Tumors of the Neurohypophysis: One Unit's Experience and Literature Review.

OBJECTIVE: To evaluate and understand the clinical behavior and radiologic correlates of tumors originating from the posterior pituitary gland. To review the management strategy for these rare tumors and add to the limited existing literature. METHODS: Retrospective review of 8 cases (5 pituicytomas, 2 spindle cell oncocytomas, and 1 granular cell tumor) managed at our institution between 2004 and 2019. The patients' clinical course, histologic features, and radiologic findings were reviewed. Their management and long-term follow-up is presented and compared with the literature. RESULTS: Long-term follow-up ranged from 1 to 9 years. There was 1 recurrence in a patient with spindle cell oncocytoma, and this was treated with radiotherapy. The endoscopically managed cases resulted in complete tumor excision with no recurrence. CONCLUSIONS: Epidemiologic data on primary tumors of the neurohypophysis is limited because of the rarity of these tumors. This study adds to the literature that these tumors behave as World Health Organization grade I tumors, although close follow-up is recommended as a few cases have shown recurrence. The endoscopic approach resulted in better gross total tumor resection rate in this series.

Dopamine D2 receptor expression in the corticotroph cells of the human normal pituitary gland.

The dopamine D2 receptor is the main dopamine receptor expressed in the human normal pituitary gland. The aim of the current study was to evaluate dopamine D2 receptor expression in the corticotroph cell populations of the anterior lobe and pars intermedia, as well as posterior lobe of the human normal pituitary gland by immunohistochemistry. Human normal pituitary gland samples obtained from routine autopsies were used for the study. In all cases, histology together with immunostaining for adrenocorticotropic hormone, melanocyte-stimulating hormone, prolactin, and neurofilaments were performed and compared to the immunostaining for D2 receptor. D2 receptor was heterogeneously expressed in the majority of the cell populations of the anterior and posterior lobe as well as in the area localized between the anterior and posterior lobe, and arbitrary defined as "intermediate zone". This zone, characterized by the presence of nerve fibers included the residual pars intermedia represented by the colloid-filled cysts lined by the remnant melanotroph cells strongly expressing D2 receptors, and clusters of corticotroph cells, belonging to the anterior lobe but localized within the cysts and adjacent to the posterior lobe, variably expressing D2 receptors. D2 dopamine receptor is expressed in the majority of the cell populations of the human normal pituitary gland, and particularly, in the different corticotroph cell populations localized in the anterior lobe and the intermediate zone of the pituitary gland.

VEGF-dependent and PDGF-dependent dynamic neurovascular reconstruction in the neurohypophysis of adult mice.

Hypothalamo-neurohypophysial system (HNS) releases arginine vasopressin (AVP) and oxytocin (OXT) from axonal terminals of the neurohypophysis (NH) into blood circulation for controlling body fluid homeostasis and lactation. Chronic osmotic and suckling stimulations have been shown to cause neurovascular and neuroglial reconstruction in the NH of adult mammals and no study has been reported for vascular dynamics. The aim of this study was to elucidate the occurrence of continuous angiogenesis and growth factor-dependent neurovascular reconstruction in the NH of adult mice. Active proliferation of endothelial cells and oligodendrocyte progenitor cells (OPCs) was observed using the immunohistochemistry of bromodeoxyuridine and Ki-67. Vascular endothelial growth factor A (VEGFA) and VEGF receptor 2 (VEGFR2 (KDR)) were highly expressed at pituicytes and endothelial cells respectively. Moreover, prominent expression of platelet-derived growth factor B (PDGFB) and PDGF receptor beta was observed at OXT-containing axonal terminals and pericytes respectively. Administration of the selective tyrosine kinase inhibitor AZD2171 for VEGFRs and STI571 for PDGFRs significantly decreased proliferation of endothelial cells and OPCs. Moreover, AZD2171 treatment decreased vascular density by facilitating apoptosis of endothelial cells and the withdrawal of its treatment led to remarkable rebound proliferation of endothelial cells, so that vascular density rapidly returned to normal levels. AZD2171 decreased the density of both AVP- and OXT-containing axonal terminals, whereas STI571 selectively decreased the density of AVP-containing ones. Thus, this study demonstrates that the signaling pathways of VEGF and PDGF are crucial mediators for determining proliferation of endothelial cells and OPCs and the density of AVP- and OXT-containing axonal terminals in the HNS.

