Isolation of Rat Leydig Cells by Density Gradient Centrifugation

Gale, Jean S.; Wakefield, J. St. J.; Ford, Henry C.

doi:10.1677/joe.0.0920293

Cited by 32 publications

(15 citation statements)

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“…Previous studies have described two peaks of hCG binding in collagenase-dispersed interstitial cells from adult rat testes separated by Percoll density gradients (2,4,11,12,41). The density range covered by fractions II and III in the present study includes the more dense of these two peaks, which has previously been shown to consist predominantly of intact Leydig cells (4,11,12,(41)(42)(43). In a previous study using similar mild dissociation conditions, a second peak of hCG binding was observed in fraction IV, which contained intact Leydig cells (12).…”

Section: Discussionsupporting

confidence: 58%

The Heterogeneity of Isolated Adult Rat Leydig Cells Separated on Percoll Density Gradients: An Immunological, Cytochemical, and Functional Analysis

Hedger

Eddy

1987

Endocrinology

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Intertubular cells, isolated from adult rat testes by collagenase dispersal under conditions designed to minimize cell damage, were fractionated on Percoll density gradients. In the gradient fractions, there was a close cellular correlation between the presence of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), determined by cytochemistry, and other Leydig cell markers (nonspecific esterase, autofluorescence, and an antigen defined by monoclonal antibody LC-1C6). As the reagents for 3 beta HSD cytochemistry are excluded by intact membranes, Leydig cells with damaged plasma membranes were identified by 3 beta HSD reactivity in suspended cell preparations, and the total number of 3 beta HSD-positive (3 beta HSD+) cells in the same preparations was determined after lysis of the cell membrane. Whole cells were differentiated from cytoplasmic fragments by counterstaining with the nuclear dye propidium iodide, and the number of intact Leydig cells in each preparation was determined subsequently by subtracting the number of damaged nucleated 3 beta HSD+ cells from the total number of nucleated 3 beta HSD+ cells. The majority of intact isolated Leydig cells were found in gradient fractions of 1.054-1.096 g/ml density. Acute (3-H) basal and hCG-stimulated testosterone production per intact Leydig cell were dependent upon the concentration of Leydig cells per assay well, indicating that there is cooperativity among Leydig cells in vitro. There was no difference in steroidogenic function among intact Leydig cells from different fractions of the above density gradient range at assay concentrations greater than 10,000 Leydig cells/well. At lower cell concentrations, Leydig cells from gradient fractions of lower density (1.054-1.064 g/ml) produced slightly less testosterone in response to hCG stimulation than Leydig cells from more dense fractions (1.070-1.096 g/ml). Prolonging the exposure of isolated cells to the dispersal conditions caused declines in the apparent buoyant density and basal testosterone and hCG-stimulated cAMP and testosterone production of all Leydig cells, without detectable changes in cell integrity. The data indicate that both the absolute steroidogenic function and the functional heterogeneity of isolated intact Leydig cells are, at least in part, dependent upon the procedures used for their isolation.

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Section: Discussionsupporting

confidence: 58%

The Heterogeneity of Isolated Adult Rat Leydig Cells Separated on Percoll Density Gradients: An Immunological, Cytochemical, and Functional Analysis

Hedger

Eddy

1987

Endocrinology

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“…After washing twice, cells were resuspended in Medium-199 and 5 ml of interstitial cell suspension (20-25 109 cells/1) was layered on the top of each vial containing a previously prepared discontin¬ uous density gradient of Percoli (0-60% v/v), as described by Gale et al. (20), and then centrifuged at 800 g for 20 min at room temperature. The fractions were collected from the bottom of the tubes with a peristaltic pump and then washed twice with isotonic Medium-199 (1:1, v/v) to remove any residual Percoli.…”

