Comparison of the cellular composition and steroidogenic properties of preparations of interstitial cells isolated from immature and mature rat testis

1987 ◽  
Vol 112 (3) ◽  
pp. 361-NP ◽  
Author(s):  
A. P. N. Themmen ◽  
R. Molenaar ◽  
W. J. Visser ◽  
J. F. Jongkind ◽  
F. F. G. Rommerts ◽  
...  
Keyword(s):  
Leydig Cells ◽  
Interstitial Cells ◽  
Cellular Composition ◽  
Percoll Gradient ◽  
Dibutyryl Cyclic Amp ◽  
Steroid Production ◽  
Immature Rat ◽  
Collagenase Treatment ◽  
Ficoll Gradient ◽  
Immature Rats

ABSTRACT The morphological and steroidogenic properties of preparations of interstitial cells isolated by collagenase treatment from testes of immature and mature rats have been compared. After additional purification on a Ficoll gradient, 80% of cells from mature rat testes were found to be Leydig cells; 20% were macrophages. Forty to sixty per cent of collagenase-dispersed cells isolated from immature rats were Leydig cells, 37% were mesenchymal cells and there were no macrophages. A preparation in which 90% cells were Leydig cells could be obtained from immature testes after further purification by centrifugation through a Percoll gradient. The distribution of steroidogenic enzymes through the gradient corresponded to the distribution of LH-dependent steroid production. The results indicate that the steroidogenic activity per Leydig cell from mature rats is fourfold greater than the activity in immature rat Leydig cells in control incubations or after stimulation with LH, dibutyryl cyclic AMP or in the presence of 22R-hydroxycholesterol. J. Endocr. (1987) 112, 361–366

FORMATION OF 5α-REDUCED PRODUCTS FROM TESTOSTERONE IN VITRO BY GERM CELLS FROM IMMATURE RATS

1972 ◽  
Vol 71 (2) ◽  
pp. 393-408 ◽  
Author(s):  
Morio Yamada ◽  
Shunji Yasue ◽  
Keishi Matsumoto
Keyword(s):  
Germ Cells ◽  
Ventral Prostate ◽  
Seminiferous Tubules ◽  
Immature Rat ◽  
Collagenase Treatment ◽  
Incubation Study ◽  
Reduced Products ◽  
Immature Rats ◽  
Tissue Homogenates

ABSTRACT In the incubation study of [3H] testosterone with tissue homogenates in the presence of NADPH, it was shown that the rate of production of the sum of all 5α-reduced products by 30-day-old rat testes (50 to 100 nmoles/g tissue or 100 mg protein/h) was of the order of twenty times that by the ventral prostate (2 to 4 nmoles/g tissue/h). The activities of the C19-steroid Δ4-5α-reductase of the seminiferous tubules, germ cells and tubular non-germ cells isolated from immature rats (7 to 10, 11 to 13 and 5 to 7 nmoles/100 mg protein/h, respectively) were of similar order to those of the prostate, while much less activity was found in the spleen and muscle. It was also noted that the activity of the 5α-androstane-3α-, and -3β-hydroxysteroid dehydrogenase seemed to be higher in each tissue of immature rat testes than in the prostate tissue. The seminiferous tubules were isolated by collagenase treatment and germ cells were separated from non-germ cells by tubular cell culture at 34°C for 2 days. Thus, germ cells of immature rat testes seem to have the characteristics of androgen responsive tissue.

Inhibitory effects of alkane sulphonates on the function of immature rat Leydig, Sertoli and peritubular cells cultured in vitro

1989 ◽  
Vol 2 (2) ◽  
pp. 145-155 ◽  
Author(s):  
G. Verhoeven ◽  
J. Cailleau ◽  
I. D. Morris
Keyword(s):  
Sertoli Cells ◽  
Leydig Cells ◽  
Interstitial Cells ◽  
Aromatase Activity ◽  
Paracrine Factor ◽  
Adult Rats ◽  
Paracrine Factors ◽  
Toxic Activity ◽  
Immature Rat ◽  
Peritubular Cells