The medio-basal hypothalamus as a dynamic and plastic reproduction-related kisspeptin-gnrh-pituitary center in fish.

Kisspeptin regulates reproductive events, including puberty and ovulation, primarily via GnRH neurons. Prolonged treatment of prepubertal striped bass females with kisspeptin (Kiss) 1 or Kiss2 peptides failed to enhance puberty but suggested a gnrh-independent pituitary control pathway. Kiss2 inhibited, but Kiss1 stimulated, FShß expression and gonadal development, although hypophysiotropic gnrh1 and gnrh receptor expression remained unchanged. In situ hybridization and immunohistochemistry on brains and pituitaries revealed a differential plasticity between the 2 kisspeptin neurons. The differences were most pronounced at the prespawning phase in 2 regions along the path of gnrh1 axons: the nucleus lateralis tuberis (NLT) and the neurohypophysis. Kiss1 neurons appeared in the NLT and innervated the neurohypophysis of prespawning males and females, reaching Lh gonadotropes in the proximal pars distalis. Males, at all reproductive stages, had Kiss2 innervations in the NLT and the neurohypophysis, forming large axonal bundles in the former and intermingling with gnrh1 axons. Unlike in males, only preovulatory females had massive NLT-neurohypophysis staining of kiss2. Kiss2 neurons showed a distinct appearance in the NLT pars ventralis-equivalent region only in spawning zebrafish, indicating that this phenomenon is widespread. These results underscore the NLT as important nuclei for kisspeptin action in 2 facets: 1) kisspeptin-gnrh interaction, both kisspeptins are involved in the regulation of gnrh release, in a stage- and sex-dependent manner, especially at the prespawning phase; and 2) gnrh-independent effect of Kiss peptides on the pituitary, which together with the plastic nature of their neuronal projections to the pituitary implies that a direct gonadotropic regulation is plausible.

Immunofluorescence evidence of melanotrophs in the pituitary of four odontocete species. An immunohistochemical study and a critical review of the literature.

Cetaceans share peculiar features of their pituitary glands, with a complete separation of pars distalis and pars nervosa by a dural septum and the absence of an intermediate lobe and cleft. In most mammals the pars intermedia is the main source of circulating α-melanocyte stimulating hormone (α-MSH), derived from a large precursor called proopiomelanocortin (POMC), which also generates adrenocorticotropic hormone (ACTH) in the adenohypophysis. The lack of an intermediate lobe in cetaceans led us to investigate whether their glands are able to produce α-MSH, and if this hormone is secreted by a distinct population of melanotrophs or by corticotrophs in the pars distalis. Immunofluorescence evidences seem to support the first assumption, with ACTH-immunoreactive (-ir) elements rarely overlapping with α-MSH-ir ones. The discovery of a population of true melanotrophs in the hypophysis of some odontocetes underscores the need for further research on the melanocortin system of cetaceans.

Pituitary melanotrope cells of Xenopus laevis are of neural ridge origin and do not require induction by the infundibulum.

Classical studies in amphibians have concluded that the endocrine pituitary and pars intermedia are derived from epithelial buccal epidermis and do not require the infundibulum for their induction. These studies also assumed that the pituitary is not subsequently determined by infundibular induction. Our extirpation, auto-transplantation and immunohistochemical studies with Xenopus laevis were initiated to investigate early presumptive pituitary development. These studies were conducted especially with reference to the pars intermedia melanotrope cell's induction, and its production and release of α-melanophore stimulating hormone (α-MSH) from the precursor protein proopiomelanocortin (POMC). Auto-transplantation studies demonstrated that the pituitary POMC-producing cells are determined at a stage prior to pituitary-infundibular contact. The results of experiments involving the extirpation of the presumptive infundibulum also indicated that the infundibulum is not essential for the differentiation of POMC-producing cells. We also demonstrated that early pituitary development involves adherence to the prechiasmatic area of the diencephalon with the pituitary placode growing in a posterior direction toward the infundibulum where contact occurs at Xenopus stage 39/40. Overall, our studies provide a model for early tissue relations among presumptive pituitary, suprachiasmatic nucleus, pars tuberalis and infundibulum during neurulation and later neural tube stages of development. It is hypothesized that the overlying chiasmatic area suppresses pituitary differentiation.

