On the mechanism of action of lead in the testis: In vitro suppresion of FSH receptors, cyclic AMP and steroidogenesis

Life Sciences, Vol. 32, pp. 1997-2005 Printed in the U.S.A.

Pergamon Press

ON THE MECHANISM OF ACTION OF LEAD IN THE TESTIS: IN VITRO SUPPRESSION OF FSH RECEPTORS, CYCLIC AMP AND STEROIDOGENESlS. John P Wiebe*, Arthur I. Salhanick** and Kathryn I. Myers Hormonal Regulatory Mechanism Laboratory, Department of Zoology, U n i v e r s i t y of Western Ontario, London, Canada N6A 5B7 (Received in final form January 31, 1983)

SUMMARY Previous evidence has shown that prenatal and neonatal exposure to low levels of Pb r e s u l t in decreased FSH binding and steroidogenesis in the testes at the onset of puberty. The purpose of the present study was to determine by in v i t r o methods, i f Pb acts by i n t e r f e r i n g d i r e c t l y with hormone binding, c y c l i c AMP production and steroidogenic enzyme a c t i v i t y . S e r t o l i c e l l s were isolated from testes of prepubertal rats and cultured in the presence of 2.64 x lO-4M of e i t h e r NaAc (control) or PbAc f o r I , 4, 24, 48, 96 or 144 hr. There was no reduction in FSH binding and in FSHinduced c y c l i c AMP a f t e r a I-4 hr exposure to Pb. A f t e r a 24-hr exposure to Pb, the c e l l s exhibited a 10-20% decrease in FSH binding and c y c l i c AMP production and a f t e r 96 hr there was a 75% decrease in these 2 parameters. The i n h i b i t i o n was greater in c e l l s from 16 day old than from 20 day old r a t s , so that in the former, a f t e r a 144 hr exposure the FSH-induced c y c l i c AMP of the Pb exposed c e l l s was only 3% of the amount produced by the NaAc exposed c e l l s ( i . e . a 97% i n h i b i t i o n ) . A f t e r in v i t r o exposure to Pb for 48 hr, the steroidogenic a c t i v i t y (progesterone conversion to steroid metabolites) of S e r t o l i c e l l s was s i g n i f i c a n t l y reduced and t h e i r steroidogenesis was no longer stimulated by FSH. A crude t e s t i c u l a r enzyme preparation containing 3B-hydroxysteroid dehydrogenase (3B-HSD) exhibited approximately 25% reduction in a c t i v i t y i f the assay b u f f e r contained PbCI2 instead of the equivalent in NaCI. Prolonged in vivo exposure to Pb resulted in approximately 50% reduction in 3B-HSD a c t i v i t y . This is the f i r s t i n d i c a t i o n that in the t e s t i s Pb may act d i r e c t l y (immediate e f f e c t ) by suppressing enzyme a c t i v i t i e s , and i n d i r e c t l y (long term e f f e c t ) by reducing gonadotropin-receptor binding and the r e s u l t a n t c y c l i c AMP production. The t o x i c e f f e c t s of lead (Pb) at high doses have been known f o r more than 2,000 years ( I ) and v i v i d descriptions of c o l i c , anemia and neurotoxic sequelae of Pb ingestion date back to the Greek philosophers. At present, a large amount of l i t e r a t u r e e x i s t s to show that high levels of Pb have serious c l i n i c a l e f f e c t s on a number of body functions including heme biosynthesis, energy metabolism, kidney and l i v e r functions, the nervous system, * To whom requests for r e p r i n t s should be addressed. ** Present address: Endocrine/Metabolism Unit, U n i v e r s i t y of Rochester Medical Centre, Rochester, New York 14642 0024-3205/83/171997-09503.00/0 Copyright (c) 1983 Pergamon Press Ltd.

