scholarly journals Localization of protamine 1 mRNA in different stages of the cycle of the rat seminiferous epithelium.

1988 ◽  
Vol 107 (2) ◽  
pp. 407-412 ◽  
Author(s):  
P Mali ◽  
M Sandberg ◽  
E Vuorio ◽  
P C Yelick ◽  
N B Hecht ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Northern Blot ◽  
Northern Blot Analysis ◽  
Cdna Probe ◽  
Seminiferous Epithelium ◽  
Seminiferous Tubules ◽  
Mrna Levels ◽  
Low Levels ◽  
Protamine 1

A mouse protamine 1 cDNA probe was used to study P1 protamine gene expression during the cycle of the seminiferous epithelium in the rat. In situ hybridization experiments showed that transcription of the P1 protamine mRNA starts in the middle of step 7 of spermiogenesis during substage VIIc. The mRNA levels stay high in steps 7-14 spermatids but decrease during steps 15-16 and are virtually undetectable in steps 17-19 spermatids. Northern blot analyses of RNAs isolated from microdissected pools of seminiferous tubules show high P1 protamine mRNA concentrations during stages VIIc-XIV-III of the cycle and lower levels during stages IV-VIIb. Owing to a post-transcriptional shortening of the poly(A) tail by 130 bases, a decrease in the size of protamine 1 mRNA from approximately 580 to 450 nucleotides was observed in stages XIII-XIV suggesting an initiation of protamine 1 synthesis in step 13-14 spermatids. In stages II-VI (steps 16-18 spermatids), only the smaller size protamine 1 mRNA was detectable. The expression of protamine 1 mRNAs has been localized in the very last phase of the haploid gene activity. Although the in situ hybridization suggests a disappearance of protamine 1 mRNA after step 16 of spermiogenesis, Northern blot analysis shows that low levels of mRNA are present during the period of final condensation of the chromatin, reflecting the association of protamine with DNA.

scholarly journals Immunohistochemistry and in situ hybridization of P-45017 alpha (17 alpha-hydroxylase/17,20-lyase).

1992 ◽  
Vol 40 (7) ◽  
pp. 903-908 ◽  
Author(s):  
T Suzuki ◽  
H Sasano ◽  
T Sawai ◽  
J I Mason ◽  
H Nagura
Keyword(s):  
In Situ Hybridization ◽  
Leydig Cells ◽  
Cellular Localization ◽  
Cdna Probe ◽  
Simultaneous Detection ◽  
Seminiferous Tubules ◽  
Zona Fasciculata ◽  
Mrna Levels ◽  
Theca Interna

Cytochrome P-45017 alpha catalyzes both 17 alpha-hydroxylation and 17,20-side-chain cleavage in steroidogenesis and lies at a key branch point in the pathways of steroid hormone biosynthesis. To obtain information on the precise localization of P-45017 alpha in swine testis, ovary, and adrenal, we undertook the simultaneous detection of P-45017 alpha mRNA and protein by combining immunohistochemistry with in situ hybridization. In situ hybridization was performed on 4% paraformaldehyde-fixed, paraffin-embedded sections by employing either a 39-base oligomer or a cDNA insert (1.7 KB) of porcine testis P-45017 alpha as DNA probe. Immunohistochemical study was performed by employing anti-P-45017 alpha. Hybridization signals were obtained in Leydig cells of the testis, theca interna of the ovarian follicle, and zona fasciculata reticularis cells of the adrenal cortex. Oligonucleotide probing yielded lower background signal than the cDNA probe. No specific signals were obtained in seminiferous tubules of the testis, medulla, and zona glomerulosa of the adrenal, and in membrana granulosa and interstitial cells of the ovary. Hybridization signals were obtained in the cells where immunoreactivity of the enzyme was observed by immunohistochemistry, except for some Leydig cells of the testis and theca interna cells of the ovary in which only immunoreactivity but not hybridization signal was observed. The present study provided detailed information about the precise cellular localization of P-45017 alpha expression at both the protein and mRNA levels in swine adrenal glands and gonads. This approach of simultaneous immunohistochemistry and in situ hybridization analysis of steroidogenic enzymes can be applied in the future to tissues exhibiting abnormal steroid metabolism and should contribute to a better understanding of steroidogenesis.

scholarly journals Acetylcholine receptor alpha-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: Northern blot analysis and in situ hybridization.

