Effect of lutropin and cycloheximide on lutropin receptors and cyclic AMP production in Leydig tumour cells in vitro.

A system to study lutropin-induced desensitization of tumour Leydig cells in vitro has been investigated. Tumour Leydig cells were purified on a Percoll gradient and then incubated for 30 min with lutropin (0-1000ng/ml). The cells were then washed and incubated in suspension media at 32 degrees C. 125I-labelled human choriogonadotropin binding and basal and lutropin-stimulated cyclic AMP production were determined at various times. Initially the cells showed a dose-dependent decrease in human choriogonadotropin binding (1.18 and 0.13fmol/10(6) cells respectively) followed by an increase at 1 h (2.32 and 0.87fmol/10(6) cells respectively). Human choriogonadotropin binding remained elevated in the cells pre-incubated without lutropin, whereas the cells pre-incubated with lutropin showed a dose-dependent decrease over the next 10 h (2.20-0.18fmol/10(6) cells respectively). Basal production of cyclic AMP initially reflected the pre-incubation conditions (1.17-21.19ng/10(6) cells per h for 0-1000ng of lutropin/ml respectively). However, by 1 h there was a marked rise in basal cyclic AMP production which returned to the initial lower values by 4 h. At all time intervals studied, lutropin-induced cyclic AMP production showed a decrease that was proportional to lutropin concentration in the pre-incubated media. The decreases in human choriogonadotropin binding produced by pre-incubations with lutropin (100ng/ml) was partially inhibited by the presence of cycloheximide in the pre-incubation media and totally prevented by the continuous presence of cycloheximide. These results demonstrate that desensitization of tumour Leydig cells occurs after exposure to lutropin in vitro. This desensitization involves both a loss of plasma membrane receptors for lutropin and lutropin-stimulated adenylate cyclase. These events can be prevented by cycloheximide and are therefore probably dependent on protein synthesis.     

The mechanism of action of lutropin on regulator protein(s) involved in Leydig-cell steroidogenesis

The dependence on lutropin of the synthesis of a proposed short-half-life protein regulator involved in Leydig-cell steroidogenesis was investigated. This was carried out by determining the effect of the protein-synthesis inhibitor cycloheximide, added before and during incubations with lutropin (and/or dibutyryl cyclic AMP), on the rate of testosterone production in suspensions of purified Leydig cells from adult rat testes. The Leydig cells were preincubated in Eagle's medium for 2.5h followed by 30min incubation with and without cycloheximide. The inhibitor was removed by washing the cells and then lutropin was added and testosterone concentrations were determined after incubation of the cells at 32 degrees C. No significant effect of cycloheximide pretreatment on lutropin-stimulated steroidogenesis was found during 60min incubation. This was in contrast with the complete inhibiting effect of cycloheximide when it was added with the lutropin. The pretreatment experiments with cycloheximide were repeated in the presence of dibutyryl cyclic AMP and elipten phosphate (to inhibit cholesterol side-chain cleavage) followed by incubation with lutropin. After 5, 10, 20 and 60min of incubation, testosterone concentrations were 61+/-3, 46+/-3, 27+/-4 and 18+/-4% lower than in the cells pretreated without cycloheximide respectively (means+/-s.e.m., n=4-6). In the cells not pretreated with cycloheximide and in the absence of lutropin, testosterone production increased from 1.36+/-0.5 to 36.5+/-1.0ng/10(6) cells during 20min of incubation, after which no further increase occurred. Pretreatment of the cells with cycloheximide decreased these testosterone concentrations by 65, 46, 42 and 36% in the 5, 10, 20 and 60min incubations respectively (mean values, n=2-4). It is apparent from these results that inhibition of steroidogenesis only occurs if protein synthesis is inhibited in the presence of lutropin or cyclic AMP. A new hypothesis is put forward to explain these findings: it is proposed that lutropin affects the stability of a precursor of a regulator protein by converting it from a stable (inactive) to an unstable (active) form with a short half-life.     

Effect of lutropin on phosphorylation of endogenous proteins in testis Leydig cells. Correlation with testosterone production

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.     

