Accumulation, spatial distribution and partial characterization of poly(A)+RNA in the developing oocytes of Xenopus laevis

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 127-140
Author(s):  
M. Wakahara
Keyword(s):  
Spatial Distribution ◽  
In Situ Hybridization ◽  
Xenopus Laevis ◽  
Binding Activity ◽  
Dnase I ◽  
Rnase A ◽  
Type Activity ◽  
Rnase T2 ◽  
Silver Grains

In situ hybridization using [3H]poly(U) was applied to developing oocytes of Xenopus laevis, which had been fixed in Bouin's solution. Tissue sections were pretreated with DNase I, annealed with [3H]poly(U) and post-treated with RNase A and TCA. After the autoradiographical processing, silver grains over the oocyte were counted. As a result of the control experiments which included RNase A, RNase T2, DNase I and Pronase E hydrolysis and Cordycepin (3'-deoxyadenosine) incubation before in situ hybridization, it was concluded that the poly(U)-binding activity detected upon the oocytes was due to the possible presence of poly(A)+RNAs. Spatial distribution of the poly(U)-binding sites changed during the development of the oocytes; in a small oocyte before the pachytene stage, silver grains developed over the nucleus, while in a larger oocyte after the diplotene the grains were concentrated over the cytoplasm. After yolk platelets were deposited in the cytoplasm, two types of poly(U)-binding activities were noted; a bound-type activity which was firmly associated with the cytoplasm, so that the positions of the silver grains were not influenced by fixation, and an unbound type which did not bind so firmly to the cytoplasm and was therefore easily influenced by inflow of fixative. The bound-type activity persisted in the cytoplasm throughout the oogenesis, but the unbound type appeared only after the vitellogenesis, especially in the yolky cytoplasm. The total poly(U)-binding activity per oocyte increased continuously with the growth of the oocyte.

Ultrastructural analysis of the spatial distribution of MRNA

1992 ◽  
Vol 50 (1) ◽  
pp. 552-553
Author(s):  
Gary Bassell ◽  
Robert H. Singer
Keyword(s):  
Electron Microscopy ◽  
Spatial Distribution ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Nucleic Acids ◽  
Colloidal Gold ◽  
Dna Probe ◽  
Nucleotide Analogs ◽  
Gold Particles

We have been investigating the spatial distribution of nucleic acids intracellularly using in situ hybridization. The use of non-isotopic nucleotide analogs incorporated into the DNA probe allows the detection of the probe at its site of hybridization within the cell. This approach therefore is compatible with the high resolution available by electron microscopy. Biotinated or digoxigenated probe can be detected by antibodies conjugated to colloidal gold. Because mRNA serves as a template for the probe fragments, the colloidal gold particles are detected as arrays which allow it to be unequivocally distinguished from background.

scholarly journals Localization of type 1 17beta-hydroxysteroid dehydrogenase mRNA and protein in syncytiotrophoblasts and invasive cytotrophoblasts in the human term villi

2000 ◽  
Vol 165 (2) ◽  
pp. 217-222 ◽  
Author(s):  
M Bonenfant ◽  
PR Provost ◽  
R Drolet ◽  
Y Tremblay
Keyword(s):  
In Situ Hybridization ◽  
Hydroxysteroid Dehydrogenase ◽  
Binding Activity ◽  
Placental Lactogen ◽  
Specific Expression ◽  
P450 Aromatase ◽  
Chain Cleavage ◽  
Extravillous Cytotrophoblasts

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) play a key role in the synthesis of sex steroids. The hallmark of this family of enzymes is the interconversion, through their oxydoreductive reactivity at position C17, of 17-keto- and 17beta-hydroxy-steroids. Because this reaction essentially transforms steroids having low binding activity for the steroid receptor to their more potent 17beta-hydroxysteroids isoforms, it is crucial to the control of the physiological activities of both estrogens and androgens. The human placenta produces large amounts of progesterone and estrogens throughout pregnancy. The placental type 1 17beta-HSD enzyme (E17beta-HSD) catalyzes the reduction of the low activity estrogen, estrone, into the potent estrogen, estradiol. We studied the cell-specific expression of type 1 17beta-HSD in human term placental villous tissue by combining in situ hybridization to localize type 1 17beta-HSD mRNA with immunohistochemistry using an antibody against human placental lactogen, a trophoblast marker. Immunolocalization of E17beta-HSD was also performed. To ascertain whether other steroidogenic enzymes are present in the same cell type, cytochrome P450 cholesterol side-chain cleavage (P450scc), P450 aromatase, and type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD) were also localized by immunostaining. Our results showed that the syncytium is the major steroidogenic unit of the fetal term villi. In fact, type 1 17beta-HSD mRNA and protein, as well as P450scc, P450 aromatase, and 3beta-HSD immunoreactivities were found in these cells. In addition, our results revealed undoubtedly that extravillous cytotrophoblasts (CTBs), e.g. those from which cell columns of anchoring villous originate, also express the type 1 17beta-HSD gene. However, CTBs lying beneath the syncytial layer, e.g. those from which syncytiotrophoblasts develop, contained barely detectable amounts of type 1 17beta-HSD mRNA as determined by in situ hybridization. These findings, along with those from other laboratories confirm the primordial role of the syncytium in the synthesis of steroids during pregnancy. In addition, our results indicate for the first time that CTBs differentiating along the invasive pathway contain type 1 17beta-HSD mRNA.

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 UNDER-REPLICATION OF RIBOSOMAL CISTRONS IN POLYTENE CHROMOSOMES OF RHYNCHOSCIARA

1974 ◽  
Vol 62 (1) ◽  
pp. 215-222 ◽  
Author(s):  
A. G. Gambarini ◽  
F. J. S. Lara
Keyword(s):  
Salivary Gland ◽  
In Situ Hybridization ◽  
Xenopus Laevis ◽  
Related Species ◽  
Polytene Chromosomes ◽  
Chromosome X ◽  
Heterologous Hybridization ◽  
Ribosomal Cistrons ◽  
Rhynchosciara Americana

DNA preparations obtained from several tissues of Rhynchosciara americana and two related species, R. milleri and R. papaveroi, were hybridized to R. americana rRNA. The percentage of hybridization was found to be higher in tissues with low polyteny than in tissues with high polyteny, suggesting a relationship between the amount of rDNA and the tissue polyteny. This could be explained by under-replication of ribosomal cistrons in polytene cells, such as those from the salivary gland. Only slight tissue-dependent changes in the percentages of hybridization can be observed in heterologous hybridization using Xenopus laevis rRNA. The possibility that these experiments could not detect differences in the amount of ribosomal cistrons among tissues is discussed. The female:male ratio for the percentages of hybridization in the salivary gland of R. americana agrees with the results obtained by in situ hybridization experiments (16, 17) which have shown that the rRNA cistrons are distributed among chromosomes other than chromosome X.

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.

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.

Immunocytochemistry and in situ hybridization of neuropeptide Y in the hypothalamus of Xenopus laevis in relation to background adaptation

Neuroscience ◽  
1993 ◽  
Vol 55 (3) ◽  
pp. 667-675 ◽  
Author(s):  
R. Tuinhof ◽  
F.Y.S.C. Laurent ◽  
R.G.E. Ebbers ◽  
W.J.A.J. Smeets ◽  
M.C.H.M. van Riel ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Xenopus Laevis ◽  
Neuropeptide Y ◽  
Background Adaptation

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

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.

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