Murine Leydig Tumor Cell Line Research Articles

Sirt3 Regulates Proliferation and Progesterone Production in Leydig Cells via Suppression of Reactive Oxygen Species.

Sirt3 is a mitochondrial protein deacetylase functioning in energy metabolism, regulation of intracellular reactive oxygen species (ROS) levels, and aging. Although Sirt3 loss has negative effects on fertility of oocytes during in vitro fertilization and on progesterone production in granulosa cells, Sirt3's function in Leydig cells remains unclear. Therefore, we investigated Sirt3 activity in Leydig cells, focusing on androgen production. To do so, we performed immunohistochemistry to confirm Sirt3 localization in gonads and observed strong Sirt3 immunostaining in Leydig cells of human testes and of Sirt3+/+ and Sirt3+/- mouse testes, while Sirt3-/- mouse testis tissue was negative. In human ovary, hilus cells were strongly Sirt3-positive, theca cells showed weak positivity, and granulosa cells showed very weak or almost no immunostaining. Next, we used the murine Leydig tumor cell line MA-10 as a model. We overexpressed Sirt3 but observed no changes in proliferation, expression of Star, Cyp11a1 (p450scc gene), and Hsd3b, or progesterone production in MA-10 cells. Sirt3 knockdown significantly reduced proliferation, suppressed expressions of steroidogenic enzymes and of transcription factors Ad4bp (Sf-1 gene) and Gata4, and decreased progesterone production. Sirt3 knockdown in MA-10 cells also increased intracellular ROS levels based on CM-H2DCFDA fluorescence dye analysis and increased the proportion of both early and late apoptotic (necrotic) cells based on Annexin V/7AAD assays. These results indicate that Sirt3 has a potential function in androgen production in Leydig cells by regulating intracellular ROS levels.

In vitro effects of the endocrine disruptor p,p'DDT on human choriogonadotropin/luteinizing hormone receptor signalling.

Dichlorodiphenyltrichloroethane (p,p'DDT) is an endocrine-disrupting chemical (EDC). Several studies showed an association between p,p'DDT exposure and reprotoxic effects. We showed that p,p'DDT was a positive allosteric modulator of human follitropin receptor (FSHR). In contrast, we demonstrated that p,p'DDT decreased the cyclic AMP (cAMP) production induced by human choriogonadotropin (hCG). This study evaluated further the effects of p,p'DDT on Gs-, β-arrestin 2- and steroidogenesis pathways induced by hCG or luteinizing hormone (LH). We used Chinese hamster ovary cells line stably expressing hCG/LHR. The effects of 10-100µM p,p'DDT on cAMP production and on β-arrestin 2 recruitment were measured using bioluminescence and time-resolved resonance energy transfer technology. The impact of 100µM of p,p'DDT on steroid secretion was analysed in murine Leydig tumor cell line (mLTC-1). In cAMP assays, 100µM p,p'DDT increased the EC50 by more than 300% and reduced the maximum response of the hCG/LHR to hCG and hLH by 30%. This inhibitory effect was also found in human granulosa cells line and in mLTC-1 cells. Likewise, 100µM p,p'DDT decreased the hCG- and hLH-promoted β-arrestin 2 recruitment down to 14.2 and 26.6%, respectively. Moreover, 100µM p,p'DDT decreased by 30 and 47% the progesterone secretion induced by hCG or hLH, respectively, without affecting testosterone secretion. This negative effect of p,p'DDT was independent of cytotoxicity. p,p'DDT acted as a negative allosteric modulator of the hCG/LHR signalling. This emphasizes the importance of analyzing all receptor-downstream pathways to fully understand the deleterious effects of EDC on human health.