Ghrelin immunoexpression in the human hypophysis.

The aim of this study was to immunohistochemically localize ghrelin in autopsy-obtained, nontumoral human pituitaries. Double immunostaining was also undertaken to determine the pituitary cell type expressing both adenohypophysial hormones and ghrelin. Results showed that ghrelin is present in the adenohypophysis, its immunoexpression being cytoplasmic, weak-to-moderate, and localized to a subset of cells. Double immunostaining showed that ghrelin is present in 51% to 90% of growth hormone-producing, luteinizing-producing, and α-subunit-producing cells. Ghrelin immunoexpression was less frequently observed in other adenohypophysial cell types, being seen in 30% of adrenocorticotropin and follicle-stimulating hormones, 15% of thyrotropin, and 10% of prolactin-immunoreactive cells. Ghrelin immunopositivity was also seen in nerve fibers and Herring bodies of the neurohypophysis and pituitary stalk. More work is needed to elucidate the role of ghrelin in adenohypophysial and neurohypophysial endocrine activity. It may well be that ghrelin exerts an autocrine/paracrine effect and can modulate hormone synthesis and release.

Architecture of the hypothalamo-posthypophyseal complex is controlled by monoamines.

The hypothalamo-neurohypophyseal system displays significant plasticity when subjected to physiological stimuli, such as dehydration, parturition, or lactation. This plasticity arises at the neurochemical and electrophysiological levels but also at a structural level. Several studies have demonstrated the role of monoaminergic afferents in controlling neurochemical and electrophysiological plasticity of the supraoptic nucleus (SON) and of the neurohypophysis (NH), but little is known about how the changes in structural plasticity are triggered. We used Tg8 mice, disrupted for the monoamine oxidase A gene, to study monamine involvement in the architecture of the SON and of the NH. SON astrocytes in Tg8 mice displayed an active status, characterized by an increase in S100ß expression and a significant decrease in vimentin expression, with no modification in glial fibrillary acidic protein (GFAP) levels. Astrocytes showed a decrease in glutamate dehydrogenase (GDH) levels, whereas glutamine synthetase (GS) levels remained constant, suggesting a reduction in astrocyte glutamate catabolism. Tenascin C and polysialic acid-neural cell adhesion molecule (PSA-NCAM) expressions were also elevated in the SON of Tg8 mice, suggesting an increased capacity for structural remodelling in the SON. In the NH, similar date were obtained with a stability in GFAP expression and an increase in PSA-NCAM immunostaining. These results establish monoamine (serotonin and noradrenaline) involvement in SON and NH structural arrangement. Monoamines therefore appear to be crucial for the coordination of the neurochemical and structural aspects of neuroendocrine plasticity, allowing the hypothalamo-neurohypopyseal system to respond appropriately when stimulated.

Distribution of 7B2 (secretogranin V)-like immunoreactivity in the Japanese red-bellied newt (Cynops pyrrhogaster) pituitary.

In this study, we examined 7B2 (secretogranin V)-like immunoreactivity (IR) in the Japanese red-bellied newt (Cynops pyrrhogaster) pituitary. Results showed that the pars nervosa was filled with immunoreactive granules. In the pars intermedia, all melanotrophs showed 7B2-IR. In the pars distalis, immunoreactive cells were dispersed, and the 7B2-immunoreactive cells were also immunopositive for the beta-subunit of bullfrog luteinizing hormone (fLHbeta). 7B2-IR co-localized with fLHbeta-IR in the same secretory granules. Our results suggest that 7B2 may participate in the secretion processes of gonadotropins in the pars distalis.

Oxytocin-induced elevation of ADP-ribosyl cyclase activity, cyclic ADP-ribose or Ca(2+) concentrations is involved in autoregulation of oxytocin secretion in the hypothalamus and posterior pituitary in male mice.

