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The mammalian pituitary gland drives highly conserved physiological processes such as somatic cell growth, pubertal transformation, fertility, and metabolism by secreting a variety of hormones. Recently, single-cell transcriptomics techniques have been used in pituitary gland research. However, more studies have focused on adult pituitary gland tissues from different species or different sexes, and no research has yet resolved cellular differences in pituitary gland tissue before and after sexual maturation. Here, we identified a total of 15 cell clusters and constructed single-cell transcriptional profiles of rats before and after sexual maturation. Furthermore, focusing on the gonadotrope cluster, 106 genes were found to be differentially expressed before and after sexual maturation. It was verified that Spp1, which is specifically expressed in gonadotrope cells, could serve as a novel marker for this cell cluster and has a promotional effect on the synthesis and secretion of follicle-stimulating hormone. The results provide a new resource for further resolving the regulatory mechanism of pituitary gland development and pituitary hormone synthesis and secretion. [ABSTRACT FROM AUTHOR]

Endothelial fenestrae are transcellular pores separated by diaphragms formed by plasmalemma vesicle-associated proteins (PLVAP) and function as channels for peptide hormones and other substances. Caveola, a key regulator of clathrin-independent endocytosis, may be involved in the invagination and fusion of plasma membranes, which are essential for fenestra formation. In this study, we first found that caveolin-1 and -2, the major components of caveolae, was localized in fenestrated endothelial cells in the anterior lobe of the rat pituitary by immunohistochemistry. As we also observed caveolae in the endothelial cells of the anterior lobe of the rat pituitary by transmission electron microscopy, we studied the relationship between the caveolae-mediated endocytosis pathway and fenestrae structure in cultured endothelial cells isolated from the anterior lobe of the rat pituitary (CECAL) by immunofluorescence staining and scanning electron microscopy. The inhibition of caveolae-mediated endocytosis by genistein enlarged the PLVAP-positive oval-shaped structure that represented the sieve plate and induced the formation of a doughnut-shaped bulge around the fenestra in CECAL. In contrast, the acceleration of caveolae-mediated endocytosis by okadaic acid induced the diffusion of PLVAP-positive signals in the cytoplasm and reduced the number of fenestrae in CECAL. These results indicate that the caveolae-mediated endocytosis pathway is involved in the fenestra homeostasis in the fenestrated endothelial cells of the rat pituitary. • Caveolae are present in fenestrated endothelial cells of the rat pituitary. • The inhibition of caveolae-mediated endocytosis by genistein induces the enlargement of the sieve plates. • The acceleration of caveolae-mediated endocytosis by okadaic acid reduces the number of the endothelial fenestrae. • Caveolae-mediated endocytosis pathway regulated by caveolin-1 and -2 is involved in the endothelial fenestra homeostasis. [ABSTRACT FROM AUTHOR]

The mammalian pituitary gland drives highly conserved physiological processes such as somatic cell growth, pubertal transformation, fertility, and metabolism by secreting a variety of hormones. Recently, single-cell transcriptomics techniques have been used in pituitary gland research. However, more studies have focused on adult pituitary gland tissues from different species or different sexes, and no research has yet resolved cellular differences in pituitary gland tissue before and after sexual maturation. Here, we identified a total of 15 cell clusters and constructed single-cell transcriptional profiles of rats before and after sexual maturation. Furthermore, focusing on the gonadotrope cluster, 106 genes were found to be differentially expressed before and after sexual maturation. It was verified that Spp1, which is specifically expressed in gonadotrope cells, could serve as a novel marker for this cell cluster and has a promotional effect on the synthesis and secretion of follicle-stimulating hormone. The results provide a new resource for further resolving the regulatory mechanism of pituitary gland development and pituitary hormone synthesis and secretion.

