Intratesticular factors and testosterone secretion: the role of luteinizing hormone in relation to changes during puberty and experimental cryptorchidism

Testicular interstitial fluid (IF) from the rat contains a nongonadotropic polypeptide factor (or factors) which can enhance human CG (hCG)-stimulated testosterone production by Percoll-purified Leydig cells in vitro. The potential importance of this factor has been investigated by measuring its effective levels in testicular IF from individual rats during sexual maturation or after the induction of unilateral cryptorchidism (UCD). In both situations, the effect of modulating LH drive to the testis was also assessed. In abdominal testes from UCD rats, levels of the IF factor were nearly doubled (P less than 0.001) when compared to levels in the contralateral scrotal testis; this was associated with more than an 85% reduction in IF testosterone levels. Further lowering of testosterone levels by the injection of an antiserum to LH beta, raised levels of the IF factor by 30-40% (P less than 0.01) in both scrotal and abdominal testes. Conversely, raising of testosterone levels by injection of hCG caused more than a 90% reduction (P less than 0.001) in levels of the IF factor in both scrotal and abdominal testes. During sexual maturation, levels of the IF factor doubled (P less than 0.01) between 30 and 40 days of age, remained high until 70 days of age, and then decreased to the lower levels found in adult rats. High pubertal levels of the IF factor were associated with the most rapid phase of testicular growth and with a steady increase in the IF levels of testosterone. Injection of pubertal or adult rats with antiserum to ovine LH (oLH) reduced testosterone levels by approximately 85% and doubled (P less than 0.01) levels of the IF factor(s) in both groups. It is concluded that levels of the IF-factor are influenced: by testosterone levels and/or LH drive and by the maturational and/or functional status of the testis. These results also suggest that changing levels of the IF factor may be of physiological significance during puberty.     

Intratesticular regulation of testosterone secretion: comparison of the effects and interactions of hCG, an LHRH agonist and testicular interstitial fluid on Leydig cell testosterone secretion in vitro

The stimulatory effects on Leydig cell testosterone secretion of a polypeptide(s) factor present in testicular interstitial fluid (IF) were compared with those of hCG and an LHRH agonist (LHRH-A). The actions of IF and LHRH-A were similar in showing (1) a delayed onset of action, (2) enhancement of testosterone production in response to a maximally stimulating concentration (5 nM) of hCG, and (3) near cessation of stimulation following their removal from the incubation medium. However, addition of an LHRH antagonist blocked only the actions of LHRH-A. Moreover, IF continued to stimulate testosterone production up to at least 20 h either on its own or in the presence of 5 nM hCG, whereas the stimulatory effects of LHRH-A disappeared beyond 6 h. IF was also able to enhance testosterone production in response to LHRH-A or in response to hCG + LHRH-A. IF enhanced testosterone production over 4-20 h in response to all doses of hCG and increasing concentrations of IF caused dose-dependent increments in the rate of hCG (5 nM) stimulated testosterone production. With submaximally stimulating doses of hCG or with LHRH-A alone, the stimulatory effect of IF was more or less additive, whereas with maximally stimulating doses of hCG the effect of IF was clearly synergistic. Thus, whereas the rate of testosterone production by Leydig cells in response to 5 nM hCG declined progressively from 4 to 20 h, addition of IF attenuated or prevented this decline. These findings have implications with respect to the physiological control of intratesticular testosterone levels and with respect to the regulation of steroidogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary culture of purified Leydig cells isolated from adult rat testes

Methods for isolating highly purified Leydig cells permit the study of acute responses and biochemical properties of Leydig cells independent of other testicular cell types. The present study describes the development of a primary culture system for purified Leydig cells from adult rats in which the cells retain their ability to secrete testosterone for at least 72 h in culture. When Leydig cells were cultured in tissue culture medium 199--0.1% BSA (M199-BSA), basal testosterone secretion declined by 72 h, whereas hCGB-stimulated testosterone secretion was reduced by 48 h. Changing the culture medium twice daily or adding 0.5% fetal calf serum (fcs) enhanced basal and gonadotropin-stimulated testosterone secretion at 72 h in culture, although responsiveness to hCG was reduced to 57% of that in freshly isolated cells. Incubation of Leydig cells in the defined culture medium Dulbecco's Modified Eagles-Ham's F-12 (1:1, vol/vol) supplemented with 15 mM Hepes buffer, transferrin, insulin, and epidermal growth factor (DHG:F12 + Hepes + TIE) in either the presence or absence of 0.5% fcs yielded functional Leydig cells for longer intervals in culture. Furthermore, testosterone secretion was greater in DHG:F12 + Hepes + TIE than in M199-BSA at all time intervals tested. In DHG:F12 + Hepes + TIE, basal and gonadotropin-stimulated testosterone production by Leydig cells were maintained for 72 h in culture. Degenerative changes in morphology were apparent in some cells at 72 h, but not at earlier times in culture. This primary culture system for isolated Leydig cells provides a valuable tool to examine the temporally regulated events in Leydig cell function.     