Curcumin protects against palmitic acid-induced apoptosis via the inhibition of endoplasmic reticulum stress in testicular Leydig cells

BackgroundPalmitic acid (PA) is a common saturated fatty acid that induces apoptosis in various types of cells, including testicular Leydig cells. There is evidence suggesting that PA is increased in patients with obesity and that PA-induced cell apoptosis may play an important role in obesity-related male infertility. Curcumin, a natural polyphenol, has been reported to exert cytoprotective effects in various cell types. However, the cytoprotective effect of curcumin against PA-induced apoptosis in Leydig cells remains unknown. Therefore, the current study was performed to investigate the protective effects of curcumin in response to PA-induced toxicity and apoptosis in murine Leydig tumor cell line 1 (MLTC-1) cells and explore the mechanism underlying its anti-apoptotic action.MethodsMLTC-1 cells were cultured in Roswell Park Institute-1640 medium and divided into five groups. First four groups were treated with 50–400 μM PA, 400 μM PA + 5–40 μM curcumin, 400 μM PA + 500 nM 4-phenylbutyric acid (4-PBA, an endoplasmic reticulum (ER) stress inhibitor), and 500 nM thapsigargin (TG, an ER stress inducer) + 20 μM curcumin, respectively, followed by incubation for 24 h. Effects of PA and/or curcumin on viability, apoptosis, and ER stress in MLTC-1 cells were then determined by cell proliferation assay, flow cytometry, and western blot analysis. The fifth group of MLTC-1 cells was exposed to 400 μM of PA and 5 IU/mL of human chorionic gonadotropin (hCG) for 24 h in the absence and presence of curcumin, followed by measurement of testosterone levels in cell-culture supernatants by enzyme-linked immunosorbent assay (ELISA). Rats fed a high-fat diet (HFD) were treated with or without curcumin for 4 weeks, and the testosterone levels were detected by ELISA.ResultsExposure to 100–400 μM PA reduced cell viability, activated caspase 3, and enhanced the expression levels of the apoptosis-related protein BCL-2-associated X protein (BAX) and ER stress markers glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP) in MLTC-1 cells. Treating cells with 500 nM 4-PBA significantly attenuated PA-induced cytotoxicity through inhibition of ER stress. Curcumin (20 μM) significantly suppressed PA- or TG-induced decrease in cell viability, caspase 3 activity, and the expression levels of BAX, CHOP, and GRP78. In addition, treating MLTC-1 cells with 20 μM curcumin effectively restored testosterone levels, which were reduced in response to PA exposure. Similarly, curcumin treatment ameliorated the HFD-induced decrease in serum testosterone level in vivo.ConclusionsThe present study suggests that PA induces apoptosis via ER stress and curcumin ameliorates PA-induced apoptosis by inhibiting ER stress in MLTC-1 cells. This study suggests the application of curcumin as a potential therapeutic agent for the treatment of obesity-related male infertility.

Arsenic compounds induce apoptosis through caspase pathway activation in MA-10 Leydig tumor cells.

The incidence of testicular cancer is increasing worldwide. Leydig cell tumors represent one type of sex cord-stromal testis malignancy, which tend to respond unfavorably to chemotherapies. Identifying more efficient treatment strategies is therefore crucial for patients. The present study aimed to investigate the apoptotic effects of arsenic compounds and their underlying mechanisms. The results indicated that sodium arsenite and dimethylarsenic acid induced apoptosis of the murine Leydig tumor cell line, MA-10. These apoptotic effects were characterized morphologically by membrane blebbing and cell detachment assays, biochemically using a cell viability assay, and cytologically by flow cytometry analysis. Western blotting demonstrated that caspases-3, -8 and -9, and poly(ADP-ribose) polymerase protein levels were increased compared with untreated MA-10 cells; however, the caspase inhibitor, Z-VAD-fmk, reversed these effects. In conclusion, the present study has shown that sodium arsenite and dimethylarsenic acid may activate the intrinsic and extrinsic caspase pathways, and induce MA-10 cell apoptosis. These results suggest that sodium arsenite and dimethylarsenic acid may represent novel approaches to treat clinically unmanageable forms of testicular cancer.

