Direct effects of propylthiouracil on testosterone secretion in rat testicular interstitial cells

Abstract

1. The aim of this study was to investigate the mechanism by which propylthiouracil (PTU) exerts its inhibitory effects on the production of testosterone by rat testicular interstitial cells.

2. The plasma testosterone concentration was decreased 60 and 120 min after an intravenous infusion of PTU (10 or 20 mg kg⁻¹), but the concentration of plasma T₄ was unaffected by the drug treatment.

3. Exposure of anterior pituitary tissue to PTU (3–12 mM) in vitro did not affect either basal or gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone (LH) release.

4. PTU (3–12 mM) inhibited both the basal and the human chorionic gonadotropin (hCG, 0.05 iu ml⁻¹)-stimulated release of testosterone from rat testicular tissue in vitro; at the highest concentration tested (12 mM), it also inhibited the forskolin or 8-bromo-adenosine 3′:5′-cyclic monophosphate (8-Br-cyclic AMP)-stimulated release of testosterone.

5. The 25-OH-cholesterol (10⁻⁷–10⁻⁵ M)-stimulated release of pregnenolone and testosterone by the testicular interstitial cells was inhibited by PTU (12 mM, P<0.05).

6. The results suggest that the inhibitory actions of PTU on testosterone secretion are exerted, at least in part, at the testicular level through a mechanism which is independent of thyroid status and which involves a reduction in P450scc activity and, hence, in the conversion of cholesterol to pregnenolone.

Introduction

Propylthiouracil (PTU, MW=170.2) is an anti-thyroid drug which inhibits both the synthesis of thyroid hormones in thyroid gland (Cooper, 1984), and the conversion of thyroxine (T₄) to its active form, triiodothyronine (T₃), in peripheral tissues (Yang & Gordon, 1997). Clinical studies have shown that the most common unwanted effect of PTU treatment in hyperthyroid patients is transient leukopenia (Cooper, 1984). In addition, the drug has been found to induce severe toxic effects on the liver (Levy, 1993; Deidiker & deMello, 1996) in a number of patients who subsequently developed jaundice, severe hepatocellular dysfunction, and hepatomegaly (Jonas & Eidson, 1988; Levy, 1993; Deidiker & deMello, 1996). However, the effects of PTU on human testicular cells are unclear.

Previous studies have shown that transient neonatal hypothyroidism, induced by treatment with PTU, increases testicular size, Sertoli cell numbers, and daily sperm production in the adult rat and mouse (Hess et al., 1993; Joyce et al., 1993), although serum testosterone is not raised (Cooke et al., 1991; Cooke & Meisami, 1991). Studies in rodents have suggested that PTU treatment in adults may also influence peripheral steroidogenesis. For example, Hardy et al. (1993) reported decreased serum testosterone levels in adult rats rendered hypothyroid with PTU, although others (Weiss & Burns, 1988) found testosterone production was unchanged by the drug treatment. Moreover, although Leydig cell activity has not been studied during thiouracil-induced hypothyroidism in vivo, studies in rats and rams suggest testosterone production is subnormal (Chandrasekhar et al., 1985; Ando et al., 1990).

We have previously shown that PTU decreases both the adrenocortical response to ACTH in vivo and the production of corticosterone by rat zona fasciculata-reticularis cells (Lo et al., 1998). However, the direct effects of PTU on the function of gonadal tissues are not known. In the present study, we examined the acute effects of PTU in vitro on (a) the production of testosterone and (b) activity of the steroidogenic enzyme in rat testicular interstitial cells.

Methods

Animals

Male Sprague-Dawley rats weighing 300–350 g were housed in a temperature controlled room (22±1°C) with 14 h of artificial illumination daily (0600–2000 h) and given food and water ad libitum.

Effects of PTU on plasma testosterone and T₄ in rats

Male rats were divided into two groups with eight rats in each group. Each animal was anaesthetized with ether and catheterized via the right jugular vein (Wang et al., 1994; Tsai et al., 1996). After 20 h, they were infused with 1 ml of saline or PTU (10 or 20 mg kg⁻¹, Sigma Chemical Co., St. Louis, MO, U.S.A.) via a peristaltic pump (Minipuls 3, Gilson, Villiers-le-Bel, France) for 30 min. Blood samples (0.3 ml each) were collected from the jugular catheter at 0, 30, 60, 120, 180 and 240 min after infusion.

