Electron-microscopic study on the anterior pituitary gland of spontaneous dwarf rats

Summary The spontaneous dwarf rat is a novel experimental model animal on the study of pituitary dwarfism. The fine structure of the anterior pituitary cells was studied in the immature and mature dwarf rats. Pituitary glands were removed from 5-, 10-, 20-day-old immature dwarfs, adult (45 days-16 weeks) dwarfs and normal 3-month-old rats and processed for electron-microscopic observation. In the control animals, growth hormone cells were readily identified by their ultrastructural characteristics, such as the presence of numerous electron-dense secretory granules, 300–350 nm in diameter, well developed rough endoplasmic reticulum and a prominent Golgi complex. In contrast, growth hormone cells were not found in the anterior pituitary gland of the spontaneous dwarf rat at any age examined. Other pituitary cell types, i.e., luteinizing hormone/ follicle stimulating hormone, thyroid stimulating hormone, adrenocorticotropic hormone and prolactin cells, appeared similar in their fine structure to those found in the control rats. In the pituitary gland of dwarf rats, a number of polygonal cells were observed either with no or relatively few secretory granules. The rough endoplasmic reticulum was arranged in parallel cisternae and the Golgi complex was generally prominent in these cells. In addition, many were found to have abundant lysosomes. A few minute secretory granules were occasionally observed; however, the immunogold technique failed to localize growth hormone or prolactin in the granules. The nature of these cells remained obscure in this study. Since their incidence and fine structural features, other than the secretory granules, were quite similar to those of the growth hormone cells in normal rats, we postulate that these cells are dysfunctional growth hormone cells. These results suggest that the cause of the growth impairment in the spontaneous dwarf rat is due to a defect in the functional growth hormone cells in the pituitary gland, and since other pituitary cell types appeared normal, the disorder seems to be analogous to the isolated growth hormone deficiency in the human.     

Glycosylated growth hormone: detection in murine pituitary gland and evidence of physiological fluctuations

We examined murine pituitary glands for glycosylated growth hormone using a lectin-binding immunoassay. Every gland tested exhibited significant concanavalin A-binding growth hormone immunoreactivity. The activity was lower in female rats than in males, and lower in lactating than in virgin females. The activity increased following ovariectomy, although total immunoreactive growth hormone was not altered. Administration of estradiol benzoate decreased the concentrations of both types of growth hormone. Progesterone administration, in contrast, did not change concanavalin A-binding growth hormone, but it lowered total growth hormone. Western blotting revealed a growth hormone immunoreactive band 4-5K heavier than the growth hormone monomer in both rat and mouse pituitary. These data suggest the existence in the murine pituitary of a glycosylated form of growth hormone that undergoes physiological fluctuations.     

Immunohistochemical localization of anterior pituitary hormones in S-100 protein-positive cells in the rat pituitary gland

In the anterior and intermediate lobes of the rat pituitary gland, non-hormone-producing cells that express S-100 protein coexist with various types of hormone-producing cells and are believed to function as phagocytes, supporting and paracrine-controlling cells of hormone-producing cells and stem cells, among other functions; however, their cytological characteristics are not yet fully understood. Using a transgenic rat that expresses green fluorescent protein under the promoter of the S100β protein gene, we immunohistochemically detected expression of the luteinizing hormone, thyroid-stimulating hormone, prolactin, growth hormone and proopiomelanocortin by S-100 protein-positive cells located between clusters of hormone-producing cells in the intermediate lobe. These findings lend support to the hypothesis that S-100 protein-positive cells are capable of differentiating into hormone-producing cells in the adult rat pituitary gland.     

Preparation of highly purified human somatotropin (growth hormone)

A method for preparation of highly purified human somatotropin on large-scale basis is described. Starting from deep-frozen pituitary gland, less time is needed to obtain highly purified hormone than with other published methods for preparation of human somatotropin. The hormone obtained in this fasion is chromatographically and electrophoretically homogeneous; it shows high biological and radioimmunological growth hormone activity and is free of other pituitary hormone activities. The effects of various experimental conditions upon aggregation of somatotropin are critically evaluated.     

