Cultures Of Anterior Pituitary Cells Research Articles

Plasma level of omentin-1, its expression, and its regulation by gonadotropin-releasing hormone and gonadotropins in porcine anterior pituitary cells

Omentin-1 (OMNT1) is an adipokine involved in the regulation of energy metabolism, insulin sensitivity, and reproduction. The present study was the first to investigate the plasma levels and expression of OMNT1 in the anterior pituitary (AP) gland on days 2–3, 10–12, 14–16, and 17–19 of the estrous cycle of normal-weight Large White (LW) and fat Meishan (MS) pigs. Next, we determined the effect of GnRH, LH, and FSH on the OMNT1 levels in cultured AP cells. The gene and protein expression of OMNT1 in AP fluctuated during the estrous cycle, with a higher expression in MS than in LW (except on days 10–12). However, plasma levels of OMNT1 were higher in LW than in MS. OMNT1 was localized in somatotrophs, lactotrophs, thyrotrophs, and gonadotrophs. In LW pituitary cells, GnRH and gonadotropins stimulated OMNT1 protein expression (except FSH on days 14–16) and had no effect on OMNT1 levels in the culture medium. In MS pituitary cells, we observed that GnRH and LH increased while FSH decreased OMNT1 protein expression. These findings showed OMNT1 expression and regulation in the porcine AP and suggested that OMNT1 could be a new player modifying the pituitary functions.

Regulation of FGF2-induced proliferation by inhibitory GPCR in normal pituitary cells.

Intracellular communication is essential for the maintenance of the anterior pituitary gland plasticity. The aim of this study was to evaluate whether GPCR-Gαi modulates basic fibroblast growth factor (FGF2)-induced proliferative activity in normal pituitary cell populations. Anterior pituitary primary cell cultures from Wistar female rats were treated with FGF2 (10ng/mL) or somatostatin analog (SSTa, 100nM) alone or co-incubated with or without the inhibitors of GPCR-Gαi, pertussis toxin (PTX, 500nM), MEK inhibitor (U0126, 100µM) or PI3K inhibitor (LY 294002, 10 μM). FGF2 increased and SSTa decreased the lactotroph and somatotroph BrdU uptak2e (p<0.05) whereas the FGF2-induced S-phase entry was prevented by SSTa co-incubation in both cell types, with these effects being reverted by PTX, U0126 or LY294002 pre-incubation. The inhibition of lactotroph and somatotroph mitosis was associated with a downregulation of c-Jun expression, a decrease of phosphorylated (p) ERK and pAKT. Furthermore, SSTa was observed to inhibit the S-phase entry induced by FGF2, resulting in a further increase in the number of cells in the G1 phase and a concomitant reduction in the number of cells in the S phases (p< 0.05), effects related to a decrease of cyclin D1 expression and an increase in the expression of the cell cycle inhibitors p27 and p21. In summary, the GPCR-Gαi activated by SSTa blocked the pro-proliferative effect of FGF2 in normal pituitary cells via a MEK-dependent mechanism, which acts as a mediator of both anti and pro-mitogenic signals, that may regulate the principal effectors of the G1 to S-phase transition.

Influence of brain plasmalogen changes on gonadotropin secretion from the cultured bovine anterior pituitary cells

