Transgenic mice with inducible dwarfism

The pituitary gland, composed of the anterior, intermediate and posterior lobe, represents a principal regulatory interface through which the central nervous system controls body physiology. The ontogeny of the growth hormone (GH) and prolactin (Prl) producing cells of the anterior pituitary has been analysed in transgenic mice, using the thymidine kinase obliteration system (TKO). Cells expressing the herpes virus 1 thymidine kinase (HSV1-TK) gene acquire pharmacological sensitivity to synthetic nucleosides such as FIAU (1-(2-deoxy-2-fluoro-beta-delta-arabinofuranosyl)-5-iodouracil), whose metabolites kill dividing cells. Consequently we created transgenic mice carrying the HSV1-TK gene under the control of either the rat growth hormone or the rat prolactin promoter. If transgenic mice expressing HSV1-TK in somatotropes (GH-producing cells) are treated with FIAU, they develop as dwarfs. The anterior pituitary in these animals is nearly devoid of both somatotropes and lactotropes (Prl-producing cells). By contrast, transgenic mice expressing HSV1-TK in the lactotropes, treated with FIAU, have anatomically and histologically normal pituitaries. Because toxicity depends on cell division, we conclude that Prl expression and lactotrope differentiation are post-mitotic events. These results indicate that both somatotropes and lactotropes derive from a common GH-expressing stem-somatotrope. Unexpectedly, the stemsomatotrope is still present in the adult animal and is capable of repopulating the pituitaries of treated animals with mature GH and Prl producing cells.     

Discrete cis-active genomic sequences dictate the pituitary cell type-specific expression of rat prolactin and growth hormone genes

The anterior pituitary gland, which is derived from a common primordium originating in Rathke's pouch, contains phenotypically distinct cell types, each of which express discrete trophic hormones: adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), prolactin, growth hormone, and follicle stimulating hormone (FSH)/luteinizing hormone (LH). The structurally related prolactin and growth hormone genes, which are evolutionarily derived from a single primordial gene, are expressed in discrete cell types--lactotrophs and somatotrophs, respectively--with their expression virtually limited to the pituitary gland. The pituitary hormones exhibit a temporal pattern of developmental expression with rat growth hormone and prolactin characteristically being the last hormones expressed. The reported co-expression of these two structurally related neuroendocrine genes within single cells prior to the appearance of mature lactotrophs, in a subpopulation of mature anterior pituitary cells, and in many pituitary adenomas raises the possibility that the prolactin and growth hormone genes are developmentally controlled by a common factor(s). We now report the identification and characterization of nucleotide sequences in the 5'-flanking regions of the rat prolactin and growth hormone genes, respectively, which act in a position- and orientation-independent fashion to transfer cell-specific expression to heterologous genes. At least one putative trans-acting factor required for the growth hormone genomic sequence to exert its effects is apparently different from those modulating the corresponding enhancer element(s) of the prolactin gene because a pituitary 'lactotroph' cell line producing prolactin but not growth hormone selectively fails to express fusion genes containing the growth hormone enhancer sequence.     

Co-localization of corticotropin-releasing factor and vasopressin in median eminence neurosecretory vesicles

Vasopressin (VP) potentiates the effect of corticotropin-releasing factor (CRF) on the secretion of adrenocorticotropic hormone (ACTH) from anterior pituitary cells in vitro, and both CRF and VP have been found in portal blood. These data support the hypothesis that VP acts synergistically with CRF to cause the secretion of ACTH in vivo but the origin of the CRF and VP, and the physiology of their release, have not been precisely defined. Parvocellular cell bodies in the paraventricular nucleus (PVN) which project to the external zone of the median eminence can be stained for both CRF and VP after adrenalectomy, and there is light microscopic immunocytochemical evidence that neurophysin (NP) may be located within some of the CRF-containing axons. Electron microscopic immunocytochemical studies have demonstrated the presence of CRF, VP and its 'carrier' protein, VP-associated neurophysin (NP-VP) in 100-nm neurosecretory vesicles (NSVs) in axons terminating near the portal capillary plexus in the external zone of the median eminence. If these peptides are extensively co-localized in the same NSVs in the median eminence, then coordinate secretion of CRF and VP in vivo is obligatory, at least in some physiological circumstances. We demonstrate in this report, using post-embedding electron microscopic immunocytochemistry on serial ultrathin sections, that CRF, VP and NP-VP are contained not only in the same axons and terminals, but in the same 100-nm NSVs in the median eminence of both normal and adrenalectomized rats. In addition, in the normal rat median eminence 44% of the CRF-positive axons and terminals stained strongly for VP and NP-VP, whereas in the adrenalectomized rat virtually all the CRF-positive structures in the median eminence showed strong staining for VP and NP-VP, indicating a transformation of one subpopulation of CRF-positive axons and terminals by adrenalectomy.     