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i n t e l l i g e n c e , and behaviour and coordination (2,3). On the other hand, there have been few studies to determine the possible e f f e c t s of Pb on the endocrine systems and u n t i l recently almost nothing was known about the action of lead on reproductive endocrinology. In the l a s t two decades, numerous studies on humans, occupationally exposed to Pb, and experiments on laboratory animals, have confirmed the reports of the l a s t century (4) that lead poisoning results in decreased reproductive a b i l i t y (5). Recently we began to examine the mechanism of action of Pb on male reproductive endocrinology. Our studies have shown that subclinical levels of Pb derived s o l e l y from the mother during gestation and/or l a c t a t i o n , s i g n i f i c a n t l y depress steroid biosynthesis and hormone-receptor binding (6) and the onset of gamete production (7) during the onset of puberty. The decrease in steroidogenesis due to prenatal or neonatal Pb exposure is evident in prepubertal testes that are f r e s h l y homogenized (6) and also in isolated S e r t o l i c e l l s maintained in culture (6). I t is known that in the male FSH receptors occur s p e c i f i c a l l y in Sertoli c e l l s (8) and that FSH e l i c i t s increases in c y c l i c AMP (9) and stimulates steroidogenesis (I0) in S e r t o l i c e l l s from prepubertal r a t s . Since the i n i t i a l event in the action of a protein hormone, such as FSH, is the binding of the hormone to receptor molecules located at the cell membrane, i t is possible t h a t the lead-induced decreases in steroidogenesis r e s u l t from the suppression of the hormone-reduced binding. On the other hand, Pb may also a f f e c t the a c t i v i t y of adenylate cyclase and/or steroidogenic enzymes by a d i r e c t i n h i b i t i o n . The possible action of Pb at the level of gonadotropin-receptor binding, adenylate cyclase or steroidogenic enzyme a c t i v i t i e s , do not appear to have been reported previously. Therefore, the purpose of t h i s in v i t r o study was to determine i f Pb i n t e r f e r e s d i r e c t l y (immediate e f f e c t ) w ~ h hormone-receptor binding, c y c l i c AMP production and/or steroidogenic enzyme a c t i v i t y , or i f i t acts via the c e l l u l a r b i o s y n t h e t i c machinery (long term e f f e c t ) to decrease receptors, and c y c l i c AMP and steroidogenic enzyme synthesis. METHODS Chemicals. P u r i f i e d rat FSH (NIAMD-rat I-3) and ovine FSH (NIH-S-IO) were g i f t s from the Endocrine Study Section, N.I.H. Chloramine-T, sodium m e t a b i s u l f i t e , lead acetate and sodium acetate were purchased from Fisher S c i e n t i f i c Co.; bovine serum albumin (BSA-fraction V), collage~ase (Type I ) , and pancreatin (Grade IV) were from Sigma Chem. Co. C a r r i e r - f r e e [1251]Na was purchased from Amersham-Searle. Preparation of S e r t o l i Cells. S e r t o l i c e l l s were isolated from testes of young rats by the method previously described ( I 0 , I I ) . The r e s u l t a n t suspensions of S e r t o l i c e l l s were e i t h e r used immediately (to determine whether Pb i n t e r f e r e d d i r e c t l y with the hormone-receptor binding) or they were placed in c u l t u r e as described ( I 0 , I I ) and maintained in the presence of NaAc or PbAc f o r I-4 hr, or I , 2, 4 or 6 days. Lead Acetate and Sodium Acetate Treatments. Lead acetate and sodium acetate were dissolved in S e r t o l i cell medium ( I I ) . To determine the e f f e c t s of various doses of Pb, concentrations ranging from 2.64 x 10-12M to 2.64 x 10-4 were employed. A consistent reduction in FSH b#nding was noted a f t e r 4day incubations with Pb concentrations of 2.93 x IO-~M or higher. Therefore, Pb concentrations of 2.93 x IO-5M to 2.64 x 10-4 M were used in all subsequent experiments. To determine i f Pb i n t e r f e r e d d i r e c t l y with hormonereceptor binding and/or c y c l i c AMP synthesis, homogenates of testes or S e r t o l i c e l l s were incubated f o r 1 hr in the presence of e i t h e r NaAc or PbAc before measuring FSH-receptor binding and c y c l i c AMP concentrations as described below.