Plasma was separated by centrifugation at 10,000×g for 1 min and stored at −20°C. The concentration of plasma T₄ was measured by the radioimmunoassay (RIA) kit from DiaSorin Inc. (Stillwater, MN, U.S.A.). To measure the concentrations of testosterone, 0.1 ml plasma was mixed with 0.5 ml diethyl ether, agitated for 20 min, centrifugated at 1000×g for 5 min, and then quick frozen in a mixture of acetone and dry ice. The organic phase was collected, dried, and reconstituted in a PBSG buffer solution (0.1% gelatin in phosphate-buffered saline, PBS, pH 7.5). The concentrations of testosterone in the reconstituted extracts were measured by RIA.

Effects of PTU on LH release by anterior pituitary glands

After decapitation, the rat anterior pituitary glands (APs) were excised, bisected, and preincubated with Locke' solution containing 10 mM glucose, 0.003% bacitracin, and 0.05% HEPES at 37°C for 30 min (Wang et al., 1994; Tsai et al., 1999). Each hemi-AP gland was assigned to a flask containing 1 ml medium, which was aerated with 95% O₂ and 5% CO₂. APs were then incubated with PTU (0, 3, 6 or 12 mM), gonadotropin-releasing hormone (GnRH, 10 nM, Sigma), or PTU plus GnRH for 30 min. At the end of incubation, the medium was collected and the tissues were weighed. The concentrations of LH in medium were measured by RIA.

Preparation of testicular interstitial cells

The method of collagenase dispersion of testicular interstitial cells was modified from the procedure described by Tsai et al. (1997). Five decapsulated testes collected postmortem were added to a 50 ml polypropylene tube containing 5 ml preincubation medium and 700 μg collagenase (Type IA, Sigma, U.S.A.). Preincubation medium was made up of 1% bovine serum albumin (BSA, Fraction V, Sigma, U.S.A.) in Hank' balanced salt solution (HBSS), with HEPES 25 mM, sodium bicarbonate 0.35 g l⁻¹, penicillin-G 100 iu ml⁻¹, streptomycin sulphate 50 μg ml⁻¹, heparin 2550 USP K units l⁻¹, pH 7.4, and aerated with 95% O₂ and 5% CO₂. The tube was placed horizontally for 15 min in a 34°C water bath and shaken continuously (100 cycles min⁻¹). The digestion was then stopped by adding 35 ml of cold preincubation medium and inverting the tube several times. The tube was allowed to stand for 5 min and then filtered through a four-layer nylon mesh. Cells were collected by centrifugation at 4°C, 100×g for 10 min. The cell pellets were washed with deionized water to disrupt red blood cells (RBCs) and the osmolarity immediately restored with 10 fold Hank' balanced salt solution (HBSS). Hypotonic shock was repeated twice for RBC disruption and cell pellets resuspended in incubation medium (substitution of HBSS in preincubation medium with Medium 199, and sodium bicarbonate 2.2 g l⁻¹). Cell concentration (1.0×10⁶ cells ml⁻¹), viability (over 97%), and the sperm cells (less than 5%) were determined by use of a hemacytometer and the trypan blue exclusion method.

Effects of PTU on testosterone release by testicular interstitial cells

Aliquots (1 ml) of cell suspensions (1.0×10⁶ cells ml⁻¹) were preincubated with incubation medium in polyethylene tubes for 1 h at 34°C under a controlled atmosphere (95% O₂ and 5% CO₂), shaken at 100 cycles min⁻¹. The supernatant fluid was decanted after centrifugation of the tubes at 100×g for 10 min. The cells were then incubated with PTU (3, 6 or 12 mM), human chorionic gonadotropin (hCG, 0.05 iu ml⁻¹, Sigma), forskolin (an activator of adenylyl cyclase, 10⁻⁵ M, Sigma), 8-bromo-adenosine 3′:5′-cyclic monophosphate (8-Br-cyclic AMP, an analogue of cyclic AMP, 10⁻⁴ M, Sigma), hCG plus PTU, forskolin plus PTU, or 8-Br-cyclic AMP plus PTU in 200 μl fresh medium. Following 1 h of incubation, 2 ml ice-cold PBSG buffer solution was added to stop the incubation. The medium was centrifuged at 100×g for 10 min and the supernatant was stored at −20°C until analysed for testosterone by RIA.

