Pituitary Hormone-producing Cells Research Articles

Single-cell transcriptomic analysis reveals rich pituitary-Immune interactions under systemic inflammation.

The pituitary represents an essential hub in the hypothalamus-pituitary-adrenal (HPA) axis. Pituitary hormone-producing cells (HPCs) release several hormones to regulate fundamental bodily functions under normal and stressful conditions. It is well established that the pituitary endocrine gland modulates the immune system by releasing adrenocorticotropic hormone (ACTH) in response to neuronal activation in the hypothalamus. However, it remains unclear how systemic inflammation regulates the transcriptomic profiles of pituitary HPCs. Here, we performed single-cell RNA-sequencing (scRNA-seq) of the mouse pituitary and revealed that upon inflammation, all major pituitary HPCs respond robustly in a cell type-specific manner, with corticotropes displaying the strongest reaction. Systemic inflammation also led to the production and release of noncanonical bioactive molecules, including Nptx2 by corticotropes, to modulate immune homeostasis. Meanwhile, HPCs up-regulated the gene expression of chemokines that facilitated the communication between the HPCs and immune cells. Together, our study reveals extensive interactions between the pituitary and immune system, suggesting multifaceted roles of the pituitary in mediating the effects of inflammation on many aspects of body physiology.

PI(4,5)P2-dependent and -independent roles of PI4P in the control of hormone secretion by pituitary cells.

Plasma membrane and organelle membranes are home to seven phosphoinositides, an important class of low-abundance anionic signaling lipids that contribute to cellular functions by recruiting cytoplasmic proteins or interacting with the cytoplasmic domains of membrane proteins. Here, we briefly review the functions of three phosphoinositides, PI4P, PI(4,5)P2, and PI(3,4,5)P3, in cellular signaling and exocytosis, focusing on hormone-producing pituitary cells. PI(4,5)P2, acting as a substrate for phospholipase C, plays a key role in the control of pituitary cell functions, including hormone synthesis and secretion. PI(4,5)P2 also acts as a substrate for class I PI3-kinases, leading to the generation of two intracellular messengers, PI(3,4,5)P3 and PI(3,4)P2, which act through their intracellular effectors, including Akt. PI(4,5)P2 can also influence the release of pituitary hormones acting as an intact lipid to regulate ion channel gating and concomitant calcium signaling, as well as the exocytic pathway. Recent findings also show that PI4P is not only a precursor of PI(4,5)P2, but also a key signaling molecule in many cell types, including pituitary cells, where it controls hormone secretion in a PI(4,5)P2-independent manner.

Disease Modeling of Pituitary Adenoma Using Human Pluripotent Stem Cells.

Simple SummaryPituitary adenoma pathophysiology has been studied mainly using murine cell lines, animal models, and pituitary tumor samples. However, the lack of human pituitary cell line is a significant limiting factor in studying the molecular mechanisms of human pituitary tumors. Recently, pituitary induction methods from human-induced pluripotent stem cells (hiPSCs) have been established. These methods can induce human pituitary hormone-producing cells that retain physiological properties. hiPSCs in which tumor-causing gene mutations are introduced using genome-editing techniques, such as CRISPR/Cas9 systems, provide great opportunities to establish in vitro human pituitary adenoma disease models. The models will be a novel platform to discover novel drugs and investigate tumorigenesis and pathophysiology. The purpose of this review is to provide an overview of the applications of iPSCs for pituitary and neoplastic disorder research and genome-editing technologies to create strategies for developing pituitary adenoma models using iPSCs.Pituitary adenomas are characterized by abnormal growth in the pituitary gland. Surgical excision is the first-line treatment for functional (hormone-producing) pituitary adenomas, except for prolactin-producing adenomas; however, complete excision is technically challenging, and many patients require long-term medication after the treatment. In addition, the pathophysiology of pituitary adenomas, such as tumorigenesis, has not been fully understood. Pituitary adenoma pathophysiology has mainly been studied using animal models and animal tumor-derived cell lines. Nevertheless, experimental studies on human pituitary adenomas are difficult because of the significant differences among species and the lack of reliable cell lines. Recently, several methods have been established to differentiate pituitary cells from human pluripotent stem cells (hPSCs). The induced pituitary hormone-producing cells retain the physiological properties already lost in tumor-derived cell lines. Moreover, CRISPR/Cas9 systems have expedited the introduction of causative gene mutations in various malignant tumors into hPSCs. Therefore, hPSC-derived pituitary cells have great potential as a novel platform for studying the pathophysiology of human-specific pituitary adenomas and developing novel drugs. This review presents an overview of the recent progresses in hPSC applications for pituitary research, functional pituitary adenoma pathogenesis, and genome-editing techniques for introducing causative mutations. We also discuss future applications of hPSCs for studying pituitary adenomas.

