Inhibition by 5-thio-d -glucopyranose of protein biosynthesis in vitro in spermatids from RAT testis

474

Biochimica et Biophysica Acta, 447 (1976) 474--483 © Elsevier/North-Holland Biomedical Press

BBA 98729 INHIBITION BY 5-THIO-D-GLUCOPYRANOSE OF P R O T E I N BIOSYNTHESIS IN VITRO IN SPERMATIDS F R O M R A T TESTIS

MASAHISA NAKAMURA and PETER F. HALL Department of Physiology, California College of Medicine, University of California, Irvine, Calif. 9271 7 (U,S.A.)

(Received March 1st, 1976)

Summary The effect of 5-thio-D-glucopyranose (thioglucose) upon protein biosynthesis in vitro was examined in testes from mature rats. Thioglucose in vitro is without demonstrable effect upon incorporation of L-[ U-14C]phenylalanine into protein by whole testis b u t inhibits this incorporation by a purified fraction of immature spermatids (stages 1--8) prepared by centrifugal elutriation; the inhibition is observed with or w i t h o u t glucose added in vitro and is concentration dependent in the range 1--50 mM. Similar inhibition is observed with three other 14C-labeled amino acids (leucine, lysine and glutamate), Mature spermatids (>stage 8) and other heterogeneous fractions of testicular cells prepared by the same m e t h o d also show inhibition by thioglucose of incorporation of phenylalanine into protein so that it is not possible to say that the effect is confined to spermatids although it is most pronounced in these cells, Inhibition of protein synthesis in vitro is also observed when thioglucose was administered in vivo (33 mg/kg b o d y wt./day). This change occurs at the minimal dose observed by other workers to produce arrest of spermatogenesis and hence infertility.

Introduction The substance 5-thio-D-glucopyranose (thioglucose) has recently attracted attention as a possible male contraceptive [ 1 ]. The c o m p o u n d causes regression of the germinal epithelium in the mouse [2] and it has been proposed that it inhibits glucose metabolism [3]. Since the testis is known to depend heavily upon glucose as a metabolic substrate [4,5], it is suggested that low doses of thioglucose may inhibit metabolism of the testis without significant effects upon most mammalian cells which are believed to be less dependent upon glucose as a source of energy. One important effect of glucose on testicular metab.olism is that the hexose stimulates protein biosynthesis by two distinct mecha-

475 nisms, namely increased amino acid transport and some subsequent action on intracellular amino acid [6]. These responses appear to result from increased production of ATP in the presence of glucose [7]. It is believed that the effects of glucose are largely, but not entirely, exerted upon spermatids although evidence on this point is indirect [8,9]. It was decided to determine whether thioglucose affects protein biosynthesis by spermatids from rat testis in vitro. Materials and Methods

Animals Rats of the Wistar strain aged 50--60 days were used in these studies. The animals were fed ad libitum before the experiments. Rats were decapitated and the testes removed and placed on ice. Preparation of tissue (a) Whole testis. The tunica was removed from each testis and the tubules were gently separated. The tissue was then cut into fragments and incubated as described below (100 mg/flask). Whole testis was kept on ice for 2 h to make results strictly comparable with the studies using testicular cells which require 2 h to prepare (see below). However, previous studies have shown that 2 h on ice makes only a small difference in testicular protein synthesis. (b) Spermatid and other cell fractions. Cells were separated by centrifugal elutriation as described by Grabske et al. [10] (see below). Most studies used immature spermatids (stages 1--8) and these cells will be referred to by that name. Immature spermatids were collected from centrifugation in our fraction 3 (Sm~ 6.2--7.2) [10]. Mature (>stage 8) spermatids (fraction 2; Smax 5--6.2) were used in some studies. Other studies used the following fractions: fraction 4 (Sm~ 7.2--10.8) consisting mostly of spermatocytes with Leydig cells, Sertoli cells, and some immature spermatids; fraction 5 (Smax 10.8--18.5) containing pachytene cells, multinucleated spermatids and some Sertoli cells; fraction 6 (Smax > 18.5), containing some spermatids, multinucleatid cells, elongated spermatozoa and unseparated cells. Our fractions can be compared with those of Grabske et al. [10] on the basis of values for S~,ax but not by fraction number because their studies used somewhat different conditions and were performed on mouse testis. To prepare the cells, testes were lightly dissected by separating tubules and incubated in a buffered saline medium prepared as described by Grabske et al. [10] except that glucose was omitted. To this medium coUagenase (0.05%. w/v) was added. Incubation was performed at 28°C for 40 min in a metabolic shaker (75 oscillations/min); 40 ml of buffer was used with 4 testes from 2 rats (approx. 6 g tissue). The tissue was then filtered through nylon mesh (61 pm gauge) and the cells which passed through the mesh were subjected to centrifugal elutriation as described by Grabske et al. [10]. Cells harvested in various fractions from centrifugal elutriation were suspended in 200 ml of buffered saline (as above) and centrifuged at 200 × g for 7 min. The pellet was resuspended in 20 ml of buffer and transferred to incubation flasks by means of a pipette. The entire preparation from removal of testis to the beginning of incubation required 2 h.

