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1.
Similarities and differences in phorbol ester- and luteinizing-hormone-induced desensitization of rat tumour Leydig-cell adenylate cyclase. 总被引:2,自引:2,他引:0
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The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was shown to mimic luteinizing hormone (LH; lutropin) in causing desensitization of LH-mediated cyclic AMP production in tumour Leydig cells. However, there were differences between LH- and TPA-induced desensitization: (1) TPA induced a more rapid effect than LH; (2) adenosine did not inhibit TPA-induced desensitization, whereas it completely inhibited the LH-induced desensitization; (3) adenylate cyclase activity in plasma membranes from TPA-desensitized cells was not decreased, whereas similar preparations from LH-desensitized cells lost their response to LH and to LH plus guanosine 5'-[beta gamma-imido]triphosphate; TPA-, but not LH-, treated cells had a decreased capacity to respond to cholera toxin and forskolin. These results indicate that LH and phorbol esters induce desensitization of adenylate cyclase in rat tumour Leydig cells by different mechanisms. 相似文献
2.
The responsiveness of the membrane bound adenylyl cyclase (AC) system to various hormones and hormone analogues in a transplantable rat Leydig cell tumor (H-540) has been investigated and compared with that of interstitial tissue from normal adult rats. The results can be summarized as follows: The AC of the Leydig cell tumors was stimulated by isoproterenol, epinephrine, norepinephrine, dopamine, glucagon, PGE1, PGF2 alpha and LH/hCG. The AC of the Leydig cell tumors exhibited a much greater response to PGE1 than the AC in normal interstitial tissue (5-fold and 2-fold stimulation, respectively). The AC of the Leydig cell tumours also revealed a somewhat higher response to isoproterenol than the AC of interstitial tissue (2.5-fold and 2-fold, respectively). Adenylyl cyclase in both tissues were equally stimulated by glucagon (2-fold). The LH response in the Leydig cell tumors was only half of that found in normal Leydig cells. This study indicates, that in spite of the fact that tumor Leydig cells respond to LH and produce testosterone, the response pattern of the AC is different from that of normal Leydig cells. 相似文献
3.
The cellular localization of beta-adrenergic and prostaglandin (PG) receptors and their effects on adenylate cyclase activity (AC) and testosterone production in vitro were investigated in a transplantable rat Leydig cell tumor (H-540). Separation of the tumor cells in Percoll gradients revealed that the specific binding of [3H]PGE1 and [125I]Cyanopindolol was found in the same fraction as that of [125I]LH. This fraction--judged by light microscopy of smears--consisted of tumor Leydig cells. In addition, [125I]cyanopindolol was found specifically bound in the red blood cell fraction. In the Leydig tumor cells, approx 25% of the beta-adrenergic receptors was identified as beta 1-receptors, whereas approx 75% of the receptors were of the beta 2-subtype. The AC in Percoll purified Leydig tumor cells was stimulated by hCG (6-fold), PGE1 (2-fold), PGE2 (1.5-fold), PGI1 (2-fold) and isoproterenol (2-fold). The AC in the red blood cell fraction was stimulated by isoproterenol whereas the PGs and hCG had little or no effect. hCG, isoproterenol and PGE1 were able to stimulate testosterone production in vitro. At 44 h incubation, PGE1 was the most potent stimulator of testosterone production. In conclusion, tumor Leydig cells possess hCG, PGE1, PGI2 and beta-adrenergic receptors coupled to the AC. PGE1 and beta-adrenergic agonists stimulate testosterone production after prolonged incubation in vitro. 相似文献
4.
