Comprehensive analysis of differences in N6-methyladenosine RNA methylomes in the rat adenohypophysis after GnRH treatment.

N6-methyladenosine is considered to be the most common and abundant internal chemical modification among the more than 150 identified chemical RNA modifications. It is involved in most biological processes and actively participates in the regulation of animal reproduction. However, the potential function of m6 A in the pituitaries of mammals is not yet clear. It is also unknown whether m6 A is involved in the secretion and regulation of FSH by GnRH, which in turn affects mammalian reproduction. In this study, rats were treated with gonadorelin to simulate physiological GnRH-mediated regulation of FSH synthesis and secretion, and m6 A-seq was used to analyze the differential m6 A modification of the rat pituitary after gonadorelin treatment. A whole-transcriptome map of m6 A in the rat pituitary gland before and after gonadorelin treatment was successfully created. A total of 6413 differential peaks were identified, of which 3764 m6 A peaks were upregulated and 2649 m6 A peaks were downregulated. Among the 709 differentially expressed genes, 250 genes were discovered with differential methylation modifications. Intriguingly, the altered m6 A peaks within mRNAs were enriched in steroid biosynthetic processes and responses to cAMP. The results of the study will lay a foundation for further exploration of the potential role of m6 A modification in the regulation of reproductive hormone secretion and provide a theoretical basis for the application of GnRH analogs in mammalian artificial reproduction.

The GnRH Antagonist Degarelix Suppresses Gonadotropin Secretion and Pituitary Sensitivity in Midgestation Sheep Fetuses.

The specific role of gonadotropin-releasing hormone (GnRH) on brain sexual differentiation remains unclear. To investigate whether gonadotropin and, in turn, testosterone (T) secretion is regulated by GnRH during the critical period for brain differentiation in sheep fetuses, we attempted to selectively suppress pituitary-testicular activation during midgestation with the long-acting GnRH antagonist degarelix. Fetuses received subcutaneous injections of the antagonist or vehicle on day 62 of gestation. After 2 to 3 weeks we examined consequences of the intervention on baseline and GnRH-stimulated plasma luteinizing hormone (LH) and T levels. In addition, we measured the effect of degarelix-treatment on messenger RNA (mRNA) expression for the pituitary gonadotropins and key gonadal steroidogenic enzymes. Baseline and GnRH-stimulated plasma LH levels were significantly suppressed in degarelix-treated male and female fetuses compared to control values. Similarly, T concentrations were suppressed in degarelix-treated males. The percentage of LHß-immunoreactive cells colocalizing c-fos was significantly reduced by degarelix treatment indicating that pituitary sensitivity was inhibited. Degarelix treatment also led to the significant suppression of mRNA expression coding for the pituitary gonadotropin subunits and for the gonadal enzymes involved in androgen synthesis. These findings demonstrate that pharmacologic inhibition of GnRH early in gestation results in suppression of LH secretion and deficits in the plasma T levels of male lamb fetuses. We conclude that GnRH signaling plays a pivotal role for regulating T exposure during the critical period of sheep gestation when the brain is masculinized. Thus, disturbance to gonadotropin secretion during this phase of gestation could have long-term consequence on adult sexual behaviors and fertility.

Alteration of Extracellular Matrix Components in the Anterior Pituitary Gland of Neonatal Rats Induced by a Maternal Bisphenol A Diet during Pregnancy.

The extracellular matrix (ECM) plays crucial roles in the anterior pituitary gland via the mechanism of cell-ECM interaction. Since bisphenol A (BPA), a well-known endocrine disruptor, can cross through the placenta from mother to fetus and bind with estrogen receptors, cell populations in the neonatal anterior pituitary gland could be the target cells affected by this chemical. The present study treated maternal rats with 5000 µg/kg body weight of BPA daily throughout the pregnancy period and then investigated the changes in ECM-producing cells, i.e., pericytes and folliculostellate (FS) cells, including their ECM production in the neonatal anterior pituitary at Day 1. We found that pericytes and their collagen synthesis reduced, consistent with the increase in the number of FS cells that expressed several ECM regulators-matrix metalloproteinase (MMP) 9 and the tissue inhibitors of metalloproteinase (TIMP) family. The relative MMP9/TIMP1 ratio was extremely high, indicating that the control of ECM homeostasis was unbalanced. Moreover, transmission electron microscopy showed the unorganized cell cluster in the BPA-treated group. This study revealed that although the mother received BPA at the "no observed adverse effect" level, alterations in ECM-producing cells as well as collagen and the related ECM balancing genes occurred in the neonatal anterior pituitary gland.

