The effect of calcium ions on testosterone production in Leydig cells from rat testis.
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ABSTRACT:
SUBMITTER: Janszen FH
PROVIDER: S-EPMC1164258 | biostudies-other | 1976 Dec
REPOSITORIES: biostudies-other
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Project description:Leydig cells in the testes produce testosterone in the presence of gonadotropins. Therefore, male testosterone levels must oscillate within a healthy spectrum, given that elevated testosterone levels augment the risk of cardiovascular disorders. We observed that the expression of death-associated protein-like 1 (DAPL1), which is involved in the early stages of epithelial differentiation and apoptosis, is considerably higher in the testes of sexually mature mice than in other tissues. Accordingly, Dapl1-null mice were constructed to evaluate this variation. Notably, in these mice, the testicular levels of steroidogenic acute regulatory protein (StAR) and serum testosterone levels were significantly elevated on postnatal day 49. The findings were confirmed in vitro using I-10 mouse testis-derived tumor cells. The in vivo and in vitro data revealed the DAPL1-regulated the expression of StAR involving altered transcription of critical proteins in the protein kinase A and CREB/CREM pathways in Leydig cells. The collective findings implicate DAPL1 as an important factor for steroidogenesis regulation, and DAPL1 deregulation may be related to high endogenous levels of testosterone.
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Project description:The effect of lutropin on phosphorylation of endogenous proteins in testis Leydig cells was investigated, by incubating purified Leydig cells with lutropin and [(32)P]P(i) followed by sodium dodecyl sulphate/polyacrylamide-slab gel electrophoresis of the [(32)P]phosphoproteins. The radioactivity of the proteins was quantified by densitometry of the radio-autograms obtained. The following results were obtained. 1. Lutropin increased the amount of (32)P incorporated into three proteins (A, B and C) with apparent mol.wts. of 14300, 57000 and 77600 respectively. 2. The increase in incorporation of (32)P into these proteins was detectable within 5min, reaching a maximum in approx. 20min. 3. The (32)P incorporated into protein B (but not proteins A and C) was significantly increased with 0.1 and 1.0ng of lutropin/ml. Incorporation of (32)P into all three proteins was significantly increased with 10ng of lutropin/ml, reaching a maximum with 100ng/ml. 4. Testosterone production was significantly increased with 1ng of lutropin/ml, and between 10 and 1000ng/ml the degree of stimulation of testosterone production and incorporation of (32)P into proteins A, B and C was similar. 5. Cyclic AMP production was significantly increased with 10ng of lutropin/ml and had not reached a maximum with 1000ng/ml. 6. In Leydig cells isolated from hypophysectomized rats 3h after injection of choriogonadotropin in vivo, phosphoproteins with the same molecular weights as proteins A, B and C were found. No further increases in incorporation of (32)P into these proteins were obtained when lutropin was added to the Leydig cells in vitro. 7. Dibutyryl cyclic AMP (but not follitropin or testosterone) also stimulated the incorporation of (32)P into proteins A, B and C in Leydig cells.
| S-EPMC1184115 | biostudies-other
Project description:Luteinizing-hormone-stimulated testosterone biosynthesis was inhibited by cycloheximide during incubation of rat testis intersitial tissue in vitro and also by puromycin and cycloheximide during incubation of Leydig-cell preparations, but not by chloramphenicol. These results suggest that a protein regualtor(s) formed by cytoplasmic protein synthesis is involved in steroidogenesis in the rat testis. The specific effect of cycloheximide and puromycin on protein synthesis rather than on other non-specific processes is suggested by the inhibition of protein synthesis and steroidogenesis with different doses of the inhibitors and the lack of effect of cycloheximide on luteinizing-hormone-induced adenosine 3':5'-cyclic monophosphate production. Stimulation of testosterone production by luteinizing hormone during superfusion of interstitial tissue was detectable within 10-20 min and reached a maximum of 120 min, and thereafter slowly decreased. Cycloheximide added at maximum steroid production caused a rapid decrease in testosterone synthesis which followed first-order kinetics (half-life 13 min), thus indicating that the protein regulator(s) has a short half-life. No effect of cycloheximide, puromycin or chloramphenicol on testosterone production in the absence of added luteinizing hormone was found, suggesting that the basal production of testosterone is independent of protein synthesis.
| S-EPMC1165756 | biostudies-other
Project description:BackgroundPerfluorooctane sulfonate (PFOS) is a synthetic material that has been widely used in industrial applications for decades. Exposure to PFOS has been associated with decreased adult testosterone level, and Leydig cell impairment during the time of adulthood. However, little is known about PFOS effects in utero on fetal Leydig cells (FLC).Methods and resultsThe present study investigated effects of PFOS on FLC function. Pregnant Sprague Dawley female rats received vehicle (0.05% Tween20) or PFOS (5, 20 mg/kg) by oral gavage from gestational day (GD) 11-19. At GD20, testosterone (T) production, FLC numbers and ultrastructure, testicular gene and protein expression levels were examined. The results indicate that exposures to PFOS have affected FLC function as evidenced by decreased T production, impaired FLC, reduced FLC number, and decreased steroidogenic capacity and cholesterol level in utero.ConclusionThe present study shows that PFOS is an endocrine disruptor of male reproductive system as it causes reduction of T production and impairment of rat fetal Leydig cells.