Studies were conducted to define further the development of the gonadotropin induced, E2 mediated steroidogenic lesion (17-alpha-hydroxylase/17,20-desmolase) in fetal Leydig cell cultures. Analysis of dispersed fetal testes purified by centrifugal elutriation demonstrated a group of cells with sedimentation velocity 12 less than to less than 16.8 mm/h.g containing a small population of adult like "transitional" Leydig cells and homogeneous "fetal" Leydig cell population collected at greater than 19.3 mm/h.g. After cells were cultured for 3 days with addition of 1 microgram oLH at 3 day intervals, the transitional cells showed testosterone accumulation comparable to the fetal cells. In contrast, transitional cells had 10-fold higher basal and hCG-stimulated aromatase activity than fetal cells, and a lack of testosterone response to acute (3 h) hCG stimulation. At day 6, transitional cells steroidogenic ability declined markedly. The fetal population maintained in culture with LH additions every 3 days, showed typical immature Leydig cell response, with enhancement of acute testosterone response to hCG at 3 day (1-fold) and at 6 day of culture (5-fold). Higher doses of LH (5 micrograms/day) or daily treatment of 1 microgram to fetal cultures, induced a lesion of 17 alpha-hydroxylase/17,20-desmolase with reduction of enzymatic activities (P less than 0.01) and impaired testosterone production (P less than 0.01) in response to acute hCG stimulation. Also aromatase was stimulated by hCG + 140% and 50% and E2 receptors were increased by 100 and 180% at 3 days and 6 days of cultures with daily or high dose LH addition, findings consistent with the observation of the E2-mediated lesion during LH action. In conclusion, the cultured fetal Leydig cell provides a useful model to elucidate molecular mechanisms involved in the development of gonadotropin-induced estradiol-mediated desensitization. Treatment of fetal Leydig cell cultures with multiple or frequent doses of LH elevate aromatase activity to necessary levels for the induction of desensitization. We have isolated small population of transitional Leydig cells with morphological characteristics of cells found in 15 day post-natal testis but functional capabilities of adult cells. We have also demonstrated the emergence of a functional adult-like population from the fetal Leydig cell. 相似文献
5.
D. R. E. Abayasekara L. O. Kurlak A. M. Band M. H. F. Sullivan B. A. Cooke 《In vitro cellular & developmental biology. Animal》1991,27(3):253-259
Summary This study examines the effects of cell purity and incubation conditions on testosterone production by rat testis Leydig cells
in short-term primary culture. Both basal and luteinizing hormone (LH)-stimulated testosterone production were affected by
the purity of the cell preparation, i.e. as the purity of the cell preparation was increased the amount of testosterone produced
per Leydig cell was also found to increase. The stimulation ratio of testosterone production, calculated as the secretion
of testosterone in the presence of LH (100 ng/ml) divided by the basal secretion of testosterone, increased with the increase
in plating density (20 000 to 200 000 cells per well). This pattern of change was independent of the vessel and volume of
incubation. In terms of the absolute amount of testosterone produced, increasing the plating density led to a decrease in
the amount of steroid produced both basally and in response to LH. Composition of the incubation medium also had an effect
on testosterone production; phenol red and sodium bicarbonate exerted negative effects. At all temperatures studied (4°, 24°,
34°, and 37° C), LH increased testosterone production and the degree of stimulation increased with temperature. We conclude
that cell purity and incubation conditions markedly affect rat Leydig cell steroidogenesis in vitro. Furthermore, the manner
in which the results are presented can affect their interpretation. 相似文献
6.
Using metrizamide gradient centrifugation two populations of Leydig cells were found in both 60-90 day-old and 24 month-old rats. Cells from both Band 2 (B2) and Band 3 (B3) responded to LH stimulation with increased cyclic AMP formation; however, only B3 cells produced significant amounts of testosterone. Cells from both B2 and B3 of the old rats synthesized less cyclic AMP and testosterone than cells from their younger counterparts. In response to LH stimulation, 0.01 - 1.0 mIU/ml, no appreciable difference of cyclic AMP formation could be detected between young and old Leydig cells. Maximal testosterone production occurred when 1 mIU/ml LH was used. Only when LH concentration was increased to 10 and 100 mIU/ml, did young Leydig cells produce significantly more cyclic AMP than old Leydig cells. After addition of 5X10(-7)M of pregnenolone or progesterone to the incubation medium, both young and old Leydig cells produced comparable amounts of testosterone. These results demonstrate no impairment of old rat Leydig cells to synthesize testosterone from pregnenolone and progesterone. 相似文献
7.
