Reporter Expression, Induced by a Growth Hormone Promoter-Driven Cre Recombinase (rGHp-Cre) Transgene, Questions the Developmental Relationship between Somatotropes and Lactotropes in the Adult Mouse Pituitary Gland

This report describes the development and validation of the rGHp-Cre transgenic mouse that allows for selective Cre-mediated recombination of loxP-modified alleles in the GH-producing cells of the anterior pituitary. Initial screening of the rGHp-Cre parental line showed Cre mRNA was specifically expressed in the anterior pituitary gland of adult Cre+/− mice and cephalic extracts of e17 Cre+/− fetuses. Heterozygote rGHp-Cre transgenic mice were crossbred with Z/AP reporter mice to generate Cre+/−,Z/AP+/− offspring. In this model system, the GH promoter-driven, Cre-mediated recombination of the Z/AP reporter leads to human placental alkaline phosphatase (hPLAP) expression that serves to mark cells that currently produce GH, in addition to cells that would have differentiated from GH cells but currently do not express the GH gene. Double immunocytochemistry of adult male and female Cre+/−,Z/AP+/− pituitary cells revealed the majority (∼99%) of GH-producing cells of the anterior pituitary also expressed hPLAP, whereas ACTH-, TSH-, and LH-producing cells were negative for hPLAP, confirming previous reports that corticotropes, thyrotropes, and gonadotropes develop independently of the somatotrope lineage. A small subset (∼10%) of the prolactin-producing cells was positive for hPLAP, consistent with previous reports showing lactotropes can arise from somatotropes during pituitary development. However, the fact that 90% of prolactin-producing cells were negative for hPLAP suggests that the majority of lactotropes in the adult mouse pituitary gland develop independently of the somatotrope lineage. In addition to developmental studies, the rGHp-Cre transgenic mouse will provide a versatile tool to study the role of a variety of genes in somatotrope function and neoplastic transformation.

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IGF-I regulates pro-opiomelanocortin and GH gene expression in the mouse pituitary gland

Journal of Endocrinology ◽

10.1677/joe.0.1780071 ◽

2003 ◽

Vol 178 (1) ◽

pp. 71-82 ◽

Cited By ~ 10

Author(s):

J Honda ◽

Y Manabe ◽

R Matsumura ◽

S Takeuchi ◽

S Takahashi

Keyword(s):

Pituitary Gland ◽

Anterior Pituitary ◽

Northern Blotting ◽

Pituitary Cells ◽

Mrna Levels ◽

Gh Treatment ◽

Gh Receptor ◽

Pomc Mrna ◽

Igf I ◽

Mouse Pituitary

IGF-I is expressed in somatotrophs, and IGF-I receptors are expressed in most somatotrophs and some corticotrophs in the mouse pituitary gland. Our recent study demonstrated that IGF-I stimulates the proliferation of corticotrophs in the mouse pituitary. These results suggested that somatotrophs regulate corticotrophic functions as well as somatotrophic functions by the mediation of IGF-I molecules. The present study aimed to clarify factors regulating pituitary IGF-I expression and also the roles exerted by IGF-I within the mouse anterior pituitary gland. Mouse anterior pituitary cells were isolated and cultured under serum-free conditions. GH (0.5 or 1 microg/ml), ACTH (10(-8) or 10(-7) M), GH-releasing hormone (GHRH; 10(-8) or 10(-7) M), dexamethasone (DEX; 10(-8) or 10(-7) M) and estradiol-17beta (e2; 10(-11) or 10(-9) M) were given for 24 h. IGF-I mRNA levels were measured using competitive RT-PCR, and GH and pro-opiomelanocortin (POMC) mRNA levels were measured using Northern blotting analysis. GH treatment significantly increased IGF-I mRNA levels (1.5- or 2.1-fold). ACTH treatment did not alter GH and IGF-I mRNA levels. IGF-I treatment decreased GH mRNA levels (0.7- or 0.5-fold), but increased POMC mRNA levels (1.8-fold). GH treatment (4 or 8 microg/ml) for 4 days increased POMC mRNA levels. GHRH treatment increased GH mRNA levels (1.3-fold), but not IGF-I mRNA levels. DEX treatment significantly decreased IGF-I mRNA levels (0.8-fold). e2 treatment did not affect IGF-I mRNA levels. GH receptor mRNA, probably with GH-binding protein mRNA, was detected in somatotrophs, and some mammotrophs and gonadotrophs by in situ hybridization using GH receptor cDNA as a probe. These results suggested that IGF-I expression in somatotrophs is regulated by pituitary GH, and that IGF-I suppresses GH expression and stimulates POMC expression at the transcription level. Pituitary IGF-I produced in somatotrophs is probably involved in the regulation of somatotroph and corticotroph functions.