Profiling of FSHR negative allosteric modulators on LH/CGR reveals biased antagonism with implications in steroidogenesis

Biased signaling has recently emerged as an interesting means to modulate the function of many G protein-coupled receptors (GPCRs). Previous studies reported two negative allosteric modulators (NAMs) of follicle-stimulating hormone receptor (FSHR), ADX68692 and ADX68693, with differential effects on FSHR-mediated steroidogenesis and ovulation. In this study, we attempted to pharmacologically profile these NAMs on the closely related luteinizing hormone/chorionic gonadotropin hormone receptor (LH/CGR) with regards to its canonical Gs/cAMP pathway as well as to β-arrestin recruitment in HEK293 cells. The NAMs' effects on cAMP, progesterone and testosterone production were also assessed in murine Leydig tumor cell line (mLTC-1) as well as rat primary Leydig cells. We found that both NAMs strongly antagonized LH/CGR signaling in the different cell models used with ADX68693 being more potent than ADX68692 to inhibit hCG-induced cAMP production in HEK293, mLTC-1 and rat primary Leydig cells as well as β-arrestin 2 recruitment in HEK293 cells. Interestingly, differential antagonism of the two NAMs on hCG-promoted steroidogenesis in mLTC-1 and rat primary Leydig cells was observed. Indeed, a significant inhibition of testosterone production by the two NAMs was observed in both cell types, whereas progesterone production was only inhibited by ADX68693 in rat primary Leydig cells. In addition, while ADX68693 totally abolished testosterone production, ADX68692 had only a partial effect in both mLTC-1 and rat primary Leydig cells. These observations suggest biased effects of the two NAMs on LH/CGR-dependent pathways controlling steroidogenesis. Interestingly, the pharmacological profiles of the two NAMs with respect to steroidogenesis were found to differ from that previously shown on FSHR. This illustrates the complexity of signaling pathways controlling FSHR- and LH/CGR-mediated steroidogenesis, suggesting differential implication of cAMP and β-arrestins mediated by FSHR and LH/CGR. Together, our data demonstrate that ADX68692 and ADX68693 are biased NAMs at the LH/CGR in addition to the FSHR. These pharmacological characteristics are important to consider for potential contraceptive and therapeutic applications based on such compounds.

GATA4 is a key regulator of steroidogenesis and glycolysis in mouse Leydig cells.

Transcription factor GATA4 is expressed in somatic cells of the mammalian testis. Gene targeting studies in mice have shown that GATA4 is essential for proper differentiation and function of Sertoli cells. The role of GATA4 in Leydig cell development, however, remains controversial, because targeted mutagenesis experiments in mice have not shown a consistent phenotype, possibly due to context-dependent effects or compensatory responses. We therefore undertook a reductionist approach to study the function of GATA4 in Leydig cells. Using microarray analysis and quantitative RT-PCR, we identified a set of genes that are down-regulated or up-regulated after small interfering RNA (siRNA)-mediated silencing of Gata4 in the murine Leydig tumor cell line mLTC-1. These same genes were dysregulated when primary cultures of Gata4(flox/flox) adult Leydig cells were subjected to adenovirus-mediated cre-lox recombination in vitro. Among the down-regulated genes were enzymes of the androgen biosynthetic pathway (Cyp11a1, Hsd3b1, Cyp17a1, and Srd5a). Silencing of Gata4 expression in mLTC-1 cells was accompanied by reduced production of sex steroid precursors, as documented by mass spectrometric analysis. Comprehensive metabolomic analysis of GATA4-deficient mLTC-1 cells showed alteration of other metabolic pathways, notably glycolysis. GATA4-depleted mLTC-1 cells had reduced expression of glycolytic genes (Hk1, Gpi1, Pfkp, and Pgam1), lower intracellular levels of ATP, and increased extracellular levels of glucose. Our findings suggest that GATA4 plays a pivotal role in Leydig cell function and provide novel insights into metabolic regulation in this cell type.

Effects of di(n-butyl) and monobutyl phthalate on steroidogenesis pathways in the murine Leydig tumor cell line MLTC-1

Di(n-butyl) phthalate (DBP) and its active metabolite monobutyl phthalate (MBP) have been shown to disrupt reproductive organ growth. The objective of this study was to evaluate the effects of DBP/MBP on steroidogenesis in the murine Leydig tumor cell line MLTC-1 in vitro. MLTC-1 cells were incubated with various concentrations of DBP (100, 1, 0.01, and 0μmol/l in DMSO) and MBP (1000, 10, 0.1, and 0μmol/l in DMSO) for 24h. Testosterone secretion was stimulated at the lowest doses and inhibited at higher treatment doses of DBP and MBP. The mRNA levels of the side-chain cleavage enzyme (P450scc), cytochrome p450c17 (P450c17) and 3β-hydroxy-steroid dehydrogenase (3βHSD) were significantly reduced in the phthalate-exposed groups, whereas, the transcription and translation of insulin-like hormone 3 (INSL3) was affected by DBP and MBP. Alterations of the steroidogenic enzymes and INSL3 in MLTC-1 cells may be involved in the biphasic effects of DBP/MBP on androgen production.