Effects of antimicrotubular agents in cAMP production and in steroidogenic response of isolated rat Leydig cells 总被引:1,自引:0,他引:1
D Saltarelli M P De la Llosa-Hermier C Tertrin-Clary C Hermier 《Biology of the cell / under the auspices of the European Cell Biology Organization》1984,52(3):259-266
In dispersed rat Leydig cells, colchicine was found to stimulate basal cAMP production and testosterone secretion in a dose and time-dependent manner, but to a lesser extent than LH. However, these drugs are unable to stimulate adenylate cyclase activity in plasma membranes isolated from these cells. The amount of testosterone secreted at 150 min under the influence of colchicine and LH added simultaneously was not different from the amount produced during stimulation by LH alone. It is only after exposure of the cells for 1 hr to colchicine that the accumulation of cAMP in response to LH was inhibited; furthermore, both intracellular and medium testosterone accumulation in response to the hormone were reduced. Similar effects were observed with two other alkaloids, vinblastine and podophyllotoxin. The three drugs also inhibited the stimulation of testosterone secretion by 8-Br-cAMP or choleratoxin. These studies suggest that the state of microtubule polymerization and/or tubulin can influence the process of steroidogenesis in rat Leydig cells. 相似文献
8.
P S Li 《The Journal of steroid biochemistry and molecular biology》1991,38(2):205-212
The direct effects of hydrocortisone (HS) and adrenocorticotropin (ACTH) on testicular testosterone production were studied in purified immature pig Leydig cells in vitro. Leydig cells were obtained from 3- to 4-week-old piglet testes by enzymatical dispersion followed by discontinuous Percoll gradient centrifugation. Leydig cells were treated with HS and ACTH in the absence or presence of luteinizing hormone (LH) after 12 h of incubation. Media were collected 48 h later for testosterone and cyclic adenosine 3',5'-monophosphate (cAMP) measurement. Treatment of Leydig cells with increasing concentrations (0.001-10.0 micrograms/ml) of HS for 48 h resulted in a dose-dependent increase in basal and LH-stimulated testosterone production. Increasing duration (6-72 h) of treatment with HS (100 ng/ml) led to a time-dependent increase in basal and LH-stimulated testosterone production, achieving statistical significance by 48 and 24 h, respectively. HS increased LH-stimulated cAMP production. HS also increased testosterone production induced by (Bu)2 cAMP. Forskolin stimulated testosterone production to an extent comparable to that attained with LH, and HS augmented forskolin-stimulated testosterone production. HS enhanced the conversion of exogenous 17 alpha-hydroxyprogesterone to testosterone, but did not affect the conversion of pregnenolone and progesterone to testosterone, suggesting a specific stimulation of 17,20-desmolase. Porcine ACTH had no influence on basal and LH-stimulated testosterone production. These results suggest that HS directly stimulates immature pig Leydig cell steroidogenesis, at least in part via an enhancement of the generation of cAMP, leading to an increase in the activity of 17,20-desmolase. 相似文献
9.
The effect of phorbol esters on calcium-activated, phospholipid-dependent kinase (protein kinase C) and luteinizing hormone (LH) secretion was examined in cultured rat anterior pituitary cells. The potent tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) stimulated LH secretion and activated pituitary protein kinase C in the presence of calcium and phosphatidylserine. The enzyme activity present in cytosol and particulate fractions was eluted at about 0.05 M NaCl during DE52-cellulose chromatography. Preincubation of pituitary cells with TPA markedly decreased cytosolic protein kinase C activity and increased enzyme activity in the particulate fraction. The maximal TPA-induced change in enzyme activity, with a 76% decrease in cytosol and a 4.3-fold increase in the particulate fraction, occurred within 10 min. The dose-dependent changes in protein kinase C redistribution in TPA-treated cells were correlated with the stimulation of LH release by the phorbol ester. These results suggest that activation of protein kinase C by TPA is associated with intracellular redistribution of the enzyme and is related to the process of secretory granule release from gonadotrophs. 相似文献
10.
