Temporal changes in inhibin subunit mRNAs during atresia of preovulatory follicles in the rat

This study aimed to investigate the time course of disappearance of the mRNAs of the various subunits of inhibin in follicles which become atretic. An animal model was used in which atresia of preovulatory follicles could be studied in a chronological order. Injection of gonadotrophin-releasing hormone (GnRH) antagonist (20 microg) at the morning of pro-oestrus (P) blocked ovulation and the 10-12 preovulatory follicles became gradually atretic. A second injection was given the next day to prevent delayed ovulation. The rate of atresia could be delayed by simultaneous administration of a subovulatory dose of human chorionic gonadotrophin (hCG) (0.5 IU) and could be advanced by administration of a fivefold larger amount of GnRH antagonist. Functional activity of follicles becoming atretic was studied by measuring oestradiol production after incubation of individual follicles for 4 h. Follicles isolated 24 h after the first injection of GnRH antagonist (P+24) already secreted significantly less oestradiol in vitro than follicles isolated at pro-oestrus, although they were morphologically not different from pro-oestrous follicles. Follicles isolated at P+24 from hCG-treated rats secreted more oestradiol compared with follicles from rats not treated with hCG. In contrast, follicles isolated at P+24 from rats that were given a fivefold larger amount of GnRH antagonist secreted less oestradiol. Once this model was validated, temporal changes in inhibin subunit mRNAs in follicles undergoing atresia were measured by in situ hybridization and RNase protection assay. In situ hybridization showed abundant alpha- and betaA-subunit mRNA in the whole granulosa layer of preovulatory follicles at P and P+24, while betaB-subunit mRNA was restricted to the antral layer and cumulus. At P+48 the amount of alpha- and betaA-subunit mRNA had declined and was restricted to the cumulus, whereas betaB-subunit mRNA was absent. In the atretic follicles present at P+72 and P+96, mRNAs of all three inhibin subunits were absent. Administration of 0.5 IU hCG delayed the decline in the amount of alpha, betaA and betaB mRNA in preovulatory follicles at P+48. RNase protection assay of inhibin subunits in isolated follicles revealed no changes between P and P+24. However, at P+48, the mRNAs of alpha- and betaA-subunits were decreased. Expression of the mRNA of betaB-subunit declined gradually from P to P+48. The present study demonstrates that in follicles which are becoming atretic, mRNAs of alpha- and betaA-subunits decline simultaneously with the appearance of pycnotic cells in the granulosa layer, while betaB-subunit mRNA declines earlier, simultaneously with the decrease in the ability to secrete oestradiol in vitro.

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Distribution and development of Gαi-2 mRNA in the rat cerebral cortex investigated with in situ hybridization and RNAse protection assay

Developmental Brain Research ◽

10.1016/0165-3806(94)00174-x ◽

1995 ◽

Vol 84 (2) ◽

pp. 208-214 ◽

Cited By ~ 2

Author(s):

Peter S. Eriksson ◽

Michael Nilsson ◽

Göran L. Matejka

Keyword(s):

Cerebral Cortex ◽

In Situ Hybridization ◽

Rnase Protection Assay ◽

Rat Cerebral Cortex ◽

Protection Assay ◽

Rnase Protection

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Integrin alpha subunit mRNAs are differentially expressed in early Xenopus embryos

Development ◽

10.1242/dev.117.4.1239 ◽

1993 ◽

Vol 117 (4) ◽

pp. 1239-1249 ◽

Cited By ~ 7

Author(s):

C.A. Whittaker ◽

D.W. DeSimone

Keyword(s):

