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.     

Effects of Cordyceps sinensis on testosterone production in normal mouse Leydig cells.

The stimulatory effect of Cordyceps sinensis (CS) on MA-10 mouse Leydig tumor cell steroidogenesis was previously demonstrated in our laboratory. In the present studies, we further determined the effect of CS on steroidogenesis in purified normal mouse Leydig cells. Different concentrations of CS (0.1-10 mg/ml) were added to Leydig cells without or with human chorionic gonadotropin (hCG) (50 ng/ml), and the steroid production was determined by radioimmunoassay (RIA). The results illustrated that CS stimulated normal mouse Leydig cell steroidogenesis in a dose-dependent relationship. CS at 3 mg/ml significantly stimulated testosterone production (p<0.05). Concerning the temporal relationship, CS at 3 mg/ml stimulated maximal testosterone production between 2 to 3 hr. Interestingly, hCG-stimulated testosterone productions were suppressed by CS in a dose-dependent relationship. CS also reduced dbcAMP-stimulated testosterone productions, which indicated that CS affected signal transduction pathway of steroidogenesis after the formation of cyclic AMP. Moreover, cycloheximide inhibited CS-treated mouse Leydig cell testosterone production, suggesting that new protein synthesis was required for CS-stimulated steroidogenesis.     

Acute stimulation of steroidogenesis in corpus luteum and adrenal cortex by peptide hormones. Rapid induction of a similar protein in both tissues

Two-dimensional electrophoresis was used to detect a protein (ic) synthesized in rat corpus luteum cells in response to acute stimulation by human chorionic gonadotropin or dibutyryl cyclic AMP. This induced protein ic is isoelectric at pH 6.5 (isoelectric focusing) and has an apparent molecular weight of 28,000 (sodium dodecyl sulfate electrophoresis). The human chorionic gonadotropin or dibutyryl cyclic AMP dose response and time course of synthesis of the protein parallel those of progesterone synthesis in stimulated luteal cells. Additionally, cycloheximide, which inhibits the increase in progesterone formation caused by human chorionic gonadotropin or cAMP, also inhibits the synthesis of ic. Proteolytic polypeptide mapping suggests that ic has a very similar primary structure to another protein (pc), which has the same molecular weight as ic, differs from ic in pI, and is synthesized only in unstimulated cells. These polypeptide maps also demonstrate the close similarity of pc and ic to two proteins p and i, synthesized in control and in adrenocorticotropic hormone-stimulated rat adrenal cortex cells, respectively (Krueger, R. J. and Orme-Johnson, N. R. (1983) J. Biol. Chem. 258, 10159-10167). In both adrenal cortex and corpus luteum, binding of a tissue-specific polypeptide hormone acts via cAMP to cause increased steroidogenesis and induction of the synthesis of protein i (ic), with the same time course and hormone dose dependence. Also in both tissues, inhibition of protein synthesis at the level of translation (e.g. by cycloheximide addition) causes inhibition of i (ic) synthesis and of stimulated steroid production. This close correlation between the two different tissues in conditions which cause induction of the synthesis of these proteins suggests that the proteins may be common intermediaries in the control by polypeptide hormones of steroidogenesis in endocrine tissues.     

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 protein-synthesis inhibitors on testosterone production in rat testis interstitial tissue and Leydig-cell preparations.

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.     

Down-regulation of gonadotropin and beta-adrenergic receptors by hormones and cyclic AMP

Loss of gonadotropin receptors in murine Leydig tumor cells and of beta-adrenergic receptors in rat glioma C6 cells occurred following exposure of the cells to human chorionic gonadotropin and isoproterenol, respectively. Down-regulation of receptors was mimicked in part by other agents that elevated cyclic AMP levels in the cells such as cholera toxin and dibutyryl cyclic AMP. Whereas agonist-mediated receptor loss was rapid and almost total, down-regulation by cyclic AMP was slower and less extensive. Down-regulation of receptors did not appear to be accompanied by loss of the regulatory and catalytic components of adenylate cyclase. Hormone-mediated down-regulation was preceded by desensitization of hormone-stimulated adenylate cyclase. In contrast, there was no evidence that cyclic AMP caused desensitization. Finally, loss of receptors induced either by agonists or cyclic AMP required protein synthesis as cycloheximide inhibited down-regulation. We conclude that down-regulation of receptors in these cells is a complex process involving both cyclic AMP-independent and -dependent events.     

Release of arachidonic acid and the effects of corticosteroids on steroidogenesis in rat testis Leydig cells.