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Mechanisms of Action of Lead

1999

Measurement of FSH-receptor binding. Rat FSH was iodinated and p u r i f i e d as described previously (12). To q u a n t i t a t e FSH binding the procedure outlined previously (12) was employed. B r i e f l y , the 20,000 x g p e l l e t of homogenized S e r t o l i c e l l s (50-100 g protein) was resuspended in Assay Buffer [ 0 . I M phosphate pH 7.5, containing 0.I M sucrose, 0.015 M MgCI2, 0.1% (w/v) BSA and p e n i c i l l i n - G (500 u n i t s / m l ) ] and added to the p u r i f i e d [1251]-rFSH (10,000-300,000 cpm) in disposable polyethylene tubes to a f i n a l volume of 400~I. Each incubation occurred for I-2 hours at 34°C in a shaking water bath (140 osc/min) and then was terminated by the addition of 1 ml of ice-cold Assay Buffer. The tubes were then centrifuged (4°C) at 20,000 x g for 45 min. The radioactive p e l l e t was washed 2X with 1 ml portions of Assay Buffer and counted in a gamma spectrometer (Beckman Gamma 4000, 80% e f f i c i e n cy). Non-specific binding was determined by running p a r a l l e l sets of tubes containing excess (20wg) unlabelled oFSH. The amount (CPM) of [1251]-rFSH s p e c i f i c a l l y bound to S e r t o l i cell p e l l e t s was corrected to lO0~g S e r t o l i cell protein. At the concentration of cell protein used in this study, a l i n e a r r e l a t i o n s h i p was found between the amount of trace s p e c i f i c a l l y bound and the amount of S e r t o l i cell protein incubated. Protein measurements were made by a modification (13) of the method of Lowry et al. (14). Measurement of c y c l i c AMP and FSH-induced c y c l i c AMP. For each experiment S e r t o l i c e l l s were isolated from prepubertal rats at a p a r t i c u l a r age (between 16 and 21 days old) and placed in culture as described previously ( I 0 , I I ) , in the presence of PbAc or NaAc for periods ranging from I hour to 6 days. At termination, the culture medium was removed and replaced with 1.0 ml Krebs-Ringer Phosphate solution without NaHCO3 (KRP; pH 7.4). In each treatment group, some dishes received only KRP to determine basal c y c l i c AMP l e v e l s , some received KRP with 0.4 mM MIX ( l - m e t h y l - 3 - i s o b u t y l x a n t h i n e ; Aldrich Chem. Co.) to i n h i b i t phosphodiesterase a c t i v i t y , and some received KRP with MIX and FSH (I0 ~g oFSH-S-IO per ml). The dishes were incubated for 1 hour at 32°C and then the KRP solution and c e l l s were separated by c e n t r i f u g a t i o n (800 x g for 5 min). The KRP solution was transferred to a fresh tube which was placed in b o i l i n g water for I0 min and then stored at -20°C. The c e l l s were resuspended in 1.0 ml ethanol and stored at -20°C. For the cyclic AMP assay, the KRP solution and the ethanolic cell extracts were thawed, vortexed and centrifuged for 15 min at 1500 x g. A 500 ~I aliquot of the supernatant was transferred to a fresh tube. In the case of the ethanolic e x t r a c t , a 500 ul a l i q u o t was dried down and redissolved in 500 ~I of 0.05 M acetate b u f f e r (pH 6.2). Acetylation of the samples and buffer blanks was accomplished with 15 ~I of a 2:1 mixture of t r i e t h y l a m i n e / acetic anhydride. For the c y c l i c AMP assay, the tubes contained I0,00012,000 cpm [1251]-tyrosine methyl ester c y c l i c AMP, c y c l i c AMP antibody ( g i f t of Dr. F. Labrie) and buffer or sample to a total volume of 350 ~I. The tubes were incubated overnight at 4°C, then 900 ~I of a 0.2% solution of Norit A charcoal in 0.I M phosphate b u f f e r with 0.25% BSA was added to each. A f t e r 15 min the tubes were centrifuged at 2000 x g f o r 15 min. The supernatant was decanted and counted in a gamma spectrometer (Beckman Gamma 4000). The s e n s i t i v i t y of the c y c l i c AMP assay was 4 fmoles, the inter-assay and intra-assay C.V. were 5-10% and 10-15% r e s p e c t i v e l y , and the c r o s s - r e a c t i v i t y with c y c l i c GMP was less than 0.5%. Measurement of Steroidogenesis. To determine the in v i t r o e f f e c t s of lead on steroidogenesis, S e r t o l i c e l l s from fourteen 17 day o ~ r a t s were isolated and placed in culture (12 p e t r i dishes) as described ( I 0 , I I ) . The c e l l s were cultured in S e r t o l i cell medium ( I I ) for 96 hours, then the dishes were divided into 4 treatment grou~s (3 dishes each). Treatments A and B had medium containing NaAc (2.64 x 10- M); treatments C and D had medium containing PbAc (2.64 x IO-4M); treatments B and D also contained FSH (5 wg FSH-NIHS-lO/ml of medium). Culturing under these conditions was continued f o r 48 hours, then medium was removed and replaced with medium containing [14C]-

