Intrapituitary regulatory system of proliferation of mammotrophs in the pituitary gland

Anterior pituitary cells produce growth factors plus cytokines and their receptors. Although some of these pituitary growth factors and cytokines are known to be involved in the control of cell differentiation, proliferation and hormone production in the pituitary gland, their physiological roles remain unknown. Lots of evidence indicates that they are involved in the regulation of prolactin-secreting mammotroph cell proliferation. The regulation of mammotroph functions is a suitable system for understanding the intrapituitary regulatory system operated by growth factors and cytokines, since mammotrophs are the most actively proliferating cells in female pituitary glands. This review discusses the possible intrapituitary regulation of mammotroph differentiation and proliferation in rat and mouse pituitaries.     

Neuronal differentiation and expression of neural epitopes in pituitary adenomas.

Neural transdifferentiation is increasingly recognized in neural crest and neural stem cell tumors. Neuronal differentiation has been anecdotally described primarily in somatotroph cell adenomas associated with acromegaly, but its prevalence in adenomas and relationship to adenoma type has not been completely established. In this study we performed a retrospective morphological and immunohistochemical analysis of neurofilament, phosphoneurofilament, Neu-N, class III tubulin, and Hu in WHO grade I pituitary adenomas. Limited numbers of cells with neuronal features and neuron-associated epitopes may be more common in pituitary adenomas than previously recognized. These may occur in many forms of adenomas including somatotroph, lactotroph, mixed somatotroph and lactotroph, null cell/gonadotroph cell and, rarely, corticotroph cell adenomas.     

Postnatal restoration of the mouse urinary bladder urothelium

Mouse urothelium is disrupted just before birth, followed by a postnatal restoration process which includes cell proliferation, death and differentiation. We assessed urothelial proliferation by the expression of proliferating cell nuclear antigen (PCNA), desquamation by electron microscopy, and apoptosis by TUNEL staining and urothelial differentiation by the expression of uroplakins and cytokeratin 20 (CK20) as well as the apical plasma membrane maturation. Our results indicated that urothelial proliferation was high from birth until about the 14th postnatal day. A majority of basal cells and even occasional superficial cells were PCNA positive during the first 5 postnatal days. Cell death occurred during the first 9 postnatal days. Between birth and day 5, single cells underwent apoptosis, whereas between days 6 and 9 cells mainly desquamated. CK20 and uroplakins were expressed in all superficial cells in postnatal urothelium. Their subcellular distribution characteristically changed in accordance with the progressive differentiation of superficial cells. During the urothelial postnatal development, proliferation activity slowly decreases to the proliferatively quiescent urothelium of the adult animal. Apoptosis is present in the first 9 postnatal days and within a few days of this period it appears simultaneously with desquamation. Superficial urothelial cells gradually differentiate, which is reflected in the changeable morphology of the apical plasma membrane.     

Pituitary corticotrophs proliferate temporarily after adrenalectomy

Summary Relationship of corticotroph proliferation answer and survival time after adrenalectomy was examined. Corticotroph proliferation rates were detected by short-term ³H-thymidine radiolabeling, then ACTH immunostaining and autoradiography. Effect of adrenalectomy on corticotroph proliferation rate was examined in vivo and an elevation was demonstrated first on the second postoperative day, increasing on the third-fourth day postoperatively and then decreasing. Effects of different secretagogues on corticotroph proliferation were examined in short-term pituitary monolayer cultures taken from ADX rats. CRF and Forskolin treatment potentiated corticotroph proliferation in cultures taken from adrenalectomized rats, but not in the controls. We suggest that croticotroph proliferation is stimulated via the cAMP-proteinkinase A pathway, while adrenalectomy plays a permissive role.     

