Gonadotropin binding and Leydig cell activation in the rat testis in vivo

Testicular LH receptor occupancy and steroidogenic responses were measured in adult male rats after intracardiac injections of [125I]iodo-hCG (0.5--5 x 10(6) cpm) mixed with known amounts of nonradioactive hCG to yield doses ranging from 10 ng to 300 micrograms. Uptake of the hormone by the testis was measured in the whole tissue or the 20,000 x g homogenate, with correction for nonspecific binding in animals injected with a 100-fold excess of unlabeled hCG. The steroidogenic response to hCG was followed by measurements of serum and testicular testosterone. Maximum specific uptake of [125I]iodo-hCG by the testes was observed 4--6 h after hormone injection. Of the specific counts, 80% were recovered in the 20,000 x g pellet of the tissue homogenate. The testicular contents of hCG-binding sites were similar when measured by in vivo occupancy of the receptors and by the in vitro receptor assay, indicating the physiological validity of the receptor measurements in tissue homogenates. Serum and testicular testosterone levels reached a maximum at 1 h, independent of the hCG dose used. When receptor occupancy in vitro after injection of hCG was compared with stimulation of steroidogenesis, a significant (P less than 0.05) 3-fold elevation of serum testosterone was seen when only 0.05% of the receptors were occupied. The maximal testosterone response was reached with 0.8% receptor occupancy. It is concluded that the same number of testicular LH receptors can be occupied by the circulating hormone in vivo and in tissue homogenates in vitro. The spare receptor concept also applied to the in vivo situation, since stimulation of steroidogenesis in the intact animal requires occupancy of only a few receptors per Leydig cell. This may be a general feature of hormonal activation of endocrine target cells in vivo.     

Factors determining whether the direct effects of an LHRH agonist on Leydig cell function in vivo are stimulatory or inhibitory

The influence of (1) repeated exposure to LHRH agonist and (2) concomitant exposure to various levels of LH or hCG, on the direct testicular effects of LHRH agonist have been investigated in hypophysectomized and intact rats. In the latter, LHRH agonist was injected intratesticularly into the right testis whilst the left testis was injected with vehicle, and the level of testosterone in interstitial fluid (IF) from left and right testes was compared 2-4 h later. In hypophysectomized rats, a single injection of 50-1000 ng LHRH agonist raised (P less than 0.001) serum levels of testosterone 2 h later to within the normal range (1-8 ng/ml) for intact rats of comparable age. Subsequent daily injections of LHRH agonist elicited progressively smaller responses and by day 5 no response was evident, this decline being unrelated to the increase in time after hypophysectomy. Comparable changes were observed in hypophysectomized rats pretreated with an LH antiserum. Daily injection of hypophysectomized rats with 10 IU hCG for 6 days raised serum levels of testosterone to supraphysiological levels on each day, whilst concomitant injection of LHRH agonist (1 microgram) decreased (P less than 0.001) this response at all times, an effect that became progressively more pronounced. In contrast, daily treatment with 1 microgram ovine LH raised serum levels of testosterone to within the physiological range, and concomitant treatment with LHRH agonist (50 ng) had either no effect (days 1-2) or significantly increased (days 4-6) the testosterone response to LH. In intact rats, a single unilateral intratesticular injection of 1 ng LHRH agonist increased the IF levels of testosterone unilaterally 3 h later, but subsequent injections on days 2 and 3 elicited progressively smaller responses. In rats given a single intratesticular injection of LHRH agonist combined with a peripheral injection of different doses of LH or hCG, the LHRH agonist induced a unilateral increase in IF levels of LH/hCG, whilst in rats treated with high doses of LH (12.5--25 micrograms) or hCG (50 IU). LHRH agonist either had no effect or significantly reduced the IF levels of testosterone unilaterally. However, LHRH agonist also had significant effects on testicular IF volume and, as this may reflect altered transport of LH and hCG to the Leydig cells, the inhibitory effects of LHRH agonist may be related to this change rather than to an effect of steroidogenesis itself.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specificity of the direct effect of an LHRH agonist on testicular 17-hydroxylase but not on 5 alpha-reductase activity in hypophysectomized adult rats

