Decreased testicular function occurs as a concomitant of aging in men and is accentuated by the p... more Decreased testicular function occurs as a concomitant of aging in men and is accentuated by the presence of systemic illness. Previous studies identified an intrinsic Leydig cell defect as reflected by high LHkestosterone ratios and impaired hCG responsiveness in older men. No quantitative impairment of gonadotropin secretion, apparent upon study of integrated LH levels, pulse amplitude, or frequency, and LHRH responsiveness has been consistently identified.2 This study questioned whether a qualitative change in LH secretion, as reflected by altered biohmmuno LH ratios, might occur during aging and be accentuated by systemic illness.
We have recently demonstrated the presence in the rat Leydig cells of a corticotropin releasing f... more We have recently demonstrated the presence in the rat Leydig cells of a corticotropin releasing factor (CRF) receptor and an inhibitory action of the peptide on human chorionic gonadotropin (hCG)-induced cAMP generation and steroidogenesis. The inhibitory action of CRF was unaffected by pertussis toxin and was completely reversed by 8-bromo-cAMP (Ulisse, S., Fabbri, A., and Dufau, M. L. (1989) J. Biol. Chem. 264, 2156-2163). In this study, we have evaluated the participation of protein kinase C in CRF action in the Leydig cells and the level of the gonadotropin signal pathway affected by CRF. Binding of 125I-labeled ovine CRF to Leydig cell membranes was reduced by GTP and guanyl-5'-yl imidodiphosphate (Gpp(NH)p), in a dose-dependent manner. Phorbol 12-myristate 13-acetate, like CRF, caused time-dependent inhibition of hCG-induced cAMP generation and steroidogenesis. This inhibitory action was reversed by 8-bromo-cAMP. Both CRF and 12-O-tetradecanoylphorbol-13-acetate did not affect 125I-hCG binding. No additive effects of CRF and the phorbol ester were observed in these studies. CRF caused a rapid translocation of protein kinase C in Leydig cells. Preincubation of cells with protein kinase C inhibitors or TPA-induced depletion of protein kinase C prevented the inhibitory actions of CRF and TPA. CRF and TPA were able to inhibit the stimulation of cAMP and testosterone production by cholera toxin and forskolin. Adenylate cyclase stimulation by Gpp(NH)p, luteinizing hormone + Gpp(NH)p, and NaF in crude membranes or by forskolin and manganese in solubilized membranes, prepared from CRF- and TPA-treated cells, was also markedly inhibited. We conclude that CRF receptors interact with a pertussis toxin-insensitive G protein (possibly Gp) in the Leydig cell and that the inhibitory action of CRF on Leydig cell function is exerted mainly on the catalytic subunit of adenylate cyclase through a direct or indirect action of protein kinase C.
Luteinizing hormone is the major regulator of Leydig cell differentiation and steroidogenic funct... more Luteinizing hormone is the major regulator of Leydig cell differentiation and steroidogenic function. A number of hormones produced by the Leydig cell (e.g. estrogen, angiotensin, CRF, vasopressin) and the tubular compartment (inhibin, TGF beta), can influence both acute and long-term actions of LH. Conversely, hormones produced in the Leydig cells modulate tubular function (e.g. androgen, beta-endorphin, oxytocin). The LH stimulatory event can be negatively influenced by the action of angiotensin II through the guanyl nucleotide inhibitory unit of adenylate cyclase. We have recently discovered an action of corticotrophin releasing hormone through specific high-affinity low-capacity receptors in the Leydig cells which involves a pertussis toxin insensitive guanyl nucleotide regulatory unit with interaction between signalling pathways and resulting inhibition of LH induced cAMP generation and consequently of steroidogenesis. In contrast to other tissues the CRF receptor in the Leydig cells did not couple to Gs. CRF action is exerted through direct or indirect action of protein kinase C, at the level of the catalytic subunit of adenylate cyclase. Physiological increases in endogenous LH cause positive regulation of membrane receptors and steroidogenesis, while major elevations in circulating gonadotropin can induce down-regulation of LH receptors and desensitization