Locally released oxytocin (OT) activates OT receptors (2.1:OXY:1:OT:) in neighboring neurons in the hypothalamus and their terminals in the posterior pituitary, resulting in further OT release, best known in autoregulation occurring during labor or milk ejection in reproductive females. OT also plays a critical role in social behavior of non-reproductive females and even in males in mammals from rodents to humans. Social behavior is disrupted when elevation of free intracellular Ca(2+) concentration ([Ca(2+)](i)) and OT secretion are reduced in male and female CD38 knockout mice. Therefore, it is interesting to investigate whether ADP-ribosyl cyclase-dependent signaling is involved in OT-induced OT release for social recognition in males, independent from female reproduction, and to determine its molecular mechanism. Here, we report that ADP-ribosyl cyclase activity was increased by OT in crude membrane preparations of the hypothalamus and posterior pituitary in male mice, and that OT elicited an increase in [Ca(2+)](i) in the isolated terminals over a period of 5 min. The increases in cyclase and [Ca(2+)](i) were partially inhibited by nonspecific protein kinase inhibitors and a protein kinase C specific inhibitor, calphostin C. Subsequently, OT-induced OT release was also inhibited by calphostin C to levels inhibited by vasotocin, an OT receptor antagonist, and 8-bromo-cADP-ribose. These results demonstrate that OT receptors are functionally coupled to membrane-bound ADP-ribosyl cyclase and/or CD38 and suggest that cADPR-mediated intracellular calcium signaling is involved in autoregulation of OT release, which is sensitive to protein kinase C, in the hypothalamus and neurohypophysis in male mice.

Oxytocin action at the lactotroph is required for prolactin surges in cervically stimulated ovariectomized rats.

Cervical stimulation induces two daily rhythmic prolactin surges, nocturnal and diurnal, which persist for several days. We have shown that a bolus injection of oxytocin initiates a similar prolactin rhythm, which persists despite low levels of oxytocin after injection. This suggests that oxytocin may trigger the cervical stimulation-induced rhythmic prolactin surges. To investigate this hypothesis, we infused an oxytocin antagonist that does not cross the blood-brain barrier for 24 h before and after cervical stimulation and measured serum prolactin. We also measured dopaminergic neuronal activity because mathematical modeling predicted that this activity would be low in the presence of the oxytocin antagonist. We thus tested this hypothesis by measuring dopaminergic neuronal activity in the tuberoinfundibular, periventricular hypophyseal, and tuberohypophyseal dopaminergic neurons. Infusion of oxytocin antagonist before cervical stimulation abolished prolactin surges, and infusion of oxytocin antagonist after cervical stimulation abolished the diurnal and significantly decreased the nocturnal surges of prolactin. The rhythmic prolactin surges returned after the clearance of the oxytocin antagonist. Hypothalamic dopaminergic activity was elevated in antiphase with prolactin surges, and the antiphase elevation was abolished by the oxytocin antagonist in the tuberoinfundibular and tuberohypophyseal dopaminergic neurons, consistent with the mathematical model. These findings suggest that oxytocin is a physiologically relevant prolactin-releasing factor. However, the cervical stimulation-induced prolactin surges are maintained even in the absence of oxytocin actions at the lactotroph, which strongly suggests the maintenance of prolactin surges are not dependent upon oxytocin actions at the pituitary gland.

Hypertrophy and increased kisspeptin gene expression in the hypothalamic infundibular nucleus of postmenopausal women and ovariectomized monkeys.

CONTEXT: Human menopause is characterized by ovarian failure, gonadotropin hypersecretion, and neuronal hypertrophy in the hypothalamic infundibular (arcuate) nucleus. Recent studies have demonstrated a critical role for kisspeptins in reproductive regulation, but it is not known whether menopause is accompanied by changes in hypothalamic kisspeptin neurons. OBJECTIVES: Our objective was to map the location of neurons expressing kisspeptin gene (KiSS-1) transcripts in the human hypothalamus and determine whether menopause is associated with changes in the size and gene expression of kisspeptin neurons. In monkeys, our objective was to evaluate the effects of ovariectomy and hormone replacement on neurons expressing KiSS-1 mRNA in the infundibular nucleus. SUBJECTS: Hypothalamic tissues were collected at autopsy from eight premenopausal and nine postmenopausal women and from 42 young cynomolgus monkeys in various endocrine states. METHODS: We used hybridization histochemistry, quantitative autoradiography, and computer-assisted microscopy. RESULTS: Examination of human hypothalamic sections revealed that KiSS-1 neurons were located predominantly in the infundibular nucleus. In the infundibular nucleus of postmenopausal women, there was a significant increase in the size of neurons expressing KiSS-1 mRNA and the number of labeled cells and autoradiographic grains per neuron. Similar to postmenopausal women, ovariectomy induced neuronal hypertrophy and increased KiSS-1 gene expression in the monkey infundibular nucleus. Conversely, in ovariectomized monkeys, estrogen replacement markedly reduced KiSS-1 gene expression. CONCLUSIONS: The cynomolgus monkey experiments provide strong evidence that the increase in KiSS-1 neuronal size and gene expression in postmenopausal women is secondary to ovarian failure. These studies suggest that kisspeptin neurons regulate estrogen negative feedback in the human.