Effects of Norepinephrine and Acetylcholine on the Development of Cultured Leydig Cells in Mice

Few data have suggested how norepinephrine (NE) and acetylcholine (Ach) regulate the development of Leydig cells in mice at prepuberty, except for data indicating endocrine effects. The present study aims to elucidate the roles of NE and Ach on the differentiation and proliferation of Leydig cells. Firstly, the expression of adrenergic receptors and muscarinic acetylcholine receptors in Leydig cells was investigated. It was found that adrenergic receptors (β1AR, β2AR, and α1D) and muscarinic acetylcholine receptors (M1 and M3) mRNA are expressed in adult Leydig cells. Then, the effects of NE and Ach on the differentiation and proliferation of Leydig cells were analyzed. The results showed that NE and Ach at 10 μM significantly increased the number of 3β-hydroxysteroid-dehydrogenase- (3β-HSD-) positive Leydig cells and improved the expression of proliferating cell nuclear antigen (PCNA) in Leydig cells on postnatal day (PD) 15 (P < 0.05). NE and Ach at 10 μM had no impact on the expression of PCNA mRNA (P > 0.05), but reduced the expression of 3β-HSD mRNA in adult Leydig cells and a murine Leydig tumor cell line (MLTC-1) (P < 0.05). Therefore, a conclusion may be reached that NE and Ach participated in stimulating the development of Leydig cells in mice from prepuberty to adult stage.

Progesterone action in a murine Leydig tumor cell line (mLTC-1), possibly through a nonclassical receptor type.

In a recent report we demonstrated that a high (micromolar) concentration of progesterone (P) specifically down-regulates LH receptor (R) expression and function in murine Leydig tumor cells. The aim of the present study was to characterize further the putative novel R, mediating these P effects in the murine Leydig tumor cell line, mLTC-1. The binding of [3H]P to these cells revealed a high (Kd, approximately 9.3 nmol/liter) and a low affinity (Kd, approximately 284 nmol/liter) component, and the binding displayed with specificity (P > dehydroepiandrosterone > 17-OHP). The binding was apparently different from that of the classical nuclear PR in the following ways. 1) The P/glucocorticoid antagonist RU 486 did not compete with [3H]P binding to the mLTC-1 cells. 2) No expression of the classical PR messenger RNA was detected, despite clear P binding to these cells, by Northern hybridization or RT-PCR. 3) An antibody against the C-terminal end of the classical PR (alpha c-262) revealed in mLTC-1 cells several molecular size protein bands between 45-57 kDa on Western hybridization, whereas these immunoreactive proteins were faintly recognized by another antibody (alpha-PR) directed toward the NH2-terminal region of the classical PR. The sizes of the immunoreactive molecules were relatively similar to those detected using the same antibodies in human sperm lysates, but were at variance with the classical PR (120, 94, and 60 kDa), detected with these antibodies in human uterus. The immunoreactive proteins bound peroxidase-labeled-P, which could be displaced in the presence of a 10-fold excess of free P. 4) An immediate increase in the intracellular free calcium level was observed after P treatment in cultured mLTC-1 cells, whereas it also increased the 45Ca2+ entry within 15 min in these cells. 5) Increasing doses of P (0.1-10 micromol/liter) demonstrated significant inhibition of LH receptor messenger RNA levels in a dose-dependent manner in mLTC-1 cells. In conclusion, a nonclassical PR is expressed and functional in these cells, and it is clearly distinct from the classical nuclear PR. It is apparent that recently reported inhibitory effects of P on LH receptor gene expression and function are mediated through this novel type PR in mouse Leydig cells.

Progesterone Action in a Murine Leydig Tumor Cell Line (mLTC-1), Possibly through a Nonclassical Receptor Type

Progesterone Action in a Murine Leydig Tumor Cell Line (mLTC-1), Possibly through a Nonclassical Receptor Type

Expression of functional leptin receptors in rodent Leydig cells.