We recently discovered that the orphan G-protein–coupled receptor (GPR) 61 colocalized with GnRH receptors (GnRHRs) on the surface of most of bovine gonadotrophs. A recent study suggested that ethanolamine plasmalogen (PI) is a ligand for GPR61 in mouse neuroblastoma. Therefore, this study evaluated the hypothesis that PI alters LH and FSH secretion from cultured bovine anterior pituitary (AP) cells. We prepared bovine AP cells from postpubertal heifers (26 mo old) and cultured the cells for 3.5 d. We treated the cells with increasing concentrations (0, 5, 50, 500, 5,000, 50,000, or 500,000 pg/mL) of phosphoethanolamine PI (PEPI) extracted from the bovine brain, or l-α-lysophosphatidylethanolamine PI (LEPI) extracted from the bovine brain, for 5 min before either no treatment or GnRH stimulation. The medium samples were harvested 2 h after culture for LH and FSH assays. Phosphoethanolamine PI (50–500 pg/mL) stimulated (P < 0.05) the basal secretion of FSH but not LH. Phosphoethanolamine PI at 50 pg/mL also enhanced (P < 0.05) GnRH-induced FSH secretion. However, higher doses (500–500,000 pg/mL) of PEPI suppressed GnRH-induced FSH secretion. Moreover, 50 to 500,000 pg/mL PEPI suppressed GnRH-induced LH secretion. None of the tested concentrations of LEPI showed any effect on basal or GnRH-induced LH or FSH secretion. Pretreatment with Sma and Mad pathway inhibitors suppressed FSH secretion induced by PEPI, whereas an extracellular signal-regulated kinase pathway inhibitor blocked the PEPI-induced suppression of GnRH-stimulated LH secretion. Therefore, PEPI, but not LEPI, extracted from the bovine brain, alters FSH and LH secretion from cultured AP cells. Further studies are required to decide whether PEPI binds to GPR61 and whether PEPI plays an important role in the control of gonadotropin secretion from gonadotrophs.

Heifers express G-protein coupled receptor 153 in anterior pituitary gonadotrophs in stage-dependent manner.

We recently found that orphan G-protein-coupled receptor (GPR)153 is expressed in the anterior pituitary (AP) of heifers, leading us to speculate that GPR153 colocalizes with gonadotropin-releasing hormone receptor (GnRHR) in the plasma membrane of gonadotrophs and is expressed at specific times of the reproductive cycle. To test this hypothesis, we examined the coexpression of GnRHR, GPR153, and either luteinizing hormone or follicle-stimulating hormone in AP tissue and cultured AP cells by immunofluorescence microscopy. GPR153 was detected in the gonadotrophs, and was colocalized with GnRHR in the plasma membrane. GPR153 was also detected in the cytoplasm of cultured gonadotrophs. Real-time PCR and western blot analyses found that expression was lower (P<0.05) in AP tissues during early luteal phase as compared to pre-ovulation or late luteal phases. The 5'-flanking region of the GPR153 gene contained a consensus response element sequence for estrogen, but not for progesterone. These data suggest that some, but not all GPR153 colocalizes with GnRHR in the plasma membrane of gonadotrophs, and its expression changes stage-dependently in the bovine AP.

Heifers express G-protein coupled receptor 61 in anterior pituitary gonadotrophs in stage-dependent manner

Gonadotropin-releasing hormone receptors (GnRHRs) colocalize with insulin and glucocorticoid receptors in lipid rafts of the gonadotroph plasma membrane, where they facilitate downstream signaling. We recently found that orphan G-protein-coupled receptor (GPR)61 is expressed in the anterior pituitary (AP) of heifers, leading us to speculate that GPR61 colocalizes with GnRHR in the plasma membrane of gonadotroph and is expressed at specific times of the reproductive cycle. To test this hypothesis, we examined the coexpression of GnRHR, GPR61, and either luteinizing hormone (LH) β subunit or follicle-stimulating hormone (FSH) β subunit in AP tissue and cultured AP cells by immunofluorescence microscopy. GPR61 was detected in gonadotrophs, with a majority of them being colocalized with GnRHR and the remainder present at other parts of the cell surface or in the cytoplasm. We obtained a strong positive overlap coefficient (0.71±0.01) between GPR61 and GnRHR on the cell-surface of cultured GnRHR-positive AP cells. Real-time PCR and western blot analyses found that expression was lower (P<0.05) in AP tissues during early luteal phase as compared to pre-ovulation or mid- or late luteal phases. Additionally, the 5ꞌ-flanking region of the GPR61 gene contained several sites with response elements similar to those of estrogen or progesterone. These data suggested that GPR61 colocalizes with GnRHR in the plasma membrane of gonadotrophs, and its expression changes stage-dependently in the bovine anterior pituitary gland.