Endothelial fenestrae are transcellular pores divided by a diaphragm consisting of plasmalemma vesicle-associated protein (PLVAP). They function as a channel for peptide hormones and other substances. Invagination of the plasma membrane is necessary for the fenestra formation. The actin cytoskeleton is essential for scission of endocytic vesicles from the invaginated plasma membrane. Therefore, we examined the involvement of the actin cytoskeleton in fenestra formation in cultured endothelial cells isolated from the anterior lobe (AL) of the rat pituitary, using immunofluorescence and scanning electron microscopy. Inhibition of polymerization and depolymerization of the actin cytoskeleton by latrunculin A and jasplakinolide, respectively, remarkably increased the PLVAP-positive sieve plate area and number of fenestrae. Jasplakinolide significantly affected the arrangement of the fenestra on the cell surface, resulting in parallel serpentine furrows of the fenestra. These results suggest that the actin cytoskeleton not only induces fenestra formation but also regulates cell arrangement. Dynamin is a scission protein of the invaginated plasma membrane and interacts with the actin cytoskeleton. We found that dynamin2 is mainly expressed in the endothelial cells of the rat AL. We then investigated the function of dynamin2 by the treatment with dyngo-4a, a potent inhibitor of dynamin1 and dynamin2, on the fenestra formation. As a result, the PLVAP-positive area is significantly increased by the treatment. These results show that the actin-dynamin2 interaction is essential for the control of the fenestra formation in endothelial cells of rat AL. In conclusion, the actin cytoskeleton and dynamin2 function as regulators of endothelial fenestra formation. [ABSTRACT FROM AUTHOR]

Pituitary endocrine cells are supplied by Sox2-expressing stem/progenitor cells in the anterior lobe of the adult pituitary gland. These SOX2-positive cells are maintained in two types of microenvironments (niches): the marginal cell layer (MCL)-niche and the parenchymal-niche. Recently, we isolated dense SOX2-positive cell clusters from the parenchymal-niche by taking advantage of their resistance to protease treatment as parenchymal stem/progenitor cell (PS)-clusters. In the present study, by analyzing these isolated PS-clusters, we attempted to identify novel structural characteristics of pituitary stem/progenitor cell niches. Quantitative real-time PCR showed that tight junction-related genes were distinctly expressed in the isolated PS-clusters. Immunocytostaining showed that the tight junction molecules, ZO-1 and occludin, were localized in the apical membrane facing the pseudo-follicle-like structure of the isolated PS-clusters regardless of the expression of S100β, which distinguishes the sub-population of SOX2-positive cells. Furthermore, immunohistochemistry of the pituitary glands of adult rats clearly demonstrated that ZO-1 and occludin were densely present in the parenchymal-niche encircling the pseudo-follicle, while they were observed in the apical membrane in the MCL-niche facing the residual lumen. Collectively, these tight junction-related proteins might be involved in the architecture and maintenance of the plasticity of pituitary stem/progenitor cell niches.

Chronic stress as one of the most significant risk factor can trigger overactivity of hypothalamic-pituitary-adrenal (HPA) axis in depression as well as anxiety. Yet, the shared and unique neurobiological underpinnings underlying the pituitary abnormality in these two disorders have not been made clear. We previously have established depression-susceptible, anxiety-susceptible and insusceptible groups using a valid chronic mild stress (CMS) model. In this work, the possible protein expression changes in the rat pituitary of these three groups were continuously investigated through the use of the comparative quantitative proteomics and bioinformatics approaches. The pituitary-proteome analysis identified totally 197 differential proteins as a CMS response. These deregulated proteins were involved in diverse biological functions and significant pathways potentially connected with the three different behavioral phenotypes, likely serving as new investigative protein targets. Afterwards, parallel reaction monitoring-based independent analysis found out that expression alterations in Oxct1 , Sec24c , Ppp1cb , Dock1 , and Coq3 ; Lama1 , Glb1 , Gapdh , Sccpdh , and Renbp ; Sephs1 , Nup188 , Spp1 , Prodh1 , and Srm were specifically linked to depression-susceptible, anxiety-susceptible and insusceptible groups, respectively, suggesting that the same CMS had different impacts on the pituitary protein regulatory system. Collectively, the current proteomics research elucidated an important molecular basis and furnished new valuable insights into neurochemical commonalities and specificities of the pituitary dysfunctional mechanisms in HPA axis underlying vulnerability and resistance to stress-induced anxiety or depression. [ABSTRACT FROM AUTHOR]