of steroid responses in the adult cell. Gonadotropin-induced desensitization in adult rat tests include an estrogen mediated steroidogenic lesion of the microsomal enzymes 17 alpha-hydroxylase/17,20-desmolase. For further understanding of the regulation of this key enzyme of the androgen pathway the rat P450(17) alpha cDNA was cloned and sequenced. This cDNA expressed in COS-1 cells 17 alpha-hydroxylase/17,20-desmolase activities. From the deduced amino acid sequence, two transmembrane regions were identified, a signal peptide for insertion in the ER, and a 2nd transmembrane region separated from the first by 122 amino acids. The carboxy terminal non-transmembrane region possesses 4 hydrophobic clefts, of which cleft II would contain the putative steroid binding site for both hydroxylase and lyase activities. The rat cDNA was employed to evaluate the hormonal regulation of mRNA levels in adult and fetal Leydig cells. Low dose hCG treatment caused an early increase in mRNA levels followed by a return to control values at later times, while with higher desensitizing doses the initial increase in mRNA was followed by a marked reduction in mRNA at 24 h and a small recovery at 48 h. Fetal rat Leydig cells treated with E2 showed a 70% decrease in P450 mRNA levels, and testosterone production closely followed the changes in mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)
The Journal of Clinical Endocrinology & Metabolism, 1985
Quantitative reduction in LH secretion resulting from hypothalamic-pituitary dysfunction is a kno... more Quantitative reduction in LH secretion resulting from hypothalamic-pituitary dysfunction is a known cause of impotence. Qualitative abnormalities of secreted LH, however, have not been described under these circumstances. During evaluation of a 39-yr-old man with impotence and a calcified pituitary mass (pituitary stone), we detected a qualitative abnormality of LH characterized by a low ratio of bio- to immunoactivity (B:I). Initial work-up revealed basal morning serum testosterone levels of 2.14, 3.18, 3.97, and 3.11 ng/ml on 4 separate days, low to low normal urinary LH (300, 200, and 478 mIU/h), and normal GH, TSH, PRL, and ACTH secretion after provocative testing. The response of impotence to testosterone but not placebo in a double blind trial confirmed the clinical significance of the borderline low androgen levels. These findings prompted a systematic analysis of 24-h LH pulses as well as clomiphene and GnRH responsiveness. By RIA, mean serum LH levels [9.1 +/- 0.3 (+/- SE) mIU/ml] and all other response parameters were normal. In striking contrast, mean serum LH by bioassay was low (9.9 +/- 0.4 mIU/ml vs. 41.4 +/- 5.7 in normal subjects), as were B:I ratios (1.0 +/- 0.03 vs. control values of 3.1 +/- 0.5 to 5.3 +/- 0.3). Only during maneuvers designed to increase GnRH were B:I ratios increased to 3.3 +/- 0.22 (exogenous GnRH) and 1.8 +/- 0.12 (clomiphene). Mean testosterone levels before and after exogenous GnRH treatment were 3.28 +/- 0.24 and 4.76 +/- 0.16, respectively (P less than 0.001). The results suggest an association between the low LH B:I ratio and the anatomical disruption of the hypothalamic-pituitary portal system by the pituitary stone. The increased B:I ratio during GnRH or clomiphene administration indicates a functional link between pituitary GnRH exposure and the greater potency of the LH secreted.
Purification of specific endocrine cells from mixed populations after dispersion of target tissue... more Purification of specific endocrine cells from mixed populations after dispersion of target tissues is important for detailed analysis of mechanisms of hormone action. A simple method for rapid isolation of endocrine cells with retention of biological integrity, has been developed by centrifugation in density gradients formed with Metrizamide. By this procedure, highly purified Leydig cells retaining morphological and biochemical characteristics were obtained. Such preparations bound 20, 300+/-3, 100 molecules of hCG per cell with affinity of 1.1+/-0.25 X 10(10) M-1. During incubation with hCG, cyclic AMP and testosterone responses of purified Leydig cells were considerably increased, and hCG concentrations as low as 0.2 pM caused activation of cAMP-dependent protein kinase.
