Regulatory mechanism of Toona sinensis on mouse leydig cell steroidogenesis

Toona sinensis (TS), a kind of arbor, widely distributes nowadays in Asia. The leaves of TS have been used as an effective nutritious food in Chinese society for a long time. It was reported that Toona sinensis can induce apoptosis of cancer cells, reduce plasma glucose in diabetic rats, and improve lipolysis of differentiated 3T3-L1 adipocyte and its uptake of glucose. It has also been shown that TS may increase dynamic activity of human sperm. Thus, we are interested to investigate whether Toona sinensis has any effect on mouse Leydig cell testosterone production, which correlates to sperm activity. Primary mouse Leydig cells were purified to conduct the in vitro experiments. Different concentrations of crude Toona sinensis were added to primary mouse Leydig cells and the testosterone production was determined. The results showed that crude TS significantly inhibited both basal and human chorionic gonadotropin (hCG)-stimulated testosterone productions in dose dependent manner, respectively (P<0.05). Crude TS also reduced the forskolin- and dibutyryl-cAMP (dbcAMP)-stimulated testosterone production (P<0.05), which indicated that crude TS might affect protein kinase A (PKA) signal transduction pathway at the site after the formation of cyclic AMP. Moreover, TS inhibited Leydig cell steroidogenesis by suppressing the activity of steroidogenic enzymes including P450 side chain cleavage enzyme, 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, 20 alpha-hydroxylase and 17 beta-hydroxysteroid dehydrogenase (P<0.05). In summary, these results suggested that TS inhibited steroidogenesis by suppressing the cAMP-PKA signaling pathway and the activities of steroidogenic enzymes in normal mouse Leydig cells.     

In vivo and in vitro stimulatory effects of Cordyceps sinensis on testosterone production in mouse Leydig cells

The in vivo and in vitro effects of Cordyceps sinensis (CS) and its extracted fractions on the secretion of testosterone in mice were studied. CS, F2 (water soluble protein), and F3 (poorly water soluble polysaccharide and protein) significantly stimulated in vitro testosterone production in purified mouse Leydig cells. However, F1 (water soluble polysaccharide) had no effect (p>0.05). F2 and F3 stimulated in vitro testosterone production in dose- and time-dependent relationships with maximal responses at 3 mg/ml for 3 h (p<0.05). An in vivo study illustrated that testosterone levels in plasma were significantly increased by CS, F2, and F3, respectively (p<0.05). Because CS, F2, and F3 stimulated both in vitro and in vivo testosterone secretions in mice, it is possible that CS might contribute to an alternative medicine for the treatment of some reproductive problems caused by insufficient testosterone levels in human males.     

Assessment of in vitro effects of metyrapone on Leydig cell steroidogenesis

Metyrapone, a specific inhibitor of 11beta-hydroxylase inhibits glucocorticoid production and it is used in the diagnosis/treatment of hypercortisolism and also to test the functional integrity of hypothalamo-pituitary-adrenal axis. To assess the impact of glucocorticoid deficiency, this drug is preferred over adrenalectomy, which eliminates all the hormonal secretions of the adrenal cortex and medulla. However, whether metyrapone has any direct effect on the extra-adrenocortical cellular or tissue functions remains to be resolved. Our previous study showed a depressed testicular Leydig cell testosterone production in rats treated with metyrapone. Therefore, the present study was designed to examine the possible direct effect of metyrapone on testicular Leydig cell steroidogenesis in vitro. Leydig cell viability and the reactive oxygen species (ROS) concentration were not altered by any of the concentration of metyrapone tested. The efficacy of Leydig cell testosterone production under basal as well as LH-stimulated condition was not altered by metyrapone treatment. Further, Leydig cellular (14)C-glucose oxidation, the activity and mRNA levels of cytochrome side chain cleavage (P(450)scc), 3beta- and 17beta-hydroxysteroid dehydrogenase (3beta-HSD and 17beta-HSD) were not altered in metyrapone-treated cells. Therefore, it is concluded from the present study that metyrapone has no direct effect on Leydig cell testosterone production and, therefore, changes recorded in the in vivo studies are exclusively due to corticosterone deficiency.     

