Localization of the sites of synthesis and action of prolactin by immunocytochemistry and in-situ hybridization within the human utero-placental unit

1991 ◽  
Vol 7 (3) ◽  
pp. 241-247 ◽  
Author(s):  
W.-X. Wu ◽  
J. Brooks ◽  
M. R. Millar ◽  
W. L. Ledger ◽  
P. T. K. Saunders ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Culture Medium ◽  
Cdna Probe ◽  
Specific Hybridization ◽  
Decidual Cells ◽  
Its Gene ◽  
First Time ◽  
Silver Grains ◽  
Prolactin Mrna

ABSTRACT While the fetal pituitary synthesizes and releases prolactin, it is also produced within the utero-placental unit during pregnancy in women and has been localized in the amnion, chorion and decidua. However, it is not clear whether prolactin is synthesized within all these non-fetal pituitary tissues. We have investigated prolactin production and its gene expression using tissue culture, immunocytochemistry and in-situ hybridization techniques. Prolactin was immunolocalized not only in the decidua but also in amnion and trophoblast cells. In contrast, the in-situ hybridization results showed that silver grains, formed by specific hybridization of a prolactin cDNA probe to prolactin mRNA, were confined to decidual cells of early and term pregnancy. The results from tissue cultures correlated well with those of in-situ hybridization, that is that only the decidua made detectable prolactin, while it was undetectable in the culture medium from trophoblast tissue, irrespective of the stage of pregnancy. This study, for the first time, establishes that only decidualized cells are involved in biosynthesis of prolactin; other prolactin-containing cells in the amnion and trophoblast appear to sequester prolactin, possibly via receptors, suggesting that prolactin may play an important paracrine role within the amnion and syncitio- and cytotrophoblast of the utero-placental unit.

In situ localization of mRNA for epidermal growth factor in the submandibular gland of the mouse.

1985 ◽  
Vol 33 (12) ◽  
pp. 1235-1240 ◽  
Author(s):  
E W Gresik ◽  
R M Gubits ◽  
T Barka
Keyword(s):  
In Situ Hybridization ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Submandibular Gland ◽  
Cdna Probe ◽  
Coding Region ◽  
Specific Hybridization ◽  
Epidermal Growth ◽  
Convoluted Tubules

Epidermal growth factor (EGF) is a polypeptide originally isolated from the mouse submandibular gland, where it is localized immunocytochemically in cells of the granular convoluted tubules (GCT). cDNAs encoding the precursor of mouse submandibular EGF have been cloned (Scott et al. Science 221:236, 1983; Gray et al. Nature 303:722, 1983). A fragment of one of these clones, pmegf10, containing the EGF coding region, was tritium-labeled by nick-translation and used as a probe for in situ hybridization to EGF mRNA. A specific hybridization signal for EGF mRNA was seen only in mature or developing GCT cells. The intensity of the signal was stronger in glands of intact males than in females or in castrated males. In glands of castrates treated with testosterone, or of intact females treated with triiodothyronine (T3), the signal was comparable to that in intact males. In glands of males treated with T3 the intensity of the signal was stronger than in untreated males. A weak to moderate signal was seen in developing GCT cells of 20-day-old males but not females. Hybridization for 3 days gave a stronger signal than that for 1 day. No signal was seen in either sex at 10 days of age, or in control preparations exposed to labeled DNA of pBR322. The presence of EGF mRNA exclusively in GCT cells provides strong evidence that these cells are the only site of synthesis of EGF in the submandibular gland. In situ hybridization with this cDNA probe will provide a sensitive method to determine possible cellular sites of EGF production outside of the submandibular gland.