The direct effect of treatment with a potent LHRH agonist on testicular steroidogenesis was studied by incubation of radioactive steroids with a testicular homogenate or with a suspension of interstitial cells obtained following 7 days of treatment of adult hypophysectomized male rats. The animals received [D-Ser(tBu)6,des-Gly-NH2(10)]LHRH ethylamide (25 micrograms) administered 3 times a day, hCG (5 or 25 IU) once daily or a combination of both drugs. The metabolism of tritiated progesterone into delta 4-metabolites by a suspension of interstitial cells was markedly reduced by treatment with the LHRH agonist (LHRH-A) alone or following combined treatment with hCG and LHRH-A. No formation of 5 alpha-reduced steroids was detected in the medium following incubation with testicular homogenate or interstitial cells. Similar findings were obtained by measurement of testicular steroid content. The present data demonstrate that the direct effect of the LHRH agonist is limited to the Leydig cells on 17-hydroxylase activity. This inhibitory effect is reflected by an accumulation of testicular pregnenolone and progesterone content and a marked inhibition of progesterone metabolism into delta 4-androgens. However, no stimulation of 5 alpha-reductase, an enzyme localized in seminiferous tubules, could be detected. Such data show clear differences between the direct and the pituitary-mediated effects of treatment with LHRH agonists on testicular steroidogenesis. While the LHRH agonist administered at high doses in the rat can directly inhibit 17-hydroxylase activity, the stimulatory effect on 5 alpha-reductase activity is regulated by another mechanism.     

Gonadotropin-releasing hormone and its agonist inhibit testicular luteinizing hormone receptor and steroidogenesis in immature and adult hypophysectomized rats

The direct effect of gonadotropin-releasing hormone (GnRH) and its agonist on testicular LH receptor and steroidogenesis was studied in hypophysectomized immature and adult rats. Hypophysectomized rats were treated daily with varying doses of GnRH or [des-Gly10,D-Leu6(N alpha Me)Leu7, Pro9-NHEt]GnRH(a potent agonist). Some animals were also treated concomitantly with FSH, PRL, GH and/or LH to prevent the hypophysectomy-induced loss of testicular LH receptor and steroidogenic capacity. At the end of 5 days of treatment, testicular LH/hCG receptor concentration was measured by a [125I]-hCG-binding assay and steroidogenic responsiveness was determinded by in vitro incubations. GnRH and the GnRH agonist reduced testicular LH receptor in control and FSH-treated hypophysectomized immature rats. As little as 0.5 microgram agonist/day induced a greater than 40% decrease in the LH receptor content, whereas GnRH was less potent, with 50 micrograms/day inducing about a 50% decrease. The inhibitory effect of GnRH was shown to be the result of decreases in the concentration of LH receptor rather than changes in the receptor affinity (Kd = 1.1 X 10(-10)M). GnRH did not interfere with the [125I]hCG receptor assay. Treatment with PRL, GH, and FSH, alone or in various combinations, increased the testicular LH receptor content. The stimulatory effect of these pituitary hormones was depressed by concomitant treatment with the GnRH agonist. Similar inhibitory effects of GnRH and the agonist on testicular LH receptor were demonstrated in adult hypophysectomized rats. In vitro studies demonstrated that treatment with the GnRH agonist in vivo inhibited both basal and hCG-stimulated androgen production in FSH-primed immature hypophysectomized rats. Associated with decreases in androgens (testosterone and androstenedione) and reduced androgens (dihydrotestosterone, androstanediol, and androsterone), there was marked suppression of 17 alpha-hydroxylated precursors and C-21 steroid intermediates in animals treated with the GnRH agonist, thus suggesting that the inhibitory effect of the GnRH agonist was associated with possible defects in 17 alpha-hydroxylase and side-chain cleavage enzymes. Likewise, treatment with the GnRH agonist inhibited in vitro testicular steroidogenic responses in adult hypopysectomized rats. These results demonstrate the extrapituitary inhibitory effect of GnRH on testicular LH receptor content and Leydig cell steroidogenesis in immature and adult hypophysectomized rats.     