Chronic stress as one of the most significant risk factor can trigger overactivity of hypothalamic-pituitary-adrenal (HPA) axis in depression as well as anxiety. Yet, the shared and unique neurobiological underpinnings underlying the pituitary abnormality in these two disorders have not been made clear. We previously have established depression-susceptible, anxiety-susceptible and insusceptible groups using a valid chronic mild stress (CMS) model. In this work, the possible protein expression changes in the rat pituitary of these three groups were continuously investigated through the use of the comparative quantitative proteomics and bioinformatics approaches. The pituitary-proteome analysis identified totally 197 differential proteins as a CMS response. These deregulated proteins were involved in diverse biological functions and significant pathways potentially connected with the three different behavioral phenotypes, likely serving as new investigative protein targets. Afterwards, parallel reaction monitoring-based independent analysis found out that expression alterations in Oxct1, Sec24c, Ppp1cb, Dock1, and Coq3; Lama1, Glb1, Gapdh, Sccpdh, and Renbp; Sephs1, Nup188, Spp1, Prodh1, and Srm were specifically linked to depression-susceptible, anxiety-susceptible and insusceptible groups, respectively, suggesting that the same CMS had different impacts on the pituitary protein regulatory system. Collectively, the current proteomics research elucidated an important molecular basis and furnished new valuable insights into neurochemical commonalities and specificities of the pituitary dysfunctional mechanisms in HPA axis underlying vulnerability and resistance to stress-induced anxiety or depression.

Endothelial fenestrae are the transcellular pores existing on the capillary walls which are organized in clusters referred to as sieve plates. They are also divided by a diaphragm consisting of plasmalemma vesicle-associated protein (PLVAP). In this study, we examined the involvement of fibronectin signaling in the formation of fenestra and diaphragm in endothelial cells. Results showed that Itga5 and Itgb1 were expressed in PECAM1-positive endothelial cells isolated from the anterior lobe (AL) of the rat pituitary, and integrin α5 was localized at the fenestrated capillaries of the rat pituitary and cultured PECAM1-positive endothelial cells isolated from AL (CECAL). Inhibition of both integrin α5β1 and FAK, a key molecule for integrin-microtubule signaling, respectively, by ATN-161 and FAK inhibitor 14, caused the delocalization of PLVAP at the sieve plates and depolymerization of microtubules in CECAL. Paclitaxel prevented the delocalization of PLVAP by the inhibition of integrin α5β1. Microtubule depolymerization induced by colcemid also caused the delocalization of PLVAP. Treatment of CECAL with ATN-161 and colcemid caused PLVAP localization at the Golgi apparatus. The localization of PLVAP at the sieve plates was inhibited by BFA treatment in a time-dependent manner and spread diffusely to the cytoplasm. These results indicate that a constant supply of PLVAP proteins by the endomembrane system via the Golgi apparatus is essential for the localization of PLVAP at sieve plates. In conclusion, the endomembrane transport pathway from the Golgi apparatus to sieve plates requires microtubule cytoskeletons, which are regulated by fibronectin-integrin α5β1 signaling. [ABSTRACT FROM AUTHOR]