Immunohistochemistry of the golden hamster pituitary during chronic administration of diethylstilbestrol: a quantitative analysis using confocal laser scanning microscopy

The aim of this study was to examine by immunohistochemistry the morphologic changes affecting pituitary cell populations in male Syrian hamsters undergoing chronic exposure (3 days to 9 months) to diethylstilbestrol (DES). Cell proliferation in the hypophysis was monitored by the immunohistochemical demonstration of S-phase cells after pulse labeling with 5-bromo-2'-deoxyuridine. Cell proliferation analysis was combined with the identification of different cell populations by immunostaining with antisera raised against hypophyseal hormones. Sections processed for double-label immunofluorescence were examined by confocal microscopy. In the adenohypophysis, the relative surface occupied by gonadotrophs and thyrotrophs decreased rapidly during the first months of treatment while corticotroph and somatotroph populations remained unaffected. Accordingly, the incidence of S-phase cells in these four cell populations was lower than or similar to control values. In contrast, lactotrophs increased gradually during the first month of exposure to DES to reach a maximum value at 2-4 months. At the beginning, this increase was primarily due to hyperplasia but later on it also involved cellular hypertrophy. Somatomammotrophs did not seem to be involved in this model. In the pars intermedia, the labeling index of melanotrophs rose rapidly to reach values 5-6 times higher than controls. After 4 months, neoplasms originating from the pars intermedia were seen invading both the neuro- and the adenohypophysis. At the end of treatment, the pituitary was markedly enlarged resulting from the development of an adenoma of the pars intermedia.     

Proliferative activity of cells of the intermediate lobe of the rat pituitary during the postnatal period.

Cellular proliferation was studied in the intermediate lobe (IL) of the pituitary gland of developing rats by labelling cells at the S-phase of the cell cycle with bromodeoxyuridine (BrdU). The number of BrdU-labelled cells in the IL decreased from birth until the 14th postnatal day and was low from that day until the end of the first month after birth. Throughout the postnatal period a large proportion of BrdU-labelled cells was found in the marginal layer (ML) of the IL, suggesting for the ML a role as a germinative layer of the IL during postnatal growth. Double immunostaining with anti-BrdU and anti-MSH showed that MSH cells actively proliferate as from the day of birth. Cells doubly immunostained with anti-BrdU and anti-S100 protein were first seen on the 14th postnatal day. From then onwards, most proliferating cells were labelled with either anti MSH or anti S-100 protein. This, together with the high proportion of proliferating cells found in the ML marks a clear difference with the pattern of cellular proliferation previously reported during a similar period in the anterior lobe of the rat pituitary.     

垂体瘤微环境免疫细胞浸润与肿瘤侵袭性的相关性研究

目的:探讨垂体瘤免疫微环境中免疫细胞浸润情况及其与垂体瘤侵袭性的相关性。方法:选取35例垂体瘤病理组织切片,通过免疫组化染色分析巨噬细胞、T细胞以及中性粒细胞的特异性标记蛋白CD68、CD4、CD8和MPO的表达情况。结合临床和影像学数据分析,分析生长激素腺瘤、泌乳素腺瘤和无功能腺瘤的侵袭性与免疫细胞浸润数量的相关性。结果:在15例生长激素腺瘤,10例泌乳素腺瘤和10例无功能性腺瘤患者中,侵袭性垂体瘤中有更多的巨噬细胞浸润(P分别为0.014,0.032和0.032)。侵袭性促生长激素腺瘤、泌乳素腺瘤和无功能性腺瘤巨噬细胞浸润数量均明显多于非侵袭性促生长激素腺瘤、泌乳素腺瘤和无功能性腺瘤(P0.05)。结论:在垂体腺瘤中,巨噬细胞是肿瘤免疫微环境的主体。巨噬细胞浸润可能促进垂体瘤的进展。     

Pituitary corticotrophs proliferate temporarily after adrenalectomy.