Intratesticular secretion of a factor(s) with major stimulatory effects on Leydig cell testosterone secretion in vitro

As intratesticular communication between the Sertoli and Leydig cells must take place via testicular interstitial fluid (IF), we have tested whether this fluid contains a factor(s) which has effect on testosterone secretion by Percoll-purified Leydig cells in vitro. Addition of increasing concentrations of charcoal-stripped IF to Leydig cells caused dose-dependent stimulation of basal testosterone secretion and greatly enhanced the response to a maximally stimulating dose of hCG. These effects were first evident after 2 h of incubation and were progressive up to 24 h. Charcoal-stripped serum from the same donor animals also had some stimulatory effect on basal and, occasionally, on hCG-stimulated testosterone secretion, but the magnitude and pattern of this effect and its limited dose-dependence were very different from the effects seen with IF. The effects of IF and serum were not altered by the addition of an antiserum to LH. It is therefore concluded that testicular IF contains a factor(s), not derived from serum, which can alter Leydig cell testosterone responsiveness. This factor(s) was heat-sensitive with a MW of greater than 10 000 daltons. IF from the abdominal testes of adult rats which had been made unilaterally cryptorchid contained much higher levels of the stimulatory factor(s) compared to the contralateral scrotal testes, and this is of interest because of the well-documented alteration in Sertoli-Leydig cell interaction that occurs in this situation. These results provide the first direct evidence that one or more factors capable of altering testosterone secretion are produced within the testis of the normal adult rat, and raises the possibility that this factors(s) may be involved in the local regulation of the intratesticular testosterone levels.     

The quantification of steroidogenesis-stimulating activity in testicular interstitial fluid by an in vitro bioassay employing adult rat Leydig cells

An in vitro bioassay for steroidogenesis-stimulating activity (SSA) in charcoal-extracted rat testicular interstitial fluid (IF) was developed. The bioassay was based upon stimulation of testosterone production by Percoll gradient-purified adult rat Leydig cells during a 20 h incubation in the presence of a maximally stimulating dose of human CG (hCG). The hCG-stimulated conditions were employed to avoid assay interference by endogenous LH in the sample preparations. The standard preparation (a pool of IF from normal adult rats) stimulated testosterone production 3- to 4-fold above that obtained with a maximal dose of either hCG or LH alone, and a linear log dose-testosterone response was observed over the range from 150% to 300% of hCG-stimulated testosterone production. The bioassay had a mean index of precision (lambda) of 0.13 (n = 10 assays), a between assay variation of 10.7-11.7%, and a useful working range from 4.9-28 microliters IF including at least three serial half-dilutions. Parallelism with the IF standard was obtained with IF collected from aged (greater than 15 months old) rats, adult rats made bilaterally cryptorchid for either 4 weeks or 12 months, or injected 6 h previously with 100 IU hCG, and with ovine testicular lymph. Testicular SSA was not affected by coincubation with rat LH antiserum and was not attributable to prevention of oxygen-mediated enzyme damage during the incubation period. Although charcoal-extracted rat serum log dose-response relationships were nonparallel with the standard, serum displayed an apparent relative bioactivity of approximately 20% that of normal IF based on ED50 comparisons. The activity of testicular IF was not affected by coincubation with serum. Untreated and charcoal-extracted rat albumin, at an assay concentration equivalent to that present in normal rat serum or IF, caused only a minor stimulation of testosterone production. Charcoal-extracted bovine albumin, ovalbumin, epidermal growth factor, insulin-like growth factor-1, bovine 31 kDa inhibin, and transforming growth factor-beta were inactive. Activity was nondetectable in rat thoracic duct lymph or high speed supernatants of adult rat testicular extracts. Testicular SSA and serum from normal rats displayed slightly different time-courses of action, with SSA active during both acute (1.0 h) and longer term (2.0-20 h) incubations, while serum stimulated testosterone production only in the longer term incubations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increase in Leydig cell responsiveness in the unilaterally cryptorchid rat testis and its relationship to the intratesticular levels of testosterone

