Pars Intermedia Research Articles

Altered expression of neprilysin family members in the pituitary gland of sleep‐disturbed rats, an animal model of severe fatigue

Alterations of the expression of some peptidases in the pituitary gland of a fatigued rat model were identified. Rats were kept in a cage filled with water to a height of 1.5 cm to disturb deep sleep. After 24-h sleep disturbance, expression of neutral endopeptidase 24.11 (neprilysin) mRNA was increased in the intermediate lobe of the pituitary gland, whereas the mRNA expression of another family member, damage-induced neuronal endopeptidase, which is normally expressed in a subgroup of anterior pituitary cells, was significantly suppressed. These alterations were demonstrated by RT-PCR, northern blotting and in situ hybridization. Other family members, such as neprilysin 2 and endothelin converting enzyme-1, did not show any change in mRNA expression. An increase of neprilysin mRNA expression was not seen in any other tissues of the sleep-disturbed rats. The enzymatic activity of neprilysin was also increased in the pituitary. The augmentation of neprilysin expression and activity was prolonged as long as the sleep disturbance continued (up to 5 days), and returned to the basal level when rats were allowed to sleep freely. These results suggest that peptide processing and degradation in the pituitary may be an influential factor in fatigued states such as sleep disturbance.

Distribution of Urocortin 2 in Various Tissues of the Rat

Urocortin (Ucn) 2 is a new member of the corticotrophin-releasing hormone (CRH) neuropeptide family that is expressed in the central nervous system and peripheral tissues. However, the expression levels of Ucn 2 in various tissues of the rat remains unclear. Thus, the aim of the present study was to characterise the expression of Ucn 2 in the various tissues of the rat. Reverse transcriptase-polymerase chain reaction analysis demonstrated that Ucn 2 mRNA is expressed in the hypothalamus, pituitary, adrenal, stomach, skin, ovary, uterus and skeletal muscle. Histologically, Ucn 2 mRNA and Ucn 2-like immunoreactivity (LI) were demonstrated in both the anterior and intermediate lobes of the pituitary, but not detected in the posterior lobe. Furthermore, all Ucn 2-positive cells in the anterior and intermediate lobes were also positive for beta-endorphin. Ucn 2 mRNA was detected in the adrenal cortex and medulla although Ucn 2-LI was only found in the adrenal medulla. High-performance liquid chromatography analysis of hypothalamic, pituitary, and adrenal extracts showed that the main Ucn 2-LI peak occurred at the same molecular size as that of synthetic Ucn 2. These results suggest that Ucn 2 is synthesised in various tissues, including the anterior and intermediate lobes of the pituitary and the adrenal.

Subfunctionalization of Expression and Peptide Domains Following the Ancient Duplication of the Proopiomelanocortin Gene in Teleost Fishes

The proopiomelanocortin gene (POMC) encodes several bioactive peptides, including adrenocorticotropin hormone, alpha-, beta-, and gamma-melanocyte-stimulating hormone, and the opioid peptide beta-endorphin, which play key roles in vertebrate physiology. In the human, mouse, and chicken genomes, there is only one POMC gene. By searching public genome projects, we have found that Tetraodon (Tetraodon nigroviridis), Fugu (Takifugu rubripes), and zebrafish (Danio rerio) possess two POMC genes, which we called POMCalpha and POMCbeta, and we present phylogenetic and mapping evidence that these paralogue genes originated in the whole-genome duplication specific to the teleost lineage over 300 MYA. In addition, we present evidence for two types of subfunction partitioning between the paralogues. First, in situ hybridization experiments indicate that the expression domains of the ancestral POMC gene have been subfunctionalized in Tetraodon, with POMCalpha expressed in the nucleus lateralis tuberis of the hypothalamus, as well as in the rostral pars distalis and pars intermedia (PI) of the pituitary, whereas POMCbeta is expressed in the preoptic area of the brain and weakly in the pituitary PI. Second, POMCbeta genes have a beta-endorphin segment that lacks the consensus opioid signal and seems to be under neutral evolution in tetraodontids, whereas POMCalpha genes possess well-conserved peptide regions. Thus, POMC paralogues have experienced subfunctionalization of both expression and peptide domains during teleost evolution. The study of regulatory regions of fish POMC genes might shed light on the mechanisms of enhancer partitioning between duplicate genes, as well as the roles of POMC-derived peptides in fish physiology.