Effects of PTU on the steroidogenesis in testicular interstitial cells

Rat testicular interstitial cells (1.0×10⁶ cells ml⁻¹) were preincubated for 1 h and then incubated for 1 h with or without PTU at 12 mM in the presence or absence of five steroidal precursors as described previously (Lin et al., 1998). These precursors included 25-hydroxy-cholesterol (membrane-permeable cholesterol, 25-OH-C), pregnenolone (Δ₅P), progesterone (P), 17α-hydroxy-progesterone (17α-OH-P), and androstenedione (Δ₄). At the end of incubation, 2 ml ice-cold PBSG buffer was added and the tubes centrifuged immediately at 100×g for 10 min at 4°C. The supernatant fluid was stored at −20°C until analysed for testosterone by RIA.

Effects of PTU on the 25-OH-cholesterol-stimulated pregnenolone production in testicular interstitial cells

In order to explore the activity of cytochrome P450 side-chain cleavage enzyme (P450scc), cell suspensions (1.0×10⁶ cells ml⁻¹) were preincubated in medium for 1 h at 34°C and then incubated for 1 h with or without PTU at 12 mM in the presence of 25-OH-cholesterol (10⁻⁷ or 10⁻⁵ M). At the end of incubation, 2 ml ice-cold PBSG buffer were added and immediately followed by centrifugation at 100×g for 10 min at 4°C. The supernatant fluid was stored at −20°C until analysed for pregnenolone by RIA.

RIA of testosterone, pregnenolone and LH

The concentrations of testosterone in plasma and medium were determined by RIA as described previously (Wang et al., 1994; Tsai et al., 1996). With anti-testosterone serum no. W8, the sensitivity of testosterone RIA was 2 pg per assay tube. The intra- and interassay coefficients of variation (CV) were 4.1% (n=6) and 4.7% (n=10), respectively.

The concentration of pregnenolone was determined by RIA with anti-regnenolone antiserum purchased from Biogenesis Inc (Sandown, NH, U.S.A.). The sensitivity of the pregnenolone RIA was 16 pg per assay tube. The intra- and interassay coefficients of variation were 2.3% (n=6) and 3.7% (n=4), respectively.

The concentration of medium LH was determined by RIA as described previously with anti-LH serum PW11-2 (Wang et al., 1994). The rat LH-I-6 used for iodination and the rat LH-RP-3 which served as standard preparations were provided by National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Child Health and Human Development, and U.S. Department of Agriculture, U.S.A. The sensitivity was 0.1 ng for LH RIA. The intra- and interassay coefficients of variability were 3.8% (n=4), and 6.6% (n=5), respectively.

Materials

Bovine serum albumin (BSA), N-2-hydroxyethylpiperazine-N′-2-ethane-sulphonic acid (HEPES), Hank' balanced salt solution (HBSS), medium 199, sodium bicarbonate, penicillin-G, streptomycin, heparin, collagenase, propylthiouracil (PTU), gonadotropin-releasing hormone (GnRH), human chorionic gonadotropin (hCG), forskolin, 8-bromo-adenosine 3′:5′-cyclic monophosphate (8-Br-cyclic AMP), and testosterone were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). [³H]-testosterone, and [¹²⁵I]-Na were obtained from Amersham International plc. (Bucks, U.K.). The doses of drugs are expressed in their final molar concentrations in the flask.

Statistical analysis

All values are given as the mean±s.e.mean. The treatment means were tested for homogeneity by analysis of variance (ANOVA) and the difference between specific means was tested for significance by Duncan' multiple-range test (Steel & Torrie, 1960). A difference between two means was considered statistically significant when P<0.05.

Results

Effects of PTU on plasma testosterone and T₄ in rats

The effects of PTU infusion on plasma testosterone are shown in Figure 1. The levels of plasma testosterone were not altered by saline infusion. The plasma concentration of testosterone decreased gradually from 60–240 min after intravenous infusion of the high dose of PTU (20 mg kg⁻¹ group, 60 min, 0.48±0.11; 120 min, 0.34±0.06; 180 min, 0.19±0.05; 240 min, 0.10±0.02 ng ml⁻¹, n=8, versus 0 min, 0.93±0.23 ng ml⁻¹, n=8, P<0.05 or 0.01). The lower dose of PTU was also effective in this regard and the plasma levels of testosterone at 120, 180 and 240 min following an intravenous infusion were significantly lower in PTU-infused rats than in saline-infused animals (120 min, 10 mg kg⁻¹ group, 0.41±0.05 ng ml⁻¹, n=8, 20 mg kg⁻¹ group, 0.34±0.06 ng ml⁻¹, n=8, versus 0.79± 0.19 ng ml⁻¹, n=8, P<0.05; 180 min, 10 mg kg⁻¹ group, 0.23±0.05 ng ml⁻¹, n=8, 20 mg kg⁻¹ group, 0.19± 0.05 ng ml⁻¹, n=8, versus 0.61±0.14 ng ml⁻¹, n=8, P<0.05 or 0.01; 240 min, 10 mg kg⁻¹ group, 0.14 ±0.03 ng ml⁻¹, n=8, 20 mg kg⁻¹ group, 0.10±0.02 ng ml⁻¹, n=8, versus 0.59±0.13 ng ml⁻¹, n=8, P<0.01).