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progestergQe (500,000 DPM/3 ~g progesterone/dish). A f t e r 7 hours in c u l t u r e with the 14C-labelled progesterone, the c e l l s and media were t r a n s f e r r e d to e x t r a c t i o n tubes, each containing 5 ml ether/chloroform (4:1). The samples were extracted 3 times with ether/chloroform ( 4 : 1 ) , then the following standards were added to the e x t r a c t : lO ~g of progesterone, 20~hydroxy-4-pregnen3-one, testosterone and 17B-hydroxy-4-pregnen-3,20-dione and 30 ~g of 5~pregnane-3,20-dione, 3~-hydroxy-5~-pregnan-20-one and 5~-pregnane-3~,20~-diol.The e x t r a c t s (containing the standards) were then run 2-dimensionally on TLC (15) and the metabolites i d e n t i f i e d and quantitated as previously described (15). Direct Effect of Pb on Steroid Enzyme A c t i v i t y . Testes from four 35-day old rats were homogenized (Polytron with PT-IO generator, f o r 30 sec at 23,500 rpm) in homogenizing buffer (0.8 mM Na2HP04, 0.2 mM NaH2PO4, 250 mM sucrose, 1,0 mM EDTA, 5% g l y c e r o l ) . The homogenate was centrifuged at 2,500x g for I0 min and the supernatant was c e n t r i f u g e d 12,000xg f o r 15 min. The 12,000xg supernatant was used as the enzyme s o l u t i o n to measure the 3Bhydroxysteroid dehydrogenase (3~-HSD) a c t i v i t y . The enzyme a c t i v i t y was measured at 30°C in a temperature-controlled Unicam SPI800 spectrophotometer using semi-micro cuvettes with I0 mm l i g h t path as previously described (6). Sample cuvettes contained 800 ~I b u f f e r , 200 ~I methanol with 50 ~g of 38hydroxy-5B-androstan-17-one, I00 ~I enzyme s o l u t i o n and I00 ~I H20 with 1.0 mg B-nicotinamide adenine dinucleotide (NAD, Sigma). Blank cuvettes lacked s t e r o i d substrate. The b u f f e r consisted of T r i s (0.05 M; pH 8.0) with 0.001 M of e i t h e r PbCI2 or NaCI. To determine i f Pb has a d i r e c t i n h i b i t o r y e f f e c t on 3~-HSD a c t i v i t y , separate sets of cuvettes were prepared with b u f f e r containing e i t h e r PbCI2 or NaCI and the reaction was i n i t i a t e d with the addition of the enzyme. To determine i f Pb i n a c t i v a t e s 3B-HSD with time, the cuvettes containing a l l the components except NAD were incubated for 15 min at 30°C before i n i t i a t i n g the reaction with NAD. Statistical

comparisons were made by Student's t t e s t . RESULTS

Exposure of t e s t i c u l a r homogenate to lead f o r 1 hour did not reduce the FSH. receptor binding (Table I ) . A 4 hour exposure to lead by isolated S e r t o l i c e l l s s i m i l a r l y did not r e s u l t in altered FSH-receptor binding nor in FSH-induced c y c l i c AMP (Table 2). However, a 24-hour exposure to lead by S e r t o l i c e l l s TABLE I.