The release of arachidonic acid by luteinizing hormone (LH) and the effects of inhibiting phospholipase A2 (PLA2) in vivo and in vitro on LH stimulated steroidogenesis in rat testis Leydig cells has been investigated. It was found that arachidonic acid is rapidly incorporated into phospholipids and is released within 1 min after addition of LH. The effects of treating adult rats with dexamethasone and human chorionic gonadotropin (hCG) in vivo on steroidogenesis and prostaglandin synthesis in Leydig cells isolated 6 h later were determined. It was found that hCG caused a marked increase in prostaglandin F2 alpha formation which was inhibited by treatment with dexamethasone. LH-stimulated testosterone production was inhibited in the hCG treated rats and dexamethasone caused a further decrease. Treatment with dexamethasone alone also caused a decrease in the response to LH. HCG, but not dexamethasone, had similar inhibitory effects on LH-stimulated cyclic AMP production. Similarly, the PLA2 inhibitors quinacrine, dexamethasone and corticosterone, added to the Leydig cells in vitro, inhibited LH-stimulated testosterone production but not cyclic AMP production. 11-Dehydrocorticosterone also inhibited LH-stimulated testosterone production, but higher concentrations were required to give 50% inhibition compared to corticosterone (50 and 25 microM, respectively). Ring A-reduced metabolites of corticosterone and progesterone were also found to inhibit LH-stimulated steroidogenesis. The results obtained in this and previous studies are consistent with the activation of PLA2, (either directly by LH and/or via cyclic AMP), which results in the release of arachidonic acid and the formation of leukotrienes, which stimulate steroidogenesis in the Leydig cell. This study also indicates that corticosteroids and their metabolites may exert inhibitory effects at other sites in the steroidogenic pathways, in addition to PLA2.     

Role of carbohydrate of human chorionic gonadotropin in the mechanism of hormone action.

The role of the carbohydrate part of human chorionic gonadotropin (hCG) was investigated by measuring the ability of hCG derivatives lacking various sugar residues to bind to rat Leydig cells and stimulate them to synthesize testosterone and cyclic adenosine 3':5'-monophosphate (cyclic AMP). Whereas sequential removal of the sialic acid, galactose, N-acetylglucosamine, and mannose residues led to a progressive increase in the effective dose of the hormone required to stimulate steroidogenesis, it resulted in a marked loss in the ability of the hormone to stimulate cyclic AMP accumulation. Low doses of the glycosidase-treated hormone derivatives were additive with hCG when their ability to stimulate testosterone synthesis was analyzed. Nevertheless, the glycosidase-treated derivatives were potent inhibitors of hCG-induced cyclic AMP accumulation, suggesting that removal of the sugars did not influence binding of the hormone to the cell as much as it reduced the ability of the bound hormone to activate adenyl cyclase. This hypothesis was further supported by our finding that the hCG derivatives were highly effective inhibitors of 125I-hGC binding to the intact cells. Removal of sialic acid and galactose enhanced the inhibition, whereas removal of all the sugar residues only decreased the inhibition slightly. The degree of these effects was comparatively small. The possibility that steroidogenesis and cyclic AMP accumulation are altered independently by hCG stimulation is discussed.     

The peripheral-type benzodiazepine receptor is functionally linked to Leydig cell steroidogenesis

Testicular mitochondria were previously shown to contain an abundance of peripheral-type benzodiazepine recognition site(s)/receptor(s) (PBR). We have previously purified, cloned, and expressed an Mr 18,000 PBR protein (Antkiewicz-Michaluk, Mukhin, A. G., Guidotti, A., and Krueger, K. E. (1988) J. Biol. Chem. 263, 17317-17321; (Sprengel, R., Werner, P., Seeburg, P. H., Mukhin, A. G., Santi, M. R., Grayson, D. R., Guidotti, A., and Krueger, K. E. (1989) J. Biol. Chem. 264, 20415-20421); and in this report, we present evidence that PBR are functionally linked to Leydig cell steroid biosynthesis. A spectrum of nine different ligands covering a range of over 4 orders of magnitude in their affinities for PBR were tested for their potencies to modulate steroidogenesis in the MA-10 mouse Leydig tumor cell line. The Ki for inhibition of [3H]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide binding and the EC50 for steroid biosynthesis for this series of compounds showed a correlation coefficient of r = 0.95. The most potent ligands stimulated steroid production by approximately 4-fold in these cells. This stimulation was not inhibited by cycloheximide, unlike human chorionic gonadotropin- or cyclic AMP-activated steroidogenesis. The action of PBR ligands was not additive to stimulation by human chorionic gonadotropin or cyclic AMP, but was additive to that of epidermal growth factor, another regulator of MA-10 Leydig cell steroidogenesis. Moreover, PBR ligands stimulated, in a dose-dependent manner, pregnenolone biosynthesis by isolated mitochondria when supplied with exogenous cholesterol. This effect was not observed with mitoplasts (mitochondria devoid of the outer membrane). Cytochrome P-450 side chain cleavage activity, as measured by metabolism of (22R)-hydroxycholesterol, was not affected by PBR ligands in intact cells. Similar results were also obtained with purified rat Leydig cells. In conclusion, PBR are implicated in the acute stimulation of Leydig cell steroidogenesis possibly by mediating the entry, distribution, and/or availability of cholesterol within mitochondria.     

