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7 records – page 1 of 1.

Application of the immunofluorescence technique and confocal laser scanning microscopy for studying the distribution of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor on rat luteal cells.

https://arctichealth.org/en/permalink/ahliterature65080
Source
J Histochem Cytochem. 1991 Apr;39(4):397-400
Publication Type
Article
Date
Apr-1991
Author
J T Lakkakorpi
H J Rajaniemi
Author Affiliation
Biocenter University of Oulu, Finland.
Source
J Histochem Cytochem. 1991 Apr;39(4):397-400
Date
Apr-1991
Language
English
Publication Type
Article
Keywords
Animals
Female
Fluorescent Antibody Technique
Image Processing, Computer-Assisted
Immunohistochemistry - methods
Lasers
Luteal Cells - metabolism - ultrastructure
Microscopy, Electron - methods
Pseudopregnancy - metabolism
Rats
Rats, Inbred Strains
Receptors, Gonadotropin - metabolism
Receptors, LH - metabolism
Research Support, Non-U.S. Gov't
Abstract
We used confocal scanning microscopy to study the semi-quantitative distribution of luteinizing hormone/chorionic gonadotropin (LH/CG) receptors on rat luteal cells at both the two- and the three-dimensional level. The receptors were visualized in 6-microns sections of pseudopregnant rat ovaries using polyclonal rabbit antiserum to hCG-affinity-purified LH/CG receptor in conjunction with rhodamine-conjugated anti-rabbit immunoglobulins. Twenty to 30 optical sections were taken at different focal planes from representative luteal cells with a confocal laser scanning microscope and then processed digitally to two- and three-dimensional pseudocolored images. Distinct differences in fluorescence intensity could be demonstrated at both the two- and the three-dimensional level on the luteal cell surfaces, suggesting an uneven distribution of the LH/CG receptors on the cell membranes. This probably results in the compartmentalization and polarization of luteal cell function.
PubMed ID
2005369 View in PubMed
Less detail

Human chorionic gonadotrophin (CG)-induced down-regulation of the rat luteal LH/CG receptor results in part from the down-regulation of its synthesis, involving increased alternative processing of the primary transcript.

https://arctichealth.org/en/permalink/ahliterature64837
Source
J Mol Endocrinol. 1993 Apr;10(2):153-62
Publication Type
Article
Date
Apr-1993
Author
J T Lakkakorpi
E M Pietilä
J T Aatsinki
H J Rajaniemi
Author Affiliation
Biocenter, University of Oulu, Finland.
Source
J Mol Endocrinol. 1993 Apr;10(2):153-62
Date
Apr-1993
Language
English
Publication Type
Article
Keywords
Animals
Base Sequence
Blotting, Northern
Chorionic Gonadotropin - physiology
DNA, Single-Stranded
Densitometry
Down-Regulation
Female
Humans
Immunoblotting
Molecular Sequence Data
Ovary - metabolism
Polymerase Chain Reaction
Pseudopregnancy - metabolism
RNA Processing, Post-Transcriptional
RNA, Messenger - metabolism
Rats
Rats, Sprague-Dawley
Receptors, LH - genetics - metabolism
Research Support, Non-U.S. Gov't
Transcription, Genetic
Abstract
To elucidate the molecular mechanisms involved in the homologous regulation of LH/chorionic gonadotrophin (CG) receptors, the receptor and its mRNA levels were analysed in the same pseudopregnant rat ovarian samples after human (h)CG-induced down-regulation using a binding assay, ligand blotting, immunoblotting and Northern blotting together with the polymerase chain reaction (PCR). Treatment of the animals with 500 IU hCG resulted in a loss of 125I-labelled hCG binding and the 90 kDa receptor on the ligand and immunoblots within 12 and 24 h respectively, followed by a transient partial recovery on days 4 and 5, while a distinct decline occurred only on day 7 in the controls. Northern blots of total ovarian RNA, as probed with a 293 bp AvaI/HindIII fragment from the extracellular domain of PCR-generated full-length rat LH/CG receptor cDNA, revealed six major mRNAs of 7.0, 4.2, 2.8, 2.0, 1.4 and 1.1 kb. The 4.2 kb mRNA, which was the most abundant, possibly encodes the 90 kDa receptor, while the smaller species probably represent alternatively spliced forms of the LH/CG receptor pre-mRNA, as also supported by the finding that PCR produced three cDNA bands of 2.1, 2.0 and 1.8 kb when oligomers derived from the N and C termini of rat LH/CG receptor cDNA were used as primers and rat ovarian total RNA as a template. Treatment with hCG led to the down-regulation of all six mRNAs in a fashion parallel to the changes in receptor protein. No smaller receptor components capable of binding radiolabelled hCG or receptor antibody appeared on the ligand or immunoblots prior to or during down-regulation or the subsequent transient period of up-regulation, suggesting that the smaller mRNA species are translated in minute amounts in vivo or are not translated at all. Laser densitometric analysis of the Northern blots revealed that the amounts of the four smallest mRNA species increased during the period of down-regulation in relation to the 4.2 kb mRNA, and correspondingly decreased during the subsequent period of up-regulation, indicating changes in the alternative splicing of the primary transcript. The data suggest that hCG-induced transient down-regulation of the LH/CG receptor results in part from down-regulation of its mRNA levels, and that changes in alternative processing of the receptor pre-mRNA may play a regulatory role in the expression of functional LH/CG receptor during down- and up-regulation.
PubMed ID
8484864 View in PubMed
Less detail