Effect of d i r e c t addition of Pb to t e s t i c u l a r homogenate on the FSH-receptor binding

Treatment Conditions I. 2. 3.

Homog. + 0.5 ml H20 Homog. + 0.5 ml H20 with NaAc Homog. + 0.5 ml H20 with PbAc

Specific Binding (cpm 1251-FSH bound) 15,449 ± 618 15,951 ± 875 16,111 ± 714

Four ml of t e s t i c u l a r homogenate from 25 day old rats was used for each incubation. Water (0.5 ml) or water containing NaAc or PbAc was added and the incubation occurred for 60 min at 34°C. The solutions were then centrifuged at 20,O00xg, each p e l l e t was washed 2X with c e n t r i f u g a t i o n at 20,O00xg and the f i n a l p e l l e t was resuspended in 2.5 ml Assay Buffer. For each FSH-receptor binding assay, 0.2 ml of suspended p e l l e t was used. The f i n a l concentration of NaAc and PbAc was 0.293 mM. Results are expressed as means (÷S.E.) of 4 r e p l i c a t e assays. E s s e n t i a l l y the same results were obtained with t e s t i c u l a r homogenates from rats aged 16, 20 or 65 days.

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TABLE 2.

Mechanisms of Action of Lead

2001

Effect of short term (4-24 hr) Pb exposure of S e r t o l i c e l l s on FSH-receptor binding and FSH-induced c y c l i c AMP.

Treatment

Time of Exposure (hr)

NaAc PbAc NaAc PbAc

4 4 24 24

FSH Bound (cpm/lOO~g) 8254 9102 8762 7930

± 870 ± 306 ± 442 ± 70

FSH-induced c y c l i c AMP (pmol/ml) 29.01 28.79 25.13 20.75

± ± ± ±

0.38 0.36 0.47 0.41"**

S e r t o l i c e l l s were obtained by our published procedure ( I I ) from 19-21 day old rats. The c e l l s were placed in petri dishes (about 1.6 x 106 c e l l s / d i s h ) and incubated with NaAc or PbAc (0.264mM) f o r 4 or 24 hr before assaying for FSH-receptor binding and c y c l i c AMP as described in Methods. Values represent mean (+ S.E.) of 3 or 4 r e p l i c a t e assays. The c y c l i c AMP is the net amount produced as a r e s u l t of incubation in the presence of lO~g FSH/ml; the amount in the presence of MIX alone has been subtracted. * * * S i g n i f i c a n t l y d i f f e r e n t from NaAc (24 hr) at p < O.OOl.

TABLE 3. Treatment NaAc PbAc

Effect of 6 day exposure to Pb on the FSH-receptor binding and the c y c l i c AMP of S e r t o l i c e l l s in c u l t u r e . FSH bound (cpm/lOOug) 8273 ± 192 1039 ± 343***

Cyclic AMP (MIX) (pmol/ml) 4.57 2.65

± 0.23 ± 0.47**

Cyclic AMP (MIX ÷ FSH) (pmol/ml) 384.00 8.99

± 53.03 ± 3.87***

S e r t o l i c e l l ~ from 16 day old rats were cultured f o r 6 days in the presence of 2.64 x ]O-"M NaAc or PbAc. Then FSH-receptor binding, c y c l i c AMP in presence of MIX and c y c l i c AMP due to FSH (MIX + FSH) were measured as outlined in Methods. Values are the means (± S.E.) of 3 observations. The experiment was repeated several times with S e r t o l i c e l l s from rats ranging in age from 16 to 21 days; e s s e n t i a l l y the same results were obtained in each experiment. * * S i g n i f i c a n t l y d i f f e r e n t from NaAc treatment at p < 0.01. * * * S i g n i f i c a n t l y d i f f e r e n t from NaAc treatment at p < 0.001.