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

International Journal of Toxicology ◽

10.1177/1091581816645797 ◽

2016 ◽

Vol 35 (4) ◽

pp. 463-475 ◽

Cited By ~ 8

Author(s):

Sonia A. Ronchetti ◽

María S. Bianchi ◽

Beatriz H. Duvilanski ◽

Jimena P. Cabilla

Keyword(s):

Oxidative Stress ◽

Pituitary Gland ◽

Anterior Pituitary ◽

Inorganic Arsenic ◽

Anterior Pituitary Gland ◽

Pituitary Cells ◽

Prolactin Release ◽

Stress Responsive Genes

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.

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Neuroendocrine Plasticity in the Anterior Pituitary: Gonadotropin-Releasing Hormone-Mediated Movement in Vitro and in Vivo

Endocrinology ◽

10.1210/en.2006-1153 ◽

2007 ◽

Vol 148 (4) ◽

pp. 1736-1744 ◽

Cited By ~ 43

Author(s):

Amy M. Navratil ◽

J. Gabriel Knoll ◽

Jennifer D. Whitesell ◽

Stuart A. Tobet ◽

Colin M. Clay

Keyword(s):

Pituitary Gland ◽

Anterior Pituitary ◽

Fluorescent Protein ◽

Cell Movement ◽

Pituitary Cells ◽

Rous Sarcoma ◽

Cytoskeleton Disruption ◽

Green Fluorescent

The secretion of LH is cued by the hypothalamic neuropeptide, GnRH. After delivery to the anterior pituitary gland via the hypothalamic-pituitary portal vasculature, GnRH binds to specific high-affinity receptors on the surface of gonadotrope cells and stimulates synthesis and secretion of the gonadotropins, FSH, and LH. In the current study, GnRH caused acute and dramatic changes in cellular morphology in the gonadotrope-derived αT3-1 cell line, which appeared to be mediated by engagement of the actin cytoskeleton; disruption of actin with jasplakinolide abrogated cell movement and GnRH-induced activation of ERK. In live murine pituitary slices infected with an adenovirus-containing Rous sarcoma virus-green fluorescent protein, selected cells responded to GnRH by altering their cellular movements characterized by both formation and extension of cell processes and, surprisingly, spatial repositioning. Consistent with the latter observation, GnRH stimulation increased the migration of dissociated pituitary cells in transwell chambers. Our data using live pituitary slices are a striking example of neuropeptide-evoked movements of cells outside the central nervous system and in a mature peripheral endocrine organ. These findings call for a fundamental change in the current dogma of simple passive diffusion of LH from gonadotropes to capillaries in the pituitary gland.

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Bradykinin stimulates phosphoinositide metabolism and prolactin secretion in rat anterior pituitary cells

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0020047 ◽

1989 ◽

Vol 2 (1) ◽

pp. 47-53 ◽

Cited By ~ 12

Author(s):

T.H. Jones ◽

B. L. Brown ◽

P. R. M. Dobson

Keyword(s):