Receptor for 1,25-dihydroxyvitamin D3 in GH3 pituitary cells

Cytosol prepared in 0.3 M KCl from pituitary GH3 cells, but not from AtT-20 cells contains a receptor-like macromolecule that binds 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) with specificity and high affinity (Kd = 2.9 x 10(-10) M). The GH3 cytosolic binding component sediments at 3.3 S in high-salt sucrose gradients and adsorbs to DNA-cellulose; its elution profile from DNA-cellulose and other biochemical properties are indistinguishable from those of classical 1,25(OH)2D3 hormone receptors. The presence of the 1,25(OH)2D3 receptor in pituitary cells which secrete primarily growth hormone and prolactin (GH3), but not in a line which secretes the 31,000-dalton ACTH precursor and its derived peptides (AtT-20), suggests that 1,25(OH)2D3 may play a regulatory role in specific pituitary cells.     

Hypophysiotropic activity of histone H3 in vitro

To assess the effect of histone H3 on pituitary hormone secretion, rat anterior pituitary (AP) cells were used and growth hormone, prolactin, thyrotropin, luteinizing hormone and follicle stimulating hormone measured by radioimmunoassay. Incubation of cells with H3 (1, 6, and 30 microM) stimulated the release of all five hormones in a dose-dependent manner. This effect was blocked by preincubation of H3 with an anti-H3 antibody. Incubation of AP cells with 6 microM H3 in the presence of specific AP hormone secretagogues (GRP-6, thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH)) showed additive effects on hormone secretion. Pharmacological experiments suggested that calcium- and diacylglycerol- (DAG) associated pathways, but not cAMP, participate in the hypophysiotropic activity of H3. Our results confirm previous evidence that histones may act as hypophysiotropic signals.     

Expression of retinaldehyde dehydrogenase (RALDH)2 and RALDH3 but not RALDH1 in the developing anterior pituitary glands of rats

Retinoic acid (RA) plays an important role in cell growth and tissue development and is also a regulating factor of pituitary function. However, whether RA is generated in the pituitary gland and plays a role as a paracrine and/or autocrine hormone is generally unknown. RA is synthesized from retinoids through oxidation processes. Dehydrogenases catalyzing the oxidation of retinal to RA are members of the retinaldehyde dehydrogenase (RALDH) family. In this study, we examined the expression of RALDH1, RALDH2, and RALDH3 mRNA in the rat embryonic pituitary gland. By in situ hybridization with digoxigenin-labeled cRNA probes, we detected mRNA expression for RALDH2 and RALDH3, but not RALDH1. The expression of RALDH2 and RALDH3 was located in Rathke’s pouch at embryonic day 12.5 (E12.5) and subsequently in the developing anterior pituitary gland. We also used quantitative real-time polymerase chain reaction to analyze RALDH2 and RALDH3 mRNA expression levels during the development of the pituitary gland. We found that pituitary RALDH2 and RALDH3 mRNA levels were high at E17.5 and decreased markedly after birth. Our study is the first to show that RALDH2 and RALDH3, but not RALDH1, are expressed in the embryonic anterior pituitary gland of the rat.     

丘脑下部促黄体素释放激素类似物(LRH-A)的作用机制 Ⅲ.对罗非鱼脑垂体促性腺激素分泌细胞超微结构的影响

前文(方永强等,1981)探讨了罗非鱼脑垂体促性腺细胞的生理活动与雌鱼性腺发育不同时期的相互关系,对比注射LRH-A后2、4和10小时对促性腺细胞的影响。根据促性腺细胞核的形态和细胞质中分泌颗粒数量的变化,以及DNA、RNA、酸碱性蛋白和酸碱性磷酸酶等一系列反应,初步阐明了LRH-A的作用机制与细胞核和细胞质的代谢变化有关。在此基础上,本文较详细观察了LRH-A对垂体促性腺细胞超微结构的影响,目的在于从亚细胞的水平上,进一步了解其作用机制。     