Several studies indicate that the size of body fat stores and the circulating levels of the adipocyte-derived hormone leptin are able to influence the activity of the hypothalamic-pituitary-gonadal axis. The leptin-hypothalamic-pituitary-gonadal interactions have been mainly studied at the level of the central nervous system. In this study, we investigated the possibility that leptin may have direct effects on the rodent Leydig cell function. To probe this hypothesis, we first analyzed the expression of leptin receptors (OB-R) in rodent Leydig cells in culture. RT-PCR studies showed that rat Leydig cells express both the long (OB-Rb) and short isoform (OB-Ra) of leptin receptor, whereas MLTC-1 cells (a murine Leydig tumor cell line) express only the long isoform. Short-term (30-90 min) incubation of rat Leydig cells with increasing concentrations ofleptin (2-500 ng/ml) led to a significant and dose-dependent inhibition of human (h)CG-stimulated testosterone (T) production (approximately 60% reduction, IC50 = 20 ng/ml) but no change in basal androgen release. Also, leptin (150 ng/ml) amplified hCG-induced intracellular cAMP formation (1- to 2-fold) without modifying basal cAMP levels. Subsequent experiments showed that leptin inhibited 8Br-cAMP-stimulated T production, indicating that leptin's effect is exerted beyond cAMP. The inhibitory effect of leptin on hCG-induced T secretion was accompanied by a significant reduction of androstenedione and a concomitant rise of the precursor metabolites pregnenolone, progesterone, and 17-OH-progesterone, conceivable with a leptin-induced lesion of 17,20 lyase activity. Separate experiments performed with the MLTC-1 cells (not expressing cytochrome P450-17alpha) showed that leptin, though amplifying hCG-stimulated cAMP production, did not modify hCG-stimulated pregnenolone and progesterone release. These results further indicate that leptin action on steroidogenesis occurs downstream of progesterone synthesis. Northern Blot experiments showed no acute effect of leptin on cytochrome P450-17alpha messenger RNA accumulation in rat Leydig cells in basal and hCG-stimulated conditions, excluding that the rapid changes observed were caused by messenger RNA degradation. In conclusion, these findings, for the first time, show that leptin has direct, receptor-mediated actions on rodent Leydig cells in culture, at concentrations within the range of obese men.

Regulation of luteinizing hormone receptor gene expression by insulin-like growth factor-I in an immortalized murine Leydig tumor cell line (BLT-1)

It is postulated that insulin-like growth factor-I (IGF-I), a 70-amino acid mitogenic polypeptide, regulates Leydig cell steroidogenesis. In the present study, we assessed the effect of IGF-I on LH receptor (LHR) gene expression in an immortalized murine Leydig tumor cell line (BLT-1). Culture of BLT-1 cells in the presence of IGF-I (0.1-100 ng/ml) for 24 or 48 h increased their [125I]iodo-hCG binding in a dose-dependent manner up to 275% of the control level. Northern hybridization analysis revealed four major transcripts of LHR mRNA in BLT-1 cells (6.9, 2.6, 1.7, and 1.2 kilobases), and treatment at 10-100 ng/ml of IGF-I increased steady-state levels of LHR mRNAs in coordinate fashion up to 2. 2-fold. IGF-I (30 ng/ml) induced a time-dependent increase in [125I]hCG binding after a lag period of 2-6 h when studied up to 48 h, with a subsequent decrease. A similar response with steady increase up to 72 h was observed in total LHR mRNA. To elucidate the molecular mechanism of IGF-I action on LHR mRNA expression, we measured the transcription rate of the LHR gene by nuclear run-off assay and assessed transcript stability by the actinomycin D blocking method. The results showed that IGF-I treatment had no effect on the transcription rate of the LHR gene, whereas the half-life (t1/2) of LHR mRNA was significantly prolonged (IGF-I-treated cells, 30 +/- 3.8 h; controls, 17 +/- 2.5 h). Furthermore, IGF-I at 30 ng/ml and 100 ng/ml increased the expression of LHR promoter-driven luciferase and cytomegalovirus-promoter driven ss-galactosidase activities in BLT-1 cells; however, the former increased only marginally more than the latter. This suggests that the increase of LHR mRNA by IGF-I in Leydig cells is mainly due to increased mRNA stability.