Desensitization of tumour Leydig cells by lutropin: evidence for uncoupling of the lutropin receptor from the guanine nucleotide-binding protein

Purified rat Leydig tumour cells were pretreated with lutropin and the effect on the subsequent response to lutropin was determined. Maximal cyclic AMP production was achieved with the same concentration of lutropin in control and lutropin-pretreated cells; however, the maximum stimulated level in pretreated cells was only 30% of controls. The sensitivity to lutropin was decreased in lutropin-pretreated cells [ED(50) (dose that produces a response that is 50% of the maximum response) 60+/-5.7ng/ml and 8+/-1.8ng/ml (mean+/-s.d., n=3) for controls], as was the rate of maximal cyclic AMP production (0.58, compared with 1.89pmol/10(6) cells per min for controls). However, cholera-toxin-stimulated cyclic AMP production was not decreased by lutropin pretreatment, and a potentiation was seen at all time points studied (up to 6h). Pre-incubation with lutropin caused a decrease in specific (125)I-labelled human choriogonadotropin binding; however, this decrease was abolished if the cells were washed under acidic conditions (pH3.0 for 2min at 4 degrees C), indicating that occupation but not loss of the lutropin receptors had taken place. The effect of pretreating the cells with lutropin on adenylate cyclase activity in purified plasma membranes was also investigated. In plasma membranes from control cells both guanosine 5'-[beta,gamma-imido]triphosphate [p(NH)ppG] plus lutropin and NaF plus lutropin caused a 50-60-fold linear increase in cyclic AMP production over 40min compared with 15-fold with p(NH)ppG and 6-fold with lutropin alone. In plasma membranes isolated from lutropin-treated cells the NaF-plus-lutropin- and the p(NH)ppG-stimulated cyclic AMP production rates were unchanged but no effect of lutropin could be demonstrated with or without added p(NH)ppG. In contrast the plasma membranes from dibutyryl cyclic AMP-treated cells had similar cyclic AMP production rates to control cells with all stimulants studied. The present evidence obtained from studies both with intact cells and with isolated plasma membranes indicates that the initial lutropin-induced desensitization of the rat Leydig tumour cell is due to a lesion in the hormone-receptor coupling to the guanine nucleotide regulatory protein. This process is apparently not mediated by cyclic AMP.     

Glucose-stimulated protein phosphorylation in the pancreatic islet

The effect of inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism on steroidogenesis in rat testis Leydig cells and rat tumour Leydig cells has been investigated. In the presence of nordihydroguaiaretic acid [NDGA; 4,4'-(2,3- dimethylbutan -1,4- diyl )bis[1,2- benzendiol ]], 5,8,11,14-eicosatetraynoic acid (ETYA), BW 755C [3-amino-1-[3-(trifluoromethyl)phenyl]-2-pyrazoline hydrochloride] and benoxaprofen [ Opren ; 2-(2-p-chlorophenyl- benzoxazol -5-yl)propionic acid)] (which inhibit lipoxygenase activity), but not indomethacin and aspirin (which inhibit cyclo-oxygenase activity), a dose-related inhibition of lutropin (LH)-stimulated testosterone and pregnenolone production was obtained (ID50 values of 2.5, 30, 25 and 30 microM for NDGA, ETYA, BW 755C and benoxaprofen were obtained, respectively). BW 755C and benoxaprofen had no significant effect on LH-stimulated cyclic AMP production except at the highest concentrations examined (330 and 380 microM, respectively), whereas NDGA and ETYA inhibited LH-stimulated cyclic AMP production in a dose-dependent manner (ID50 7.0 and 22 microM respectively). However, NDGA and ETYA also caused a dose-dependent inhibition of dibutyryl cyclic AMP-stimulated testosterone and pregnenolone production. The metabolism of exogenous ( 22R )-hydroxycholesterol or pregnenolone to testosterone by Leydig cells was not inhibited by either NDGA, ETYA or indomethacin. At low concentrations of NDGA and ETYA a significant increase in the conversion of both pregnenolone and ( 22R )-hydroxycholesterol to testosterone was obtained. Studies in which the metabolism of [14C]arachidonic acid by purified rat tumour Leydig cells was investigated indicate that products are formed by tumour Leydig cells that have similar mobilities in a thin layer chromatography system to 5-L-hydroxy-6,8,11,14-eicosatetraenoic acid, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and leukotriene B4. The formation of these products was inhibited to varying degrees by NDGA, BW 755C and benoxaprofen but not by aspirin and indomethacin. These studies demonstrate for the first time that inhibition of lipoxygenase activity but not cyclo-oxygenase activity causes an inhibition of LH- and dibutyryl cyclic AMP-stimulated steroid production and suggest a stimulatory role for products of the lipoxygenase pathway of arachidonic acid metabolism in steroidogenesis. The site of this stimulation is apparently distal to the production of cyclic AMP and before the side chain cleavage of cholesterol.     