Structure-activity relationships of mammalian, chicken, and salmon gonadotropin releasing hormones in vivo in goldfish

Mammalian, chicken, and salmon gonadotropin releasing hormones (GnRHs), and anlogs of each peptide, were injected either alone or in combination with pimozide into goldfish, and the changes in serum gonadotropin (GtH) levels determined. The native peptides had similar potencies in terms of magnitude and duration of the GtH response. Analogs of LHRH that are superactive in mammals are also superactive in goldfish; although [(imBzl)-D-His6, Pro9-NEt]-LHRH is very highly superactive in mammals it has activity similar to [D-Ala6, Pro9-NEt]-LHRH in goldfish. D-Ala6 or (imBzl)-D-His6 substitutions of [Trp7, Leu8, Pro9-NEt]-LHRH are not superactive in goldfish, whereas the D-Arg6 substitution is highly superactive, indicating that there are differences in the factors that make salmon and mammalian GnRH superactive. These results also indicate that the structural modifications that determine superactivity of GnRHs in goldfish differ from what is known for mammals.     

Inhibition of the pituitary-gonadal axis in nude male mice by continuous administration of LHRH agonists and antagonists

Analogues of LHRH can be used for the treatment of sex hormone-dependent tumours. The nude mouse is a valuable model for the investigation of transplanted human cancers, but there is a body of literature reporting that the function of the pituitary-gonadal axis in normal (immunocompetent) and nude (immunocompromised) mice, unlike that of other species, cannot be suppressed by the administration of LHRH agonists and antagonists. To explore this view further, long-term experiments were carried out in nude male mice, in which sustained-release formulations of the agonist [D-Trp6]-LHRH and of two new potent antagonists were used which permitted a continuous release of the peptides into the circulation. Nude male mice were treated for 28-30 days with 50 micrograms of antagonists [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Trp3,D-Cit6, D-Ala10]-LHRH (SB-30) or [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Pal(3)3, D-Cit6,D-Ala10]-LHRH (SB-75)/day delivered by osmotic minipumps. Some mice were injected twice a day with 25 micrograms SB-75. Other groups received microcapsule preparations of the agonist [D-Trp6]-LHRH, releasing 25 or 12.5 micrograms/day for 30 days. At autopsy, in mice which received 50 micrograms SB-30 or SB-75/day by minipumps, there was a significant decrease in weights of testes, ventral prostate and seminal vesicles compared with controls. [D-Trp6]-LHRH microcapsules at either dose also reduced weights of testes and accessory sex organs. Serum LH and testosterone were significantly reduced in all groups treated with analogues. There was a greater decrease in testicular weights and serum testosterone in nude mice which received SB-75 in a continuous fashion from minipumps than in animals injected twice a day.(ABSTRACT TRUNCATED AT 250 WORDS)     

Do testicular opiates regulate Leydig cell function?

beta-Endorphin is believed to be synthesized in testicular Leydig cells. To gain more information about the role of this and other endogenous opioid peptides in the testis, opiate antagonists (naloxone and nalmefene, 100 micrograms/testis) were administered intratesticularly to hemicastrated adult rats. Leydig cell function was evaluated by measurement of serum testosterone and testosterone production in vitro. Estimation of androgen binding protein (rABP) was used as an index of Sertoli cell function. Serum testosterone was reduced significantly by intratesticular administration of naloxone and nalmefene in treated animals. Systemic administration of these antagonists had no effect at the doses used. Testes from treated animals incubated in vitro with or without hCG produced significantly less testosterone than vehicle-treated control testes. Hemicastration reduced rABP synthesis and secretion; however, treatment with opiate antagonists did not alter the amount of this protein in the serum or epididymides of these rats. These observations suggest that endogenous testicular opiates modulate testosterone secretion by Leydig cells.     

Testicular steroidogenesis in the testicular feminized (Tfm) mouse: loss of 17 alpha-hydroxylase activity.