Paracrine Signalling From SOX2-Expressing Pituitary Embryonic Cells Is Required for Terminal Differentiation of Hormone-Producing Cells

The pituitary gland is the master regulator of the endocrine system, housing six major hormone producing cell types. This gland is derived from Rathke’s Pouch, an invagination of the oral ectoderm. Hormone-producing pituitary cell lineages are derived from a population of embryonic cells expressing SOX2. ZFP36L1/Butyrate Response Factor 1 (BRF1) is an RNA binding protein that binds and targets mRNAs of various cytokines and chemokines for degradation prior to translation, attenuating secretion of inflammatory factors (Herranz et al. 2015). Here, we show that BRF1 is a novel marker expressed in SOX2+ cells in human and mouse pituitaries, suggesting that these cells may have a secretory profile. To investigate this possibility, we have combined molecular and genetic studies in vivo. We have used a novel mouse model, R26lsl-mBRF1 that allows the expression of a mutant, constitutively active BRF1 protein upon Cre-mediated recombination, alongside our lab’s models (Hesx1Cre/+ and Sox2CreERT2/+), to express mutant BRF1 in HESX1+ and SOX2+ cells during development and postnatally. This approach results in pituitary hypoplasia and severe hypopituitarism due to a failure of cell-lineage specified cells to differentiate into hormone-producing cells. Hormone production in these mutant cells, however, can be rescued in vitro through co-culture with WT pituitaries and in vivo in chimeric pituitaries, highlighting a cell non-autonomous mechanism underlying the phenotype. Single cell RNA sequencing of WT and Sox2CreERT2/+;R26lsl-mBRF1 murine embryonic pituitaries, as well as use publicly available human pituitary single cell datasets, have allowed us to identify specific cytokines and chemokines secreted by SOX2+ cells, as well as downstream intracellular signalling pathways in differentiating cells (Zhang et al. 2020), which may be responsible for controlling terminal differentiation of hormone-producing cells within the developing pituitary. Together with our recently published data, these results support the notion that SOX2+ pituitary stem cells play a critical paracrine role in controlling progenitor cell proliferation and terminal differentiation (Russell et al. 2021).

Congenital pituitary hypoplasia model demonstrates hypothalamic OTX2 regulation of pituitary progenitor cells

Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone-producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.

Aldolase C is a novel molecular marker for folliculo-stellate cells in rodent pituitary.

The anterior pituitary gland is composed of five types of hormone-producing cells and folliculo-stellate cells. Folliculo-stellate cells do not produce anterior pituitary hormones but they are thought to play important roles as stem cells, phagocytes, or supporting cells of hormone-producing cells in the anterior pituitary. S100β protein has been used as a folliculo-stellate cell marker in some animals, including rats. However, since no reliable molecular marker for folliculo-stellate cells has been reported in mice, genetic approaches for the investigation of folliculo-stellate cells in mice are not yet available. Aldolase C/Zebrin II is a brain-type isozyme and is a fructose-1,6-bisphosphate aldolase. In the present study, we first used immunohistochemistry to verify that aldolase C was produced in the anterior pituitary of rats. Moreover, using transgenic rats expressing green fluorescent protein under the control of the S100β gene promoter, we identified aldolase C-immunoreactive signals in folliculo-stellate cells and marginal cells located in the parenchyma of the anterior pituitary and around Rathke's cleft, respectively. We also identified aldolase C-expressing cells in the mouse pituitary using immunohistochemistry and in situ hybridization. Aldolase C was not produced in any pituitary hormone-producing cells, while aldolase C-immunopositive signal co-localized with E-cadherin- and SOX2-positive cells. Using post-embedding immunoelectron microscopy, aldolase C-immunoreactive products were observed in the cytoplasm of marginal cells and folliculo-stellate cells of the mouse pituitary. Taken together, aldolase C is a common folliculo-stellate cell marker in the anterior pituitary gland of rodents.

Colocalization of GPR120 and anterior pituitary hormone-producing cells in female Japanese Black cattle.