Does repeated gold-nanoparticles administration affect pars distalis hormonal and folliculo-stellate cells in adult male albino rats?

INTRODUCTION: Worldwide, nanoparticles especially gold-nanoparticles (Au-NPs) are widely used in medicine, cancer treatment and cosmetic industry. They are easily conjugated with different biomedical and biological agents and effortlessly absorbed with few side effects. The pars distalis of the pituitary gland is considered as the maestro of the endocrine peripheral glands since it secrets trophic hormones that controls their functions. 5-10% of the non-granular pars distalis cells are folliculo-stellate cells (FSCs) that support the granular cells' functions. The aim of the study was to explore the histological and the biochemical effects of repeated exposure to Au-NPs on the pars distalis in adult male albino rats with highlighting the impact on FSCs. MATERIAL AND METHODS: Thirty-six adult male albino rats were divided equally into control group and Au-NPs group (received 40 µg/kg/day of 11 ± 2 nm spherical Au-NPs orally for 2 weeks). Then, rats were euthanized and deposition of Au-NPs in pars distalis was investigated. Biochemical investigations and histological studies including hematoxylin and eosin staining, periodic acid Schiff's reaction, immunohistochemistry (IHC) for S-100, connexin 43 (Cx43) and Cytochrome-C (Cyt-C) as well as electron-microscopic and morphometric studies were carried out. RESULTS: The Au-NPs group demonstrated structural disorganization in the pars distalis, inflammation, congestion and increased extracellular PAS-positive colloid deposition due to the accumulation of Au-NPs. A significant increase in the immunoreactivity of S-100, Cx43 and Cyt-c, along with a significant increase in TNF-a, MDA, and bFGF content in the pituitary homogenates, was noted as compared to the control group. Ultrastructurally, degenerative changes were observed in the secretory cells. FSCs showed proliferation and increased phagocytic activity. CONCLUSIONS: Repetitive exposure of adult male albino rats to Au-NPs prompted the accumulation of these nanoparticles in the pars distalis that was accompanied by cellular degeneration and dysfunction of the secretory cell and proliferation of FSCs. Thus, monitoring of the pars distalis hormonal levels might be useful for early detection of some hazardous effects possibly associated with the use of gold-nanoparticles.

Differential distribution and potential regulatory roles of estrogen receptor 2a and 2b in the pituitary of ricefield eel Monopterus albus.

Estrogens play important regulatory roles in the pituitary of vertebrates. Two forms of estrogen receptor 2 (Esr2), namely Esr2a and Esr2b, are identified in teleosts, but their differential roles remain to be fully elucidated. In the present study, expression and potential functional roles of Esr2a and Esr2b were characterized in ricefield eels. esr2a and esr2b mRNA were broadly distributed in tissues, with high levels observed in the brain, pituitary, and gonads. In order to examine the cellular localization of Esr2a and Esr2b in the pituitary, specific antisera against ricefield eel Esr2a and Esr2b were generated, respectively. Interestingly, immunohistochemistry and Western blot analysis revealed that Esr2a and Esr2b were differentially distributed in the pituitary, with the former localized to the adenohypophysis while the latter to the neurohypophysis. Dual fluorescent immunostaining showed that immunoreactive Esr2a was present in Gh and Prl cells, but not in Lh and Fsh cells. Estradiol (E2) stimulated lhb and prl gene expression in dispersed pituitary cells of intersexual ricefield eels, but had no effects on gh, fshb, and gnrhr2 gene expression and Gh release. Results of the present study are helpful for further understanding the roles and mechanisms of estrogen signals in the pituitary.

Lithocholic acid inhibits P2X2 and potentiates P2X4 receptor channel gating.

