Functional activity of rat testicular cells in culture

Testicular cells from adult hypophysectomized rats were cultured for 10 or 12 days, and the effect of treatment with hCG (10 ng/ml) on testosterone and progesterone production and the activity of the Leydig cell enzyme, 3 beta-hydroxysteroid dehydrogenase, were studied. Regardless of hormone treatment, on 4th day in culture a decline in the steroidogenic activity of cultured cells could be observed. Treatment with hCG resulted in stimulation of steroidogenesis on days 6 to 10 in culture, as measured by testosterone and progesterone production. Hormone treatment stimulated or inhibited the enzyme activity depending on the presence or absence in the culture medium of 10(-6) M spironolactone, an inhibitor of 17 alpha-hydroxylase, or an anti-androgen, cyproterone acetate.     

Studies with purified immature porcine Leydig cells in primary culture

The steroidogenic capacity of purified immature porcine Leydig cells in culture was studied over several days. The cells were obtained by fractionating crude testicular interstitial cell suspensions on a discontinuous Percoll gradient (d = 1.037, 1.042, 1.052, 1.098 g/ml), and characterized by specific binding of 125I-human chorionic gonadotropin (hCG), testosterone (T) and cyclic adenosine 3':5'-monophosphate (cAMP) production in response to hCG, and the enzymatic determination of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. The Leydig cells were recovered in a density band between 1.052-1.068 g/ml and grown in a chemically defined medium (Mather et al., 1981). In the absence of hCG, T production was low throughout the 6 days of culture. However, in response to hCG (10 mIU/ml), the cultured Leydig cells showed a progressive increase in T synthesis, which reached a maximum at Days 3-4. 8-Br-cAMP (1 mM) induced a comparable rise in T production to that obtained with hCG throughout the culture period. In contrast, 8-Br-cAMP induced a near maximal increase in dehydroepiandrosterone (DHEA) production from Day 1. This paper demonstrates that purified immature porcine Leydig cells in primary culture are a valuable model to study the ontogeny of Leydig cell function.     

Leydig cell number and function in the adult cynomolgus monkey (Macaca fascicularis) is increased by daily hCG treatment but not by daily FSH treatment

Daily treatment of adult cynomolgus monkeys with 450 i.u. hCG for 16 days resulted in a significant 163% increase in the number of Leydig cells, and a 9-fold rise in plasma testosterone concentrations. The number of proliferating Leydig cells was very low, even after 16 days of treatment with hCG. Daily FSH administration (2 injections of 15 i.u. per day) did not have any effect on the number of Leydig cells or plasma testosterone values. It can be concluded, therefore, that in adult cynomolgus monkeys daily hCG treatment results in an increase in the number of Leydig cells, which is mainly caused by the differentiation of precursor cells. Since plasma testosterone concentrations were increased to an even higher extent, the steroid production per Leydig cell was also stimulated.     

Regulation of 3 beta-hydroxysteroid dehydrogenase activity by human chorionic gonadotropin, androgens, and anti-androgens in cultured testicular cells

delta 5-3 beta-Hydroxysteroid dehydrogenase is a key enzyme for testicular androgen biosynthesis and a marker for the Leydig cells. The hormonal regulation of this enzyme was studied in cultured rat testicular cells. Human chorionic gonadotropin (hCG) increased testosterone production in vitro while time course studies indicated a biphasic action of the gonadotropin on 3 beta-hydroxysteroid dehydrogenase activity. An initial stimulation (51%) of the enzyme was detected between 3 and 12 h of culture when medium testosterone was low. This is followed by an inhibition of 3 beta-hydroxysteroid dehydrogenase activity on days 2 and 3 of culture when medium testosterone was elevated. Concomitant treatment with a synthetic androgen (R1881) inhibited 3 beta-hydroxysteroid dehydrogenase activity and testosterone production in hCG-treated cultures while an anti-androgen (cyproterone acetate) increased 3 beta-hydroxysteroid dehydrogenase activity and testosterone biosynthesis. Addition of 10(-5) M spironolactone, an inhibitor of 17 alpha-hydroxylase, blocked the hCG stimulation of testosterone production but increased medium progesterone. In the absence of the secreted androgen, hCG stimulated 3 beta-hydroxysteroid dehydrogenase activity in a time- and dose-related manner. Furthermore, hCG stimulation of 3 beta-hydroxysteroid dehydrogenase activity and progesterone accumulation in spironolactone-supplemented cultures was decreased by concomitant treatment with R1881 but was not affected by cyproterone acetate. The inhibitory effect of R1881 was blocked by the anti-androgen. In the absence of hCG, treatment with testosterone, dihydrotestosterone, or R1881, but not promegestone, alone also inhibited 3 beta-hydroxysteroid dehydrogenase activity while the inhibitory effect of testosterone was blocked by cyproterone acetate. Thus, hCG stimulates 3 beta-hydroxysteroid dehydrogenase activity in cultured testicular cells. The androgenic steroidogenic end products, in turn, inhibit this enzyme. The hormonal regulation of 3 beta-hydroxysteroid dehydrogenase activity may be important in the ultrashort loop autoregulation of androgen biosynthesis.     