Quantitative in-situ hybridization histochemistry in the rat pituitary gland: effect of bromocriptine on prolactin and pro-opiomelanocortin gene expression

1988 ◽  
Vol 118 (2) ◽  
pp. 205-NP ◽  
Author(s):  
A. Levy ◽  
S. L. Lightman
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Messenger Rna ◽  
Cdna Probe ◽  
Intermediate Lobe ◽  
Hybridization Histochemistry ◽  
In Situ Hybridization Histochemistry ◽  
Pomc Mrna ◽  
Prolactin Mrna

ABSTRACT The temporal effect of orally administered bromocriptine on pro-opiomelanocortin (POMC) and prolactin gene expression in male Sprague–Dawley rats was examined using in-situ hybridization histochemistry. Messenger RNA in the anterior and intermediate lobes could be clearly delineated in each section. Administration of bromocriptine resulted in a reduction in hybridization of 35S-labelled cDNA probe to prolactin mRNA from 0·69 × 1012 to 0·29 × 1012 copies bound/g after 150 h. POMC mRNA in the anterior lobe remained unchanged with 0·08 × 1012 copies of probe bound/g for the duration of the experiment, while in the intermediate lobe it decreased from 2·44 × 1012 to 0·44 × 1012 copies of probe bound/g at 150 h. The rate of reduction in intermediate lobe POMC mRNA was similar to that of prolactin mRNA for the first 24 h but was subsequently more rapid and more profound, falling to 20% of the control value at 84 h and to 18% at 150 h. J. Endocr. (1988) 118, 205–210

scholarly journals Electron microscopic autoradiographic localization of prolactin mRNA in rat pituitary.

1989 ◽  
Vol 37 (5) ◽  
pp. 567-571 ◽  
Author(s):  
Y Tong ◽  
H F Zhao ◽  
J Simard ◽  
F Labrie ◽  
G Pelletier
Keyword(s):  
In Situ Hybridization ◽  
Secretory Granules ◽  
Cdna Probe ◽  
Cell Types ◽  
Hybridization Technique ◽  
Experimental Conditions ◽  
Thin Sections ◽  
Rat Pituitary ◽  
Silver Grains

Recent immunoelectron microscopic studies have shown that immunoreactive prolactin (PRL) in rat pituitary can be detected not only in typical PRL cells, characterized by large secretory granules, but also in another type of cell, which contains small secretory granules. To determine whether or not these two cell types are involved in PRL biosynthesis, we developed a procedure to investigate PRL gene expression by using in situ hybridization at the ultrastructural level. Rat pituitary was fixed and vibratome sections were incubated with a PRL [35S]-cDNA probe and subsequently flat-embedded in Araldite. Semi-thin and ultra-thin sections were processed for autoradiography. The results indicate that only the two PRL cell types were labeled. When immunolabeling for PRL was applied to ultra-thin sections, only immunopositive cells were seen to contain silver grains. In these cells the silver grains were associated with the rough endoplasmic reticulum and nucleus. When a growth hormone (GH) [35S]-cDNA probe was used as a control, only GH-secreting cells were labeled. This study confirms that the two PRL cell types are involved in biosynthesis of PRL. Moreover, this simple in situ hybridization technique provides a new approach to accurately localize mRNA in complex tissue and to investigate the subcellular distribution of mRNA under differing experimental conditions.

scholarly journals In situ detection of vitamin D-induced calcium-binding protein (9-kDa CaBP) messenger RNA in rat duodenum.

1986 ◽  
Vol 34 (2) ◽  
pp. 277-280 ◽  
Author(s):  
M Warembourg ◽  
O Tranchant ◽  
C Perret ◽  
C Desplan ◽  
M Thomasset
Keyword(s):  
Vitamin D ◽  
In Situ Hybridization ◽  
Calcium Binding ◽  
Binding Protein ◽  
Cdna Probe ◽  
Calcium Binding Protein ◽  
Pbr322 Dna ◽  
Rat Duodenum ◽  
Columnar Cells