Endothelial fenestrae are transcellular pores that pierce the capillary walls in endocrine glands such as the pituitary. The fenestrae are covered with a thin fibrous diaphragm consisting of the plasmalemma vesicle–associated protein (PLVAP) that clusters to form sieve plates. The basal surface of the vascular wall is lined by basement membrane (BM) composed of various extracellular matrices (ECMs). However, the relationship between the ECMs and the endothelial fenestrae is still unknown. In this study, we isolated fenestrated endothelial cells from the anterior lobe of the rat pituitary, using a dynabeads-labeled antibody against platelet endothelial cell adhesion molecule 1 (PECAM1). We then analyzed the gene expression levels of several endothelial marker genes and genes for integrin α subunits, which function as the receptors for ECMs, by real-time polymerase chain reaction (PCR). The results showed that the genes for the integrin α subunit, which binds to collagen IV, fibronectin, laminin-411, or laminin-511, were highly expressed. When the PECAM1-positive cells were cultured for 7 days on collagen IV-, fibronectin-, laminins-411-, or laminins-511-coated coverslips, the sieve plate structures equipped with probably functional fenestrae were maintained only when the cells were cultured on fibronectin. Additionally, real-time PCR analysis showed that the fibronectin coating was effective in maintaining the expression pattern of several endothelial marker genes that were preferentially expressed in the endothelial cells of the fenestrated capillaries. These results indicate that fibronectin functions as the principal factor in the maintenance of the sieve plate structures in the endothelial cells of the fenestrated capillary. [ABSTRACT FROM AUTHOR]

• Cu-GnRH activates IP 3 /PKC and cAMP/PKA-induced gonadotrope gene transcription. • Cu-GnRH transcriptional effectiveness depends on pulse frequency. • Cu-GnRH operates via GnRHR and PAC1 receptor activation. Unlike gonadotropin-releasing hormone (GnRH) analogues characterized by amino acid replacement in decapeptide primary structure, Cu-GnRH molecule preserves the native sequence but contains a Cu2+ ion stably bound to the nitrogen atoms including that of the imidazole ring of His2. Cu-GnRH can operate via cAMP/PKA signalling in anterior pituitary cells, suggesting that it may affect selected gonadotropic network gene transcription in vivo. We analysed pituitary mRNA expression of Egr-1 , Nr5a1, and Lhb based on their role in luteinizing hormone (LH) synthesis; and Nos1, Adcyap1, and Prkaca due to their dependence on cAMP/PKA activity. In two independent experiments, ovariectomized rats received intracerebroventricular pulsatile (one pulse/h or two pulses/h over 5 h) microinjections of 2 nM Cu-GnRH; 2 nM antide (GnRH antagonist) + 2 nM Cu-GnRH; 100 nM PACAP 6–38 (PACAP receptor antagonist) + 2 nM Cu-GnRH. Relative expression of selected mRNAs was determined by qRT-PCR. LH serum concentration was examined according to RIA. All examined genes responded to Cu-GnRH stimulation with increased transcriptional activity in a manner dependent on pulse frequency pattern. Increased expression of Nr5a1 , Lhb, Nos1, Adcyap1, and Prkaca mRNA was observed solely in rats receiving the complex with frequency of two pulses/h over 5 h. Egr-1 transcription was up-regulated for both applied Cu-GnRH pulsatile patterns. The stimulatory effect of Cu-GnRH on gene transcription was dependent on both GnRH receptor and PAC-1 activation. In conclusion, obtained results indicate that Cu-GnRH complex is a GnRH analogue able to induce both IP 3 /PKC and cAMP/PKA-dependent gonadotrope network gene transcription in vivo. [ABSTRACT FROM AUTHOR]