resulted in a s i g n i f i c a n t reduction in FSH-induced c y c l i c AMP (Table 2), and a six day exposure (Table 3) markedly decreased the FSH-receptor binding (87% reduction), the basal c y c l i c AMP (42% reduction) and the FSH-induced c y c l i c AMP (97.6% reduction). The values in Table 3 are i n d i c a t i v e of the results obtained in several s i m i l a r experiments with S e r t o l i c e l l s from rats at several prepubertal ages. These results c l e a r l y show that prolonged exposure to lead results in decreased FSH-receptor binding and c y c l i c AMP and they prompted a more detailed i n v e s t i g a t i o n into the e f f e c t of time of exposure. Figure 1 shows that the i n h i b i t o r y e f f e c t s of lead on FSH-receptor binding and FSH stimulation of c y c l i c AMP are beginning to be apparent a f t e r a

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--'••.'•

FIGURE I. Effect of Pb exposure, as a function of time on the FSH-receptor binding ~ - Z ~ 1251-FS H BOUND and on FSH-induced c y c l i c AMP 8O -= FSH INDUCED cAMP of S e r t o l i c e l l s in c u l t u r e . 0 nS e r t o l i c e l l s were obtained Z 0 from f i f t y 19-21 day old 60 rats. Thirty-eight petri dishes were seeded with approx. 1.5xlO 6 c e l l s each. ~ 40 z The c e l l s were incubated with o 2.6xlO-~M of e i t h e r NaAc or PbAc f o r 4, 24, Pb AC ( c o n t r o l ) ~ 20 50, 96 or 144 hours and were then used to measure 1251-FSH binding (~--A) and FSH-induI I / I I I ,, I ced c y c l i c AMP (m---B). Each 0 24 48 72 96 120 144 point and v e r t i c a l l i n e repEXPOSURE TIME (HOURS) resents the mean (±S.E.) of 3 or 4 r e p l i c a t e determinations. The determinations were made on a per mg protein basis and the values in the f i g u r e are plotted as percent of control values (NaAc = 100%). 100

T

24-hour exposure and the i n h i b i t i o n increases markedly i f the in v i t r o exposure continues for 50 hours (60% i n h i b i t i o n ) , 96 hours (75% i n h i b i t i o n ) and 144 hours (81% i n h i b i t i o n ) . The S e r t o l i c e l l s used to obtain the results of Figure 1 came from 20 day old r a t s ; two separate experiments using S e r t o l i c e l l s from 16 day old (Table 3) and 17 day old rats (not shown) indicated that at the younger age, exposure to Pb f o r 6 days (144 hours) resulted in a 97% reduction (compared to NaAc treatment) in FSH-induced c y c l i c AMP. Figure 1 also demonstrates that the t i m e - r e l a t e d i n h i b i t o r y actions of lead on FSH-receptor binding and on hormone induced c y c l i c AMP are e s s e n t i a l l y identical. We have previously shown (6) that S e r t o l i c e l l s from 13 day old rats exposed to Pb p r e n a t a l l y and neonatally e x h i b i t reduced steroidogenic a c t i v i t y in c u l t u r e . However, no attempts had been made to determine i f the increases in steroidogenesis due to FSH which normally occur in young S e r t o l i c e l l s in c u l t u r e (I0) were also suppressed by the Pb treatments. Figure 2 shows that S e r t o l i c e l l s ~om 17 day old rats in c u l t u r e for 6 days exhibited a s i g n i f i c a n t increase in " " C - p r o g e s t e r o n e conversion as a r e s u l t of a 48 hour exposure to FSH (in the presence of NaAc). However, S e r t o l i c e l l s exposed to Pb f o r 48 hours have s i g n i f i c a n t l y less steroidogenic a c t i v i t y and FSH is not able to increase steroidogenesis in these c e l l s (Figure 2). Thus, a 2-day exposure to Pb resulted in a 37% reduction of steroidogenic a c t i v i t y in S e r t o l i c e l l s which were not treated with FSH, and a 66% reduction in c e l l s treated with FSH for 2 days (Figure 2). The steroidogenic enzyme, 3~HSD, present in t e s t i c u l a r microsomes, was assayed spectrophotometrically to see i f Pb has a d i r e c t e f f e c t on the enzyme activity. The 3~-HSD a c t i v i t y was immediately decreased by 26.3% i f the assay solution contained PbCI2 instead of an equimolar amount of NaCl (Table 4). Preincubation with PbCI2 for 15 min did not r e s u l t in f u r t h e r attenuation of the a c t i v i t y (24.7%; Table 4).