Adipokines (Leptin, Adiponectin, Resistin) Differentially Regulate All Hormonal Cell Types in Primary Anterior Pituitary Cell Cultures from Two Primate Species

Adipose-tissue (AT) is an endocrine organ that dynamically secretes multiple hormones, the adipokines, which regulate key physiological processes. However, adipokines and their receptors are also expressed and regulated in other tissues, including the pituitary, suggesting that locally- and AT-produced adipokines might comprise a regulatory circuit that relevantly modulate pituitary cell-function. Here, we used primary pituitary cell-cultures from two normal nonhuman-primate species [Papio-anubis/Macaca-fascicularis] to determine the impact of different adipokines on the functioning of all anterior-pituitary cell-types. Leptin and resistin stimulated GH-release, a response that was blocked by somatostatin. Conversely, adiponectin decreased GH-release, and inhibited GHRH-, but not ghrelin-stimulated GH-secretion. Furthermore: 1) Leptin stimulated PRL/ACTH/FSH- but not LH/TSH-release; 2) adiponectin stimulated PRL-, inhibited ACTH- and did not alter LH/FSH/TSH-release; and 3) resistin increased ACTH-release and did not alter PRL/LH/FSH/TSH-secretion. These effects were mediated through the activation of common (AC/PKA) and distinct (PLC/PKC, intra-/extra-cellular calcium, PI3K/MAPK/mTOR) signaling-pathways, and by the gene-expression regulation of key receptors/transcriptional-factors involved in the functioning of these pituitary cell-types (e.g. GHRH/ghrelin/somatostatin/insulin/IGF-I-receptors/Pit-1). Finally, we found that primate pituitaries expressed leptin/adiponectin/resistin. Altogether, these and previous data suggest that local-production of adipokines/receptors, in conjunction with circulating adipokine-levels, might comprise a relevant regulatory circuit that contribute to the fine-regulation of pituitary functions.

Rasd1 is an estrogen-responsive immediate early gene and modulates expression of late genes in rat anterior pituitary cells.

Dexamethasone-induced Ras-related protein 1 (Rasd1) is a member of the Ras superfamily of monomeric G proteins that have a regulatory function in signal transduction. Here we investigated the role of Rasd1 in regulating estrogen-induced gene expression in primary cultures of rat anterior pituitary cells. Rasd1 mRNA expression in anterior pituitary cells decreased after treatment with forskolin or serum and increased after treatment with 17β-estradiol (E2). Increases in Rasd1 mRNA expression occurred as early as 0.5 h after E2 treatment, peaked at 1 h and were sustained for as long as 96 h. This rapid and profound increase in Rasd1 mRNA expression induced by E2 was also seen in GH4C1 cells, an estrogen receptor-positive somatolactotroph cell line. Among pituitary estrogen-responsive late genes studied, basal mRNA expression of Pim3 and Igf1 genes was decreased by RNA interference-mediated knockdown of Rasd1 expression, whereas basal expression of the Giot1 gene was increased. Moreover, Rasd1 knockdown enhanced stimulation of Pim3 mRNA expression and attenuated inhibition of Fosl1 mRNA expression 24 h after E2 treatment. These changes in mRNA expression were accompanied by enhanced activity of promoters containing CRE, AP-1 and SRE binding sequences. These results suggest that Rasd1 is an estrogen-responsive immediate early gene and modulates E2 induction of at least several late genes in anterior pituitary cells.

Notch Signaling and Maintenance of SOX2 Expression in Rat Anterior Pituitary Cells.

After publication of reports describing the presence of stem/progenitor cells among non-hormone-producing cells in the pituitary, the mechanism responsible for proliferation and differentiation generated considerable interest. Several studies have suggested that Notch signaling is involved. In the present study, we examined the histochemical relationship between Notch signaling molecules and the transcription factor SOX2 in rat pituitary. Combined in situ hybridization and immunohistochemistry showed that Notch2 mRNA and SOX2 were co-expressed at embryonic day 14.5 in most cells in the adenohypophyseal primordium. In adult rat pituitary, double immunohistochemistry showed that SOX2 and either Notch2 or the Notch signaling target HES1 were co-localized within cells with large oval nuclei in both the marginal cell layer and cell aggregates in the main part of the anterior lobe, which are believed to be stem cell niches. Furthermore, when the Notch signaling inhibitor DAPT was added to a primary culture of adult rat anterior pituitary cells, the proportion of SOX2-expressing cells within Notch2-positive cells was approximately 30% lower. These findings suggest that Notch signaling has a role in maintaining the stemness of precursor cells in the adult rat pituitary gland.