1989 ◽  
Vol 108 (5) ◽  
pp. 1823-1832 ◽  
Author(s):  
O Horovitz ◽  
D Knaack ◽  
T R Podleski ◽  
M M Salpeter
Keyword(s):  
Ascorbic Acid ◽  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Alpha Subunit ◽  
Mrna Levels ◽  
Surface Receptors ◽  
Subunit Mrna

Ascorbic acid is the major factor in brain extract responsible for increasing the average acetylcholine receptor (AChR) site density on the cloned muscle cell line L5. In the present study, we show that this effect of ascorbic acid requires mRNA synthesis, and that the mRNA level for the AChR alpha-subunit is increased to about the same level as are the surface receptors. We have found no increase in the mRNA levels of the beta-, gamma-, and delta-subunits, or in the mRNAs of other muscle-specific proteins, such as that of light chain myosin 2, alpha-actin, and creatine kinase. By in situ hybridization, we further show that the increase in alpha-mRNA in response to ascorbic acid is exclusively in myotubes and is located near clusters of nuclei. mRNA levels for the alpha-subunit in mononucleated cells are very low and do not significantly increase in response to ascorbic acid. The mononucleated cells are thus excluded as a possible source for the increase in alpha-subunit mRNA detected by Northern blot analysis. Our results indicate that there is a very specific action of ascorbic acid on the regulation of AChR alpha-mRNA in the L5 muscle cells, and that the expression of surface receptors in these cells is limited by the amount of AChR alpha-subunit mRNA.

scholarly journals Towards Quantitative In Situ Hybridization

1997 ◽  
Vol 45 (3) ◽  
pp. 413-423 ◽  
Author(s):  
Ard Jonker ◽  
Piet A. J. de Boer ◽  
Maurice J. B. van den Hoff ◽  
Wouter H. Lamers ◽  
Antoon F. M. Moorman
Keyword(s):  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Photographic Emulsion ◽  
Mrna Levels ◽  
Intestinal Tissue ◽  
Tissue Sections ◽  
Silver Grains

In situ hybridization analysis of tissue mRNA concentrations remains to be accepted as a quantitative technique, even though exposure of tissue sections to photographic emulsion is equivalent to Northern blot analysis. Because of the biological importance of in situ quantification of RNA sequences within a morphological context, we evaluated the quantitative aspects of this technique. In calibrated microscopic samples, autoradiographic signal (density of silver grains) was proportionate to the radioactivity present, to the exposure time, and to time of development of the photographic emulsion. Similar results were obtained with tissue sections, showing that all steps of the in situ hybridization protocol, before and including the detection of the signal, can be reproducibly performed. Furthermore, the integrated density of silver grains produced in liver and intestinal sections by the in situ hybridization procedure using 35S-labeled riboprobes is directly proportionate to the signal obtained by quantitative Northern blot analysis. The significance of this finding is that in situ quantification of RNA can be realized with high sensitivity and with the additional advantage of the possibility of localizing mRNA within the cells of interest. Application of this procedure on fetal and adult intestinal tissue showed that the carbamoylphosphate synthetase (CPS)-expressing epithelial cells of both tissues accumulated CPS mRNA to the same level but that whole-organ CPS mRNA levels decreased four- to fivefold in the same period, owing to a comparable decrease in the number of CPS-expressing cells in total intestinal tissue. (J Histochem Cytochem 45:413–423, 1997)

Cell-specific metallothionein gene expression in mouse decidua and placentae

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 611-621 ◽  
Author(s):  
S.K. De ◽  
M.T. McMaster ◽  
S.K. Dey ◽  
G.K. Andrews
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Embryo Implantation ◽  
Normal Pregnancy ◽  
Mrna Levels ◽  
Metallothionein Gene ◽  
Visceral Yolk Sac ◽  
Rna In Situ Hybridization ◽  
Low Levels

Oligodeoxyribonucleotide excess solution hybridization, Northern blot and in situ hybridization were used to analyze metallothionein gene expression in mouse decidua and placentae during gestation. Metallothionein (MT) -I and -II mRNA levels were constitutively elevated, 11- and 13-fold, respectively, relative to the adult liver, in the deciduum (D8), and decreased coordinately about 6-fold during the period of development when the deciduum is replaced by the developing placenta (D10-16). Coincident with this decline, levels of MT mRNA increased dramatically in the visceral yolk sac endoderm. In situ hybridization established that MT-I mRNA was present at low levels in the uterine luminal epithelium (D4), but was elevated at the site of embryo implantation exclusively in the primary decidual zone by D5, and then in the secondary decidual zone (D6-8). Although low levels of MT mRNA were detected in total placental RNA, in situ hybridization revealed constitutively high levels in the outer placental spongiotrophoblasts. Analysis of pulse-labeled proteins from decidua and placentae established that these tissues are active in the synthesis of MT. The constitutively high levels of MT mRNA in decidua were only slightly elevated following injection of cadmium (Cd) and/or zinc (Zn), whereas in placentae they increased several-fold. MT mRNA levels were equally high in decidua and experimentally induced deciduomata (D8) which establishes that decidual MT gene expression is not dependent on the presence of the embryo or some embryo-derived factor. Although the functional role of MT during development is speculative, these results establish the concept that, from the time of implantation to late in gestation, the mouse embryo is surrounded by cells, interposed between the maternal and embryonic environments, which actively express the MT genes. This suggests that MT plays an important role in the establishment and maintenance of normal pregnancy.