Effect of phorbol ester and phospholipase C on LH-stimulated steroidogenesis in purified rat Leydig cells 总被引:1,自引:0,他引:1
When the phorbol ester, 4 beta-phorbol-12-myristate-13-acetate (PMA) or bacterial phospholipase C (PL-C) is added to a preparation of purified adult rat Leydig cells, containing 2 mM CaCl2, a time- and dose-dependent decreases of LH-stimulated testosterone production is observed. After a 3 h stimulation with oLH (100 ng/ml), PMA (100 ng/ml) and PL-C (1.6 U/ml) do not affect the cell viability or the hCG specific binding, while cAMP accumulation is significantly reduced; cAMP-stimulated steroidogenesis is diminished only in the presence of PL-C. These observations suggest that in vitro: (i) activated Ca2+- and phospholipid-dependent protein kinase is implicated in the regulation of rat Leydig cell steroidogenesis by LH at a step before the adenylate cyclase; (ii) phospholipids play an important role in cAMP-stimulated testosterone synthesis. 相似文献
11.
Differences between the regulation of cholesterol side-chain cleavage in Leydig cells from mice and rats 总被引:1,自引:0,他引:1
A O Brinkmann F G Leemborg F F Rommerts H J van der Molen 《Journal of steroid biochemistry》1984,21(3):259-264
A Leydig cell culture system has been used to study the in vitro modulation by luteinizing hormone (LH) of steroidogenesis in Leydig cells isolated from mice and immature rats. Mouse Leydig cells precultured for 24 h in the presence of increasing concentrations of LH (1 ng-1 microgram/ml) showed a dose-dependent decrease of the maximal LH-stimulated testosterone production. After pretreatment with 1 microgram LH/ml, maximal LH-stimulated testosterone production. After production in the presence of excess 20 alpha-hydroxycholesterol (a cholesterol side-chain cleavage substrate) were reduced to approx. 50% of control values. The possible site of action of LH is probably prior to pregnenolone, because testosterone production in the presence of excess pregnenolone was not affected by the LH pretreatment. Immature rat Leydig cells showed no decrease of maximal steroid production after 24 h culture in the presence of 1 microgram LH/ml. These results indicate that the regulation of the cholesterol side-chain cleavage activity during long-term LH action is different in mouse and rat Leydig cells. The properties of the cholesterol side-chain cleavage enzyme in mouse and rat Leydig cells were further investigated with different hydroxylated cholesterol derivatives as substrates. Steroid production by mouse Leydig cells in the presence of (22R)-22 hydroxycholesterol was similar as in the presence of LH. In contrast, steroidogenesis in rat Leydig cells in the presence of (22R)-22 hydroxycholesterol was at least 10-fold higher than in the presence of LH. It is concluded that the cholesterol side-chain cleaving enzyme in the mouse Leydig cell operates at its maximal capacity during short-term LH stimulation and can be inhibited after long-term LH action, whereas in the rat Leydig cell only a fraction of the potential activity is used during short-term LH stimulation, which is not affected during long-term LH action. 相似文献
12.
Luteinizing hormone (LH) interacts with its plasma membrane receptor to activate the formation of cyclic AMP via the regulatory GTP binding protein (Gs). This is followed by a desensitization of that same hormonal response which is caused by an uncoupling of the LH receptor from Gs. The coupling between Gs and the adenylate cyclase catalytic unit remains intact. Treatment of Leydig and other cell types with phorbol esters mimics hormone-induced desensitization. However, differences between hormone- and phorbol ester-induced desensitization have been found. In testis Leydig cells phorbol esters, as well as uncoupling the LH receptor from Gs, also inactivates the subunit of the inhibitory GTP binding protein (Gi). These studies suggested that activation of protein kinase may be involved in the hormone-induced desensitization of adenylate cyclase. Paradoxically, it has also been found that two inhibitors of protein kinase C, sphingosine and psychosine also inhibited LH-induced cyclic AMP production. These effects were mainly found during the initial stimulatory period with LH. It is suggested that activation of adenylate cyclase may require a protein kinase C-mediated phosphorylation step which is followed by further phosphorylation resulting in uncoupling of the receptor from Gs. No direct stimulation of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3), diacylglycerol and/or activation of protein kinase C by LH in Leydig cells has been demonstrated. An alternative mechanism of protein kinase C activation has been proposed for brain cells, i.e. that involving arachidonic acid activation of protein kinase C instead of diacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
Rothschild G Sottas CM Kissel H Agosti V Manova K Hardy MP Besmer P 《Biology of reproduction》2003,69(3):925-932
