ToxSci Advance Access originally published online on March 28, 2006
Toxicological Sciences 2006 91(2):550-556; doi:10.1093/toxsci/kfj178
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Radiation Exposure Impairs Luteinizing Hormone Signal Transduction and Steroidogenesis in Cultured Human Leydig Cells
* Department of Endocrinology, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai-600 113, Tamil Nadu, India; Department of Urology, Madras Medical College and Hospital, Chennai-600 002, Tamil Nadu, India; and Department of Radiation Oncology, Government General Hospital, Chennai-600 014, Tamil Nadu, India
1 To whom correspondence should be addressed at Department of Endocrinology, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai-600 113, Tamil Nadu, India. Fax: +91-44-24926709. E-mail: kbala82{at}hotmail.com.
Received August 24, 2005; accepted October 7, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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Therapeutic, accidental, and experimental radiation exposures decreased serum testosterone in males, leading to various sexual problems. Since testicular Leydig cells are the predominant source of circulating testosterone, findings on the direct effects of radiation on Leydig cell steroidogenesis and the mechanism behind such effects would be of greater importance to the use of safer radiation doses in cancer therapy and to adopt preventive or therapeutic measures to alleviate postirradiation lesions, respectively. Therefore, this study was undertaken to explore the same using cultured human Leydig cells. Testicles removed from advanced prostatic carcinoma patients were used for isolation and purification of Leydig cells. Purified Leydig cells were cultured and then exposed to different doses (2, 4, 6, 8, and 10 Gy) of fractioned gamma radiation. Normal and irradiated cells were used for luteinizing hormone (LH) receptor quantification or total RNA isolation to study LH receptor mRNA expression or LH/cyclic AMP (cAMP) stimulation test. While LH-stimulated cells were used for cAMP assay, LH- and cAMP-stimulated cells were used for the estimation of steroidogenic enzymes, testosterone and estradiol production. Radiation exposure caused adverse effects on Leydig cell steroidogenesis in a dose-dependent manner. While lower doses (2 and 4 Gy) were ineffective, higher doses (6 Gy and above) drastically decreased LH receptor, basal and LH-stimulated cAMP generation, and basal, LH-, and cAMP-stimulated steroidogenesis. While 2 Gy of radiation exposure increased the LH receptor mRNA level, other doses did not induce any significant change. Therefore, it is concluded that higher doses of radiation impair Leydig cell steroidogenesis by affecting LH signal transduction at the level of both pre- and post-cAMP generation. Decreased level of LH receptors following higher doses of radiation exposure is not coupled with impaired expression of its mRNA.
Key Words: gamma radiation; human Leydig cell; LH signaling; LH receptor expression; steroidogenesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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Tremendous advancement in oncological research leads to the development of effective therapeutic regimens to treat various cancers. Radiotherapy has been in practice to control the progression of cancers effectively. However, an important concern over radiotherapy is its side effects on other organs including gonads. Testis is one of the most important radiosensitive tissues because even very low doses of radiation deteriorate testicular functions. Leydig cells reside in the interstitium of the testes and contribute about 75% of the total testosterone produced by normal adult male to support spermatogenesis and to maintain masculinity. Therefore, any defect in Leydig cell functions is expected to have a significant effect upon the quality of life manifested by sexual and psychosocial problems. Various clinical and experimental reports reveal the adverse effects of radiation on Leydig cell function (Izard, 1995
). In boys with acute lymphoblastic leukemia, the main site of leukemic relapse is the testis, and direct testicular irradiation being the effective treatment, these children often fail to undergo normal pubertal development due to Leydig cell dysfunction (Brauner et al., 1988; Didi et al., 1994; Sather et al., 1981). Leydig cell damage was reported in lymphoblastic leukemia patients following radiation therapy of 24 Gy in 10 fractions for 2 weeks (Blatt et al., 1985; Brauner et al., 1983). Decreased response of Leydig cells to human chorionic gonadotropin (hCG) was also reported in lymphoblastic leukemia patients treated with 20 Gy of testicular irradiation for leukemic infiltration (Carrascosa et al., 1984). In healthy adult men, a single dose of 600 rad of radiation directed to the testis decreased testosterone levels and raised LH levels in serum due to Leydig cell dysfunction (Rowley et al., 1974).
