113

Biochimica et Biophysica Acta, 588 ( 1 9 7 9 ) 1 1 3 - - 1 1 9 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 29097

SELENOPROTEINS FROM RAT TESTIS CYTOSOL

K E N N E T H P. M c C O N N E L L *, R O B E R T M. B U R T O N , T I M O T H Y K U T E ** a n d P A T R I C K J. H I G G I N S

The Veterans Administration Hospital, 800 Zorn Avenue, and Department of Obstetrics and Gynecology, and Department of Biochemistry, University of Louisville School of Medicine, Health Sciences Center, Louisville, K Y 40232 (U.S.A.) ( R e c e i v e d M a r c h 23rd, 1 9 7 9 )

Key words: Selenoprotein; Spermatogenesis; (Spermatozoa, Testes, Epididymidis)

Summary Radioactive inorganic selenium, administered intrapefitoneally at 1 mg/kg body weight to young adult rats, accumulates in testes for 7 days or longer, whereas liver, kidney and serum levels fall more rapidly. 3--4 h after administration of [TSSe]selenite, 55--60% of the radioactivity in the testes was found in the cytosol, associated with protein. Ultragel ACA-22 chromatography of testis cytosol prepared 4 h after 7SSe treatment revealed a major selenoprotein having an apparent molecular weight of 59 000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated extensive heterogeneity of radioactivity with apparent molecular weights of about 57 000 and 45 000 and 15 000. Cytosol from rats treated 4 weeks earlier showed predominance of the 15 000 molecular weight [ 7sSe] selenoprotein. Sucrose density gradient ultracentrifugation at either low or high ionic strength demonstrated a single 7 S selenoprotein. Chromatography with Blue-Sepharose indicated that the radioactivity was not associated with albumin. Strong 7SSe binding to protein was demonstrated by overnight dialysis against water, 2 M NaC1, ~-mercaptoethanol~ 8 M urea, selenite. However, 85% of the 7SSe was removed by dialysis against 0.5 M NaOH. This stability contrasts with the lability of disulfide reagents of selenite-protein complexes formed in vitro. The fact that selenium is incorporated in substantial amounts into a discrete and stable protein suggests a physiological role for this essential trace element in the testes.

* Present address: D e p a r t m e n t of Biochemistry, Health Sciences Center, P.O. Box 35260, Louisville, KY 40232, U,S.A. ** Present address: D e p a r t m e n t of Medicine, Bowman-Gray Medics/ Center, Wake Forest University, Winston-Salem, NC 27103, U.S.A,

114 Introduction In studies on the distribution of selenium in reproductive organs, Rosenfeld [1] found that after repeated administration of trace dose of 7SSe to rats, the testes contained the highest concentration of selenium of any organ except the kidneys. Later, Gunn et al. [2] reported that, after a single subcutaneous injection of a tracer dose of 7SSe to mice, the testes, which ranked low in 7SSe uptake at 1 h, continued to accumulate 7SSe, whereas all other tissues tested showed diminishing levels; by 7 days the male gonads ranked third to the liver and kidney in 75Se concentration. Burk et al. [3] showed that in profound selenium deficiency, small doses of selenite were preferentially taken up by the testes and thymus. Chen et al. [ 4] reported a selenoprotein in the rat testicular soluble fraction which was sensitive to cadmium exposure, having an Mr of about 115 000. Prohaska et al. [5] in studies of cadmium-selenium interactions, have separated glutathione peroxidase activity from the major testicular selenoprotein(s) by gel filtration. The present study was designed to investigate the properties of the testicular soluble fraction selenoprotein which was labeled after the injection of trace amounts of 7SSe to rats on an adequate selenium diet. Methods and Procedures Male rats of the Sprague Dawley strain were obtained from Laboratory Supply (Indianapolis, IN). They were maintained on Purina Laboratory Chow No. 5001 (0.36 ppm Se according to manufacturer) and ranged in weight from 105--175 g (age, 6--8 weeks). Rat livers, testes and epididymides were sliced into sections and placed in twice their weight of 10 mM Tris-HC1 buffer (pH 7.4)/1.5 mM EDTA/10 mM ~-mercaptoethanol. The cytosol was prepared by centrifugation at 105 000 × g for 30 min. Chemicals for polyacrylamide ge1 electrophoresis were purchased from BioRad, while standard proteins (bovine serum albumin, ovalbumin, ribonuclease A, ~-gaiactosidase) dithioerythritol, chymotrypsinogen A and buffers were obtained from Sigma Chemical Company (St. Louis, MO). Glucose oxidase was purchased from Boehringer-Mannheim (Indianapolis, IN). Human gamma globulin (Cohn fraction II) was obtained from Miles Laboratories, (Kankakee, IL), and Ultragel ACA 22 from LKB Instruments, (Rockville, MD). [TSSe]Selenious acid (42--97 Ci/g) was a product of the International Chemical and Nuclear Corporation. All other chemicals were of reagent grade or equivaient. Deionized, distilled water was used throughout. Sucrose gradient ultracentrifugation was performed as described earlier [6]. Before SDS-polyacrylamide gel electrophoresis [7], the samples were incubated at 65°C for 35 min in 0.50% sodium dodecyl sulfate (SDS)/20 mM dithioerythritol/0.50 mM EDTA/8 mM glycine/10 mM Tris-HC1 (pH 8.0). Solid sucrose was added to 10--20 pl samples of cytosol; these were applied to gels containing 0.25% SDS and were electrophoresed at room temperature at a constant current of 4 mA per tube. Buffer containing 0.25% sodium dodecyl sulfate (SDS) was employed. The gels were frozen immediately and sliced into