Relationship of corticotroph proliferation answer and survival time after adrenalectomy was examined. Corticotroph proliferation rates were detected by short-term 3H-thymidine radiolabeling, then ACTH immunostaining and autoradiography. Effect of adrenalectomy on corticotroph proliferation rate was examined in vivo and an elevation was demonstrated first on the second postoperative day, increasing on the third-fourth day postoperatively and then decreasing. Effects of different secretagogues on corticotroph proliferation were examined in short-term pituitary monolayer cultures taken from ADX rats. CRF and Forskolin treatment potentiated corticotroph proliferation in cultures taken from adrenalectomized rats, but not in the controls. We suggest that croticotroph proliferation is stimulated via the cAMP-proteinkinase A pathway, while adrenalectomy plays a permissive role.     

Nitric oxide stimulates embryonic somatotroph differentiation and growth hormone mRNA and protein expression through a cyclic guanosine monophosphate-independent mechanism

In the pituitary gland, NO is locally synthesized by gonadotroph and folliculo-stellate cells. Many reports have shown that NO can modulate the growth hormone (GH) secretion. However, its role on mice embryo GH regulation remains unclear. In addition, it is unknown whether the regulation is associated with the proliferation of pituitary cells. In this study, we have investigated the regulatory effects of NO on somatotroph differentiation, proliferation and GH mRNA and protein expression using primary cell cultures of mice fetal pituitaries (embryonic days 16.5, ED 16.5). Our results show that incubation of pituitary cells in the presence of sodium nitroprusside (SNP; 1 mM), a NO donor, for 4.5 h resulted in a significant increase in GH mRNA and protein expression (P < 0.05) and the stimulation of SNP can be inhibited by hemoglobin, a NO scavenger. But the addition of cyclic guanosine monophosphate (cGMP; 3.0 mM), the second messenger of multiple NO actions cannot influence GH mRNA and protein expression. The cyclic nucleotide cellular efflux pumps existed in the pituitary cells can transport the majority of de novo-produced cGMP and effectively block cGMP accumulation. For maintaining intracellular concentration of cGMP, probenecid (0.5 mM), a blocker of cGMP efflux pump, combined with cGMP (3.0 mM) was used to treat the pituitary cells. This also cannot influence GH mRNA and protein expression. In addition, the ratio of GH-positive cells is increased significantly after the stimulation of SNP (P < 0.05). However, SNP cannot modulate the pituitary cell proliferation. From these results we conclude that NO can increase GH mRNA and protein expression in fetal pituitary cells and cGMP is not involved in this hormonal regulation. Stimulation of NO on the somatotroph differentiation does not occur due to pituitary cell proliferation.     

Suppression of cell proliferation and programmed cell death by dexamethasone during postnatal lung development

Prematurely born babies are often treated with glucocorticoids. We studied the consequences of an early postnatal and short dexamethasone treatment (0.1-0.01 microg/g, days 1-4) on lung development in rats, focusing on its influence on peaks of cell proliferation around day 4 and of programmed cell death at days 19-21. By morphological criteria, we observed a dexamethasone-induced premature maturation of the septa (day 4), followed by a transient septal immatureness and delayed alveolarization leading to complete rescue of the structural changes. The numbers of proliferating (anti-Ki67) and dying cells (TdT-mediated dUTP nick end labeling) were determined and compared with controls. In dexamethasone-treated animals, both the peak of cell proliferation and the peak of programmed cell death were reduced to baseline, whereas the expression of tissue transglutaminase (transglutaminase-C), another marker for postnatal lung maturation, was not significantly altered. We hypothesize that a short neonatal course of dexamethasone leads to severe but transient structural changes of the lung parenchyma and influences the balance between cell proliferation and cell death even in later stages of lung maturation.     

Substance P, neurokinin A and calcitonin gene-related peptide during development of the rat gastrointestinal tract.

