1,721,055 research outputs found
Commonalities in the endocrinology of stem cell biology and organ regeneration (In special issue: Stem cells and progenitors in organ maintenance, regeneration and replacement: the role of hormones and growth factors in health and disease)
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The human fetal adrenal cortex and the window of sexual differentiation
No full textUnderstanding normal development is fundamental to appreciating postnatal morphology, physiology and, in some instances, pathophysiology. Developmental biology tends to interrogate models in nonprimate species, for instance the mouse, where genetic manipulation gives privileged insight into the function of particular genes. Some human developmental processes, as occur in the adrenal gland, are not faithfully reproduced in these rodent models, yet have an impact on the pathophysiology and treatment of endocrine disorders, such as congenital adrenal hyperplasia. In this setting, in vitro research of normal human development complements clinical investigation of patients born with congenital disorder
Age-specific changes in sex steroid biosynthesis and sex development
No full textNormal male sex development requires the SRY gene on the Y chromosome, the regression of Mullerian structures via anti-Mullerian hormone (AMH) signalling, the development of the Wolffian duct system into normal male internal genital structures consequent to testosterone secretion by the testicular Leydig cells, and finally, sufficient activation of testosterone to dihydrotestosterone by 5alpha-reductase. All these events take place during weeks 8-12 of gestation, a narrow window of sexual differentiation. Recent studies in human fetal development have demonstrated the early fetal expression of the adrenocorticotrophic hormone (ACTH) receptor and all steroidogenic components necessary for the biosynthesis of cortisol. These findings provide compelling evidence for the assumed pathogenesis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, diminished feedback to the pituitary due to glucocorticoid deficiency, subsequent ACTH excess, and up-regulation of adrenal androgen production with subsequent virilization. Another CAH variant, P450 oxidoreductase deficiency, manifests with 46,XX disorder of sex development (DSD), i.e., virilized female genitalia, despite concurrently low circulating androgens. This CAH variant illustrates the existence of an alternative pathway toward the biosynthesis of active androgens in humans which is active in human fetal life only. Thus CAH teaches important lessons from nature, providing privileged insights into the window of human sexual differentiation, and particularly highlighting the importance of steroidogenesis in the process of human sexual differentiatio
The adrenal cortex and sexual differentiation during early human development
No full textHuman sexual differentiation is a critical process whereby a strict dimorphism is established that enables future reproductive success as phenotypic males and females. Significant components of this differentiation pathway unfold during the first three months of gestation when they are sensitive to disruption by abnormal hormonal influences. Excessive exposure of female development to androgens in conditions such as congenital adrenal hyperplasia causes virilization. However, recently we have suggested that female development normally takes place in the presence of low, yet significant, levels of androgen, implying a need for strict regulation to avoid virilization and the potential for a biological role of androgens in females that has not been fully elucidated. Here, we review androgen-dependent male differentiation of the external genitalia in humans, and link this to current understanding of female development and steroidogenesis in the developing adrenal cortex
The self-orientation of mammalian cells in optical tweezers - the importance of the nucleus
No full textHere we present the first evidence showing that eukaryotic cells can be stably trapped in a single focused Gaussian beam with an orientation that is defined by the nucleus. A mammalian eukaryotic cell (in suspension) is trapped and is re-oriented in the focus of a linearly polarized Gaussian beam with a waist of dimension smaller than the radius of the nucleus. The cell reaches a position relative to the focus that is dictated by the nucleus and nuclear components. Our studies illustrate that the force exerted by the optical tweezers at locations within the cell can be predicted theoretically; the data obtained in this way is consistent with the experimental observations
Phospholamban and sarcolipin are maintained in the endoplasmic reticulum by retrieval from the ER-Golgi intermediate compartment
No full textObjectivePhospholamban and sarcolipin are small transmembrane proteins that modulate cardiac contractility through their interaction with the sarcoplasmic reticulum (SR) calcium pumps (SERCAs). We have examined the hypothesis that phospholamban and sarcolipin are maintained in the SR by a process of retrieval from post-SR compartments and the role of their transmembrane domains in targeting.MethodsAntibodies directed against phospholamban and protein markers of the endoplasmic reticulum/Golgi intermediate compartment (ERGIC) and the trans-Golgi were used in fluorescence microscopy studies of cultured human fetal cardiac myocytes. In addition, sarcolipin and phospholamban were tagged at the N-terminus with enhanced-green-fluorescent protein (EGFP) and expressed in COS 7 cells. The EGFP-tagged constructs were localised using fluorescence microscopy and cell fractionation. The length of the transmembrane domains of phospholamban and sarcolipin were extended and the effect on cellular location was also examined.ResultsIn fetal cardiac myocytes phospholamban was located in the SR and the ERGIC, but did not migrate to the trans-Golgi network. Tagged-phospholamban and sarcolipin were located in the endoplasmic reticulum (ER) of COS 7 cells indicating that their targeting was unaffected by the EGFP tag. Significant proportions of the tagged phospholamban and sarcolipin were also located in the ERGIC but not in the trans-Golgi. Increasing the length of the transmembranous domains of EGFP-tagged phospholamban and sarcolipin resulted in their mis-targeting to the plasma membrane.ConclusionsPhospholamban and sarcolipin are maintained in the SR/ER by a process that includes their retrieval from the ERGIC following their passage from the SR/ER into the ERGIC. The transmembrane domains of phospholamban and sarcolipin are involved in the retrieval process