Adult rats were made unilaterally cryptorchid (UCD) and 6-7 weeks later Leydig cells were isolated from the scrotal and abdominal testes and their capacity to secrete testosterone in vitro was compared. Basal testosterone production by Leydig cells from the abdominal testes of UCD rats was lowered, compared with cells from the contralateral scrotal testes, whilst their responsiveness to both human chorionic gonadotrophin and an LH releasing hormone agonist was enhanced two- to threefold (P less than 0.001) compared both with cells from the contralateral scrotal testes and with cells isolated from untreated rats of the same age. In the UCD rats, concentrations of testosterone in testicular interstitial fluid (IF) were reduced (P less than 0.001) by 70-90% in abdominal, compared with scrotal, testes. A similar reduction was evident in the levels of testosterone in spermatic venous blood, and both this decrease and that in IF levels of testosterone varied according to the degree of testicular involution. The ontogeny of the above changes was investigated. After induction of unilateral cryptorchidism, the weight of the abdominal compared with the scrotal testis declined slowly, such that by day 5 there was only a 25% reduction in weight compared with a 70% reduction by day 40. In contrast, the levels of testosterone in IF from abdominal testes declined rapidly, such that by day 5 an 80% reduction was attained, compared with scrotal testes, with little further change by day 40.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-2 is a potent inhibitor of Leydig cell steroidogenesis

Interstitial tissue of the testis consists of Leydig cells, macrophages, lymphocytes, plasma cells, mast cells and fibroblasts. Previously we have reported that interleukin-1 (IL-1) inhibits Leydig cell androgen production. In the present study, the effect of IL-2 was investigated. Leydig cells (10(5) cells/ml) from adult Sprague-Dawley rats were cultured with or without IL-2 for 24 h. After medium changes, human CG (hCG), 8-bromo-cAMP, or forskolin was added with or without IL-2. Cultures were continued for an additional 24 h, and testosterone and cAMP levels were measured. IL-2 up to 100 U/ml had no effect on basal testosterone production. hCG-stimulated testosterone formation was inhibited in a dose-dependent manner by the addition of IL-2. IL-2 in a concentration of 100 U/ml decreased hCG-induced testosterone formation from 49.6 +/- 3.6 ng/ml (mean +/- SE) to 8.5 +/- 4.2 ng/ml. The hCG dose-response curve was shifted to the right by the addition of IL-2. Maximal testosterone production in response to hCG was reduced 40% in the presence of IL-2 (50 U/ml) without alteration of median effective dose (ED50). IL-2 also inhibited hCG-induced cAMP formation and 8-bromo cAMP- and forskolin-stimulated testosterone production. However, IL-2 did not alter the binding of [125I]hCG to purified Leydig cells. Furthermore, IL-2 significantly inhibited the conversion of 20-OH-cholesterol, 22-OH-cholesterol, pregnenolone, progesterone, 17 alpha-hydroxypregnenolone, and 17 alpha-hydroxyprogesterone to testosterone but did not alter the conversion of dehydroepiandrosterone and androstenedione to testosterone. Our results suggest that a T cell growth factor, IL-2, is a potent inhibitor of steroidogenesis. IL-2 may play a paracrine role in modulating Leydig cell function.     