Dopaminergic stimulation of prolactin release

Prolactin (PRL) secretion from anterior pituitary is believed to be under tonic inhibitory control of dopamine (DA) released from the tubero-infundibular dopaminergic neurones into the hypophysial portal blood. Inhibition of PRL release by DA seems to be mediated by sereospecific DA receptors located in PRL cells. Apomorphine and various ergot alkaloids such as bromocryptine mimic the inhibitory effect of DA both in vivo and in vitro, presumably by a direct agonist action on these 'inhibitory' receptors. We now report that PRL secretion in primary cultures of rat pituitary cells can be stimulated by DA when concentrations a thousand times lower than those required for inhibition are used. Secretion rates above basal release can also be induced by apomorphine and bromocryptine when the 'inhibitory' receptors are blocked with certain DA receptor antagonists.     

Binding sites for growth hormone releasing factor on rat anterior pituitary cells

Growth hormone releasing factors (GRFs) have been isolated from human pancreatic tumours (hGRF) and rat hypothalamus (rhGRF). The response to GRF at the pituitary level can be modulated by other factors, including glucocorticoids, thyroid hormones, somatostatin and other neuropeptides and somatomedins. Glucocorticoids enhance GRF-induced growth hormone (GH) secretion in primary cultures of rat anterior pituitary cells, and the synthetic glucocorticoid dexamethasone has recently been shown to increase the amounts of GH released in freely moving rats in response to submaximal doses of intravenous GRF. To investigate whether somatotroph sensitivity to GRF is modulated at its receptor level, we have developed a radioreceptor assay using an iodinated analogue of hGRF as radioligand. We report here that the relative binding affinities of rGRF, hGRF and the two analogues are correlated with their in vitro biological potencies. Further, the number of GRF binding sites is drastically decreased in cells deprived of glucocorticoids either in vivo or in vitro.     

Identification of a potent Xenopus mesoderm-inducing factor as a homologue of activin A

The first inductive interaction in amphibian development is mesoderm induction, when a signal from the vegetal hemisphere of the blastula induces mesoderm from overlying equatorial cells. Recently, several 'mesoderm-inducing factors' (MIFs) have been discovered. These cause isolated Xenopus animal caps to form mesodermal cell types such as muscle, instead of their normal fate of epidermis. The MIFs fall into two classes. One comprises members of the fibroblast growth factor (FGF) family, and the other members of the transforming growth factor type beta (TGF-beta) family. Of the latter group, the most potent is XTC-MIF, a protein produced by Xenopus XTC cells. Here we show that XTC-MIF is the homologue of mammalian activin A. Activins modulate the release of follicle-stimulating hormone from cultured anterior pituitary cells and cause the differentiation of two erythroleukaemia cell lines. Our results indicate that these molecules may also act in early development during formation of the mesoderm.     

Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth

The neurohumoral regulation of growth hormone secretion is mediated in part by two hypothalamic peptides that reach the anterior pituitary via the hypothalamo-hypophysial portal blood system. Somatostatin inhibits the release of growth hormone, whereas growth hormone-releasing factor (GRF) positively regulates both growth hormone synthesis and secretion. Two forms of human GRF, 40 and 44 amino acids long, have been characterized from extra-hypothalamic tumours as well as from the hypothalamus. Analysis of human GRF complementary DNA and genomic clones indicates that the GRF peptides are first synthesized as a 107- or 108-amino-acid precursor protein. To examine the physiological consequences of GRF expression, we have established strains of transgenic mice containing a fusion gene including the promoter/regulatory region of the mouse metallothionein-I (MT-I) gene and the coding region of the human GRF gene. We report that expression of the human GRF precursor protein in these animals results in measurable levels of human GRF and increased levels of mouse growth hormone in plasma and accelerated growth rates relative to control littermates. These results demonstrate a direct role for GRF in the positive regulation of somatic growth. Unexpectedly, female transgenic mice carrying the MT-GRF fusion gene are fertile, in contrast to female transgenic mice expressing human or rat growth hormone, which are generally infertile. These transgenic mouse strains should provide useful animal models for the study of several types of human growth disorders.     

Sodium and calcium action potential in pituitary cells

Several endocrine cells or their neoplastic derivatives generate action potentials similar to those seen in neurones, and in the adrenal chromaffin cell such regenerative potentials depend primarily on a sodium mechanism. Kidokoro has described action potentials in the GH3 rat pituitary cell line which seem to depend on a calcium mechanism. We have re-investigated the action potential in GH3 cells and found that it results from combined Na and Ca mechanisms in physiological conditions. In addition, we have recorded similar electrical excitability from two human pituitary tumours grown in vitro.     