High-pressure freezing followed by cryosubstitution as a tool for preserving high-quality ultrastructure and immunoreactivity in the Xenopus laevis pituitary gland

Subcellular localisation of proteins and peptides yields fundamental information about cell functioning. Immunoelectron microscopy is a powerful tool to achieve this goal, but combining good tissue preservation with strong immunoreactivity is a great challenge in electron microscopy. We have applied a novel approach, using high-pressure freezing (HPF) followed by cryosubstitution, to prepare the pituitary gland of the amphibian Xenopus laevis for immunogold-electron microscopy. In this way, we investigated the subcellular distribution of brain-derived neurotrophic factor and the amphibian neurohormone mesotocin in the pituitary neural lobe, and the peptide hormone α-melanophore-stimulating hormone and its protein precursor proopiomelanocortin in melanotrope cells of the pituitary intermediate lobe. In contrast to conventional chemical fixation (followed by cryosubstitution), HPF not only revealed strong immunoreactivity of the secretory products, but also provided excellent ultrastructural preservation of cell organelles, including secretory granule subtypes. We conclude that HPF followed by cryosubstitution provides a preparation technique of choice when both optimal tissue ultrastructure and strong immunoreactivity are required.

Alpha-melanocyte stimulating hormone release in response to thyrotropin releasing hormone in healthy horses, horses with pituitary pars intermedia dysfunction and equine pars intermedia explants

Thyrotropin releasing hormone (TRH) stimulates an increase in plasma cortisol in horses with pars intermedia dysfunction (PPID, Cushing's disease). A similar phenomenon is observed in humans with Cushing's disease or Nelson's syndrome. The mechanism of the response in humans is not known, but an alteration in receptor expression, selectivity or responsiveness in abnormal corticotropes has been proposed. Horses with PPID, unlike humans, almost exclusively have adenomas of pars intermedia (PI) rather than pars distalis (PD) origin. Therefore, the mechanism responsible for the TRH response observed in horses likely differs. We proposed that TRH directly stimulates the PI in normal and PPID-affected horses to release proopiomelanocortin (POMC) derived peptides. Using α-melanocyte stimulating hormone (α-MSH) as a marker of a PI response and ACTH as a marker of a PD response, we were able to demonstrate a marked increase in plasma concentration of α-MSH and a modest, but significant increase in ACTH after TRH treatment in normal horses. The ability of TRH to directly stimulate release of POMC peptides was confirmed using PI and PD tissue explants. The presence of TRH receptor mRNA in PI tissue from both normal and PPID horses was confirmed using reverse transcriptase polymerase chain reaction. We conclude that TRH triggers the release of POMC-derived peptides from the PI through the direct stimulation of TRH receptors normally expressed on melanotropes. The increase in plasma cortisol following TRH in horses with PPID is likely attributable to the release of ACTH from the hyperplastic PI.

Induced expression of c-fos in the diencephalon and pituitary gland of goats following transportation1