Testicular feminized (Tfm) mice are totally insensitive to androgen and may be used to study the role of the androgen receptor in normal development and function. We have examined testicular and Leydig cell steroidogenesis in Tfm mice. Serum bioactive LH was high in Tfm mice but serum testosterone was low and this was associated with a severe reduction in testicular testosterone production in vitro. Examination of [3H]pregnenolone metabolism by testes of Tfm mice indicated that progesterone, rather than testosterone, was the major steroid produced. Leydig cells were isolated from normal and Tfm mice and from normal mice in which testicular descent was surgically prevented before puberty. As in whole testes, androgen production in response to human chorionic gonadotrophin was severely reduced in Leydig cells from testes of Tfm mice compared with normal or cryptorchid groups. In contrast, progesterone production by Leydig cells from testes of Tfm mice was markedly increased in comparison with other groups. Total steroid production (progesterone plus androstenedione plus testosterone), however, was only 24% of normal in Leydig cells from Tfm mice. The pattern of steroid production by Leydig cells from cryptorchid testes was similar to control, although total steroid production was reduced to about 50% (this was significantly higher than the Tfm group, P less than 0.05). The high progesterone/androgen ratio in testes from Tfm mice suggested that 17 alpha-hydroxylase was depleted in these animals. To confirm this, activity of the four major steroidogenic enzymes associated with the smooth endoplasmic reticulum was measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulatory effect of LHRH and its agonists on Leydig cell steroidogenesis in vitro

Short-term (4 h) incubation of collagenase-dispersed Leydig cells from adult rats in the presence of an LHRH agonist caused a 2-3-fold stimulation (P less than 0.001) of testosterone production. This effect was dose-dependent and as little as 5 x 10(-11) M LHRH agonist caused significant stimulation whilst maximal effects were achieved with 10(-9) M concentrations. Stimulation of steroidogenesis by LHRH agonist was prevented by addition of an antiserum specific for the peptide, but was exaggerated in the presence of the phosphodiesterase inhibitor MIX, suggesting the involvement of cyclic AMP in the response of the Leydig cells to the agonist. Native LHRH caused a similar degree of stimulation of testosterone secretion to LHRH agonist but concentrations 1000 times greater than those of the agonist were required to achieve this, a finding consistent with the known affinities of these 2 peptides for the Leydig cell LHRH-receptor. The addition of LHRH agonist also enhanced (P less than 0.001) testosterone secretion by adult rat Leydig cells in response to hCG or dibutyryl cyclic AMP, and this effect was still evident in the presence of maximally-stimulating concentrations of these factors. LHRH agonist also stimulated testosterone secretion by Leydig cells from immature rats, but this effect differed from that in the adult in being of smaller magnitude and being restricted to effects on basal secretion or secretion elicited by low concentrations of hCG. These results show for the first time (a) that LHRH and its agonists can exert effects on Leydig cell steroidogenesis during short-term incubation, and (b) that these effects are stimulatory, which contrasts with the inhibitory effects reported after long-term (2-3 days) exposure of Leydig cells to LHRH agonists in vivo and in vitro. The availability of this simple and rapid measure of a biological action of LHRH on the Leydig cell should enable its precise mode of action to be determined, and should throw light on the physiological role of endogenously produced testicular 'LHRH'.     

Gonadotropin-releasing hormone agonist analog-induced steroidogenic lesion in the neonatal rat testis: evidence for direct gonadal action

In this study we sought to determine whether a GnRH agonist analog (GnRH-A) could influence steroidogenesis by a direct effect on the neonatal rat testis. Five-day-old male rats were given a single sc injection of hCG (600 IU/kg BW), GnRH-A [D-Ser(tBu)6]des-Gly10-GnRH N-ethylamide (4 micrograms/kg BW), or their combination. Testicular testosterone (T) was increased (3-fold) only 6 h post-GnRH-A treatment, whereas after hCG administration testicular T remained elevated (3 to 6-fold) for 48 h. Testicular progesterone (P) increased by 40% 6-72 h after hCG treatment, but was not raised after GnRH-A injection. In vitro T production by testes from control and GnRH-A-treated (injection 24 h earlier) animals was stimulated 5 to 8-fold by hCG (7.9 nM) or 8-bromo-cAMP (8-Br-cAMP; 1 mM). hCG and 8-Br-cAMP did not further stimulate T production from testes of animals treated with hCG in vivo 24 h earlier. While hCG and 8-Br-cAMP had only a small stimulatory effect (1.5 to 2-fold) on in vitro P production by testes from control or hCG-treated animals, their stimulation of P production from testes of GnRH-A-treated animals was dramatic (20 to 30-fold). In vitro P production from testes of animals receiving combined treatment with hCG and GnRH-A in vivo reached a high hCG-stimulated rate similar to that found after GnRH-A treatment alone; the unstimulated values were also considerably elevated (5-fold) compared to those of untreated animals. The ability of GnRH-A treatment to stimulate testicular P production in the presence of a high concentration of hCG strongly suggests a direct gonadal action of the peptide. The possibility of such action was corroborated by the finding of abundant GnRH receptors in the neonatal testis. These results indicate that the steroidogenic lesion seen in adult rat testis after gonadotropic stimulation (blockade of C21 steroid side-chain cleavage with compensatory accumulation of P) can be reproduced in neonatal rat testes by a direct action of GnRH-A, but not by hCG.     