The family of ATP-gated purinergic P2X receptors comprises seven bunits (P2X1-7) that are unevenly distributed in the central and peripheral nervous systems as well as other organs. Endogenous modulators of P2X receptors are phospholipids, steroids and neurosteroids. Here, we analyzed whether bile acids, which are natural products derived from cholesterol, affect P2X receptor activity. We examined the effects of primary and secondary bile acids and newly synthesized derivatives of lithocholic acid on agonist-induced responses in HEK293T cells expressing rat P2X2, P2X4 and P2X7 receptors. Electrophysiology revealed that low micromolar concentrations of lithocholic acid and its structural analog 4-dafachronic acid strongly inhibit ATP-stimulated P2X2 but potentiate P2X4 responses, whereas primary bile acids and other secondary bile acids exhibit no or reduced effects only at higher concentrations. Agonist-stimulated P2X7 responses are significantly potentiated by lithocholic acid at moderate concentrations. Structural modifications of lithocholic acid at positions C-3, C-5 or C-17 abolish both inhibitory and potentiation effects to varying degrees, and the 3α-hydroxy group contributes to the ability of the molecule to switch between potentiation and inhibition. Lithocholic acid allosterically modulates P2X2 and P2X4 receptor sensitivity to ATP, reduces the rate of P2X4 receptor desensitization and antagonizes the effect of ivermectin on P2X4 receptor deactivation. Alanine-scanning mutagenesis of the upper halve of P2X4 transmembrane domain-1 revealed that residues Phe48, Val43 and Tyr42 are important for potentiating effect of lithocholic acid, indicating that modulatory sites for lithocholic acid and ivermectin partly overlap. Lithocholic acid also inhibits ATP-evoked currents in pituitary gonadotrophs expressing native P2X2, and potentiates ATP currents in nonidentified pituitary cells expressing P2X4 receptors. These results indicate that lithocholic acid is a bioactive steroid that may help to further unveil the importance of the P2X2, and P2X4 receptors in many physiological processes.

The Impact of Photoperiod on the Leptin Sensitivity and Course of Inflammation in the Anterior Pituitary.

Leptin has a modulatory impact on the course of inflammation, affecting the expression of proinflammatory cytokines and their receptors. Pathophysiological leptin resistance identified in humans occurs typically in sheep during the long-day photoperiod. This study aimed to determine the effect of the photoperiod with relation to the leptin-modulating action on the expression of the proinflammatory cytokines and their receptors in the anterior pituitary under physiological or acute inflammation. Two in vivo experiments were conducted on 24 blackface sheep per experiment in different photoperiods. The real-time PCR analysis for the expression of the genes IL1B, IL1R1, IL1R2, IL6, IL6R, IL6ST, TNF, TNFR1, and TNFR2 was performed. Expression of all examined genes, except IL1ß and IL1R2, was higher during short days. The leptin injection increased the expression of all examined genes during short days. In short days the synergistic effect of lipopolysaccharide and leptin increased the expression of IL1B, IL1R1, IL1R2, IL6, TNF, and TNFR2, and decreased expression of IL6ST. This mechanism was inhibited during long days for the expression of IL1R1, IL6, IL6ST, and TNFR1. The obtained results suggest the occurrence of leptin resistance during long days and suggest that leptin modulates the course of inflammation in a photoperiod-dependent manner in the anterior pituitary.

Gonadotropin-releasing hormone-Cu complex (Cu-GnRH) transcriptional activity in vivo in the female rat anterior pituitary gland.

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 PACAP6-38 (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 IP3/PKC and cAMP/PKA-dependent gonadotrope network gene transcription in vivo.

Effects of Melatonin on Anterior Pituitary Plasticity: A Comparison Between Mammals and Teleosts.

Melatonin is a key hormone involved in the photoperiodic signaling pathway. In both teleosts and mammals, melatonin produced in the pineal gland at night is released into the blood and cerebrospinal fluid, providing rhythmic information to the whole organism. Melatonin acts via specific receptors, allowing the synchronization of daily and annual physiological rhythms to environmental conditions. The pituitary gland, which produces several hormones involved in a variety of physiological processes such as growth, metabolism, stress and reproduction, is an important target of melatonin. Melatonin modulates pituitary cellular activities, adjusting the synthesis and release of the different pituitary hormones to the functional demands, which changes during the day, seasons and life stages. It is, however, not always clear whether melatonin acts directly or indirectly on the pituitary. Indeed, melatonin also acts both upstream, on brain centers that control the pituitary hormone production and release, as well as downstream, on the tissues targeted by the pituitary hormones, which provide positive and negative feedback to the pituitary gland. In this review, we describe the known pathways through which melatonin modulates anterior pituitary hormonal production, distinguishing indirect effects mediated by brain centers from direct effects on the anterior pituitary. We also highlight similarities and differences between teleosts and mammals, drawing attention to knowledge gaps, and suggesting aims for future research.