Negative energy balance in domestic animals suppresses their reproductive function. These animals commonly use long-chain fatty acids (LCFAs) from adipocytes as an energy source under states of malnutrition. The G-protein coupled receptor, GPR120, is a specific receptor for LCFAs, but its role in reproductive function remains unknown in domestic animals. The purpose of this study was to examine whether GPR120 is involved in the reproductive system of cattle. GPR120 mRNA expression was evaluated in brain, pituitary, and ovarian tissue samples by RT-PCR. GPR120 gene expression was detected with high intensity only in the anterior pituitary sample, and GPR120-immunoreactive cells were found in the anterior pituitary gland. Double immunohistochemistry of GPR120 in the anterior pituitary hormone-producing cells, such as gonadotropes, thyrotropes, lactotropes, somatotropes, and corticotropes, was performed to clarify the distribution of GPR120 in the anterior pituitary gland of ovariectomized heifers. Luteinizing hormone β subunit (LHβ)- and follicle-stimulating hormone β subunit (FSHβ)-immunoreactive cells demonstrated GPR120 immunoreactivity at 80.7% and 85.9%, respectively. Thyrotropes, lactotropes, somatotropes, and corticotropes coexpressed GPR120 at 21.1%, 5.4%, 13.6%, and 14.5%, respectively. In conclusion, the present study suggests that GPR120 in the anterior pituitary gland might mediate LCFA signaling to regulate gonadotrope functions, such as hormone secretion or production, in cattle.

Congenital pituitary hypoplasia model demonstrates hypothalamic OTX2 regulation of pituitary progenitor cells.

Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone-producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.

Application of pluripotent stem cells for treatment of human neuroendocrine disorders.

The neuroendocrine system is composed of many types of functional cells. Matured cells are generally irreversible to progenitor cells and it is difficult to obtain enough from our body. Therefore, studying specific subtypes of human neuroendocrine cells in vitro has not been feasible. One of the solutions is pluripotent stem cells, such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. These are unlimited sources and, in theory, are able to give rise to all cell types of our body. Therefore, we can use them for regenerative medicine, developmental basic research and disease modeling. Based on this idea, differentiation methods have been studied for years. Recent studies have successfully induced hypothalamic-like progenitors from mouse and human ES/iPS cells. The induced hypothalamic-like progenitors generated hypothalamic neurons, for instance, vasopressin neurons. Induction to adenohypophysis was also reported in the manner of self-formation by three-dimensional floating cultures. Rathke's pouch-like structures, i.e., pituitary anlage, were self-organized in accordance with pituitary development in embryo. Pituitary hormone-producing cells were subsequently differentiated. The induced corticotrophs secreted adrenocorticotropic hormone in response to corticotropin-releasing hormone. When engrafted in vivo, these cells rescued systemic glucocorticoid levels in hypopituitary mice. These culture methods were characterized by replication of stepwise embryonic differentiation. It is based on the idea of mimicking the molecular environment of embryogenesis. Thanks to these improvements, these days, we can generate hormone-secreting neuroendocrine cells from pluripotent stem cells. The next problems that need to be solved are improving differentiation efficiency even further and structuring networks.

The Morpho-Functional Parameters of Rat Pituitary Hormone Producing Cells After Genistein Treatment

Abstract Phytoestrogens are a diverse group of steroid–like compounds that occur naturally in many plants. There are various types of phytoestrogens, including the best-researched isoflavones which are commonly found in soy. The consumption of soy products has many health benefits, including protection against breast cancer, prostate cancer, menopausal symptoms, heart disease and osteoporosis. In contrast, use of hormonally active compounds-isoflavones may unfortunately interfere with the endocrine system and can have far-reaching consequences. Genistein, the most abundant soy-bean derived isoflavone, possesses a ring system similar to estrogens and acts through an estrogen receptor (ER)-mediated mechanism, by increasing or decreasing the transcription of ER-dependent target genes. Also, genistein can act on cells through ER non-dependent mechanisms, such as tyrosine kinase inhibitor. The neuroendocrine systems are responsible for the control of homeostatic processes in the body, including reproduction, growth, metabolism and energy balance, and stress responsiveness. It is well known, that estrogen is important for development of the neuroendocrine system in both sexes. At the pituitary level, estrogen is known to affect the regulation of all hormone producing (HP) cells, by direct and/or indirect mechanisms. Due to structural and functional resemblance to estrogen, the question may arise of whether and how genistein affects the morphofunctional features of pituitary HP cells. This review deals with the consequences of genistein’s effects on morphological, stereological and hormonal features of HP cells within the anterior pituitary gland. Transparency on this issue is needed because isoflavones are presently highly consumed. Inter alia, genistein as well as other isoflavones, are present in various dietary supplements and generally promoted as an accepted alternative to estrogen replacement therapy. Potential isoflavone biomedical exploitation is not only limited to estrogen replacement therapy, so it should be treated in a wider context of different ageing symptoms remediation.