In Situ Hybridization ◽

Rnase Protection Assay ◽

Early Embryo ◽

Mrna Levels ◽

Rnase Protection ◽

Dorsal Midline ◽

Transmembrane Receptors ◽

Early Embryos ◽

Whole Mount

Adhesion of cells to extracellular matrix proteins is mediated, in large part, by transmembrane receptors of the integrin family. The identification of specific integrins expressed in early embryos is an important first step to understanding the roles of these receptors in developmental processes. We have used polymerase chain reaction methods and degenerate oligodeoxynucleotide primers to identify and clone Xenopus integrin alpha subunits from neurula-stage (stage 17) cDNA. Partial cDNAs encoding integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and an alpha IIb-related subunit were cloned and used to investigate integrin mRNA expression in early embryos by RNase protection assay and whole-mount in situ hybridization methods. Considerable integrin diversity is apparent early in development with integrins alpha 2, alpha 3, alpha 4, alpha 5 and alpha 6 each expressed by the end of gastrulation. Both alpha 3 and alpha 5 are expressed as maternal mRNAs. Zygotic expression of alpha 2, alpha 3, alpha 4 and alpha 6 transcripts begins during gastrulation. Integrin alpha 5 is expressed at relatively high levels during cleavage, blastula and gastrula stages suggesting that it may represent the major integrin expressed in the early embryo. We demonstrated previously that integrin beta 1 protein synthesis remains constant following induction of stage 8 animal cap cells with activin (Smith, J. C., Symes, K., Hynes, R. O. and DeSimone, D. W. (1990) Development 108, 289–298.). Here we report that integrin alpha 3, alpha 4 and alpha 6 mRNA levels increase following induction with 10 U/ml activin-A whereas alpha 5, beta 1 and beta 3 mRNA levels remain unchanged. Whole-mount in situ hybridization reveals that alpha 3 mRNAs are expressed by cells of the involuting mesoderm in the dorsal lip region of early gastrulae. As gastrulation proceeds, alpha 3 expression is localized to a stripe of presumptive notochordal cells along the dorsal midline. In neurulae, alpha 3 mRNA is highly expressed in the notochord but becomes progressively more restricted to the caudalmost portion of this tissue as development proceeds from tailbud to tadpole stages. In addition, alpha 3 is expressed in the forebrain region of later stage embryos. These data suggest that integrin-mediated adhesion may be involved in the process of mesoderm involution at gastrulation and the organization of tissues during embryogenesis.

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Developmental expression of tissue inhibitor of metalloproteinase (TIMP) RNA

Development ◽

10.1242/dev.105.3.575 ◽

1989 ◽

Vol 105 (3) ◽

pp. 575-583 ◽

Cited By ~ 1

Author(s):

S. Nomura ◽

B.L. Hogan ◽

A.J. Wills ◽

J.K. Heath ◽

D.R. Edwards

Keyword(s):

Rnase Protection Assay ◽

Tissue Inhibitor Of Metalloproteinase ◽

Developmental Expression ◽

Corpora Lutea ◽

Tissue Inhibitor ◽

Rnase Protection ◽

Rna Probes ◽

Order Of Magnitude

Single-stranded antisense RNA probes have been used to study the expression of the metalloproteinase inhibitor TIMP (tissue inhibitor of metalloproteinases), during mouse embryogenesis and in adult tissues. Using a sensitive RNase protection assay, low levels of transcript can be detected in a variety of tissues, including maternal deciduum, embryonic kidney, lung and amnion. Higher levels are seen in osteogenic tissues such as calvaria, while the highest level in any tissue is found in the ovary, though even here expression is an order of magnitude below that observed in growth factor-treated fibroblasts in vitro. Using the technique of in situ hybridization, TIMP transcripts can first be detected in osteogenic tissues in the head and limb at about 15.5 days post coitum, and increase in amount until birth. The high levels of TIMP RNA in the ovary are localized to cells of the corpora lutea.

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Expression and localization of aquaporins in rat gastrointestinal tract

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.3.c621 ◽

1999 ◽

Vol 276 (3) ◽

pp. C621-C627 ◽

Cited By ~ 134

Author(s):

Yu Koyama ◽

Tadashi Yamamoto ◽

Tatsuo Tani ◽

Kouei Nihei ◽

Daisuke Kondo ◽

...