Polarity and fusion of JAR choriocarcinoma cells as assessed by enveloped viral glycoproteins.

https://arctichealth.org/en/permalink/ahliterature24023
Source
Exp Cell Res. 1993 Jun;206(2):276-82
Publication Type
Article
Date
Jun-1993
Author
R A Olli
H J Rajaniemi
R. Rydbeck
K. Metsikkö
Author Affiliation
Department of Anatomy, University of Oulu, Finland.
Source
Exp Cell Res. 1993 Jun;206(2):276-82
Date
Jun-1993
Language
English
Publication Type
Article
Keywords
Animals
Antibodies, Monoclonal
Cell Fusion
Choriocarcinoma - pathology - physiopathology
Female
Giant Cells - cytology - physiology
Humans
Parainfluenza Virus 3, Human - genetics
Pregnancy
Tumor Cells, Cultured
Uterine Neoplasms - pathology - physiopathology
Vero Cells
Vesicular stomatitis-Indiana virus - genetics
Viral Envelope Proteins - biosynthesis - isolation & purification
Abstract
Placental trophoblasts are epithelial cells which undergo physiological fusion and generate syncytia. In this study, a placental choriocarcinoma cell line JAR was infected with two enveloped viruses, Parainfluenza type 3 (P3) or vesicular stomatitis virus (VSV). Both viruses possess a fusion glycoprotein known to be able to induce polykaryon formation in nonpolarized cells. The P3 virus fusion protein was localized on the apical as well as on the basal plasma membrane domains of the infected JAR cells. Infection of the JAR cell monolayer with P3 virus, whose fusion protein is active at pH 7.0, resulted in syncytia formation. Furthermore, the actin ring structure surrounding individual cells disappeared during the P3 virus induced cell-cell fusion. On the contrary, the VSV glycoprotein was found preferentially on the apical plasma membrane domain. To activate the VSV fusogen, the cells were subjected to pH 5.0. However, no syncytia formation peculiar to VSV-infected fibroblasts was observed, and the actin ring structures remained intact. We conclude that in JAR cells the VSV fusion protein exhibits a polarized expression while the P3 virus fusion glycoprotein is distributed between the two membrane domains. Our results suggest that an apically situated fusogen is not sufficient to mediate cell-cell fusion of JAR cells.
PubMed ID
8388801 View in PubMed
Less detail

Processing of the LH/CG receptor and bound hormone in rat luteal cells after hCG-induced down-regulation as studied by a double immunofluorescence technique in conjunction with confocal laser scanning microscopy.