Section: Methodsmentioning

confidence: 99%

Erythropoietin and testicular steroidogenesis: the role of second messengers

Mioni¹,

Bordon²,

Gottardello³

et al. 1995

European Journal of Endocrinology

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It has been demonstrated that erythropoietin (EPO) influences rat and human Leydig cell steroidogenesis, stimulating testosterone production through a direct and specific receptor-mediated mechanism. The aim of this study was to investigate the mechanism by which recombinant human erythropoietin (rHuEPO) exerts its stimulatory effect on rat Leydig cells. Recombinant human EPO did not induce, at any dose tested (10(-10) to 10(-13) mol/l), an increase in either cAMP or cGMP, suggesting that in Leydig cells the effect of rHuEPO does not involve the adenylate or guanylate-cyclase systems. The role of transmembrane calcium flux in rHuEPO-stimulated steroidogenesis was studied by evaluating the effect of calcium channel blocker, verapamil, and by the 45Ca2+ uptake method. Verapamil did not influence rHuEPO-induced testosterone secretion and rHuEPO did not modify calcium recycling, indicating that calcium transmembrane flux is not involved in the rHuEPO effect. The protein kinase C inhibitor staurosporine (10, 30, 100 and 300 nmol/l) inhibited rHuEPO-stimulated testicular steroidogenesis in a dose-dependent manner. This indirect evidence suggests that the stimulatory effect of rHuEPO on rat Leydig cells may involve protein kinase C activation.

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“…Of the criteria available to identify a Leydig cell, it is only their characteristic morphology that is universally accepted as corresponding to the classical histological definition of these cells as described in 1850 by Leydig (22). Yet, in most studies, the histochemical demonstration of 3/3-hydroxysteroid dehydrogenase (3/5-HSD) (1, 3-6, 10-17, 19) or phenyl esterase (1,5), the presence of LH/hCG receptors (7,16,17) and, less frequently, the observation of yellow haloes seen with phase contrast microscopy (6)(7)(8) have each been considered acceptable markers for identification of isolated Leydig cells. Although limited morphological descriptions of en-PERCOLL GRADIENT SEPARATION OF MOUSE LEYDIG CELLS 1031 riched Leydig cell suspensions are available (1,2,4,6,14,15,17), details of the quantitative methods used to determine Leydig cell yield have not been given (2,6,14,15).…”

mentioning

confidence: 99%

Morphological and Functional Characterization of Interstitial Cells from Mouse Testes Fractionated on Percoll Density Gradients*

1985

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Cell fractions were obtained by separation on Percoll density gradients after dissociation of mature mouse testes by mechanical or collagenase dispersion, and the ultrastructure, hCG receptor properties, and hCG-stimulated testosterone (T) production of these fractions were compared. Gradients were fractionated according to specific gravity, and all cell types were quantitated using morphometric techniques. Three peaks of specific [125I]iodo-hCG binding corresponding to densities of 1.0667 g/cm3 (fractions 2-3), 1.045 g/cm3 (fractions 6-7), and 1.0365-1.0215 g/cm3 (fraction 9) were obtained after collagenase dispersion, but the second peak of binding (fractions 6-7) was not observed after mechanical dispersion. Morphometric studies were performed by light microscopy on the cells present in the fractions corresponding to the first and second hCG binding peaks obtained by either mechanical or collagenase dispersion; the third representing germ cells and membranous debris was not studied further. Regardless of the method of preparation, morphologically intact Leydig cells represented 60-80% and 7-10% of the cells in fractions 2-3 and 6-7 that were associated with the first and second peaks of hCG binding Leydig cells obtained from fractions 6-7 contained greater numbers of lipid inclusions than Leydig cells from fractions 2-3. Mechanically dispersed Leydig cells exhibited similar numbers of hCG receptors in the dense and light Leydig cells, but hCG stimulated T production per Leydig cell was significantly greater in the dense Leydig cells containing few lipid inclusions. T production by dense and light collagenase-dispersed Leydig cells was not significantly different. The second hCG binding peak in collagenase-dispersed cell fractions 6-7 was associated with an increase in the number of an indeterminate connective cell type released from the testes by collagenase treatment, whose ultrastructure and limited hCG-binding capacity suggested that they may represent Leydig cell precursors. It is concluded that the identification and quantitation of different cell types in isolated testicular cell fractions is, therefore, of fundamental importance in the interpretation of receptor and secretory capacities of enriched preparations of Leydig cells.

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Isolation of Rat Leydig Cells by Density Gradient Centrifugation

Cited by 32 publications

References 0 publications

The Heterogeneity of Isolated Adult Rat Leydig Cells Separated on Percoll Density Gradients: An Immunological, Cytochemical, and Functional Analysis

The Heterogeneity of Isolated Adult Rat Leydig Cells Separated on Percoll Density Gradients: An Immunological, Cytochemical, and Functional Analysis

Erythropoietin and testicular steroidogenesis: the role of second messengers

Morphological and Functional Characterization of Interstitial Cells from Mouse Testes Fractionated on Percoll Density Gradients*

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