Cloning and widespread distribution of the rat rod-type cyclic nucleotide-gated cation channel

1997 ◽  
Vol 272 (4) ◽  
pp. C1335-C1344 ◽  
Author(s):  
C. Ding ◽  
E. D. Potter ◽  
W. Qiu ◽  
S. L. Coon ◽  
M. A. Levine ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
In Situ Hybridization ◽  
Pineal Gland ◽  
Northern Blot ◽  
Blot Analysis ◽  
Cyclic Nucleotide ◽  
Northern Blot Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

We used Northern blot analysis, ribonuclease protection assay (RPA), reverse transcriptase-polymerase chain reaction, and in situ hybridization to investigate the hypothesis that the CNG1 isoform of the cyclic nucleotide-gated nonselective cation channel may be widely distributed in tissues of the rat. A cDNA encoding the CNG1 isoform was isolated from rat eye and human retina, and partial sequences were isolated from rat pineal gland and human kidney. Northern blot analysis revealed a 3.1-kilobase (kb) CNG1 transcript in rat eye, pineal gland, pituitary, adrenal gland, and spleen, and a larger transcript of 3.5 kb was found in testis. RPA confirmed the identity of CNG1 mRNA in rat eye, lung, spleen, and brain. Polymerase chain reaction-based detection of the mRNA for CNG1 indicates that the channel is expressed in lower abundance in many other tissues, including thymus, skeletal muscle, heart, and parathyroid gland. The cellular distribution of CNG1 was further studied by in situ hybridization, which demonstrated expression of mRNA in lung, thymus, pineal gland, hippocampus, cerebellum, and cerebral cortex but not in heart or kidney.

Presence and distribution of endothelin-1 gene expression in human kidney

1994 ◽  
Vol 267 (4) ◽  
pp. F679-F687 ◽  
Author(s):  
C. Pupilli ◽  
M. Brunori ◽  
N. Misciglia ◽  
C. Selli ◽  
L. Ianni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
In Situ Hybridization ◽  
Renal Cortex ◽  
Cdna Probe ◽  
Renal Medulla ◽  
Human Kidney ◽  
Renal Tumors ◽  
Mrna Levels

To investigate the presence and the distribution of preproendothelin-1 (prepro-ET-1) mRNA in human kidney, eight human kidneys obtained at surgery from patients affected by localized renal tumors were studied. Northern blot analysis using a human prepro-ET-1 cDNA probe labeled with 32P showed the presence of a single band of approximately 2.3 kb that was present both in the renal cortex and medulla of all the kidneys studied. Densitometric analysis of hybridization signals demonstrated that prepro-ET-1 mRNA levels in the renal medulla were 2.2-fold higher than those in the renal cortex. The distribution of prepro-ET-1 mRNA in human kidney was investigated by in situ hybridization using a human prepro-ET-1 RNA probe labeled with 35S. The greatest density of prepro-ET-1 mRNA was observed in the renal medulla, where hybridization signal was demonstrated in vasa recta bundles and capillaries and in collecting ducts. By combining in situ hybridization with immunohistochemical detection of von Willebrand factor, we demonstrated that 93 +/- 2.5% of nontubular medullary cells containing prepro-ET-1 mRNA were endothelial cells. In the cortex, prepro-ET-1 mRNA was localized in the endothelial layer of arcuate and interlobular arteries and veins and in the endothelial cells of afferent arterioles. The results of the present study demonstrate that ET-1 gene expression is present in vascular and tubular structures of the human kidney. It is possible that ET-1 synthesized locally in the human kidney represents a local system affecting renal hemodynamics and functions through paracrine and/or autocrine actions on different renal structures.