Effect of cell purity,cell concentration,and incubation conditions on rat testis leydig cell steroidogenesis

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.     

Inhibitory and stimulatory effects of limb ectoderm on in vitro chondrogenesis

Previous studies have indicated possible dual effects of the limb ectoderm in cartilage differentiation. On one hand, explants from early (stage 15) wing buds are dependent on contact with the limb ectoderm for cartilage differentiation (Gumpel-Pinot, J. Embryol. Exp. Morph. 59:157-173, 1980). On the other hand, limb ectoderm from stage 23/24 wing buds inhibits cartilage differentiation by cultured limb mesenchyme cells even without direct contact (Solursh et al., Dev. Biol. 86:471-482, 1981). In the present study, ectoderms from both stage 15/16 and stage 23/24 wings are cultured under the same conditions, and ectoderms from each source are shown to have two effects. Each stimulates chondrogenesis in stage 15 wing bud mesenchyme, and each inhibits chondrogenesis in older wing mesenchyme. The results suggest that the limb ectoderm has at least dual effects on cartilage differentiation, depending on the stage of the mesenchyme. One effect involves an early mesenchymal dependence on the ectoderm. This effect requires contact between the ectoderm and mesoderm (Gumpel-Pinot, J. Embryol. Exp. Morphol. 59:157-173, 1980) but also can be observed at a distance from the ectoderm. Later, the ectoderm can act without direct contact between the ectoderm and mesoderm to inhibit chondrogenesis over some distance.     

Granulosa cell steroidogenesis before in vitro fertilization

Endocrine and gametogenic functions of the ovulatory follicle may be linked. To verify this, we studied granulosa cell steroidogenesis in relation to oocyte fertilization and preimplantation embryo development in vitro. Multiple follicles were stimulated in in vitro fertilization patients with clomiphene citrate and ovulation was induced with human chorionic gonadotropin (hCG). Oocytes were fertilized with husband's sperm and normal embryos were replaced 48 h later. Granulosa cells were separated from follicular fluid from 64 follicles and incubated for 3 h with and without aromatase substrate (1 microM testosterone). Progesterone and estradiol levels were measured in follicular fluid and incubation medium. Follicular fluid steroid levels and granulosa cell steroidogenesis showed no significant differences for oocytes which cleaved normally and those which did not. Granulosa cell aromatase activity was high in all follicles, suggesting that the low periovulatory follicular fluid estradiol level is not explained by a fall in granulosa cell aromatase after hCG. High granulosa cell progesterone production and follicular fluid progesterone were consistent with advanced granulosa cell luteinization. Oocytes undergoing polyspermic activation were from larger follicles with elevated follicular fluid progesterone levels, suggesting that follicular size and follicular fluid progesterone are correlated with "over-ripeness" and polyspermy. No simple relationship exists between oocyte function and the present indices of granulosa cell steroid metabolism.     

The avoidance of stimulatory artifacts in cell cycle determinations in vitro

Myogenic cultures exhibit a predictable pattern of growth characterized by an initially exponential accumulation of DNA, which declines to a plateau level after fusion. This pattern is related to cell-mediated modifications of the culture medium and can be altered by deviations from the routine feeding schedule. The data demonstrate that fresh medium stimulates DNA accumulation in older cultures while the medium removed from such older, fusing cultures reduces the rate of DNA accumulation in younger cultures and also promotes fusion. Under experimental conditions which require some replacement of medium (e.g., pulse-chase labeling), such effects are potential sources of experimental artifact. Such artifacts can be eliminated, however, by the proper choice of medium. The least perturbation of the cultures occurs when medium withdrawn from sister cultures of the same age is employed.     