Gonadotropin induction of a regulatory mechanism of steroidogenesis in fetal Leydig cell cultures

Studies were conducted to define further the development of the gonadotropin induced, E2 mediated steroidogenic lesion (17-alpha-hydroxylase/17,20-desmolase) in fetal Leydig cell cultures. Analysis of dispersed fetal testes purified by centrifugal elutriation demonstrated a group of cells with sedimentation velocity 12 less than to less than 16.8 mm/h.g containing a small population of adult like "transitional" Leydig cells and homogeneous "fetal" Leydig cell population collected at greater than 19.3 mm/h.g. After cells were cultured for 3 days with addition of 1 microgram oLH at 3 day intervals, the transitional cells showed testosterone accumulation comparable to the fetal cells. In contrast, transitional cells had 10-fold higher basal and hCG-stimulated aromatase activity than fetal cells, and a lack of testosterone response to acute (3 h) hCG stimulation. At day 6, transitional cells steroidogenic ability declined markedly. The fetal population maintained in culture with LH additions every 3 days, showed typical immature Leydig cell response, with enhancement of acute testosterone response to hCG at 3 day (1-fold) and at 6 day of culture (5-fold). Higher doses of LH (5 micrograms/day) or daily treatment of 1 microgram to fetal cultures, induced a lesion of 17 alpha-hydroxylase/17,20-desmolase with reduction of enzymatic activities (P less than 0.01) and impaired testosterone production (P less than 0.01) in response to acute hCG stimulation. Also aromatase was stimulated by hCG + 140% and 50% and E2 receptors were increased by 100 and 180% at 3 days and 6 days of cultures with daily or high dose LH addition, findings consistent with the observation of the E2-mediated lesion during LH action. In conclusion, the cultured fetal Leydig cell provides a useful model to elucidate molecular mechanisms involved in the development of gonadotropin-induced estradiol-mediated desensitization. Treatment of fetal Leydig cell cultures with multiple or frequent doses of LH elevate aromatase activity to necessary levels for the induction of desensitization. We have isolated small population of transitional Leydig cells with morphological characteristics of cells found in 15 day post-natal testis but functional capabilities of adult cells. We have also demonstrated the emergence of a functional adult-like population from the fetal Leydig cell.     

Effects of whole-body 50-Hz magnetic field exposure on mouse Leydig cells

The main goal of this study was to evaluate the possible effect of whole-body magnetic field (MF) exposure on the steroidogenic capacity of Leydig cells in vitro. In four separate experiments, male CFLP mice were exposed to sinusoidal 50-Hz, 100-microT MF. The duration of exposure was 23.5 h/day over a period of 14 days. At the end of the exposure, interstitial (Leydig) cells were isolated from the testicles of the sham-exposed and exposed animals. The cells were cultured for 48 h in the presence or absence of 1, 10, or 100 mIU/ml human chorionic gonadotropin (hCG). The luteinizing hormone (LH) analog hCG was used to check the testosterone (T) response of the sham-exposed controls and to evaluate the possible effect of the whole-body MF exposure on the steroidogenic capacity of Leydig cells in vitro. Testosterone content of the culture media and blood sera was measured by radioimmunoassay (RIA). In the cultures obtained from MF-exposed animals, the hCG-stimulated T response was significantly higher (p < 0.01) compared with the sham-exposed controls, while the basal T production of cells and the level of serum T remained unaltered. No MF exposure-related histopathological alterations were found in testicles, epididymes, adrenals, prostates, and pituitary glands. The MF exposure did not affect the animal growth rate and the observed hematologic and serum chemical variables. Our results indicate a presumably direct effect of whole-body MF exposure on the hCG-stimulated steroidogenic response of mouse Leydig cells.     