Substance P receptor expression in intestinal epithelium inClostridium difficile toxin A enteritis in rats

1998 ◽  
Vol 275 (1) ◽  
pp. G68-G75 ◽  
Author(s):  
Charalabos Pothoulakis ◽  
Ignazio Castagliuolo ◽  
S. E. Leeman ◽  
Chi-Chung Wang ◽  
Hanzong Li ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Substance P ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Rat Intestine ◽  
Toxin A

We previously reported that the inflammatory effects of Clostridium difficile toxin A on rat intestine can be significantly inhibited with a specific neurokinin-1 receptor (NK-1R) antagonist. In this study we investigated the localization and expression of NK-1R mRNA and protein in rat intestine by in situ hybridization, Northern blot analysis, and immunohistochemistry, respectively, after exposure to toxin A. Northern blot analysis showed increased mucosal levels of NK-1R mRNA starting 30 min after toxin A administration. In situ hybridization showed that toxin A increased NK-1R mRNA expression in intestinal epithelial cells after 30, 120, and 180 min. In rats pretreated with the NK-1R antagonist CP-96345 the increase in NK-1R mRNA levels after exposure to toxin A was inhibited, indicating that NK-1R upregulation is substance P (SP) dependent. One hour after exposure to toxin A many of the intestinal epithelial cells showed staining for NK-1R compared with controls. Specific 125I-SP binding to purified epithelial cell membranes obtained from ileum exposed to toxin A for 15 min was increased twofold over control and persisted for 4 h. This report provides evidence that NK-1R expression is increased in the intestinal epithelium shortly after exposure to toxin A and may be important in toxin A-induced inflammation.

scholarly journals Expression and localization of mRNA for the 16 KD subunit of V-ATPase in the rat embryo.

1995 ◽  
Vol 43 (8) ◽  
pp. 761-769 ◽  
Author(s):  
M Numata ◽  
S Ohkuma ◽  
S Iseki
Keyword(s):  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Mammalian Development ◽  
Mesenchymal Differentiation ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation

Vacuolar H(+)-ATPase (V-ATPase), an enzyme composed of multisubunits, is located in the membrane of intracellular organelles (e.g., lysosomes, and endosomes) and maintains the intraorganellar acidic pH by pumping protons across the membrane. Although there is growing evidence for some important role of V-ATPase in cell proliferation and differentiation, the functional significance of V-ATPase in vivo during mammalian development remains obscure. In the present study we investigated the expression and localization of mRNA for the 16 KD subunit of V-ATPase, an essential sector for enzymatic activity, in prenatal rat by Northern blot analysis and in situ hybridization with a specific oligonucleotide probe. With Northern blot analysis, consistent expression of the mRNA was observed in the embryos throughout the period examined (E14-E20). On in situ hybridization, mRNA signal was distributed with various intensities in both the epithelial and mesenchymal tissues at embryonic day 14 (E14). In E17 and E20 embryos, localization of strong signal became more restricted to distinct mesenchymal cells such as fibroblasts adjacent to the epithelia of skin, lung, and intestine, the cells of perichondrium, and myoblasts in the process of fusion. These results suggest that V-ATPase performs specific functions during the later stages of embryogenesis, especially at sites of mesenchymal differentiation and epithelium-mesenchyme interaction.

scholarly journals Expression of a testis-specific hsp70 gene-related RNA in defined stages of rat seminiferous epithelium.

1988 ◽  
Vol 107 (4) ◽  
pp. 1317-1323 ◽  
Author(s):  
Z Krawczyk ◽  
P Mali ◽  
M Parvinen
Keyword(s):  
Cellular Localization ◽  
Living Condition ◽  
Seminiferous Epithelium ◽  
Seminiferous Tubules ◽  
Hsp70 Gene ◽  
Late Pachytene ◽  
Gene Coding ◽  
Contrast Microscopy ◽  
Low Levels

Changes in the level of a testis-specific hsp70 gene-related transcript (hst70 RNA) and its cellular localization during the cycle of rat seminiferous epithelium have been investigated. Segments of seminiferous tubules at defined stages of the cycle were isolated in living condition by transillumination-assisted microdissection and the exact stages identified by phase-contrast microscopy of live cell squashes. The levels of the hst70 RNA were determined by Northern and slot blotting of whole cell lysates. High levels were found in stages XII-XIV and I to early VII of the cycle, and low levels were found in other stages, i.e., late VII (VIId) through VIII-XI of the cycle. The in situ hybridization revealed that the hst70 gene was activated in late pachytene primary spermatocytes during stage XII of the cycle, and that mRNA was then present in cells during differentiation through diakinesis, meiotic divisions, and early spermiogenesis (steps 1 through early 7). The activation of the gene coding for hst70 RNA shortly before meiotic divisions may indicate that the gene product is needed either during differentiation of late spermatocytes into spermatids or later during spermiogenesis, and that the mRNA may be stored in early spermatids.

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