Are ovine Leydig cells able to aromatize androgens?

1997 ◽  
Vol 9 (2) ◽  
pp. 193 ◽  
Author(s):  
Barbara Biliska ◽  
Marcin Le´sniak ◽  
Barbara Schmalz
Keyword(s):  
Aromatase Inhibitor ◽  
Incubation Period ◽  
Immunohistochemical Staining ◽  
Leydig Cells ◽  
Culture Medium ◽  
Aromatase Activity ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Testosterone Synthesis

The conversion of testosterone into oestradiol by ovine Leydig cells culturedin vitrowas studied using the non-steroidal aromatase inhibitor CGS 16949A. Additionally, aromatase activity was detected by immunohistochemical staining of cultured Leydig cells or cryosections. The cells were obtained from testes of Polish Mountain rams 5–6 months old (immature) or 12–15 months old (mature). Leydig cells were cultured alone (controls) or incubated for 6 h in the presence of testosterone. Aromatase inhibitor was then added to the cultures which were incubated for a further 18 h. After a 24-h incubation period, testosterone and oestradiol secretion were determined by testing the culture medium using radioimmunological methods. The addition of testosterone to the culture medium enhanced oestradiol synthesis, suggesting that exogenous testosterone could also be aromatized to oestradiol by ovine Leydig cells in vitro. In the presence of CGS 16949A, the conversion of testosterone to oestradiol was significantly suppressed in a dose-dependent manner. All Leydig cells obtained from testes of mature rams and stained immunohistochemically were positive for aromatase, whereas Leydig cells from immature males were negative. The localization of immunoreactive aromatase appeared to be dependent on the age of the donor ram. It is suggested therefore, that mature Leydig cells in the ram are not only the site for testosterone synthesis, they are also capable of converting androgens into oestrogens.

Regulation of oxytocin production by purified adult rat Leydig cells in vitro: effects of LH, testosterone and lipoproteins

1994 ◽  
Vol 143 (2) ◽  
pp. 325-332 ◽  
Author(s):  
J Frayne ◽  
H D Nicholson
Keyword(s):  
Leydig Cells ◽  
Interstitial Fluid ◽  
Culture Medium ◽  
Culture Period ◽  
Dependent Manner ◽  
Paracrine Factor ◽  
Adult Rat ◽  
In Vitro Effects ◽  
Dose Dependent

Abstract The aim of the present study was to determine whether LH stimulates oxytocin production by adult rat Leydig cells directly or indirectly via testosterone. Purified adult rat Leydig cells were cultured in the presence or absence of 0·1 ng/ml LH or 1, 10 or 100 ng/ml testosterone for 22 h. Culture medium was collected at 2-hourly intervals and assayed for oxytocin and testosterone. In the presence of LH, Leydig cells produced significantly higher levels of both testosterone (basal production 1·4± 0·13 ng, LH-stimulated 4·1 ±0·13 ng/106 cells per 2 h) and oxytocin (basal production 8·3± 1·2 pg, LH-stimulated 20·2± 1·3 pg/106 cells per 2 h). Testosterone also stimulated oxytocin secretion. However, the increase was smaller compared with that seen with LH and was not found to be dose-dependent. Furthermore, testosterone production was only significantly increased by LH during the first 10 h of the 22-h culture period whereas LH stimulated oxytocin production throughout the whole culture period. To further determine the effect of LH on oxytocin production, cultures were performed in the presence of LH and/or 400 μm aminoglutethimide. In the presence of aminoglutethimide both the basal and LH-stimulated production of testosterone was significantly reduced. However, in the same cultures aminoglutethimide did not alter either the basal or LH-stimulated production of oxytocin. These data show that LH does not act via testosterone to stimulate oxytocin production and therefore acts directly or by some alternative indirect mechanism. In this study it was found that two other factors, cell density and lipoprotein, also influenced oxytocin production by isolated Leydig cells. Decreasing the density at which Leydig cells were cultured from 106 to 10 cells/well significantly increased both their basal and LH-stimulated production of oxytocin. Lipoproteins were also found to stimulate oxytocin production in a dose-dependent manner and to synergize with LH to further increase LH-stimulated oxytocin production. The results of this study show the production of oxytocin by Leydig cells to be regulated not only by the gonadotrophin LH but also by lipoproteins which are known to be present in interstitial fluid. These data add to the accumulating evidence that intratesticular oxytocin may be a paracrine factor. Journal of Endocrinology (1994) 143, 325–332