The effects of actinomycin D and cordycepin on neurite formation and acetylcholinesterase activity in mouse neuroblastoma cells

Actinomycin D (actD) (0.003–0.10 μg/ml) and cordycepin (3–30 μg/ml) were used to examine the requirement of de novo RNA synthesis in the pH 6.6-induced expression of neurites and acetylcholinesterase activity in C-1300 mouse neuroblastoma cells. ActD at 0.03 and 0.10 μg/ml caused a pronounced stimulation in neurite formation following 20 h of treatment, although by 30 h exposure to actD (0.01–0.10 μg/ml), neurite formation had rapidly declined. Cordycepin (3–30 μg/ml) also inhibited neurite formation in a concentration- and time-dependent manner, although it did not produce an initial stimulation in neurite formation. The pH 6.6-induced increase in acetylcholinesterase activity was inhibited by both actD and cordycepin in a concentration- and time-dependent manner. Cell viabilities in the presence of actD and cordycepin were 90% or greater throughout the course of these studies.The effects of actD on [³H]uridine and [³H]leucine transport into cells and on incorporation into acid-insoluble material showed that actD inhibited RNA synthesis to a greater extent than it inhibited protein synthesis. Cordycepin caused only minor effects on [³H]uridine and [³H]leucine transport into cells and incorporation into acid-insoluble material; these effects were variable and neither concentration- nor time-dependent. The results of this study show that actD can inhibit the pH 6.6-induced expression of neurites and acetylcholinesterase activity in mouse neuroblastoma cells at concentrations which were relatively non-toxic and which caused a greater inhibition of RNA synthesis than of protein synthesis. This suggests that de novo RNA synthesis is required for the expression and maintenance of neurites and acetylcholinesterase activity in mouse neuroblastoma cells. Experiments with cordycepin were consistent with this conclusion.     

The effects of dimebon on mouse brain cell endoplasmic reticulum membrane viscosity in vivo

The neuroprotective effects of dimebon on the microviscosity of endoplasmic reticulum membranes isolated from the mouse brain after chronic drug administration were studied. This study revealed that dimebon decreases the microviscosity of the near-protein regions of the microsomal membranes. This neuroprotector showed no effect on thermally induced structural transitions, as well as on the activation energy of the corresponding structural states. This apparently may be due to the insufficient neuroprotector concentration in the brain cells.     

Effects of cytokines on porcine granulosa cell steroidogenesis in vitro

Recent evidence indicates that factors produced by immune cells (cytokines) may play a role in ovarian function. To explore this possibility, we examined the effects of conditioned medium obtained from cultures of either unstimulated splenocytes (splenocyte-conditioned medium; SCM) or concanavalin A-stimulated splenocytes (CAS) on estrogen and progesterone production by porcine granulosa cells. Granulosa cells were obtained from small (less than 3 mm) or large (greater than 7 mm) follicles and treated with increasing doses of SCM or CAS in the presence or absence of pFSH (100 ng/ml) for 24 h at 37 degrees C. In granulosa cells obtained from small follicles it was found that both SCM and CAS evoked a dose-dependent increase in estrogen but not progesterone production. Estrogen production was no further enhanced by the presence of FSH. Additionally, SCM was able to augment FSH-stimulated progesterone production by these cells, whereas CAS had no effect. Identical treatment of granulosa cells obtained from large follicles demonstrated that both SCM and CAS caused dose-dependent increases in estrogen as well as progesterone production. In response to CAS, FSH augmented progesterone production but exerted a biphasic on estrogen production (inhibiting at lower doses while stimulating at higher doses). In contrast, SCM had no effect on FSH-stimulated estrogen production. Additional controls indicated that the above results could not be attributed to either concanavalin A or serum. Taken together, these findings suggest that cytokines can exert significant effects over granulosa cell steroidogenesis and further imply that these factors may play an important role in the differentiation and developmental regulation of granulosa cell function.     

The inhibitory effects of lead on steroidogenesis in MA-10 mouse Leydig tumor cells