476

Number, Mentity and condition o f cells Cells were counted in a h e m o c y t o m e t e r and protein was determined separately on aliquots from each flask by the m e t h o d of Lowry et al. [11]. It was discovered that relative values for incorporation of amino acids into protein were not significantly different when expressed according to cell number or to protein/flask; in the accompanying studies values are expressed per mg protein. Each flask contained approx. 800 000 cells (100 pg protein). Spermatids prepared by this m e t h o d exclude trypan blue (>98%), consume oxygen and produce lactate (Nakamura, M., Romrell, L.J. and Hall, P.F., unpublished) and incorporate amino acids into protein (see below). Samples of each preparation of cells were examined by phase-contrast microscopy; other samples were stained by the periodic acid/Schiff, counterstained with hematoxylin and the percentage of cells which excluded trypan blue was determined with each preparation. Each gradient was loaded with 1.2 × l 0 s cells (2 × 107 immature spermatids) and the final yield of immature spermatids was 9 × 106 cells (45%) which provided sufficient cells for 10 flasks.

Epididymal sperm Rats were decapitated and epididymes were removed, cut open and washed with the buffer used for preparation of spermatids. The buffer was delivered from a syringe with a 22 gauge needle. Buffer was collected and cells were incubated w i t h o u t further treatment. The cells were collected in this manner from both caput and cauda of the epididymis. Microscopic examination of cell fractions revealed typical epididymal sperm without significant amounts of free cytoplasmic droplets.

Incu ba tion Cell suspensions were pipetted into 25-ml Erlenmeyer flasks on ice, containing buffer and the various additions except radioactively labeled amino acids. Cells were then preincubated for 10 min at 34°C. Incubation was started by addition of radioactive amino acid and continued for 60 min in a final volume of 3 ml at 75 oscillations/min and 34°C. It was observed that incorporation of amino acids into testicular proteins was linear for more than 60 min. The standard incubation involved the use of L-[U-laC]phenylalanine (1 pCi:2.5 nmol/flask). Incubation was stopped by adding thrichloroacetic acid to a final concentration of 10%.

Administration o f thioglucose Thioglucose was dissolved in drinking water and control animals were given the same dose of D-glucose by the same route. These compounds were administered on two consecutive days; rats were killed on the third day. Spermatids were prepared as described above and incubated with L-[U-l~C]phenylalanine.

Miscellaneous Incorporation of amino acids into protein was measured as described previously [ 12]. In preliminary experiments, a number of features of the incorporation of [14C]phenylalanine into trichloroacetic acid-precipitable material were examined to determine whether this incorporation involves peptide bond for-

477

mation. The relevant methods have been reported elsewhere [12]. Oxygen consumption was determined polarographically using a Clarke electrode (Gilson) [13]. Lactate was measured by a method based upon reduction of NAD ÷ using lactate dehydrogenase [14]. Liquid scintillation spectrometry was performed as described elsewhere [12]. Efficiency for counting ~4C was 82%. An experiment refers to studies performed on one day with pooled testicular cells from four testes (2 rats). No comparisons are made with other preparations of cells. In every experiment each condition was studied in duplicate, i.e. four experiments means two determinations on each of four preparations of cells.

Materials Amino acids were obtained as follows: L-[U-]4C]phenylalanine (Lot No.: 892-013; 418 Ci/mol) and L-[U-~4C]leucine (Lot No.: 892-125; 325 Ci/mol) were purchased from New England Nuclear; L-[U-J4C]lysine (Lot No.: 1907191Y; 240 Ci/mol) was obtained from International Chemical and Nuclear Corporation, City of Industry, California and L-[U-~4C]glutamic acid (Lot No.: ZR-1344; 260 Ci/mol) from Schwarz-Mann. Before use in the present studies, ~4C-labeled amino acids were purified by two systems of paper chromatography [15]. Thioglucose (5-thio-D-glucose) was of A.R. grade and was purchased from Pfanstiehl Laboratories, Inc., Waukegan, Illinois~ Dithiothreitol and 2mercaptoethanol were obtained from Sigma Chemical Corporation. Nylon mesh was obtained from Corth Plastics. The elutriation rotor (JE-6) is manufactured by Beckman Instruments, Inc., Palo Alto, California and centrifugation was performed in a refrigerated centrifuge (J-21B) made by the same company. Collagenase (Type IV) was obtained from Worthington Biochemical Corp., Freehold, N.J. Results