Papaya shoot tip associated endophytic bacteria isolated from in vitro cultures and host–endophyte interaction in vitro and in vivo

2007 ◽  
Vol 53 (3) ◽  
pp. 380-390 ◽  
Author(s):  
Pious Thomas ◽  
Sima Kumari ◽  
Ganiga K. Swarna ◽  
T.K.S. Gowda
Keyword(s):  
Culture Medium ◽  
Carica Papaya ◽  
In Vitro Cultures ◽  
Dependent Manner ◽  
16S Rdna Sequence ◽  
16S Rdna Sequence Analysis ◽  
Single Organism ◽  
Dose Dependent

Fourteen distinct bacterial clones were isolated from surface-sterilized shoot tips (~1 cm) of papaya (Carica papaya L. ‘Surya’) planted on Murashige and Skoog (MS)-based papaya culture medium (23/50 nos.) during the 2–4 week period following in vitro culturing. These isolates were ascribed to six Gram-negative genera, namely Pantoea ( P. ananatis ), Enterobacter ( E. cloacae ), Brevundimonas ( B. aurantiaca ), Sphingomonas , Methylobacterium ( M. rhodesianum ), and Agrobacterium ( A. tumefaciens ) or two Gram-positive genera, Microbacterium ( M. esteraromaticum ) and Bacillus ( B. benzoevorans ) based on 16S rDNA sequence analysis. Pantoea ananatis was the most frequently isolated organism (70% of the cultures) followed by B. benzoevorans (13%), while others were isolated from single stocks. Bacteria-harboring in vitro cultures often showed a single organism. Pantoea, Enterobacter, and Agrobacterium spp. grew actively on MS-based normal papaya medium, while Microbacterium, Brevundimonas, Bacillus, Sphingomonas, and Methylobacterium spp. failed to grow in the absence of host tissue. Supplying MS medium with tissue extract enhanced the growth of all the organisms in a dose-dependent manner, indicating reliance of the endophyte on its host. Inoculation of papaya seeds with the endophytes (20 h at OD550 = 0.5) led to delayed germination or slow seedling growth initially. However, the inhibition was overcome by 3 months and the seedlings inoculated with Pantoea, Microbacterium, or Sphingomonas spp. displayed significantly better root and shoot growths.

Inhibitory Effect of Tetrandrine on the Proliferation of Human Dermal Fibroblasts Derived from Hypertrophic Scars

2011 ◽  
Vol 268-270 ◽  
pp. 838-840
Author(s):  
De Wu Liu ◽  
Xiang Hu ◽  
De Ming Liu ◽  
Ping Zou
Keyword(s):  
Hypertrophic Scar ◽  
Culture Medium ◽  
Inhibitory Effect ◽  
Dermal Fibroblasts ◽  
Dependent Manner ◽  
Human Dermal Fibroblasts ◽  
Hypertrophic Scars ◽  
Result Show ◽  
Dose Dependent

Tetrandrine can inhibit the proliferation and collagen synthesis of fibroblasts in lung and liver tissue confirmed by a series of clinical research. In this chapter, we investigated the effect of Tetrandrine on the proliferation of human dermal fibroblasts derived from hypertrophic scars. The dermal fibroblasts were isolated from human hypertrophic scar tissues and cultured in vitro. Tetrandrine with different concentration were added to culture medium respectively. The proliferative activities were determined. The result show that when the concentration of added Tetrandrine increased from 5μg/ml to 80μg/ml, the proliferative activities of cultured dermal fibroblasts were decreased gradually in dose-dependent manner. It conclusions that Tetrandrine can obviously inhibit the proliferation of human dermal fibroblasts derived from hypertrophic scars.