ABSTRACT Ethane 1,2-dimethane sulphonate (EDS) selectively destroys Leydig cells in the interstitium of the testis of adult rats. The toxic activity of this compound is much less obvious in the immature rat testis. We examined the effects of EDS, its monomethyl derivative and busulphan on cultured interstitial cells, percoll-purified Leydig cells, Sertoli cells and peritubular cells derived from immature rats. The studies with interstitial cells and Leydig cells showed that EDS (40–160 μg/ml) blocked the conversion of C21 and androgen precursors into testosterone and androstenedione. Higher concentrations of this compound also inhibited the production of C21 steroids and the LH-induced production of cyclic AMP (cAMP). The observed effects required a latent period of at least 8 h and were slowly reversible. Isolated cells were more sensitive to EDS than monolayer cultures. Reaggregation cultures were even less sensitive. EDS was markedly more effective on immature Leydig cells than its monomethyl derivative and busulphan. In cultured Sertoli cells FSH-inducible aromatase activity, cAMP production, androgen-binding protein (ABP) production and the secretion of a paracrine factor with Leydig cell-stimulatory activity were markedly reduced by busulphan. In these cells, busulphan was clearly more active than EDS and its monomethyl derivative. The production of paracrine factors which increase ABP production and decrease FSH-inducible aromatase activity in Sertoli cells was studied as a parameter of the effects of alkane sulphonates on peritubular cells. Only busulphan markedly decreased the production of these paracrine factors. It is concluded that EDS displays a selective toxicity to Leydig cells derived from immature animals and that, apart from its effects on germ cells, busulphan may also directly impair the function of Sertoli cells and peritubular cells.

scholarly journals Inhibition of steroid production in Leydig cells by non-steroidal anti-inflammatory and related compounds: evidence for the involvement of lipoxygenase products in steroidogenesis

1984 ◽  
Vol 219 (2) ◽  
pp. 529-537 ◽  
Author(s):  
C J Dix ◽  
A D Habberfield ◽  
M H F Sullivan ◽  
B A Cooke
Keyword(s):  
Arachidonic Acid ◽  
Cyclic Amp ◽  
Leydig Cells ◽  
Acid Metabolism ◽  
Arachidonic Acid Metabolism ◽  
Eicosatetraenoic Acid ◽  
Dibutyryl Cyclic Amp ◽  
Steroid Production ◽  
Oxygenase Activity ◽  
Dose Dependent

The effect of inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism on steroidogenesis in rat testis Leydig cells and rat tumour Leydig cells has been investigated. In the presence of nordihydroguaiaretic acid [NDGA; 4,4′-(2,3- dimethylbutan −1,4- diyl)bis[1,2- benzendiol]], 5,8,11,14-eicosatetraynoic acid (ETYA), BW 755C [3-amino-1-[3-(trifluoromethyl)phenyl]-2-pyrazoline hydrochloride] and benoxaprofen [Opren; 2-(2-p-chlorophenyl- benzoxazol −5-yl)propionic acid)] (which inhibit lipoxygenase activity), but not indomethacin and aspirin (which inhibit cyclo-oxygenase activity), a dose-related inhibition of lutropin (LH)-stimulated testosterone and pregnenolone production was obtained (ID50 values of 2.5, 30, 25 and 30 microM for NDGA, ETYA, BW 755C and benoxaprofen were obtained, respectively). BW 755C and benoxaprofen had no significant effect on LH-stimulated cyclic AMP production except at the highest concentrations examined (330 and 380 microM, respectively), whereas NDGA and ETYA inhibited LH-stimulated cyclic AMP production in a dose-dependent manner (ID50 7.0 and 22 microM respectively). However, NDGA and ETYA also caused a dose-dependent inhibition of dibutyryl cyclic AMP-stimulated testosterone and pregnenolone production. The metabolism of exogenous (22R)-hydroxycholesterol or pregnenolone to testosterone by Leydig cells was not inhibited by either NDGA, ETYA or indomethacin. At low concentrations of NDGA and ETYA a significant increase in the conversion of both pregnenolone and (22R)-hydroxycholesterol to testosterone was obtained. Studies in which the metabolism of [14C]arachidonic acid by purified rat tumour Leydig cells was investigated indicate that products are formed by tumour Leydig cells that have similar mobilities in a thin layer chromatography system to 5-L-hydroxy-6,8,11,14-eicosatetraenoic acid, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and leukotriene B4. The formation of these products was inhibited to varying degrees by NDGA, BW 755C and benoxaprofen but not by aspirin and indomethacin. These studies demonstrate for the first time that inhibition of lipoxygenase activity but not cyclo-oxygenase activity causes an inhibition of LH- and dibutyryl cyclic AMP-stimulated steroid production and suggest a stimulatory role for products of the lipoxygenase pathway of arachidonic acid metabolism in steroidogenesis. The site of this stimulation is apparently distal to the production of cyclic AMP and before the side chain cleavage of cholesterol.