Nitric oxide inhibits Leydig cell steroidogenesis.

Testicular macrophages as well as endothelial cells, which are intimately associated with Leydig cells, constitute a potential source of paracrine nitric oxide (NO) in the testis. In the present study, we investigated the effect of NO donors on MA-10 murine Leydig tumor cell line and rat Leydig cell steroidogenesis. We show that NO donors inhibit human CG-induced steroidogenesis in both type of cells. We also studied NO mechanism of action. Contrary to what is observed in many other systems, NO inhibitory effect on Leydig cell steroidogenesis is not mediated by cyclic GMP (cGMP) because NO fails to increase cGMP production, and cGMP analogs do not reproduce NO effect. NO does not modify the production of cAMP, the main second messenger that mediates gonadotropin action. When we studied NO effect over the steroidogenic pathway in MA-10 cells, we found that NO was inhibiting the conversion of cholesterol to pregnenolone. Taken together these results show an inhibitory effect of NO donors on Leydig cell steroidogenesis, and suggest that NO can be directly inhibiting cholesterol side-chain cleavage enzyme (cytochrome P450scc) as it does with other heme proteins, including different cytochromes P450.

Nitric oxide inhibits Leydig cell steroidogenesis

Testicular macrophages as well as endothelial cells, which are intimately associated with Leydig cells, constitute a potential source of paracrine nitric oxide (NO) in the testis. In the present study, we investigated the effect of NO donors on MA-10 murine Leydig tumor cell line and rat Leydig cell steroidogenesis. We show that NO donors inhibit human CG-induced steroidogenesis in both type of cells. We also studied NO mechanism of action. Contrary to what is observed in many other systems, NO inhibitory effect on Leydig cell steroidogenesis is not mediated by cyclic GMP (cGMP) because NO fails to increase cGMP production, and cGMP analogs do not reproduce NO effect. NO does not modify the production of cAMP, the main second messenger that mediates gonadotropin action. When we studied NO effect over the steroidogenic pathway in MA-10 cells, we found that NO was inhibiting the conversion of cholesterol to pregnenolone. Taken together these results show an inhibitory effect of NO donors on Leydig cell steroidogenesis, and suggest that NO can be directly inhibiting cholesterol side-chain cleavage enzyme (cytochrome P450scc) as it does with other heme proteins, including different cytochromes P450.

Prolactin and MA-10 Leydig cell steroidogenesis: biphasic effects of prolactin and signal transduction.

The present investigation was designed to study the direct role of PRL on testicular Leydig cell steroidogenesis, using the MA-10 murine Leydig tumor cell line as a model system. We have previously reported on the presence of specific PRL binding sites in those cells, and we now demonstrate the functionality of those sites and the biological responses induced by the binding of PRL. When cultured MA-10 cells were exposed for 24 h to increasing concentrations of PRL, washed, and then subjected to a 3-h human CG (hCG) stimulation test, a clear dose-dependent biphasic effect of PRL on the steroidogenic response was observed, even though PRL had no effect on MA-10 cell proliferation: at low PRL concentrations (0.1-10 ng/ml), hCG-induced steroidogenesis was stimulated (maximal stimulation by 1 ng/ml PRL being 200-250% of control); at higher concentrations, hCG-induced steroidogenesis was inhibited (60% inhibition was achieved by 1000 ng/ml PRL). When steroidogenesis was induced with various concentrations of cholera toxin, instead of hCG, no effect of the prior exposure to increasing concentrations of PRL was observed, indicating that PRL acts either at the level of the LH/hCG receptor or at some stage proximal to adenylate cyclase. Indeed, further study revealed that 24 or 72 h exposure of MA-10 cells to PRL caused a dose-dependent reduction in hCG binding. Thus, the maximal inhibition of 62% after 72 h with 500 ng/ml PRL, may explain, at least in part, the inhibitory effects of high PRL concentrations on hCG-induced progesterone secretion. Evidence demonstrating possible involvement of a pertussis toxin-(PT-)sensitive G protein in the signal transduction mechanism of PRL receptors is also presented: 1. GTP caused a dose-dependent reduction in affinity (Ka) of PRL binding by its receptors (from Ka = 1.66 +/- 0.2 x 10(9) M(-1) for control MA-10 cell membranes to Ka 3.03 +/- 0.6 x 10(8) M(-1) for membranes incubated with 8 mM GTP). 2. Prior exposure of MA-10 cells to PRL (10 pg/ml) caused a significant reduction in the ability of a 44-kDa membrane protein to undergo PT-induced [32P]ADP-ribosylation. These results demonstrate that MA-10 Leydig cells possess highly specific and biologically functional PRL receptors mediating direct and dose-dependent biphasic effects of PRL on hCG-induced progesterone secretion. These cells thus offer a suitable model to study the mechanism(s) of PRL action and signal transduction of its receptor on a physiologically relevant differentiated function.