GPR30 mediates estrone, estriol, and estradiol to suppress gonadotropin-releasing hormone-induced luteinizing hormone secretion in the anterior pituitary of heifers.

Recent studies demonstrated that G-protein-coupled receptor 30 (GPR30) on the plasma membrane of gonadotroph cells mediates picomolar, but not nanomolar, levels of estradiol (E2) to rapidly suppress gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion in the anterior pituitary (AP). While estrone (E1) and estriol (E3) are considered “weak” estrogens that exert suppressive effects through estrogen receptors α and β, it is conceivable that they also strongly suppress GnRH-induced LH secretion via GPR30. Both E1 and E3 are likely present within the blood at picomolar or nanomolar concentrations, indicating that such concentrations are sufficient to suppress GnRH-induced LH secretion. To evaluate this possibility, bovine AP cells were cultured under steroid-free conditions and then incubated with various concentrations (0.01 pM to 10 nM) of E2, E1, or E3, prior to stimulation with GnRH. Notably, GnRH-induced LH secretion from AP cells was inhibited by 1–100 pM E2, 1–10 pM E1, and 1–100 pM E3. GnRH-induced LH secretion from AP cells was not inhibited by lower (0.01–0.1 pM) or higher (1–10 nM) concentrations of E2, E1, and E3. These suppressive effects were inhibited by pre-treatment of AP cells with the GPR30 antagonist G36, but not with the estrogen receptor alpha antagonist. Treatment with E1 or E3 also yielded decreased cytoplasmic cAMP levels in cultured AP cells pre-treated with dopamine and phosphodiesterase inhibitors. Therefore, these results suggest that GPR30 mediates the suppressive effects of E1, E3, and E2 on GnRH-induced LH secretion from bovine AP.

In Vivo and In Vitro Arsenic Exposition Induces Oxidative Stress in Anterior Pituitary Gland.

Inorganic arsenic (iAs) is at the top of toxic metalloids. Inorganic arsenic-contaminated water consumption is one of the greatest environmental health threats worldwide. Human iAs exposure has been associated with cancers of several organs, neurological disorders, and reproductive problems. Nevertheless, there are no reports describing how iAs affects the anterior pituitary gland. The aim of this study was to investigate the mechanisms involved in iAs-mediated anterior pituitary toxicity both in vivo and in vitro. We showed that iAs administration (from 5 to 100 ppm) to male rats through drinking water increased messenger RNA expression of several oxidative stress-responsive genes in the anterior pituitary gland. Serum prolactin levels diminished, whereas luteinizing hormone (LH) levels were only affected at the higher dose tested. In anterior pituitary cells in culture, 25 µmol/L iAs significantly decreased prolactin release in a time-dependent fashion, whereas LH levels remained unaltered. Cell viability was significantly reduced mainly by apoptosis evidenced by morphological and phosphatidylserine externalization studies. This process is characterized by early depolarization of mitochondrial membrane potential and increased levels of reactive oxygen species. Expression of some key oxidative stress-responsive genes, such as heme oxygenase-1 and metallothionein-1, was also stimulated by iAs exposure. The antioxidant N-acetyl cysteine prevented iAs-induced effects on the expression of oxidative stress markers, prolactin release, and apoptosis. In summary, the present work demonstrates for the first time that iAs reduces prolactin release both in vivo and in vitro and induces apoptosis in anterior pituitary cells, possibly resulting from imbalanced cellular redox status.

Central effect of vasotocin 4 receptor (VT4R/V1aR) antagonists on the stress response and food intake in chicks given neuropeptide Y (NPY)