Radiotherapy for testicular seminoma, unilateral germ cell cancer, and carcinoma in situ of the testis showed depressed serum testosterone with a concomitant increase in LH (Petersen et al., 2002, 2003; Tsatsoulis et al., 1990). Pelvic radiotherapy for prostatic carcinoma with doses between 10 and 25 Gy drastically decreased testosterone and increased LH in serum (Shapiro et al., 1985; Tomic et al., 1983). An elevated level of gonadotropins (both follicle stimulating hormone [FSH] and LH) was recorded following radiation treatment between 4.5 and 6 Gy over 7–8 weeks for the carcinoma of the prostate (Grigsby and Perez, 1986). Total body irradiation for bone marrow transplantation is also associated with Leydig cell dysfunction as incidental elevation of serum LH was recorded in these cases (Bakker et al., 2000). Radioiodine therapy for thyroid carcinoma resulted in transient impairment of testicular functions (Pacini et al., 1994) and is also associated with decreased testosterone/LH ratio (Wichers et al., 2000). In experimental studies, radiation has been shown to induce both acute and chronic damage to Leydig cells of prepubertal and adult rats, as decreased testosterone secretion and increased gonadotropin release are reported (Delic et al., 1985, 1986a,b).
Though clinical and experimental studies revealed the adverse effects of radiation on testicular steroidogenesis, diverse opinion exists on the sensitivity of Leydig cells to radiation. Studies on the molecular mechanisms behind radiation-induced Leydig cell dysfunction would be of great interest to adopt therapeutic or preventive measures to preserve Leydig cell function in postirradiated state. None of the previous reports describe the plausible molecular mechanism involved in radiation-induced Leydig cell dysfunction. Based on the existing information, it is hypothesized that gamma radiation impairs Leydig cell steroidogenesis by affecting LH receptor expression and its signal transduction. The present study was designed to test this hypothesis. The outcome of this study is expected to enlighten the radiologist to employ the safer dose during radiotherapy.
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METHODS AND MATERIALS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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Isolation and Culture of Leydig Cells
After getting clearance from the local ethical committee and consent from the patients, testicles were obtained from men aged between 60 and 70 years, who were undergoing orchidectomy for prostatic carcinoma. Testes from the patients who did not receive any treatment and did not have any endocrine or metabolic diseases or systemic illness known to affect Leydig cell function were only used. As soon as testicles were removed from the patients, they were transported to the laboratory in an ice-cold condition and decapsulated aseptically. Leydig cells were isolated by the procedure described by Lejeune et al. (1993)
. Decapsulated testes were incubated for 1–2 h with constant agitation at 37°C in 200 ml Dulbecco's modified Eagle's medium (DME) F 12 with 0.5 mg/ml type IV collagenase, 5 µg/ml DNase, and 2 µg/ml soybean trypsin inhibitor (Sigma-Aldrich, St Louis, MO). At the end of incubation, digested tissue was filtered through nylon gauze (400 mesh), and the cells were pelleted by centrifugation at 200 x g for 5 min. The cell pellet was resuspended in DME and loaded onto five-layer discontinuous Percoll gradient (21, 26, 34, 40, and 60% Percoll in F 12 DME) (Amersham Biosciences, Kwai Chung, Hong Kong). After centrifugation at 1500 x g for 30 min, three cell bands were seen; "L1" at 26%, "L2" at 34%, and "L3" at 40% Percoll. Cell bands L2 and L3 were collected and diluted with two volumes of DME and centrifuged at 200 x g for 10 min. Cells were then resuspended in fresh medium and counted using hemocytometer. The percentage of Leydig cells in air-dried cell smear was determined histochemically by staining for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) as described previously (Sharpe and Fraser, 1983). Viability of the purified Leydig cells was determined by trypan blue exclusion method (Aldred and Cooke, 1983), and about 95% of the cells were viable. Leydig cells were plated (16 h) in culture tubes containing DME F 12 nutrient mixture with 10% fetal bovine serum (FBS) (Sigma-Aldrich) at a density of 1 x 106 cells/tube. After the incubation period, the medium was replaced with FBS-free fresh medium, and the cells were exposed to different doses of gamma radiation (2, 4, 6, 8, and 10 Gy) (Theratron Phoenix, Ontario, Canada). Each dose was delivered in three equal fractions for 3 consecutive days and then the cells were used for cell-surface LH receptor quantification or total RNA isolation to study LH receptor mRNA expression by RT-PCR or stimulated with LH (100 ng/ml)/cyclic AMP (cAMP) (3mM) for 24 h along with normal cells. The cell viability was assessed in some of the culture tubes after radiation exposure and stimulation test to find out the viability loss, if any. While LH-stimulated cells were used for the assay of cAMP, LH- and cAMP-stimulated cells were used for the estimation of steroidogenic enzymes and testosterone and estradiol production.