Stimulatory effect of lactate on testosterone production by rat Leydig cells

Previously we found that the increased plasma testosterone levels in male rats during exercise partially resulted from a direct and luteinizing hormone (LH)-independent stimulatory effect of lactate on the secretion of testosterone. In the present study, the acute and direct effects of lactate on testosterone production by rat Leydig cells were investigated. Leydig cells from rats were purified by Percoll density gradient centrifugation subsequent to enzymatic isolation of testicular interstitial cells. Purified rat Leydig cells (1 x 10(5) cells/ml) were in vitro incubated with human chorionic gonadotropin (hCG, 0.05 IU/ml), forskolin (an adenylyl cyclase activator, 10(-5) M), or 8-bromo-adenosine-3':5'-cyclic monophosphate (8-Br-cAMP, 10(-4) M), SQ22536 (an adenylyl cyclase inhibitor, 10(-6)-10(-5) M), steroidogenic precursors (25-hydroxy-cholesterol, pregnenolone, progesterone, and androstenedione, 10(-5) M each), nifedipine (a L-type Ca(2+) channel blocker, 10(-5)-10(-4) M), or nimodipine (a potent L-type Ca(2+) channel antagonist, 10(-5)-10(-4) M) in the presence or absence of lactate at 34 degrees C for 1 h. The concentration of medium testosterone was measured by radioimmunoassay. Administration of lactate at 5-20 mM dose-dependently increased the basal testosterone production by 63-187% but did not alter forskolin- and 8-Br-cAMP-stimulated testosterone release in rat Leydig cells. Lactate at 10 mM enhanced the stimulation of testosterone production induced by 25-hydroxy-cholesterol in rat Leydig cells but not other steroidogenic precursors. Lactate (10 mM) affected neither 30- nor 60-min expressions of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein. The lactate-stimulated testosterone production was decreased by administration of nifedipine or nimodipine. These results suggested that the physiological level of lactate stimulated testosterone production in rat Leydig cells through a mechanism involving the increased activities of adenylyl cyclase, cytochrome P450scc, and L-type Ca(2+) channel.     

The different mechanisms for suppression of pituitary and testicular function

The differential mechanisms reducing androgen secretion by LHRH agonists are discussed with relevance to clinical therapy. LH secretion can be desensitised by exposure to agonists using high doses, frequent injections or sustained release/constant infusion. The desensitized pituitary is refractory to hypothalamic stimulation. Pituitary receptor suppression is associated with depletion of pituitary gonadotrophin content, and a decline of LH and FSH secretion to a basal rate. Recovery of LH responsiveness to endogenous LHRH stimulation requires restitution of gonadotrophin content (about 7 days in rats). After long-term infusions in normal men, testosterone secretion recovers within 7-10 days. The binding capacity of testicular LH/hCG receptors is reduced in rats after supraphysiological gonadotrophin stimulation, by agonists or directly by hCG, concomitantly the steroidogenic capacity of the testis in vitro is impaired. Qualitative changes in androgen biosynthesis are a marked fall in testosterone production and dose-dependent enhancement of progesterone production. After 12 months of buserelin injections, the changes in hCG-stimulated rat testes are an increased ratio of progesterone/17-OH-progesterone (inhibition of 17-hydroxylase), a reduced capacity for secretion of androstenedione and testosterone (block of 17,20-desmolase), and increased 5 alpha-pregnane-3,20-dione (this steroid inhibits the 17,20-desmolase, similarly to progesterone). After treatment, Leydig cell function recovers completely. Leydig cell hyperplasia is observed as a result of the steroidogenic changes. These findings in rats have not been observed in dogs, monkeys or in humans.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a Sertoli cell, FSH-suppressible inhibiting factor(s) of testicular steroidogenic activity