Previous studies identified SR-49059 as a most effective antagonist of the avian vasotocin 4 receptor (VT4R) compared to other candidate blockers including the Manning compound using in silico 3 dimensional (3D) modeling/docking analysis of the chicken VT4R and an in vitro anterior pituitary cell culture study. The present experiments were designed to validate whether SR-49059 and the Manning compound would likewise be effective in vivo in blocking the VT4R when applied intracerebroventricularly (ICV) to chicks. Two treatments were tested, a stressor (immobilization) and administration of neuropeptide Y (NPY), a potent orexigenic compound. In the first experiment, birds were given the Manning compound, SR-49059 or physiological saline ICV followed by immobilization stress. Blood samples were taken and corticosterone (CORT) was determined by radioimmunoassay. It was hypothesized that both antagonists would reduce the stress response. A second experiment examined the role of the VT4R in food intake regulation. The Manning compound, SR-49059 or physiological saline was administered prior to NPY and food intake was monitored for 1h. It was hypothesized that each of the two antagonists coupled with NPY would augment food intake above the intake resulting from saline plus NPY administration. Related to the second experiment was a third that examined the difference between the effect of central administration of NPY versus SR-49059 in releasing CORT. Results of the first study showed that the Manning compound or SR-49059 prior to stress decreased CORT levels compared to controls while the second experiment showed that SR-49059 or the Manning compound plus NPY, enhanced food intake above that of the experimental group given saline and NPY. The last study showed that NPY increased plasma CORT above birds given SR-49059 centrally or saline administered controls. Taken together, results suggest that the avian VT4R is involved in the central neuroendocrine stress response as well as functions in appetite regulation by mediating an anorexigenic effect similar to what has been reported in mammals for the V1aR. In conclusion, similar to the past in silico and in vitro tests, the current in vivo experiments showed SR-49059 to be a most efficacious avian vasotocin receptor antagonist. Therefore based upon results of functional tests utilizing a highly specific mammalian antagonist, SR-49059, to the mammalian V1aR that likewise was most effective in blocking the avian VT4R and past reported high sequence homology between the mammalian V1aR and the VT4R, it is recommended that the chicken VT4R be renamed the avian V1aR to facilitate better communication among scientists involved in comparative studies.

Retinoic acid and retinaldehyde dehydrogenase are not involved in the specific induction of the follicle-stimulating hormone β subunit by trichostatin A, a selective inhibitor of histone deacetylase

The selective histone deacetylase inhibitor, trichostatin A (TSA), increases follicle-stimulating hormone β subunit (FSHβ) mRNA expression but not α- and luteinizing hormone β (LHβ)-subunits in both the pituitary gonadotrophic cell line LβT2 and primary cultures of rat anterior pituitary cells. TSA increased histone acetylation in whole cell lysates in both cells. In addition, retinaldehyde dehydrogenases (RALDHs), which are retinoic acid (RA)-synthesizing enzymes, were induced by TSA in these cells. Anacardic acid, a histone acetyltransferase inhibitor that prevents histone acetylation, significantly inhibited TSA-induced FSHβ mRNA expression as well as TSA-induced RALDH2 and RALDH3 mRNA expression. Similar to the effect of TSA, gonadotropin-releasing hormone stimulated RALDH expression in LβT2 cells. RA directly applied to the pituitary cells stimulated the transcriptional activity of the FSHβ promoter. In addition, α- and LHβ-subunit promoters were also activated by RA. Our results suggest that TSA specifically increases FSHβ expression with a concomitant increase in RALDHs; however, RALDH and RA are not directly involved in the specific regulation of FSHβ by TSA.

The nonsteroidal mycoestrogen zearalenone and its five metabolites suppress LH secretion from the bovine anterior pituitary cells via the estradiol receptor GPR30 in vitro