https://arctichealth.org/en/permalink/ahliterature64715
Source
J Histochem Cytochem. 1994 Jun;42(6):727-32
Publication Type
Article
Date
Jun-1994
Author
J T Lakkakorpi
M. Yang
H J Rajaniemi
Author Affiliation
Biocenter Oulu and Department of Anatomy, University of Oulu, Finland.
Source
J Histochem Cytochem. 1994 Jun;42(6):727-32
Date
Jun-1994
Language
English
Publication Type
Article
Keywords
Animals
Antibodies, Monoclonal
Chorionic Gonadotropin - metabolism - pharmacology
Corpus Luteum - cytology - drug effects - metabolism
Down-Regulation - drug effects
Female
Fluorescent Antibody Technique
Gonadotropins, Equine - pharmacology
Immunohistochemistry
Mice - immunology
Microscopy - methods
Ovary - cytology - drug effects - metabolism
Pseudopregnancy
Rabbits - immunology
Rats
Rats, Sprague-Dawley
Receptors, LH - biosynthesis - metabolism
Research Support, Non-U.S. Gov't
Abstract
We developed a double immunofluorescence technique for detection of the rat luteinizing hormone/choriogonadotropin (LH/CG) receptor and bound hCG in the same rat ovarian section and used it in conjunction with confocal laser scanning microscopy (CLSM) to study the fate of the receptor-hormone complex in luteal cells during the hCG-induced down-regulation. Pseudopregnant immature females rats were perfusion-fixed before (0 hr) and 2, 6, 12, 24, or 36 hr after a down-regulating dose of hCG (500 IU IV). The cryosections were stained for the LH/CG receptor and bound hormone by sequential incubations with a polyclonal rabbit antiserum to purified rat LH/CG receptor and a mouse monoclonal antibody (MAb) to hCG, followed by sequential incubation with TRITC- and FITC-conjugated secondary antibodies to rabbit and mouse immunoglobulins, respectively. The results were semiquantitatively analyzed by a pseudo-three-dimensional (3D) plotting of the intensities of the receptor and hormone-specific fluorescence in luteal cells by CLSM. The analysis suggested that the majority of the LH/CG receptors are located on the luteal cells before induction of the down-regulation and that their content seem to vary not only among cells but also on the surface of single cells, thus supporting the previous concept of the functional heterogeneity among the cells and their functional compartmentation. At 2 hr after injection of the down-regulating dose of hCG, the LH/CG receptor-specific and hCG-specific fluorescences clearly co-localized on the luteal cells. Both the LH/CG receptor- and hCG-specific fluorescences disappeared from the luteal cell surfaces in a parallel fashion within 36 hr without a detectable accumulation of either fluorescence deep in the cell interior. These results suggest that the LH/CG receptor and bound hCG do not differ in their manner of in vivo processing in luteal cells. Therefore, the disappearance of the receptor and bound hormone occurs in a parallel fashion and without detectable internalization.
PubMed ID
8189034 View in PubMed
Less detail

Production and characterization of polyclonal antiserum to rat luteinizing hormone/chorionic gonadotropin receptor and its immunohistochemical application for studying receptor location and down-regulation.

https://arctichealth.org/en/permalink/ahliterature65176
Source
Endocrinology. 1990 Aug;127(2):513-22
Publication Type
Article
Date
Aug-1990
Author
J T Lakkakorpi
K P Keinänen
H J Rajaniemi
Author Affiliation
Biocenter, University of Oulu, Finland.
Source
Endocrinology. 1990 Aug;127(2):513-22
Date
Aug-1990
Language
English
Publication Type
Article
Keywords
Animals
Antigen-Antibody Complex
Chorionic Gonadotropin - metabolism
Corpus Luteum - cytology - metabolism
Down-Regulation
Electrophoresis, Polyacrylamide Gel
Female
Histocytochemistry
Immune Sera
Kinetics
Molecular Weight
Ovary - metabolism
Pseudopregnancy
Rabbits - immunology
Rats
Rats, Inbred Strains
Receptors, LH - analysis - isolation & purification - metabolism
Research Support, Non-U.S. Gov't
Abstract
A polyclonal antiserum against the affinity-purified nondenatured rat 90K LH/CG receptor polypeptide was raised in rabbits, characterized, and used to study the location of the LH/CG receptor in pseudopregnant rat luteal cells and the fate of the receptor-hCG complex together with the specific anti-hCG serum during hCG-induced down-regulation by immunochemical techniques. Even at a 1:3000 dilution, the antiserum recognized a single 90K polypeptide on Western blots of both the affinity-purified receptor and the initial detergent extract of the pseudopregnant rat ovarian membranes. It recognized sodium dodecyl sulfate-denatured and reduced, sodium dodecyl sulfate-denatured, and native forms of the receptor on dot blots; the immunoreaction was the most intense with the native receptor. The antiserum also contained antibodies that recognized the hormone-binding site, or a region near to it, and the occupied receptor. The majority of the LH/CG receptors were located on the luteal cells in pseudopregnant rat ovaries before the induction of down-regulation. The receptor content seemed to vary among the luteal cells, however, and on single cells, suggesting both functional heterogeneity and a functional polarization of the luteal cells. Upon induction of down-regulation with hCG both the receptors and the bound hormone disappeared from the luteal cell surfaces at a very slow rate, without any simultaneous appearance of receptor- or hCG-specific immunostaining in the luteal cell interior. No accumulation of receptor degradation products capable of [125I]iodo-hCG or antibody binding could be detected on Western blots of the tissue. The polyclonal LH/CG receptor antiserum described here is useful for studying the structure and function of this receptor, particularly for immunohistochemical investigations into receptor location and regulation.
PubMed ID
2373047 View in PubMed
Less detail