Morphine sulfate was found to have a direct inhibitory effect on both basal and GnRH-stimulated L... more Morphine sulfate was found to have a direct inhibitory effect on both basal and GnRH-stimulated LH release by cultured rat pituitary cells. The inhibitory effect of morphine on LH release was prevented by the opiate antagonist naltrexone, and treatment of cells with naltrexone or beta-endorphin antiserum significantly increased basal LH release. Also, incubation of pituitary cells with CRF caused a significant decrease in basal LH release, an effect that was reversed by naltrexone. Saturable opiate-binding sites were demonstrated in enriched gonadotrophs by [3H]etorphine binding studies. The ability of morphine to inhibit gonadotropin secretion through a direct action on pituitary opiate receptors suggests that long term exposure to exogenous opiates may suppress reproductive function at the hypophyseal level. In addition, the converse effects of CRF and naltrexone or beta-endorphin antiserum on LH release indicate that intrapituitary opioid peptides could exert a paracrine inhibitory action on the gonadotroph.
The beta-adrenergic antagonist propranolol binds to serotonin (5HT) receptors (5HT1B > 5HT... more The beta-adrenergic antagonist propranolol binds to serotonin (5HT) receptors (5HT1B > 5HT1A > 5HT2) in brain membranes. We have recently demonstrated that 5HT acts through 5HT2 receptors in rat Leydig cells to release CRF, which, in turn, inhibits hCG-stimulated cAMP production and steroidogenesis. These observations prompted us to study the effects of propranolol on CRF secretion and cAMP and testosterone production in cultured rat Leydig cells. Treatment with (-)propranolol increased CRF release and inhibited basal and hCG-stimulated cAMP and steroidogenesis, with effects evident at 0.1 microM, an IC50 of 6 microM, and reduction of stimulated levels to near basal at 100 microM. These effects of the drug were prevented by pretreatment of cultures with the 5HT2 receptor antagonist ketanserin or a CRF antagonist or antiserum. Furthermore, propranolol increased the level of 5HT in the incubation medium of cultured Leydig cells. The (+)isomer of propranolol had minor effects on these parameters. Increasing concentrations of (-)propranolol displaced the binding of [125I] +/- 1-[2,5-dimethoxy-4-iodophyryl]-2-amino propane hydrochloride (DOI), a selective 5HT2 ligand, to Leydig cell membranes (IC50, 0.2 microM), and (+)propanolol showed weaker potency. The inhibitory actions of propranolol were exerted through its blockade of the Leydig cell 5HT2 low affinity receptor, which has functional properties of an autoreceptor, with consequent increases in 5HT and stimulation of CRF release through 5HT action at the high affinity site. The serotonergic actions of propranolol were prevented by DOI, an inhibitor of 5HT actions at the high affinity site. In addition, the propranolol-induced blockade of the low affinity site further increased the cAMP and steroidogenic responses to gonadotropin over those observed with DOI treatment alone. These studies demonstrate that propranolol acts as an antagonist at the Leydig cell low affinity 5HT2 receptor and stimulates CRF release via a serotonergic mechanism, with consequent inhibition of cAMP generation and steroidogenesis. This serotonergic action of the drug could contribute to the impairment of sexual function reported during propranolol treatment in man.
GH-releasing hormone (GHRH) is present in the interstitial and germ cells of the rat testis. In p... more GH-releasing hormone (GHRH) is present in the interstitial and germ cells of the rat testis. In previous studies we found that GHRH is secreted from rat adult Leydig cells, in which it stimulates basal and LH-induced cAMP formation and steroidogenesis. In other studies cAMP production in Sertoli cells was found to be stimulated by GHRH. In the present report, we describe a potential paracrine action of GHRH in the Sertoli cell, with stimulation of cAMP formation in cultured adult and pubertal Sertoli cells. GHRH increased FSH-stimulated cAMP production in adult and pubertal cultures in a time-dependent manner. GHRH stimulation of basal and FSH-induced extracellular cAMP formation was more prominent in pubertal than in adult cultures. Immunocytochemical studies demonstrated the presence of GHRH-like immunoreactivity in rat interstitial cells from day 4 to adult life and in the acrosomal region of early and intermediate spermatids at stages III-VI of the seminiferous epithelium cycle. Immunoreactive GHRH was not observed in late spermatids and mature sperm or in Sertoli cells at any age. These results indicate that GHRH acts synergistically with FSH to promote cAMP production in Sertoli cells in culture. Testicular GHRH of Leydig and germ cell origin may be an important paracrine regulator of Sertoli cell function. Alternatively, GHRH present in germ cells may exert stage-specific intracrine functions.