| S-EPMC3891643 | biostudies-literature
Project description:This study investigated the acute effects of green tea extract (GTE) and its polyphenol constituents, (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin (EC), on basal and stimulated testosterone production by rat Leydig cells in vitro. Leydig cells purified in a Percoll gradient were incubated for 3 h with GTE, EGCG or EC and the testosterone precursor androstenedione, in the presence or absence of either protein kinase A (PKA) or protein kinase C (PKC) activators. The reversibility of the effect was studied by pretreating cells for 15 min with GTE or EGCG, allowing them to recover for 1 h and challenging them for 2 h with human chorionic gonadotropin (hCG), luteinizing hormone releasing hormone (LHRH), 22(R)-hydroxycholesterol or androstenedione. GTE and EGCG, but not EC, inhibited both basal and kinase-stimulated testosterone production. Under the pretreatment conditions, the inhibitory effect of the higher concentration of GTE/EGCG on hCG/LHRH-stimulated or 22(R)-hydroxycholesterol-induced testosterone production was maintained, whereas androstenedione-supported testosterone production returned to control levels. At the lower concentration of GTE/EGCG, the inhibitory effect of these polyphenols on 22(R)-hydroxycholesterol-supported testosterone production was reversed. The inhibitory effects of GTE may be explained by the action of its principal component, EGCG, and the presence of a gallate group in its structure seems important for its high efficacy in inhibiting testosterone production. The mechanisms underlying the effects of GTE and EGCG involve the inhibition of the PKA/PKC signalling pathways, as well as the inhibition of P450 side-chain cleavage enzyme and 17beta-hydroxysteroid dehydrogenase function.
| S-EPMC3735300 | biostudies-other
Project description:Previous studies have demonstrated that rodent stem Leydig cell (SLC) transplantation can partially restore testosterone production in Leydig cell (LC)-disrupted or senescent animal models, which provides a promising approach for the treatment of hypogonadism. Here, we isolated human SLCs prospectively and explored the potential therapeutic benefits of human SLC transplantation for hypogonadism treatment. In adult human testes, p75 neurotrophin receptor positive (p75+) cells expressed the known SLC marker nestin, but not the LC lineage marker hydroxysteroid dehydrogenase-3β (HSD3β). The p75+ cells which were sorted by flow cytometry from human adult testes could expand in vitro and exhibited clonogenic self-renewal capacity. The p75+ cells had multi-lineage differentiation potential into multiple mesodermal cell lineages and testosterone-producing LCs in vitro. After transplantation into the testes of ethane dimethane sulfonate (EDS)-treated LC-disrupted rat models, the p75+ cells differentiated into LCs in vivo and secreted testosterone in a physiological pattern. Moreover, p75+ cell transplantation accelerated the recovery of serum testosterone levels, spermatogenesis and reproductive organ weights. Taken together, we reported a method for the identification and isolation of human SLCs on the basis of p75 expression, and demonstrated that transplanted human p75+ SLCs could replace disrupted LCs for testosterone production. These findings provide the groundwork for further clinical application of human SLCs for hypogonadism.
| S-EPMC5680910 | biostudies-literature
Project description:A systematic phytochemical study on the components in the seeds of Allium tuberosum was performed, leading to the isolation of 27 steroidal glycosides (SGs 1-27). The structures of SGs were identified mainly by nuclear magnetic resonance and mass spectrometries as well as the necessary chemical evidence. In the SGs, 1-10 and 22-26 are new steroidal saponin analogues. An in vitro bioassay indicates that 1, 2, 7, 8, 10, 13-15, 20, 23, and 26 display promotional roles in testosterone production of rat Leydig cells with the EC50 values of 1.0 to 4.5 μM, respectively.
| S-EPMC7700350 | biostudies-literature
Project description:BackgroundBenzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), is a ubiquitous environmental pollutant that is currently suspected of being an endocrine disruptor. The testis is an important target for PAHs, yet insufficient attention has been paid to their effects on steroidogenesis in Leydig cells.ObjectiveWe hypothesized that long-term exposure to low concentrations of B[a]P might disrupt testosterone production in Leydig cells via an alteration of steroidogenic proteins.ResultsOral exposure to B[a]P reduced serum and intratesticular fluid testosterone levels in rats. However, we did not observe serious testicular atrophy or azoospermia, although spermatogonial apoptosis was significantly increased. Compared with control cells, Leydig cells primed with B[a]P in vivo produced less testosterone in response to human chorionic gonadotropin (hCG) or dibutyl cyclic adenosine monophosphate in vitro. Of note, the reduction of testosterone levels was accompanied by decreased expression of steroidogenic acute regulatory protein (StAR) and 3?-hydroxysteroid dehydrogenase (3?-HSD), as well as increased levels of cytochrome P450 side chain cleavage (P450scc), in Leydig cells. The up-regulation of P450scc expression after exposure to B[a]P appears to be associated with a compensatory mechanism for producing the maximum amount of pregnenolone with the minimum amount of transported cholesterol by StAR; the down-regulation of 3?-HSD may occur because B[a]P can negatively target 3?-HSD, which is required for testosterone production.ConclusionsB[a]P exposure can decrease epididymal sperm quality, possibly by disturbing testosterone levels, and StAR may be a major steroidogenic protein that is targeted by B[a]P or other PAHs.