The role of LH in regulation of Leydig cell responsiveness to an LHRH agonist

The short-term relationship between deprivation of LH and changes in the responsiveness of isolated Leydig cells to an LHRH agonist was studied in adult rats injected with a potent antiserum to oLH. This treatment suppressed serum and intra-testicular levels of testosterone within 4 h of injection by 80-90% and 70%, respectively, with further suppression to levels found in hypophysectomized rats by 20 h. At 12-20 h after antiserum injection there was no major consistent change from control values in the number of Leydig cell LH (hCG)-receptors or in the testosterone response of the cells to hCG-stimulation. In the same cells the number of LHRH-receptors and the maximal testosterone response to LHRH agonist were increased (P less than 0.001) by 50% or more, although the magnitude of increase varied considerably between experiments. The ability of LHRH agonist to enhance the testosterone response of cells to hCG-stimulation was also increased significantly (P less than 0.001) in antiserum-treated rats. Comparable changes in testosterone responsiveness to LHRH agonist were observed following purification of Leydig cells on Percoll gradients. Although there was some evidence for changes in Leydig cell LHRH-receptor numbers and responsiveness to LHRH agonist at 4 h after intravenous antiserum injection, these changes were small compared with changes seen at later times. It is concluded that Leydig cell responsiveness to an LHRH agonist is negatively regulated by LH, and this has implications with respect to a possible role of 'testicular LHRH' in regulation of the intra-testicular levels of testosterone.     

Testicular blood flow, vascular permeability, and testosterone production after stimulation of unilaterally cryptorchid adult rats with human chorionic gonadotropin

Testicular blood flow, testosterone production, and the formation of testicular interstitial fluid (IF) were studied in unilaterally cryptorchid rats, basally, 8 h and 24 h after treatment with 200 IU human CG (hCG). Testicular blood flow was lower in the abdominal testis in both control rats and hCG-treated rats than in the scrotal testis within the same treatment group. The scrotal testicular blood flow increased significantly 24 h after hCG treatment, but not after 8 h. In the abdominal testis, there was a significant increase of blood flow 8 h after hCG, but not 24 h after. The formation of IF was subnormal in the abdominal testes of control rats, but this was corrected in hCG-treated rats, where there was a significant increase of IF in both abdominal and scrotal testes. The total endothelial surface of small blood vessels was decreased in abdominal testes. Testosterone concentration in the spermatic vein was significantly lower on the abdominal side than on the scrotal side in both control and hCG-treated rats. The concentration of testosterone was lower in IF on the abdominal side in control rats, but after hCG the testosterone concentration was similar in both scrotal and abdominal testes, indicating a trapping of testosterone in the abdominal testis. The outflow of testosterone in the spermatic vein was significantly increased at both 8 and 24 h after hCG from both the scrotal and abdominal testes, although it was always smaller from the abdominal testis. The lower secretion of testosterone from the abdominal testis after hCG was mainly due to reduced blood flow and not to any disability of the Leydig cells of abdominal testes to produce testosterone.     

Comparison of the effects on purified Leydig cells of four hormones (oxytocin, vasopressin, opiates and LHRH) with suggested paracrine roles in the testis

Four hormones have been identified by various authors as possible paracrine regulators of testicular Leydig cells. The aim of this study was to evaluate their effects on purified adult rat Leydig cells under various conditions in vitro, and then to assess whether comparable effects occurred in vivo. In agreement with previous findings, an LHRH agonist (LHRH-A) exerted clear-cut effects on testosterone secretion by Leydig cells both in vitro and in vivo. On its own, LHRH-A stimulated testosterone production by Leydig cells for up to 24 h in culture but inhibited testosterone production stimulated by human chorionic gonadotrophin (hCG) between 24 and 72 h of culture. In-vivo, unilateral intratesticular injection of adult rats with 1 ng LHRH-A resulted 5 h later in a significant increase in testosterone concentrations in testicular interstitial fluid (IF). Vasopressin exerted effects in vitro which were similar to those of LHRH-A. On its own, vasopressin stimulated testosterone production for up to 5 h of culture, but not thereafter, while in the presence of hCG, vasopressin inhibited testosterone production beyond 24 h of culture. The initial stimulatory effect of vasopressin on testosterone production occurred with concentrations of 1 nmol/l and higher, but the magnitude of stimulation (threefold or less) was considerably less than that induced by LHRH-A (ninefold) over the same time period. In contrast to LHRH-A, unilateral intratesticular injection of vasopressin in high doses (20 and 2 ng) had no effect on IF testosterone levels 5 h later.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo chorionic gonadotropin administration reverses the testosterone secretory defect of Leydig cells from old rats

