Ultrastructural and immunohistochemical analysis of the 8-20 week human fetal pancreas

Development of the human pancreas is well-known to involve tightly controlled differentiation of pancreatic precursors to mature cells that express endocrine- or exocrine-specific protein products. However, details of human pancreatic development at the ultrastructural level are limited. The present study analyzed 8–20 week fetal age human pancreata using scanning and transmission electron microscopy (TEM), TEM immunogold and double or triple immunofluorescence staining. Primary organization of islets and acini occurred during the developmental period examined. Differentiating endocrine and exocrine cells developed from the ductal tubules and subsequently formed isolated small clusters. Extracellular matrix fibers and proteins accumulated around newly differentiated cells during their migration and cluster formation. Glycogen expression was robust in ductal cells of the pancreas from 8–15 weeks of fetal age; however, this became markedly reduced at 20 weeks, with a concomitant increase in acinar cell glycogen content. Insulin secretory granules transformed from being dense and round at 8 weeks to distinct geometric (multilobular, crystalline) structures by 14–20 weeks. Initially many of the differentiating endocrine cells were multihormonal and contained polyhormonal granules; by 20 weeks, monohormonal cells were in the majority. Interestingly, certain secretory granules in the early human fetal pancreatic cells showed positivity for both exocrine (amylase) and endocrine proteins. This combined ultrastructural and immunohistochemical study showed that, during early developmental stages, the human pancreas contains differentiating epithelial cells that associate closely with the extracellular matrix, have dynamic glycogen expression patterns and contain polyhormonal as well as mixed endocrine/ exocrine granules

Conserved Usage of Alternative 5′ Untranslated Exons of the GATA4 Gene

BACKGROUND:GATA4 is an essential transcription factor required for the development and function of multiple organs. Despite this important role, our knowledge of how the GATA4 gene is regulated remains limited. To better understand this regulation, we characterized the 5' region of the mouse, rat, and human GATA4 genes. METHODOLOGY/PRINCIPAL FINDINGS:Using 5' RACE, we identified novel transcription start sites in all three species. GATA4 is expressed as multiple transcripts with varying 5' ends encoded by alternative untranslated first exons. Two of these non-coding first exons are conserved between species: exon 1a located 3.5 kb upstream of the GATA4 ATG site in exon 2, and a second first exon (exon 1b) located 28 kb further upstream. Expression of both mRNA variants was found in all GATA4-expressing organs but with a preference for the exon 1a-containing transcript. The exception was the testis where exon 1a- and 1b-containing transcripts were similarly expressed. In some tissues such as the intestine, alternative transcript expression appears to be regionally regulated. Polysome analysis suggests that both mRNA variants contribute to GATA4 protein synthesis. CONCLUSIONS/SIGNIFICANCE:Taken together, our results indicate that the GATA4 gene closely resembles the other GATA family members in terms of gene structure where alternative first exon usage appears to be an important mechanism for regulating its tissue- and cell-specific expression

Erratum: A Case-Control Study of Maternal Polybrominated Diphenyl Ether (PBDE) Exposure and Cryptorchidism in Canadian Populations

[This corrects the article DOI: 10.1289/EHP522.]

Direct effects of tamoxifen on growth hormone secretion by pituitary cells in vitro

There is now strong evidence to suggest that insulin-like growth factor I (IGF-I) plays an important role in breast cancer proliferation. Recently we observed that tamoxifen-treated stage I breast cancer patients have serum IGF-I levels significantly lower than placebo-treated patients. Since IGF-I is growth hormone (GH) dependent, we have tested the hypothesis that tamoxifen alters serum IGF-I levels through direct inhibition of GH secretion. Immature lamb pituitary cultures were examined for acute (3 h) or chronic (1-6 day) effects of the drug, using doses (0.1-10 μmol/l) based on known steady state levels in patients on tamoxifen therapy (0.31-3.1 μmol/l). Tamoxifen had a direct, dose-related, inhibitory effect on GH release from pituitary somatotropes, during acute as well as chronic treatment. The 10 μmol/l dose consistently decreased both basal and growth hormone releasing factor stimulated GH release. These in vitro data are consistent with our hypothesis that tamoxifen suppresses serum IGF-I levels by acting at the pituitary to inhibit GH release

Expression and activation of caspase-6 in human fetal and adult tissues.

Caspase-6 is an effector caspase that has not been investigated thoroughly despite the fact that Caspase-6 is strongly activated in Alzheimer disease brains. To understand the full physiological impact of Caspase-6 in humans, we investigated Caspase-6 expression. We performed western blot analyses to detect the pro-Caspase-6 and its active p20 subunit in fetal and adult lung, kidney, brain, spleen, muscle, stomach, colon, heart, liver, skin, and adrenals tissues. The levels were semi-quantitated by densitometry. The results show a ubiquitous expression of Caspase-6 in most fetal tissues with the lowest levels in the brain and the highest levels in the gastrointestinal system. Caspase-6 active p20 subunits were only detected in fetal stomach. Immunohistochemical analysis of a human fetal embryo showed active Caspase-6 positive apoptotic cells in the dorsal root ganglion, liver, lung, kidney, ovary, skeletal muscle and the intestine. In the adult tissues, the levels of Caspase-6 were lower than in fetal tissues but remained high in the colon, stomach, lung, kidney and liver. Immunohistological analyses revealed that active Caspase-6 was abundant in goblet cells and epithelial cells sloughing off the intestinal lining of the adult colon. These results suggest that Caspase-6 is likely important in most tissues during early development but is less involved in adult tissues. The low levels of Caspase-6 in fetal and adult brain indicate that increased expression as observed in Alzheimer Disease is a pathological condition. Lastly, the high levels of Caspase-6 in the gastrointestinal system indicate a potential specific function of Caspase-6 in these tissues

Regulation of growth hormone secretion from human fetal pituitaries : interactions between growth hormone releasing factor and somatostatin

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Integrin α3, but not β1, regulates islet cell survival and function via PI3K/Akt signaling pathways

β1-Integrin is a well-established regulator of β-cell activities; however, the role of its associated α-subunits is relatively unknown. Previously, we have shown that human fetal islet and INS-1 cells highly express α3β1-integrin and that collagens I and IV significantly enhance their survival and function; in addition, blocking β1 function in the fetal islet cells decreased adhesion on collagen I and increased apoptosis. The present study investigates the effect of blocking α3. Using α3 blocking antibody or small interfering RNA, the effects of α3-integrin blockade were examined in isolated human fetal or adult islet cells or INS-1 cells, cultured on collagens I or IV. In parallel, β1 blockade was analyzed in INS-1 cells. Perturbing α3 function in human islet or INS-1 cells resulted in significant decreases in cell function (adhesion, spreading, proliferation and Pdx1 and insulin expression/secretion), primarily on collagen IV. A significant decrease in focal adhesion kinase and ERK1/2 phosphorylation and increased caspase3 cleavage were observed on both collagens. These effects were similar to changes after β1 blockade. Interestingly, only α3 blockade reduced expression of phospho-Akt and members of its downstream signaling cascades (glycogen synthase kinase β and X-linked inhibitor of apoptosis), demonstrating a specific effect of α3 on the phosphatidyl-inositol 3-kinase/Akt pathway. These results suggest that α3- as well as β1-integrin-extracellular matrix interactions are critical for modulating β-cell survival and function through specialized signaling cascades and enhance our understanding of how to improve islet microenvironments for cell-based treatments of diabetes. Copyright © 2011 by The Endocrine Society