Enrichment of ovine gonadotropes via adenovirus gene targeting enhances assessment of transcriptional changes in response to estradiol-17 beta†.

Gonadotropes represent approximately 5-15% of the total endocrine cell population in the mammalian anterior pituitary. Therefore, assessing the effects of experimental manipulation on virtually any parameter of gonadotrope biology is difficult to detect and parse from background noise. In non-rodent species, applying techniques such as high-throughput ribonucleic acid (RNA) sequencing is problematic due to difficulty in isolating and analyzing individual endocrine cell populations. Herein, we exploited cell-specific properties inherent to the proximal promoter of the human glycoprotein hormone alpha subunit gene (CGA) to genetically target the expression of a fluorescent reporter (green fluorescent protein [GFP]) selectively to ovine gonadotropes. Dissociated ovine pituitary cells were cultured and infected with an adenoviral reporter vector (Ad-hαCGA-eGFP). We established efficient gene targeting by successfully enriching dispersed GFP-positive cells with flow cytometry. Confirming enrichment of gonadotropes specifically, we detected elevated levels of luteinizing hormone (LH) but not thyrotropin-stimulating hormone (TSH) in GFP-positive cell populations compared to GFP-negative populations. Subsequently, we used next-generation sequencing to obtain the transcriptional profile of GFP-positive ovine gonadotropes in the presence or absence of estradiol 17-beta (E2), a key modulator of gonadotrope function. Compared to non-sorted cells, enriched GFP-positive cells revealed a distinct transcriptional profile consistent with established patterns of gonadotrope gene expression. Importantly, we also detected nearly 200 E2-responsive genes in enriched gonadotropes, which were not apparent in parallel experiments on non-enriched cell populations. From these data, we conclude that CGA-targeted adenoviral gene transfer is an effective means for selectively labeling and enriching ovine gonadotropes suitable for investigation by numerous experimental approaches.

AMP-activated protein kinase activation reduces the transcriptional activity of the murine luteinizing hormone ß-subunit gene.

Malnutrition is one of the factors that induces reproductive disorders. However, the underlying biological processes are unclear. AMP-activated protein kinase (AMPK) is an enzyme that plays crucial role as a cellular energy sensor. In the present study, we examined the effects of AMPK activation on the transcription of the murine gonadotropin subunit genes Cga, Lhb, and Fshb, and the gonadotropin-releasing hormone receptor Gnrh-r. Real-time PCR and transcription assay using LßT2 cells demonstrated that 5-amino-imidazole carboxamide riboside (AICAR), a cell-permeable AMP analog, repressed the expression of Lhb. Next, we examined deletion mutants of the upstream region of Lhb and found that the upstream regulatory region of Lhb (-2527 to -2198 b) was responsible for the repression by AICAR. Furthermore, putative transcription factors (SP1, STAT5a, and TEF) that might mediate transcriptional control of the Lhb repression induced by AICAR were identified. In addition, it was confirmed that both AICAR and a competitive inhibitor of glucose metabolism, 2-deoxy-D-glucose, induced AMPK phosphorylation in LßT2 cells. Therefore, the upstream region of Lhb is one of the target sites for glucoprivation inducing AMPK activation. In addition, AMPK plays a role in repressing Lhb expression through the distal -2527 to -2198 b region.

Hypothalamic-Pituitary and Adipose Tissue Responses to the Effect of Resistin in Sheep: The Integration of Leptin and Resistin Signaling Involving a Suppressor of Cytokine Signaling 3 and the Long Form of the Leptin Receptor.