Angiotensin II receptors in testes

Receptors for angiotensin II (AII) were identified and characterized in testes of rats and several primate species. Autoradiographic analysis of the binding of 125I-labeled [Sar1,Ile8]AII to rat, rhesus monkey, cebus monkey, and human testicular slide-mounted frozen sections indicated specific binding to Leydig cells in the interstitium. In rat collagenase-dispersed interstitial cells fractionated by Percoll gradient, AII receptor content was parallel to that of hCG receptors, confirming that the AII receptors are in the Leydig cells. In rat dispersed Leydig cells, binding was specific for AII and its analogs and of high affinity (Kd, 4.8 nM), with a receptor concentration of 15 fmol/10(6) cells. Studies of AII receptors in rat testes during development reveals the presence of high receptor density in newborn rats which decreases toward the adult age (4934 +/- 309, 1460 +/- 228, 772 +/- 169, and 82 +/- 12 fmol/mg protein at 5, 15, 20, and 30 days of age, respectively) with no change in affinity. At all ages receptors were located in the interstitium, and the decrease in binding was parallel to the decrease in the interstitial to tubular ratio observed with age. AII receptor properties in membrane-rich fractions from prepuberal testes were similar in the rat and rhesus monkey. Binding was time and temperature dependent, reaching a plateau at 60 min at 37 C, and was increased by divalent cations, EGTA, and dithiothreitol up to 0.5 mM. In membranes from prepuberal monkey testes, AII receptors were specific for AII analogs and of high affinity (Kd, 4.2 nM) with a receptor concentration of 7599 +/- 1342 fmol/mg protein. The presence of AII receptors in Leydig cells in rat and primate testes in conjunction with reports of the presence of other components of the renin-angiotensin system in the testes suggests that the peptide has a physiological role in testicular function.     

Studies of the biological activity of LHRH analogs in the rainbow trout, landlocked salmon, and the winter flounder

Studies of gonadotropic hormone (GtH) release bioactivity by mammalian and submammalian varieties of LHRH and LHRH analog were primarily conducted in vivo in testosterone-primed yearling (TPY) rainbow trout, a convenient test animal for LHRH bioassays in fish. Validation of these results, using sexually mature fish, was accomplished by examining LHRH agonist activities on release of GtH in vivo in spermiating landlocked salmon and by studying LHRH peptide hormone binding affinities using a flounder pituitary LHRH radioreceptor assay. Our surveys of LHRH analog bioactivity in vivo in TPY trout and salmon demonstrated that all types of fish, bird, and mammalian LHRH agonists possess superactive properties on the fish pituitary. The most active group of LHRH analogs, based upon both LHRH receptor binding affinity and in vivo release of gonadotropin, was judged to include [D-Nal(2)6,Pro9-NHEt]LHRH, [D-Nal(2)6-AzaGly10]LHRH, [D-Ala6,Pro9-NHEt]-LHRH, and the fish LHRH analogs, [D-Arg6,Trp7,Leu8,Pro9-NHEt]LHRH, [D-hArg(Et2(6),Trp7,Leu8,Pro9-NHEt]LHRH.     