Sox2-expressing stem/progenitor cells in the anterior lobe of the pituitary gland form two types of micro-environments (niches): the marginal cell layer and dense cell clusters in the parenchyma. In relation to the mechanism of regulation of niches, juxtacrine signaling via ephrin and its receptor Eph is known to play important roles in various niches. The ephrin and Eph families are divided into two subclasses to create ephrin/Eph signaling in co-operation with confined partners. Recently, we reported that ephrin-B2 localizes specifically to both pituitary niches. However, the Ephs interacting with ephrin-B2 in these pituitary niches have not yet been identified. Therefore, the present study aims to identify the Ephs interacting with ephrin-B2 and the cells that produce them in the rat pituitary gland. In situ hybridization and immunohistochemistry demonstrated cell type-specific localization of candidate interacting partners for ephrin-B2, including EphA4 in cells located in the posterior lobe, EphB1 in gonadotropes, EphB2 in corticotropes, EphB3 in stem/progenitor cells and EphB4 in endothelial cells in the adult pituitary gland. In particular, double-immunohistochemistry showed cis-interactions between EphB3 and ephrin-B2 in the apical cell membranes of stem/progenitor cell niches throughout life and trans-interactions between EphB2 produced by corticotropes and ephrin-B2 located in the basolateral cell membranes of stem/progenitor cells in the early postnatal pituitary gland. These data indicate that ephrin-B2 plays a role in pituitary stem/progenitor cell niches by selective interaction with EphB3 in cis and EphB2 in trans. [ABSTRACT FROM AUTHOR]

The pituitary gland, an indispensable endocrine organ that synthesizes and secretes pituitary hormones, develops with the support of many factors. Among them, neuronatin (NNAT), which was discovered in the neonatal mouse brain as a factor involved in neural development, has subsequently been revealed to be coded by an abundantly expressing gene in the pituitary gland but its role remains elusive. We analyze the expression profile of Nnat and the localization of its product during rat pituitary development. The level of Nnat expression was high during the embryonic period but remarkably decreased after birth. Immunohistochemistry demonstrated that NNAT appeared in the SOX2-positive stem/progenitor cells in the developing pituitary primordium on rat embryonic day 11.5 (E11.5) and later in the majority of SOX2/PROP1 double-positive cells on E13.5. Thereafter, during pituitary embryonic development, Nnat expression was observed in some stem/progenitor cells, proliferating cells and terminally differentiating cells. In postnatal pituitaries, NNAT-positive cells decreased in number, with most coexpressing Sox2 or Pit1, suggesting a similar role for NNAT to that during the embryonic period. NNAT was widely localized in mitochondria, peroxisomes and lysosomes, in addition to the endoplasmic reticulum but not in the Golgi. The present study thus demonstrated the variability in expression of NNAT-positive cells in rat embryonic and postnatal pituitaries and the intracellular localization of NNAT. Further investigations to obtain functional evidence for NNAT are a prerequisite. [ABSTRACT FROM AUTHOR]

The copper-gonadotropin-releasing hormone molecule (Cu-GnRH) is a GnRH analog, which preserves its amino acid sequence, but which contains a Cu 2+ ion stably bound to the nitrogen atoms including that of the imidazole ring of Histidine 2 . A previous report indicated that Cu-GnRH was able to activate cAMP/PKA signaling in anterior pituitary cells in vitro, but raised the question of which intracellular mechanism(s) mediated the Cu-GnRH-induced cAMP synthesis in gonadotropes. To investigate this mechanism, in the present study, female rat anterior pituitary cells in vitro were pretreated with 0.1 μM antide, a GnRH antagonist; 0.1 μM cetrorelix, a GnRH receptor antagonist; 0.1 μM PACAP 6–38 , a PAC-1 receptor antagonist; 2 μM GF109203X, a protein kinase C inhibitor; 50 mM PMA, a protein kinase C activator; the protein kinase A inhibitors H89 (30 μM) and KT5720 (60 nM); factors affecting intracellular calcium activity: 2.5 mM EGTA; 2 μM thapsigargin; 5 μM A23187, a Ca 2+ ionophore; or 10 μg/ml cycloheximide, a protein synthesis inhibitor. After one of the above pretreatments, cells were incubated in the presence of 0.1 μM Cu-GnRH for 0.5, 1, and 3 h. Radioimmunoassay analysis of cAMP confirmed the functional link between Cu-GnRH stimulation and cAMP/PKA signal transduction in rat anterior pituitary cells, demonstrating increased intracellular cAMP, which was reduced in the presence of specific PKA inhibitors. The stimulatory effect of Cu-GnRH on cAMP production was partly dependent on GnRH receptor activation. In addition, an indirect and Ca 2+ -dependent mechanism might be involved in intracellular adenylate cyclase stimulation. Neither activation of protein kinase C nor new protein synthesis was involved in the Cu-GnRH-induced increase of cAMP in the rat anterior pituitary primary cultures. Presented data indicate that conformational changes of GnRH molecule resulting from cooper ion coordination affect specific pharmacological properties of Cu-GnRH molecule including specific pattern of intracellular activity induced by complex in anterior pituitary cells in vitro . [ABSTRACT FROM AUTHOR]