Selenoprotein T is a novel OST subunit that regulates UPR signaling and hormone secretion.

Selenoprotein T (SelT) is a recently characterized thioredoxin-like protein whose expression is very high during development, but is confined to endocrine tissues in adulthood where its function is unknown. We report here that SelT is required for adaptation to the stressful conditions of high hormone level production in endocrine cells. Using immunofluorescence and TEM immunogold approaches, we find that SelT is expressed at the endoplasmic reticulum membrane in all hormone-producing pituitary cell types. SelT knockdown in corticotrope cells promotes unfolded protein response (UPR) and ER stress and lowers endoplasmic reticulum-associated protein degradation (ERAD) and hormone production. Using a screen in yeast for SelT-membrane protein interactions, we sort keratinocyte-associated protein 2 (KCP2), a subunit of the protein complex oligosaccharyltransferase (OST). In fact, SelT interacts not only with KCP2 but also with other subunits of the A-type OST complex which are depleted after SelT knockdown leading to POMC N-glycosylation defects. This study identifies SelT as a novel subunit of the A-type OST complex, indispensable for its integrity and for ER homeostasis, and exerting a pivotal adaptive function that allows endocrine cells to properly achieve the maturation and secretion of hormones.

Functional anterior pituitary generated in self-organizing culture of human embryonic stem cells.

Anterior pituitary is critical for endocrine systems. Its hormonal responses to positive and negative regulators are indispensable for homeostasis. For this reason, generating human anterior pituitary tissue that retains regulatory hormonal control in vitro is an important step for the development of cell transplantation therapy for pituitary diseases. Here we achieve this by recapitulating mouse pituitary development using human embryonic stem cells. We find that anterior pituitary self-forms in vitro following the co-induction of hypothalamic and oral ectoderm. The juxtaposition of these tissues facilitated the formation of pituitary placode, which subsequently differentiated into pituitary hormone-producing cells. They responded normally to both releasing and feedback signals. In addition, after transplantation into hypopituitary mice, the in vitro-generated corticotrophs rescued physical activity levels and survival of the hosts. Thus, we report a useful methodology for the production of regulator-responsive human pituitary tissue that may benefit future studies in regenerative medicine.

Making pituitary hormone-producing cells in a dish [Review

The hypothalamic-pituitary system is essential for maintaining life and controlling systemic homeostasis. The functional disorder makes patients suffer from various symptoms all their lives. Pluripotent stem cells, such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, differentiate into neuroectodermal progenitors when cultured as floating aggregates under serum-free conditions. Recent results have shown that strict removal of exogenous patterning factors during the early differentiation period induces rostral hypothalamic-like progenitors from mouse ES cells. The use of growth factor-free, chemically defined medium was critical for this induction. The ES cell-derived hypothalamic-like progenitors generated rostral-dorsal hypothalamic neurons, in particular magnocellular vasopressinergic neurons. We subsequently reported self-formation of adenohypophysis in three-dimensional floating cultures of mouse ES cells. The ES cell aggregates were stimulated to differentiate into both non-neural head ectoderm and hypothalamic neuroectoderm in adjacent layers. Self-organization of Rathke's pouch-like structures occurred at the interface of the two epithelia in vitro. Various pituitary endocrine cells including corticotrophs and somatotrophs were subsequently produced from the Rathke's pouch-like structures. The induced corticotrophs efficiently secreted ACTH in response to CRH. Furthermore, when engrafted in vivo, these cells rescued systemic glucocorticoid levels in hypopituitary mice. Our latest study aimed to prepare hypothalamic and pituitary tissues from human pluripotent stem cells. We succeeded in establishing the differentiation method using human ES/iPS cells. The culture method is characterized by replication of stepwise embryonic differentiation. Therefore, these methods could potentially be used as developmental and disease models, as well as for future regenerative medicine.