Old male rats have decreased serum testosterone, luteinizing hormone (LH), follicle -stimulating hormone (FSH), Leydig cell testosterone secretory responsiveness to chorionic gonadotropin (hCG), and gonadotropin binding capacity. Although prolonged in vivo hCG administration restores serum testosterone, in vitro hCG exposure of Leydig cells does not reverse their age-related hyporesponsiveness. To explore this discrepancy, we studied Leydig cell function before and after hCG administration in vivo (treatment). Before treatment, old rats had lower serum testosterone, in vitro testosterone and cyclic adenosine monophosphate (cAMP) production, both basally and in response to hCG, and reduced hCG binding capacity. After treatment, old and young rats did not differ in any of these parameters. hCG binding and cAMP responses were reduced in both old and young rats. Thus, prolonged in vivo exposure to hCG appeared to reverse both the in vivo and in vitro age-related Leydig cell secretory defect, despite gonadotropin receptor down-regulation. This suggests that the "aging" defect(s) are caused by chronic gonadotropin deprivation.     

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)     

Isolation of human Leydig cells which are highly responsive to human chorionic gonadotropin

Leydig cells were purified on discontinuous Percoll gradients after collagenase digestion of human or rat testes. Leydig cells from both species were found in three bands. As determined by positive staining for 3 beta-hydroxysteroid dehydrogenase, band 1 (lowest density cells) from both species contained only 12-28% Leydig cells. However, while band 3 was the most Leydig cell-enriched fraction in rat cell preparations (70-90% Leydig cells), human band 2 (48-70% Leydig cells) was consistently more Leydig cell enriched than was band 3 (30-56% Leydig cells). Despite their slightly different fractionation pattern, Leydig cells prepared from five men with prostatic carcinoma were similar to those from the rat, both in terms of the amount of testosterone produced basally per 10(6) Leydig cells (80-234 ng/20 h) and in terms of the magnitude of their response to hCG (764-1342 ng/10(6) Leydig cells X 20 h; 5- to 17-fold stimulation above basal). Cells prepared from five other men with prostatic carcinoma produced much lower amounts of testosterone, but still had up to a 6-fold response to hCG. Plasma LH, FSH, and testosterone concentrations in the latter group could not be distinguished from those in the group whose Leydig cells produced large amounts of testosterone in vitro. Morphologically, the testes from the latter group appeared to contain more darkly staining than lightly staining Leydig cells than did the former group. Rat Leydig cells responded in a dose-dependent fashion to hCG over the range 0.03-0.5 mU/mL, whereas human Leydig cells were 10- to 100-fold less sensitive, responding to hCG in the range 0.4-100 mU/mL. The number and affinity of Leydig cell LH (hCG) receptors were assessed from Scatchard analysis of [125I]hCG binding. Compared with rat cells, human Leydig cells contained approximately 20% of the number of LH receptors, while the affinity of the receptors (Kd, approximately 10(-10) M) was similar to that in rats. In conclusion, a method for the isolation of highly responsive human Leydig cells has been developed. The results so far suggest that the function of human Leydig cells may be more similar to that of the rat than thought previously.     

Assessment of the contribution of Leydig cells to the secretion of inhibin by the rat testis

Cultured Leydig cells secreted 1.3-4.3 ng 1-26 alpha-inhibin/10(6) cells/24 h, and although this was unaffected by human chorionic gonadotrophin (hCG), these cells could contribute to the intratesticular and blood levels of inhibin. The present study evaluated this contribution in rats in which the Leydig cells were destroyed by injection of ethane dimethane sulphonate (EDS). In these animals, inhibin levels increased in testicular interstitial fluid (IF), and in testicular (TV) and spermatic (SV) venous blood. In EDS-treated rats supplemented for 21 days with 1 or 25 mg testosterone esters to maintain full spermatogenesis and/or suppress the elevated follicle-stimulating hormone (FSH) levels and prevent Leydig cell regeneration, significant changes occurred in the levels of inhibin in IF, in TV and SV plasma and in the route of secretion of inhibin from the testis (i.e. via IF or seminiferous tubule fluid). However, none of these changes was related to the presence or absence of Leydig cells. It is concluded that Leydig cells make little contribution to the intratesticular and blood levels of inhibin in the adult rat.     

Interstitial fluid volume in the rat testis: androgen-dependent regulation by the seminiferous tubules?