To identify regions of the caprine diencephalone and pituitary gland related to transportation stress, the expression of c-fos protein was examined immunohistochemically as an indicator of neural activation. Ten castrated Shiba goats (Capra hircus), five transported and five controls, were used. Transported goats were trucked for 1 h and killed by transcardiac perfusion 1 h after the end of transportation. Control goats were housed in single pens killed in the same manner and at the same time as the transported goats. The diencephalon and the pituitary gland were removed after perfusion and used for immunostaining. Plasma cortisol concentrations during and after transportation also were investigated. During transportation, plasma cortisol concentrations increased (P < 0.05) compared with those in the controls. In the diencephalon, c-fos immunoreactive cells were detected in the subcallosa, the lateral septal area, the bed nucleus of stria terminalis (BNST), the preoptic hypothalamic area (POA), the suprachiasmatic nucleus (SCN), the supraoptic nucleus, the paraventricular hypothalamic nucleus parvocellular (PVNp), the paraventricular hypothalamic nucleus magnocellular (PVNm), the arcuate nucleus (ARC), the paraventricular thalamic nucleus, and the stria medullaris in both control and transported goats. The numbers of c-fos immunoreactive cells were increased (P < 0.05) by transportation in the PVNm, the PVNp, the BNST, the POA, the ARC, and the SCN (P < 0.10). In the anterior pituitary gland, the number of c-fos immunoreactive cells in transported goats was 4 to 30 times as much as in control goats; however, there were no differences in the intermediate and posterior lobes between control and transported goats. This study has identified regions in the caprine diencephalon and pituitary gland that show transport-induced increases in c-fos immunoreactive cells. In conclusion, the PVNm, the PVNp, the BNST, the POA, the SCN in the diencephalons, and the anterior lobe of pituitary gland may be involved in the stress responses of goats to transportation.

Intermedin Functions as a Pituitary Paracrine Factor Regulating Prolactin Release

Calcitonin, alpha- and beta-calcitonin gene-related peptides, amylin, and adrenomedullin belong to a unique group of peptide hormones important for homeostasis maintenance. We recently identified intermedin (IMD) as a novel member of the calcitonin/calcitonin gene-related peptide family expressed in the pituitary, digestive tract, and other organs of vertebrates. Real-time PCR and immunohistochemical analysis of pituitaries from rats at different stages of development showed that IMD is expressed in the intermediate lobe and select adrenocorticotrophs in the anterior lobe, suggesting that IMD could function as a paracrine factor regulating anterior pituitary hormone secretion. In support of a paracrine role for IMD in the pituitary, quantitative and in situ hybridization analyses showed the expression of IMD receptor transcripts including the calcitonin receptor-like receptor and receptor activity-modifying proteins in the pituitary. Treatment with IMD leads to a dose-dependent increase of prolactin release in cultured rat pituitary cells. In contrast, IMD treatment has negligible effects on the release of GH, FSH, or ACTH. Likewise, in vivo treatment with IMD leads to an elevation of plasma prolactin levels in conscious rats. Based on these functional characteristics, we hypothesized that IMD could represent one of the intermediate lobe-derived prolactin-releasing factors important for prolactin regulation during reproduction. In support of this hypothesis, studies of IMD expression in lactating and ovariectomized rats showed that pituitary IMD transcripts in lactating animals increased to more than 2-fold over nonlactating controls whereas ovariectomy leads to a 90% reduction of IMD expression in the pituitary. Of importance, subsequent treatment with 17beta-estradiol or diethylstilbestrol increased pituitary IMD expression in ovariectomized rats. In addition, analysis of the proximate region of the IMD gene promoter showed that the IMD gene promoter contains consensus estrogen response element sequences, and estrogen treatments up-regulate the promoter reporter activity in transfected pituitary cells. Collectively, the present study indicates that IMD represents a novel estrogen-dependent intermediate lobe-derived prolactin-releasing factor and could play important roles in the regulation of prolactin release during reproduction in females.

Structures for the proopiomelanocortin family genes proopiocortin and proopiomelanotropin in the sea lamprey Petromyzon marinus