Cultured juxtaglomerular cells: production and localization of renin

1987 ◽  
Vol 65 (7) ◽  
pp. 1409-1415 ◽  
Author(s):  
J. Lacasse ◽  
C. Kreft ◽  
J. Gutkowska ◽  
J. Genest ◽  
M. Cantin
Keyword(s):  
Culture Medium ◽  
Renin Release ◽  
Juxtaglomerular Cells ◽  
Dependent Manner ◽  
Time Intervals ◽  
Cortical Cells ◽  
Newborn Mice ◽  
In Vitro System ◽  
Dose Dependent

Trypsin- or collagenase-dispersed renal cortical cells from newborn mice were filtered and cultured. The cultures comprised 25% juxtaglomerular cells as identified by immunocytochemistry. Renin was measured by radioimmunoassay in the culture medium and in the cells at various time intervals. This in vitro system was responsive to isoproterenol, which stimulated renin release in a dose-dependent manner.

Immunization of male rabbits with sheep luteal receptor to LH results in production of antibodies exhibiting hormone-agonistic and -antagonistic activities

1996 ◽  
Vol 150 (3) ◽  
pp. 431-443 ◽  
Author(s):  
M Jeyakumar ◽  
N R Moudgal
Keyword(s):  
Leydig Cells ◽  
Serum Testosterone ◽  
Fold Increase ◽  
Dependent Manner ◽  
Primary Immunization ◽  
Testosterone Production ◽  
Dose Dependent ◽  
Receptor Antibodies

Abstract Antibodies to LH/chorionic gonadotrophin receptor (LH/CG-R; molecular weight 67 000), isolated in a homogenous state (established by SDS-PAGE and ligand blotting) from sheep luteal membrane using human CG (hCG)–Sepharose affinity chromatography, were raised in three adult male rabbits (R-I, R-II and R-III). Each of the rabbits received 20–30 μg of the purified receptor in Freund's complete adjuvant at a time. Primary immunization was followed by booster injection at intervals. Production of receptor antibodies was monitored by (1) determining the dilution of the serum (IgG fraction) that could specifically bind 50% of 125I-LH/CG-R added and (2) analysing sera for any change in testosterone levels. Following primary immunization and the first booster, all three rabbits exhibited a 2·5- to 6·0-fold increase in serum testosterone over basal levels and this effect was spread over a period of time (∼40 days) coinciding with the rise and fall of receptor antibodies. The maximal antibody titre (ED50) produced at this time ranged from 1:350 to 1:100 to below detectable limits for R-I, R-II and R-III respectively. Subsequent immunizations followed by the second booster resulted in a substantial increase in anti-body titre (ED50 of 1:5000) in R-I, but this was not accompanied by any change in serum testosterone over preimmune levels, suggesting that with the progress of immunization the character of the antibody produced had also changed. Two pools of antisera from R-I collected 10 days following the booster (at day 70 (bleed I) and day 290 (bleed II)) were used in further experiments. IgG isolated from bleed I but not from bleed II antiserum showed a dose-dependent stimulation of testosterone production by mouse Leydig cells in vitro, thus confirming the in vivo hormone-mimicking activity of antibodies generated during the early immunization phase. The IgG fractions from both bleeds were, however, capable of inhibiting (1) 125I-hCG binding to crude sheep luteal membrane (EC50 of 1:70 and 1:350 for bleed I and II antisera respectively) and (2) ovine LH-stimulated testosterone production by mouse Leydig cells in vitro, indicating the presence of antagonistic antibodies irrespective of the period of time during which the rabbits were immunized. The fact that bleed I-stimulated testosterone production could be inhibited in a dose-dependent manner by the addition of IgG from bleed II to the mouse Leydig cell in vitro assay system showed that the agonistic activity is intrinsic to the bleed I antibody. The receptor antibody (bleed II) was also capable of blocking LH action in vivo, as rabbits passively (for 24 h with LH/CG-R antiserum) as well as actively (for 430 days) immunized against LH/CG-R failed to respond to a bolus injection of LH (50 μg). At no time, however, was the serum testosterone reduced below the basal level. This study clearly shows that, unlike with LH antibody, attempts to achieve an LH deficiency effect in vivo by resorting to immunization with holo LH receptor is difficult, as receptor antibodies exhibit both hormone-mimicking (agonistic) as well as hormone-blocking (antagonistic) activities. Journal of Endocrinology (1996) 150, 431–443