Evidence for the requirement of proteolysis in LH stimulated cyclic AMP production and steroidogenesis in Leydig cells.

We have investigated the effect of protease activity on cyclic AMP production and steroidogenesis in rat testis, mouse testis and mouse tumour Leydig (MA10) cells. LH-, dibutyryl cyclic AMP-, and forskolin-stimulated steroidogenesis, but not 22R(OH) cholesterol conversion to pregnenolone, was inhibited by protease inhibitors. In mouse Leydig cells, LH but not forskolin or cholera toxin stimulated cyclic AMP production was inhibited by protease inhibitors. These results suggest that steroidogenesis in Leydig cells requires proteolysis before the conversion of cholesterol to pregnenolone. In the mouse but not rat Leydig cells, LH-stimulated cyclic AMP production is also dependent on proteolysis.     

Regulation of the synthesis of lutropin-induced protein in rat testis Leydig cells

The mechanism of action of lutropin on the stimulation of the synthesis of a specific lutropin-induced protein in rat testis Leydig cells was investigated. Lutropin-induced protein has a mol.wt. of approx. 21000 and is detected by labelling the Leydig-cell proteins with [³⁵S]methionine, followed by separation by polyacrylamide-gel electrophoresis and radioautography of the dried gel. The incorporation of ³⁵S into lutropin-induced protein was used as an estimate for the synthesis of the protein. Incubation of Leydig cells with dibutyryl cyclic AMP or cholera toxin also resulted in the stimulation of synthesis of the protein. Synthesis of lutropin-induced protein, when maximally stimulated with 100ng of lutropin/ml, could not be stimulated further by addition of dibutyryl cyclic AMP. Addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, further increased synthesis of the protein in the presence of a submaximal dose of lutropin (10ng/ml) but not in the absence of lutropin or with maximal amounts of lutropin (100 and 1000ng/ml). Actinomycin D prevented the effect of lutropin on the stimulation of lutropin-induced protein synthesis when added immediately or 1h after the start of the incubation, but not when added after 5–6h. This is interpreted as reflecting that, after induction of mRNA coding for lutropin-induced protein, lutropin had no influence on the synthesis of the protein in the presence of actinomycin D. Synthesis of the protein was also stimulated in vivo by injection of choriogonadotropin into rats 1 day after hypophysectomy, and the time course of this stimulation of lutropin-induced protein synthesis in vivo was similar to that obtained by incubating Leydig cells in vitro with lutropin. From these results it is concluded that stimulation of lutropin-induced protein synthesis by lutropin is most probably mediated by cyclic AMP and involves synthesis of mRNA.     

Lutropin increases phosphorylation of a 33 000-dalton ribosomal protein in rat tumour Leydig cells.