Cell Viability Assay
Leydig cell viability was assessed by trypan blue exclusion method (Aldred and Cooke, 1983). Immediately after trypsinization, 100 µl of Leydig cell suspension (
50,000 cells) was incubated with 100 µl of 0.4% trypan blue solution containing 1% bovine serum albumin for 5 min at 37°C. The cells were then washed twice with saline and resuspended. Ten microliters of cell suspension was placed in the hemocytometer and viewed under microscope. The number of unstained and stained cells represents viable and damaged cells, respectively. The cells were counted, and the percentage of viable cells was calculated.
LH/hCG Receptor Assay
Highly purified hCG (NIDDK, Bethesda, MD) was radioiodinated using lactoperoxidase (Thorell and Johansson, 1971). Cell surface LH/hCG receptor was quantified by a previously described method (Habberfield et al., 1986). For LH/hCG receptor assay, both normal and irradiated cells were incubated with saturating concentration of 125I-hCG (50,000 cpm) plus or minus unlabeled hormone for 16 h at 4°C. Nonspecific binding was determined by incubating the cells with 125I-hCG in the presence of 10 µg of unlabeled hCG. At the end of incubation, cells were washed twice with PBS and lysed with 0.1 N sodium hydroxide. The cell-bound radioactivity was measured in the LKB gamma counter (Wallac OY, Turku, Finland), and the data were subjected to Scatchard analysis. LH/hCG receptor concentration was expressed as fmol/106 cells.
cAMP Assay
cAMP levels in the basal and LH-stimulated normal and irradiated cells were assayed using 3H-cAMP assay system (Amersham Biosciences). The sensitivity of the assay was 0.05 pmol/tube.
Steroidogenic Enzyme Assays
Cytochrome P450 side-chain cleavage enzyme.
P450 side-chain cleavage (P450 scc) enzyme activity was determined radiometrically as per the method of Georgiou et al. (1987) by measuring the conversion of 26,27-3H-25-hydroxycholesterol to 3H-labeled, 4-hydroxy-4-methylpentanoic acid. Cultured Leydig cells were washed twice with fresh medium to remove endogenous substrates. Then the enzyme activity was determined by incubating the cells with a saturating concentration of [26,27-3H]-25-hydroxycholesterol (5 µmol; 0.5 µCi) (NEN Life Science Products, Boston, MA) in 1 ml culture medium containing 100mM dimethyl sulfoxide at 32°C for 1 h in the CO2 incubator. The enzyme reaction was stopped by the addition of 0.1 ml of 1 N NaOH, and 14C-isocaproic acid (3000 cpm) (Amersham Biosciences) was added as recovery standard. The medium was removed to an extraction tube, and the culture tube was washed with 1 ml of alkalinized medium, which was combined with 1 ml original medium and extracted with 10 ml chloroform. One and half milliliter of extracted aqueous phase was vortexed with 0.8 g neutral alumina for 1 min, followed by centrifugation at 1200 x g for 25 min. The aqueous-phase (0.4 ml) supernatant was transferred to scintillation vials containing cocktail toluene, and radioactivity was measured in a liquid scintillation counter (Wallac OY).
3ß-Hydroxysteroid dehydrogenase.
3ß-HSD enzyme activity was measured based on an established spectrophotometric method (Bergmeyer, 1974). Briefly, cultured Leydig cells were sonicated in ice-cold Tris-HCl buffer (0.1µM; pH 7.2) and centrifuged at 16,000 x g for 5 min at 4°C. The supernatant was taken as enzyme extract. The reaction mixture contained 0.6 ml pyrophosphate buffer (100µM), 0.2 ml of 0.5µM nicotinamide adenine dinucleotide, 2 ml distilled water, and 0.1 ml of pregnenolone (0.1µM) (Sigma-Aldrich). After the addition of enzyme extract, the absorbance at 340 nm was measured at 20 sec intervals for 5 min in a spectrophotometer against blank.