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Mechanisms

2°° I

4

m

TABLE 4.

Pb

C FSH

Pb FSH

Effect of Pb on 3#-HSD A c t i v i t y Enzyme A c t i v i t y No preincubation

NaCl PbCI

2003

FIGURE 2. Effect of in v i t r o Pb exposure on S e r t o l i c e l l steroidogenesis. S e r t o l i c e l l s were isolated from 17 day old rats and placed in culture f o r 4 days. On day 4 six p e t r i dishes received medium containing lead acetate (Pb; 300 ~g/ml) and six control (C) dishes received medium with equimolar amounts of sodium acetate. Three dishes of each group also received FSH (15ug NIH-S-19 dish). Two days l a t e r (day 6), the media were replaced with medium containing 14Cprogesterone (5xlO 5 DPM/dish) and incubation was continued for 7 hours. Cells and media were then extracted and metabolites i d e n t i f i e d and quantitated. The bars show the mean sum (~S.E.) in ng of the major metab o l i t e s (5~-pregnane-3,20-dione, 3~-hydroxy 5~-pregnan-20-one, 20~-dydroxy-4-pregnen-3one, and 3~-hydroxy-4-pregnen-20-one) formed from progesterone during the 7 hr incubation.

300

C

of Action of Lead

(ng/mg/min)

Preincubation (15 min)

369 • 27 272 ± 23*

365 ± 17 275 ± 25*

The 12,000 g supernatant from homogenized testes of 35 day old rats was used to measure 3B-HSD a c t i v i t y in the presence of e i t h e r NaCl or PbCI 2 as outlined in Methods. The a c t i v i t y was measured e i t h e r immediately a f t e r adding the enzyme (no preincubation) or a f t e r preincubating the enzyme preparation for 15 min with NaCI or PbCI 2 . The values are the means (±S.E.) of 3-4 r e p l i c a t e assays and represent the amount of 3B-hydroxy steroid converted to 3-keto steroid per mg protein per min. * S i g n i f i c a n t l y d i f f e r e n t from NaCI at p < 0.05.

DISCUSSION There is now good evidence that the S e r t o l i c e l l is a target for FSH (8, 16, 17). FSH binds to the plasma membranes of S e r t o l i c e l l s (8, 17), a c t i vates adenylate cyclase (17), increases c y c l i c ~,IP production (19, 17), protein kinase a c t i v i t y (17) and androgen binding protein (17) and stimulates steroidogenesis (I0) in Sertoli c e l l s from prepubertal rats. FSH also appears to play a role in i n i t i a t i n g spermatogenesis, probably by inducing S e r t o l i c e l l s to provide the appropriate milieu (18). The sequence of biochemical events between FSH stimulation of c y c l i c ~ P and the increases in steroidogenesis is not f u l l y understood, but appears to involve stimulation