Effect of thioglucose on incorporation of [~4C]phenylalanine into testicular proteins (i) Whole testis. Thioglucose (20 raM) was without detectable effect on incorporation of phenylalanine into protein by whole testis whether glucose was present in the medium or not (data not shown). (ii) Immature spermatids. Table I shows the results of six experiments in which incorporation of [l~C]phenylalanine into protein by immature spermatids was measured. Glucose stimulated incorporation and thioglucose inhibits the response to glucose and caused some inhibition in the absence of added glucose (P ~ 0.02). Intense inhibition caused by puromycin (0.17 raM) in the presence of glucose is also seen. The effect of various concentrations of thioglucose with a fixed concentration of glucose (10 mM) is seen in Fig. 1. Inhibition is seen both with and without glucose and in both cases significant inhibition is observed with 20 mM thioglucose (P ~ 0.02 for both cases). The effect of thioglucose (20 mM) with various concentrations of glucose (0.5--20 raM) upon incorporation o f [ 14C]phenylalanine into protein by immature spermatids is shown in Fig. 2. A curvilinear relationship is observed between incorporation of phenylalanine and concentration of glucose; inhibition

478 TABLE I INCORPORATION OF FROM RAT TESTES

[14C]PHENYLALANINE

INTO

PROTEIN

BY I M M A T U R E S P E R M A T I D S

I m m a t u r e s p e r m a t i d s w e r e i n c u b a t e d w i t h L - [ U - 1 4 C ] p h e n y l a l a n i n e a n d t h e a d d i t i o n s s h o w n for 60 rain. S a m p l e s w e r e s u b j e c t e d to p r e c i p i t a t i o n w i t h t r i c h l o r o a c e t i e acid a n d i n c o r p o r a t i o n o f [ 1 4 C ] p h e n . y l a l a n i n c i n t o p r o t e i n w a s m e a s u r e d b y liquid scintillation s p e c t r o m e t r y . E a c h v a l u e r e p r e s e n t s the m e a n s +- S,E.M. f r o m 6 d i f f e r e n t e x p e r i m e n t s . [U-14C] L-phenylalanine incorporation (cpm/mg protein)

Addition

Saline Glucose (10 mM) T h i g l u c o s e ( 2 0 raM) G l u c o s e ( 1 0 m M ) + t h i o g l u c o s e (20 m M ) G l u c o s e ( 1 0 m M ) + p u r o m y c i n ( 0 . 1 7 raM)

1768 2838 * 1420 ** 1 9 3 8 127

+ 84 -+ 171 + 61 -+ 97 -+ 8

* P < 0 . 0 2 f o r p a i r e d c o m p a r i s o n s w i t h saline. ** P < 0.01 f o r p a i r e d c o m p a r i s o n s w i t h glucose ( 1 0 m M ) .

by thioglucose is seen at all concentrations tested. (iii) Other cell fractions. Similar studies revealed that with mature (>stage 8) spermatids (fraction 2), incorporation of phenylalanine in the presence of glucose is inhibited by thioglucose (20 mM) but not in the absence of glucose (P > 0.2) (Table II). With fraction 4 some inhibition is seen both with and without glucose (Table III). The same is true for fraction 6; in the case of fraction 5 in-

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479

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T A B L E II INCORPORATION OF L-[U-14C]PHENYLALANINE FROM RAT TESTES

INTO PROTEIN

BY MATURE

SPERMATIDS

E x p e r i m e n t s w e r e p e r f o r m e d a s d e s c r i b e d u n d e r T a b l e I. D a t a are r e p o r t e d as m e a n ± S.E.M. f o r 3 different experiments, Addition

Incorporation of L-[U-14C] phenylalanine (cpm/mg protein)

Saline Glucose (10 raM) Thioglucose (20 mM) Glucose (10 mM) + thioglucose (20 raM)

1447 2600 * 1306 ** 2 1 1 9

+ 92 + 149 ± 139 +- 1 8 2

* P ~ 0 . 2 , p a i r e d c o m p a r i s o n s w i t h saline. ** P ~ 0 . 0 5 , p a i r e d c o m p a r i s o n s w i t h g l u c o s e ( 1 0 m M ) .