Decreased testicular function occurs as a concomitant of aging in men and is accentuated by the p... more Decreased testicular function occurs as a concomitant of aging in men and is accentuated by the presence of systemic illness. Previous studies identified an intrinsic Leydig cell defect as reflected by high LHkestosterone ratios and impaired hCG responsiveness in older men. No quantitative impairment of gonadotropin secretion, apparent upon study of integrated LH levels, pulse amplitude, or frequency, and LHRH responsiveness has been consistently identified.2 This study questioned whether a qualitative change in LH secretion, as reflected by altered biohmmuno LH ratios, might occur during aging and be accentuated by systemic illness.
We have recently demonstrated the presence in the rat Leydig cells of a corticotropin releasing f... more We have recently demonstrated the presence in the rat Leydig cells of a corticotropin releasing factor (CRF) receptor and an inhibitory action of the peptide on human chorionic gonadotropin (hCG)-induced cAMP generation and steroidogenesis. The inhibitory action of CRF was unaffected by pertussis toxin and was completely reversed by 8-bromo-cAMP (Ulisse, S., Fabbri, A., and Dufau, M. L. (1989) J. Biol. Chem. 264, 2156-2163). In this study, we have evaluated the participation of protein kinase C in CRF action in the Leydig cells and the level of the gonadotropin signal pathway affected by CRF. Binding of 125I-labeled ovine CRF to Leydig cell membranes was reduced by GTP and guanyl-5'-yl imidodiphosphate (Gpp(NH)p), in a dose-dependent manner. Phorbol 12-myristate 13-acetate, like CRF, caused time-dependent inhibition of hCG-induced cAMP generation and steroidogenesis. This inhibitory action was reversed by 8-bromo-cAMP. Both CRF and 12-O-tetradecanoylphorbol-13-acetate did not affect 125I-hCG binding. No additive effects of CRF and the phorbol ester were observed in these studies. CRF caused a rapid translocation of protein kinase C in Leydig cells. Preincubation of cells with protein kinase C inhibitors or TPA-induced depletion of protein kinase C prevented the inhibitory actions of CRF and TPA. CRF and TPA were able to inhibit the stimulation of cAMP and testosterone production by cholera toxin and forskolin. Adenylate cyclase stimulation by Gpp(NH)p, luteinizing hormone + Gpp(NH)p, and NaF in crude membranes or by forskolin and manganese in solubilized membranes, prepared from CRF- and TPA-treated cells, was also markedly inhibited. We conclude that CRF receptors interact with a pertussis toxin-insensitive G protein (possibly Gp) in the Leydig cell and that the inhibitory action of CRF on Leydig cell function is exerted mainly on the catalytic subunit of adenylate cyclase through a direct or indirect action of protein kinase C.
Luteinizing hormone is the major regulator of Leydig cell differentiation and steroidogenic funct... more Luteinizing hormone is the major regulator of Leydig cell differentiation and steroidogenic function. A number of hormones produced by the Leydig cell (e.g. estrogen, angiotensin, CRF, vasopressin) and the tubular compartment (inhibin, TGF beta), can influence both acute and long-term actions of LH. Conversely, hormones produced in the Leydig cells modulate tubular function (e.g. androgen, beta-endorphin, oxytocin). The LH stimulatory event can be negatively influenced by the action of angiotensin II through the guanyl nucleotide inhibitory unit of adenylate cyclase. We have recently discovered an action of corticotrophin releasing hormone through specific high-affinity low-capacity receptors in the Leydig cells which involves a pertussis toxin insensitive guanyl nucleotide regulatory unit with interaction between signalling pathways and resulting inhibition of LH induced cAMP generation and consequently of steroidogenesis. In contrast to other tissues the CRF receptor in the Leydig cells did not couple to Gs. CRF action is exerted through direct or indirect action of protein kinase C, at the level of the catalytic subunit of adenylate cyclase. Physiological increases in endogenous LH cause positive regulation of membrane receptors and steroidogenesis, while major elevations in circulating gonadotropin can induce down-regulation of LH receptors and desensitization