We have previously described the molecular cloning of a cDNA fragment synthesized from rat duodenal mRNA coding for a 9000-dalton vitamin D-induced calcium-binding protein (9-kDa CaBP) (3). We now report the use of this cloned cDNA to study the cytological distribution of 9-kDa CaBP mRNA in rat duodenum by in situ hybridization. Tissue sections, fixed in ethanol:acetic acid, were hybridized to the 3H-cDNA probe and processed for autoradiography. The specificity of the CaBP mRNA-DNA hybrid formation was checked using 3H-labeled plasmid pBR322 DNA as a control probe. 9k-Da CaBP mRNA, visualized by silver grains, was found only in the absorptive epithelial cells, and the concentration was greater in the cells at the villous tips than in those of the crypts. The 9k-Da CaBP mRNA was observed mainly in the cytoplasm of the columnar cells and less frequently in the nucleus. Labeling was not seen in the brush border and goblet cells. The submucosa, with Brunner's glands and muscularis, also showed no specific 9-kDa CaBP mRNA concentration. This demonstration of 9-kDa CaBP gene activity in the columnar cells of the rat duodenum illustrates the usefulness of in situ hybridization for characterization of specific cells involved in the expression of 1,25(OH)2 D3 activity.

scholarly journals Quantitation of erythropoietin-producing cells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal erythropoietin mRNA, and serum erythropoietin concentration

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 645-651 ◽  
Author(s):  
ST Koury ◽  
MJ Koury ◽  
MC Bondurant ◽  
J Caro ◽  
SE Graber
Keyword(s):  
In Situ Hybridization ◽  
Blood Loss ◽  
Mrna Levels ◽  
Outer Cortex ◽  
Acute Anemia ◽  
Small Clusters ◽  
Inner Cortex ◽  
Silver Grains ◽  
Serum Erythropoietin

Abstract In situ hybridization was used to quantitate the cells that produce erythropoietin (EP) in the renal cortices of mice with varying severities of acute anemia and of mice recovering from severe, acute anemia. The number of EP-producing cells in the renal cortex increased in an exponential manner as hematocrit was decreased. Individual EP- producing cells had very similar densities of silver grains in autoradiograms regardless of whether they were from normal mice or from slightly, moderately or severely anemic animals. With increasingly severe anemia, total renal EP mRNA levels and serum EP concentrations showed increases that correlated with the number of renal EP-producing cells. These results indicate that as mice become more anemic, additional cells are recruited to produce EP rather than the cells already producing EP being stimulated to increase their individual production. In mildly and moderately anemic animals, small clusters of EP-producing cells were found in the inner cortex with large areas of cortex containing no EP-producing cells. In severely anemic mice, EP- producing cells were found throughout the inner cortex with only a very few found scattered in the outer cortex and outer medulla. The data indicate that only a subset of total renal interstitial cells produce EP. During recovery from severe, acute anemia, the numbers of EP- producing cells decreased exponentially as hematocrits rose and correlated with decreases in total renal EP mRNA and serum EP concentrations. These results suggest that following an acute blood loss and during the recovery from a blood loss, the capacity to deliver oxygen, as represented by hematocrit, is the major regulator of EP production.

scholarly journals Fluorescence In Situ Hybridization (FISH) on Human Chromosomes Using Photoprobe Biotin-labeled Probes

2003 ◽  
Vol 51 (4) ◽  
pp. 549-551 ◽  
Author(s):  
Anja Weise ◽  
Peter Harbarth ◽  
Uwe Claussen ◽  
Thomas Liehr
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Dna Probes ◽  
Human Chromosomes ◽  
First Time ◽  
Established Technique ◽  
Whole Chromosome Painting

Fluorescence in situ hybridization (FISH) on human chromosomes in meta-and interphase is a well-established technique in clinical and tumor cytogenetics and for studies of evolution and interphase architecture. Many different protocols for labeling the DNA probes used for FISH have been published. Here we describe for the first time the successful use of Photoprobe biotin-labeled DNA probes in FISH experiments. Yeast artificial chromosome (YAC) and whole chromosome painting (wcp) probes were tested.

scholarly journals Autoradiographic visualization of 35S-labeled cRNA probes combined with immunoperoxidase detection of choleragenoid: a double-labeling light microscopic method for in situ hybridization and retrograde tract tracing.