Paired-related homeobox transcription factors, PRRX1 and PRRX2, are known to be important factors for craniofacial and limb morphogenesis. We recently cloned Prrx2 from the porcine adult pituitary cDNA library and found that only PRRX1 is present in the rat embryonic pituitary. In this study, we re-investigated the temporospatial expression and localization of PRRX1 and PRRX2 in the rat pituitary throughout life. The persistent expression of Prrx1 was ascertained after the middle stage of embryonic development, whereas significant expression of Prrx2 was found only in the postnatal pituitary. Immunohistochemistry confirmed that PRRX1-positive cells appeared inside the pituitary on embryonic day 16.5 in the marginal cell layer (MCL), a pituitary stem/progenitor cell niche, and the expanding parenchyma of the anterior pituitary. In contrast, PRRX2-positive cells first appeared in the anterior lobe and intermediate lobe sides of the MCL around postnatal day 30 when the postnatal pituitary growth wave had almost terminated. Immunostaining for PRRX1 with a stem/progenitor cell marker SOX2, a pituitary progenitor marker PROP1, or pituitary hormones revealed that PRRX1 localized in cells in the transition process from the multipotent progenitor stage to the early stage of terminal differentiation throughout life. PRRX2 emerged in cells positive for SOX2 but negative for PROP1 in the anterior and intermediate lobe sides of the postnatal MCL. Thus, PRRX1 and PRRX2 might participate distinctly in pituitary organogenesis and the postnatal cell-supply system. [ABSTRACT FROM AUTHOR]

Pituitary endocrine cells are supplied by Sox2-expressing stem/progenitor cells in the anterior lobe of the adult pituitary gland. These SOX2-positive cells are maintained in two types of microenvironments (niches): the marginal cell layer (MCL)-niche and the parenchymal-niche. Recently, we isolated dense SOX2-positive cell clusters from the parenchymal-niche by taking advantage of their resistance to protease treatment as parenchymal stem/progenitor cell (PS)-clusters. In the present study, by analyzing these isolated PS-clusters, we attempted to identify novel structural characteristics of pituitary stem/progenitor cell niches. Quantitative real-time PCR showed that tight junction-related genes were distinctly expressed in the isolated PS-clusters. Immunocytostaining showed that the tight junction molecules, ZO-1 and occludin, were localized in the apical membrane facing the pseudo-follicle-like structure of the isolated PS-clusters regardless of the expression of S100β, which distinguishes the sub-population of SOX2-positive cells. Furthermore, immunohistochemistry of the pituitary glands of adult rats clearly demonstrated that ZO-1 and occludin were densely present in the parenchymal-niche encircling the pseudo-follicle, while they were observed in the apical membrane in the MCL-niche facing the residual lumen. Collectively, these tight junction-related proteins might be involved in the architecture and maintenance of the plasticity of pituitary stem/progenitor cell niches.