of steroid responses in the adult cell. Gonadotropin-induced desensitization in adult rat tests include an estrogen mediated steroidogenic lesion of the microsomal enzymes 17 alpha-hydroxylase/17,20-desmolase. For further understanding of the regulation of this key enzyme of the androgen pathway the rat P450(17) alpha cDNA was cloned and sequenced. This cDNA expressed in COS-1 cells 17 alpha-hydroxylase/17,20-desmolase activities. From the deduced amino acid sequence, two transmembrane regions were identified, a signal peptide for insertion in the ER, and a 2nd transmembrane region separated from the first by 122 amino acids. The carboxy terminal non-transmembrane region possesses 4 hydrophobic clefts, of which cleft II would contain the putative steroid binding site for both hydroxylase and lyase activities. The rat cDNA was employed to evaluate the hormonal regulation of mRNA levels in adult and fetal Leydig cells. Low dose hCG treatment caused an early increase in mRNA levels followed by a return to control values at later times, while with higher desensitizing doses the initial increase in mRNA was followed by a marked reduction in mRNA at 24 h and a small recovery at 48 h. Fetal rat Leydig cells treated with E2 showed a 70% decrease in P450 mRNA levels, and testosterone production closely followed the changes in mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)
The Journal of Clinical Endocrinology & Metabolism, 1985
Quantitative reduction in LH secretion resulting from hypothalamic-pituitary dysfunction is a kno... more Quantitative reduction in LH secretion resulting from hypothalamic-pituitary dysfunction is a known cause of impotence. Qualitative abnormalities of secreted LH, however, have not been described under these circumstances. During evaluation of a 39-yr-old man with impotence and a calcified pituitary mass (pituitary stone), we detected a qualitative abnormality of LH characterized by a low ratio of bio- to immunoactivity (B:I). Initial work-up revealed basal morning serum testosterone levels of 2.14, 3.18, 3.97, and 3.11 ng/ml on 4 separate days, low to low normal urinary LH (300, 200, and 478 mIU/h), and normal GH, TSH, PRL, and ACTH secretion after provocative testing. The response of impotence to testosterone but not placebo in a double blind trial confirmed the clinical significance of the borderline low androgen levels. These findings prompted a systematic analysis of 24-h LH pulses as well as clomiphene and GnRH responsiveness. By RIA, mean serum LH levels [9.1 +/- 0.3 (+/- SE) mIU/ml] and all other response parameters were normal. In striking contrast, mean serum LH by bioassay was low (9.9 +/- 0.4 mIU/ml vs. 41.4 +/- 5.7 in normal subjects), as were B:I ratios (1.0 +/- 0.03 vs. control values of 3.1 +/- 0.5 to 5.3 +/- 0.3). Only during maneuvers designed to increase GnRH were B:I ratios increased to 3.3 +/- 0.22 (exogenous GnRH) and 1.8 +/- 0.12 (clomiphene). Mean testosterone levels before and after exogenous GnRH treatment were 3.28 +/- 0.24 and 4.76 +/- 0.16, respectively (P less than 0.001). The results suggest an association between the low LH B:I ratio and the anatomical disruption of the hypothalamic-pituitary portal system by the pituitary stone. The increased B:I ratio during GnRH or clomiphene administration indicates a functional link between pituitary GnRH exposure and the greater potency of the LH secreted.
Purification of specific endocrine cells from mixed populations after dispersion of target tissue... more Purification of specific endocrine cells from mixed populations after dispersion of target tissues is important for detailed analysis of mechanisms of hormone action. A simple method for rapid isolation of endocrine cells with retention of biological integrity, has been developed by centrifugation in density gradients formed with Metrizamide. By this procedure, highly purified Leydig cells retaining morphological and biochemical characteristics were obtained. Such preparations bound 20, 300+/-3, 100 molecules of hCG per cell with affinity of 1.1+/-0.25 X 10(10) M-1. During incubation with hCG, cyclic AMP and testosterone responses of purified Leydig cells were considerably increased, and hCG concentrations as low as 0.2 pM caused activation of cAMP-dependent protein kinase.
