Rat Pituitary Gland Research Articles

18F-labelling of A-ring substituted 16α-fluoro-estradiols as potential radiopharmaceuticals for PET imaging

The 2-methoxy derivative of estradiol is currently in Phase II clinical trial as an anticancer agent while the 4-methyl derivative has been shown to interact with cytoplasmic and nuclear estrogen receptors in rat pituitary gland and hypothalamus. We hypothesize that the 16α- 18F-analogs of these estrogens could be suitable radiotracers to evaluate action mechanisms of the parent compounds. In this study we report the synthesis of the 16α- 18F and 16α- 19F-analogs of the A-ring substituted estradiols in high yield via stereoselective opening of the intermediate 16β,17β-O-cyclic sulfones with [ 18F]F − or F − followed by deprotection.

The spatial and temporal expression of delta-like protein 1 in the rat pituitary gland during development

An analysis of secreted proteins by the signal sequence trap method using a cDNA library of the rat pituitary anlage at embryonic days (E) 13.5 revealed the abundant expression of delta-like protein 1 (Dlk1) in the pituitary gland. Dlk1, an epidermal growth factor-like repeat protein in preadipocytes, functions in maintaining the preadipose state. Expression of Dlk1 mRNA in the pituitary at E13.5 and in the adult pituitary was confirmed by in situ hybridization. The expression pattern of Dlk1 during pituitary development was also studied by immunohistochemistry. Dlk1 protein first appeared in Rathke's pouch and the infundibulum at E11.5; as development proceeded, expression became restricted to the pars distalis and pars tuberalis (PT). Dlk1 was expressed in most ACTH cells during the embryonic stages, but its expression was limited to only a few ACTH cells in the adult pituitary. It was also expressed in a small population of TSH, GTH, and PRL cells throughout development, whereas it was present in the cytoplasm of most GH cells at all developmental stages. Similarly, Dlk1 was localized in the cytoplasm of PT cells during development. These findings provide new insights into the mechanism of Dlk1 regarding its regulation of pituitary hormone-secreting cells during development.

Effects of Vasopressin V1b Receptor Deficiency on Adrenocorticotropin Release from Anterior Pituitary Cells in Response to Oxytocin Stimulation

Oxytocin (OT) is one of the secretagogues for stress-induced ACTH release. OT-induced ACTH release is reported to be mediated by the vasopressin V1b receptor in the rat pituitary gland, which contains both OT and V1b receptors. We examined OT-induced ACTH release using primary cultures of anterior pituitary cells from wild-type (V1bR+/+) and V1b receptor knockout (V1bR-/-) mice. OT stimulated similar levels of ACTH release from pituitary cells of V1bR+/+ and V1bR-/- mice. OT-induced ACTH release was significantly inhibited by the selective V1b receptor antagonist SSR149415 and the OT receptor antagonist CL-14-26 in V1bR+/+ mice. In addition, cotreatment with SSR149415 at 10(-6) m and CL-14-26 at 10(-6) m inhibited OT-induced ACTH release to the control level inV1bR+/+ mice. In V1bR-/- mice, OT-induced ACTH release was significantly inhibited by CL-14-26 at 10(-8) m and completely inhibited at 10(-7)m. These results indicate that OT induces the ACTH response via OT and V1b receptors inV1bR+/+ mice but via only OT receptors in V1bR-/- mice. The gene expression level of the OT receptor was significantly higher in the anterior pituitary gland of V1bR-/- mice than in that of V1bR+/+ mice, suggesting that the OT receptor is up-regulated to compensate for ACTH release under conditions of V1b receptor deficiency.

An Involvement of Estrogen Receptors in the Transcriptional and Translational Induction of Growth Hormone Evoked by Xenoestrogens in Rat Pituitary Gland.

An Involvement of Estrogen Receptors in the Transcriptional and Translational Induction of Growth Hormone Evoked by Xenoestrogens in Rat Pituitary Gland.

Proteomic Analysis of Diaminochlorotriazine (DACT) Adducts in Wister Rat Pituitary Glands and LβT2 Rat Pituitary Cells.