Systemic administration of alcohol to adult rats inhibits leydig cell activity: Time course of effect and role of nitric oxide

BACKGROUND: Alcohol has been shown to interfere with testosterone (T) release from Leydig cells. However, the mechanisms responsible for this phenomenon, which may include decreased activity of the luteinizing hormone-releasing hormone (LHRH)-LH axis, as well as a direct influence of the drug on the testes, are not fully understood. In this work, we investigated the influence of alcohol, administered intragastrically (i.g.) or delivered via vapors, on Leydig cell activity and T release. Leydig cell function was studied by measuring changes in the levels of the steroidogenic proteins steroidogenic acute regulatory (StAR), the peripheral-type benzodiazepine receptor (PBR), and the cytochrome P450 side-chain cleavage enzyme (P450scc). Testosterone release was studied under basal conditions or in response to human chorionic gonadotropin (hCG). Finally, to identify potential factors mediating the influence of alcohol, we measured the testicular variant of the neuronal nitric oxide (NO) synthase (NOS), TnNOS, in semipurified Leydig cells. METHODS: Adult male Sprague-Dawley rats were either injected with alcohol i.g. once or exposed to alcohol vapors (4 h/d) for 1 or 5 days. Controls received the vehicle (i.g. model) or were kept in boxes through which no vapors were circulated. Following these treatments, one series of experiments was devoted to investigate Leydig cell responsiveness by measuring plasma T levels before or after the intravenous injection of hCG (1 U/kg). In another series of experiments, we used semipurified Leydig cell preparations to measure StAR, PBR, P450scc, and TnNOS by Western blot analysis. RESULTS: In the i.g. model, the T response to hCG was blunted for 12 hours following alcohol injection, but showed a rebound at 48 hours. Levels of StAR protein and of PBR, but not of P450scc, were significantly decreased within 10 minutes of drug administration. While StAR then remained depressed for 24 hours, PBR values were variable over this time course. By 48 hours, StAR, PBR, and P450scc levels had increased above control values. Both StAR and PBR levels showed correlations with plasma T levels. In the alcohol vapor models, both regimens of the drug also significantly depressed StAR and PBR protein concentrations, blunted the T response to hCG, and did not alter P450scc. Finally, we observed that alcohol delivered i.g. or via vapors up-regulated TnNOS levels in Leydig cells, but that blockade of NO formation failed to restore a normal T response to hCG. CONCLUSIONS: Collectively, these results suggest that (a) the ability of Leydig cells to release T does not show a simple correlation with changes in StAR, PBR, and P450scc levels; (b) the time course of the alcohol-induced changes were protein-specific; and (c) despite the ability of alcohol to stimulate TnNOS expression, NO does not appear to mediate the inhibitory influence of this drug on testicular steroidogenesis in the models that we studied.     

Regulation of testicular P-450 cholesterol side-chain cleavage and P-450 C17-20 lyase/C17 hydroxylase enzymes in the neonatal and adult rat

Adult Leydig cells respond to LH or hCG with an initial stimulation of testosterone secretion followed by LH receptor down-regulation and blockade of androgen biosynthesis. In contrast, fetal Leydig cells respond with increased LH receptor number and enhanced steroidogenesis. In this study, the molecular mechanisms of high-dose hCG treatment on steroidogenesis in adult and neonatal testes (containing predominantly the fetal generation of Leydig cells) were examined using two recombinant DNA clones specific for enzymes of the rat steroidogenic pathway (P-450 cholesterol side-chain cleavage enzyme, P-450scc and P-450 17 alpha-hydroxylase/C17-20 lyase, P-450c17). We treated adult (60 days of age) and neonatal (2 days of age) rats with a single high dose of hCG (600 IU/kg), sc. The high dose of hCG caused neonatal testicular P450scc and P450c17 mRNA levels to increase, and stimulated adult testicular P450scc mRNA levels, but caused a decrease in adult P450c17 mRNA levels. These studies suggest that high doses of hCG regulate testosterone production differently in adult and fetal Leydig cells at a pretranslational level of the P450c17 enzyme, while mRNA for P450scc is stimulated in both the adult and fetal Leydig cell.     

