Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure

Depriving rats of luteinizing hormone (LH) causes Leydig cells to lose smooth endoplasmic reticulum and diminishes their P450 C17-hydroxylase/C17,20-lyase activity (Wing et al., 1984). LH administration to hypophysectomized rats prevents these changes in Leydig cell structure and function (Ewing and Zirkin, 1983). We adopted a multistep procedure of rat Leydig cell isolation to study the trophic effects of LH on steroidogenesis in the Leydig cell. Our method employs vascular perfusion, enzymatic dissociation, centrifugal elutriation, and Percoll gradient centrifugation. The purified Leydig cell fraction obtained after Percoll density-gradient centrifugation contains 95% well-preserved 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)-staining cells with ultrastructural characteristics of Leydig cells. These Leydig cells produced 248 and 29 ng of testosterone/10(6) Leydig cells when incubated for 3 h with and without a maximally stimulating concentration of ovine LH. Purified Leydig cells obtained from control rats and rats treated with testosterone-estradiol (T-E) implants for 4 days to inhibit LH production were incubated with a saturating concentration (2 microns) of pregnenolone. Leydig cells from control and T-E-implanted rats produced 537 and 200 ng of testosterone/10(6) Leydig cells X 3 h, respectively, suggesting a defect in the steroidogenic reactions converting pregnenolone to testosterone in Leydig cells from T-E-implanted rats. By using rabbit antibodies to the P450 C17-hydroxylase/C17,20-lyase pig microsomal enzyme, immunoblots of one-dimensional sodium dodecyl sulfate polyacrylamide gels of Leydig cell microsomal protein from control and 4- and 12-day T-E implanted rats revealed a continued loss of enzyme as the period of LH withdrawal continues. These results show that Leydig cells from animals deprived of LH had diminished capacity to convert pregnenolone to testosterone and reduced P450 C17-hydroxylase/C17,20-lyase content.     

Effects and interactions of LH and LHRH agonist on testicular morphology and function in hypophysectomized rats

The effects of single or combined daily treatment with an LHRH agonist and low or high doses of LH upon the testes of adult hypophysectomized rats were studied for up to 2 weeks in which changes in testicular histology, particularly the interstitial tissue, were examined by morphometry and related to functional assessment of the Leydig cells in vivo and in vitro. Compared to saline-treated controls, LHRH agonist treatment did not alter testis volume or the composition of the seminiferous epithelium or any of the interstitial tissue components although serum testosterone and in-vitro testosterone production by isolated Leydig cells were significantly reduced. With 2 micrograms LH for treatment, testis volume was increased, spermatogenesis was qualitatively normal, total Leydig cell volume was increased, serum testosterone values were initially elevated but subsequently declined and in-vitro testosterone production was enhanced. Testis volume with 20 micrograms LH treatment was unchanged compared to saline treatment, the seminiferous epithelium exhibited severe disruption but total Leydig cell volume was greatly increased due to interstitial cell hyperplasia. This group showed elevated serum testosterone concentrations and major increases in testosterone production in vitro. Treatment with LHRH agonist with either dose of LH resulted in reduced testis volume, moderate to very severe focal spermatogenic disruption and increased total Leydig cell volume although serum testosterone values and in-vitro testosterone production were markedly reduced compared to control rats. It is concluded that, in the absence of the pituitary, LHRH agonist fails to disrupt spermatogenesis and the previously described antitesticular action of LHRH agonists in intact rats is therefore dependent upon the presence of LH, which alone or in combination with LHRH agonist, may focally disrupt spermatogenesis in hypophysectomized rats whereas the Leydig cells undergo hyperplasia. The findings show that impairment of spermatogenesis is accompanied by alterations of the interstitial tissue and suggest that communication between these two compartments is involved in the regulation of testicular function.     

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.     