Atrazine (ATRA) is the most commonly applied herbicide in the United States and is frequently detected in drinking water at significant levels. After oral exposure, ATRA metabolism yields diaminochlorotriazine (DACT), an electrophilic molecule that has been shown to form covalent protein adducts. This research was designed to identify ATRA-induced protein adducts formed in the pituitary gland of ATRA-exposed rats and in DACT-exposed LβT2 rat pituitary cells. Immunohistochemistry showed diffuse cytoplasmic and nuclear staining in both pituitary sections and LβT2 cells indicating the formation of DACT protein adducts. Protein targets from both rat pituitaries and LβT2 cell culture were identified following two-dimensional electrophoresis (2DE), immunodetection, and matrix-assisted laser desorption ionization−time of flight mass spectrometry analysis. Western blots from both exposed rats and LβT2 cells revealed over 30 DACT-modified spots that were not present in control animals. Protein spots were matched t...

Histopathological effects of 4- tert-octylphenol treatment through the pregnancy period, on the pituitary, adrenal, pancreas, thyroid and parathyroid glands of offspring rats at adulthood

The present study was carried out to investigate the effects of 4- tert-octylphenol (OP) exposure at fetal period on adrenal, pituitary, thyroid + parathyroid and pancreas tissues of male and female offsprings. Pregnant rats were treated with OP (100 or 250 mg/(kg day)) in vehicle (corn oil) or vehicle alone daily from day 1 to 20 of pregnancy. After birth, young rats were allowed to growth until adulthood. While there were no differences in data of organ weight between control and treatment groups, in contrast, a decrease of relative organ weights of thyroid + parathyroid and adrenal in high dose treatment group in male rats, otherwise an increase of final body weights was found in 250 mg/(kg day) treatment group in all rats. Also, a lot of histopathological findings were observed in investigated tissues. The results of this study suggest that, the octylphenol which was applied in fetal period causes negative effects on the adrenal, pituitary gland, thyroid + parathyroid and pancreas in rats.

Proteomic Analysis of Diaminochlorotriazine Adducts in Wister Rat Pituitary Glands and LβT2 Rat Pituitary Cells

Atrazine (ATRA) is the most commonly applied herbicide in the United States and is frequently detected in drinking water at significant levels. After oral exposure, ATRA metabolism yields diaminochlorotriazine (DACT), an electrophilic molecule that has been shown to form covalent protein adducts. This research was designed to identify ATRA-induced protein adducts formed in the pituitary gland of ATRA-exposed rats and in DACT-exposed LbetaT2 rat pituitary cells. Immunohistochemistry showed diffuse cytoplasmic and nuclear staining in both pituitary sections and LbetaT2 cells indicating the formation of DACT protein adducts. Protein targets from both rat pituitaries and LbetaT2 cell culture were identified following two-dimensional electrophoresis (2DE), immunodetection, and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. Western blots from both exposed rats and LbetaT2 cells revealed over 30 DACT-modified spots that were not present in control animals. Protein spots were matched to concurrently run 2DE gels stained with Sypro Ruby, excised, and in-gel-digested with trypsin. Mass spectrometry analysis of digest peptides resulted in the identification of 19 spots and 8 unique proteins in the rats and 21 spots and 19 unique proteins in LbetaT2 cells. The identified proteins present in both sample types included proteasome activator complex subunit 1, ubiquitin carboxyl-terminal hydrolase isozyme L1, tropomyosin, ERp57, and RNA-binding proteins. Each of these proteins contains active-site or solvent-exposed cysteine residues, making them viable targets for covalent modification by DACT.

Pharmacological investigations of the unique herbal formula Menoprogen in rats: Estrogenic activity and mechanism

The present study aimed to evaluate the pharmacological effects of Menoprogen in the management of menopausal symptoms for aged female rats. Menoprogen was supplemented to a group of elderly female rats for 8 weeks. Subsequently, histopathological examinations were conducted in the isolated uterine and ovary tissues and pituitary glands of the rats. Serum levels of estradiol (E2), progesterone (P), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were determined. The ultrastructure of the rat ovarian granulocytes was analyzed. The histopathological examinations revealed no statistical difference in the quantity of follicles in the ovary and of acidophil and basophil cells in the anterior pituitary gland of the Menoprogen-fed rats versus healthy normal rats. Moreover, the cellular morphogenesis was in a healthy state for the Menoprogen-fed rats. Menoprogen significantly increased the levels of serum E2 and P but reduced FSH and LH levels. The electron microscopic analysis showed that Menoprogen significantly retarded apoptosis of the ovarian granulocytes of the rats. Further investigation of Menoprogen for the alternative treatment of menopausal symptoms is warranted.