We hypothesized that resistin is engaged in the development of leptin central insensitivity/resistance in sheep, which is a unique animal model to explore reversible leptin resistance. Thirty Polish Longwool ewes, which were ovariectomized with estrogen replacement, were used. Treatments consisted of the intravenous injection of control (saline) or recombinant bovine resistin (rbresistin): control (Control; n = 10), a low dose of rbresistin (R1; 1.0 µg/kg body weight (BW); n = 10), and a high dose of rbresistin (R2; 10.0 µg/kg BW; n = 10). The studies were performed during short-day (SD) and long-day (LD) photoperiods. Leptin and resistin concentrations were determined. Expression levels of a suppressor of cytokine signaling (SOCS)-3 and the long form of the leptin receptor (LeptRb) were determined in selected brain regions, including in the anterior pituitary (AP), hypothalamic arcuate nucleus (ARC), preoptic area (POA), and ventro- and dorsomedial nuclei (VMH/DMH). The results indicate that resistin induced a consistent decrease in LeptRb (except in POA) and an increase in SOCS-3 expression during the LD photoperiod in all selected brain regions. In conclusion, the results demonstrate that the action of resistin appears to be strongly associated with photoperiod-driven changes in the leptin signaling pathway, which may underlie the phenomenon of central leptin resistance.

Nonclassical actions of estradiol-17beta are not detectable in the alphaT3-1 and LbetaT2 immortalized gonadotrope cell lines†.

The immortalized mouse gonadotrope cell lines alphaT3-1 and LbetaT2 cells have been a substitute model for primary gonadotropes. These cell lines have provided a homogeneous cell population, as compared to the dissociated anterior pituitaries, which contain a heterogeneous population of cells potentially responsive to estradiol-17beta (E2). Nonclassical actions of E2 assumed to occur through the plasma membrane estrogen receptor 1 (ESR1, also known as ERalpha). These actions have included inhibition of gonadotropin-releasing hormone (GnRH)-induced increases in intracellular calcium concentrations and phosphorylation of p44/42 mitogen-activated protein kinase (ERK-1/2) in ovine pituitaries including primary gonadotropes in vitro. The objective of the present experiment was to determine if alphaT3-1 and LbetaT2 are cell models with limitations to examine the nonclassical actions of E2 occurring in gonadotropes. Experiments were conducted to determine if the cells have ESR1 at the plasma membrane using biotinylation cell and isolation of surface protein and staining with a fluorescently labeled E2 conjugate. The alphaT3-1 cells contain ESR1 associated with but not enriched within lipid rafts of the plasma membrane and do not translocate to lipid rafts upon binding of E2. In contrast, LbetaT2 cells lack ESR1 associated with the plasma membrane. Pretreatment with E2 did not cause inhibition of GnRH-stimulated increases in intracellular concentrations of calcium for either cell type. Phosphorylation of ERK-1/2 was not stimulated by E2 in either cell type. Although these cells lines have been used extensively to study GnRH signaling, in vitro or in vivo effects of nonclassical actions of E2 cannot be replicated in either cell line.

Selenium protection against mercury neurotoxicity: Modulation of apoptosis and autophagy in the anterior pituitary.

AIMS: The aim of the present study is to shed light on the modulating action of selenium on two of the most crucial cellular pathways; apoptosis and autophagy and the possible interplay between them in determining the pituitary fate in the context of mercury intoxication through demonstration of the molecular, histopathological, immunohistochemical, and ultrastructural features of selenium mercury-treated adenohypophysis. METHODS: Thirty adult Sprague Dawley male albino rats were assigned into control group, mercury-treated group and mercury­selenium concomitantly-treated group. The adenohypophysis was subjected to structural, molecular and protein expression assessment of autophagy and apoptotic markers and western blotted analysis of Beclin 1 as a key cross-regulator of autophagy and apoptosis. KEY FINDINGS: Selenium treatment ameliorated the mercury-induced apoptosis detected by improvement in PCR and immunohistochemical expression of the apoptotic markers Bax, Bcl-2 and Caspase-3. Selenium also improved mercury-induced autophagic dysfunction with statistically significant improvement in western blotted levels of the autophagy markers LC3I, LC3II and Beclin1. The histopathological and ultrastructural studies strongly confirmed those findings. SIGNIFICANCE: The crosstalk between the apoptotic Bcl-2 family of proteins and the autophagic Beclin-1LC3 pathway in the context of mercury intoxication paves the way for developing novel effective treatment strategies for several mercury-induced pituitary diseases.

Transcriptomic profile of anterior pituitary cells of pigs is affected by adiponectin.