Taenia taeniaeformis: inhibition of rat testosterone production by excretory-secretory product of the cultured metacestode

In 3- to 5-month-old male Sprague-Dawley rats infected with the hepatic metacestode, Taenia taeniaeformis, the serum testosterone level was significantly lower than in comparable uninfected controls. By transmission electron microscopy, testicular Leydig cells of infected rats had less smooth endoplasmic reticulum than control Leydig cells. Cultured metacestodes isolated from the hepatic cysts secreted or excreted substances into the incubation medium. The effect of the excretory-secretory product on testosterone concentration in the sera and testes of 15-day-old rats was examined. Subcutaneous injection of 50-200 micrograms of excretory-secretory product/0.1 ml saline/rat for 2 days significantly reduced human chorionic gonadotropin-stimulated serum and testicular testosterone concentrations. Furthermore, the effect of the excretory-secretory product on isolated rat Leydig cell testosterone production was examined. Rat Leydig cells produced testosterone in vitro and, in the presence of 50 IU human chorionic gonadotropin/ml incubation medium, they responded with approximately 100% increase in testosterone production. Addition of 2-10 micrograms excretory-secretory product protein/ml of culture medium significantly reduced the testosterone production by rat Leydig cells in vitro. These results indicate that excretory-secretory product of cultured T. taeniaeformis metacestodes has a direct inhibitory effect on Leydig cell testosterone production under stimulation with human chorionic gonadotropin.     

Differential effects of the destruction of Leydig cells by administration of ethane dimethane sulphonate to postnatal rats

Ethane dimethane sulphonate (EDS) is a cytotoxic drug that selectively destroys Leydig cells in adult testes. This study has examined the effect of a single injection of EDS on the Leydig cell populations present in the testes of rats aged 5, 10, or 20 days. Microscopic examination of the tissue demonstrated that the fetal Leydig cell population was destroyed at all ages, but that subsequent development of the adult population of Leydig cells was not affected. Whilst the destruction of the fetal Leydig cells in this acute phase of EDS on 5-day-old rats was accompanied by a decline in serum testosterone levels, there was no apparent effect on this hormone when EDS administered at 10 or 20 days of age, despite the destruction of fetal Leydig cells in these rats. The long-term effects of EDS on Day 5 of age resulted in proliferation of the intertubular tissue in which more Leydig cells were observed, but serum testosterone and testosterone levels in response to human chorionic gonadotropin stimulation in vitro were normal despite moderate or severe disruption of the seminiferous epithelium. These data show that the fetal Leydig cells of immature testes are sensitive to the cytotoxic effects of EDS in the adult, but the response of the testes differs depending on the age at which the drug is administered.     

Chronic corticosterone treatment impairs Leydig cell 11beta-hydroxysteroid dehydrogenase activity and LH-stimulated testosterone production.

The effects of excess corticosterone on luteinizing hormone (LH)-stimulated Leydig cell testosterone production and activity of 11beta-HSD was studied. Adult male rats (200-250 g body weight) were treated with corticosterone-21-acetate (2 mg/100 g body weight, i.m., twice daily) for 15 days. Another set of rats was treated with corticosterone (dose as above) plus LH (ovine LH 100 microg/kg body weight, s.c., daily) for 15 days. Corticosterone administration significantly increased serum and testicular interstitial fluid (TIF) corticosterone but decreased testosterone levels. Administration of LH with corticosterone partially prevented the decrease in serum and TIF testosterone. The oxidative activity of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) was significantly decreased in Leydig cells of rats treated with corticosterone alone and in combination with LH. The direct effect of corticosterone on Leydig cell steroidogenic potency was also studied in vitro. Addition of corticosterone to Leydig cell culture showed a dose dependent effect on LH-stimulated testosterone production. Corticosterone at 50 and 100 ng/ml did not alter LH-stimulated testosterone production, but at high doses (200-400 ng/ml), decreased basal and LH-stimulated testosterone production. Basal and LH-stimulated cAMP production was not altered by corticosterone in vitro. It is concluded from the present study that elevated levels of corticosterone decreased the oxidative activity of 11beta-HSD and thus resulting in impaired Leydig cell steroidogenesis and the inhibitory effects of corticosterone on testosterone production appear to be mediated through inhibition of LH signal transduction at post-cAMP level.     