Picomolar concentrations of estradiol produce nongenomic suppression of GnRH-induced LH secretion from the anterior pituitary (AP) of cattle via G-protein–coupled receptor 30 (GPR30). Zearalenone (ZEN) is the nonsteroidal mycoestrogen produced by Fusarium fungi and has been detected in cereal grains, animal feed, and ruminant urine worldwide. Zearalenone has a prolonged blood half-life that results from enterohepatic cycling. There are five metabolites of ZEN: α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), and zearalanone, which may persist for long periods in animals and humans after consumption of ZEN-contaminated feed. We recently reported that GPR30 bound with α-ZAL decreases cytoplasmic cAMP but not gene expression of LHα and LHβ subunits, and GPR30 bound with α-ZAL suppresses GnRH-induced LH release in bovine AP cells in vitro. We tested the hypothesis that GPR30 bound with ZEN or one of the four previously untested metabolites suppresses GnRH-induced LH release from the bovine AP cells in vitro. Anterior pituitary cells were cultured for 3 days under steroid-free conditions and were then incubated with various concentrations (0.001–10 nM) of estradiol or one of the ZEN analogs for 5 minutes before GnRH stimulation. Gonadotropin-releasing hormone–stimulated LH secretion from AP cells was inhibited by all of the test concentrations of ZEN, 0.001 to 1 nM of α-ZAL, and 0.001 to 0.1 nM of the remaining four analogs. Pretreatment for 5 minutes with a GPR30-specific antagonist, G36, inhibited estradiol- and the ZEN analog–induced suppression of LH secretion from cultured AP cells. G36 alone had no significant effect on LH secretion. The estimated order of the nongenomic inhibiting effect was ZEN, α-ZAL, zearalanone, α-ZOL, β-ZOL, and β-ZAL, which is quite different from the reported order for their genomic effects. Therefore, ZEN and all of its metabolites suppress LH secretion from the bovine AP cells via GPR30 in vitro.

The non-steroidal mycoestrogen zeranol suppresses luteinizing hormone secretion from the anterior pituitary of cattle via the estradiol receptor GPR30 in a rapid, non-genomic manner

Picomolar concentrations of estradiol produce rapid suppression of GnRH-induced luteinizing hormone (LH) secretion from the anterior pituitary (AP) of cattle via G-protein-coupled receptor 30 (GPR30). Zeranol is a strong estrogenic metabolite derived from zearalenone, a non-steroidal mycoestrogen produced by Fusarium that induces reproductive disorders in domestic animals. The hypothesis was tested that zeranol suppresses GnRH-induced LH release from the AP of cattle via GPR30 in a rapid, non-genomic manner. The AP cells (n=15) were cultured for 3 days in steroid-free conditions and then treated them with estradiol (0.001–10nM) or zeranol (0.001–100nM) for 5min before GnRH stimulation. Pre-treatment with 0.001–0.1nM estradiol suppressed GnRH-stimulated LH secretion. Pre-treatment with zeranol at concentrations of 0.001nM (P<0.01), 0.01nM (P<0.01), 0.1nM (P<0.05), and 1nM (P<0.05), but not at concentrations of 10 and 100nM, also inhibited GnRH-stimulated LH secretion from AP cells. Pre-treatment for 5min with a GPR30-specific antagonist, G36, inhibited estradiol or zeranol suppression of LH secretion from cultured AP cells. Cyclic AMP measurements and quantitative PCR analyses revealed that pre-treatment with small amounts of estradiol (P<0.05) or zeranol (P<0.01) decreased cAMP, but not gene expressions of the LHα, LHβ, or FSHβ subunits in the AP cells. Hence, zeranol may suppress luteinizing hormone secretion from the AP of cattle via GPR30 in a rapid, non-genomic manner.

Prolactin anterior pituitary expression and circulating levels are reduced in obese and diabetic rats: role of TGF-β and TNF-α.

The levels of the hormone prolactin (PRL) are reduced in the circulation of patients with Type 2 diabetes and in obese children, and lower systemic PRL levels correlate with an increased prevalence of diabetes and a higher risk of metabolic syndrome. The secretion of anterior pituitary (AP) PRL in metabolic diseases may be influenced by the interplay between transforming growth factor β (TGF-β) and tumor necrosis factor α (TNF-α), which inhibit and can stimulate AP PRL synthesis, respectively, and are known contributors to insulin resistance and metabolic complications. Here, we show that TGF-β and TNF-α antagonize the effect of each other on the expression and release of PRL by the GH4C1 lactotrope cell line. The levels of AP mRNA and circulating PRL decrease in high-fat diet-induced obese rats in parallel with increased and reduced AP levels of TGF-β and TNF-α mRNA, respectively. Likewise, AP expression and circulating levels of PRL are reduced in streptozotocin-induced diabetic rats and are associated with higher AP expression and protein levels of TGF-β and TNF-α. The opposing effects of the two cytokines on cultured AP cells, together with their altered expression in the AP of obese and diabetic rats suggest they are linked to the reduced PRL production and secretion characteristics of metabolic diseases.