Pituitary Hormone-producing Cells After Estradiol Application in Rat Models of Menopause

Abstract Female ageing represents the biological process of structural and functional changes in endocrine cells and tissues, as well as in pituitary hormone-producing cells. In addition to the hypothalamic releasing hormones, estradiol plays a significant role in the regulation of the synthesis/secretion of pituitary hormones and is still used therapeutically for menopausal symptoms. The effects of ageing or ovariectomy and synthetic estradiol application under these circumstances were evaluated in pituitary hormone-producing cells of female rats (animal models of menopause); i.e., the following cells were observed: gonadotropes (FSH and LH), thyrotropes (TSH), somatotropes (GH), mammotropes (PRL) and corticotropes (ACTH). The cells were immunostained and histologically analysed. The ELISA method was used for hormonal analyses. Ageing was found to cause diverse, commonly reductive changes regarding the volume, number and secretion of menopausal rat pituitary hormone- producing cells, except for PRL cells that exhibit significantly increased numbers and intensified secretion. After the treatment of middle-aged female rats wiThestradiol, the absolute and relative pituitary weights significantly increased in comparison with the control females. Histological parameters such as the cell and volume density of PRL and ACThcells were significantly increased compared with the control values. The mentioned parameters of FSH, LH, GH, and occasionally TSH cells after estradiol treatment significantly decreased in comparison with the controls. The corresponding hormone levels followed the changes in the histological parameters. These data indicate that the application of estradiol to menopausal females may specifically, in two directions, modify the histological characteristics and secretory activities of different pituitary-hormone producing cells..

Role of Ras, ERK, and Akt in glucocorticoid-induced differentiation of embryonic rat somatotropes in vitro

This study investigated the roles of Ras, ERK, and Akt in the glucocorticoid-induced differentiation of growth hormone-producing pituitary cells in vitro. Pituicytes isolated from day-18 rat embryos were cultured with 50 mM dexamethasone in addition to specific inhibitors of Ras (manumycin; 0.5, 5, 50 nM), ERK (U0126, 10 μM), or Akt (LY294002, 25 μM). Differentiation was assessed using immunofluorescent staining of intracellular growth hormone. Radioimmunoassay and Western blot analyses were used to determine levels of secreted and intracellular growth hormone, respectively. Manumycin reduced the fraction of growth hormone-positive cells and dexamethasone-induced growth hormone secretion in a dose-dependent manner (both P < 0.001). In the absence of dexamethasone, LY294002 and U0126 did not alter the fraction of growth hormone-positive cells or intracellular growth hormone protein expression or secretion. Both LY294002 and U0126 alone significantly attenuated the fraction of dexamethasone-treated GH-positive cells and the secretion of GH compared to those of cells treated only with dexamethasone (50 nM for 44 h or 48 h) (all P < 0.05). Dexamethasone treatment alone did not change GH protein levels. Treatment of cells with a combination of LY294402 and U0126 significantly attenuated the fraction of dexamethasone-treated GH-positive cells, GH protein levels, and GH secretion compared to cells treated with dexamethasone alone (all P < 0.05). Moreover, dexamethasone-induced phosphorylation of GTP-Ras, ERK, and Akt was significantly attenuated by exposure to the respective inhibitors (P < 0.05). Taken together, our results indicate that Ras, ERK, and Akt are key effectors in the glucocorticoid-induced differentiation of growth hormone-secreting cells.

Pituitary hormone-producing cells after estradiol application in rat models of menopause

Pituitary hormone-producing cells after estradiol application in rat models of menopause

Specification of Functional Cranial Placode Derivatives from Human Pluripotent Stem Cells

Cranial placodes are embryonic structures essential for sensory and endocrine organ development. Human placode development has remained largely inaccessible despite the serious medical conditions caused by the dysfunction of placode-derived tissues. Here, we demonstrate the efficient derivation of cranial placodes from human pluripotent stem cells. Timed removal of the BMP inhibitor Noggin, a component of the dual-SMAD inhibition strategy of neural induction, triggers placode induction at the expense of CNS fates. Concomitant inhibition of fibroblast growth factor signaling disrupts placode derivation and induces surface ectoderm. Further fate specification at the preplacode stage enables the selective generation of placode-derived trigeminal ganglia capable of invivo engraftment, mature lens fibers, and anterior pituitary hormone-producing cells that upon transplantation produce human growth hormone and adrenocorticotropic hormone invivo. Our results establish a powerful experimental platform to study human cranial placode development and set the stage for the development of human cell-based therapies in sensory and endocrine disease.