Adiponectin is thought to be involved in the regulation of metabolic homeostasis and reproductive processes. It also has an important role in the modulation of female reproductive functions, both directly and by affecting the secretory functions of the hypothalamic-pituitary-gonadal axis. The main aim of this study was to determine the effect of adiponectin on global gene expression and on differentially expressed genes (DE-genes) regulated by adiponectin in anterior pituitary (AP) cells of pigs. The changes in the transcriptomic profile of AP cells of pigs were examined using the Porcine (V2) two-colour gene expression microarray, 4 × 44. An analysis of data from the microarray experiment indicated there were 716 DE-genes. A total of 466 genes (220 up-regulated and 246 down-regulated) with fold change greater than 1.2 (P < 0.05) were subsequently selected for further analysis. Gene ontology was analysed based on a list of DE-genes. A list of biological processes was generated for both up-regulated and down-regulated DE-genes. The products of up-regulated DE-genes were involved in 60 biological processes, whereas for down-regulated products there were 18 processes. An analysis of the interactions between DE-genes indicated that adiponectin interacted with genes that potentially encode intracellular signalling pathways and factors which regulate reproductive functions. Furthermore, nine genes were selected from the list of DE-genes to confirm microarray results by quantitative PCR. The results enhance the knowledge about adiponectin's role in the pituitary functions of pigs and provide valuable insights for further studies into adiponectin's mechanism of action in the pituitary.

Identification of TGFß-induced proteins in non-endocrine mouse pituitary cell line TtT/GF by SILAC-assisted quantitative mass spectrometry.

TtT/GF is a mouse cell line derived from a thyrotropic pituitary tumor and has been used as a model of folliculostellate cells. Our previous microarray data indicate that TtT/GF possesses some properties of endothelial cells, pericytes and stem/progenitor cells, along with folliculostellate cells, suggesting its plasticity. We also found that transforming growth factor beta (TGFß) alters cell motility, increases pericyte marker transcripts and attenuates endothelial cell and stem/progenitor cell markers in TtT/GF cells. The present study explores the wide-range effect of TGFß on TtT/GF cells at the protein level and characterizes TGFß-induced proteins and their partnerships using stable isotope labeling of amino acids in cell culture (SILAC)-assisted quantitative mass spectrometry. Comparison between quantified proteins from TGFß-treated cells and those from SB431542 (a selective TGFß receptor I inhibitor)-treated cells revealed 51 upregulated and 112 downregulated proteins (|log2| > 0.6). Gene ontology and STRING analyses revealed that these are related to the actin cytoskeleton, cell adhesion, extracellular matrix and DNA replication. Consistently, TGFß-treated cells showed a distinct actin filament pattern and reduced proliferation compared to vehicle-treated cells; SB431542 blocked the effect of TGFß. Upregulation of many pericyte markers (CSPG4, NES, ACTA, TAGLN, COL1A1, THBS1, TIMP3 and FLNA) supports our previous hypothesis that TGFß reinforces pericyte properties. We also found downregulation of CTSB, EZR and LGALS3, which are induced in several pituitary adenomas. These data provide valuable information about pericyte differentiation as well as the pathological processes in pituitary adenomas.

Gonadotropin-releasing hormone and kisseptin-10 regulate nuclear receptor subfamily 5 group a member 1/catenin beta 1/ nuclear receptor subfamily 0 group B member 1 activity in female rat anterior pituitary gland.

In this study, we tested the hypothesis that modulation of endogenous gonadotropin-releasing hormone (Gnrh) neuronal network activity alters the mRNA expression of nuclear receptor subfamily 5 group A member 1 (Nr5a1), through one of the component of Wnt pathway signaling - catenin beta 1 (Ctnnb1) (its co-activator), and its co-repressor nuclear receptor subfamily 0, group B member 1 (Nr0b1) in the female rat pituitary gland in vivo. Adult ovariectomized rats were given a serial infusion of Gnrh, kisspeptin-10, Gnrh + Gnrh antagonist (Antide), or kisspeptin-10 + kisspeptin antagonist (kisspeptin-234) into the third ventricle of the brain. The anterior pituitary and blood was used to mRNA and protein expression analysis. We demonstrated that Gnrh up-regulates Nr5a1 mRNA expression in the anterior pituitary and induces NR5A1 depletion in gonadotropes. Gnrh administration increased both Ctnnb1 mRNA expression and protein synthesis, and induced activation of cellular Ctnnb1 via translocation from the gonadotropes cytoplasm to nucleus. After kisspeptin-10 treatment, up-regulation of Nr0b1 mRNA and protein expression in the anterior pituitary was observed. These data indicate that Gnrh-neuron-mediated network activity alters Nr5a1 gene transcription and translation in gonadotrope cells and this effect may result from the changes induced in the Ctnnb1 and Nr0b1 gene/protein expression balance.