Immunocytochemical Localization and Endogenous Synthesis of Apolipoprotein E in Testicular Leydig Cells1

Apolipoprotein E (apoE) is synthesized by a wide variety of tissues, including those involved in steroidogenesis; its function in most extrahepatic tissues is unknown. Significant amounts of apoE mRNA have been detected in the testis, but the cellular origin of this material has not yet been determined. The purpose of the present study was to determine whether the steroidogenic cells of the testis synthesize apoE. We localized apoE in the testis of mice by an avidin-biotin peroxidase technique using a cross-reactive anti-rat apoE antibody. ApoE immunoreactivity was strongest in interstitial cells but was also diffusely localized throughout the seminiferous tubules. Acute treatment of mice with hCG diminished apoE immunoreactivity in the testis. A murine Leydig tumor cell line (I-10 cells) also demonstrated apoE immunoreactivity, suggesting that at least one source of interstitial apoE is the Leydig cell. Normal Leydig cells were subsequently isolated from control and hCG-treated mice using Percoll density gradients. Isolated hepatocytes, I-10 cells, and Leydig cells (with or without hCG in vitro) were incubated in the presence of [35S]methionine. A 35S-labeled protein of approximately 33-35 kDa was immunoprecipitated from the cells and media of all three types of preparations using whole antiserum or affinity-purified antibody. Preincubating the antibodies with apoE-containing murine very low-density lipoprotein or purified rat apoE eliminated these bands. Leydig cell apoE synthesis and secretion were decreased by hCG treatment in vivo and/or in vitro. These data suggest that apoE is synthesized by normal and transformed Leydig cells and may play a role in sterol transport in the testis.

Endothelin-1 promotes steroidogenesis and stimulates protooncogene expression in transformed murine Leydig cells.

The effects of endothelin-1 (ET-1), a potent vasoconstrictor and mitogen to various cell types, on proliferation and differentiated functions of the murine Leydig tumor cell line MA-10 were investigated. Kinetic binding experiments at room temperature showed that [125I] ET-1 bound to MA-10 cells and reached equilibrium in 2 h. The data from competitive binding experiments yielded an apparent single class of high affinity binding sites characterized by a Kd and maximum binding capacity of 1 nM and 59 fmol/10(6) cells, respectively. For steroidogenic assays, cells were incubated with ET-1 (1 pM to 1 microM) and with epidermal growth factor (10 ng/ml) for 4 h at 37 C, and the progesterone levels in the medium were measured by RIA. Like epidermal growth factor, ET-1 caused about a 6-fold increase in progesterone production. ET-1 also enhanced the transient expression of the protooncogenes c-jun and c-myc by 3- and 2-fold, respectively. For proliferation studies, ET-1 (1 pM to 1 microM) was added to quiescent MA-10 cells for 24 h, and cell counts were performed; no increase in cell number was observed. The results of this study demonstrate that MA-10 cells possess high affinity binding sites for ET-1 and that ET-1 stimulates progesterone production and protooncogene expression, but not mitosis in this cell line.

Conversion of lysine 91 to methionine or glutamic acid in human choriogonadotropin alpha results in the loss of cAMP inducibility.