1994 ◽  
Vol 42 (6) ◽  
pp. 827-831 ◽  
Author(s):  
O Hermanson ◽  
H Ericson ◽  
G Sanchez-Watts ◽  
A G Watts ◽  
A Blomqvist
Keyword(s):  
In Situ Hybridization ◽  
Cobalt Acetate ◽  
Reaction Products ◽  
Double Labeling ◽  
Cholera Toxin Subunit B ◽  
Efferent Projections ◽  
Labeled Rna ◽  
Subunit B ◽  
Silver Grains

We describe a protocol for simultaneous light microscopic visualization of a neuron's efferent projections and its expression of mRNA. We have combined immunohistochemical visualization of the retrograde marker cholera toxin subunit B (CTb) with autoradiographic visualization of 35S-labeled cRNA probes. Injections of CTb were made into rat brain. Immunoreactivity for CTb was demonstrated by modification of the peroxidase-anti-peroxidase immunohistochemical technique, with DAB and nickel ammonium sulfate or cobalt acetate as chromogen. On the same sections, in situ hybridization was performed with a 35S-labeled RNA probe complementary to preproenkephalin mRNA or tyrosine hydroxylase mRNA. Many double-labeled neurons were detected. These neurons contained peroxidase reaction product and were covered by an accumulation of silver grains in the overlaying emulsion layer. The present method has several advantages over double-labeling methods using the combination of fluorescent tracers and oligonucleotide probes. Both reaction products are permanent and can be visualized simultaneously by light microscopy. Furthermore, both CTb and cRNA probes are very sensitive markers. In addition, the sections can be counterstained.

scholarly journals Catalyzed Reported Deposition-Fluorescence In Situ Hybridization Protocol To Evaluate Phagotrophy in Mixotrophic Protists

2005 ◽  
Vol 71 (11) ◽  
pp. 7321-7326 ◽  
Author(s):  
Juan M. Medina-Sánchez ◽  
Marisol Felip ◽  
Emilio O. Casamayor
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Signal Amplification ◽  
Cell Attachment ◽  
Cell Loss ◽  
Alexa Fluor ◽  
Hybridization Protocol ◽  
Card Fish ◽  
First Time

ABSTRACT We describe a catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH) protocol particularly suited to assess the phagotrophy of mixotrophic protists on prokaryotes, since it maintains cell and plastid integrity, avoids cell loss and egestion of prey, and allows visualization of labeled prey against plastid autofluorescence. This protocol, which includes steps such as Lugol's-formaldehyde-thiosulfate fixation, agarose cell attachment, cell wall permeabilization with lysozyme plus achromopeptidase, and signal amplification with Alexa-Fluor 488, allowed us to detect almost 100% of planktonic prokaryotes (Bacteria and Archaea) and, for the first time, to show archaeal cells ingested by mixotrophic protists.

scholarly journals EnVision+, a New Dextran Polymer-based Signal Enhancement Technique for In Situ Hybridization (ISH)

2001 ◽  
Vol 49 (9) ◽  
pp. 1067-1071 ◽  
Author(s):  
Klaus Hermann Wiedorn ◽  
Torsten Goldmann ◽  
Christof Henne ◽  
Heike Kühl ◽  
Ekkehard Vollmer
Keyword(s):  
In Situ Hybridization ◽  
Signal Amplification ◽  
Detection System ◽  
Lower Sensitivity ◽  
The Novel ◽  
Visualization System ◽  
Integrated Optical ◽  
Assay Time ◽  
First Time

Seventy paraffin-embedded cervical biopsy specimens and condylomata were tested for the presence of human papillomavirus (HPV) by conventional in situ hybridization (ISH) and ISH with subsequent signal amplification. Signal amplification was performed either by a commercial biotinyl-tyramide-based detection system [GenPoint (GP)] or by the novel two-layer dextran polymer visualization system EnVision+ (EV), in which both EV–horseradish peroxidase (EV–HRP) and EV–alkaline phosphatase (EV–AP) were applied. We could demonstrate for the first time, that EV in combination with preceding ISH results in a considerable increase in signal intensity and sensitivity without loss of specificity compared to conventional ISH. Compared to GP, EV revealed a somewhat lower sensitivity, as measured by determination of the integrated optical density (IOD) of the positively stained cells. However, EV is easier to perform, requires a shorter assay time, and does not raise the background problems that may be encountered with biotinyl–tyramide-based amplification systems. (J Histochem Cytochem 49:1067–1071, 2001)

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