Gnathostomes express a common proopiomelanocortin ( POMC) gene in the pars distalis (PD) and the pars intermedia (PI) of the pituitary gland. In contrast, the sea lamprey Petromyzon marinus expresses one distinct gene in each lobe; proopiocortin ( POC) encoding adrenocorticotropic hormone (ACTH) and β-endorphin (END) is expressed in the PD and proopiomelanotropin ( POM) encoding melanophore-stimulating hormone (MSH), and a different β-END is expressed in the PI. We characterized the genomic structure of the sea lamprey POC and POM genes including their 5′-flanking regions. Both genes have two introns at positions similar to those of gnathostomes. Each exon encodes genetic information seen in the gnathostome POMC gene: exon 1 encodes an untranslated nucleotide sequence, exon 2 encodes a signal peptide and the N-terminal short part of POC or POM, and exon 3 encodes all other parts including ACTH, MSHs or β-END. Intron-A of POM (2289 bp) is six times longer than that of POC (379 bp). The POM intron-A has three transposon-like sequences (TnL-1, -2, -3), the total length of which is 1781 bp, suggesting that it has expanded via the insertion of TnLs. The 5′-flanking region of the POC gene contains two TATA boxes, a CCAAT box, eight E boxes, STAT, RAIE, and one binding site each for Ptx1, Pit-1, and Tpit. The POM gene contains four TATA boxes, eight E boxes, three STATs, two RAIEs, two CRE-like elements, and one binding site for Pit1. However, there is virtually no similarity between the two genes in the distribution of the elements. The transcriptional regulation of POC and POM may have diverged with the functional differentiation of the two genes.

Intercellular communications within the rat anterior pituitary: XI: An immunohistochemical study of distributions of S-100 positive cells in the anterior pituitary of the rat

The distribution of the S-100 protein cell (folliculo-stellate cell) is very important to our understanding of the regulation of the anterior pituitary. In this study, 10 intact 60-day-old male Wistar–Imamichi rats, were separated equally into two groups. One was used for immunohistochemical study, and the other for electron microscopic analysis. Immunostained pituitary sections with S-100 protein antibody were photographed using a CCD camera equipped with a computer. The S-100 protein cells were then measured using NIH image software, and the three-dimensional distribution of the cells was analyzed. The distribution of the cells observed in each serial section showed that S-100 protein cells were dense at the basal zone of the gland and at the “transitional zone” where the pars tuberalis adjoined the anterior and intermediate lobes, where they represented over 50% of the total cell population. They then decreased in number with distance from this region to mid-way towards the sagittal axis before increasing again in the tail of the gland. The population of cells also decreased with increasing distance from the “transitional zone” to the wing and with distance from the basal zone. Portal vessels entered the anterior lobe through the “transitional zone” as thick capillaries, ran through the basal surface and penetrated into the central area of the anterior lobe. In all planes, S-100 protein cells encircled the capillaries. Ultrastructural observations confirmed the light microscopic findings indicating that clusters of agranular cells were densely located at the “transitional zone” and in the pars tuberalis. The distribution pattern of the folliculo-stellate cells and the capillaries showed good agreement and the spatial relationship between these two is detailed so as to better understand hypophyseal histophysiology.

ACTH, α-MSH, and control of cortisol release: cloning, sequencing, and functional expression of the melanocortin-2 and melanocortin-5 receptor in Cyprinus carpio

Cortisol release from fish head kidney during the acute phase of the stress response is controlled by the adrenocorticotropic hormone (ACTH) from the pituitary pars distalis (PD). Alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin, from the pars intermedia (PI), have been implicated in cortisol release during the chronic phase. The present study addresses the regulation of cortisol release by ACTH and alpha-MSH in common carp (Cyprinus carpio) and includes characterization of their receptors, namely, the melanocortin-2 and melanocortin-5 receptors (MC2R and MC5R). We could not demonstrate corticotropic activity of alpha-MSH, beta-endorphin, and combinations of these. We do show a corticotrope in the PI, but its identity is as yet uncertain. Carp restrained for 1 and 7 days showed elevated plasma cortisol and alpha-MSH levels; cortisol is still elevated but lower at day 7 than day 1 of restraint. Interrenal response capacity is unaffected, as estimated by stimulation with a maximum dose ACTH in a superfusion setup. MC2R and MC5R appear phylogenetically well conserved. MC2R is predominantly expressed in head kidney; a low abundance was found in spleen and kidney. MC5R is expressed in brain, pituitary PD, kidney, and skin. Quantitative PCR analysis of MC2R and MC5R expression in the head kidney of restrained fish reveals MC2R mRNA downregulation after 7 days restraint, in line with lower plasma cortisol levels seen. We discuss regulation of corticosteroid production from a phylogenetic perspective. We propose that increased levels of alpha-MSH exert a positive feedback on hypothalamic corticotropin-releasing hormone release to sustain a mild stress axis activity.