Müllerian-inhibiting substance inhibits rat Leydig cell regeneration after ethylene dimethanesulphonate ablation

The postnatal development of Leydig cell precursors is postulated to be controlled by Sertoli cell secreted factors, which may have a determinative influence on Leydig cell number and function in sexually mature animals. One such hormone, Mullerian inhibiting substance (MIS), has been shown to inhibit DNA synthesis and steroidogenesis in primary Leydig cells and Leydig cell tumor lines. To further delineate the effects of MIS on Leydig cell proliferation and steroidogenesis, we employed the established ethylene dimethanesulphonate (EDS) model of Leydig cell regeneration. Following EDS ablation of differentiated Leydig cells in young adult rats, recombinant MIS or vehicle was delivered by intratesticular injection for 4 days (Days 11-14 after EDS). On Days 15 and 35 after EDS (1 and 21 days post-MIS injections), endocrine function was assessed and testes were collected for stereology, immunohistochemistry, and assessment of proliferation and steroidogenesis. Although serum testosterone and luteinizing hormone (LH) were no different, intratesticular testosterone was higher on Day 35 in MIS-treated animals. At both time points, intratesticular 5alpha-androstan-3alpha,17beta-diol concentrations were much higher than that of testosterone. MIS-treated animals had fewer mesenchymal precursors on Day 15 and fewer differentiated Leydig cells on Day 35 with decreased numbers of BrdU+ nuclei. Apoptotic interstitial cells were observed only in the MIS-treated testes, not in the vehicle-treated group on Day 15. These data suggest that MIS inhibits regeneration of Leydig cells in EDS-treated rats by enhancing apoptotic cell death as well as by decreasing proliferative capacity.     

Onset of compensatory hypertrophy of interstitial tissue and Leydig cells in rats hemicastrated around the time of puberty

When rats were unilaterally castrated at 20, 30, and 40 days of age, only those rats hemicastrated at 40 days showed compensatory hypertrophy of the interstitial tissue and Leydig cells when killed 30 days after hemicastration. At the time of death, volume densities of interstitial tissue, Leydig cells, and vascular components were greater in 70-day-old hemicastrated rats than in intact rats of the same age. The total number of Leydig cells per testis in hemicastrated and intact rats was always the same at any age. Estimated Leydig cell volume in 70-day-old rats was twice that in intact rats. By contrast, the testes of 50- and 60-day-old rats at the time of death displayed essentially the same morphological features, regardless of whether animals were hemicastrated. The concentration of plasma testosterone was higher in 50-day-old controls than in hemicastrated rats. Seventy-day-old hemicastrated rats showed higher levels of plasma testosterone than controls. The level of plasma dihydrotestosterone in 60- and 70-day-old hemicastrated rats exceeded that in the controls. A significant increase in follicle-stimulating hormone was noted in 50- and 70-day-old hemicastrated rats compared to normal rats, while levels of luteinizing hormone were basically the same. The increase in Leydig cell volume, interstitial tissue volume, vascular component volume, and plasma testosterone level caused by hemicastration at 40 days of age differed from that at 20 and 30 days of age.     

Chronic administration of corticosterone impairs LH signal transduction and steroidogenesis in rat Leydig cells

The mechanism involved in the inhibitory actions of chronic corticosterone treatment on Leydig cell steroidogenesis was studied in adult Wistar rats. Rats were treated with corticosterone-21-acetate (2 mg/100 g body weight, i.m., twice daily) for 15 days and another set of rats was treated with corticosterone plus ovine luteinizing hormone (oLH) (100 microg/kg body weight, s.c., daily) for 15 days. Chronic treatment with corticosterone increased serum corticosterone but decreased serum LH, testosterone, estradiol and testicular interstitial fluid (TIF) testosterone and estradiol concentrations. Administration of LH with corticosterone partially prevented the decrease in serum and TIF testosterone and estradiol. Leydig cell LH receptor number, basal and LH-stimulated cAMP production were diminished by corticosterone treatment which remained at control level in the corticosterone plus LH treated rats. Activities of steroidogenic enzymes, 3beta- and 17beta-hydroxysteroid dehydrogenase (3beta-HSD and 17beta-HSD) were significantly decreased in corticosterone treated rats. LH plus corticosterone treatment did not affect 3beta-HSD activity but decreased 17beta-HSD activity, indicating a direct inhibitory effect of excess corticosterone on Leydig cell testosterone synthesis. The indirect effect of corticosterone, thus, assume to be mediated through lower LH which regulates the activity of 3beta-HSD. Basal, LH and cAMP-stimulated testosterone production by Leydig cells of corticosterone and corticosterone plus LH treated rats were decreased compared to control suggesting the deleterious effect of excess corticosterone on LH signal transduction and thus steroidogenesis.     