T A B L E III INCORPORATION

OF L-[U-14C]PHENYLALANINE

I N T O P R O T E I N BY C E L L S F R O M R A T T E S T E S

F o r e x p e r i m e n t a l details, see T a b l e I. Addition

Saline Glucose (10 raM) Thioglucose (20 raM) Glucose (10 mM) + thioglucose (20 mM)

I n c o r p o r a t i o n o f [ 14 C] p h e n y l a l a n i n e (cpm/mg protein) Fraction 4

Fraction 5

Fraction 6

1096 1529 752 1103

386 599 327 595

1283 1976 984 1369

480 T A B L E IV I N F L U E N C E O F T H I O G L U C O S E IN V I V O U P O N I N C O R P O R A T I O N N I N E I N T O P R O T E I N BY I M M A T U R E R A T S P E R M A T I D S I N V I T R O

OF L - [ U - 1 4 C ] P H E N Y L A L A -

T h i o g l u c o s e (33 m g / k g b o d y w e i g h t / d a y ) , or glucose in t h e case of c o n t r o l , w e r e a d m i n i s t e r e d for t w o d a y s and r a t s w e r e killed o n t h e t h i r d d a y . I m m a t u r e s p e r m a t i d s w e r e p r e p a r e d b y the m e t h o d o f centrifugal c l u t r i a t i o n (see M e t h o d s a n d M a t e r i a l s ) a n d i n c u b a t e d w i t h o u t glucose in v i t r o . I n c o r p o r a t i o n of [ 1 4 C ] p h e n y l a l a n i n e w a s m e a s u r e d as d e s c r i b e d u n d e r M e t h o d s a n d Materials. Values s h o w m e a n s a n d s t a n d a r d e r r o r s f o r t h r e e o b s e r v a t i o n s o n t e s t e s f r o m e a c h o f f o u r r a t s in the t w o g r o u p s (4 c o n t r o l ; 4 treated). I n c o r p o r a t i o n of [ 14C] p h e n y l a l a n i n e (cpm/mg protein) Control Thioglucose

1 8 2 5 -+ 131 1 5 8 4 + 88 (P < 0 . 0 1 )

hibition does not occur or is extremely small (Table III). It will be seen that glucose stimulates incorporation of phenylalanine in all these fractions.

Effect of thioglucose on incorporation of various 14C-labeled amino acids into protein by immature spermatids Thioglucose caused similar inhibition of incorporation of a number of amino acids into protein by immature spermatids, namely [14C]leucine, [~4C]lysine and [14C]glutamate (data not shown). Effect of thioglucose in vivo Spermatids were prepared from rats receiving thioglucose (treated) or glucose (control) in drinking water for two days (33 mg/kg body wt./day). The animals were killed at the end of 48 h after the first exposure to these agents. It can be seen from Table IV that incorporation of [ 14C]phenylalanine into spermatid protein is inhibited by treatment with thioglucose. Protein synthesis by epididymal sperm Incorporation of L-[U-~4C]phenylalanine into protein by epididymal sperm was neither stimulated by glucose nor inhibited by thioglucose. Protein synthesis by spermatocytes A fraction enriched in spermatocytes was used to study the effect of thioglucose in vitro on protein synthesis by these cells. Thioglucose was without significant effect on protein synthesis by these cells (data not shown). Studies with other thiols Two other thiol compounds (dithiothreitol and 2-mercaptoethanol) were without effect upon incorporation of [ 14C]phenylalanine into protein by immature spermatids in the dose range of 5--20 mM {data not shown). Discussion

The present studies reveal that spermatids, both immature and mature, show increased incorporation of amino acids into trichloroacetic acid-precipitable