Substance P, neurokinin A and calcitonin gene-related peptide (CGRP) were determined in the stomach and small intestine of rats during late foetal development and up to 35 days postnatal life. Concentrations of substance P in stomach and intestine increased from 14 gestational days to 3 days postpartum, and declined thereafter. Concentrations of neurokinin A in stomach declined from 14 days gestation over the period 3-35 postnatal days. In the intestine, concentrations of neurokinin A increased steadily from 14 days gestation to 21-35 postnatal days. Concentrations of CGRP in stomach and intestine declined from 14 days gestation to 7 postnatal days. Thereafter, concentrations of CGRP increased in both stomach and intestine. Total contents of each of the three peptides increased progressively with gestational and postnatal age in parallel with increasing stomach and intestinal weights. The results demonstrate different patterns of change in the concentrations of substance P, neurokinin A and CGRP during the dynamic phases of growth and maturation of the gastrointestinal tract in the foetal and postnatal rat.     

Silencing of RASSF3 by DNA Hypermethylation Is Associated with Tumorigenesis in Somatotroph Adenomas

The pathogenic mechanisms underlying pituitary somatotroph adenoma formation, progression are poorly understood. To identify candidate tumor suppressor genes involved in pituitary somatotroph adenoma tumorigenesis, we used HG18 CpG plus Promoter Microarray in 27 human somatotroph adenomas and 4 normal human adenohypophyses. RASSF3 was found with frequent methylation of CpG island in its promoter region in somatotroph adenomas but rarely in adenohypophyses. This result was confirmed by pyrosequencing analysis. We also found that RASSF3 mRNA level correlated negatively to its gene promoter methylation level. RASSF3 hypermethylation and downregulation was also observed in rat GH3 and mouse GT1.1 somatotroph adenoma cell lines. 5-Aza-2′ deoxycytidine and trichostatin-A treatment induced RASSF3 promoter demethylation, and restored its expression in GH3 and GT1.1 cell lines. RASSF3 overexpression in GH3 and GT1.1 cells inhibited proliferation, induced apoptosis accompanied by increased Bax, p53, and caspase-3 protein and decreased Bcl-2 protein expression. We also found that the antitumor effect of RASSF3 was p53 dependent, and p53 knockdown blocked RASSF3-induced apoptosis and growth inhibition. Taken together, our results suggest that hypermethylation-induced RASSF3 silencing plays an important role in the tumorigenesis of pituitary somatotroph adenomas.     

Developmental changes of hypothalamic, pituitary and striatal tachykinins in response to testosterone: influence of prenatal melatonin.

Substance P (SP) and neurokinin A (NKA), members of the family of mammalian tachykinins, are involved in the regulation of many physiological functions and are widely distributed in mammalian tissues. In this report, the effects of prenatal melatonin on the postnatal developmental pattern of NKA, and SP, and on testosterone secretion were investigated. Also, tachykinin response to the administration of testosterone propionate (TP) was studied. The brain areas studied were medio-basal-hypothalamus, pituitary gland and striatum. Male rat offspring of control or melatonin treated mother rats were studied at different ages of the sexual development: infantile, juvenile or prepubertal periods, and pubertal period. Both groups received exogenous TP (control-offspring+TP and MEL-offspring+TP), or the vehicle (control-offspring+placebo and MEL-offspring+placebo). Hypothalamic concentrations of all peptides studied in control-offspring+placebo remained at low levels until the juvenile period, days 30-31 of age. After this age, increasing concentrations of these peptides were found, with peak values at puberty, 40-41 days of age, then declining until adulthood. In the MEL-offspring+placebo a different pattern of development was observed; hypothalamic concentrations of NKA and SP from the infantile period until the end of juvenile period were significantly higher than in control-offspring+placebo. TP administration exerted a more marked influence on MEL-offspring than on control-offspring and prevented the elevation in tachykinin concentrations associated with prenatal melatonin treatment. TP administration to control-offspring resulted in significantly reduced (P < 0.05) tachykinin concentration only at 40-41 days of age, and increased (P < 0.01) during infantile period as compared to control-offspring+placebo. Pituitary NKA concentrations were lower than in the hypothalamus. In control-offspring+placebo pituitary NKA levels did not show significant changes throughout sexual development. A different developmental pattern was observed in MEL-offspring+placebo, with significantly increased (P < 0.05) pituitary NKA concentrations at 35-36 days of age than in control-offspring+placebo. TP administration to control-offspring influenced pituitary NKA levels at the end of the infantile and pubertal periods, showing at both stages significantly higher (P < 0.05) NKA levels as compared to control-offspring+placebo. NKA levels in MEL-offspring+TP were only affected at 21-22 days of age, showing significantly increased (P < 0.01) values as compared to MEL-offspring+placebo. Striatal tachykinin concentrations in control-offspring did not undergo important modifications throughout sexual development, but during the prepubertal period they started to increase. Maternal melatonin and TP injections produced short-lived alterations during the infantile period. The results showed that prenatal melatonin delayed the postnatal testosterone secretion pattern until the end of the pubertal period and postnatal peptide secretion in brain structures. Consequently, all functions depending of the affected areas will in turn, be affected.     