17ß-Hydroxysteroid dehydrogenase.
17ß-Hydroxysteroid dehydrogenase (17ß-HSD) activity was determined based on the radiometric method described by Murono (1990). Briefly, the cells were preincubated for 30 min with fresh culture media and then washed two times with fresh media to remove endogenous substrate. For the actual assay, the cells were incubated with 3H-androstenedione (10µM/0.5 µCi) (Amersham Biosciences) dissolved in culture medium containing 0.3% dimethyl sulfoxide (final concentration) for 1 h at 37°C in a humidified atmosphere of 95% air and 5% CO2. The reaction was terminated by the addition of 0.1 ml of 1 mol/l NaOH. One hundred micrograms of each androstenedione and testosterone was added as carriers. To estimate recovery, 4000 cpm of 3H-testosterone (Amersham Biosciences) was processed separately. Samples were extracted with five volumes of diethyl ether, and the residue of ether extracts was chromatographed by thin layer chromatography (TLC) using chloroform:methanol (99.5:0.5, vol/vol) as solvent system. The product testosterone was localized using anisaldehyde spray, cut out, and counted in a liquid scintillation counter (Wallac OY).
Hormone Assays
Testosterone.
Testosterone in the culture media was extracted in diethyl ether and then assayed using solid-phase radioimmunoassay (RIA) kit (Diasorin, Saluggia, Italy). The sensitivity of the assay was 0.05 ng/ml. The percentages of cross-reactivity of testosterone antibody to other steroids such as 5
-dihydrotestosterone and androstenedione was 6.9 and 1.1%, respectively. Intra- and interassay coefficients of variation were 6.9–13.2 and 10.3–13, respectively.
Estradiol.
Estradiol in the culture media was extracted in diethyl ether and then assayed using the solid-phase RIA kit (Diasorin). The sensitivity of the assay was 4.0 pg/ml. The percentage of cross-reactivity of estradiol antibody to other steroids was minimal, i.e., 1.3% with estrone and 0.65% with estriol. Intra- and interassay coefficients of variation were 3–15 and 4–14, respectively.
Quantification of LH Receptor mRNA Expression
To quantify LH receptor mRNA expression, total RNA isolated (Total RNA Isolation Reagent, ABgene, Surrey, United Kingdom) from normal and irradiated Leydig cells was subjected to RT-PCR using the one-step RT-PCR kit (Qiagen, Hilden, Germany) and gene-specific primers (forward 5'-CTT GGA TAT TTC TTC CAC CAA A-3' and reverse 5'-TGG CAT GGT TAT AGT ACT GGC-3') selected based on previous literature (Minegishi et al., 1990; Dirnhofer et al., 1998). An internal control human ß-actin mRNA was coamplified using specific primers (forward 5'-CCC AGG CAC CAG GGC GAT AT-3' and reverse 5'-TCA AAC ATG ATC TGG GTC AT-3') designed according to Licht et al. (2003) to normalize the amplified LH receptor cDNA. To find out the saturation point, different concentrations (100 pg–3 µg) of total RNA from normal cells were used for RT-PCR, and it was found that band intensity was saturated at the 2.5-µg level after optimizing various parameters. Therefore, 2 µg of total RNA was taken for each reaction. The RT reaction was done at 50°C for 30 min followed by initial PCR activation at 95°C for 15 min. The three-step PCR cycles include the following: denaturation at 95°C for 1.5 min, annealing at 57°C for 1.5 min, and extension at 72°C for 1.5 min. The product was amplified 40 times and then finally extended at 72°C for 10 min. Ten microliters of RT-PCR mixture was resolved on 2% agarose gel containing ethidium bromide (0.5 µg/ml) along with a 100-bp DNA ladder in a separate well for 2 h. Then the gel was subjected to densitometric scanning to find out the optical density (OD) units of LHR cDNA bands after normalization against ß-actin.
Statistical Analysis
Data are presented as the mean ± SEM. Data were analyzed by ANOVA. When the F ratio was significant, multiple comparisons were done by the Student-Newman-Keuls test using SPSS statistical package to determine the differences among various group means.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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Dose-Dependent Effects of Gamma Radiation on the Viability of Cultured Human Leydig Cells
Leydig cell viability was not significantly altered under any dose employed in this study as shown in Figure 1.