Biphasic effect of gonadotropin-releasing hormone and its agonist analog (HOE766) on in vitro testosterone production by purified rat Leydig cells

GnRH and GnRH agonists have stimulatory and inhibitory effects on testicular testosterone secretion both in vivo and in vitro. To determine whether they are exerted directly on the Leydig cells and to explore the temporal relationships, we examined the effects of acute (3 h) and chronic (24-72 h) exposure of purified (greater than or equal to 80%) rat Leydig cells to GnRH and its agonist analog HOE766 (D-Ser-t-BU6,des-Gly-NH2 10LHRH ethylamide; Hoechst, Frankfurt, Germany) on testosterone production. GnRH and HOE766 enhanced basal testosterone secretion by freshly isolated or cultured Leydig cells. HOE766 was at least 100 times more potent than GnRH. However, exposure of Leydig cells to HOE766 for 24 h or longer lead to a significant reduction in hCG responsiveness without altering basal testosterone secretion. Both the stimulatory and inhibitory effects were dose related, with a maximal response elicited by 10(-9) M HOE766. HOE766 reduced Leydig cell sensitivity to hCG (ED50) stimulation, but did not alter the slope of the dose-response curves. Thus, GnRH and its agonist appear to have a dual and biphasic effect on the Leydig cells. Acute exposure stimulates basal testosterone secretion (and occasionally the hCG response), whereas chronic exposure decreases the response to hCG stimulation. These data provide additional evidence that GnRH has a direct effect on Leydig cell steroidogenesis.     

Differences between dispersed Leydig cells and intact testes in their sensitivity to gonadotrophin-stimulation in vitro after alteration of LH-receptor numbers

The sensitivity to hCG-stimulation in vitro of intact hemi-testes and collagenase-dispersed Leydig cells has been compared directly following either an hCG-induced loss of LH-receptors or after a hyperprolactinaemia-induced increase in LH-receptors. Injection of hCG 65 h previously, reduced hCG-binding to dispersed Leydig cells by over 84%. The sensitivity of the steroidogenic response of these cells to hCG-stimulation in vitro was reduced 22-fold whereas intact testes from the same animals showed only a 3-fold reduction in sensitivity to hCG. Dispersed Leydig cells from control rats were 8 times more sensitive to hCG-stimulation than intact testes from the same rats, a difference not evident with hCG-injected rats. In contrast, there was no difference between intact testes and dispersed Leydig cells from control and hCG-injected rats in their sensitivity to stimulation with dibutyryl cyclic AMP in vitro. Induction of hyperprolactinaemia increased hCG-binding to dispersed Leydig cells by 55%. The sensitivity of these cells to hCG-stimulation in vitro was increased by a factor of 4.5, a difference not found with intact testes from the same animals. These results show that experimental manipulation of LH-receptor numbers alters the sensitivity of dispersed Leydig cells, but not of the intact testis, to hCG-stimulation in vitro, a difference which appears to reside at the receptor level. Possible explanations for these findings are discussed together with their implications with respect to the distribution of LH-receptors over the Leydig cell surface.     

Effect of a second injection of human chorionic gonadotropin on the desensitized Leydig cells

Experimental evidence has demonstrated that multiple doses of LH will increase the steroidogenic capacity of Leydig cells. This work was undertaken with the aim of defining the effect of a second hCG administration on the desensitized state, measuring binding of the gonadotropin and the steroidogenic capacity of rat testes. A single injection of 200 IU hCG induced a sharp increase of plasma testosterone which was still evident 24 h later. A second peak was observed at 72 h. The in vivo refractoriness of Leydig cells between 24 and 72 h after the single injection was proved by the fact that a second administration of hCG, 2 h before sacrifice, did not induce any increase in plasma testosterone. A second administration of 200 IU hCG, 48 h after the first injection, showed a similar pattern but on the 5th day there was an increased stimulation of testosterone production with respect to that obtained after a single dose of hCG. The in vitro studies on testicular binding capacity and steroidogenic responsiveness showed that the second administration of hCG, 48 h after the first injection, maintained the testicular binding capacity at the lowest level and the 'adenylate cyclase desensitization' but restored the steroidogenic capacity to even supramaximal values, compared to normal rats, 3 days after this second hCG administration. These results would support a dissociation between receptor loss and maximal testosterone synthesis as well as possibly indicating an alternative pathway different from the classical.     

Effects of FSH on Leydig cell morphology and function in the hypogonadal mouse.