Postnatal maturation of the parenchymal cell types in the rabbit pineal gland.

An ultrastructural study on the maturation of the parenchymal rabbit pineal cell types from the first postnatal day up to 120 days is presented. Two main cell types are distinguished from the first 24h of postnatal life. Pinealocytes of the types I and II display different developmental degrees. Both immature cell types are arranged in groups. In addition, type II pinealocytes form rosette-like structures. Both cell types progressively become isolated and display cell processes. The nucleus and the cytoplasm of type I pinealocytes are barely electrondense. During the postnatal period, the number of cytoplasmic organelles, cell processes and terminal clubs increase progressively. Terminal clubs are frequently seen near blood vessels. After 30 days, type I pinealocytes show characteristics of adult pinealocytes. However, the maturation of most type I pinealocytes does not complete until the 90th postnatal day. Type II pinealocytes present a fairly electrondense nucleus and cytoplasm. Mature forms can be seen after the 5th postnatal day. During the postnatal period, a close relationship is determined among type II pinealocytes and cell processes and terminal clubs of type I pinealocytes.     

Estrogen-induced transformation of somatotrophs into mammotrophs in the rat

Summary In the normal male rat pituitary tritiated thymidine labeled mainly STH cells (somatotrophs), no labeled prolactin cell was found. Following estradiol treatment for 21 days tritiated thymidine labeled mainly prolactin cells (mammotrophs). To determine the origin of these mammotrophs tritiated thymidine was given before the estradiol treatment started, thus labeling many somatotrophs. After 21 days of estradiol, out of 42 labeled cells, 14 were mammotrophs and 13 were somatotrophs; these results suggest that there might be a true transformation of somatotrophs into mammotrophs under the influence of estradiol or that there exist two types of somatotrophs: 1) a committed somatotroph which is not transformed by estrogen treatment, and 2) an uncommitted mammosomatotroph, which under normal conditions bears the features of a somatotroph, but which transforms into a mammotroph under the influence of estradiol.This work was supported by grants MA-552 and MT-2701 from the Medical Research Council of Canada. The authors wish to thank Dr. G. M. Brown, Clarke Institute of Psychiatry, University of Toronto, for the radioimmunoassays of growth hormone and prolactin. Reagents for both radioimmunoassays were kindly provided by the National Institute of Arthritis and Metabolic Diseases, through the Rat Pituitary Hormone Distribution Program. — We are also thankful to Dr. L. Endrenyi, Department of Pharmacology, University of Toronto, for the statistical analysis of our data.Fellow of the Medical Research Council of Canada.     