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Dose-Dependent Effects of Gamma Radiation on LH Receptor Concentration in Cultured Human Leydig Cells
Leydig cell surface LH/hCG receptor concentration was significantly decreased following higher doses of radiation exposure (6 Gy and above). However, lower doses (2 and 4 Gy) did not significantly affect the receptor concentration (Fig. 2).
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Dose-Dependent Effects of Gamma Radiation on Basal and LH-Stimulated cAMP Production in Cultured Human Leydig Cells
Gamma radiation was found to have an inhibitory effect on both basal and LH-stimulated cAMP production in a dose-dependent manner. While the lower doses (2 and 4 Gy) did not show any appreciable change in the generation of cAMP, higher doses (6 Gy and above) brought down the cAMP generation in both basal and LH-stimulated conditions as represented in Figure 3.
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Basal, 
LH-stimulated.Dose-Dependent Effects of Gamma Radiation on Basal, LH-, and cAMP-Stimulated Steroidogenic Enzymes (P450 scc, 3ß-HSD, and 17ß-HSD) Activity in Cultured Human Leydig Cells
cAMP stimulated all the enzymes effectively than LH in normal as well as irradiated cells. Higher doses (6, 8, and 10 Gy) of radiation drastically decreased the steroidogenic enzymes in basal and stimulated conditions in a dose-dependent manner. However, lower doses did not induce any significant change in the enzyme activities (Figs. 4–6
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cAMP-stimulated.
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Dose-Dependent Effects of Gamma Radiation on Basal, LH-, and cAMP-Stimulated Testosterone Production in Cultured Human Leydig Cells
The efficacy of cAMP was more than LH in terms of inducing testosterone production in normal and irradiated cells. While lower doses of radiation did not affect the basal and stimulated testosterone production significantly, higher doses deteriorated the same. The adverse effect of radiation was more pronounced with increasing doses of radiation (Fig. 7).
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Dose-Dependent Effects of Gamma Radiation on Basal, LH-, and cAMP-Stimulated Estradiol Production in Cultured Human Leydig Cells
LH and cAMP were found to stimulate estradiol production equally. Radiation depressed estradiol production in a dose-dependent fashion similar to that of testosterone production under basal and stimulated conditions. While lower doses were ineffective in bringing any change, higher doses decreased the same (Fig. 8).
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Dose-Dependent Effects of Gamma Radiation on LH Receptor mRNA Expression in Cultured Human Leydig Cells
LH receptor mRNA expression was found to be increased following 2 Gy of radiation exposure. However, the mRNA expression was not altered under any other doses tested (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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In the present study, Leydig cell–surface LH/hCG receptor concentration decreased following higher doses of radiation exposure (6, 8, and 10 Gy). However, lower doses such as 2 and 4 Gy did not induce any significant change. In accordance with our findings, Delic et al. (1985)
reported the decreased testicular LH receptor number following higher doses of radiation (5–15 Gy) exposure to prepubertal rat testis. LH is the primary hormonal factor that controls Leydig cell function through its specific receptor, which is coupled to both adenylate cyclase and phospholipase-C pathways (Cooke, 1996; Misrahi et al., 1998). It is therefore suggested that radiation-induced decrease in Leydig cell LH receptors could be responsible for the steroidogenic lesions recorded after radiation exposure in clinical and experimental studies. Since the effects of LH/hCG on Leydig cells are exerted predominantly through cAMP-mediated events (Saez, 1994), it was intended to estimate the basal and LH-stimulated cAMP generation in normal and irradiated Leydig cells. Radiation exposure decreased the basal and LH-stimulated cAMP generation in a dose-dependent manner. While lower doses (2 and 4 Gy) did not influence cAMP generation, higher doses (6, 8, and 10 Gy) diminished the same in a dose-dependent way. cAMP is the classical second messenger involved in LH-induced Leydig cell testosterone production, and its generation solely depends on LH and its receptor interaction and effector activation (Dufau, 1998). Therefore, the depressed LH-stimulated cAMP production in higher doses treated cells may be due to subnormal Leydig cell surface LH receptors and associated impairment in downstream events such as transmembrane signaling and activation of effector system. In this regard, it is worth to recall the findings of Morel et al. (2002), who have shown decreased secretory activity of colon to vasoactive intestinal peptide (VIP) as a result of reduced cAMP, decreased adenylate cyclase activity, and diminution of VIP receptor numbers in rats exposed to 10 Gy of abdominal irradiation. In addition to