Addition of lutropin (luteinizing hormone, 'LH') and 3-isobutyl-1-methylxanthine to tumour Leydig cells stimulated phosphorylation of five proteins, of 17 000, 22 000, 24 000, 33 000 and 57 000 Da. Phosphorylation of these proteins coincided with increased pregnenolone production. Phosphorylation of a 33 000-Da protein was lutropin-dependent in Leydig cells isolated from a Leydig-cell tumour, from immature testes or from mature testes. In tumour Leydig cells this protein was present in the small ribosomal subunit. Incubation of tumour Leydig cells with either cycloheximide or puromycin inhibited both basal and lutropin-dependent pregnenolone production, by approx. 90% and 98% respectively. In contrast, basal pregnenolone production in Leydig cells from immature and mature testes was insensitive to cycloheximide or puromycin. Cycloheximide or puromycin increased phosphorylation of the 33 000-Da phosphoprotein by approx. 130% and 80% respectively (effect of lutropin/3-isobutyl-1-methylxanthine on phosphorylation: 100%). The molecular mass, the subcellular localization and the sensitivity to phosphorylation in the presence of inhibitors of protein synthesis indicate that the 33 000-Da protein could be similar to ribosomal protein S6.     

Multistep regulation of Leydig cell function

LH controls Leydig cell steroidogenesis by interaction with specific membrane receptors initiating membrane coupling events. Stimulation of the androgen pathways occurs mainly through cAMP mediated mechanism including LH induced guanyl nucleotide binding, membrane phosphorylation and adenylate cyclase activation. cAMP dependent kinase activation presumably causes phosphorylation of key proteins of the steroidogenic pathway and consequent increase in testosterone production. The hormone also appears to facilitate the androgen stimulus by a cyclic AMP independent mechanism located at the plasma membrane or intracellular sites. The stimulatory event can be negatively influenced by the action of certain peptide hormones (i.e. angiotensin II) through the guanyl nucleotide inhibitory subunit of adenylate cyclase (Gi). In recent studies we have presented evidence for a Ca2+ sensitive kinase system present in purified cell membranes. Gpp(NH)p, GTP, and phospholipid in presence of nanomolar Ca2+ induce phosphate incorporation into Mr 44,500 substrate with marked inhibition at microM Ca2+. Similarly a biphasic pattern of activation was observed with adenylate cyclase activity. Membrane phosphorylation may be a modifier of LH-stimulated adenylate cyclase activity and possibly other LH induced actions in the activated Leydig cell membrane. Furthermore we have defined the stimulatory effects of forskolin on all Leydig cell cyclic AMP pools and have provided additional evidence of functional compartmentalization and/or cAMP independent facilitory stimulus of steroidogenesis by the trophic hormone. The demonstration of a novel high affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation mediated by the Gi subunit of adenylate cyclase has provided a new approach for direct evaluation of functional inhibitory influence of Gi subunit in the Leydig cell. The cultured fetal Leydig cell system has provided a useful model to elucidate mechanisms involved in the development of gonadotropin induced estradiol mediated desensitization of steroidogenesis. We have isolated from the fetal testis a small population (2-5% of total) of transitional cells with morphological characteristics of cells found in 15 day postnatal testis but functional capabilities of the adult cell. We have also demonstrated after appropriate treatment (i.e. estrogen, and frequent or a high gonadotropin dose) the emergence of a functional adult-like cell type from the fetal Leydig cell population.     

The role of calcium in luteinizing hormone-releasing hormone agonist (ICI 118630)-stimulated steroidogenesis in rat Leydig cells.

The luteinizing hormone-releasing hormone (LHRH) agonist ICI 118630 was found to increase testosterone production in purified rat testis Leydig cells in a concentration- and time-dependent manner, but no consistent changes in cyclic AMP levels were detectable. The stimulation of steroidogenesis by LHRH agonist was found to be dependent on the concentration of Ca2+ in the incubation medium; at least 1 mM was required. The calcium ionophore A23187 mimicked the effects of the LHRH agonist on steroidogenesis, and addition of both compounds together did not further increase testosterone production. The calcium ionophore caused a small increase in cyclic AMP which was independent of the concentration of the ionophore and of the calcium concentrations. The evidence obtained in this study indicates that LHRH agonist-stimulated steroidogenesis in rat testis Leydig cells is primarily mediated by calcium and not cyclic AMP.     

Resensitization of lutropin-desensitized tumour Leydig-cell adenylate cyclase with human erythrocyte membranes.