In-vitro DNA synthesis in Leydig and other interstitial cells of the rat testis

1992 ◽  
Vol 134 (2) ◽  
pp. 247-255 ◽  
Author(s):  
A. Moore ◽  
K. Findlay ◽  
I. D. Morris
Keyword(s):  
Dna Synthesis ◽  
Germ Cell ◽  
Leydig Cell ◽  
Small Proportion ◽  
Leydig Cells ◽  
Interstitial Cell ◽  
Interstitial Cells ◽  
Immature Rat ◽  
In Cells

ABSTRACT Replicative DNA synthesis (125I-labelled iododeoxy-uridine incorporation) was measured in interstitial cells prepared from rat testes and separated by Percoll density gradient centrifugation. Leydig cells were identified by 125I-labelled human chorionic gonadotrophin (hCG) binding and 3β-hydroxysteroid dehydrogenase histochemistry. Continuous density gradients indicated that interstitial cell DNA synthesis was not associated with Leydig cells, and was greater in cells from the immature than from the mature rat testis. Fractionation of cells by discontinuous density gradients into Leydig cell-rich and -depleted pools did not result in a similar enrichment of DNA synthesis. Treatment of the adult rat with hCG increased DNA synthesis into both fractions but oestrogen had no effect. DNA synthesis was greater in cells from the immature rat but, in contrast to the adult, in-vivo hCG treatment had no effect, whilst oestrogen decreased synthesis. To characterize the cells synthesizing DNA further, interstitial cells were prepared from testes in which the Leydig cells were depleted by in-vivo treatment with ethane dimethanesulphonate (EDS) or depleted in their germ cells by treatment in utero with busulphan. EDS treatment had no effect on DNA synthesis by the interstitial cells in spite of the 125I-labelled hCG binding being markedly reduced. Similarly, busulphan treatment was also without effects upon DNA synthesis. Fluorescence-activated cell cycle analysis of cells from both fractions from germ cell-depleted testes indicated that only a small proportion (3%) of the interstitial cells were actively dividing and this was almost doubled in cells from the germ cell-depleted immature rat testes. The experiments showed that the majority of cells in the interstitium of the rat testes do not synthesize DNA and are not undergoing cell division. The small proportion of cells that are dividing are probably not Leydig cells. The experiments have identified a dividing interstitial cell population and, in consideration of the changes in the immature and mature rat as well as the effects of hormone treatment, they may be regarded as putative Leydig cell precursors. Journal of Endocrinology (1992) 134, 247–255

Non-specific esterase: a specific and useful marker enzyme for Leydig cells from mature rats

1986 ◽  
Vol 108 (3) ◽  
pp. 329-NP ◽  
Author(s):  
R. Molenaar ◽  
F. F. G. Rommerts ◽  
H. J. van der Molen
Keyword(s):  
Leydig Cell ◽  
Esterase Activity ◽  
Leydig Cells ◽  
Room Temperature ◽  
Peritoneal Macrophages ◽  
Human Chorionic Gonadotrophin ◽  
Marker Enzyme ◽  
Steroid Production ◽  
Fluorescent Beads ◽  
Immature Rats

ABSTRACT The presence of non-specific esterase activity is correlated with different Leydig cell characteristics: 3β-hydroxysteroid dehydrogenase (3β-HSD), human chorionic gonadotrophin binding and LH-stimulated steroid production. This indicates that esterase can be used as a marker enzyme for Leydig cells. Esterase, however, has also been used as a marker enzyme for macrophages. We have compared, using biochemical and histochemical techniques, the esterase activity of Leydig cell preparations from mature and immature rats and of preparations enriched in testicular or peritoneal macrophages. Leydig cells were identified by staining for 3β-HSD, and macrophages by phagocytosis of fluorescent beads. Leydig cell preparations from mature rats showed an approximately 400-fold higher esterase activity than peritoneal macrophage preparations and an approximately 50-fold higher activity than testicular macrophage preparations. Leydig cell preparations from mature rats showed a 60-fold higher esterase activity than Leydig cell preparations from immature rats. Differences in esterase activity were also demonstrated histochemically. Leydig cells from mature rats showed positive esterase staining after 30 s at room temperature. Testicular macrophages showed esterase activity after staining for 3 min. Only approximately 25% of the 3β-HSD-positive cells from immature rats showed esterase activity after staining for 6 min. Esterase is therefore a useful marker enzyme for Leydig cells from mature rats and can be of help in studies concerning the development of these cells. J. Endocr. (1986) 108, 329–334

EFFECT OF SERUM GONADOTROPHIN ON DEHYDROGENASES OF THE CITRIC ACID CYCLE IN THE OVARY OF THE RAT

1963 ◽  
Vol 42 (4) ◽  
pp. 480-484 ◽  
Author(s):  
B. Eckstein ◽  
R. Landsberg
Keyword(s):  
Citric Acid ◽  
Citric Acid Cycle ◽  
Age Groups ◽  
Succinic Dehydrogenase ◽  
Immature Rat ◽  
Acid Cycle ◽  
Immature Rats ◽  
Rat Ovary

ABSTRACT The succinic, malic and isocitric dehydrogenases in the ovary of immature and mature, normal and serum gonadotrophin injected rats were examined. The Qo2 of these enzymes were markedly enhanced in the gonadotrophin injected rats of both age groups, except in the case of succinic dehydrogenase in the ovary of the immature rats, where a slight non-significant decrease was noted. It is concluded that in the mature rat ovary, gonadotrophin administration stimulates the activity of all the examined dehydrogenases of the citric acid cycle, whereas in the immature rat ovary, at least the isocitric- and malic dehydrogenases are thus stimulated.