In Vivo Manganese MR Imaging of Calcium Influx in Spontaneous Rat Pituitary Adenoma

Rapid uptake of the calcium analog manganese (Mn2+) into spontaneous pituitary adenoma during MR imaging of aged rats generated the hypothesis that neuroendocrine tumors may have a corresponding increase in calcium influx required to trigger hormonal release. A goal of this study was to investigate the potential for clinical evaluation of pituitary adenoma by MR imaging combined with administration of Mn2+ (Mn-MR imaging). Mn-MR imaging was used to characterize the dynamic calcium influx in normal aged rat pituitary gland as well as spontaneous pituitary adenoma. To confirm the validity of Mn2+ as a calcium analog, we inhibited Mn2+ uptake into the olfactory bulb and pituitary gland of normal rats by using the calcium channel blocker verapamil. Rats with adenomas received fluorodeoxyglucose-positron-emission tomography (FDG-PET) scanning for characterization of tumor metabolism. Mn2+ influx was characterized in cultured pituitary adenoma cells. Volume of interest analysis of the normal aged pituitary gland versus adenoma indicated faster and increased calcium influx in adenoma at 1, 3, 11, and 48 hours. Mn2+ uptake into the olfactory bulb and pituitary gland of normal rats was inhibited by calcium channel blockers and showed dose-dependent inhibition on dynamic MR imaging. FDG-PET indicated correlation between tumor energy metabolism and Mn2+ influx as well as tumor size. These results indicate that adenomas have increased activity-dependent calcium influx compared with normal aged pituitary glands, suggesting a potential for exploitation in the clinical work-up of pituitary and other neuroendocrine tumors by developing Mn-MR imaging for humans.

The classical and a non-classical pathways associated with NF-κB are involved in estrogen-medicated regulation of Calbindin-D9k gene in rat pituitary cells

Calbindin-D9k (CaBP-9k) is a high affinity calcium binding protein that is highly expressed in the duodenum, kidney, uterus, and pituitary glands. Previous studies have shown that CaBP-9k expression is regulated by several steroid hormones, such as 1,25-dihydroxyvitamin D3, glucocorticoids, 17β-estradiol (E2), and progesterone (P4), in a tissue-specific manner. However, the promoter elements that mediate transcriptional regulation by these steroid hormones are not clearly understood, mainly due to the lack of an appropriate cell line expressing CaBP-9k. Recently it was shown that CaBP-9k was constitutively expressed in the rat pituitary gland, and is expressed in an E2-dependent manner in a pituitary gland tumor-derived cell line, GH3. In the current study, we examined the activity of the estrogen responsive element (ERE) in rat CaBP-9k gene in GH3 cells, using a luciferase gene reporter assay, electrophoretic mobility shift assay, and mutagenesis. A nuclear factor-κB (NF-κB) binding site in the CaBP-9k promoter region was identified (nucleotides −848 to −834 from the transcriptional start site), and we demonstrated that addition of an NF-κB blocker to GH3 cells reduced E2-induced CaBP-9k transcription. In the present study, we further showed a previously reported imperfect ERE (nucleotides +51 to +65) between exon I and intron A of CaBP-9k, indicating that the interaction of estrogen receptor (ER) α with this region is involved in the regulation of CaBP-9k promoter activity and its expression. Taken together, these results suggest that in GH3 cells, both the classical ERα-ERE pathway and a non-classical pathway involving NF-κB are involved in E2-mediatd regulation of CaBP-9k expression in the pituitary gland.