IGF-I is distinctly located in the bony fish pituitary as revealed for Oreochromis niloticus, the Nile tilapia, using real-time RT-PCR, in situ hybridisation and immunohistochemistry.

In bony fish, IGF-I released from the liver under the control of pituitary GH is the main endocrine regulator of growth, maintenance and development, and the amount of circulating IGF-I regulates synthesis and release of GH. In mammals and amphibia, evidence indicates that anterior pituitary endocrine cells also contain IGF-I. However, only preliminary and conflicting data exist on IGF-I gene expression in bony fish pituitary. Thus, we investigated the presence of IGF-I in the tilapia (Oreochromis niloticus) pituitary by quantitative real-time RT-PCR, in situ hybridisation and immunohistochemistry. The absolute amount of IGF-I mRNA in the whole pituitary (7.4+/-3.3 x 10(-3)pg/microg total RNA) was 1000-times lower than in liver (7.5+/-3.1 pg/microg total RNA). IGF-I peptide occurred in both neuro- and adenohypophysis but IGF-I gene expression was mainly restricted to the adenohypophysis. In the neurohypophysis, only few cells, probably pituicytes, contained IGF-I mRNA whereas IGF-I peptide was found also in numerous axons in the pars nervosa. In the adenohypophysis, both IGF-I mRNA and peptide were present in the majority of ACTH cells in all individuals investigated. In alpha-MSH cells, only IGF-I mRNA but no IGF-I peptide was detected likely suggesting an immediate release of IGF-I after synthesis. IGF-I mRNA and peptide were further observed in GH cells but their presence showed pronounced inter-individual differences likely due to the physiological, e.g., nutritional, status of the individual. IGF-I released from the GH cells may serve as auto/paracrine mediator of a negative feedback mechanism in addition to liver-derived endocrine IGF-I. Generally, the constitutive synthesis of IGF-I in ACTH cells and the varying content in GH and alpha-MSH cells suggest particular roles for IGF-I. Local IGF-I may regulate synthesis and release of pituitary hormones in an autocrine and/or paracrine manner as well as prevent apoptosis and stimulate proliferation of endocrine cells.

Evidence of haematopoiesis within the developing human diencephalon. Correlations with vertebrate phylogeny.

AIM: The sites of haematopoiesis during human ontogeny can be correlated to the sites where haematopoiesis occurs in vertebrate phylogeny. As haematopoiesis has been described in the diencephalon and pituitary gland of water-inhabiting vertebrates we wanted to find out whether such a phenomenon also occurs in human embryos. MATERIAL AND METHODS: Paraffin-embedded specimens from the diencephalon and pituitary gland of human embryos at the 7th to 22nd gestational week and from adults were investigated by conventional histology and immunohistology for the presence of haematopoietic cells. RESULTS: Cellular accumulations predominantly of erythroid and megakaryocytic lineage were identified in the floor of the developing diencephalon of the 7th/8th gestational week. At the older developmental stages of the 18th to 22nd gestational week loose aggregates of haematopoietic cells within the leptomeningeal spaces adjacent to the hypophyseal infundibulum were detected in 2 out of 7 cases analyzed. CONCLUSIONS: As it has been proposed that lymphohaematopoietic clusters occasionally occur within the brain in bone marrow-less vertebrates as a response to noxious agents, we speculate that this temporal appearance of haematopoietic cell clusters within the diencephalon floor in early human ontogeny could also be due to fetal immunomodulations.

Oligodendrocyte precursor cells generate pituicytes in vivo during neurohypophysis development.