Purified rat tumour Leydig cells were pretreated with or without lutropin (1 h at 32 degrees C). The plasma membranes were then isolated and the adenylate cyclase activity measured in the presence of freshly prepared or heat-inactivated (1 h at 60 degrees C) human erythrocyte membranes. In plasma membranes from control cells in the presence of heat-inactivated human erythrocyte membranes both guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) plus lutropin and NaF caused a 45--50-fold increase in cyclic AMP production over 30 min compared with 12--13 fold p[NH[ppG and 2--3-fold with lutropin alone. In plasma membranes isolated from lutropin-pretreated cells the NaF- and the p[NH]ppG-stimulated cyclic AMP production rates were unchanged, but no effect of lutropin could be demonstrated with or without added p[NH]ppG. However, after mixing lutropin-desensitized Leydig tumour-cell plasma membranes with freshly prepared human erythrocyte plasma membranes, the adenylate cyclase activity in the presence of lutropin, p[NH]ppG, lutropin plus p[NH]ppG and NaF were similar to those of control cell plasma membranes treated in the same manner. The possible mechanisms of this reversal of lutropin-induced desensitization by human erythrocytes are discussed.     

Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase.

The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase involves a cyclic AMP-dependent lesion that is after the G-protein.     

Lutropin-dependent protein phosphorylation and steroidogenesis in rat tumour Leydig cells

Tumour Leydig cells have been incubated in the presence or absence of lutropin (luteinizing hormone, ;LH'). Stimulation of cells with lutropin (1000ng/ml) in the presence of 1-methyl-3-isobutylxanthine (0.25mm) resulted in increased steroid production and increased protein phosphorylation. When pregnenolone metabolism was inhibited, basal pregnenolone production was 26.9+/-7.4ng/60min per 10(6) cells; stimulated production was 156.1+/-39.5ng/60min per 10(6) cells (means+/-s.d., n=4). Lutropin-dependent phosphorylated proteins of molecular mass 17000, 22000, 24000, 33000 and 57000Da were detected. A significant increase of [(32)P]P(i) incorporation into these phosphorylated proteins was observed concomitant with the increased pregnenolone production. The occurrence of the phosphoproteins in nuclei, mitochondria and postmitochondrial-supernatant was investigated. The 17000Da phosphoprotein was found in the nuclear fraction, whereas the 22000, 24000, 33000 and 57000Da phosphoproteins were localized in the postmitochondrial-supernatant fraction. Of the cholesterol-side-chain-cleavage activity, 80.3+/-6.1% (mean+/-s.d., n=5) was present in the mitochondrial fraction isolated from tumour Leydig cells, and this activity was 2.5-fold increased when cells had been preincubated with lutropin/1-methyl-3-isobutylxanthine (basal production: 194.6+/-28.6ng/30min per mg of protein; lutropinstimulated production: 498.8+/-91.5ng/30min per mg of protein; means+/-s.d., n=3). The similarities in the kinetics of the phosphorylation of proteins and the pregnenolone production after addition of lutropin/1-methyl-3-isobutylxanthine indicate that the phosphoproteins could be involved in the lutropin-dependent increase in steroidogenesis in tumour Leydig cells. It remains to be demonstrated, however, to what extent the phosphoproteins outside the mitochondria can influence the cholesterol-side-chain-cleavage activity inside the mitochondria.     

The adiponectin paralog C1q/TNF-related protein 3 (CTRP3) stimulates testosterone production through the cAMP/PKA signaling pathway

CTRP3, a paralog of adiponectin, is a member of the C1q and tumor necrosis factor (TNF)-related protein (CTRP) superfamily. It is expressed at high levels in adipose tissue and has recently emerged as a novel adipokine. In the present study, we provide the first evidence for a physiological role of the new adipokine, CTRP3, in the reproductive system. CTRP3 was specifically expressed in interstitial Leydig cells, where testosterone is produced, in the adult mouse testis. CTRP3 increased testosterone production by TM3 mouse Leydig cells in a dose-dependent manner. The increased testosterone production was linked to upregulation of steroidogenic proteins expression, such as steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage cytochrome P450 (P450scc). Moreover, increases in intracellular cyclic AMP (cAMP) concentrations and the phosphorylation of cAMP-response element binding protein (CREB) in CTRP3-stimulated TM3 Leydig cells were observed. Inhibition of this signaling pathway by a specific protein kinase A (PKA) inhibitor, H89, blocked testosterone production in CTRP3-stimulated Leydig cells, suggesting that the stimulatory effect of CTRP3 on testosterone production is associated with activation of the cAMP/PKA signaling pathway. Thus, our results demonstrate a physiological role for CTRP3 in testicular steroidogenesis and provide novel insights in the intracellular mechanisms activated by this protein.     