Figure 1.

Plasma testosterone concentrations at various times after an intravenous infusion of saline or PTU via right jugular vein. The horizontal line indicates time of infusion. +P<0.05, ++P<0.01 compared to basal release. ^* P<0.05, ^** P<0.01 compared with the saline group. Each value represents mean±s.e.mean.

The mean plasma levels of T₄ at all time points were 18.1±3.1 ng ml⁻¹ for the saline-infused animals, and 20.9±2.2 ng ml⁻¹ for the PTU-infused rats, respectively. The concentrations of plasma T₄ were not altered by the acute administration of PTU in rats.

Effects of PTU on LH release by rat anterior pituitary glands

The effects of PTU on LH release by rat APs in vitro were examined. As compared to the control group, neither basal LH release (4.34±0.36 to 5.13±1.23 ng mg⁻¹ 30 min⁻¹, n=7, versus control group 4.39±0.91 ng mg⁻¹ 30 min⁻¹, n=7) nor GnRH-stimulated LH release (9.86±1.17 to 8.91 ± 1.65 ng mg⁻¹ 30 min⁻¹, n=7, versus GnRH-treated group 11.21±1.58 ng mg⁻¹ 30 min⁻¹, n=7) by rat APs was altered by the administration of PTU (3–12 mM).

Effects of PTU on testosterone release by rat testicular interstitial cells

The effects of PTU on testosterone release by rat testicular interstitial cells in vitro are shown in Figure 2. Following 1 h of preincubation, testicular interstitial cells (1.0×10⁶ cells) were incubated with or without human chorionic gonadotropin (hCG, 0.05 iu ml⁻¹), combined with PTU (0, 3, 6 or 12 mM) for 1 h. As compared to the control group, administration of PTU (3–12 mM) inhibited testosterone release by the testicular interstitial cells (2.39±0.26 to 1.83±0.34 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, versus basal level 6.76±0.88 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, P<0.01). Incubation of testicular interstitial cells with hCG for 1 h increased testosterone secretion (hCG-treated group 60.0±11.2 ng 1.0×10⁶  cells⁻¹ h⁻¹, n=8, versus basal group, P<0.01). A combination of hCG with PTU of 3–12 mM resulted in an inhibition of the hCG-stimulated release of testosterone (31.9±4.9 to 12.6±1.5 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, versus hCG-treated group, P<0.05 or 0.01).

Figure 2.

Effects of PTU (3–12 mM) on the release in vitro of testosterone by rat testicular interstitial cells in the presence or absence of hCG. +P<0.05 and ++P<0.01 compared to PTU at 0 mM. ^** P<0.01 compared with the vehicle group. Each value represents mean±s.e.mean.

Figure 3 demonstrates the effects of PTU on forskolin (10⁻⁵ M) and 8-Br-cyclic AMP (10⁻⁴ M)-stimulated testosterone release by testicular interstitial cells. Treatment with hCG, forskolin, and 8-Br-cyclic AMP all produced significant (P<0.01) increases in testosterone release versus vehicle. PTU (3–12 mM) inhibited the basal (1.98±0.43 to 1.51±0.29 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, versus basal level 3.74±0.35 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, P<0.01) and hCG-stimulated (37.6±4.8 to 19.8±2.3 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, versus hCG-treated group 56.9±8.6 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, P<0.05 or 0.01) testosterone release. Administration of high dose of PTU (12 mM) inhibited testosterone release in response to forskolin (21.6±2.1 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, versus 56.0±11.0 ng 1.0×10⁶ cells⁻¹ h⁻¹ at PTU=0 mg ml⁻¹, n=8, P<0.01) and 8-Br-cyclic AMP (16.4±1.6 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, versus 34.2±3.4 ng 1.0×10⁶ cells⁻¹ h⁻¹ at PTU=0 mg ml⁻¹, n=8, P<0.01).