Pituitary Gland ◽

Anterior Pituitary ◽

Inositol Phosphate ◽

Prolactin Secretion ◽

Male Rats ◽

Pituitary Cells ◽

Phosphoinositide Metabolism ◽

Phosphate Accumulation ◽

Anterior Pituitary Cells ◽

Inositol Phosphate Accumulation

ABSTRACT Bradykinin stimulated prolactin secretion from monolayer cultures of rat anterior pituitary cells, the stimulation being greater from the cells of male rats. This stimulated secretion was accompanied by a rise in total inositol phosphate accumulation, suggesting that the action of bradykinin is mediated by phosphoinositide hydrolysis. The increase in inositol phosphate accumulation was biphasic; a further sharp rise occurred when the concentration of bradykinin exceeded 1 μmol/l. This may indicate that bradykinin acts on other cell types in the pituitary gland. Bradykinin had no effect on growth hormone secretion from cells of normal pituitary glands, or on prolactin secretion and phosphoinositide metabolism in GH3 rat pituitary tumour cells. Bradykinin receptor antagonists (both B1 and B2) had no effect on either bradykinin-stimulated inositol phosphate accumulation or prolactin secretion. Kallikreins, the enzymes responsible for the generation of kinins, are known to be present in the adenohypophysis. Therefore, the results presented here would suggest that kinins may have a role as paracrine agents in the pituitary gland.

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EFFECT OF EXOGENOUS THYROXINE ON 3H-THYMIDINE UPTAKE IN MOUSE PITUITARY GLAND

Acta Endocrinologica ◽

10.1530/acta.0.0610133 ◽

1969 ◽

Vol 61 (1) ◽

pp. 133-136 ◽

Cited By ~ 4

Author(s):

B. Messier

Keyword(s):

Thyroid Gland ◽

Oral Administration ◽

Pituitary Gland ◽

Intact Animal ◽

Thymidine Uptake ◽

Pituitary Cells ◽

Thyroxine Treatment ◽

Before And After ◽

Mouse Pituitary

ABSTRACT Destruction of the thyroid gland in the mouse induces proliferation of pituitary thyrotropes. This phenomenon was studied quantitatively with 3H-thymidine, before and after oral administration of thyroxine. The frequency of radioactive cells in the intact animal is 3.4 per 10 000 pituitary cells. This value rises to 19.6, 18.4 and 56.0 four, eight, and twelve months after thyroidectomy, respectively. All these increased values are brought back to normal levels after 5 to 10 days of thyroxine treatment. Thus, the intense proliferation of pituitary thyrotropes following thyroidectomy is still responsive to the homeostatic action of thyroxine.

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Immunocytochemical evidence that S-100-positive cells of the mouse anterior pituitary contain interleukin-6 immunoreactivity.

Journal of Histochemistry & Cytochemistry ◽

10.1177/41.2.8419456 ◽

1993 ◽

Vol 41 (2) ◽

pp. 151-156 ◽

Cited By ~ 60

Author(s):

H Vankelecom ◽

P Matthys ◽

J Van Damme ◽

H Heremans ◽

A Billiau ◽

...

Keyword(s):

Interleukin 6 ◽

Anterior Pituitary ◽

Tumor Cell Line ◽

Polyclonal Antiserum ◽

Positive Staining ◽

Pituitary Cells ◽

S 100 ◽

Mouse Pituitary ◽

Acth Secreting

We have previously shown that bioactive interleukin-6 (IL-6) is produced by rat and mouse (anterior) pituitary cells in vitro. Since the amount produced correlated with the presence of S-100-containing folliculostellate (FS) cells, these cells were suggested to be a source of IL-6 in the anterior pituitary (AP) lobe. In the present study we used immunocytochemical techniques to confirm this presumption. Freshly isolated mouse pituitary cells were subjected to immunocytochemical procedures whereby two different (neutralizing) monoclonal antibodies (MAb) against mouse IL-6 (6B4 and 20F3) and a polyclonal antiserum raised against bovine S-100 were used as primary antibodies. Single immunostaining revealed a small portion of mouse pituitary cells (about 6.5%) to be positive for IL-6 immunoreactivity with both antibodies. Importantly, the same proportion of cells was found to be IL-6 positive if only the AP was used as the cell source. About 7.5% of the pituitary cells stained for the presence of S-100 immunoreactivity. Positive staining for IL-6 was also found in pituitary cell samples from 2-day-old monolayer cultures and from redispersed 9-day-old histotypic aggregates, which both secreted bioassayable IL-6. In contrast, no IL-6 staining was found in AtT-20 cells, an established ACTH-secreting tumor cell line of the mouse pituitary which did not secrete bioactive IL-6. The specificity of the IL-6 immunostaining was demonstrated by a total loss of staining when MAb 6B4 was omitted or replaced by irrelevant rat IgG. Conclusively, pre-adsorption of the anti-IL-6 MAb (6B4) with recombinant mouse IL-6 totally abolished staining of pituitary cells. Double immunostaining for IL-6 and S-100 revealed that most if not all of the IL-6-containing pituitary cells were positive for S-100. Few of the S-100-containing cells did not stain for IL-6. These results confirm our previous hypothesis that FS cells, characterized by immunostaining of S-100 protein, contain bioactive and immunoreactive IL-6 and therefore are very likely producers of IL-6 in the AP. Furthermore, our results suggest that IL-6 is implicated in the local regulatory role ascribed to FS cells in the pituitary gland.