Regulation of testicular interstitial fluid (IF) volume has been investigated in adult male rats in which the Leydig cells were selectively destroyed with a single i.p. injection of ethane dimethane sulphonate (EDS). Following this treatment, some animals also received testosterone supplementation by s.c. injection every 3 days, beginning either from the time of EDS injection, or 3-12 days afterwards. The volume of IF obtained by drip collection was determined, and testosterone and gonadotrophin concentrations measured in blood and in IF. Testosterone levels in IF and serum became undetectable by 3 days after EDS treatment. IF volume was reduced by 50% (P less than 0.01) to reach a minimum level between 6 and 9 days after treatment. However, this decline was prevented in the absence of Leydig cells by supplementation with testosterone from the time of EDS injection, a treatment which also kept gonadotrophins at minimum or undetectable levels. Furthermore, the reduced IF volume seen up to 9 days after treatment with EDS alone could be restored to control levels within 3 days by a single injection of testosterone. The results obtained demonstrate that androgens, but not Leydig cells or gonadotrophins, are required for the maintenance of interstitial fluid volume in the adult rat testis. It is suggested that the seminiferous tubules may mediate this response, through an androgen-dependent mechanism.     

Regulation of testosterone production in Leydig cells from fetal mice under dynamic conditions: effect of human chorionic gonadotrophin and 8-bromo-cyclic AMP

The temporal release of testosterone by Leydig cells from 18-day-old mouse fetuses in response to human chorionic gonadotrophin (hCG) and to 8-bromocyclic AMP (8-bromo-cAMP) was investigated under short-term incubation (180 min) conditions. A rapid and large increase in testosterone release was induced by a 5-min exposure to hCG (20 i.u./l) or 8-bromo-cAMP (10 mmol/l). The testosterone response of fetal Leydig cells to the two gonadotrophic stimuli was Gaussian in distribution with a peak value of testosterone by 15-20 min. Repeated exposure to hCG resulted in a reduced testosterone response but an increased accumulation of cAMP. The apparent resistance of fetal Leydig cells to hCG could not be overcome either by increasing the hCG concentration (to 2000 i.u./l) or by exposing the cells to 8-bromo-cAMP (10 mmol/l). Continuous exposure to hCG (200 i.u./l) divided into multiple small doses (each 8 i.u./l) induced testosterone secretion with different kinetic characteristics: a three-fold longer time-lag between hormone exposure and the peak value; a twofold greater testosterone response (P less than 0.001) and a gradual decrease of testosterone secretion. Oestradiol significantly reduced basal and hCG-stimulated testosterone production only at a high concentration (10 mumol/l). These results indicate that continuous or pulsatile exposure to hCG can induce refractoriness of fetal Leydig cells. The similarity between the actions of hCG and 8-bromo-cAMP on fetal steroidogenesis suggests that this rapid defect is not primarily due to a depletion of gonadotrophin receptors but results from disruption of regulatory mechanisms at the post-receptor level.     

Identification of an inhibitory factor from a Sertoli clonal cell line (TM4) that modulates adult rat Leydig cell steroidogenesis

Sertoli cell produces several biological factors that modulate Leydig cell steroidogenic function by either stimulating or inhibiting its testosterone production. We have evaluated the effect of an inhibitory factor in the spent media of a Sertoli clonal cell line (TM4) which inhibits Leydig cell steroidogenesis. The presence of such an inhibitory factor in TM4 media was bioassayed using Percoll purified Leydig cells isolated from adult rats with purity of greater than 95%. TM4 media inhibited both human chorionic gonadotropin (hCG)-stimulated testosterone and cAMP production by purified Leydig cells dose-dependently but had no apparent effect on 8-bromo-cAMP- and forskolin-stimulated testosterone production. Also it did not interfere with the binding of [¹²⁵I]hCG to Leydig cells. TM4 media inhibited cholera toxin-stimulated testosterone production as well as forskolin- and cholera toxin-stimulated cAMP production. The mechanism of action of this factor in TM4 media appears to be different from transforming growth factor (TGF-β) which inhibited both 8-bromo-cAMP- and forskolin-stimulated testosterone production and inhibited the binding of [¹²⁵I]hCG binding to Leydig cells. The inhibitory factor contained in TM4 media has been partially purified by sequential preparative anion exchange and C-18 reversed-phase high-performance liquid chromatography. In summary, the Sertoli TM4 cell line produces at least one potent inhibitory factor which decreases the responsiveness of purified Leydig cells to hCG stimulation with a dramatically different mechanism from other currently known Leydig cell inhibitory factors; this protein may serve as a valuable tool to study testicular paracrine regulation.     