The hypogonadal (hpg) mouse has a congenital deficiency in gonadotrophin-releasing hormone and the gonads consequently lack exposure to endogenous gonadotrophins during development. To determine the effect of FSH on Leydig cell function in these animals adult hpg mice were injected twice daily with FSH (2 micrograms injections) or LH (40 ng injections, the presumed LH contamination of FSH used). Following FSH treatment there was a clear stimulation of the seminiferous epithelium and in animals injected with FSH plus [3H]thymidine, the incorporation of label was largely confined to the germ cells with no apparent uptake by the Sertoli cells. In FSH-treated testes the Leydig cells contained numerous large lipid droplets, similar to the unstimulated hpg testis. There was no evidence of the interstitial hyperplasia which is observed following injection of high doses of LH (2 micrograms twice daily). There was no change in basal androgen content of the testis in vivo following FSH treatment but injection of a maximal dose of human chorionic gonadotrophin (hCG), 1 h before death, markedly increased testicular androgen content only in the FSH-treated group. Testicular androgen production in vitro was significantly increased following FSH treatment both under basal conditions (FSH-treated, 17.4 pmol/testis; control, 1.46 pmol/testis) and during stimulation by hCG (FSH-treated, 940 pmol/testis; control, 81 pmol/testis). Associated with the increased androgen production following FSH treatment there were significant increases in the activities of three steroidogenic enzymes; cholesterol side-chain cleavage (186-fold increase over control), 17 alpha-hydroxylase (103-fold increase) and 17-ketosteroid reductase (177-fold increase).(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin and steroid secretory patterns during chronic treatment with a luteinizing hormone-releasing hormone agonist analog in men

LHRH agonist analogs induce hypogonadism in man but the mechanism is uncertain. To evaluate this, we treated 13 normal men with 50 micrograms/day D-Trp6,Pro9-NEt LHRH (LHRHA) for periods up to 8 weeks and measured (1) patterns of endogenous gonadotropin and testosterone secretion, (2) gonadal response to exogenous human LH infusions, and (3) gonadotropin and testosterone responses to hourly bolus doses of LHRH. Seven men were evaluated with frequent sampling for 12-h periods every 4 week during treatment with LHRHA. Before treatment, all had three to four spikes of LH in 12 h. On the first day of treatment, the response to LHRHA was tested in four of the men, and there were significant increases in LH, FSH, and testosterone. After 4 weeks, all men had dramatic decreases in mean testosterone levels and blunted or absent gonadotropin responses to acute injection of LHRHA. Mean gonadotropin levels at 4 and 8 weeks were variable; values lower than pretreatment basal levels were found in three men, while unchanged or higher values were found in the remaining four. The pulsatile pattern of LH secretion, characteristic among these men before treatment, was lost during LHRHA therapy. Forty-eight-hour constant infusion of human LH in four other analog-treated men resulted in increases in serum testosterone comparable to those in untreated men. Pulsatile administration of native sequence LHRH to two other men during chronic treatment with LHRHA failed to elicit demonstrable responses in serum gonadotropin or testosterone levels. LHRHA produced a qualitative change in the pattern of LH release from the pituitary. Mean basal LH levels varied during treatment, but the normal pulsatile pattern was diminished. The gonadotropin response to pulsatile administration of LHRH was lost during chronic treatment with LHRHA, but the Leydig cell remained responsive to exogenous human LH. Thus, the locus of action of the analog appears to be at the level of the pituitary in man.     

RNA synthesis in the Leydig cells of the mature rat: effect of oestradiol-17 beta

In view of the evidence that there may be an effect of high concentrations of oestradiol on testicular steroidogenic function, we have investigated the effect of this steroid on [3H]uridine incorporation into RNA by testicular cells. Our results have shown that oestradiol in vitro induced a marked dose-dependent inhibition of RNA synthesis by purified Leydig cells. The concentrations of oestradiol tested varied from 2 to 40 mumol/l; these concentrations also impaired net testosterone synthesis in vitro after human chorionic gonadotrophin (hCG) stimulation. Under the effect of oestradiol, the kinetics of [3H]nucleoside incorporation into RNA were impaired early and the inhibition of RNA synthesis was specific for oestrogenic compounds. It was concluded that, in Leydig cells, oestradiol, in addition to its known inhibitory action on the response of testosterone to hCG, triggers a more extensive response that also includes RNA synthesis in vitro.