Figure 3.

Effects of PTU (3–12 mM) on the release in vitro of testosterone by rat testicular interstitial cells after incubation with vehicle, hCG, forskolin or 8-Br-cyclic AMP. +P<0.05 and++P<0.01 compared with PTU at 0 mM. The levels of testosterone in response to hCG, forskolin, and 8-Br-cyclic AMP groups all are significantly (P<0.01) higher than the corresponding vehicle group. Each value represents mean±s.e. mean.

Effects of PTU on the steroidogenesis in testicular interstitial cells

Four of the testosterone precursors tested (Δ₅P, P, 17α-OH-P and Δ₄, 10⁻⁵–10⁻⁷ M) produced significant concentration-dependent increases in testosterone production in vitro. 25-OH-C also stimulated testosterone release at the higher concentration tested (10⁻⁵ M), but at a lower concentration (10⁻⁷ M), it was without effect (Figure 4). PTU (12 mM) decreased both basal testosterone release (P<0.01) and the production of testosterone facilitated by the low (10⁻⁷ M) concentration of each precursor (1.69±0.23 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, versus 25-OH-C group, 3.59±0.51 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, P<0.01; 2.33±0.31 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, versus Δ₅P group, 5.49±0.58 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, P<0.01; 2.50±0.19 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, versus P group, 5.67±0.68 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, P<0.01; 3.25±0.17 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, versus 17α-OH-P group, 5.12±0.41 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, P<0.01 and 3.23±0.16 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, versus Δ₄ group, 5.67±0.70 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, P<0.01). PTU (12 mM) also decreased the production of testosterone facilitated by the high concentration (10⁻⁵ M) of 25-OH-C (3.25±0.19 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, versus 25-OH-C group, 6.03±0.73 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=8, P<0.01). However, it did not affect the production of testosterone induced by the high concentration (10⁻⁵ M) of the other four precursors tested.

Figure 4.

Effects of PTU (12 mM) on the release in vitro of testosterone from rat testicular interstitial cells treated with vehicle or testosterone precursors. The precursors were 25-hydroxy-cholesterol (25-OH-C), pregnenolone (Δ₅P), progesterone (P), 17α-hydroxy-progesterone (17α-OH-P) and androstenedione (Δ₄). ^** P<0.01 compared with vehicle group. Each value represents mean±s.e. mean.

Effects of PTU on the 25-OH-cholesterol-stimulated pregnenolone production in testicular interstitial cells

Figure 5 demonstrates effects of PTU on 25-OH-C-stimulated pregnenolone release by testicular interstitial cells. PTU (12 mM) decreased the production of pregnenolone facilitated by the 25-OH-C (10⁻⁷ and 10⁻⁵ M, 10⁻⁷ M group, 0.09±0.03 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=7, versus vehicle group, 0.24±0.05 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=7, P<0.05 and 10⁻⁵ M group, 0.18±0.03 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=7, versus vehicle group, 0.38±0.09 ng 1.0×10⁶ cells⁻¹ h⁻¹, n=7, P<0.05).

Figure 5.

Effects of PTU (12 mM) on the release in vitro of pregnenolone after incubation of rat testicular interstitial cells with 25-OH-cholesterol. ^* P<0.05 compared with vehicle group. Each value represents mean±s.e. mean.

Discussion

The present results demonstrate that exposure of rat testicular cells to PTU in vitro diminishes the resting and evoked secretion of testosterone by a mechanism involving decreased activity of the P450scc enzyme. By contrast, PTU does not affect the secretion of LH by pituitary tissue in vitro.

Previous studies have shown that both thyroidectomy (Aruldhas et al., 1982; Biswas et al., 1994) and PTU-treatment (Hardy et al., 1993) decrease serum testosterone concentrations in the rat. This response has been attributed to the decreased levels of thyroid hormones in serum but the underlying mechanisms are ill-defined. Clinical studies have shown that PTU has toxic effects on the liver (Levy, 1993; Deidiker & de Mello, 1996) and the immune system (Cooper, 1984). However, the possibility that PTU exerts unwanted pharmacological effects on PTU on other tissues has scarcely been evaluated. Recently we found that PTU, given acutely to euthyroid rats decreases the rise in plasma corticosterone concentration induced by ACTH and that, in vitro, it decreases corticosterone production in rat zona fasciculata-reticularis cells (Lo et al., 1998), suggesting that PTU may directly regulate adrenal steroidogenesis. There is also evidence that neonatal administration of PTU impairs testicular steroidogenesis in the adult but this is the first study to examine the direct effects of PTU on the endocrine function of rat testicular interstitial cells.