Gonadotropin-releasing hormone (GnRH) receptors of cattle aggregate on the surface of gonadotrophs and are increased by elevated GnRH concentrations

The presence of gonadotropin-releasing hormone (GnRH) receptors (GnRHRs) on gonadotrophs in the anterior pituitary (AP) is an important factor for reproduction control. However, little is known regarding GnRHR gene expression in gonadotrophs of cattle owing to the lack of an appropriate anti-GnRHR antibody. Therefore, an anti-GnRHR antibody for immunohistochemistry, flow cytometry, and immunocytochemistry assays was developed to characterize GnRHR gene expression in gonadotrophs. The anti-GnRHR antibody could suppress GnRH-induced LH secretion from cultured AP cells of cattle. The GnRHR, luteinizing hormone (LH), and follicle stimulating hormone (FSH) in the AP tissue was analyzed by fluorescence immunohistochemistry. The GnRHRs were aggregated on a limited area of the cell surface of gonadotrophs, possibly localized to lipid rafts. The LH secretion was stimulated with increasing amounts of GnRH; however, excessive concentrations (>1nM) resulted in a decrease in LH secretion. A novel method to purify gonadotrophs was developed using the anti-GnRHR antibody and fluorescence-activated cell sorting. Flow cytometric analysis using the anti-GnRHR antibody for cultured bovine AP cells, however, failed to support the hypothesis that GnRH induces GnRHR internalization and decreases GnRHR on the surface of GnRHR-positive AP cells. In contrast, immunocytochemistry using primary antibodies for cultured bovine AP cells showed that 10nM (P<0.05) and 100nM (P<0.01) GnRH, but not 0.01–1nM GnRH, increased GnRHR in the cytoplasm of LH-positive cells. In conclusion, these data suggested that GnRHRs were aggregated on the surface of gonadotrophs and GnRHR inside gonadotrophs increased with elevated concentrations of GnRH.

Proton receptor GPR68 expression in dendritic-cell-like S100β-positive cells of rat anterior pituitary gland: GPR68 induces interleukin-6 gene expression in extracellular acidification

S100β-positive cells, which do not express the classical pituitary hormones, appear to possess multifunctional properties and are assumed to be heterogeneous in the anterior pituitary gland. The presence of several protein markers has shown that S100β-positive cells are composed of populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. Recently, we succeeded in separating S100β-positive cells into round-cell (dendritic-cell-like) and process-cell types. We also found the characteristic expression of anti-inflammatory factors (interleukin-6, Il-6) and membrane receptors (integrin β-6) in the round type. Here, we further investigate the function of the subpopulation of S100β-positive cells. Since IL-6 is also a paracrine factor that regulates hormone producing-cells, we examine whether a correlation exists among extracellular acid stress, IL-6 and hormone production by using primary cultures of anterior pituitary cells. Dendritic-cell-like S100β-positive cells notably expressed Gpr68 (proton receptor) and Il-6. Furthermore, the expression of Il-6 and proopiomelanocortin (Pomc) was up-regulated by extracellular acidification. The functional role of IL-6 and GPR68 in the gene expression of Pomc during extracellular acidification was also examined. Small interfering RNA for Il-6 up-regulated Pomc expression and that for Gpr68 reversed the down-regulation of Il-6 and up-regulated Pomc expression by extracellular acidification. Thus, S100β-positive dendritic-like cells can sense an increase in extracellular protons via GPR68 and respond by the production of IL-6 in order to suppress the up-regulation of Pomc expression.

Regulation of gene expression of vasotocin and corticotropin-releasing hormone receptors in the avian anterior pituitary by corticosterone