Correlation of protein kinase activation and testosterone production after stimulation of Leydig cells with luteinizing hormone.

The effect of different doses of luteinizing hormone on activation of protein kinases, cyclic AMP and testosterone production was studied in purified rat testis Leydig-cell preparations in the presence of 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor). In addition, the nature of the protein kinases present in these cells and other tissues was investigated. The following results were obtained. 1. With all the amounts of luteinizing hormone used (0.1-1000 ng/ml), both activation of protein kinase and stimulation of testosterone production were demonstrated. With the lowest amount of luteinizing hormone (0.1 ng/ml), an 8.4+/-0.9% (S.E.M.,n=6) stimulation of protein kinase activation occurred, increasing to 100% with 1000 ng/ml, compared with 3.2+/-1.0%(S.E.M.,n=7) and 100% stimulation of testosterone production with 0.1 and 100 ng/ml respectively. 2. With amounts of luteinizing hormone up to 1 ng/ml (which gave half-maximal stimulation of testosterone production) no detectable increases in net cyclic AMP production were obtained. With higher amounts of luteinizing hormone, cyclic AMP production increased, but maximal production was not reached with 1000 ng/ml. 3. Two isoenzymic forms of protein kinase were present in Leydig cells and seminiferous tubules; type I was eluted with 0.075 M-and type II with 0.22-0.25 m-NaCl from DEAE-cellulose columns. 4. The protein kinase activity was not affected by the presence of erythrocytes in the Leydig-cell preparation, but varied depending on the type of histone used as substrate (histone F2b greater than mixed greater than histone F1).     

Gonadotropin binding and stimulation of cyclic adenosine 3':5'-monophosphate and testosterone production in isolated Leydig cells.

Gonadotropin binding and stimulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) formation and testosterone synthesis were studied in collagenase-dispersed interstitial cells from the adult rat testis. Binding of 125I-human chorionic gonadotropin (hCG) by isolated Leydig cells was of high affinity (Ka = 10(10) M-1) and low capacity, equivalent to approximately 6000 sites/cell. The binding data were consistent with the presence of a single order of receptors, with no interaction between binding sites. Stimulation of testosterone synthesis by increasing concentrations of hCG was completely dissociated from changes in cyclic AMP formation, and maximum activation of steroidogenesis was induced by hCG concentrations which had no effect upon cyclic AMP production. Kinetic analysis of gonadotropin-induced responses in dispersed Leydig cells also showed a marked dissociation between steroidogenesis and cyclic nucleotide formation. Low concentrations of hCG caused maximum stimulation of testosterone production which was not accompanied by a rise in cyclic AMP formation at any time after addition of gonadotropin. Higher concentrations of hCG caused marked elevations of cyclic AMP at progressively earlier time intervals, but did not alter the 20 to 30 min lag period required for induction of testosterone synthesis. These observations indicated that occupancy of gonadotropin receptors occurs over a much wider range of hCG concentration than that required for maximum steroidogenesis.     

Stimulation of cholesterol side-chain cleavage by a luteinizing-hormone-releasing hormone (luliberin) agonist (ICI 118630) in rat Leydig cells.