Ghrelin increases intracellular Ca2+ concentration in the various hormone-producing cell types of the rat pituitary gland

Ghrelin, isolated from the stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), has potent growth hormone release ability in vivo and in vitro. Although GHS-R is abundantly expressed in the pituitary gland, there is no direct evidence of a relationship between hormone-producing cells and functional GHS-R in the pituitary gland. The aim of this study was to determine which anterior pituitary cells respond to ghrelin stimulation in male rats. We performed Fura-2 Ca2+ imaging analysis using isolated pituitary cells, and performed immunocytochemistry to identify the type of pituitary hormone-producing cells. In Fura-2 Ca2+ imaging analysis, ghrelin administration increased the intracellular Ca2+ concentration in approximately 50% of total isolated anterior pituitary cells, and 20% of these cells strongly responded to ghrelin. Immunocytochemical analysis revealed that 82.9±1.3% of cells that responded to ghrelin stimulation were GH-immunopositive. On the other hand, PRL-, LH-, and ACTH-immunopositive cells constituted 2.0±0.3%, 12.6±0.3%, and 2.5±0.8% of ghrelin-responding pituitary cells, respectively. TSH-immunopositive cells did not respond to ghrelin treatment. These results suggest that ghrelin directly acts not only on somatotrophs, but also on mammotrophs, gonadotrophs, and corticotrophs in the rat pituitary gland.

Paracrine Control of Gonadotrophs by Somatolactotrophs through TRHinduced Follistatin Production

There is increasing evidence for the existence of local regulation of hormone secretion among pituitary cells. Hormone-producing pituitary cells may communicate with each other and with folliculostellate cells. Activin is one of the regulators of follicle stimulating hormone (FSH) secretion and gene expression, whereas follistatin negatively regulates FSH production by binding to and bioneutralizing the effects of activin. In prolactin-secreting GH3 cells, hypothalamic thyrotropin-releasing hormone (TRH) has been known to stimulate prolactin synthesis and secretion. Follistatin is produced in GH3 cells and is up-regulated by TRH in an ERK dependent manner. Prolactin mRNA levels in GH3 cells are not affected by increasing the dose of exogenous follistatin, and TRH-induced prolactin expression is not modulated in the presence of follistatin. Follistatin produced in somatolactotroph GH3 cells does not affect prolactin production in these cells. In pituitary gonadotroph cell line, LT2, activin increases FSHpromoter activity and mRNA expression, and follistatin completely inhibits this activin-increased FSHgene expression. On the other hand, activin reduces the basal activity of prolactin transcription and follistatin prevents these effects. When GH3 cells and LT2 cells are co-cultured, activin-induced FSHpromoter activity is completely inhibited in the presence of follistatin. In addition, FSHmRNA is not detected in LT2 cells, when co-cultured with GH3 cells. These observations using the model for somatolactotroph and gonadotroph suggest the possibility that somatolactotrophs and gonadotrophs interact with each other, and TRH might indirectly affect gonadotropin production though follistatin.

Immunohistochemical Localization of Aquaporin-4 in the Rat Pituitary Gland

Aquaporin-4 (AQP4) is an isoform of membrane water channel aquaporins. It is most abundant in the brain, and is believed to be expressed by astrocytes and ependymal cells. We have reported previously that rat pituitary glands express some aquaporin subtypes, including AQP4, but the precise distribution of AQP4 in the pituitary gland is not known. The present work investigated whether AQP4 immunoreactivity exists in various pituitary cell types in adult rats, using double immunofluorescent staining and confocal microscopy. In the adenohypophysis, co-labeling for S-100 protein indicated that folliculo-stellate (FS) cells and marginal layer cells in Rathke's residual pouch have extensive labeling for AQP4. Some AQP4-expressing cells also contained glial fibrillary acidic protein (GFAP) in parenchymal tissue of the anterior lobe, and cytokeratin in marginal layer cells. AQP4 was not coexpressed in any hormone-producing pituitary cells, however. In the neurohypophysis, AQP4 was expressed in some pituicytes, which have been identified as GFAP-positive. These results suggest that AQP4, which is expressed in FS cells, in marginal layer cells and in pituicytes, but not in hormone-producing cells, is important in water transfer within the pituitary gland; astrocytes and ependymal cells do the same in the brain.