Human choriogonadotropin (hCG) is a heterodimeric glycoprotein hormone. The alpha subunit comprises 92 amino acids, of which 6 are Lys residues (Morgan, F.G., Birken, S., and Canfield, R.E. (1975) J. Biol. Chem. 250, 5247-5258). Our photoaffinity-labeling studies indicate that several of these Lys residues make contact with the lutropin receptor and are covalently cross-linked to the receptor. Lys-91 of the alpha subunit is of interest because deletion of the two alpha C-terminal residues, Lys-91 and Ser-92, results in a significant reduction in the bioactivity of lutropin and thyrotropin (Cheng, K.-W., Glazer, A.N., and Pierce, J.G. (1973) J. Biol. Chem. 248, 7930-7937). To determine the importance of Lys-alpha 91, we substituted it with Arg, Met, or Glu. The resulting mutant alpha cDNA constructs were co-transfected into CHO cells with the wild type hCG beta cDNA construct. Secreted hCG dimers were assayed for binding to receptors on porcine granulosa cells and stimulation of cAMP synthesis in a murine Leydig tumor cell line. The natural hCG, wild type hCG, and all mutant hCGs recognized the receptor, although with somewhat divergent affinities. However, there was a striking difference in the ability of cAMP induction. The natural hCG, wild type hCG, and Lys-91----Arg mutant hCG induced cAMP synthesis, whereas the Lys-91----Met and Lys-91----Glu mutants did not. These results demonstrate that Lys-91 is important for receptor modulation in the stimulation of cAMP synthesis.

N-Linked Oligosaccharides Are not Required for Hormone Binding of the Lutropin Receptor in a Leydig Tumor Cell Line and Rat Granulosa Cells

We have examined roles of carbohydrates of the lutropin receptor in a murine Leydig tumor cell line (MLTC) and primary cultures of rat granulosa cells. We approached this issue by deglycosylating mature receptors with glycosidases and by preventing glycosylation of nascent receptors with tunicamycin B2, an inhibitor of protein glycosylation but not protein synthesis. Deglycosylation of mature receptors with neuraminidase, N-glycanase or both did not affect ligand binding capacity. Regardless of glycosidase treatment, the number of hormone binding sites was similar. The Kas for native receptors, asialoreceptors and aglycoreceptors, are also comparable, being 2.0 x 10(9) M-1, 1.9 x 10(9) M-1 and 1.7 x 10(9) M-1 respectively. In contrast, cells treated with tunicamycin B2 failed to bind the hormone. These results demonstrate that N-oligosaccharides of mature lutropin receptors are not required for ligand binding. In addition, our data suggest, for the first time, that N-glycosylation of the receptor may be necessary for expressing functional receptors on the cell surface and that there exist striking similarities in roles of oligosaccharides of lutropin and its receptor.

Molecular cloning and expression of murine guanylate cyclase/atrial natriuretic factor receptor cDNA.

The potent diuretic and natriuretic peptide hormone atrial natriuretic factor (ANF), with vasodilatory activity also stimulates steroidogenic responsiveness in Leydig cells. The actions of ANF are mediated by its interaction with specific cell surface receptors and the membrane-bound form of guanylate cyclase represents an atrial natriuretic factor receptor (ANF-R). To understand the mechanism of ANF action in testicular steroidogenesis and to identify guanylate cyclase/ANF-R that is expressed in the Leydig cells, the primary structure of murine guanylate cyclase/ANF-R has been deduced from its cDNA sequence. A cDNA library constructed from poly(A+) RNA of murine Leydig tumor (MA-10) cell line was screened for the membrane-bound form of ANF-R/guanylate cyclase sequences by hybridization with a rat brain guanylate cyclase/ANF-R cDNA probe. The amino acid sequence deduced from the cDNA shows that murine guanylate cyclase/ANF-R cDNA consists of 1057 amino acids with 21 amino acids comprising the transmembrane domain which separates an extracellular ligand-binding domain (469 amino acid residues) and an intracellular guanylate cyclase domain (567 amino acid residues). Upon transfection of the murine guanylate cyclase/ANF-R cDNA in COS-7 cells, the expressed protein showed specific binding to 125I-ANF, stimulation of guanylate cyclase activity and production of intracellular cGMP in response to ANF. The expression of guanylate cyclase/ANF-R cDNA transfected in rat Leydig tumor cells stimulated the production of testosterone and intracellular cGMP after treatment with ANF. The results presented herein directly show that ANF can regulate the testicular steroidogenic responsiveness in addition to its known regulatory role in the control of cardiovascular homeostasis.