| S-EPMC3226498 | biostudies-literature
Project description:Background: Propofol is a widely used anesthetic. Whether propofol inhibits androgen production by rat Leydig cells and the underlying mechanism remains unclear. The objective of the current study was to examine the effects of propofol exposure to rat primary immature Leydig cells and to define propofol-induced inhibition of steroidogenic enzymes in both rat and human testes in vitro. Methods: Immature Leydig cells were purified from 35-day-old male Sprague-Dawley rats and were exposed to propofol for 3 h. The androgen production by Leydig cells under basal, luteinizing hormone, 8bromo-cAMP, and steroid-substrate stimulated conditions and gene expression of Leydig cells after exposure to propofol were measured. Immature Leydig cells were treated with propofol for 3 h and switched to propofol-free medium for additional 3 and 9 h to test whether propofol-induced inhibition is reversible. 3H-Steroids were used to evaluate the direct action of propofol on cytochrome P450 cholesterol side chain cleavage (CYP11A1), 3β-hydroxysteroid dehydrogenase (HSD3B), cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1), and 17β-hydroxysteroid dehydrogenase 3 (HSD17B3) activities in rat and human testes in vitro. Results: Propofol significantly lowered luteinizing hormone and 8bromo-cAMP stimulated androgen production by Leydig cells after 3-h exposure. Further investigation showed that propofol down-regulated the expression of Cyp11a1 and Cyp17a1 and their proteins at 5 and 50 µM, although it up-regulated Lhcgr expression at 50 µM. Propofol significantly suppressed phosphorylation of ERK1/2 and induced ROS production in immature Leydig cells at 5 and 50 µM. Propofol significantly induced apoptosis of immature Leydig cells at 50 µM. Propofol specifically inhibited rat and human testis HSD3B activities in vitro. The half maximal inhibitory concentrations of propofol for rat and human HSD3B enzymes were 1.011 ± 0.065 and 3.498 ± 0.067 µM, respectively. The mode of action of propofol of inhibiting HSD3B was competitive when pregnenolone was added. At 50 µM, propofol did not directly inhibit rat and human testis CYP11A1, CYP17A1, and HSD17B3 activities in vitro. Conclusion: Propofol inhibits androgen production via both directly inhibiting HSD3B activity and down-regulating Cyp11a1 and Cyp17a1 expression in Leydig cells. Suppression of steroidogenic enzymes is presumably associated with the lower production of androgen by Leydig cells after propofol treatment. However, propofol-induced inhibition on androgen production is reversible.
| S-EPMC6624235 | biostudies-literature
Project description:BackgroundMAMLD1 is known to be a causative gene for hypospadias. Although previous studies have indicated that MAMLD1 mutations result in hypospadias primarily because of compromised testosterone production around the critical period for fetal sex development, the underlying mechanism(s) remains to be clarified. Furthermore, although functional studies have indicated a transactivation function of MAMLD1 for the non-canonical Notch target Hes3, its relevance to testosterone production remains unknown. To examine these matters, we performed Mamld1 knockdown experiments.Methodology/principal findingsMamld1 knockdown was performed with two siRNAs, using mouse Leydig tumor cells (MLTCs). Mamld1 knockdown did not influence the concentrations of pregnenolone and progesterone but significantly reduced those of 17-OH pregnenolone, 17-OH progesterone, dehydroepiandrosterone, androstenedione, and testosterone in the culture media. Furthermore, Mamld1 knockdown significantly decreased Cyp17a1 expression, but did not affect expressions of other genes involved in testosterone biosynthesis as well as in insulin-like 3 production. Hes3 expression was not significantly altered. In addition, while 47 genes were significantly up-regulated (fold change >2.0×) and 38 genes were significantly down-regulated (fold change <0.5×), none of them was known to be involved in testosterone production. Cell proliferation analysis revealed no evidence for compromised proliferation of siRNA-transfected MLTCs.Conclusions/significanceThe results, in conjunction with the previous data, imply that Mamld1 enhances Cyp17a1 expression primarily in Leydig cells and permit to produce a sufficient amount of testosterone for male sex development, independently of the Hes3-related non-canonical Notch signaling.
| S-EPMC3084764 | biostudies-literature