Keyword(s):

Small Intestine ◽

In Situ Hybridization ◽

Gastrointestinal Tract ◽

Epithelial Cells ◽

Cellular Localization ◽

Basolateral Membrane ◽

Rnase Protection Assay ◽

Rnase Protection ◽

Basolateral Membranes

A family of water-selective channels, aquaporins (AQP), has been demonstrated in various organs and tissues. However, the localization and expression of the AQP family members in the gastrointestinal tract have not been entirely elucidated. This study aimed to demonstrate the expression and distribution of several types of the AQP family and to speculate on their role in water transport in the rat gastrointestinal tract. By RNase protection assay, expression of AQP1–5 and AQP8 was examined in various portions through the gastrointestinal tract. AQP1 and AQP3 mRNAs were diffusely expressed from esophagus to colon, and their expression was relatively intense in the small intestine and colon. In contrast, AQP4 mRNA was selectively expressed in the stomach and small intestine and AQP8 mRNA in the jejunum and colon. Immunohistochemistry and in situ hybridization demonstrated cellular localization of these AQP in these portions. AQP1 was localized on endothelial cells of lymphatic vessels in the submucosa and lamina propria throughout the gastrointestinal tract. AQP3 was detected on the circumferential plasma membranes of stratified squamous epithelial cells in the esophagus and basolateral membranes of cardiac gland epithelia in the lower stomach and of surface columnar epithelia in the colon. However, AQP3 was not apparently detected in the small intestine. AQP4 was present on the basolateral membrane of the parietal cells in the lower stomach and selectively in the basolateral membranes of deep intestinal gland cells in the small intestine. AQP8 mRNA expression was demonstrated in the absorptive columnar epithelial cells of the jejunum and colon by in situ hybridization. These findings may indicate that water crosses the epithelial layer through these water channels, suggesting a possible role of the transcellular route for water intake or outlet in the gastrointestinal tract.

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Developmental regulation of epithelial sodium channel subunit mRNA expression in rat colon and lung

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1998.275.6.g1227 ◽

1998 ◽

Vol 275 (6) ◽

pp. G1227-G1235 ◽

Cited By ~ 14

Author(s):

Shigeru Watanabe ◽

Kazumichi Matsushita ◽

John B. Stokes ◽

Paul B. McCray

Keyword(s):

In Situ Hybridization ◽

Epithelial Cells ◽

Mrna Expression ◽

Developmental Regulation ◽

Rat Colon ◽

Rnase Protection ◽

Quantitative Expression ◽

Subunit Mrna ◽

Lung And Kidney

Na+absorption via amiloride-sensitive Na+ channels is of critical importance in the transition between fetal and neonatal life in several tissues, including the colon, lung, and kidney. To characterize and contrast the mRNA expression of each of the three epithelial Na+ channel complex (ENaC) subunits, we conducted RNase protection assays (RPA) and in situ hybridization in colon and lung in fetal (17, 19, 20, and 21 days) and postnatal (1, 3, 9, 15, and 30 days) rats (r). In the colon the α-, β-, and γ-rENaC subunits showed quantitatively different but qualitatively similar expression. All three subunits gradually increased in abundance from fetal day 19 through day 30 of life. The amount of each subunit on day 30 was approximately three times the amount at day 1. In situ hybridization showed that each subunit was localized to the surface epithelial cells with minimal expression in the crypts. The lung showed a completely different pattern. In contrast to the colon, the total amount of α-rENaC mRNA (by RPA) in the lung increased dramatically from fetal day 19 to 21, whereas β- and γ-rENaC showed modest prenatal increases. The amounts of all three mRNAs fell after birth through day 9 (to about 75% of the day 1 value). On days 15 and 30 the amount of mRNA rose to approach the values on day 1. α-rENaC mRNA abundance always exceeded β- and γ-rENaC, and the quantitative expression was different for α- than for β- and γ-rENaC. In situ hybridization studies showed that all three subunits were expressed in epithelial cells of the bronchi, bronchioles, and alveoli and not in blood vessels. These studies show striking developmental heterogeneity in rENaC mRNA expression between lung and colon, probably reflecting different developmental regulatory mechanisms in these organs.