Kit and its ligand, Kitl, function in hematopoiesis, melanogenesis, and gametogenesis. In the testis, Kitl is expressed by Sertoli cells and Kit is expressed by spermatogonia and Leydig cells. Kit functions are mediated by receptor autophosphorylation and subsequent association with signaling molecules, including phosphoinositide (PI) 3-kinase. We previously characterized the reproductive consequences of blocking Kit-mediated PI 3-kinase activation in KitY(719F)/Kit(Y719F) knockin mutant male mice. Only gametogenesis was affected in these mice, and males are sterile because of a block in spermatogenesis during the spermatogonial stages. In the present study, we investigated effects of the Kit(Y719F) mutation on Leydig cell development and steroidogenic function. Although the seminiferous tubules in testes of mutant animals are depleted of germ cells, the testes contain normal numbers of Leydig cells and the Leydig cells in these animals appear to have undergone normal differentiation. Evaluation of steroidogenesis in mutant animals indicates that testosterone levels are not significantly reduced in the periphery but that LH levels are increased 5-fold, implying an impairment of steroidogenesis in the mutant animals. Therefore, a role for Kit signaling in steroidogenesis in Leydig cells was sought in vitro. Purified Leydig cells from C57Bl6/J male mice were incubated with Kitl, and testosterone production was measured. Kitl-stimulated testosterone production was 2-fold higher than that in untreated controls. The Kitl-mediated testosterone biosynthesis in Leydig cells is PI 3-kinase dependent. In vitro, Leydig cells from mutant mice were steroidogenically more competent in response to LH than were normal Leydig cells. In contrast, Kitl-mediated testosterone production in these cells was comparable to that in normal cells. Because LH levels in mutant males are elevated and LH is known to stimulate testosterone biosynthesis, we proposed a model in which serum testosterone levels are controlled by elevated LH secretion. Leydig cells of mutant males, unable to respond effectively to Kitl stimulation, initially produce lower levels of testosterone, reducing testosterone negative feedback on the hypothalamic-pituitary axis. The consequent secretion of additional LH, under this hypothesis, causes a restoration of normal levels of serum testosterone. Kitl, acting via PI 3-kinase, is a paracrine regulator of Leydig cell steroidogenic function in vivo. 相似文献
14.
To examine whether immature rat Sertoli cells in culture secrete a factor(s) which stimulates testosterone production by mature mouse Leydig cells, Sertoli cell-enriched cultures were prepared from 3-week-old male rats with trypsin and collagenase. Sertoli cells were plated at an initial density of 3-5 x 10(6) cells/35 mm well and cultured in 3 ml serum free media supplemented with insulin (10 micrograms/ml). Sertoli cell culture medium (SCCM) collected every 3rd day was added to Leydig cells (10(6) cells in 1 ml of MEM with 2% steroid-free FCS) prepared from 10-week-old mice by mechanical separation and incubated for 3 h at 34 degrees C. Secreted testosterone was determined by RIA. SCCM 15 times concentrated by Amicon YM10 membrane demonstrated a dose-dependent stimulation of testosterone production, whereas there was no effect on testosterone secretion when Leydig cells were maximally stimulated by LH. Leydig cell stimulating activity was retained by both a dialysis membrane with a pore size of 24 A and an ultrafiltration membrane with a molecular weight cut-off of 10 kDa. However, activity was reduced by heating at 60 degrees C for 30 min and almost lost after incubation with 0.1% trypsin for 1 h at 37 degrees C. This activity was not retained by means of a Con A-Agarose column and was demonstrated only in break-through fractions. HPLC gel filtration of a 15 times concentrated SCCM preparation on a TSK gel G3000SW revealed Leydig cell-stimulating activity at approximately 13 kDa.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
A P Themmen J W Hoogerbrugge F F Rommerts H J van der Molen 《Biochemical and biophysical research communications》1985,128(3):1164-1172
Two adenylate cyclase inhibitors: 9-(tetrahydro-2-furyl)adenine and 2'5'-dideoxyadenosine decreased cAMP levels in LH-stimulated immature rat Leydig cells by 20-40%, independent of the concentration of LH. Steroid production was not correlated with this decrease in cAMP, but was increased (146%). The phorbol ester 4 beta-phorbol-12-myristate-13-acetate stimulated steroidogenesis and the phosphorylation of a 17 kD and a 33 kD protein, which was also stimulated by LH, whereas the inactive phorbol ester 4 beta-phorbol-12,13-diacetate did not have any effects. Moreover, the Ca2+-channel blocker diltiazem inhibited LH effects, but had no direct effects on the cholesterol side chain cleavage enzyme. It is concluded that cAMP may not be the only second messenger in LH action, and that other second messenger systems are probably also involved. 相似文献
16.