Morphometry and cytochemistry of Leydig cells in experimental diabetes

Leydig cell ultrastructure and function in diabetic rats were studied by concurrent cytochemistry, morphometry, and testosterone assay. The streptozotocin (Stz) model was modified to include nondiabetic Stz-injected rats, an insulin-treated diabetic group, and semistarved animals in addition to controls and untreated diabetic rats. The separation of the effects of diabetes, Stz, semistarvation, and insulin treatments was achieved by application of orthogonal contrast statistics. After 3 months of treatments, testes were perfusion-fixed, incubated for delta 5,3 beta-hydroxysteroid dehydrogenase (HSD) activity, and processed for electron microscopy. Diabetes increased Leydig cell smooth endoplasmic reticulum (SER), increased mitochondrial and lipid content, decreased HSD staining, and decreased serum testosterone levels. Insulin treatment reduced SER and increased testosterone concentrations. Semistarvation also increased SER and reduced testosterone levels but did not alter HSD staining. Stz had no significant effect on these variables. The results suggested that the hypoandrogen state was due to a primary Leydig cell compromise and not solely to malnutrition and that it was correctable by insulin treatment.     

Testosterone restores testicular inhibin content in cryptorchid rats

The effects of testosterone administration on testicular inhibin content and histology were studied in bilaterally cryptorchid rats, in which a marked decrease in testicular inhibin content had been observed. Mature male Wistar rats weighing approximately 300 g were made bilaterally cryptorchid by placing the testes in the abdominal cavity. Testosterone in oil, 0.1, 1.0 or 10 mg, was given i.m. each week. Testicular inhibin and testosterone content, histology and plasma LH, FSH and testosterone were studied 2 weeks later. Abnormally decreased testicular inhibin in cryptorchidism was restored toward normal by testosterone in a dose dependent manner in 2 weeks after surgery. Sertoli cell structure also recovered toward normal with increasing amount of testosterone. Decreased testicular testosterone content and Leydig cell atrophy were observed with suppressed plasma LH and FSH after testosterone. These results showed that the increased plasma concentration of testosterone had a stimulatory effect on the Sertoli cell function in cryptorchidism, in which compensated Leydig cell failure was demonstrated.     

Crystalloid formation in Leydig cells of rats (Rattus fuscipes)

Summary The ultrastructure of Leydig cells in a seasonally breeding rodent, Rattus fuscipes, was studied in the breeding and non-breeding season and compared with Leydig cell morphology after suppression of gonadotrophin secretion induced by hypophysectomy or chronic administration of testosterone. Serum luteinizing hormone (LH) and testosterone (T) were measured and in-vitro T production by testes was assessed by stimulation with human chorionic gonadotrophin (hCG). In non-breeding wild-trapped rats and rats with experimental suppression of gonadotrophins, the Leydig cells were atrophied and exhibited variable amounts of cytoplasmic lipid and crystalloid inclusions, the latter commonly dominating the cytoplasmic area. Compared with fertile rats, serum LH and hCG-stimulated T production of experimentally regressed rats was significantly reduced, confirming structural features indicative of Leydig cell inactivity. Atrophy of Leydig cell nuclei was accompanied by the formation of unusual intranuclear vesicles sometimes containing small crystalloids. Ultrastructural analysis suggested transfer of the vesicles to the cytoplasm where their unification gave rise to much larger crystalloid bodies. Crystalloids occurred when serum LH was depressed and with either full (T treatment) or arrested spermatogenesis (hypophysectomy) suggesting that their formation is governed by pituitary function and is not dependent upon the degree of spermatogenic activity.     

Ultrastructure and morphometry of testicular Leydig cells and the interstitial components correlated with testosterone in aging rats

The ultrastructure of testicular interstitium in young and aged adult rats was analysed using morphometric methods, and the plasma testosterone concentration was measured. With increasing age there was an augumentation in the volume of collagen fibrils in the intercellular matrix and in blood vessels. During the aging process (approximately two years) the average volume of the Leydig cell decreased from 1364 m³ to 637 m³, but the number of Leydig cells in paired testes increased from 53x10⁶ to 113x10⁶. The absolute volume of smooth surfaced endoplasmic reticulum (SER) per Leydig cell amounted in aged rats to 78% of that in young adult rats. The total amount of SER in paired testes increased by 62% with aging. The present analysis suggests that the ability of SER to maintain peripheral testosterone concentration decreases with age. In young adult rats the absolute volume of peroxisomes per Leydig cell correlated significantly with the concentration of testosterone in blood and also with the absolute volume of SER per Leydig cell. These results combined with ultrastructural observations of close apposition of peroxisomes and SER suggest that peroxisomes have a role in testosterone secretion by Leydig cells.Visiting scientist to Laboratory of Electron Microscopy (Director: Prof. L.J. Pelliniemi)     