人树突状细胞的体外培养和鉴定

目的:探索在体外分离培养人外周血树突状细胞的方法,同时为进一步研究树突状细胞工作奠定实验基础.方法:采用Ficoll、Precoll和Panning法分离和纯化人外周血DC,再加GM-CSF(100μg/mL)和IL-4(10μg/mL)诱生出大量树突状细胞,并进行免疫组化鉴定.结果:培养到第3天的DC扫描电镜可见DC伸出的树突状突起,呈毛刺状.培养到第7天的DC扫描电镜可见细胞胞体体积变大.这些毛刺状细胞表面有丰富的呈分叉的树突状胞质突起,某些突起形成薄片状结构,培养后的DC细胞呈S-100蛋白阳性染色.结论:人外周血树突状细胞可以在体外大量培养.     

Self-formation of functional adenohypophysis in three-dimensional culture

The adenohypophysis (anterior pituitary) is a major centre for systemic hormones. At present, no efficient stem-cell culture for its generation is available, partly because of insufficient knowledge about how the pituitary primordium (Rathke's pouch) is induced in the embryonic head ectoderm. Here we report efficient self-formation of three-dimensional adenohypophysis tissues in an aggregate culture of mouse embryonic stem (ES) cells. ES cells were stimulated to differentiate into non-neural head ectoderm and hypothalamic neuroectoderm in adjacent layers within the aggregate, and treated with hedgehog signalling. Self-organization of Rathke's-pouch-like three-dimensional structures occurred at the interface of these two epithelia, as seen in vivo, and various endocrine cells including corticotrophs and somatotrophs were subsequently produced. The corticotrophs efficiently secreted adrenocorticotropic hormone in response to corticotrophin releasing hormone and, when grafted in vivo, these cells rescued the systemic glucocorticoid level in hypopituitary mice. Thus, functional anterior pituitary tissue self-forms in ES cell culture, recapitulating local tissue interactions.     

Division and differentiation of isolated CNS blast cells in microculture

The mechanism of transformation of the overtly similar cells of the neural plate into the numerous and diverse cell types of the mature vertebrate central nervous system (CNS) can better be understood by studying the clonal development of isolated CNS precursor cells. Here I describe a culture system in which blast cells (cells capable of division) isolated from embryonic day 13.5-14.5 rat forebrain can divide and differentiate into a variety of clonal types. Most clones contain only neurons or glia; 22% contain both neurons and non-neuronal cells. For the division of blast cells, live conditioning cells need to be present indicating that environmental signals influence proliferation. Heterogeneous clones develop in homogeneous culture conditions, so factors intrinsic to the blast cells are probably important in determining the number and type of clonal progeny.     

Programmed death of autoreactive thymocytes

T lymphocytes bearing high-affinity T-cell receptors (TCR) for self-antigens are clonally deleted during thymus development. Several recent studies have identified variable domains of the beta-chain of the TCR that are specifically deleted in vivo in mouse strains that express major histocompatibility complex class II molecules in addition to poorly defined self-antigens, including those encoded by the Mls-1a and Mls-2a loci. Deletion of autoreactive cells in these systems occurs in the thymus, and antibody blocking experiments in vivo have implicated the phenotypically immature CD4+CD8+ 'cortical' subset as the target population for clonal deletion. Similarly, studies with transgenic mice bearing autoreactive TCR have provided independent evidence that clonal deletion occurs at the CD4+CD8+ stage of development. But none of these studies directly identified dying autoreactive cells, and the circumstances leading to deletion remain unclear. Here we report that neonatal thymus contains a significant population of phenotypically mature CD4+CD8- cells bearing autoreactive TCR. When placed in short-term culture, a large proportion (60%) of these autoreactive cells die selectively. Furthermore, their death can be prevented by inhibitors of macromolecule (RNA and protein) synthesis, as is the case for glucocorticoid-induced death of thymocytes. These data indicate that physiological clonal deletion of autoreactive cells involves 'programmed' cell death, and that it can occur in cells with a mature (CD4+CD8-) surface phenotype.     