In comparison to the doses used clinically (200–600 mg person⁻¹ day⁻¹, p.o., Astwood, 1967; Gwinup 1978; McMurry et al., 1975; Clark et al., 1992), the dose of PTU used in our in vivo study (10–20 mg kg⁻¹, i.v. i.e. ≈ 0.12 mmoles kg⁻¹ i.v.) was high. However it was in the region of that used previously in rodent studies to induce hypothyroidism (Hwang et al., 1974). The finding that this dose did not reduce the serum T₄ levels over the time course studies is not surprising as the t_1/2 of T₄ is in the region of 7 days. The concentrations of PTU used in our in vitro (e.g. 12 mM) studies are almost certainly lower (one tenth) than those attained in our in vivo model.

It is evident from the present study that PTU causes a significant decrease in the serum testosterone concentration when infused intravenously in rats. Our in vitro studies, which showed that PTU does not affect the basal or GnRH stimulated release of LH from rat pituitary tissue, suggest that this effect was not a consequence of a direct action of the drug on the pituitary gland and may therefore reflect an action on the testis. To investigate this possibility, we used an established in vitro model based on the ability of hCG to stimulate testosterone secretion by rat Leydig cells (Saez & Forest, 1979; Padron et al., 1980; Wang et al., 1994; Simpson et al., 1987; Nakhla et al., 1989; Liao et al., 1991) via a mechanism involving cyclic AMP production (Avallet et al., 1987; Petersson et al., 1988; Sakai et al., 1989; Wang et al., 1994). Our data show that PTU inhibits both the basal and the hCG-stimulated release of testosterone in vitro. It also decreases forskolin- and 8-Br-cyclic AMP-induced testosterone release suggesting that the drug acts directly on the rat testicular cells to regulate testosterone production at a point distal to the formation of cyclic AMP. Since these actions of PTU were observed in vitro in tissue removed from control rats, they were clearly independent of any effects of the drug on gonadotropin or thyroid hormone production.

We also examined the effects of PTU on the activities of steroidogenic enzymes in testicular interstitial cells by challenging the cells in vitro with the following testosterone precursors, including 25-OH-cholesterol (25-OH-C, substrate of P450scc), pregnenolone (Δ₅P, substrate of 3β-HSD), progesterone (P, substrate of 17α-hydroxylase), 17α-OH-progesterone (17α-OH-P, substrate of C17-20 lyase) and androstenedione (Δ₄, substrate of 17β-HSD). The results showed that PTU inhibits the increase in testosterone production induced by a low concentration (10⁻⁷ M) of each of these precursors. PTU also decreased testosterone production evoked by a higher concentration 25-OH-cholesterol (10⁻⁵ M) but failed to influence the steroidogenic responses to this concentration of pregnenolone-, progesterone-, 17α-OH-progesterone- and androstenedione. In addition, it inhibited the conversion of 25-OH-cholesterol to pregnenolone, a process which is catalyzed by P450scc, the rate limiting enzyme in gonadal steroidogenesis, and which is regulated by the gonadotropins (Iida et al., 1989).

Taken together these results suggest that PTU may act at the level of P450scc to inhibit testosterone production.

In conclusion, our results provide new evidence that, in high doses, PTU may act directly on the testis to repress steroidogenesis, via a mechanism involving up inhibition of P450scc activity. This action may explain the attenuation in serum testosterone concentration observed in vivo following intravenous infusion of high doses of PTU.

Abbreviations

AP

anterior pituitary gland

8-Br-cyclic AMP

8-bromo-adenosine 3′:5′-cyclic monophosphate

BSA

bovine serum albumin

GnRH

gonadotropin-releasing hormone

3β-HSD

3β-hydroxysteroid dehydrogenase

17β-HSD

17β-hydroxysteroid dehydrogenase

HBSS

Hank' balanced salt solution

hCG

human chorionic gonadotropin

HEPES

N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulphonic acid]

LH

luteinizing hormone

25-OH-C

25-hydroxy-cholesterol

17α-OH-P

17α-hydroxy-progesterone

P

progesterone

Δ₅P

pregnenolone

P450scc

cytochrome P450 side-chain cleavage

PTU

propylthiouracil

RBC

red blood cell

Δ₄

androstenedione