Effects of continuous and intermittent exposure of lactating mothers to aroclor 1242 on testicular steroidogenic function in the adult male offspring

Polychlorinated biphenyls (PCBs) are worldwide pollutants and have caused hazardous effects on many animal species including humans. They have been detected in human milk and therefore exposure of newborns to PCBs is unavoidable if they are breast-fed. We present our findings on two experiments performed to test the effects of intermittent and continuous exposure of lactating rats to two different doses (80 microg and 8 microg) of Aroclor 1242 (a PCB congener) on testicular steroidogenic function of their adult male offspring. In experiment I, three groups of lactating dams received daily subcutaneous (SC) injections of either corn oil, 80 microg of Aroclor 1242 and 8 microg of Aroclor 1242 in corn oil, respectively. In experiment II, three groups of lactating dams received two SC injections per week of either corn oil or Aroclor 1242 (80 microg and 8 microg) in corn oil, respectively. Pups in all groups (n=8 per group) were weaned at day 21 and were raised on a normal diet until sacrificed at 90 days. Experiment I: Leydig cell number per testis was significantly (P<0.05) increased and the average volume of a Leydig cell was significantly (P<0.05) reduced in both groups of Aroclor-exposed rats compared to corn oil controls. Both doses of Aroclor resulted in reduced (P<0.05) serum testosterone levels compared to corn oil-treated controls. LH-stimulated testosterone production per testis and per Leydig cell was lower in Aroclor-exposed rats compared to controls. Experiment II: No changes were observed in Leydig cell size and number per testis among the three groups. Serum LH, testosterone and LH-stimulated testicular testosterone production in offspring rats of Aroclor-treated dams were not significantly different (P>.05) from the offspring of corn oil-treated dams. However, these parameters were lower in value in the offspring of dams treated with Aroclor 80 microg compared to the other two groups. LH-stimulated testosterone secretory capacity per Leydig cell was significantly lower in offspring of dams treated with Aroclor compared to controls. Serum T4 and T3 levels were not significantly different among the Aroclor-exposed and control rats in both experiments. These results demonstrate that continuous exposure of lactating mothers to 8 and 80 microg of Aroclor 1242 causes hypotrophy and malfunctioning of Leydig cells in the adult male offspring resulting in a hypoandrogenic status. Intermittent treatment of lactating mothers with 80 microg of Aroclor (but not with 8 microg of Aroclor) also produced malfunctioning of Leydig cells and a hypoandrogenic status in the absence of Leydig cell hypotrophy. However, the Aroclor 8 microg dose was ineffective to produce the above effects.     

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.     

Cell-cell interactions in the control of spermatogenesis as studied using Leydig cell destruction and testosterone replacement

This review centers around studies which have used ethane dimethane sulphonate (EDS) selectively to destroy all of the Leydig cells in the adult rat testis. With additional manipulations such as testosterone replacement and/or experimental induction of severe seminiferous tubule damage in EDS-injected rats, the following questions have been addressed: 1) What are the roles and relative importance of testosterone and other non-androgenic Leydig cell products in normal spermatogenesis and testicular function in general? 2) What are the factors controlling Leydig cell proliferation and maturation? 3) Is it the Leydig cells or the seminiferous tubules (or both) which control the testicular vasculature? The findings emphasize that in the normal adult rat testis there is a complex interaction between the Leydig cells, the Sertoli (and/or peritubular) cells, the germ cells, and the vasculature, and that testosterone, but not other Leydig cell products, plays a central role in many of these interactions. The Leydig cells drive spermatogenesis via the secretion of testosterone which acts on the Sertoli and/or peritubular cells to create an environment which enables normal progression of germ cells through stage VII of the spermatogenic cycle. In addition, testosterone is involved in the control of the vasculature, and hence the formation of testicular interstitial fluid, presumably again via effects on the Sertoli and/or peritubular cells. When Leydig cells regenerate and mature after their destruction by EDS, it can be shown that both the rate and the location of regenerating Leydig cells is determined by an interplay between endocrine (LH and perhaps FSH) and paracrine factors; the latter emanate from the seminiferous tubules and are determined by the germ cell complement. Taken together with other data on the paracrine control of Leydig cell testosterone secretion by the seminiferous tubules, these findings demonstrate that the functions of all of the cell types in the testis are interwoven in a highly organized manner. This has considerable implications with regard to the concentration of research effort on in vitro studies of the testis, and is discussed together with the need for a multidisciplinary approach if the complex control of spermatogenesis is ever to be properly understood.     