Expression of retinaldehyde dehydrogenase 1 in the anterior pituitary glands of adult rats

Retinoic acid (RA) plays a critical role in cell growth and tissue development and is also a regulatory factor of pituitary function. However, whether RA is generated in the pituitary gland and plays a role as a paracrine and/or autocrine factor is generally unknown. RA is synthesized from retinoids through oxidation processes. Dehydrogenases that catalyze the oxidation of retinal to RA are members of the retinaldehyde dehydrogenase (RALDH) family. Recently, we demonstrated that RALDH2 and RALDH3, but not RALDH1, were expressed in the developing anterior pituitary gland of rats, but the expression of RALDHs in the adult pituitary gland was not determined. Therefore, we have now examined the expression of RALDH1, RALDH2, and RALDH3 mRNAs in the pituitary gland of adult rats. Analysis by quantitative real-time polymerase chain reaction of adult pituitary glands has revealed a high level of RALDH1 mRNA but not of RALDH2 mRNA or RALDH3 mRNA. We have also detected mRNA expression for RALDH1 in the anterior pituitary gland by in situ hybridization with digoxigenin-labeled cRNA probes. Double-staining for RALDH1 mRNA and pituitary hormones or S-100 protein, a marker of folliculo-stellate cells (FS-cells), has revealed RALDH1 mRNA expression in a portion of prolactin-producing cells, marginal layer cells, and FS-cells. Our results suggest that RA is generated in the adult anterior pituitary gland, and that it may act locally on pituitary cells.

Differential regulation of thyroid hormone receptor-mediated function by endocrine disruptors

It is well known that endocrine disruptors (EDs) act as anti-estrogenic agents and affect the function of reproductive organ. EDs are also thought to affect thyroid hormone (TH) system which is important for biological functions such as growth, development and metabolism. However, it is still not clear how EDs are able to regulate TH receptor (TR)-mediated functions. In this study, therefore, the modulatory effects of representative EDs such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyl (Aroclor 1254) and bisphenol A (BPA) were examined using TR-expressing GH3 cells (a rat pituitary gland epithelial tumor cell line) activated by triiodothyronine (T3). EDs tested significantly blocked T3 binding to TR in a dose-dependent manner. Biochemical characterization by Scatchard and Lineweaver-Burk plot analyses indicated that TCDD and aroclor 1254 bound to TH receptors in a competitive inhibitory manner, whereas BPA bound to TH receptors in a non-competitive pattern. The different inhibitory mode of action by EDs was also found in regulating TR-mediated production of prolactin (PRL). Aroclor 1254 exposure for 48 h enhanced T3-mediated PRL production, but BPA down-regulated. These results suggest that the EDs (TCDD, Aroclor 1254 and BPA) could differentially bind to TR and distinctly regulate the action of TR function, even though EDs are structurally similar.

Tyrosine Hydroxylase and Dopamine Transporter Expression in Lactotrophs from Postlactating Rats: Involvement in Dopamine-Induced Apoptosis

Cessation of lactation causes a massive loss of surplus lactotrophs in the rat pituitary gland. The factors and mechanisms involved in this phenomenon have not yet been elucidated. Besides its inhibitory control on prolactin secretion and lactotroph proliferation, evidence suggests that dopamine (DA) may be a proapoptotic factor for lactotrophs. We therefore tested the proapoptotic effect of DA on pituitary glands from virgin, lactating, and postlactating rats. By measuring mitochondrial membrane potential loss, caspase-3 activation, and nuclear fragmentation, we show that DA induces apoptosis specifically in lactotrophs from postlactating rats. We then determined that this effect was partly mediated by the DA transporter (DAT) rather than the D(2) receptor, as corroborated by the detection of DAT expression exclusively in lactotrophs from postlactating rats. We also observed tyrosine hydroxylase (TH) expression in postlactating lactotrophs that was accompanied by an increase in DA content in the anterior pituitary gland of postlactating compared with virgin rats. Finally, we observed that cells expressing TH coexpressed DAT and cleaved caspase-3. These findings show that DA may play a role in lactotroph regression during the postlactation period by inducing apoptosis. The fact that this process requires DAT and TH expression by lactotrophs themselves suggests that it may be "autocrine" in nature.