Albumin inhibits adipogenesis and stimulates cytokine release from human adipocytes

2006 ◽  
Vol 291 (1) ◽  
pp. C27-C33 ◽  
Author(s):  
Janet B. Schlesinger ◽  
Vanessa van Harmelen ◽  
Catherine E. Alberti-Huber ◽  
Hans Hauner
Keyword(s):  
Culture Medium ◽  
Dependent Manner ◽  
Human Adipocytes ◽  
Tnf Α ◽  
Proliferation And Differentiation ◽  
Human Preadipocytes ◽  
Isolated Adipocytes ◽  
Adipose Differentiation ◽  
Dose Dependent

Bovine serum albumin (BSA) is commonly used in adipocyte experiments as a binding protein for fat-soluble substances. Therefore, it is of interest to investigate whether BSA per se is influencing the functioning of human adipocytes in vitro. In the present study, we investigated the potential of BSA to affect the proliferation and differentiation capacity of human preadipocytes. BSA was found to inhibit adipose differentiation in a dose-dependent manner (being significant at concentrations of 2.5 μM), whereas proliferation was not affected. We further investigated the effect of BSA on the secretory function of adipocytes focusing on the release of selected cytokines. Preadipocytes and freshly isolated adipocytes incubated with BSA secreted significantly higher amounts of IL-6, -8, and -10, and TNF-α compared with cells incubated without BSA. The effects on cytokine secretion seemed to reside at the level of gene expression because BSA increased TNF-α and IL-6 mRNA in a dose-dependent manner. The results of the present study indicate that the presence of BSA in the culture medium has considerable effects on adipocyte function in vitro. These effects should be carefully considered for in vitro studies of adipose tissue.

Thrombosomes Show Dose-Dependent Increase in Thrombus Formation in a Model of Deep Arterial Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2319-2319
Author(s):  
Nikhil Vilas Joshi ◽  
Jennifer Raftis ◽  
AJ Lucking ◽  
Narendra Tandon ◽  
M Fitzpatrick ◽  
...  
Keyword(s):  
Whole Blood ◽  
Immunohistochemical Staining ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Arterial Injury ◽  
Dependent Manner ◽  
Flow Conditions ◽  
Structural And Functional Properties ◽  
Dose Dependent

Abstract Abstract 2319 Introduction Thrombosomes are novel lyophilized human platelet derivatives that retain a number of key platelet structural and functional properties. Building on preliminary in vitro studies, we here sought to investigate whether Thrombosomes, suspended in platelet free plasma (PFP), would enhance and be incorporated in thrombus generated under flow conditions within a validated and well-characterised model of deep arterial injury. Methods PFP was obtained by centrifuging citrated whole blood from six healthy non-smoking volunteers and filtering with a 0.22μm filter. Thrombosomes were suspended in 60 mL of PFP to generate final concentrations of 20 and 200 × 106Thrombosomes /mL. Immediately prior to use, 1.2 mL of 1M CaCl2 was added to permit fibrin generation. Thrombus formation was assessed using the Badimon Chamber at low (212 s−1) and high (1690 s−1) shear rates with porcine aortic tunica media as the thrombogenic substrate. Total thrombus area and platelet-rich area were measured histomorphometrically using conventional and immunohistochemical staining respectively. Fluorescent labeled Thrombosomes were added to the extracorporeal circuit in the Badimon chamber to study the ex vivo thrombus generation in the whole blood. Electron microscopy of Thrombosomes and platelets was undertaken. Results Thrombosomes contributed towards thrombus formation in whole human blood as evidenced by incorporation of fluorescent-labeled Thrombosomes into the thrombus. Immunohistochemical staining of the glycoprotein IIb/IIIa receptor confirmed incorporation of Thrombosomes into the thrombus. In the high shear chamber, mean thrombus area increased in a dose-dependent manner following the addition of Thrombosomes (704 μm2 [95% CI, 224 – 1184 μm2], 1511 μm2 [95% CI, 687 – 2336 μm2] and 2378 μm2 [95% CI, 1567 – 3189 μm2] for PFP and Thrombosomes at concentrations of 20 and 200 × 106/mL respectively [P= 0.003]). In the low shear chamber, total thrombus area for the PFP was 4962 μm2 (95% CI, 2489 – 7434 μm2). The addition of Thrombosomes at concentrations of 20 and 200 × 106/mL led to a numerical increase in mean thrombus area to 6170 μm2 (95% CI, 3944 – 8397 μm2) and 7504 μm2 (95% CI, 3864 – 11144 μm2) respectively, although this was not statistically significant (P= 0.2969). Conclusions Thrombosomes suspended in PFP and exposed to injured arterial tissue under physiologically relevant flow conditions are incorporated into thrombus and enhance thrombus formation in a dose dependent manner. These findings act as impetus to undertake clinical studies of this rehydrated, lyophilized infusible hemostatic platelet product. Disclosures: Fitzpatrick: Cellphire Inc.: Employment, Equity Ownership. Feuerstein:Cellphire: Consultancy. Newby:Cellphire: Research Funding.