Hormonal regulation of four urea cycle enzymes in postnatal rat liver in organ culture

The administration of hydrocortisone to 3- to 15-day-old rats increased the levels of hepatic argininosuccinate synthetase (ASS) and arginase. In 13-day-old rat liver explants maintained in organ culture, ornithine carbamoyltransferase (OTC), carbamoylphosphate synthetase (CPS) and arginase were stimulated by betamethasone. Actinomycin D prevented the responses of the latter two enzymes. Dibutyryl cyclic AMP raised OTC, CPS, ASS and arginase in vitro. The responses of the latter three enzymes were blocked by cycloheximide and puromycin and partially inhibited by actinomycin D. The simultaneous presence of betamethasone and dibutyryl cyclic AMP in the culture medium raised CPS and OTC in an additive manner. The sequential treatment of the cultures with betamethasone followed by dibutyryl cyclic AMP increased CPS and arginase synergistically and amplified the response of ASS to dibutyryl cyclic AMP.     

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.     

Inhibitory action of forskolin on adenylate cyclase activity and cyclic AMP generation

In testicular Leydig cells, forskolin causes the expected stimulation of cAMP and testosterone production and potentiates gonadotropin-induced responses, when present in concentrations of 1-10 microM. In addition, when added at lower doses that did not affect cAMP generation and testosterone responses (100 nM), forskolin caused an increase in sensitivity to hormonal stimulation for all cAMP pools (extracellular, intracellular, and receptor-bound) and a 70% reduction in the ED50 for human chorionic gonadotropin (hCG) stimulation of testosterone production. Forskolin-induced increases in receptor-bound cAMP were less effective than those elicited by hCG in stimulating steroidogenesis. In contrast to the well-known stimulatory actions of forskolin, low doses of the diterpene (in the picomolar to nanomolar range) markedly inhibited the production of cAMP and testosterone. Such inhibitory actions of low-dose forskolin were prevented by preincubation of Leydig cells with pertussis toxin before addition of forskolin and/or hCG. Low concentrations of forskolin also inhibited adenylate cyclase activation by GTP and luteinizing hormone, and this effect was prevented by pretreatment of cell membranes with pertussis toxin. These studies have defined the stimulatory effects of forskolin on Leydig-cell cAMP pools, including potentiation of the hormonal increase in receptor-bound cyclic AMP by forskolin, and have provided additional evidence for the functional importance of cAMP compartmentalization during hormonal stimulation of steroidogenesis. We have also demonstrated a novel, high-affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation, an effect that appears to be mediated by the Ni guanine nucleotide regulatory subunit of adenylate cyclase.     

Steroid production by the isolated rabbit ovarian follicle.

Graafian follicles from New Zealand white rabbits were incubated at 37 degrees C for various periods of time with air as the gas phase. Media were changed every 15 min and stored at -15 degrees C until analysed for progestins, 17 beta-hydroxyandrogens and oestrogens using established radioimmunoassay procedures. At fixed times after the start of the incubations, media containing various test substances were added with subsequent replacement by medium alone. Addition of 5 mug LH/ml for 1 sec caused a dramatic increase in the synthesis and secretion of androgen with lesser increases in progestin and oestrogen. Puromycin and cycloheximide but not actinomycin D, inhibited LH-induced steroidogenesis. Cycli AMP, dibutyryl cycli AMP, cyclic CMP, 5'-AMP, and theophylline also caused an increase in androgen production which rapidly ceased when media without nucleotides were added. Sodium fluoride had no effect on steroidogenesis. From these data it was concluded that (i) the rabbit follicle is the major source of ovarian androgen; (ii) the binding of LH to the follicular cells is a rapid process; (iii) the events following LH binding do not require the presence of LH in the medium; (iv) cyclic nucleotides which may act as second messengers also stimulate steroidogenesis; (v) the effects of LH and cyclic nucleotides on steroidogenesis are different; and (vi) the action of LH on follicular steroidogenesis probably occurs in the translational level.