Deregulated E2F Activity Induces Hyperplasia and Senescence-Like Features in the Mouse Pituitary Gland

The retinoblastoma gene, RB1, is one of the most frequently mutated genes in human cancer. Rb heterozygous mice develop pituitary tumors with 100% incidence, and the E2F transcription factors are required for this. To assess whether deregulated E2F activity is sufficient to induce pituitary tumors, we generated transgenic mice expressing an inducible E2F3 protein in the intermediate lobe of the pituitary gland. We found that short-term deregulation of E2F activity, similar to the earliest stages of Rb loss, is able to induce abnormal proliferation of otherwise quiescent melanotrophs. However, while long-term exposure to deregulated E2F activity results in hyperplasia of the intermediate lobe, it did not lead to tumor formation. In fact, melanotrophs become insensitive to sustained E2F stimulation and enter an irreversible senescence-like state. Thus, although deregulated E2F activity results in hyperproliferation, it is not sufficient to mimic loss of Rb, sustain proliferation of melanotrophs, and ultimately induce pituitary tumors. Similarly, we found that primary cells in tissue culture become insensitive to sustained E2F3 activation and undergo premature senescence in a pRB-, p16Ink4a-, and p19Arf-dependent manner. Thus, we conclude that deregulated E2F activity is not sufficient to fully mimic loss of Rb due to the engagement of a senescence response.

Brain‐Derived Neurotrophic Factor in the Hypothalamo‐Hypophyseal System of Xenopus laevis

Brain-derived neurotrophic factor (BDNF) is immunocytochemically demonstrated in the magnocellular nucleus and neural lobe of the pituitary gland of the amphibian Xenopus laevis. Immunoelectron microscopy shows BDNF in secretory granules of type A neurohemal axon terminals in the neural lobe of pituitary gland. It is proposed that BDNF released from the neural lobe acts as a neurohormone stimulating the secretory activity of the melanotrope cells in the intermediate pituitary lobe.

In Situ Hybridization Localization of TRH Precursor and TRH Receptor mRNAs in the Brain and Pituitary of Xenopus laevis

We examined the distribution of the mRNAs encoding proTRH and the three TRH receptor subtypes (xTRHR1, xTRHR2, and xTRHR3) in the Xenopus laevis CNS and pituitary. A positive correlation was generally observed between the expression patterns of proTRH and xTRHR mRNAs. xTRHRs were widely expressed in the telencephalon and diencephalon, where two or even three xTRHR mRNAs were often simultaneously observed within the same brain structures. In the pituitary, xTRHR2 was selectively expressed in the distal lobe, and xTRHR3 was found exclusively in the intermediate lobe of white background-adapted animals, indicating that, in amphibians, the effect of TRH on alpha-melanotropin (alpha-MSH) secretion from melanotrope cells is mediated through the novel receptor subtype xTRHR3.

Evidence that urocortin I acts as a neurohormone to stimulate αMSH release in the toad Xenopus laevis