Lead is an environmental and occupational pollutant. It has been reported that lead affects the male reproductive system in humans and animals. However, the cellular mechanism of the adverse effect of lead on Leydig cell steroidogenesis remains unknown. To clarify whether lead has a direct effect on Leydig cells and how lead affects Leydig cells, MA-10 cells, a mouse Leydig tumor cell line, were exploited in this study. Lead acetate significantly inhibited hCG- and dbcAMP-stimulated progesterone production in MA-10 cells at 2 h. Steroid production stimulated by hCG or dbcAMP were reduced by lead. The mechanism of lead in reducing MA-10 cell steroidogenesis was further investigated. The expression of Steroidogenic Acute Regulatory (StAR) protein and the activities of P450 side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzymes were detected. Cells were treated with dbcAMP, 22R-hydroxycholesterol or pregnenolone alone or in combination with lead acetate ranging from 10(-8) to 10(-5) M for 2 h. The expression of StAR protein stimulated by dbcAMP was suppressed by lead at about 50%. Progesterone productions treated with 22R-hydroxycholesterol or pregnenolone were reduced 30-40% in lead-treated MA-10 cells. These data suggest that lead directly inhibited steroidogenesis by decreasing StAR protein expression and the activities of P450scc and 3beta-HSD enzymes with a dose-response trend in MA-10 cells. Moreover, cadmium, a calcium channel blocker, abolished inhibitory effect of lead on MA-10 cell steroid production. This indicates that lead might act on calcium channel to regulate MA-10 cell steroidogenesis.     

The effects of calcium ionophore A23187 on interstitial cell steroidogenesis

The present study was performed to evaluate the effects of calcium ionophore A23187 on adenosine 3',5'-monophosphate (cyclic AMP) and testosterone production in rat interstitial cells. Interstitial cells were incubated in Krebs-Ringer solution with varying amounts of luteinizing hormone, pregnenolone, or A23187. Cyclic AMP and testosterone were measured in the incubation medium after 4 h incubation. A23187 (0.01--10 microgram/ml) caused progressive increases of cyclic AMP formation (from 0.18 +/- 0.02 (S.E.) pmol/10(6) cells for the control of 0.42 +/- 0.02 pmol/10(6) cells, P less than 0.025), while testosterone production remained unaltered. When varying amounts of A23187 were added concomitantly with luteinizing hormone (5 IU/l), A23187 inhibited luteinizing hormone-induced steroidogenesis in a dose-dependent manner, but it had no effect on luteinizing hormone-induced cyclic AMP formation. When pregnenolone (10(-6) M) was added to the cells, testosterone formation increased from 1.50 +/- 0.22 to 8.46 +/- 1.65 ng/10(6) cells. A23187 (1 microgram/ml) had no discernable effect on the conversion of pregnenolone to testosterone. The main effect of increased cytosol calcium on steroidogenesis seems to be at the steps beyond adenylate cyclase-cyclic AMP. These results suggest that calcium is important for the conversion of cholesterol to pregnenolone, while the steps beyond pregnenolone are relatively independent of Ca2+.     

Short-term stimulatory effect of Sertoli cell conditioned medium on Leydig cell steroidogenesis is not mediated by inhibin

Addition of concentrated rat Sertoli cell conditioned medium (rSCCM) to isolated Leydig cells from immature rats stimulated steroid production more than 13-fold within 4 h. LH-stimulated steroidogenesis was not enhanced by addition of rSCCM. The biological activity of the concentrated rSCCM was higher after incubation of Sertoli cells with FSH, whereas FSH alone did not stimulate steroid production. This effect of rSCCM was not due to inhibin, since highly purified 32 kDa rat inhibin, in doses equivalent to those present in rSCCM, had no effect on steroidogenesis during the 4 h incubation period. Furthermore, inhibin could be separated from the Leydig cell stimulating factor by anion-exchange chromatography. These results indicate a short-term paracrine control of Leydig cell steroidogenesis by Sertoli cell derived factors, which differ from inhibin.     

The in vitro and in vivo effects of anti-galactose antibodies on endothelial cell activation and xenograft rejection