481

material when glucose is added to the incubation medium. Since this incorporation shows features which indicate that the radioactive amino acids become involved in peptide bonds (see Methods and Materials), incorporation of amino acids in this system will be referred to as protein synthesis although the enhanced incorporation brought about by glucose i s n o t necessarily caused b y increased rate of synthesis of peptides. These findings could have been' predicted on the basis of earlier work with the whole testis [7,8,16] but constitute the first direct demonstration of this response in spermatids. Moreover, protein biosynthesis by immature spermatids is inhibited by thioglucose whether glucose is present or not. It cannot be determined from these studies whether thioglucose produces inhibition by the same mechanism with and without glucose; preincubation for ten minutes may not entirely deplete endogenous substrates. It is of interest to notice that thioglucose, in the doses used here, does not inhibit protein synthesis in whole testis. Interest in thioglucose lies in the possibility that it inhibits synthesis of a limited number of proteins by a limited number of testicular cells. If thioglucose does have such a selective action, fractions of testicular cells would provide a way of revealing this action which would be difficult to demonstrate in whole testis. Since it appears that thioglucose inhibits transport and subsequent metabolism of glucose [3,17] and in view of the dependence of the metabolism of spermatids on glucose [7--9], it would not be surprising if the effect of the inhibitor on protein synthesis results from these effects on glucose metabolism. However, inhibition by thioglucose was not overcome by high concentrations of glucose (Fig. 2) so that the drug may not act by simple competition with that hexose. It is not possible to conclude that the action of thioglucose is: confined to spermatids because the other cell fractions all contain some spermatids in one form or another and in any case the striking dependence upon glucose for protein synthesis in whole testis does not appear to be entirely confined to spermatids [8] (see Results). The effective dose range of thioglucose is also. of considerable interest. In vivo the minimal effective dose o f this agent is 33 mg/kg body weight/day in the mouse [2]; this dose inhibits fertility after continuous use for a period of several weeks [2]. After maintenance of rats on this dose for two days, small but significant inhibition of amino acid incorporation by spermatids in vitro was observed (Table IV). If this dose were completely absorbed from the gut and uniformly distributed throughout body water, it would provide a concentration of approx. 0.275 mM. The smallest effective dose in vitro under the conditions used here was 20 mM. Clearly conditions in vitro cannot be used to determine dosage of the drug in vivo, a state of affairs which is well known with other drugs, Moreover, the present studies do not exclude effects of thioglucose in vivo exerted outside the testis, e.g. on the pituitary. However, no evidence of extratesticular effects were observed by Zysk et al. [2] with prolonged administration of thioglucose. T h e fact that inhibition of incorporation of amino acids b y thioglucose reaches a maximum at about 25--45% of uninhibited incorporation (i,e. decrease to values of 75--55% of control values) (Fig. 2) is also significant. Either the inhibitor affects a particular population within ourspermatid fraction aad

482 affects this population severely or it affects all spermatids to a limited degree. We believe that the first explanation is unlikely. Although spermatids are found in other fractions of testicular cells and these fractions also show inhibition by thioglucose, we do not find immature spermatids in significant numbers in these fractions. It is likely then that thioglucose produces only limited inhibition of protein synthesis in spermatids. It will be of interest to determine whether the proteins affected in this way are specific and can be identified, e.g. by electrophoresis on polyacrylamide gel. It is conceivable that thioglucose inhibits maturation of spermatids by preventing the synthesis of certain proteins required for differentiation. It is worth noticing that stimulation of protein synthesis produced in the testis by glucose, does not increase synthesis of specific proteins [18]; there is, of course, no reason why the same need apply to inhibition by thioglucose. If the action of thioglucose does involve a limited number of proteins, this effect may be largely confined to the testis in which case thioglucose could prove to be an effective contraceptive, although it has been pointed out that the influence of this substance upon protein synthesis by other organs, especially those heavily dependent upon glucose, e.g. brain, must be carefully examined [19]. In any case the system described here may prove valuable as a means of studying such agents since the effects of inhibitors can be examined in vitro with the inhibitor administered in vivo or added in vitro to the cells. The mechanism by which thioglucose inhibits incorporation of amino acids into protein is at present under investigation in this laboratory.

Acknowledgments Supported by the National Science Foundation, grant No. BG43493. The au. thors would like to express their gratitude to Dr. Barton L. Gledhill and his colleagues at the Lawrence Livermore Laboratory for invaluable assistance in the use of centrifugal elutriation. Dr. Roger Howard of the Walter Rees Hospital, Chicago, gave generous advice and instruction in methods for preparing testicular cells from the whole organ. References

1 2 3 4 5 6 7 8 9 1O 11 12 13 14

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483 15 Block, R.J., Durrum, E.L. and Zweig, G. (1955) A Manual of Paper Chromatography and Paper Electrophoresis, pp. 75--113, Academic Press, New York 16 Davis, J.R. and Morris, R.N. (1963) Am. J. Physiol. 205, 833--836 17 Whistler, R.L. and Lake, W.C. (1972) Bioehem. J. 130, 919---925 18 Hollhiger, M.A. and Hwang0 F. (1972) Biochim. Biophys. Acta 281,652---657 19 Kalat, J.W. (1975) Science 186, 1074