The possible role of protein kinase C and phospholipids in the regulation of steroid production in rat Leydig cells 总被引:1,自引:0,他引:1
We have studied the possible involvement of the activation of calcium-dependent phospholipid-activated protein kinase (PK-C) in the stimulatory action of LHRH on Leydig cells, using 4 beta-phorbol-12-myristate-13-acetate (PMA) and phospholipase C (PL-C). LHRH agonist (LHRH-A) and PL-C had a large synergistic effect on LH-stimulated steroid production, whereas PMA inhibited the effect of LH. However, PMA always caused an increase in steroid production stimulated by various doses of dibutyryl cAMP. LH and PMA stimulated the phosphorylation of 17 and 33 kDa proteins, whereas LHRH-A and PL-C had no effect. Of all effectors used, LH had the most pronounced effect on the synthesis of 14, 27 and 30 kDa proteins. The present results suggest that the mechanisms of action of LHRH-A and PL-C on steroid production in Leydig cells may be similar and different from PMA, and may involve stimulation of a specific type of PK-C or hydrolysis of a specific pool of phospholipids. 相似文献
17.
Steroidogenic effect of atrial natriuretic factor in isolated mouse Leydig cells is mediated by cyclic GMP. 总被引:2,自引:1,他引:1
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The effects of different atrial natriuretic peptides on cyclic GMP formation and steroidogenesis have been studied in Percoll-purified mouse Leydig cells. Rat atrial peptides rANP (rat atrial natriuretic peptide), rAP-I (rat atriopeptin I) and rAP-II (rat atriopeptin II), in the presence of a phosphodiesterase inhibitor, stimulated cyclic GMP formation in a concentration-dependent manner. In the presence of saturating concentrations of the peptides, a 400-600 fold stimulation of cyclic GMP accumulation was observed. Among the peptides, rAP-II appeared to be the most potent. ED50 values (concentration causing half-maximal effect) for rAP-II, rANP and rAP-I were 1 X 10(-9) M, 2 X 10(-9) M and 2 X 10(-8) M respectively. A parallel stimulation of cyclic GMP formation and testosterone production by the cells was observed after incubation of the cells with various concentrations of rAP-II. In the presence of a saturating concentration of rAP-II (2 X 10(-8) M), maximum stimulation of intracellular cyclic GMP content was obtained within 5 min of incubation. Testosterone production by mouse Leydig cells could be stimulated by 8-bromo cyclic GMP in a concentration-related manner. At a 10 mM concentration of the cyclic nucleotide, steroidogenesis was stimulated to a similar extent as that obtained with a saturating concentration of human chorionic gonadotrophin (5 ng/ml). On the basis of these results we conclude that cyclic GMP acts as a second messenger in atrial-peptide-stimulated steroidogenesis in mouse Leydig cells. The steroidogenic effect of atrial peptides appears to be species-specific, since none of these peptides stimulated testosterone production by purified Leydig cells of rats, though in these cells a 40-60-fold stimulation of cyclic GMP formation in response to each of the three peptides was observed. However, 8-bromo cyclic GMP could stimulate testosterone production in rat Leydig cells. Therefore we conclude that the lack of steroidogenic response in rat Leydig cells to atrial-natriuretic-factor-stimulation results from an insufficient formation of cyclic GMP in these cells. This species difference would appear to result from a lower guanylate cyclase activity in rat Leydig cells. 相似文献
18.