We have previously produced a series of antigalactose (anti-Gal) hybridomas and characterized their heavy chain gene usage. Here we have quantified the affinity of these Abs for the alpha-Gal epitope and characterized their in vitro effects on endothelial cell activation and apoptosis. We report that anti-Gal mAbs derived from Gal(-/-) mice show a range of affinity for the alpha-Gal epitope, and that affinity was generally increased as the V(H) gene usage transitioned from germline sequences to sequences exhibiting somatic maturation. Despite an 85-fold range in affinity, all the anti-Gal mAbs examined induced alpha-Gal-specific endothelial cell activation, and after prolonged exposure induced endothelial cell apoptosis in a complement-independent manner. Only murine anti-Gal mAbs of the IgM or IgG3 subclass, but not IgG1, were effective at initiating complement-dependent cell lysis. Using a novel rat to mouse xenograft model, we examined the in vivo ability of these mAbs to induce xenograft rejection and characterized the rejection using histology and immunohistochemistry. Infusion of complement-fixing IgG3 mAbs resulted in either hyperacute rejection or acute vascular rejection of the xenograft. Surprisingly, infusion of an equal amount of a high affinity anti-Gal IgG1 mAb, that fixed complement poorly also induced a rapid xenograft rejection, which we have labeled very acute rejection. These studies emphasize the importance of in vivo assays, in addition to in vitro assays, in understanding the role of anti-Gal IgG-mediated tissue injury and xenograft rejection.     

Contrasting effects of eplerenone and spironolactone on adrenal cell steroidogenesis

Spironolactone and eplerenone are widely used as mineralocorticoid antagonists. Spironolactone has several nonspecific actions including inhibition of androgen receptor and steroid hormone biosynthesis. While studies have shown that eplerenone does not exhibit nonspecific actions on androgen receptor, its effects on steroid hormone production have not been reported. Herein, the effects of eplerenone (0.1-30 microM) and spironolactone (0.1-30 microM) on steroid production were examined in human adrenocortical H295R cells. Spironolactone inhibited basal production of cortisol (91%) and aldosterone (53%). Treatment of H295R cells with angiotensin II (Ang II) for 24 h increased aldosterone production by 11-fold. Spironolactone inhibited Ang II stimulation of aldosterone production by 80%. Addition of pregnenolone increased aldosterone (9-fold) and cortisol (3-fold) production. Spironolactone inhibited pregnenolone metabolism to aldosterone (67%) and cortisol (74%). The inhibitory effects of spironolactone occurred at concentrations far higher than those needed to block mineralocorticoid receptor, suggesting an action directly on the enzymes involved in steroid production. In contrast, eplerenone did not inhibit basal, Ang II, forskolin, pregnenolone-stimulated cortisol, or aldosterone production. Together, these data demonstrate that opposed to spironolactone, pharmacologic concentrations of eplerenone do not inhibit adrenal cell aldosterone or cortisol production.     

Prostacyclin and steroidogenesis in goat ovarian cell types in vitro

Granulosa, theca and corpus luteum cells of the goat ovary were isolated and incubated separately for 6 hours, with or without various modulators. Arachidonic acid (AA, 10 ng to 100 micrograms/ml), the precursor for prostaglandin synthesis, produced a dose-dependent increase in progesterone (P4) and estradiol-17 beta (E2) production by all the cell types. Prostaglandin synthetase inhibitors, aspirin (10(-6)-10(-3)M) and indomethacin (100 ng-1 mg/ml), produced a dose-dependent decrease in arachidonic acid-stimulated (100 micrograms/ml) steroid production. Prostacyclin synthetase stimulators, trapidil (1.6 micrograms- 1 mg/ml) and dipyridamole (10(-6)-10(-3)M), when added alone or along with AA, did not affect steroid production. Up to 100 micrograms/ml of U-51605 (9,11-azoprosta-5,13-dienoic acid), a prostacyclin synthetase inhibitor, did not inhibit basal or AA-stimulated steroid production. Prostacyclin (PGI2) and its stable analog 6 beta PGI1 (0.01-10 micrograms/ml) produced a dose-dependent increase in P4 and E2 production in all the three cell types. Increase at 1 and 10 micrograms/ml was significant in all cases. 6-keto-PGE1 (an active metabolite of PGI2 in certain systems) produced an increase in steroid production which was significant in theca at greater than or equal to 1 microgram/ml concentrations but had no significant effect on granulosa and corpus luteum cells at any dose level. 6-keto-PGF1 alpha (stable metabolite of PGI2) was without effect in the present system. The lack of effect of PGI2 at lower concentrations was not altered by either differentiation of the cells with FSH and testosterone or addition of steroid precursors, testosterone and pregnenolone. The present results indicate that AA-stimulated steroid production in the goat ovarian cell type is mediated by prostaglandins other than PGI2 though PGI2 itself can positively modulate the steroid production.