Expression of Aquaporin Water Channels in the Rat Pituitary Gland

We investigated the expression of aquaporin (AQP) subtypes (AQP1-AQP9) in the rat pituitary gland by the reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Specific amplification by RT-PCR was observed in AQP1, AQP3, AQP4 and AQP5 but not in the others. AQP1 immunoreactivity was detected in the endothelial cells of blood vessels. No positive reaction to AQP3 was observed by immunohistochemistry, in spite of the detection of mRNA. AQP4 was localized in the supporting cells of the adenohypophysis and neurohypophysis. AQP5 was expressed in parts of the marginal cells in Rathke's residual pouch. These observations suggest that several subtypes of the AQP family are involved in regulation of water homeostasis in the rat pituitary gland.

Accumulated fatigue induces over-activation and subsequent degeneration of melanotrophs in rat pituitary gland

Accumulated fatigue induces over-activation and subsequent degeneration of melanotrophs in rat pituitary gland

高感度ｍＲＮＡ検出法で細胞を同定する―ｉｎ ｓｉｔｕ ＲＴ‐ＰＣＲ法―

Recently, in situ reverse transcription (RT)-PCR has been developed to detect weak mRNA signals on histological sections by amplifying the relevant molecular mRNA, because RT-PCR allows one mRNA to be amplified a billionfold. This method was performed using rTth DNA polymerase that transcripts cDNA to mRNA, and amplifying the cDNA. We applied this technique to detect prohormone convertases PC1 and PC2 in the rat pituitary gland, and vascular endothelial growth factor (VEGF) in the developing pituitary gland, where these mRNAs were not detectable usual in situ hybridization techniques. Therefore, this in situ RT-PCR method is useful for identifying specific cells expressing weak signals in histological sections.

Calcitonin is expressed in the chicken pituitary gland: influence of gonadal steroids and sexual maturation

Calcitonin (CT) is primarily produced by the thyroid C cells in mammals or by the ultimobranchial gland in chickens. CT is also expressed by the pituitary gland in rats in which it functions as a paracrine factor causing decreased lactotroph proliferation and prolactin (PRL) secretion. Gonadal steroids influence CT expression in the rat pituitary gland. However, the expression of the CT gene in the pituitary gland of chickens or of any other avian species has not previously been reported. We have tested the hypotheses that CT is expressed in the chicken pituitary gland, and that its expression is influenced by sexual maturation or in response to ovarian steroid administration. We have detected robust expression of CT cDNA in the chicken pituitary gland by reverse transcription/polymerase chain reaction (PCR). The sequence of the pituitary-derived CT cDNA is identical to that of the ultimobranchial gland. CT-immunoreactive (ir) cells have been observed throughout the anterior pituitary gland by confocal microscopy. Many of the PRL-ir cells show co-localization with CT-ir cells. Quantitative real-time PCR analysis has revealed an inverse relationship between the quantities of PRL mRNA and CT mRNA in the pituitary gland: sexually mature hens contain lower amounts of CT mRNA but larger quantities of PRL mRNA compared with sexually immature chickens. Estradiol and/or progesterone treatment of sexually immature chickens leads to a significant decrease in the quantity of pituitary CT mRNA relative to that in the vehicle-treated chickens. We conclude that pituitary CT plays an important paracrine/autocrine role in the control of lactotroph function and PRL secretion in the chicken.