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Developmental expression of human angiotensinogen in transgenic mice

AJP Renal Physiology ◽

10.1152/ajprenal.1998.274.5.f932 ◽

1998 ◽

Vol 274 (5) ◽

pp. F932-F939 ◽

Cited By ~ 2

Author(s):

Gongyu Yang ◽

Curt D. Sigmund

Keyword(s):

In Situ Hybridization ◽

Transgenic Mice ◽

Developmental Regulation ◽

Rnase Protection Assay ◽

Late Gestation ◽

Developmental Expression ◽

Rnase Protection ◽

Adult Mice ◽

Specific Regulation

Transgenic mice containing the human angiotensinogen ( HAGT) gene were utilized to determine the developmental regulation of HAGT expression. RNase protection assay on total RNA obtained from whole transgenic fetuses revealed that HAGT expression was first detected at embryonic day 8.5( E8.5) and was abundant from E9.5 onward. The earliest expression of the HAGT transgene appeared to precede the earliest expression of the endogenous mouse AGT gene by 1–2 days. Northern blot analysis revealed moderate levels of HAGT mRNA in liver and kidney and low levels of HAGT mRNA in heart and brain from E16.5 ( day 16.5 of gestation) onward. HAGT mRNA in liver, although abundant during late gestation and in 2-wk-old and adult mice, decreased transiently around birth. In situ hybridization performed on sections from whole fetuses revealed that HAGTmRNA was restricted to the developing liver and heart between E9.5 and E11.5 but became more widespread to include the developing aorta, brain, subcutaneous tissues, and vertebra at E13.5. In situ hybridization analysis on fetal kidneys from late gestation, newborn, and 2-wk-old mice demonstrated a progressive restriction of HAGT mRNA to developing cortical proximal tubular cells. These data illustrate the developmental tissue-specific regulation of HAGTexpression and demonstrate that sequences present in the transgene can confer an appropriate developmental expression profile.

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Characterization of an Intermediate Filament Protein from the Platyhelminth, Dugesia japonica

Protein and Peptide Letters ◽

10.2174/0929866526666191025102902 ◽

2020 ◽

Vol 27 (5) ◽

pp. 432-446

Author(s):

Akiko Yamamoto ◽

Ken-ichiro Matsunaga ◽

Toyoaki Anai ◽

Hitoshi Kawano ◽

Toshihisa Ueda ◽

...

Keyword(s):

In Situ Hybridization ◽

Immunohistochemical Analysis ◽

Protein Characterization ◽

Intermediate Filament Protein ◽

Tail Domain ◽

Animal Cells ◽

Dugesia Japonica ◽

Function Relationship

Background: Intermediate Filaments (IFs) are major constituents of the cytoskeletal systems in animal cells. Objective: To gain insights into the structure-function relationship of invertebrate cytoplasmic IF proteins, we characterized an IF protein from the platyhelminth, Dugesia japonica, termed Dif-1. Method: cDNA cloning, in situ hybridization, immunohistochemical analysis, and IF assembly experiments in vitro using recombinant Dif-1, were performed for protein characterization. Results: The structure deduced from the cDNA sequence showed that Djf-1 comprises 568 amino acids and has a tripartite domain structure (N-terminal head, central rod, and C-terminal tail) that is characteristic of IF proteins. Similar to nuclear IF lamins, Djf-1 contains an extra 42 residues in the coil 1b subdomain of the rod domain that is absent from vertebrate cytoplasmic IF proteins and a nuclear lamin-homology segment of approximately 105 residues in the tail domain; however, it contains no nuclear localization signal. In situ hybridization analysis showed that Djf-1 mRNA is specifically expressed in cells located within the marginal region encircling the worm body. Immunohistochemical analysis showed that Djf-1 protein forms cytoplasmic IFs located close to the microvilli of the cells. In vitro IF assembly experiments using recombinant proteins showed that Djf-1 alone polymerizes into IFs. Deletion of the extra 42 residues in the coil 1b subdomain resulted in the failure of IF formation. Conclusions: Together with data from other histological studies, our results suggest that Djf- 1 is expressed specifically in anchor cells within the glandular adhesive organs of the worm and that Djf-1 IFs may play a role in protecting the cells from mechanical stress.

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Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrio parahaemolyticus in Oysters and Mussels

Foods ◽

10.3390/foods10071502 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1502

Author(s):

Jorge García-Hernández ◽

Manuel Hernández ◽

Yolanda Moreno

Keyword(s):

In Situ Hybridization ◽

Vibrio Parahaemolyticus ◽

Fluorescent In Situ Hybridization ◽

Potential Method ◽

Viable Count ◽

Hybridization Technique ◽

Fish Technique ◽

Viable Cells

Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 μg/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 °C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.

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