The action of a luliberin (luteinizing-hormone-releasing hormone) agonist (ICI 118630) and lutropin (luteinizing hormone) on the activity of the cytochrome P-450 cholesterol side-chain cleavage enzyme in rat Leydig cells has been investigated. This has been carried out by studying the metabolism of exogenous (22R)-22- and 25-hydroxycholesterol to testosterone. It was found that both hydroxycholesterols increased testosterone production to higher levels than achieved by lutropin alone. Addition of luliberin agonist but not lutropin was found to increase further the metabolism of the hydroxycholesterol to testosterone; this occurred in the presence of saturating and subsaturating levels of the hydroxycholesterols. This effect of luliberin agonist was potentiated in the presence of lutropin. The protein synthesis inhibitor, cycloheximide, inhibited the luliberin agonist-induced stimulation of the hydroxycholesterol metabolism. At low calcium levels (1.1 microM), testosterone production was increased by addition of (22R)-22-hydroxycholesterol but the luliberin agonist effect was negated. The calmodulin inhibitor trifluoperazine inhibited (22R)-22-hydroxycholesterol-stimulated steroidogenesis and negated the luliberin agonist effect. These results indicate that luliberin agonist specifically increases the synthesis of the cytochrome P-450 cholesterol side-chain cleavage enzyme in rat testis Leydig cells.     

The role of Ca2+ in steroidogenesis in Leydig cells. Stimulation of intracellular free Ca2+ by lutropin (LH), luliberin (LHRH) agonist and cyclic AMP.

The requirements of purified rat Leydig cells for intra- and extra-cellular Ca2+ during steroidogenesis stimulated by LH (lutropin), cyclic AMP analogues and LHRH (luliberin) agonist were investigated. The intracellular Ca2+ concentrations ([Ca2+]i) were measured by using the fluorescent Ca2+ chelator quin-2. The basal [Ca2+]i was found to be 89.4 +/- 16.6 nM (mean +/- S.D., n = 25). LH, 8-bromo cyclic AMP and dibutyryl cyclic AMP increased [Ca2+]i, by 300-500 nM at the highest concentrations of each stimulator, whereas LHRH agonist only increased [Ca2+]i by a maximum of approx. 60 nM. Low concentrations of LH (less than 1 pg/ml) and all concentrations of LHRH agonist increased testosterone without detectable changes in cyclic AMP. With amounts of LH greater than 1 pg/ml, parallel increases in cyclic AMP and [Ca2+]i occurred. The steroidogenic effect of the LHRH agonist was highly dependent on extracellular Ca2+ concentration ([Ca2+]e), whereas LH effects were only decreased by 35% when [Ca2+]e was lowered from 2.5 nM to 1.1 microM. No increase in [Ca2+]i occurred with the LHRH agonist in the low-[Ca2+]e medium, whereas LH (100 ng/ml) gave an increase of 52 nM. It is concluded that [Ca2+]i can be modulated in rat Leydig cells by LH via mechanisms that are both independent of and dependent on cyclic AMP, whereas LHRH-agonist action on [Ca2+]i is independent of cyclic AMP. The evidence obtained suggests that, at sub-maximal rates of testosterone production, Ca2+, rather than cyclic AMP, is the second messenger, whereas for maximum steroidogenesis both Ca2+- and cyclic-AMP-dependent pathways may be involved.     

Lutropin stimulates de novo synthesis of short-lived proteins required for lutropin-dependent steroid production in tumour Leydig cells

Continuous protein synthesis is required for the hormonal regulation of cholesterol side chain cleavage activity. A protein with a short half-life (t1/2 = 2-13 min) is believed to play an important role, but the regulation of the synthesis of this putative rapidly-turning-over protein is largely unknown. The steroid production rate in tumour Leydig cells can be increased more than 4-fold after addition of lutropin. However, steroid production by cells preincubated for 60 min with medium containing cycloheximide (89 microM) could not be stimulated when lutropin was added to the medium. Thus, the putative protein with the short half-life is apparently not derived from a stable precursor protein. Moreover, in tumour Leydig cells incubated with low concentrations of cycloheximide (0.2-0.8 microM), inhibition of steroid production was significantly greater in lutropin-stimulated cells than in control cells. These results support the hypothesis that lutropin regulates the de novo synthesis of rapidly-turning-over proteins by increasing the rate of initiation of the translation step of protein synthesis.