scholarly journals Inhibition of Cyclooxygenase-2 Activity Enhances Steroidogenesis and Steroidogenic Acute Regulatory Gene Expression in MA-10 Mouse Leydig Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (8) ◽  
pp. 3368-3375 ◽  
Author(s):  
XingJia Wang ◽  
Matthew T. Dyson ◽  
Youngah Jo ◽  
Douglas M. Stocco
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Leydig Cells ◽  
Promoter Activity ◽  
Regulatory Gene ◽  
Steroid Production ◽  
Star Protein ◽  
Cox Inhibitor ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

Abstract To study the mechanism for the regulatory effect of arachidonic acid (AA) on steroidogenesis, the role of cyclooxygenase (COX) in steroid production and steroidogenic acute regulatory (StAR) gene expression was investigated. Although stimulation with 0.05 mm dibutyryl cAMP (Bt2cAMP) did not increase StAR protein or progesterone in MA-10 mouse Leydig cells, the addition of 1 μm of the COX inhibitor indomethacin increased StAR protein expression and progesterone production by 5.7-fold and 34.3-fold, respectively. In the presence of indomethacin, the level of Bt2cAMP required for maximal steroidogenesis was reduced from 1.0 mm to 0.25 mm. Similar results were obtained in studies on StAR promoter activity and in Northern blot analyses of StAR mRNA expression, suggesting that inhibition of COX activity enhanced StAR gene transcription. COX2 (an inducible isoform of COX) was constitutively detected in MA-10 cells. Although SC560, a selective COX1 inhibitor, did not affect steroidogenesis, the COX2 inhibitor NS398 significantly enhanced Bt2cAMP-stimulated StAR protein expression and steroid production. Overexpression of the COX2 gene in COS-1 cells significantly inhibited StAR promoter activity. The results of the present study suggest that inhibition of COX2 activity increases the sensitivity of steroidogenesis to cAMP stimulation in MA-10 Leydig cells.

EFFECT OF OESTRADIOL ON THE INDUCTION OF PEROXIDASE IN VARIOUS TISSUES OF THE IMMATURE RAT

1978 ◽  
Vol 76 (2) ◽  
pp. 373-374 ◽  
Author(s):  
P. H. JELLINCK ◽  
ANNE-MARIE NEWCOMBE
Keyword(s):  
Rat Uterus ◽  
Immature Rat ◽  
Immature Rats ◽  
Queen's University

Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada (Received 14 September 1977) It has been shown previously (Lyttle & Jellinck, 1972) that an enzyme that catalyses the binding of oestradiol-17β to protein in the presence of H2O2 is absent from the uteri of immature rats, but can be induced by physiological doses of oestradiol-17β or gonadotrophin. The induction of peroxidase in the rat uterus under various endocrine conditions has also been investigated (Jellinck & Newcombe, 1977a) and good correlation has been obtained between the ability of compounds to induce the enzyme and their oestrogenic activity (Jellinck & Newcombe, 1977b). In this paper we report on the activity of peroxidase in tissues other than the uterus and the effect of ovariectomy, hypophysectomy and treatment with oestradiol-17β on the levels of this enzyme. Peroxidase was prepared from tissues of immature (26–29 days old) Holtzman rats weighing 70–90 g as described

scholarly journals Purification, Morphology, and Progesterone Production and Content of Three Cell Types Isolated from the Corpus Luteum of the Sheep

1982 ◽  
Vol 35 (4) ◽  
pp. 441 ◽  
Author(s):  
RJ Rodgers ◽  
JD O'Shea
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Corpus Luteum ◽  
Cell Types ◽  
Cellular Composition ◽  
Isolation And Purification ◽  
Luteal Cells ◽  
Ficoll Gradient ◽  
Luteal Tissue ◽  
Cell Fractions

A method is presented for the isolation and purification of three cell types, endothelial cells, small luteal cells and large luteal cells, from the ovine corpus luteum. The method involves enzymatic dispersion of luteal tissue followed by centrifugation of separated cells on a Ficoll gradient. The three purified cell types and others, particularly fibrocytes and smooth muscle cells, that were removed during purification, were identified by their morphology. The cell yield, the cellular composition and cellular progesterone content of each fraction from the Ficoll gradient were measured. The endothelial cell fractions were relatively free of contamination by other cell types and had negligible progesterone. Fractions of small luteal cells and those of large luteal cells contained endothelial cells but were relatively free of other cell types. Large luteal cells contained significantly more progesterone, produced more progesterone when incubated in culture, but were less responsive to luteinizing hormone than small luteal cells.

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