Expression profiles of SF-1, DAX1, and CYP17 in the human fetal adrenal gland: potential interactions in gene regulation
No full textCytochrome P450 17?-hydroxylase/17–20 lyase (P450C17) is a critical branchpoint enzyme for steroid hormone biosynthesis. During human gestation, P450C17 is required for the production of dehydroepiandrostenedione sulfate by the fetal adrenal cortex and for testicular production of androgens that mediate male sexual differentiation. In this study, we investigate the regulation of the human CYP17 gene by two orphan nuclear receptors, steroidogenic factor 1 (SF-1) and DAX1. In human embryos, SF-1 and DAX1 are expressed throughout the developing adrenal cortex from its inception at 33 days post conception (dpc). In contrast, P450C17 expression, which commences between 41 and 44 dpc, is limited to the fetal zone. The 5'-flanking region of the human CYP17 gene contains three functional SF-1 elements that collectively mediate a ?25-fold induction of promoter activity by SF-1. In constructs containing all three functional SF-1 elements, DAX1 inhibited this activation by ?55%. In the presence of only one or two SF-1 elements, DAX1 inhibition was lost even though SF-1 transactivation persisted. These data suggest that efficient repression of SF-1-mediated activation of the human CYP17 gene by DAX1 requires multiple SF-1 elements. Opposing effects of SF-1 and DAX1 may fine tune the differential responses of various SF-1 target genes in different endocrine tissues
Embryonic stem cells to beta-cells by understanding pancreas development
Full text linkInsulin injections treat but do not cure Type 1 diabetes (T1DM). The success of islet transplantation suggests cell replacement therapies may offer a curative strategy. However, cadaver islets are of insufficient number for this to become a widespread treatment. To address this deficiency, the production of beta-cells from pluripotent stem cells offers an ambitious far-sighted opportunity. Recent progress in generating insulin-producing cells from embryonic stem cells has shown promise, highlighting the potential of trying to mimic normal developmental pathways. Here, we provide an overview of the current methodology that has been used to differentiate stem cells toward a beta-cell fate. Parallels are drawn with what is known about normal development, especially regarding the human pancrea
Subcellular localization of ALMS1 supports involvement of centrosome and basal body dysfunction in the pathogenesis of obesity, insulin resistance, and type 2 diabetes
No full textAlström syndrome is a rare autosomal recessive disorder caused by mutations in a novel gene of unknown function, ALMS1. Central features of Alström syndrome include obesity, insulin resistance, and type 2 diabetes, and therefore investigating ALMS1 function stands to offer new insights into the pathogenesis of these common conditions. To begin this process, we have analyzed the subcellular localization and tissue distribution of ALMS1 by immunofluorescence. We show that ALMS1 is widely expressed and localizes to centrosomes and to the base of cilia. Fibroblasts with disrupted ALMS1 assemble primary cilia and microtubule cytoskeletons that appear normal, suggesting that the Alström syndrome phenotype results from impaired function rather than abnormal development. Coupled with recent data on the complex phenotype of Bardet-Biedl syndrome, our findings imply an unexpected central role for basal body and centrosome dysfunction in the pathogenesis of obesity, insulin resistance, and type 2 diabetes. Unraveling the molecular mechanisms underlying the Alström syndrome phenotype will be important in the search for new therapeutic targets for these conditions
Hypoxia inducible factors regulate pluripotency and proliferation in human proliferation in human embryonic stem cells cultured at reduced oxygen tensions
No full textHuman embryonic stem (hES) cells are routinely cultured under atmospheric, 20% oxygen tensions but are derived from embryos which reside in a 3-5% oxygen (hypoxic) environment. Maintenance of oxygen homeostasis is critical to ensure sufficient levels for oxygen dependent processes. This study investigates the importance of specific hypoxia inducible factors (HIFs) in regulating the hypoxic responses of hES cells. We report that culture at 20% oxygen decreased hES cell proliferation and resulted in a significantly reduced expression of SOX2, NANOG and OCT4 mRNA as well as OCT4 protein compared to hypoxic conditions. HIF-1alpha protein was not expressed at 20% oxygen and displayed only a transient, nuclear localization at 5% oxygen. HIF-2alpha and HIF-3alpha displayed a cytoplasmic localization during initial hypoxic culture but translocated to the nucleus following long term culture at 5% oxygen and were significantly upregulated compared to cells cultured at 20% oxygen. Silencing of HIF-2alpha resulted in a significant decrease in both hES cell proliferation and OCT4, SOX2 and NANOG protein expression while the early differentiation marker, SSEA1 was concomitantly increased. HIF-3alpha up-regulated HIF-2alpha and prevented HIF-1alpha expression with knock-down of HIF-3alpha resulting in the reappearance of HIF-1alpha protein. In summary, these data demonstrate that a low oxygen tension is preferential for the maintenance of a highly proliferative, pluripotent population of hES cells. While HIF-3alpha was found to regulate the expression of both HIF-1alpha and HIF-2alpha, it is HIF-2alpha which regulates hES cell pluripotency as well as proliferation under hypoxic condition
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