乌龟脑垂体显微及其腺垂体超微结构的研究

乌龟脑垂体由柄形神经垂体和椭圆形腺垂体两部分组成,神经垂体位于腺垂体后部上方呈背腹型排列。神经垂体中神经叶不发达,腺垂体分为远侧部和中间部,特殊空泡结构成为垂体门脉系统的特征。远侧部细胞分为嗜酸性细胞、嗜碱性细胞和嫌色细胞3种。通过透射电镜观察,腺垂体远侧部主要有5种分泌激素细胞:即生长激素(GH)分泌细胞、催乳激素(PRL)分泌细胞、促甲状腺激素(TSH)分泌细胞、促肾上腺皮质激素(ACTH)分泌细胞、促性腺激素(GTH)分泌细胞和非分泌类型滤泡-星形细胞(FS)。生长激素分泌细胞核大、分泌颗粒少的特征成为乌龟与其他动物最大的区别,可能与乌龟具有生长慢、寿命长的生物学特性有关。       

乌脑龟垂体显微及其腺垂体超微结构的研究

乌龟脑垂体由柄形神经垂体和椭圆形腺垂体两部分组成,神经垂体位于腺垂体后部上方呈背腹型排列。神经垂体中神经叶不发达,腺垂体分为远侧部和中间部,特殊空泡结构成为垂体门脉系统的特征。远侧部细胞分为嗜酸性细胞、嗜碱性细胞和嫌色细胞3种。通过透射电镜观察,腺垂体远侧部主要有5种分泌激素细胞：即生长激素（GH）分泌细胞、催乳激素（PRL）分泌细胞、促甲状腺激素（TSH）分泌细胞、促肾上腺皮质激素（ACTH）分泌细胞、促性腺激素（GTH）分泌细胞和非分泌类型滤泡．星形细胞（Fs）。生长激素分泌细胞核大、分泌颗粒少的特征成为乌龟与其他动物最大的区别,可能与乌龟具有生长慢、寿命长的生物学特性有关。     

Alternative splicing model for the synthesis and secretion of the 20 kilodalton form of rat growth hormone

The characterization of a 20 kilodalton (20 kD) variant of rat growth hormone is reported. The 20 kD variant from rat pituitary gland extracts was identified on Western immunoblots of polyacrylamide gels. It was also shown that pituitary tissue maintained in culture secretes the 20 kD form. A rat growth hormone cDNA fragment was used as a probe in S1 nuclease mapping experiments of rat pituitary poly (A) mRNA to detect the presence of two growth hormone mRNAs in the rat pituitary gland. The protected mRNAs correspond to the predicted sizes that would encode the 22 kD and 20 kD forms of growth hormone. The site of variation between the mRNAs maps to a potential alternative 3' splice site in the 5' end of exon 3 of the coding sequence. The results support the hypothesis that the 20 kD variant in rat is the product of an mRNA alternatively spliced in exon 3, as is the case for the human growth hormone.     

Cellular localization of somatolactin in the pars intermedia of some teleost fishes

Summary We report here on the cellular localization in the fish pituitary of somatolactin (SL), a putative new pituitary hormone related to growth hormone and prolactin, which has been recently identified in the piscine pituitary gland. Immunocytochemical staining, using anti-cod SL serum, revealed that in the cod pituitary gland, SL is produced by cells in the intermediate lobe, bordering the neural tissue. These cells, staining weakly with periodic-acid-Schiff (PAS), are distinct from the melanocyte stimulating hormone (MSH) cells which, as in all teleosts, are PAS-negative. SL-immunoreactivity was observed in the same location in all other teleost species examined: flounder, rainbow trout, killifish, molly, catfish and eel. In most fish the SL-immunoreactive cells are either strongly or weakly PAS-positive but in rainbow trout are chromophobic, indicating that the SL protein can probably exist in glycosylated and non-glycosylated forms. Thus, in demonstrating the cellular localization of SL, this study provides the first identification of the enigmatic, second cell-type of the fish pars intermedia.     