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LOCALIZATION OF PHOSPHATASE ACTIVITIES IN THE RAT ANTERIOR PITUITARY GLAND

Journal of Histochemistry & Cytochemistry ◽

10.1177/20.1.1 ◽

1972 ◽

Vol 20 (1) ◽

pp. 1-12 ◽

Cited By ~ 31

Author(s):

GEORGES PELLETIER ◽

ALEX B. NOVIKOFF

Keyword(s):

Golgi Apparatus ◽

Pituitary Gland ◽

Anterior Pituitary ◽

Secretory Cell ◽

Cell Types ◽

Anterior Pituitary Gland ◽

Pituitary Cells ◽

Blood Capillaries ◽

Phosphatase Activities ◽

Thiamine Pyrophosphatase

All five known secretory cell types of the rat anterior pituitary gland display nucleoside diphosphatase (NDPase) activity throughout the endoplasmic reticulum (ER), including the nuclear envelope but not the specialized region of ER at the inner aspect of the Golgi apparatus known as GERL. The functions of the ER diphosphatase are currently unknown. However, speculations concerning its association with glucuronyl transferase may focus on the metabolic roles of the ER in pituitary cells other than those directly related to secretory protein transport. The gonadotrophs have been studied for thiamine pyrophosphatase and acid phosphatase activities as well as NDPase activity. The results suggest that the secretory granules of gonadotrophs arise from GERL and not from the inner element of the Golgi apparatus. The innermost Golgi element of this cell type shows NDPase and thiamine pyrophosphatase activities and appears to be composed, in part at least, of anastomosing tubules. Nucleoside phosphatase activity is also present at the surfaces of all five secretory cell types and between the cells and adjacent blood capillaries.

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Imaging of Endoplasmic Reticulum Ca2+ in the Intact Pituitary Gland of Transgenic Mice Expressing a Low Affinity Ca2+ Indicator

Frontiers in Endocrinology ◽

10.3389/fendo.2020.615777 ◽

2021 ◽

Vol 11 ◽

Author(s):

Jonathan Rojo-Ruiz ◽

Paloma Navas-Navarro ◽

Lucía Nuñez ◽

Javier García-Sancho ◽

María Teresa Alonso

Keyword(s):