In the vertebrate brain, much remains to be understood concerning the origin of glial cell diversity and the potential lineage relationships between the various types of glia. Besides astrocytes and myelin-forming oligodendrocytes, other macroglial cell populations are found in discrete areas of the central nervous system (CNS). They share functional features with astrocytes and oligodendrocytes but also display specific characteristics. Such specialized cells, called pituicytes, are located in the neurohypophysis (NH). Our work focuses on the lineage of the pituicytes during rodent development. First, we show that cells identified with a combination of oligodendrocyte precursor cell (OPC) markers are present in the developing rat NH. In culture, neonatal NH progenitors also share major functional characteristics with OPCs, being both migratory and bipotential, i.e. able to give rise to type 2 astrocytes and oligodendrocytes. We then observe that, either in vitro or after transplantation into myelin-deficient Shiverer brain, pieces of NH generate myelinating oligodendrocytes, confirming the oligodendrogenic potentiality of NH cells. However, no mature oligodendrocyte can be found in the NH. This led us to hypothesize that the OPCs present in the developing NH might be generating other glial cells, especially the pituicytes. Consistent with this hypothesis, the OPCs appear during NH development before pituicytes differentiate. Finally, we establish a lineage relationship between olig1+ cells, most likely OPCs, and the pituicytes by fate-mapping experiments using genetically engineered mice. This constitutes the first demonstration that OPCs generate glial cells other than oligodendrocytes in vivo.

Differential effects of water and saline intake on water deprivation-induced c-Fos staining in the rat.

We studied c-Fos staining in adult male rats after 48 h of water deprivation and after 46 h of water deprivation with 2 h of access to water or physiological saline. Controls were allowed ad libitum access to water and physiological saline. For immunocytochemistry, anesthetized rats were perfused with a commercially available antibody for c-Fos. Dehydration significantly increased plasma vasopressin (AVP), osmolality, plasma renin activity (PRA), hematocrit, and sodium concentration and decreased urinary volume. Fos staining was significantly increased in the median preoptic nucleus, organum vasculosum of the lamina terminalis, supraoptic nucleus (SON), and magnocellular and parvocellular paraventricular nucleus (PVN), as well as the area postrema, nucleus of the solitary tract (NTS), and rostral ventrolateral medulla (RVL). Rehydration with water significantly decreased AVP levels and Fos staining in the SON, PVN, and RVL and significantly increased Fos expression in the perinuclear zone of the SON, NTS, and parabrachial nucleus. Rehydration with water was associated with decreased urinary sodium concentration and hypotonicity, and hematocrit and PRA were comparable to levels seen after dehydration. After rehydration with saline, plasma osmolality, hematocrit, and PRA were not different from control, but plasma AVP and urinary sodium concentration were increased. In the SON, Fos staining was significantly increased, with a great percentage of the Fos cells also stained for oxytocin compared with water deprivation. Changes in Fos staining were also observed in the NTS, RVL, parabrachial nucleus, and PVN. Rehydration with water or saline produces differential effects on plasma AVP, Fos staining, and sodium concentration.

Immunohistochemical study of somatotrophs in pituitary pars distalis of male viscacha (Lagostomus maximus maximus) in relation to the gonadal activity.

Somatotrophs were identified and quantified in pituitary pars distalis of male viscachas (Lagostomus maximus maximus) during the annual reproductive cycle, after the administration of melatonin, after castration and in different growth stages by immunohistochemistry and morphometric analysis. In adult male viscachas, the somatotrophs were distributed throughout the pars distalis during the reproductive cycle. They were oval, pyramidal or round in shape with a large round nucleus. The percentage immunopositive area, the major cellular diameter and the number of cells decreased during the gonadal regression period in relation to the values found in the reproductive period. The administration of melatonin did not provoke any variations of the morphometric parameters studied. On the contrary, a significant decrease in the percentage immunopositive area, in the major cellular diameter and in the number of somatotrophs in castrated viscachas was observed. The study of different growth stages showed that these morphometric parameters increased from immature to adult animals in the reproductive period. The results obtained suggested that the variations of the morphometric parameters of somatotrophs are more related to the gonadal development and activity than to a direct effect of melatonin.

Angiopoietins are expressed in the normal rat pituitary gland.