We have raised the hypothesis that in the South African clawed toad Xenopus laevis, urocortin 1 (UCN1), a member of the corticotropin-releasing factor (CRF) peptide family, functions not only within the brain as a neurotransmitter/neuromodulator but also as a neurohormone, promoting the release of α-melanophore-stimulating hormone (αMSH) from the neuroendocrine melanotrope cells in the intermediate lobe of the pituitary gland. This hypothesis has been investigated by (1) assessing the distribution of UCN1 and CRF by light immunocytochemistry, (2) determining the subcellular presence of UCN1 in the neural lobe of the pituitary gland by immuno-electron microscopy applying high-pressure freezing and cryosubstitution, and (3) testing the effect of UCN1 on MSH release from toad melanotrope cells using in vitro superfusion. In the X. laevis brain, the main site of UCN1-positive somata was found to be the Edinger–Westphal nucleus. UCN1 immunoreactivity (ir) also occurs in the nucleus posteroventralis tegmenti, central gray, nucleus reticularis medius, nucleus motorius nervi facialis, and nucleus motorius nervi vagi. UCN1 occurs together with CRF in the nucleus motorius nervi trigemini, and in the magnocellular nucleus, which send a UCN1- and CRF-containing fiber tract to the median eminence. Strong UCN1-ir and CRF-ir were found in the external zone of the median eminence. From the internal zone of the median eminence, UCN1-ir fibers, but few CRF-ir fibers, were found to project to the pituitary neural lobe, where they form numerous neurohemal axon terminals. Ultrastructurally, two types of terminal containing UCN1-ir secretory granules were distinguished: type A contains large, moderately electron-dense, round secretory granules and type B is filled with smaller, strongly electron-dense, ellipsoid secretory granules. In vitro superfusion studies showed that UCN1 stimulated the release of αMSH from melanotrope cells in a dose-dependent manner. Our results support the hypothesis that in X. laevis, UCN1 released from neurohemal axon terminals in the pituitary neural lobe functions as a stimulatory neurohormone for αMSH release from melanotrope cells of the pituitary intermediate lobe.

GABAB receptor subunit mRNAs are differentially regulated in pituitary melanotropes during development and detection of functioning receptors coincides with completion of innervation

This study examines the developmental expression of GABAB receptor subunits (GABAB(1a), GABAB(1b), GABAB(2)) in the pituitary intermediate lobe using in situ hybridization, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blots. Receptor functionality was studied by baclofen-stimulated GTPγS binding.In the adult rat pituitary all three transcripts were detected in melanotropes, but not in glia, of the intermediate lobe. No transcripts of any subunit were detected in the neural lobe. Transcripts of GABAB(1a) and GABAB(1b), but not of GABAB(2), were detected in specific subpopulations of cells in the anterior lobe. All three transcripts were detected in melanotropes on gestational day 18 using in situ hybridization. Reverse transcriptase-polymerase chain reactions comparing postnatal day 2 and adult transcript levels in the neurointermediate lobe support in situ hybridization data that GABAB(1a) mRNA levels do not change, GABAB(1b) levels increase, and GABAB(2) levels decrease as the rat matures. Thus, GABAB receptor subunit transcripts are differentially regulated in melanotropes during development. In the adult rat both GABAB(1) and GABAB(2) proteins were detected in the neurointermediate lobe using Western blotting and in melanotropes by immunohistochemistry. Developmentally, GABAB(1) protein was not detected until postnatal day 7, but was clearly expressed by postnatal day 15 while GABAB(2) protein could not be detected until postnatal day 15. Functional receptors were found in the intermediate lobe at postnatal day 15 and in the adult. The demonstration of transcripts for GABAB(1a), GABAB(1b) and GABAB(2) subunits at gestational day 18 contrasted with the failure to detect any protein before postnatal day 7, suggesting that the regulation of GABAB subunit isoforms occurs differentially at both the transcriptional and translational level as development progresses. The disparity in the regulation of the receptor subunits may suggest that GABAB(1) could have other functions besides being part of the GABAB receptor heterodimer.

Systematic Pathogenesis and Replication of Avian Hepatitis E Virus in Specific-Pathogen-Free Adult Chickens