Cellular localization of cytochrome P450IIA1 in testes of mature Sprague-Dawley rats.

Previous studies have shown that a prominent site of extrahepatic cytochrome P450IIA1 in male rats is the testis. We investigated the cellular location of cytochrome P450IIA1 in the testes of adult rats. Using specific isolation of testicular compartments and individual cell types, as well as in vivo removal of Leydig cells by ethane dimethyl sulfonate, we determined the cellular location of cytochrome P450IIA1 using testosterone hydroxylation assay, Western immunoblotting, and immunohistochemical analysis. Enriched Leydig cell fractions had the greatest testosterone 7 alpha-hydroxylase activity as well as immunoreactivity. Immunohistochemical analysis confirmed that the cellular location of cytochrome P450IIA1 was specific to Leydig cells. The specific localization of enzyme systems that are involved in xenobiotic activation may have important implications for inducing specific cell toxicity by compounds that exert their effects in the testes.     

Regulation of Leydig cell steroidogenic function during aging

This article summarizes a talk on Leydig cell aging presented at the 1999 Annual Meeting of the Society for the Study of Reproduction. In the Brown Norway rat, serum testosterone levels decrease with aging, accompanied by increases in serum FSH. The capacity of Leydig cells to produce testosterone is higher in young than in old rats. Binding studies with hCG revealed reduced receptor number in old vs. young Leydig cells. In response to incubation with LH, cAMP production was found to be reduced in old vs. young Leydig cells, indicating that signal transduction mechanisms in the old cells are affected by aging. Steroidogenic acute regulatory protein and mRNA levels are reduced in old Leydig cells, suggesting that there may be deficits in the transport of cholesterol to the inner mitochondrial membrane of aged cells. The activity of P450 side-chain cleavage enzyme is reduced in old vs. young cells, as are the activities of each of 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase/C17-20 lyase, and 17-ketosteroid reductase. Serum LH levels do not differ between young and old rats, and the administration of LH failed to induce old Leydig cells to produce high (young) testosterone levels, suggesting that the cause of age-related reductions in steroidogenesis is not LH deficits. We hypothesized that reactive oxygen, produced as a by-product of steroidogenesis itself, might be responsible for age-related reductions in testosterone production by the Leydig cells. Consistent with this, long-term suppression of steroidogenesis was found to prevent or delay the reduced steroidogenesis that accompanies Leydig cell aging. A possible explanation of this finding is that long-term suppression of steroidogenesis prevents free radical damage to the cells by suppressing the production of the reactive oxygen species that are a by-product of steroidogenesis itself.     

Functional properties of developing rat Leydig cells after treatment with ethylene dimethanesulphonate (EDS)

The development of a new population of Leydig cells after specific elimination of existing Leydig cells in mature rats by ethylene dimethanesulphonate (EDS) was characterized by investigating the testicular activities of 5 alpha-reductase and non-specific esterase, the serum concentrations of 3 alpha-androstanediol and testosterone and the Leydig cell morphology. Plasma concentrations of both androgens were strongly reduced up to 15 days after administration of EDS. Thereafter, in contrast to the gradual and continuous increase of serum testosterone values, the changes in serum 3 alpha-androstanediol were transient, with the highest level on Day 35. The temporal pattern of testicular 5 alpha-reductase activity was almost similar to that of serum 3 alpha-androstanediol. The testicular esterase activity increased gradually from Day 25 until Day 76. The temporal changes in steroid concentrations and enzyme activities after EDS administration indicate that the development of the Leydig cells in EDS-treated rats occurs in a fashion similar to that in pubertal rats. However, the numerous lipid droplets and large nuclei in these Leydig cells indicate that these cells may also be classified as fetal cells. It is concluded that, after treatment with EDS, fetal and pubertal characteristics are present in Leydig cells. It is, however, unknown whether both characteristics are present in one or in two distinct cell populations.