Interaction of vascular endothelial growth factor (VEGF) with the angiopoietins (Ang) is an essential component of angiogenesis. Localization of VEGF in the anterior pituitary raises the possibility that Ang must be present in the pituitary gland as well. In this study Ang expression was detected in the normal rat pituitary gland at the gene level by reverse-transcriptase polymerase chain reaction and at the protein level by immunohistochemistry. The latter was analyzed by both light and confocal microscopy. Constitutive expression of Ang1, Ang2, and their receptor Tie2 was detected at both the mRNA and protein level in all the pituitary glands studied. Of interest was the localization of both Ang1 and Ang2 in scattered PAS positive adenohypophysial cells rather than in endothelial cells. Confocal microscopy showed colocalization of both Ang1 and Ang2 proteins within the same adenohypophysial cells. Dual immunostaining for Ang1 and the anterior pituitary hormones that show PAS positivity demonstrated colocalization of Ang1 with follicle stimulating hormone and luteinizing hormone. In the posterior pituitary, strong Ang1 signal observed in vascular endothelial cells masked the weak Ang2 signal, a pattern that is similar to that reported in brain endothelial cells. The presence of both angiopoietins and VEGF in the pituitary gland suggest that these ligands interact during angiogenesis as they are known to do in other systems to maintain the rich vascular network of the gland. This first report of angiopoietin localization in the rat pituitary gland opens a new line of investigation on angiogenesis in pituitary glands that will impact human endocrinology in the future.

Coexistence of salusin and vasopressin in the rat hypothalamo-hypophyseal system.

Salusins are two newly discovered TOR-related peptides consisting of 28 and 20 amino acids and designated salusin-alpha and salusin-beta, respectively. Using immunohistochemistry techniques, salusin-like immunoreactivity was detected in the rat hypothalamo-neurohypophyseal tract and immunopositive cells were distributed in the suprachiasmatic, supraoptic and paraventricular nucleus. In the paraventricular nucleus, salusin-like immunoreactivity was observed both in parvocellular and magnocellular neurons. Many salusin-positive nerve fibers and their terminals were identified in the internal layer of the median eminence and posterior pituitary. Less intense salusin-positive staining of fibers and terminals was found in the suprachiasmatic nucleus and external layer of the median eminence. Dual immunostaining was performed to determine if salusin coexisted with vasopressin or oxytocin in the hypothalamus. Most of the salusin-like immunoreactivity was detected in vasopressin- but not in oxytocin-containing neurons in these nuclei. The functional significance of the coexistence of salusin with vasopressin is discussed, including the possibility that salusin participates in the regulation of blood pressure.

Search for neural substrates mediating inhibitory effects of oestrogen on pulsatile luteinising hormone-releasing hormone release in vivo in ovariectomized female rhesus monkeys (Macaca mulatta).

Neural substrates mediating the negative feedback effects of oestrogen on luteinising hormone-releasing hormone (LHRH) release were studied using the in vivo push-pull perfusion method in female rhesus monkeys. Twelve long-term ovariectomized female monkeys were implanted with Silastic capsules containing 17beta-oestradiol 2 weeks before the experiments and, on the day of the experiment, oestradiol benzoate (EB, 50 microg/kg) or oil was subcutaneously injected. Push-pull perfusate samples from the stalk-median eminence were collected in 10-min fractions from 4 h before to 18-20 h after EB or oil injection. LHRH and neuropeptide Y (NPY) levels in the same perfusates were measured by radioimmunoassay, and glutamate and GABA in the same perfusates were assessed by high-performance liquid chromatography (HPLC). The results indicate that EB significantly suppressed LHRH release (P < 0.005) starting within 2 h after EB, and continued for 18 h or until the experiment was terminated. Pulse analysis suggested that oestrogen suppressed the pulse amplitude, but not pulse frequency, of LHRH release. By contrast, EB did not alter any parameters (mean release, pulse amplitude or frequency) of pulsatile NPY release throughout the experiment. HPLC analysis further suggested that neither glutamate nor GABA levels in the stalk-median eminence were changed with oestrogen-induced LHRH suppression. Oil treatment did not alter LHRH, NPY, GABA and glutamate levels. It is concluded that oestrogen induces suppression of pulsatile LHRH release within 2 h, but the inhibitory effect of oestrogen on LHRH release does not appear to be mediated by NPY, GABAergic, or glutamatergic neurones.