Hepatitis E virus (HEV) is an important human pathogen. Due to the lack of a cell culture system and a practical animal model for HEV, little is known about its pathogenesis and replication. The discovery of a strain of HEV in chickens, designated avian HEV, prompted us to evaluate chickens as a model for the study of HEV. Eighty-five 60-week-old specific-pathogen-free chickens were randomly divided into three groups. Group 1 chickens (n=28) were each inoculated with 5 x 10(4.5) 50% chicken infectious doses of avian HEV by the oronasal route, group 2 chickens (n=29) were each inoculated with the same dose by the intravenous (i.v.) route, and group 3 chickens (n=28) were not inoculated and were used as controls. Two chickens from each group were necropsied at 1, 3, 5, 7, 10, 13, 16, 20, 24, 28, 35, and 42 days postinoculation (dpi), and the remaining chickens were necropsied at 56 dpi. Serum, fecal, and various tissue samples, including liver and spleen samples, were collected at each necropsy for pathological and virological testing. By 21 dpi, all oronasally and i.v. inoculated chickens had seroconverted. Fecal virus shedding was detected variably from 1 to 20 dpi for the i.v. group and from 10 to 56 dpi for the oronasal group. Avian HEV RNA was detected in serum, bile, and liver samples from both i.v. and oronasally inoculated chickens. Gross liver lesions, characterized by subcapsular hemorrhages or enlargement of the right intermediate lobe, were observed in 7 of 28 oronasally and 7 of 29 i.v. inoculated chickens. Microscopic liver lesions were mainly lymphocytic periphlebitis and phlebitis. The lesion scores were higher for oronasal (P=0.0008) and i.v. (P=0.0029) group birds than for control birds. Slight elevations of the plasma liver enzyme lactate dehydrogenase were observed in infected chickens. The results indicated that chickens are a useful model for studying HEV replication and pathogenesis. This is the first report of HEV transmission via its natural route in a homologous animal model.

Immunocytochemical localization and ontogenic development of ?-melanocyte-stimulating hormone (?-MSH) in the brain of a pleuronectiform fish, barfin flounder

Alpha-melanocyte-stimulating hormone (alpha-MSH) is a pituitary hormone derived by post-translational processing from proopiomelanocortin and is involved in background adaptation in teleost fish. It has also been reported to suppress food intake in mammals. Here, we examined the immunocytochemical localization of alpha-MSH in the brain and pituitary of a pleuronectiform fish, the barfin flounder (Verasper moseri), as a first step in unraveling the possible function of alpha-MSH in the brain. The ontogenic development of the alpha-MSH system was also studied. In the pituitary, alpha-MSH-immunoreactive (ir) cells were preferentially detected in the pars intermedia. In the brain, alpha-MSH-ir neuronal somata were located in the nucleus tuberis lateralis of the basal hypothalamus, and alpha-MSH-ir fibers were located mainly in the telencephalon, hypothalamus, and midbrain. Alpha-MSH-ir neuronal somata did not project their axons to the pituitary. The alpha-MSH-ir neurons differed from those immunoreactive to melanin-concentrating hormone. Alpha-MSH cells in the pituitary and alpha-MSH-ir neuronal somata in the brain were first detected 1 day and 5 days after hatching, respectively. The distribution of alpha-MSH-ir cells, neuronal somata, and fibers showed a pattern similar to that in adult fish 30 days after hatching. These results indicate that the functions of alpha-MSH in the brain and pituitary are different and that alpha-MSH plays physiological roles in the early development of the barfin flounder.

Axonal projections from the hypothalamus to the pituitary intermediate lobe in rats during ontogenesis: DiI tracing study

This study has determined ontogenetic schedule of axonal arrival from the hypothalamus in the pituitary intermediate lobe (IL) in rats using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) as a retrograde tracer. The brains with attached pituitaries were dissected in rats from the 20th embryonic day (E20) to the 20th postnatal day (P20). Anterior lobe was mechanically detached from the IL, material fixed in paraformaldehyde, and DiI crystals were applied on the IL laying on the posterior lobe (PL). The labeling of IL + PL resulted in staining of hypothalamic magnocellular neurons, which send their axons to the PL, and hypothalamic parvocellular neurons contributing to the innervation of the IL. Therefore, the magnocellular neurons were not taken into account when identifying the neurons projecting axons to the IL. Rare fluorescent neurons projecting their axons to the IL were detected as early as on E20 in the ventral part of the periventricular nucleus (Pe) and in the rostral part of the arcuate nucleus. Few DiI-labeled neurons were seen in Pe from P1 to P3. At P5, the fluorescent neurons were accumulated giving rise to the prominent cluster in the Pe, which was enlarged on later stages and occupied all the Pe. In addition to the Pe, fluorescent neurons first appeared in the retrochiasmatic region and around the ventromedial nucleus in young rats. Thus, the axons of hypothalamic neurons of the Pe and mediobasal hypothalamus first arrive in the IL in rats at the end of intrauterine development, although the principal innervation of the IL is the postnatal event.