Influence of chronic treatment with the growth hormone secretagogue Ipamorelin,in young female rats: somatotroph response in vitro

Growth hormone (GH) is secreted in the anterior pituitary gland by the somatotroph cells. Secretion is regulated by growth hormone releasing hormone (GHRH) and somatostatin. Morever, GH secretagogues (GHS) can exert a considerable effect on GH secretion. In order to determine the effects of chronic treatment with the GHS Ipamorelin on the composition of the somatotroph cell population and on somatotroph GH content, an in vitro analysis was performed of the percentage of somatotroph cells (% of total), the ratio of different GH cell types (strongly/weakly-staining) and individual GH content, in pituitary cell cultures obtained from young female rats receiving Ipamorelin over 21 days (Ipamorelin group) and the effects were compared with those of GHRH (GHRH group) or saline (saline group). The ultrastructure of somatotroph cells did not change, but the volume density of secretion granules was increased (P<0.05) by previous in vivo Ipamorelin or GHRH treatment. In 3-day basal pituitary cell monolayer cultures, the percentage of somatotroph cells showed no modifications between groups, nor was there any change in the ratio of strongly/weakly immunostaining GH cells. In the Ipamorelin group alone, in vitro treatment with Ipamorelin (10(-8) M), or GHRP 6 (10(-8) M), or GHRH (10(-8) M) for 4 hours, increased the percentage of somatotroph cells, without modifying the ratio of strongly/weakly immunostained GH cells. Basal intracellular GH content in somatotroph cells over 4 hours was lower in the Ipamorelin group and the GHRH group than in the saline group. Only in the Ipamorelin group did Ipamorelin (10(-8) M), GHRP 6 (10(-8) M) and GHRH (10(-8) M) prompt increased intracellular GH content. These data suggest that, at least in the young female rat, the GHS Ipamorelin is able to exert a dynamic control effect on the somatotroph population and on GH hormone content.     

Spatial distribution of thiamine pyrophosphatase and arylsulfatase activities in the area of the Golgi apparatus of the adenohypophyseal secretory cells in the white rat

Specimens of albino rat pituitary glands were processed consecutively for demonstration of thiamine pyrophosphatase (TPPase) and aryl sulfatase (ArSase) activities. To differentiate between the structures associated with each particular enzyme studied within a single cell, either somatotroph or mammotroph, ultrathin sections were exposed for 2 minutes to 2--50% H2SO4 which removed the reaction products for TPPase rather than those for ArSase. The comparative study of pairs of micrographs of the same area taken before and after the etching with H2SO4 has shown TPPase and ArSase to reside in the Golgi apparatus and GERL system, respectively. Transitional elements have also been discovered, thus supporting the idea that GERL may be a derivative of the Golgi apparatus.     

Increasing myocardial contraction and blood pressure in C57BL/6 mice during early postnatal development

Knowledge of the developmental changes of cardiovascular parameters in the genetic background of a mouse strain is important for understanding phenotypic changes in transgenic or knockout mouse models for heart disease. We studied arterial blood pressure and myocardial contractility in mice of the common background strain C57BL/6, aged 21 days [postnatal day 21 (P21)] to 580 days. Heart rate increased during maturation from 396 beats/min at P21 to 551 beats/min at postnatal day 50 (P50), and mean arterial blood pressure increased in parallel from 86 to 110 mmHg and remained constant afterward. Echocardiographically determined left ventricular myocardial wall dimensions (R = 0.79, P < 0.0001) and left ventricular mass calculated using the area-length algorithm correlated strongly with histomorphometrical measurements (R = 0.93, P < 0.001). Sarcomere shortening records from isolated ventricular myocytes used as a measure for myocardial contractility revealed a negative shortening-frequency relation under a pacing frequency of 2 Hz and a positive relation above 2 Hz. Shortening amplitudes recorded from P21 myocytes were smaller, and the shortening-frequency relation was less steep than in adult myocytes. A stimulation pause was followed by a negative "staircase" at pacing frequency of < or =6 Hz and a positive staircase at > or =6 Hz. P21 myocytes developed positive staircases at 8 and 10 Hz, and adult myocytes also developed them at 6 Hz. Blood pressure increase during maturation until P50 may originate from increasing single cardiomyocyte contractility.