Increase of corticotropin-releasing factor staining in rat paraventricular nucleus neurones by depletion of hypothalamic adrenaline

In response to stress, adrenocorticotropic hormone (ACTH) is released by corticotrophs in the anterior pituitary under the control of several central and peripheral factors including corticotropin-releasing factor (CRF), which was recently isolated from the brain and sequenced. Immunocytochemical studies have shown that most of the CRF-containing cell bodies that project to the median eminence are present in the hypothalamic paraventricular nucleus (PVN). A dense PNMT(phenylethanolamine-N-methyltransferase)-containing fibre network was also observed in the same region--PNMT is the final enzyme in the biosynthesis of adrenaline and has been demonstrated in the brain. In the present study we found an association of adrenergic nerve fibres and CRF neurones by immunohistochemistry using antisera to PNMT and CRF. To examine the functional significance of the adrenergic projection to the PVN, we blocked the synthesis of adrenaline using a specific inhibitor of PNMT. The depletion of adrenaline resulted in an increase in CRF immunoreactivity. The present results suggest that, as well as catecholamines which regulate ACTH release at the anterior pituitary level via a beta 2-adrenergic receptor mechanism, central catecholamines (mainly adrenaline) also affect ACTH release through their action on CRF cells. Peripheral catecholamines seem to have a direct stimulatory effect on the pituitary corticotroph cells, whereas the present findings suggest that central adrenaline-containing neurones have an inhibitory role in the physiological response to stress.     

Molecular cloning and expression of a rat V1a arginine vasopressin receptor.

The neurohypophyseal hormone arginine vasopressin has diverse actions, including the inhibition of diuresis, contraction of smooth muscle, stimulation of liver glycogenolysis and modulation of adrenocorticotropic hormone release from the pituitary. Arginine vasopressin receptors are G protein-coupled and have been divided into at least three types; the V1a (vascular/hepatic) and V1b (anterior pituitary) receptors which act through phosphatidylinositol hydrolysis to mobilize intracellular Ca2+, and the V2 (kidney) receptor which is coupled to adenylate cyclase. We report here the cloning of a complementary DNA encoding the hepatic V1a arginine vasopressin receptor. The liver cDNA encodes a protein with seven putative transmembrane domains, which binds arginine vasopressin and related compounds with affinities similar to the native rat V1a receptor. The messenger RNA corresponding to the cDNA is distributed in rat tissues known to contain V1a receptors.     

Pituitary hyperplasia and gigantism in mice caused by a cholera toxin transgene.

Cyclic AMP is thought to act as an intracellular second messenger, mediating the physiological response of many cell types to extracellular signals. In the pituitary, growth hormone (GH)-producing cells (somatotrophs) proliferate and produce GH in response to hypothalamic GH-releasing factor, which binds a receptor that stimulates Gs protein activation of adenylyl cyclase. We have now determined whether somatotroph proliferation and GH production are stimulated by cAMP alone, or require concurrent, non-Gs-mediated induction of other regulatory molecules by designing a transgene to induce chronic supraphysiological concentrations of cAMP in somatotrophs. The rat GH promoter was used to express an intracellular form of cholera toxin, a non-cytotoxic and irreversible activator of Gs. Introduction of this transgene into mice caused gigantism, elevated serum GH levels, somatotroph proliferation and pituitary hyperplasia. These results support the direct triggering of these events by cAMP, and illustrate the utility of cholera toxin transgenes as a tool for physiological engineering.     

卵泡刺激素促进卵巢颗粒细胞增殖分化的信号通路的研究进展

卵泡刺激素（FSH）是一种垂体前叶合成和分泌的糖蛋白激素,是卵巢发育的重要激素,可与卵巢颗粒细胞膜上的特异性受体结合从而促进细胞的增殖分化,激活细胞内芳香化酶生成雌激素和孕激素。FSH在卵巢颗粒细胞中主要依靠cAMP—PKA信号通路促进细胞增殖,同时也发生多种信号通路,ERK1／2、ERK5、p38MAPK、P13K／Akt、VEGF均参与FSH促进卵巢颗粒细胞的增殖分化;它们之间相互作用促进卵巢颗粒细胞的增殖。     

Somatostatin selectively inhibits noradrenaline release from hypothalamic neurones

Somatostatin in a hypothalamic peptide hormone which inhibits growth hormone release from the anterior pituitary. However, biochemical and morphological investigations have revealed that somatostatin is located not only in the hypothalamus but also in other brain areas (for example the cerebral cortex) where it occurs and in nerve cell bodies and fibres from which it can be released in a Ca2+-dependent manner. It has therefore been suggested that the neuropeptide may have functions in the central nervous system other than its effect on growth hormone release; one possible action is that of a neuromodulator. Therefore, hypothalamic and cerebral cortical slices of the rat were used to examine whether somatostatin modifies the electrically or CaCl2-evoked release of tritiated monoamines from monoaminergic neurones. it is reported here that somatostatin inhibits 3H-noradrenaline release from the hypothalamus (but not from the cerebral cortex) but does not affect the release of 3H-dopamine and 3H-serotonin.