Abstract: We recently described patterns of GnRH and GnRH receptor (GnRH-R) expression in the hypothalamus, pituitary and ovary throughout the rat estrus cycle. Here, we wished to distinguish between regulatory effects of ovarian factors and underlying circadian rhythmicity. We quantified GnRH and GnRH-R mRNA in the pituitary and hypothalamus of long-term ovariectomized (OVX) rats, at different times of day, using real-time PCR. Furthermore, we expanded our previous study of hypothalamic and pituitary GnRH and GnRH-R expression in intact rats by including more time points throughout the estrus cycle. We found different daily patterns of GnRH and GnRH-R expression in intact versus OVX rats, in both tissues. In the hypothalamus of OVX rats, GnRH mRNA peaked at 12, 16 and 20h, whereas in the hypothalamus of intact rats we observed somewhat higher GnRH mRNA concentrations at 19h on every day of the estrus cycle except proestrus, when the peak occurred at 17h. In this tissue, GnRH-R fluctuated less significantly and peaked at 16h in OVX rats. During the estrus cycle, we observed higher levels in the afternoon of each day except on estrus. In OVX rats, pituitary GnRH mRNA rose sharply at 9h, with low levels thereafter. In these animals, pituitary GnRH-R also peaked at 9h followed by a second rise at 22h. In intact rats pituitary GnRH was high at noon of diestrus-II and on estrus, whereas GnRH-R mRNA was highest in the evening of diestrus-II. This is the first demonstration of daily GnRH and GnRH-R mRNA expression patterns in castrated animals. The observed daily fluctuations hint at underlying tissue-specific circadian rhythms. Ovarian factors probably modulate these rhythms, yielding the observed estrus cycle patterns. [Copyright &y& Elsevier]

Leptin and neuropeptide-Y (NPY) are indicated to play a role in hypothalamo-pituitary-thyroid (HPT) axis in relation to regulation of energy homeostasis mediated through acting at hypothalamic synthesis and release of thyrotropin (TSH)-releasing hormone (TRH). Whether leptin and NPY also act at pituitary level in HPT axis remains unknown. This study aimed at investigating whether or not leptin and NPY exert actions at pituitary in modulation of transcript levels of TSHβ and the common pituitary glycoprotein hormone α (PGHα) subunits. The dispersed pituitary cells from 6 wk old male Wistar rats were incubated with or without TRH, leptin or NPY of 10−8 M and 10−10 M for 6 h at 37 °C in medium-199 under aeration of 95% O2 and 5% CO2. The mRNA levels of TSHβ and PGHα subunits of the incubated cells were measured by reverse transcription-polymerase chain reaction. The results revealed that leptin stimulated, while NPY inhibited, TSHβ mRNA levels in a dose-related manner. Both leptin and NPY increased α subunit mRNA levels. It is demonstrated for the first time that both leptin and NPY exert a direct action at rat pituitary affecting steady-state levels of mRNA of TSHβ and PGHα subunits. The present study supports that both leptin and NPY act at the pituitary as well besides the hypothalamus in HPT axis in relation to regulation of energy homeostasis. [Copyright &y& Elsevier]

We use a flow-through cell perifusion system allowing continuous sampling of Luteinizing Hormone (LH) release from excised pituitary tissue. With this system, we are able to sample from viable tissue in primary culture for three days as opposed to a more typical of primary culture of 6–12 hours. Rat pituitary tissue was perifused and challenged with a physiological dose of LHRH. Samples were collected for 3.5 hours on each of 3 consecutive days in a refrigerated fraction collector and assayed for LH concentration by radioimmunoassay. In these experiments we tested two types of media, each with or without fetal calf serum supplementation, for their ability to support pituitary tissue for extended lengths of perifusion. In addition, we broadened the use of aseptic technique, sterile media and equipment to limit microbial contamination. Our results indicate that perifusion of pituitary tissue can be done successfully for 3 or more days at a modest cost, greatly increasing the amount of information that can be collected from each piece of tissue harvested. [ABSTRACT FROM AUTHOR]