Peptide YY (PYY)3–36 modulates thyrotropin secretion in rats

Peptide YY (PYY)3-36 is a gut-derived hormone, with a proposed role in central mediation of postprandial satiety signals, as well as in long-term energy balance. In addition, recently, the ability of the hormone to regulate gonadotropin secretion, acting at pituitary and at hypothalamus has been reported. Here, we examined PYY3-36 effects on thyrotropin (TSH) secretion, both in vitro and in vivo. PYY3-36-incubated rat pituitary glands showed a dose-dependent decrease in TSH release, with 44 and 62% reduction at 10(-8) and 10(-6) M (P < 0.05 and P < 0.001 respectively), and no alteration in TSH response to thyrotropin-releasing hormone. In vivo, PYY3-36 i.p. single injection in the doses of 3 or 30 cg/kg body weight, administered to rats fed ad libitum, was not able to change serum TSH after 15 or 30 min. However, in fasted rats, PYY3-36 at both doses elicited a significant rise (approximately twofold increase, P < 0.05) in serum TSH observed 15 min after the hormone injection. PYY3-36 treatment did not modify significantly serum T4, T3, or leptin. Therefore, in the present paper, we have demonstrated that the gut hormone PYY3-36 acts directly on the pituitary gland to inhibit TSH release, and in the fasting situation, in vivo, when serum PYY3-36 is reduced, the activity of thyroid axis is reduced as well. In such a situation, systemically injected PYY3-36 was able to acutely activate the thyrotrope axis, suggesting a new role for PYY3-36 as a regulator of the hypothalamic-pituitary-thyroid axis.

Prorenin and Its Ancient Receptor

Prorenin, the precursor of renin, has for a long time been considered to be functionally inert, despite high circulating concentrations. Plasma levels of prorenin usually exceed renin levels and are even further increased under pathological conditions, such as in diabetes. First support of a functional significance of prorenin came from studies in transgenic rats, which overexpress a renin gene in the liver resulting in high circulating prorenin levels.1 Without systemic renin–angiotensin system activation and with normal blood pressure, these animals developed a vascular phenotype reminiscent of malignant hypertension. Moreover, in a series of elegant experiments, Ichihara et al2–4 supported the concept of prorenin as a relevant factor in the pathophysiology of diseases, such as hypertension and diabetes, by using a peptide that binds and inhibits the activation of the precursor. Thus, this peptide attenuated nephropathy in streptozotocin-induced diabetes2 and cardiac fibrosis in stroke-prone spontaneously hypertensive rats.3 By successfully repeating the streptozotocin experiment in mice deficient for the angiotensin II type 1a (AT1a) receptor, they reported recently that these effects are independent of renin activation or angiotensin II generation and concluded that the peptide, in fact, inhibits the interaction of prorenin with a receptor.4 In this issue of Hypertension , Saris et al5 contribute further evidence for an angiotensin-independent action of prorenin on cells by showing that neonatal cardiomyocytes respond to purified prorenin by p38 mitogen-activated protein (MAP) kinase phosphorylation and broad changes in gene expression partially also caused by the MAP kinase activation. Because the authors had shown before that these cells are not able to synthesize angiotensin II after the addition of prorenin, they consider the observed effects as being …

Cellular localization of apelin and its receptor in the anterior pituitary: evidence for a direct stimulatory action of apelin on ACTH release

Apelin is a bioactive peptide recently identified as the endogenous ligand of the human orphan G protein-coupled receptor APJ. The presence of apelin-immunoreactive nerve fibers, together with the detection of apelin receptor mRNA in the parvocellular part of the paraventricular nucleus and the stimulatory action of apelin on corticotropin-releasing hormone release, indicate that apelin modulates adrenocorticotropin (ACTH) release via an indirect action on the hypothalamus. However, a direct action of apelin in the anterior pituitary cannot be excluded. Here, we provided evidence for the existence of an apelinergic system within the adult male rat pituitary gland. Double immunofluorescence staining indicated that apelin is highly coexpressed in the anterior pituitary, mainly in corticotrophs (96.5 +/- 0.3%) and to a much lower extent in somatotropes (3.2 +/- 0.2%). Using in situ hybridization combined with immunohistochemistry, a high expression of apelin receptor mRNA was also found in corticotrophs, suggesting a local interaction between apelin and ACTH. In an ex vivo perifusion system of anterior pituitaries, apelin 17 (K17F, 10(-6) M) significantly increased basal ACTH release by 41%, whereas apelin 10 (R10F, 10(-6) M), an inactive apelin fragment, was ineffective. In addition, K17F but not R10F induced a dose-dependent increase in K(+)-evoked ACTH release, with maximal increase being observed for a 10(-6) M concentration. Taken together, these data outline the potential role of apelin as an autocrine/paracrine-acting peptide on ACTH release and provide morphological and neuroendocrine basis for further studies that explore the physiological role of apelin in the regulation of anterior pituitary functions.