Oxygenation of 18-hydroxycorticosterone as the final reaction for aldosterone biosynthesis

1984 ◽  
Vol 107 (3) ◽  
pp. 395-400 ◽  
Author(s):  
Itaru Kojima ◽  
Etsuro Ogata ◽  
Hiroshi Inano ◽  
Bun-ichi Tamaoki
Keyword(s):  
Carbon Monoxide ◽  
Hydroxysteroid Dehydrogenase ◽  
Dependent Manner ◽  
In Vitro Production ◽  
Mitochondrial Preparation ◽  
Final Reaction ◽  
Vitro Production ◽  
Dose Dependent ◽  
Cytochrome P 450

Abstract. Incubation of 18-hydroxycorticosterone with the sonicated mitochondrial preparation of bovine adrenal glomerulosa tissue leads to the production of aldosterone, as measured by radioimmunoassay. The in vitro production of aldosterone from 18-hydroxycorticosterone requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide. Cytochrome P-450 inhibitors such as metyrapone, SU 8000. SU 10603, SKF 525A, amphenone B and spironolactone decrease the biosynthesis of aldosterone from 18-hydroxycorticosterone. These results support the conclusion that the final reaction in aldosterone synthesis from 18-hydroxycorticosterone is catalyzed by an oxygenase, but not by 18-hydroxysteroid dehydrogenase. By the same preparation, the production of [3H]aldosterone but not [3H]18-hydroxycorticosterone from [1,2-3H ]corticosterone is decreased in a dose-dependent manner by addition of non-radioactive 18-hydroxycorticosterone.

Appraisal of Immunological Impacts of Melaleuca leucadendra Extract over Macrophage Performance in Vitro

2020 ◽  
Keyword(s):  
Nitric Oxide ◽  
Interleukin 2 ◽  
Dependent Manner ◽  
Reduced Pressure ◽  
Medical Plant ◽  
Dose Dependent ◽  
Level Production ◽  
Melaleuca Leucadendra ◽  
Phagocytic Killing

This trial research was performed to discuss the immune-influence of Melaleuca leucadendra ‘paper-bark tree’ dried leaves which is an important medical plant known in many regions in the world. The leaves were dissolved in a mixture of (ethanol + water) (3:1) mixture, then filtered, evaporated and dried under reduced pressure to obtain leaves extract. The macrophages of blood derived origin were provided from rats and mixed with three different leaves extracts doses in tissue culture plates and incubated then stained with fluorescent acridine orange and examined under fluorescent microscope to assess the phagocytic and killing potency. The wells contents were aspirated and assayed for nitric oxide and interleukin-2 levels. The results displayed an obvious increase in phagocytic, killing performance as well as nitric oxide and IL-2 level production than control in a dose dependent manner. The obtained results suggested the immune-stimulant impact of the paper-bark tree leaves.

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