Solubilization of luteinizing hormone receptor from human corpora lutea in a stable form and identification of the hormone-binding unit by ligand blotting.

https://arctichealth.org/en/permalink/ahliterature12450
Source
J Clin Endocrinol Metab. 1988 Aug;67(2):228-33
Publication Type
Article
Date
Aug-1988
Author
K P Keinänen
H J Rajaniemi
Author Affiliation
Biocenter, University of Oulu, Finland.
Source
J Clin Endocrinol Metab. 1988 Aug;67(2):228-33
Date
Aug-1988
Language
English
Publication Type
Article
Keywords
Binding Sites - drug effects
Corpus Luteum - analysis
Detergents - pharmacology
Electrophoresis, Polyacrylamide Gel
Female
Glycerol - pharmacology
Humans
Ligands
Receptors, LH - isolation & purification
Research Support, Non-U.S. Gov't
Solubility
Abstract
LH receptors were solubilized from human corpora lutea in phosphate-buffered saline containing 1% Triton X-100, 20% glycerol, and protease inhibitors. The presence of 20% (vol/vol) glycerol was necessary for quantitative preservation of [125I]hCG-binding activity in detergent solution. The solubilized receptors were stable for several weeks at -20 C and at -80 C and for at least 18 h at 4 C. Binding of [125I]hCG to the soluble LH receptors was time and temperature dependent and varied linearly with the amount of soluble protein. Equilibrium binding studies revealed a single class of high affinity [125I]hCG-binding sites with an equilibrium dissociation constant (Kd) of 4.3 x 10(-10) mol/L (at 20 C). The molecular size of the human LH receptors was analyzed by ligand blotting. Solubilized receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and transferred to nitrocellulose. Incubation of the protein blot with [125I]hCG to the 85/90K mol wt species was inhibited by unlabeled hCG. These results indicate that LH receptors can be solubilized in nonionic detergent while maintaining their hormone-binding activity and demonstrate that the receptors contain 85/90K hormone-binding species.
PubMed ID
3392160 View in PubMed
Less detail

Two polyclonal antisera to rat luteal LH/CG receptor with different ligand binding inhibition and immunohistochemical receptor detection capabilities.

https://arctichealth.org/en/permalink/ahliterature57762
Source
Acta Endocrinol (Copenh). 1991 Sep;125(3):305-12
Publication Type
Article
Date
Sep-1991
Author
J T Lakkakorpi
K P Keinänen
H J Rajaniemi
Author Affiliation
Biocenter, University of Oulu, Finland.
Source
Acta Endocrinol (Copenh). 1991 Sep;125(3):305-12
Date
Sep-1991
Language
English
Publication Type
Article
Keywords
Animals
Blotting, Western
Chromatography, Affinity
Electrophoresis, Polyacrylamide Gel
Female
Immune Sera - immunology
Immunohistochemistry
Rats
Rats, Inbred Strains
Receptors, Gonadotropin - immunology - metabolism
Receptors, LH - immunology - metabolism
Research Support, Non-U.S. Gov't
Abstract
Polyclonal antisera to a SDS-denatured and partially renatured rat luteal 90 K LH/CG receptor were raised in rabbits, characterized, and their applicability for immunohistochemical location of the receptor examined. The LH/CG receptor was purified by hCG-affinity chromatography and subjected either to a preparative SDS-PAGE or Western blotting. Gel slices containing the SDS-denatured or nitrocellulose strips containing the renatured 90 K LH/CG receptor were used for immunization. The antisera, termed ARS-2 and ARS-3, respectively, possessed similar antibody titres. Both antisera were able to recognize the native, SDS-denatured, and SDS-denatured and reduced forms of the LH/CG receptor on dot blots, but only ARS-3 contained antibodies to the hormone binding site or a region near to it, as it was able to inhibit the hCG binding to the membrane-bound LH/CG receptor in a dilution-dependent manner. Both antisera recognized the receptor-hCG complex, but ARS-2 stained the complex with about 50% less intensity than the free receptor. ARS-3 located the LH/CG receptor distinctly on the luteal cell surfaces in immunohistochemical staining with peroxidase antiperoxidase complex method, but ARS-2, although it possessed similar antibody titre, revealed negligible staining. Thus, the antisera readily recognize the native receptor, but differ in their capability for inhibiting hormone binding. Only ARS-3, produced against the renatured receptor, contains sufficient amounts of antibodies capable of recognizing free and occupied receptors in immunohistochemistry.
PubMed ID
1950343 View in PubMed
Less detail

7 records – page 1 of 1.