By using a model of immature porcine Leydig and Sertoli cells cultured in serum free defined medium, we evidenced a paracrine control of Leydig cell steroidogenic activity by Sertoli cells via a secreted inhibiting protein(s). This protein(s), partially purified using gel filtration (M.W. 20,000-30,000) suppresses the steroidogenic responsiveness to LH/hCG by decreasing the specific LH/hCG binding (52% decrease) and hormone steroid biosynthesis (73% decrease) at a level(s) located between cAMP production and pregnenolone formation. The suppression of this inhibitor(s) by FSH, in a dose dependent manner, is one mechanism by which FSH "sensitizes" Leydig cell response to LH/hCG stimulation.     

Steroid product-induced, oxygen-mediated damage of microsomal cytochrome P-450 enzymes in Leydig cell cultures. Relationship to desensitization

Treatment of Leydig cells with 1 mM 8-Br-cAMP for 48 h decreased microsomal cytochrome P-450 activities, 17 alpha-hydroxylase and C17-20 lyase, by 60-75% and resulted in desensitization of the steroidogenic response. Reduction of the oxygen tension from 19 to 1% O2 prevented the decrease in P-450 activities but not the reduction in steroidogenic capacity. The decrease in activity was also prevented by blocking steroid synthesis with aminoglutethimide. Treatment of cultures with steroid products, androstenedione or testosterone, or product analogs, epitestosterone or 17 alpha-methyltestosterone, at a concentration of 2 microM, which is equivalent to the concentration of testosterone resulting from stimulation with cAMP, also caused oxygen tension-sensitive decreases in hydroxylase activity. Treatment of cultures with 2 microM cortisol, estradiol, or methyltrienolone, an androgen receptor agonist, did not decrease hydroxylase activity, nor did treatment with an androgen receptor antagonist prevent the cAMP- or testosterone-induced decreases in hydroxylase activity. Reductions in hydroxylase and lyase activities resulting from testosterone- or epitestosterone-treatment had little or no effect on acute cAMP-stimulated testosterone production, whereas desensitization with cAMP caused an 80-90% reduction in steroidogenic capacity. 22R-Hydroxycholesterol-supported testosterone synthesis was decreased by both cAMP- and steroid-treatment at 19% O2, but not below the cAMP-stimulated level. Reduction of the oxygen tension partially prevented this decrease. These data are consistent with the hypothesis that the decline in microsomal P-450 enzymes in desensitized Leydig cells results from product (pseudosubstrate)-induced, oxygen-derived, free-radical damage rather than a steroid receptor-mediated process.     

Direct regulating effects of transforming growth factor beta on the Leydig cell steroidogenesis in primary culture

The effect of transforming growth factor beta on testicular steroidogenesis was studied by using a model of immature porcine Leydig cells cultured in a chemically defined medium. Leydig cells were cultured in the presence of human or porcine purified TGF beta and the following parameters were measured: cell proliferation, LH/hCG binding, and hCG-stimulated steroid hormone productions (DHEA, DHEAS and testosterone). Whereas TGF beta from the two sources had no effect on Leydig cell multiplication, it markedly inhibited LH/hCG-stimulated DHEA and DHEAS in a time- and dose-dependent manner. The maximal inhibitory effect of this peptide on LH/hCG binding (65% decrease), hCG-stimulated DHEA (77% decrease) and DHEAS (92% decrease) productions was observed with 2 ng/ml for 48 h of treatment. In contrast, TGF beta exerted a biphasic effect on hCG-stimulated testosterone production: stimulating (110% increase) until 2 ng/ml and inhibiting (35% decrease) for higher concentrations. [125I]TGF beta was cross-linked to Leydig cells using disuccinimidyl suberate; cells affinity labelled with [125I]TGF beta exhibit a major labelled band of approx 280 kDa, which has the properties expected from a TGF beta receptor. These data demonstrate that TGF beta is a direct potent regulator of Leydig cell steroidogenic function and its effects are probably mediated via a specific receptor.     