Endoplasmic Reticulum ◽

Pituitary Gland ◽

Transgenic Mouse ◽

Calcium Release ◽

Cell Types ◽

Thyrotropin Releasing Hormone ◽

Pituitary Cells ◽

Releasing Hormone ◽

Pituitary Glands ◽

Calcium Induced Calcium Release

The adenohypophysis contains five secretory cell types (somatotrophs, lactotrophs, thyrotrophs, corticotrophs, and gonadotrophs), each secreting a different hormone, and controlled by different hypothalamic releasing hormones (HRHs). Exocytic secretion is regulated by cytosolic Ca2+ signals ([Ca2+]C), which can be generated either by Ca2+ entry through the plasma membrane and/or by Ca2+ release from the endoplasmic reticulum (ER). In addition, Ca2+ entry signals can eventually be amplified by ER release via calcium-induced calcium release (CICR). We have investigated the contribution of ER Ca2+ release to the action of physiological agonists in pituitary gland. Changes of [Ca2+] in the ER ([Ca2+]ER) were measured with the genetically encoded low-affinity Ca2+ sensor GAP3 targeted to the ER. We used a transgenic mouse strain that expressed erGAP3 driven by a ubiquitous promoter. Virtually all the pituitary cells were positive for the sensor. In order to mimick the physiological environment, intact pituitary glands or acute slices from the transgenic mouse were used to image [Ca2+]ER. [Ca2+]C was measured simultaneously with Rhod-2. Luteinizing hormone-releasing hormone (LHRH) or thyrotropin releasing hormone (TRH), two agonists known to elicit intracellular Ca2+ mobilization, provoked robust decreases of [Ca2+]ER and concomitant rises of [Ca2+]C. A smaller fraction of cells responded to thyrotropin releasing hormone (TRH). By contrast, depolarization with high K+ triggered a rise of [Ca2+]C without a decrease of [Ca2+]ER, indicating that the calcium-induced calcium-release (CICR) via ryanodine receptor amplification mechanism is not present in these cells. Our results show the potential of transgenic ER Ca2+ indicators as novel tools to explore intraorganellar Ca2+ dynamics in pituitary gland in situ.

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Regulation of PIT-1 Expression By Ghrelin and GHRP-6 Through the GH Secretagogue Receptor

Molecular Endocrinology ◽

10.1210/mend.15.9.0694 ◽

2001 ◽

Vol 15 (9) ◽

pp. 1484-1495 ◽

Cited By ~ 32

Author(s):

Angel García ◽

Clara V. Alvarez ◽

Roy G. Smith ◽

Carlos Diéguez

Keyword(s):

Pituitary Gland ◽

Transcriptional Activation ◽

Anterior Pituitary ◽

Primary Cultures ◽

Physiological Role ◽

Luciferase Expression ◽

Established Cell Line ◽

Pituitary Cells ◽

Endogenous Ligand ◽

Infant Rat

Abstract GH secretagogues are an expanding class of synthetic peptide and nonpeptide molecules that stimulate the pituitary gland to secrete GH through their own specific receptor, the GH-secretagogue receptor. The cloning of the receptor for these nonclassical GH releasing molecules, together with the more recent characterization of an endogenous ligand, named ghrelin, have unambiguously demonstrated the existence of a physiological system that regulates GH secretion. Somatotroph cell-specific expression of the GH gene is dependent on a pituitary-specific transcription factor (Pit-1). This factor is transcribed in a highly restricted manner in the anterior pituitary gland. The present experiments sought to determine whether the synthetic hexapeptide GHRP-6, a reference GH secretagogue compound, as well as an endogenous ligand, ghrelin, regulate pit-1 expression. By a combination of Northern and Western blot analysis we found that GHRP-6 elicits a time- and dose-dependent activation of pit-1 expression in monolayer cultures of infant rat anterior pituitary cells. This effect was blocked by pretreatment with actinomycin D, but not by cycloheximide, suggesting that this action was due to direct transcriptional activation of pit-1. Using an established cell line (HEK293-GHS-R) that overexpresses the GH secretagogue receptor, we showed a marked stimulatory effect of GHRP-6 on the pit-1 −2,500 bp 5′-region driving luciferase expression. We truncated the responsive region to −231 bp, a sequence that contains two CREs, and found that both CREs are needed for GHRP-6-induced transcriptional activation in both HEK293-GHS-R cells and infant rat anterior pituitary primary cultures. The effect was dependent on PKC, MAPK kinase, and PKA activation. Increasing Pit-1 by coexpression of pCMV-pit-1 potentiated the GHRP-6 effect on the pit-1 promoter. Similarly, we showed that the endogenous GH secretagogue receptor ligand ghrelin exerts a similar effect on the pit-1 promoter. These data provide the first evidence that ghrelin, in addition to its previously reported GH-releasing activities, is also capable of regulating pit-1 transcription through the GH secretagogue receptor in the pituitary, thus giving new insights into the physiological role of the GH secretagogue receptor on somatotroph cell differentiation and function.

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