in the putative feeding centers of the ovine hypothalamus. Expression of preproorexin was localized to the dorsomedial hypothalamic nucleus, perifornical area and lateral hypothalamic area. Cells expressing prodynorphin were localized to the periventricular, supraoptic, paraventricular, ventromedial hypothalamic nuclei and the thalamus. Small numbers of single scattered cells were seen in other brain areas. A few scattered prodynorphin-expressing cells were found in the lateral hypothalamic area but, in contrast to observations in the rat, there was no colocalization with preproorexin. Long-term alterations in the bodyweight did not influence the level of expression of preproorexin or prodynorphin. These findings suggest that orexin and dynorphin may not play a direct role in appetite regulation in sheep, although regulation at the level of the receptors for these peptides remains a possibility.

Genetic load caused by variation in the amount of rDNA in a wasp.

Extensive variation in the size of the short (heterochromatic) arm of chromosome 14 was found in the wasp Trypoxylon (Trypargilum) albitarse. Ten different variants were differentiated by size and C-banding pattern. Fluorescent in-situ hybridization (FISH) revealed that ribosomal DNA in this species is clustered in the darkly C-banded parts of the heterochromatic short arm of chromosome 14. On this basis, we got an indirect estimate of the amount of rDNA from the area of these dark C-bands. The significant absence in males of the three chromosome variants with lower amounts of rDNA indicates that these three variants are lethal in this sex, and suggests the existence of a threshold marking the minimum amount of rDNA which is tolerable in haploidy. This implies about 4% genetic load in the population caused by variation in rDNA amount.

Integration of a B chromosome into the A genome of a wasp, revisited.

A previous study showed that in the haplodiploid solitary wasp Trypoxylon albitarse, most individuals carry one B chromosome per haploid genome, the same dosage as the standard (A) chromosomes, indicating a possible regularization of B-chromosome meiotic behaviour and its integration into the A genome. In a new sampling, we have analysed 15 populations (including 9 out of the 10 previously analysed) to test the evolution of this integration process. The new results provide a direct report of the invasion process in the Porto Firme population, where B frequency has dramatically increased in only four generations. In the populations from the Viçosa region, however, B frequency has remained stable, although the principal B type, the metacentric one, has increased in frequency at the expense of the acrocentric one in several populations. The implications of these new results on the hypothesis of the integration of these B chromosomes, as regular members of the A genome, are discussed.

Cells of the arcuate nucleus and ventromedial nucleus of the ovariectomized ewe that respond to oestrogen: a study using Fos immunohistochemistry.

Oestrogen produces a positive feedback effect on the secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) when implanted into the ventromedial/arcuate nucleus of the ovariectomized (OVX) ewe. This has led to the belief that it is in this area of the hypothalamus that oestrogen causes the preovulatory surge in GnRH/LH. To date, however, the cell types that are integral to this response have not been identified. The present study aimed to examine cellular responsiveness to oestrogen in this region of the brain using Fos immunohistochemistry and further aimed to determine the cell type that shows an acute response to oestrogen. OVX ewes (n = 4-6 per group) were given i.m. injections of oestradiol benzoate or oil (vehicle) and were killed 1-6 h later. Brains were perfused for immunohistochemistry. The number of cells in the arcuate nucleus which were immunopositive for Fos was greater (two- to fourfold) in the oestradiol benzoate-treated OVX ewes (n = 5) 1 h after injection. The number of Fos-positive cells in the ventromedial hypothalamic nucleus was 10-fold greater in the oestradiol benzoate-treated ewes 1 h after injection. Because there were high levels of Fos-immunoreactive cells in oil-treated ewes, we repeated the experiment with i.v. injection of 50 microg oestrogen or vehicle (n = 5). With this latter procedure, we found that oestrogen injection caused a significant increase in the number of Fos immunoreactive cells in the arcuate nucleus within 1 h, but there was no response in the ventromedial hypothalamus. To further characterize the types of cells that might respond to oestrogen, we double-labelled cells for Fos and either adrenocorticotropin hormone, neuropeptide Y or tyrosine hydroxylase (a marker for dopaminergic cells). These cell types could account for less than 30% of the total number of cells that were Fos-positive and oestrogen treatment did not cause an increase in the Fos labelling of any of these types of cell. These data show that oestrogen activates cells of the arcuate/ventromedial hypothalamus within 1 h of injection and that this response could relate to the feedback effects of this gonadal hormone. The majority of cells that produce Fos following oestrogen injection are of unknown phenotype. The data further suggest that induction of cells of the ventromedial hypothalamic nucleus require more prolonged oestrogen stimulus than cells of the arcuate nucleus.

Evidence that orexin-containing neurones provide direct input to gonadotropin-releasing hormone neurones in the ovine hypothalamus.