Hormonal regulation of testosterone production in short-term primary culture of fetal mouse leydig cells

Short-term primary culture of Leydig cells were prepared from 18 day old fetal mouse testes. The cells were cultured in a defined medium supplemented with 1% fetal calf serum, EGF and Insulin. The cells rapidly attached to the plastic culture dish. Seventy to eighty percent of the firmly attached cells stained positively for 3β-HSD activity and gradually assumed a flattened epitheloid appearance. The functional activity of these cells in terms of testosterone production and hCG-responsiveness was maintained for 2 days. There was a significant effect of plating density. Pre-culture (24 h) of fetal Leydig cells in the presence of 100 mIU hCG desensitized these cells to a subsequent stimulation by hCG. This is the first report of a short-term primary culture of fetal Leydig cells which demonstrates the maintenance of androgenic activity of these cells in vitro.     

The aging Leydig cell: 1. Testosterone and adenosine 3',5'-monophosphate responses to gonadotropin stimulation in rats

Plasma testosterone levels before and after a single injection of hCG were significantly lower in 24-month old rats than 60--90 day old animals (p less than 0.001). Even with repeated hCG administration for three weeks, plasma testosterone levels of old rats could not be restored to levels present in unstimulated young rats. In response to in vitro LH and 8-bromo-cyclic AMP stimulation, purified young Leydig cells produced significantly higher amounts of testosterone than Leydig cells from old rats. Maximal testosterone formation of the young Leydig cells in response to LH was 42.0 +/- 6.88 ng/10(6) cells, while cells from old rats produced only 16.8 +/- 3.69 ng/10(6) cells (p less than 0.01). However, the dose of LH at which one half maximal response (ED50) occurred was 0.1 mIU/ml for young Leydig cells and 0.05 mIU/ml for old Leydig cells. Basal and 1.0 mIU LH-stimulated cyclic AMP formation were comparable in both groups, but cyclic AMP formation in response to 10 mIU of LH was significantly less in the old rats (p less than 0.05). Present results demonstrate impaired steroidogenic capacity of old rats both in vivo and in vitro. Decreased testosterone response in old rats most likely is the consequence of understimulation of Leydig cells by gonadotropin; however, there appear to be additional intrinsic defects in old Leydig cells.     

The aging Leydig cell: 2. Two distinct populations of Leydig cells and the possible site of defective steroidogenesis

Using metrizamide gradient centrifugation two populations of Leydig cells were found in both 60-90 day-old and 24 month-old rats. Cells from both Band 2 (B2) and Band 3 (B3) responded to LH stimulation with increased cyclic AMP formation; however, only B3 cells produced significant amounts of testosterone. Cells from both B2 and B3 of the old rats synthesized less cyclic AMP and testosterone than cells from their younger counterparts. In response to LH stimulation, 0.01 - 1.0 mIU/ml, no appreciable difference of cyclic AMP formation could be detected between young and old Leydig cells. Maximal testosterone production occurred when 1 mIU/ml LH was used. Only when LH concentration was increased to 10 and 100 mIU/ml, did young Leydig cells produce significantly more cyclic AMP than old Leydig cells. After addition of 5X10(-7)M of pregnenolone or progesterone to the incubation medium, both young and old Leydig cells produced comparable amounts of testosterone. These results demonstrate no impairment of old rat Leydig cells to synthesize testosterone from pregnenolone and progesterone.     

Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis

This study investigates the relationship between the high density lipoprotein (HDL) receptor (scavenger receptors, SR-BI and SR-BII), selective lipoprotein-cholesteryl ester uptake, and testosterone production in Leydig cells of control, hypocholesterolemic and gonadotrophic hormone (hCG) treated rats. Leydig cells from mature control rats show poor efficiency in incorporation of labeled HDL-cholesteryl esters into testosterone, poor selective uptake of lipoprotein lipids overall, and a dramatic reduction of circulating levels of lipoproteins has no apparent effect on testosterone production or expression of intracellular enzymes synthesizing cholesterol. Leydig cells from control rats show minimal levels of SR-BI and SR-BII. However, similarly aged rats treated with hCG for several days undergo changes consistent with hormone-desensitization. Despite the resulting low levels of testosterone production, SR-BI levels are dramatically increased, Leydig cells now efficiently internalize HDL-supplied cholesteryl esters by the selective cholesterol uptake process, and various other cholesterol-sensitive genes of the cells are up-regulated. Only SR-BII expression remains negligible and unchanged throughout this period. It is of interest that Leydig cell SR-BI of hCG-treated rats is localized in surface microvilli, but is present also in an elaborate and complex channel system within the cytoplasm of the cells. In summary, Leydig cells differ from other rat steroidogenic cells in not depending on exogenous lipoprotein-cholesterol during periods of normal steroid hormone production. However, trophic hormone desensitization is accompanied by increased Leydig cell SR-BI expression and increased selective HDL-cholesteryl ester uptake, presumably in preparation for renewed testosterone production.     