Suppression of Melanotroph Carcinogenesis Leads to Accelerated Progression of Pituitary Anterior Lobe Tumors and Medullary Thyroid Carcinomas in Rb+/− Mice

Abstract Mice with a single copy of the retinoblastoma gene (Rb+/−) develop a syndrome of multiple neuroendocrine neoplasia. They usually succumb to fast-growing, Rb-deficient melanotroph tumors of the pituitary intermediate lobe, which are extremely rare in humans. Thus, full assessment of Rb role in other, more relevant to human pathology, neoplasms is complicated. To prevent melanotroph neoplasia while preserving spontaneous carcinogenesis in other types of cells, we have prepared transgenic mice in which 770-bp fragment of pro-opiomelanocortin promoter directs expression of the human RB gene to melanotrophs (TgPOMC-RB). In three independent lines, transgenic mice crossed to Rb+/− background are devoid of melanotroph tumors but develop the usual spectrum of other neoplasms. Interestingly, abrogation of melanotroph carcinogenesis results in accelerated progression of pituitary anterior lobe tumors and medullary thyroid carcinomas. A combination of immunologic tests, cell culture studies, and tumorigenicity assays indicates that α-melanocyte–stimulating hormone, which is overproduced by melanotroph tumors, attenuates neoplastic progression by decreasing cell proliferation and inducing apoptosis. Taken together, we show that cell lineage–specific complementation of Rb function can be successfully used for refining available models of stochastic carcinogenesis and identify α-melanocyte–stimulating hormone as a potential attenuating factor during progression of neuroendocrine neoplasms.

Nestin-Immunoreactive Cells in Rat Pituitary Are neither Hormonal nor Typical Folliculo-Stellate Cells

Nestin is an intermediate filament protein that has originally been identified as a marker of neuroepithelial stem/progenitor cells. The present study explored whether nestin immunoreactivity (nestin-ir) is present in the rat pituitary and in which cell type(s). Nestin-ir was observed in scattered cells in the anterior, intermediate, and neural lobes. Nestin-ir cells were predominantly of stellate shape and were more numerous in immature than in adult animals. Nestin-ir did not colocalize with any pituitary hormone, and did not colocalize or only very sporadically with the folliculo-stellate cell marker S100. In the intermediate lobe, nestin-ir cells contained glial fibrillary acidic protein in an age-dependent manner. Nestin-ir cells were closely associated with endothelial and fibronectin-ir cells, but did mostly not coincide. Nestin-ir was not found in alpha-smooth muscle actin-ir myofibroblasts or in microglial cells. Regardless of age, nestin-ir was detected in some unidentifiable cells that border the pituitary cleft. Nestin-ir remained present in pituitary cultured as three-dimensional aggregates. Treatment with basic fibroblast growth factor or leukemia inhibitory factor increased the number of nestin-ir cells. Starting from anterior lobe cell monolayer cultures, nestin-ir cells could be selected and propagated to a virtually pure population. These nestin-ir cells displayed remarkable motility and proliferative activity, and did not express hormones, glial fibrillary acidic protein, or S100, but contained vimentin-, fibronectin-, and alpha-smooth muscle actin-ir. In conclusion, nestin-ir is present in the pituitary in cells that are neither hormonal nor typical folliculo-stellate. The expression pattern depends on age and lobe examined. Pericapillar localization suggests a pericyte phenotype for some of them. Whether the heterogeneous nestin-ir population also contains pituitary progenitor cells remains to be explored.