Orexins A and B (ORX) have been added recently to the growing list of neuropeptides implicated in feeding and drinking behaviour as well as neuroendocrine function. In the present study, we have used single and dual labelling immunohistochemistry and a rabbit polyclonal anti-orexin-A antibody, which recognizes both ORX A and B, to examine ORX pathways in the sheep hypothalamus. ORX immunoreactive cells were distributed in the dorsomedial hypothalamic nucleus, lateral hypothalamic area, zona incerta and perifornical area; a few cells were also observed in the anterior hypothalamic area. In contrast to distribution in the rat brain, most of the ORX immunoreactive cells are localized to the dorsomedial hypothalamic nucleus and perifornical area; scattered cells are found in lateral hypothalamic area. ORX immunoreactive fibres were widely distributed throughout the hypothalamus and preoptic area with dense innervation of the medial preoptic area and bed nucleus of stria terminalis. Dual labelling demonstrated widespread expression of the long form of the leptin receptor within all ORX cells that were examined. Thirty percent of the gonadotropin releasing hormone (GnRH) cells that were examined had ORX immunoreactive terminals in close contact with no regional or sex differences. FluoroGold injections into the preoptic area retrogradely labelled a subpopulation of ORX cells in the lateral hypothalamic/perifornical area, showing ORX cells of this region project to the preoptic and could potentially provide input to GnRH cells. These findings suggest an integral role for ORX in the regulation of GnRH cells in the sheep and thus provide evidence of a novel mechanism whereby leptin can influence reproductive neuroendocrine function.

Immunohistochemical characterization of localization of long-form leptin receptor (OB-Rb) in neurochemically defined cells in the ovine hypothalamus.

Leptin, a hormone secreted from the adipose tissue, is involved in the regulation of food intake and neuroendocrine function, by modulation of the expression and/or function of various neuropeptides in the hypothalamus. The long isoform (OB-Rb) is the major signaling form of the leptin receptor in the hypothalamus. We have used double-labeling immunohistochemistry to examine the extent of OB-Rb expression in neurochemically defined cell types in the ovine hypothalamus. OB-Rb-like immunoreactivity was widespread within cells localized to the periventricular, paraventricular, supraoptic, dorsomedial hypothalamic, ventromedial hypothalamic and arcuate nuclei, as well as the median eminence, perifornical, anterior hypothalamic and lateral hypothalamic areas and the zona incerta. Double-labeling showed expression of OB-Rb in 59.6+/-6.0% neuropeptide Y-containing cells, 60.8+/-4.7% galanin-containing cells, 89.8+/-2.65% pro-opiomelanocortin-containing cells, 73.4+/-3.5% tyrosine hydroxylase-containing cells and 31.8+/-2.8% corticotropin-releasing factor-containing cells. Interestingly 100% of melanin-concentrating hormone and orexin positive cells were also OB-Rb immunoreactive. These data provide semi-quantitative information on the extent to which various cell types express OB-Rb in the hypothalamus. Expression of OB-Rb within specific neuropeptidergic neurons provides evidence for the direct action of leptin upon the various neurochemical systems that regulate food intake, neuroendocrine and autonomic function in the brain.

The percentage of pituitary gonadotropes with immunoreactive oestradiol receptors increases in the follicular phase of the ovine oestrous cycle.

During the oestrous cycle, there is an alteration in gonadotrope responsiveness to gonadotropin releasing hormone (GnRH). One cellular mechanism that may be involved in these changes at the pituitary level is the hormonal regulation of oestrogen receptor (ER) expression. Using double-label immunohistochemistry, we examined the proportion of gonadotropes, lactotropes and somatotropes with immunoreactive (ir) oestrogen receptor alpha (ERalpha) in pituitary sections from ewes at three stages of the ovine oestrous cycle (n = 8 per group). The percentage of ERalpha positive cells that also stained positive for luteinizing hormone (LH) increased in the transition from the luteal phase to the follicular phase (n = 8), with no further increase at the time of oestrus (n = 8). In the pituitaries from the luteal phase sheep, only a small number (15%) of lactotropes and 4% of somatotropes were found to contain ir-ERalpha and there were no alterations across the oestrous cycle. When we examined pituitaries from ovariectomized (OVX) ewes treated (i.m.) with either oestradiol benzoate (50 microg) or oil vehicle for 2, 4, 6 or 16 h (n = 4 per group), there was no effect of treatment. In fact, the percentage of gonadotropes that were ERalpha-positive in OVX ewes was similar to that observed in the pituitaries from the follicular phase ewes, both of which display a high frequency of pulsatile GnRH secretion. We conclude that the number of gonadotropes that contain ir-ERalpha increases in the follicular phase of the oestrous cycle and this may enhance the responsiveness of these cells to oestrogen and GnRH. We suggest that this may be due to increased pulsatile GnRH input rather than rising oestrogen levels.