Age-related changes in responsiveness of rat Leydig cells to hCG

The responsiveness of decapsulated testes and isolated Leydig cell preparations from rats (30-80 days of age) to a constant dose of 3 ng hCG/2 ml was assessed by comparison of the production of testosterone and "total 17beta-hydroxy androgen" (17beta-HA). When testosterone secretion was used as the index of response, there was a marked increase in the production with age by decapsulated testes and also by equal numbers of Leydig cells. When 17beta-HA was taken as the response parameter this increase was only marginal for the decapsulated testes and there was an age-dependent decrease when expressed per 10(6) cells. These differences probably reflect changes in the metabolism of testosterone to 5alpha-reduced products with increasing age because 80% of androgen secreted at 30 days is 3alpha-androstanediol and 86% is secreted as testosterone at 80 days. We conclude that for studies on hCG responsiveness and the steroidogenic capacity of immature rat Leydig cells (a) testosterone is an inappropriate response parameter and (b) this response undergoes a decrease rather than an increase during prepubertal development.     

Effects of insulin and somatomedin C on the function of cultured Leydig cells

Porcine cultured Leydig cells (LC) lose hCG receptors and hCG responsiveness (cAMP and testosterone) when they are cultured for three days in a defined medium without insulin or somatomedin C (Sm-C) (Insulin-like growth factor I). In the presence of insulin (50 ng/ml) or of Sm-C (10 ng/ml) the loss of the hCG receptor number and the decreased cAMP response to hCG were prevented, but the steroidogenic response to hCG was only partially prevented. This parameter became normal when cells were pretreated with either Sm-C (10 ng/ml) plus insulin (50 ng/ml) or with insulin alone at high concentrations (5 micrograms/ml). These results indicate that both Sm-C and insulin acting through their own receptors increase Leydig cell steroidogenic capacity by increasing hCG receptor number and improving some step beyond cAMP formation.     

Site-directed mutagenesis of the human chorionic gonadotropin beta-subunit: bioactivity of a heterologous hormone, bovine alpha-human des-(122-145)beta

Human CG contains an alpha-subunit, common to the pituitary glycoprotein hormones, and a hormone-specific beta-subunit, but unlike the pituitary beta-subunits, hCG beta is characterized by an O-glycosylated carboxy-terminal extension. A mutant beta-subunit, des-(122-145)hCG beta, was prepared using site-directed mutagenesis, and the pRSV expression plasmids were transfected into Chinese hamster ovary cells that produce the bovine alpha-subunit (b alpha). The mutant beta-subunit binds to b alpha, and the heterologous gonadotropin, b alpha-des-(122-145)hCG beta, was capable of stimulating steroidogenesis in cultured Leydig tumor cells (MA-10) to the same extent as standard hCG. When compared with the heterologous gonadotropin, b alpha-hCG beta wild type, the hybrid hormone with the truncated hCG beta exhibited equal potency, within the accuracy of the RIAs used to determine hormone concentrations, and gave a similar time course of steroidogenesis. Interestingly, these transformed Leydig cells do not distinguish between the steroidogenic potencies (as measured by progesterone production) of hCG and human LH (hLH) as do some preparations of normal rodent Leydig cells (as measured by testosterone production). However, the MA-10 cells were able to distinguish hCG from hLH based on their cAMP response; the latter produced a greater response at both maximal and submaximal gonadotropin concentrations. The two expressed heterologous gonadotropins were equipotent in their abilities to stimulate cAMP and gave similar time courses of cAMP accumulation in MA-10 cells. Thus, the carboxy-terminal extension of hCG beta is not required for association with the alpha-subunit nor for functional receptor binding, as judged by cAMP accumulation and progesterone production in MA-10 cells.