R K Stuart L L Sensenbrenner R K Shadduck A Waheed C Caramatti 《Journal of cellular physiology》1983,117(1):30-38
Tumor promoting phorbol esters, such as 12-0-tetradecanoyl-phorbol-13-acetate (TPA), stimulate colony formation in vitro by murine granulocyte-macrophage progenitors (GM-CFC) without added colony stimulating factors (CSF). To determine whether TPA induces CSF production in vitro, marrow cells were cultured for 1 to 7 days in liquid medium with or without TPA. No CSF was detected in any sample by a double antibody radioimmunoassay (sensitivity = 2 units/0.1 ml), however, colony-stimulating activity was detected in supernatant fluid from all TPA containing cultures by bioassay. This activity appeared to result from a direct effect of TPA rather than from production of CSF, as equivalent activity was found in TPA-containing medium incubated in the absence of marrow cells. Rabbit antiserum to purified L-cell CSF inhibited colony formation stimulated by L-cell CSF and WEHI-3 CSF, but had no effect on colony formation induced by TPA. Cells from long-term marrow cultures responded to TPA with colony formation, despite culture conditions and cell fractionation procedures that reduced the frequency of CSF-producing macrophages to less than 1.0%. TPA inhibited binding of radioiodinated L-cell CSF to marrow cells, especially if the cells were first exposed to TPA. These results do not support induction of CSF production as the major mechanism of phorbol ester stimulation of myelopoiesis. Phorbol esters may directly stimulate GM-CFC and/or enhance their response to CSF by a mechanism involving CSF binding sites. 相似文献
19.
Plasma testosterone levels before and after a single injection of hCG were significantly lower in 24-month old rats than 60--90 day old animals (p less than 0.001). Even with repeated hCG administration for three weeks, plasma testosterone levels of old rats could not be restored to levels present in unstimulated young rats. In response to in vitro LH and 8-bromo-cyclic AMP stimulation, purified young Leydig cells produced significantly higher amounts of testosterone than Leydig cells from old rats. Maximal testosterone formation of the young Leydig cells in response to LH was 42.0 +/- 6.88 ng/10(6) cells, while cells from old rats produced only 16.8 +/- 3.69 ng/10(6) cells (p less than 0.01). However, the dose of LH at which one half maximal response (ED50) occurred was 0.1 mIU/ml for young Leydig cells and 0.05 mIU/ml for old Leydig cells. Basal and 1.0 mIU LH-stimulated cyclic AMP formation were comparable in both groups, but cyclic AMP formation in response to 10 mIU of LH was significantly less in the old rats (p less than 0.05). Present results demonstrate impaired steroidogenic capacity of old rats both in vivo and in vitro. Decreased testosterone response in old rats most likely is the consequence of understimulation of Leydig cells by gonadotropin; however, there appear to be additional intrinsic defects in old Leydig cells. 相似文献
20.
Puberty in the male is dependent upon the elevated production of testosterone by the Leydig cells. LH affects this increase in testosterone output by increasing the total number of Leydig cells in the testis and by stimulating the steroidogenic pathway in these cells. Since Leydig cell proliferation is a prerequisite for the onset of puberty, we have examined the ability of LH and growth factors known to be present in the testis to promote DNA synthesis. Leydig cells were isolated from 21-day-old rats, cultured in serum-free medium for 48 h to become quiescent, and then treated with LH and growth factors for 18 h. [3H]Thymidine incorporation into DNA was assessed over the subsequent 4-h incubation period. Cells in control cultures incorporated low levels of [3H]thymidine into DNA and were stimulated after treatment with LH (100 ng/ml). Insulin/insulin-like growth factor-1 (IGF-1) and transforming growth factor-alpha (TGF-alpha), previously localized in Leydig cells by immunohistochemistry, also stimulated [2H]thymidine incorporation into DNA. The responses of the Leydig cells to maximum levels of insulin and TGF-alpha were dependent on the cell density. Insulin and TGF-alpha alone and in combination increased the number of cells labeled with [3H]thymidine, as assessed by autoradiography. TGF-beta, known to be secreted by Sertoli cells, also stimulated DNA synthesis under basal conditions, but the maximum response was significantly lower than that achieved in the presence of TGF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献