The effect of chronic stress (CS) on gene expression of the chicken arginine vasotocin (AVT) and corticotropin-releasing hormone (CRH) receptors [VT2R, VT4R, CRH-R1, and CRH-R2] was examined by measuring receptor mRNA levels in the anterior pituitary gland of the chicken after chronic immobilization stress compared to acute stress (AS). Radioimmunoassay results showed that blood circulating corticosterone (CORT) levels in the CS group were significantly decreased compared to that of birds in the AS group (P<0.05). The VT2R and CRH-R2 mRNA in CS birds were significantly decreased to that of controls. The VT4R mRNA was significantly decreased compared to controls in AC birds and was further decreased in the CS group compared to controls (P<0.05). The CRH-R1 mRNA was significantly decreased in the AS birds compared to controls. However, there was no significant difference of CRH-R1 mRNA between acute stress and chronic stress birds. Using primary anterior pituitary cell cultures, the effect of exogenous CORT on VT/CRH receptor gene expression was examined. Receptor mRNA levels were measured after treatment of CORT followed by AVT/CRH administration. The CORT pretreatment resulted in a dose-dependent decrease of proopiomelanocortin heteronuclear RNA, a molecular marker of a stress-induced anterior pituitary. Without CORT pretreatment of anterior pituitary cell cultures, the VT2R, VT4R and CRH-R1mRNA levels were significantly increased within 15min and then decreased at 1h and 6h by AVT/CRH administration (P<0.05). Pretreatment of CORT in anterior pituitary cells induced a dose-dependent increase of VT2R, VT4R and CRH-R2 mRNA levels, and a significant decrease of CRH-R1 mRNA levels at only the high dose (10ng/ml) of CORT (P<0.05).Taken together, results suggest a modulatory role of CORT on the regulation of VT/CRH receptor gene expression in the avian anterior pituitary gland dependent upon CORT levels.

Cellular Sensitivity to Ferrotoxicity is Associated with Specific Clinical Disorders

Copyright: © 2014 Weinberg ED. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. During the past five decades, scores of medical studies have described the association of ferrotoxicity with development of a great variety of diseases [1]. Increasingly, specific types of body cells are being reported to be injured or killed by low concentrations of iron. In the initial account, cultures of anterior pituitary cells were observed to be killed by 2 μM irons; in this system, hepatocytes remained healthy in 10-100 μM iron [2]. Thus not surprisingly, thalassemic-major children who load iron early in life are deprived of growth hormone. Persons who absorb excessive iron in late teens or early adulthood can suffer from low levels of gonadotrophic hormones. Subsequently, osteoblasts have been recognized to be unusually sensitive to low concentrations of iron [3,4]. In contrast, osteoclasts (cells of macrophage origin) are highly resistant to iron. Thus in iron loaded persons, bone rebuilding by osteoblasts is surpassed by osteoclast destruction of bone. Accordingly, osteoporosis is a common disorder in patients who load iron for genetic, environmental or behavioral reasons [5].

Expression of gonadotropin-inhibitory hormone receptors in mouse pituitary gonadotroph LβT2 cells and hypothalamic gonadotropin-releasing hormone-producing GT1-7 cells

Gonadotropin-inhibitory hormone (GnIH) was first identified in quail as a novel neurohormone that acts directly on the anterior pituitary to inhibit gonadotropin release. GnIH inhibits not only gonadotropin release from the pituitary gland but also inhibits the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. In this study, we examined how GnIH receptors were regulated in pituitary gonadotroph cells and GnRH-producing neurons in the hypothalamus. In the mouse pituitary gonadotroph cell line LβT2, GnRH increased expression of the GnIH receptor, G-protein coupled receptor 74 (GPR74). GnRH also stimulated the expression of GPR74 and GPR147 in primary cultures of rat anterior pituitary cells. In addition, when GnRH was administered to LβT2 cells in a pulsatile manner, low frequency GnRH pulse stimulation stimulated GPR74 and GPR147 expression more than did high frequency GnRH pulses. In the mouse hypothalamic GnRH-producing cell line GT1-7, hypothalamic kisspeptin did not significantly increase the expression of GnIH receptors. However, the intermittent administration of kisspeptin to GT1-7 cells significantly increased GPR74 and GPR147 mRNA expression. The overexpression of either constitutively active MEK kinase (MEKK) or protein kinase A (PKA) in LβT2 cells increased the expression of GPR74 mRNA. Conversely, in GT1-7 cells, although the overexpression of either MEKK or PKA failed to stimulate GnIH receptor expression, the combined overexpression of both kinases together increased GPR74 and GPR147 mRNA levels. Our current observations suggest that two central controllers of reproductive function, GnRH and kisspeptin, stimulate the expression of GnIH receptors in pituitary gonadotroph cells and hypothalamic GnRH neurons.