The anterior pituitary gap junctions: potential targets for toxicants.

The anterior pituitary regulates endocrine organs and physiological activities in the body. Environmental pollutants and drugs deleterious to the endocrine system may affect anterior pituitary activity through direct action on anterior pituitary cells. Within the gland, endocrine and folliculostellate cells are organized into and function as individual tridimensional networks, each network regulating its activity by coordinating the connected cells' responses to physiological or pathological cues. The gap junctions connecting endocrine cells and/or folliculostellate cells allow transmission of information among cells that is necessary for adequate network function. Toxicants may affect gap junctions as well as the physiology of the anterior pituitary. However, whether toxicants effects on anterior pituitary hormone secretion involve gap junctions is unknown. The folliculostellate cell gap junctions are sensitive to hormones, cytokines and growth factors. These cells may be an interesting experimental model for evaluating whether toxicants target anterior pituitary gap junctions.

Participation of membrane progesterone receptor α in the inhibitory effect of progesterone on prolactin secretion.

The membrane progesterone receptors (mPRα, mPRß, mPRγ, mPRδ and mPRε) are known to mediate rapid nongenomic progesterone functions in different cell types. However, the functions of these receptors in the pituitary have not been reported to date. In the present study, we show that the expression of mPRα was the highest among the mPRs in the rat anterior pituitary gland. Immunostaining of mPRα was detected in somatotrophs, gonadotrophs and lactotrophs. Interestingly, 63% of mPRα-positive cells within the pituitary were lactotrophs, suggesting that mPRα is involved in controlling prolactin (PRL) secretion in the pituitary. To test this hypothesis, rat pituitaries were incubated (1 hour) with either progesterone (P4) or the mPRα-specific agonist Org OD 02-0. PRL secretion was then measured by radioimmunoassay. The results of this experiment revealed that both P4 and Org OD 02-0 decreased PRL secretion. Moreover, the results from the GH3 cell line (CCL-82.1) showed that P4 and Org OD 02-0 inhibited PRL release, although the nuclear PR agonist R5020 was ineffective. Our investigation of the cellular mechanisms behind mPRα activity indicated that both P4 and Org OD 02-0 decreased cAMP accumulation, whereas R5020 was ineffective. In addition, the Org OD 02-0-effect on PRL release was blocked by pretreatment with pertussis toxin, an inhibitor of Go/Gi proteins. Because transforming growth factor (TGF)ß1 is a potent inhibitor of PRL secretion in lactotrophs, we lastly evaluated whether TGFß1 was activated by progesterone and whether this effect was mediated by mPRα. Our results showed that P4 and Org OD 02-0, but not R5020, increased active TGFß1 levels. This effect was not observed when cells were transfected with mPRα-small interfering RNA. Taken together, these data provide new evidence suggesting that mPRα mediates the progesterone inhibitory effect on PRL secretion through both decreases in cAMP levels and activation of TGFß1 in the lactotroph population.

Diet-Induced Growth Is Regulated via Acquired Leptin Resistance and Engages a Pomc-Somatostatin-Growth Hormone Circuit.

Anorexigenic pro-opiomelanocortin (Pomc)/alpha-melanocyte stimulating hormone (αMSH) neurons of the hypothalamic melanocortin system function as key regulators of energy homeostasis, also controlling somatic growth across different species. However, the mechanisms of melanocortin-dependent growth control still remain ill-defined. Here, we reveal a thus-far-unrecognized structural and functional connection between Pomc neurons and the somatotropic hypothalamo-pituitary axis. Excessive feeding of larval zebrafish causes leptin resistance and reduced levels of the hypothalamic satiety mediator pomca. In turn, this leads to reduced activation of hypophysiotropic somatostatin (Sst)-neurons that express the melanocortin receptor Mc4r, elevated growth hormone (GH) expression in the pituitary, and enhanced somatic growth. Mc4r expression and αMSH responsiveness are conserved in Sst-expressing hypothalamic neurons of mice. Thus, acquired leptin resistance and attenuation of pomca transcription in response to excessive caloric intake may represent an ancient mechanism to promote somatic growth when food resources are plentiful.