cAMP-mediated pathway, LH also employs calcium signaling in rat Leydig cells (Sullivan and Cooke, 1986). Calmodulin inhibitors, such as calmidazolium, trifluoperazine, and chlorpromazine, have been shown to inhibit LH-stimulated steroidogenesis in rat Leydig cells (Sullivan and Cooke, 1985). Therefore, it is suggested that impaired calcium signaling as a result of decreased Leydig cell surface LH receptors might be the additional mechanism involved in radiation-induced Leydig cell dysfunction. To explore the effect of radiation on LH signaling at pre- and post-cAMP levels, both normal and irradiated Leydig cells were stimulated with the maximum effective dose of LH or cAMP and then steroidogenic potency was assessed by estimating steroidogenic enzymes, testosterone, and estradiol production. The response of Leydig cells to cAMP stimulation attests the higher efficacy of cAMP in terms of inducing steroidogenesis than LH. Basal, LH-, and cAMP-stimulated steroidogenic enzyme activities and testosterone and estradiol production were reduced following higher doses of radiation exposure. Since the binding of LH to its receptor and the activation of effector system are the important steps involved in the activation of steroidogenesis, the observed decrease in LH/hCG receptors, cAMP production, and steroidogenic enzymes are the factors that account for the subnormal testosterone recorded in the clinical cases after radiation exposure. Under basal or unstimulated condition also radiation impairs Leydig cellular steroidogenesis as evinced by the decreased cAMP, steroidogenic enzymes, testosterone, and estradiol production. Therefore, it is suggested that radiation may affect the steroidogenic machineries directly, in addition to impairing LH signal transduction. In the experiments involving cell culture, an important factor that could influence the quality of experiment is the cell viability. In the present study, viability of cultured Leydig cells tested during the experimental period did not show any significant difference between normal and irradiated cells. It is therefore suggested that the changes observed in various parameters are not due to the loss of cell viability.
In the present study, higher doses of radiation decreased the cell-surface LH receptor concentration significantly. Since synthesis of new receptors is one of the means by which the required number of LH/hCG receptors on Leydig cells are maintained, it was intended to assess the LH receptor mRNA expression under irradiated condition to find out adverse effects, if any, at the level of LH receptor gene transcription. LH receptor mRNA expression was increased slightly in Leydig cells following 2 Gy of radiation exposure. However, other doses of radiation did not induce any change. The increased expression of LH receptor mRNA in Leydig cells at 2 Gy of exposure may be the adaptive response of Leydig cells to radiation. In accordance with the present study, expression of transforming growth factor-ß (TGF-ß1) mRNA in endothelial cells and fibroblast growth factor-2 (FGF-2) and procollagen type I mRNAs in fibroblasts were shown to be elevated between 4 and 48 h after a dose of 2 Gy ionizing radiation (Boerma et al., 2002). In the present study, higher doses of radiation reduced the Leydig cell-surface LH receptor without affecting its mRNA level. However, radiation has been shown to downregulate histone H1 expression at both transcriptional and posttranscriptional level; hence, it is suggested that radiation might have impaired the LH receptor expression at posttranscriptional level (Datta, 1993). In addition to that, radiation has been shown to induce the pinocytotic and phagocytic activity in macrophages and glial cells and increase the number, volume fraction, and activity of lysosomes in various cells (Somosy, 2000). It is therefore suggested that radiation might have enhanced the internalization and lysosomal degradation of LH receptor and thereby hampered the recycling of internalized receptors.
Based on the results obtained in this study, it is concluded that gamma radiation has dose-dependent inhibitory effects on basal and LH/cAMP-stimulated human Leydig cell steroidogenesis. Subnormal concentration of cell surface LH receptors and impaired signaling events of LH at both pre- and post-cAMP generations are the plausible mechanisms by which radiation induces lesions in human Leydig cell steroidogenesis. Radiation-induced decrease in Leydig cell surface LH receptors is not coupled with impaired expression of its mRNA.
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NOTES |
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Disclaimer: The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
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ACKNOWLEDGMENTS |
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Financial support by Department of Atomic Energy, Government of India, is acknowledged. The kind gift of oLH and hCG by National Institute of Diabetes, Digestive and Kidney diseases (NIDDK), Bethesda, MD, is acknowledged with thanks.
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