The release of neuropeptides and other proteinaceous hormones by exocytosis was studied in some detail by means of tannic acid in combination with glutaraldehyde (TAGO-method). This method strongly enhances the electron density of extracellular proteins including exocytosed secretory products, and therefore facilitates visualization of the release process in qualitative and quantitative respects. This study included a variety of neurosecretory cell types in the CNS of the freshwater snail L. stagnalis, the storage and glandular lobes of the corpora cardiaca of the locust L. migratoria, and the posterior pituitary of the rat. In addition, peptide secreting cells in the anterior pituitary were examined. The cytochemical specificity of the TAGO-method as well as the significance of 'frozen' contents of exocytosing secretory granules in mammotrophs and somatotrophs of the rat is discussed. [ABSTRACT FROM AUTHOR]

The central catecholamine innervation of the pituitary neural lobe and pars intermedia of the rat have been identified ultrastructurally and their organization has been investigated in a combined fluorescence histochemical and electron microscopical study. The dopamine analogues, 5-hydroxydopamine and 6-hydroxydopamine, were used to 'label' the catecholamine terminals, and to enable the direct correlation between the fluorescence microscopical and the electron microscopical pictures. The fibre type that was identified as catecholamine-containing was ultrastructurally chiefly characterized by dense-cored vesicles, 500-1200 Å in diameter, intermingled with varying numbers of small empty vesicles. 5-hydroxydopamine was selectively accumulated in these fibres and caused an increased electron density of the granular vesicles as well as of some small normally agranular vesicles, and systemically administered 6-hydroxydopamine caused a selective degeneration of these fibres, most prominently within the neural lobe. The dopaminergic terminals of the neural lobe showed frequent close contacts (80-120 Å), without real membrane thickenings, to neurosecretory axons and to pituicyte processes. It is suggested that these close contacts might signify a direct dopaminergic influence on the neurosecretory axons and/or on the pituicyte processes. The identified central catecholamine fibres were also found to make common synapse-like contacts on the pars intermedia cells, whereas the innervation by neurosecretory fibres was very rare. This suggests that the direct central nervous control of the rat pars intermedia is exerted by the catecholamine neurons. A very special feature of the catecholamine fibres in the pituitary is the occurrence of peculiar, large dopamine-filled droplet-like swellings. Electron microscopically, such large axonal swellings (more than 2 μ in diameter) were found to contain, in addition to the characteristic vesicles and organelles, strongly osmiophilic lamellated membrane complexes resembling myelin bodies and multivesicular bodies encircling disintegrated vesicles, suggesting that these 'droplet fibres' represent dilated stumps of spontaneously degenerating dopaminergic axons. It is suggested that the dopaminergic neural lobe fibres are undergoing continuous reorganization through degeneration-regeneration cycles, a phenomenon previously suggested for the neurosecretory axons of the neural lobe. [ABSTRACT FROM AUTHOR]

Using indomethacin (Ind), a prostaglandin, synthesis inhibitor, in vivo experiments in rats and in vitro experiments with perifusion systems of rat thyroids and pituitaries were conducted. After 35 days of intragastric infusion of Ind, serum TSH levels were markedly increased, the thyroid was swollen and, as a consequence, T and T levels were normal. The T release from perifused rat thyroids under continuous stimulation with 10 mU/ml TSH was inhibited significantly (p<0.01) by 1.0×10 M Ind. On the other hand, the TSH release from perifused rat pituitaries under TRH stimulation was enhanced conspicuously by Ind. It was concluded that Ind decelerated thyroid hormone release from the thyroid and accelerated TSH release from the pituitary in perifusion systems. [ABSTRACT FROM AUTHOR]

The relationship between the pineal gland and the pituitary gland was investigated in male rats. The results indicate that the hypothalamo-adenohypophysial-gonadal axis is affected by the pineal gland, but the appearance of castration cells following gonal ablation may be only slightly modified by alterations in pineal gland function. [ABSTRACT FROM AUTHOR]