Evidence for Leydig cell dysfunction in rats with seminiferous tubule damage.

To study the effects of seminiferous tubule damage on Leydig cell function and morphology, rats were treated by fetal irradiation (to induce Sertoli cell-only syndrome, SCO), 3 months administration of hydroxyurea (HU), or chronic feeding of a vitamin A-deficient diet (VAD). Leydig cell function was assessed by the measurement of serum LH and testosterone and the response of serum testosterone to hCG stimulation, while morphology was studied by electron microscopy after perfusion fixation. Serum LH was significantly elevated in each experimental group, while basal serum testosterone was significantly lower only in SCO rats. In all treatment groups, the serum testosterone response to hCG was significantly decreased when measureed as the area under the response curve. Despite a decreased response to hCG, the Leydig cells were larger than normal and showed striking increases in quantities of smooth endoplasmic reticulum, mitochondria and Golgi complex. Leydig cell dysfunction has been demonstrated in animals with varying degrees of seminiferous tubule damage, but paradoxically the cytological features of the Leydig cells were indicative of hypertrophy.     

5 alpha-reductase activity regulates testosterone accumulation in two bands of immature cultured Leydig cells isolated on Percoll density gradients

The 5 alpha-reductase inhibitor, 4-methyl-4-aza-3-oxo-5 alpha-pregnan-20(s)-carboxylate was utilized to examine maturational changes in testosterone synthesizing capacity of Leydig cells localizing in Band 2 and Band 3 of Percoll density gradients. Immature Band 2 Leydig cells (from 25- to 39-day-old rats) cultured without the 5 alpha-reductase inhibitor, produced increased testosterone in response to hCG or 8-br-cAMP; however, basal, hCG- or 8-br-cAMP-stimulated testosterone accumulation was 4- to 12-fold higher when cells were cultured in the presence of inhibitor. Band 2 cells from older rats produced much less testosterone in response to hCG or 8-br-cAMP (less than 3.5 pmol/10(5], even when cultured with the inhibitor. Although immature Band 3 cells cultured in the absence of 5 alpha-reductase inhibitor produced increased testosterone in response to hCG or 8-br-cAMP, testosterone levels were relatively low, because elevated 5 alpha-reductase prevented appreciable androgen accumulation; however, Band 3 cells from older rats (over 39 days old) accumulated progressively more testosterone, because of the age-dependent decline in 5 alpha-reductase activity. Immature Band 3 cells cultured with the 5 alpha-reductase inhibitor accumulated 20- to 44-fold more testosterone in response to hCG of 8-br-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lutropin/choriogonadotropin stimulate the proliferation of primary cultures of rat Leydig cells through a pathway that involves activation of the extracellularly regulated kinase 1/2 cascade

Primary cultures of progenitor and immature rat Leydig cells were established from the testes of 21- and 35-d-old rats, respectively. The cell population remained homogeneous after 4-6 d in culture as judged by staining for 3beta-hydroxysteroid dehydrogenase, but the cells were unable to bind 125I-human chorionic gonadotropin (hCG) or to respond to hCG with classical LH receptor (LHR)-mediated responses, including cAMP and inositol phosphate accumulation, steroid biosynthesis, or the phosphorylation of ERK1/2. Infection of primary cultures with recombinant adenovirus coding for beta-galactosidase showed that approximately 65% of the cells are infected. Infection with adenovirus coding for the human LHR (hLHR) allowed for expression of the hLHR at a density of approximately 25,000 receptors per cell and allowed the cells to respond to hCG with increases in cAMP and inositol phosphate accumulation, steroid biosynthesis, and the phosphorylation of ERK1/2. Although progenitor and immature cells were able to respond to hCG with an increase in progesterone, only the immature cells responded with an increase in testosterone. In addition to these classical LHR-mediated responses, the primary cultures of progenitor or immature rat Leydig cells expressing the recombinant hLHR proliferated robustly when incubated with hCG, and this proliferative response was sensitive to an inhibitor of ERK1/2 phosphorylation. These studies establish a novel experimental paradigm that can be used to study the proliferative response of Leydig cells to LH/CG. We conclude that activation of the LHR-provoked Leydig cell proliferation requires activation of the ERK1/2 cascade.