Pituitary somatostatin receptors. Characterization by binding with a nondegradable peptide analogue

Somatostatin receptors in the rat pituitary gland were characterized by binding analysis with a radioiodinated high affinity somatostatin analogue, 125I-Tyr1[D-Trp8]somatostatin. Receptor binding of this derivative reached equilibrium at 30 min and was maintained at a plateau for at least 60 min. Two L-Trp8- labeled somatostatin analogues. 125I-Tyr1- and [125I-Tyr11]somatostatin, displayed less stable and lower specific uptake and higher nonspecific binding. In contrast to the rapid degradation of the L-Trp8 ligands during binding assay, 125I-Tyr1]D-Trp8]somatostatin retained more than 80% of its binding activity after 90 min of incubation with pituitary particles. Pituitary particles bound 125I-Tyr1]D-Tyr8]somatostatin with high affinity (Ka = 8.6 +/- 1.2 X 10(9) M-1) and capacity of 54.4 +/- 2.6 fmol/mg. These binding sites showed specificity for the native peptide and its active analogues, and other peptide hormones, including angiotensin II, thyrotropin-releasing hormone, vasopressin, oxytocin, substance P, and gonadotropin-releasing hormone, did not inhibit tracer binding. A good correlation was observed between the binding affinities of several somatostatin analogues and their potencies as inhibitors of growth hormone release in rat pituitary cells. These findings emphasize the physiological importance of the pituitary somatostatin receptor in mediating the inhibitory action of the peptide on growth hormone release. The use of Tyr1[d-Trp8]somatostatin as a labeled ligand permits accurate determinations of the binding affinity and concentration of receptors for somatostatin in the normal pituitary gland and provides a basis for further studies of somatostatin receptor regulation and receptor-mediated cellular effects of the tetradecapeptide.     

Changes in thyrotroph and somatotroph cell populations induced by stimulation and inhibition of their secretory activity

Summary The populations of cells which produce immunoreactive growth hormone (GH) and thyroid stimulating hormone (TSH) in the rat pituitary gland do not occur in fixed percentages but vary greatly under different physiological and experimental conditions. These variations can be directly correlated to the levels of stimulation and/or inhibition of the specific secretory activity. In both types of cell, sustained stimulation with trophic hormones or blockage of the feedback mechanisms induces remarkable growth in the specific cell population. Conversely, the interruption or inhibition of the stimulus thwarted the hormonal secretion and caused a massive degeneration of redundant cells. The stimulation of both GH and TSH cells is accompanied by an enhanced secretory activity as judged by their higher concentrations in serum and hypertrophy of the cytoplasmic organelles involved in synthesis and intracellular processing of the hormones. By contrast, interruption of the stimulus is followed by a variable degree of disruption of the cytoplasmic organization, including a sizable degeneration of cells. In stimulated rats, the concentrations of both GH and TSH decreased significantly in pituitary tissue due to mobilization of the hormonal stores contained in secretory granules. On the other hand, the withdrawal of stimuli blocked the hormonal release; this is reflected by the accumulation of both hormones and secretory granules in pituitary tissue. The strict correlation between the size of the GH and TSH populations with stimulation and inhibition of hormonal secretory activity reported in this investigation further supports the critical role played by the cell renewal process in endocrine secretion.     

Isolation and properties of the electrophoretic components of human growth hormone by Sephadex-gel filtration and preparative polyacrylamide-gel electrophoresis

1. Human growth hormone was prepared from acetone-dried residues after extraction of gonadotrophins from pituitary glands. 2. Crude growth hormone was purified by gel filtration on Sephadex, resulting in a product that is soluble in water or 0.5% sodium chloride. It is painless on injection and shows a twofold increase in biological potency. Aggregation of growth hormone on Sephadex columns can be avoided by the addition of urea (6m) and EDTA (1mm) to the buffer. 3. Growth hormone appeared as a single component from Sephadex and ion-exchange columns and sedimented as a single boundary in the ultracentrifuge. In the circular disk electrophoresis, however, the growth hormone showed one faster and two slower-moving anionic components. 4. These components were isolated by preparative electrophoresis on polyacrylamide columns. The purified growth hormone and its three components sedimented as single boundaries with coefficients 2.62, 2.66, 2.66 and 2.83s respectively. 5. Amino acid analyses of the purified growth hormone and its components were closely related. End-group analysis of purified growth hormone and its components showed only phenylalanine at both N- and C-terminals. 6. The purified growth hormone and its components were essentially free of other pituitary hormones, but contained significant prolactin activity. The biochemical similarities among the electrophoretic components of human growth hormone and the presence of the same three components in the growth hormone prepared from a single human pituitary gland suggest polymorphism of a biologically active protein molecule.