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of RNA and protein synthesis (17). Therefore, any decrease in FSH binding and in steroidogenesis might be due to i n t e r f e r e n c e at one or several of the following s i t e s : FSH-receptor binding, receptor-adenylate cyclase coupling, i n h i b i t i o n or kinase a c t i v a t i o n of enzymes, synthesis of RNA, steroidogenic enzymes and/or FSH receptor proteins, or the positioning of the receptors w i t h i n the cell membrane s t r u c t u r e . Our previous i n v e s t i g a t i o n s (6) showed that in vivo administration of Pb results in decreased S e r t o l i c e l l steroidogenesis and FSH binding. The present results indicate that Pb does not act by a d i r e c t physicochemical interference in e i t h e r the binding of FSH to i t s receptors (Table I) or the a c t i v i t y of adenylate cyclase (Table 2). Instead of an immediate in v i t r o e f f e c t , the c e l l s show no change in FSH binding and c y c l i c AMP production a f t e r a 4 hour exposure to Pb (Figure I ) ; a f t e r 24 hours exposure to Pb a small i n h i b i t i o n in FSH binding and c y c l i c ~,IP has become evident and this i n h i b i t i o n has increased to about 80% f o l l o w i n g a 96 hour exposure to the metal (Figure I ) . The e f f e c t of Pb on hormone-receptor binding has been b r i e f l y examined with respect to e s t r a d i o l receptor a c t i v i t y in u t e r i of pregnant mice (19). These workers found a decrease in e s t r a d i o l binding when Pb was added in v i t r o to the cytosol. However, the reduced binding was an immediate e f f e c t and was considered to be due to c o p r e c i p i t a t i o n of Pb and receptor proteins. Our present r e s u l t s , showing a reduction in FSH binding and c y c l i c AMP synthesis only a f t e r many hours of exposure to Pb, suggest that at these sites Pb is not acting by a p r e c i p i t a t i o n reaction or a d i r e c t interference but probably by i n h i b i t i n g the synthesis of s p e c i f i c proteins. However, the immediate in v i t r o reduction in 3~-HSD a c t i v i t y due to Pb (Table 4) suggests that this ~-eta--~ may also i n t e r f e r e d i r e c t l y with the a c t i v i t y of enzymes already present in the c e l l s . I t may be that in vivo, Pb causes a marked decrease in steroidogenesis because both mechanisms are involved, i . e . i n h i b i t i o n of steroid enzyme synthesis and a c t i v i t y . Our previous results (6) showed that the 3~-HSD a c t i v i t y is reduced by about 50% when animals are exposed to Pb for a number of days, whereas the present results (Table 4) show that d i r e c t in v i t r o addition of Pb to the enzyme reduces the a c t i v i t y only by about 25%. The additional reduction in steroidogenic a c t i v i t y which is seen with long in vivo exposure to Pb, could then be explained on the basis of reduced steroid enzyme synthesis. The e f f e c t s of Pb on c y c l i c AMP production have been examined in several tissues and the results range from s i g n i f i c a n t stimulation to marked suppression, depending upon the t i s s u e , the mode of Pb administration ( i n vivo or in v i t r o ) and on the time of exposure to the metal (20). To our knowledge, ours is the f i r s t study which attempts to examine the mechanism of action of Pb by c o r r e l a t i n g the e f f e c t s on the following sequence: hormone binding, hormone-stimulated c y c l i c AMP, and hormone stimulated response (steroidogenesis). I t appears from these in v i t r o experiments and our previous in vivo r e s u l t s (6) that Pb may act-on t e s t i c u l a r c e l l s by i n h i b i t ing the a c t i v i t y of steroidogenic enzymes and the synthesis of enzymes and hormone receptor proteins. The net r e s u l t is a decrease in gonadotropin binding, c y c l i c AI,IP and steroidogenic enzyme a c t i v i t i e s . Since these a c t i v i t i e s are cardinal f o r the proper development and functioning of the t e s t i s , t h e i r suppression by Pb is expected to r e s u l t in the reduced ~eproductive functions observed a f t e r exposure to this metal. The present results thus provide the f i r s t evidence of the biochemical events which appear to be affected by Pb in the t e s t i s .

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Mechanisms of Action of Lead

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ACKNOWLEDGEMENTS This work was supported by N.S.E.R.C. of Canada Strategic Grant G0538. We are grateful f o r the technical assistance of K. Buckingham and K. Barr and thank C. Cesarini f o r assistance in the preparation of the manuscript. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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