Morphine sulfate was found to have a direct inhibitory effect on both basal and GnRH-stimulated L... more Morphine sulfate was found to have a direct inhibitory effect on both basal and GnRH-stimulated LH release by cultured rat pituitary cells. The inhibitory effect of morphine on LH release was prevented by the opiate antagonist naltrexone, and treatment of cells with naltrexone or beta-endorphin antiserum significantly increased basal LH release. Also, incubation of pituitary cells with CRF caused a significant decrease in basal LH release, an effect that was reversed by naltrexone. Saturable opiate-binding sites were demonstrated in enriched gonadotrophs by [3H]etorphine binding studies. The ability of morphine to inhibit gonadotropin secretion through a direct action on pituitary opiate receptors suggests that long term exposure to exogenous opiates may suppress reproductive function at the hypophyseal level. In addition, the converse effects of CRF and naltrexone or beta-endorphin antiserum on LH release indicate that intrapituitary opioid peptides could exert a paracrine inhibitory action on the gonadotroph.
The beta-adrenergic antagonist propranolol binds to serotonin (5HT) receptors (5HT1B > 5HT... more The beta-adrenergic antagonist propranolol binds to serotonin (5HT) receptors (5HT1B > 5HT1A > 5HT2) in brain membranes. We have recently demonstrated that 5HT acts through 5HT2 receptors in rat Leydig cells to release CRF, which, in turn, inhibits hCG-stimulated cAMP production and steroidogenesis. These observations prompted us to study the effects of propranolol on CRF secretion and cAMP and testosterone production in cultured rat Leydig cells. Treatment with (-)propranolol increased CRF release and inhibited basal and hCG-stimulated cAMP and steroidogenesis, with effects evident at 0.1 microM, an IC50 of 6 microM, and reduction of stimulated levels to near basal at 100 microM. These effects of the drug were prevented by pretreatment of cultures with the 5HT2 receptor antagonist ketanserin or a CRF antagonist or antiserum. Furthermore, propranolol increased the level of 5HT in the incubation medium of cultured Leydig cells. The (+)isomer of propranolol had minor effects on these parameters. Increasing concentrations of (-)propranolol displaced the binding of [125I] +/- 1-[2,5-dimethoxy-4-iodophyryl]-2-amino propane hydrochloride (DOI), a selective 5HT2 ligand, to Leydig cell membranes (IC50, 0.2 microM), and (+)propanolol showed weaker potency. The inhibitory actions of propranolol were exerted through its blockade of the Leydig cell 5HT2 low affinity receptor, which has functional properties of an autoreceptor, with consequent increases in 5HT and stimulation of CRF release through 5HT action at the high affinity site. The serotonergic actions of propranolol were prevented by DOI, an inhibitor of 5HT actions at the high affinity site. In addition, the propranolol-induced blockade of the low affinity site further increased the cAMP and steroidogenic responses to gonadotropin over those observed with DOI treatment alone. These studies demonstrate that propranolol acts as an antagonist at the Leydig cell low affinity 5HT2 receptor and stimulates CRF release via a serotonergic mechanism, with consequent inhibition of cAMP generation and steroidogenesis. This serotonergic action of the drug could contribute to the impairment of sexual function reported during propranolol treatment in man.
GH-releasing hormone (GHRH) is present in the interstitial and germ cells of the rat testis. In p... more GH-releasing hormone (GHRH) is present in the interstitial and germ cells of the rat testis. In previous studies we found that GHRH is secreted from rat adult Leydig cells, in which it stimulates basal and LH-induced cAMP formation and steroidogenesis. In other studies cAMP production in Sertoli cells was found to be stimulated by GHRH. In the present report, we describe a potential paracrine action of GHRH in the Sertoli cell, with stimulation of cAMP formation in cultured adult and pubertal Sertoli cells. GHRH increased FSH-stimulated cAMP production in adult and pubertal cultures in a time-dependent manner. GHRH stimulation of basal and FSH-induced extracellular cAMP formation was more prominent in pubertal than in adult cultures. Immunocytochemical studies demonstrated the presence of GHRH-like immunoreactivity in rat interstitial cells from day 4 to adult life and in the acrosomal region of early and intermediate spermatids at stages III-VI of the seminiferous epithelium cycle. Immunoreactive GHRH was not observed in late spermatids and mature sperm or in Sertoli cells at any age. These results indicate that GHRH acts synergistically with FSH to promote cAMP production in Sertoli cells in culture. Testicular GHRH of Leydig and germ cell origin may be an important paracrine regulator of Sertoli cell function. Alternatively, GHRH present in germ cells may exert stage-specific intracrine functions.
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Papers by M. Dufau