115 2-mm sections for radioactivity determinations. 7SSe was determined on a Packard Gamma Counter 3011 with an efficiency of 45%. Protein concentrations were determined by the method of Lowry et al. [8] using crystallized human serum albumin as standard. Results Following intraperitoneal administration of 7SSeO]-(1 mg/kg) to young adult rats, radioactivity accumulates in the testes for 7 days or longer, whereas liver, kidney and serum levels fall rapidly (Fig. 1). 3 h after administration of [TSSe]selenite, 50--60% of the 75Se radioactivity in the testes was found in the cytosol fraction, predominantly in protein. Time~listribution studies of the uptake of 7SSe into the testes cytosol fraction showed a rapid uptake of 7SSe during the first day and then a gradual further increase up to 7 days. Gel filtration on ACA-22 (LKB) of testis cytosol after 4 h treatment revealed an excluded high molecular weight fraction and a major selenoprotein having an apparent molecular weight of 59 000 (Fig. 2). SDS-polyacrylamide gel electrophoresis indicated heterogeneity of radioactivity but showed a major selenoprotein with apparent molecular weight of 62 000 as well as lower molecular weight components. Further experiments comparing in vivo labeling periods up to 1 week (Fig. 3) showed that the selenoprotein in the rat testis cytosol consisted of three electrophoretic species: a slow, an intermediate and a fast component with mean molecular weights of 57 000 ± 900 (S.E.) 45 600 ± 700 and 14 700 ± 200, respectively. The approximately 60 000 and 45 000 molecular weight labeled components predominate at the shorter labeling times, i.e., at 1 h, 4 h and 1 day; however, an increase in the low molecular weight species is

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HOURS F i g . 1. A c c u m u l a t i o n of injected 75Se in rat t e s t e s . Radioactivity expressed i n p e r c e n t o f d o s e p e r g w e t

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Fig. 2. Gel filtration o f testicular c y t o s o l o b t a i n e d 7 d a y s after i n j e c t i o n o f [ T S S e ] s e l e n i t e o n L K B A C A 22 Ultragel c o l u m n ( 1 . 5 X 6 3 c m , V 0 = 31 m l ) at 4 ° C . 1 m l o f c y t o s o l w a s applied.

evident at 1 week and correspondingly, the proportion of larger selenoproteins is less at the later times. Using gel filtration under non-dissociative conditions, this molecular size shift is also evident (compare Fig. 4 and Fig. 2). There was no significant difference of apparent molecular weights within a size category, e.g., for the approx. 15 000 dalton species as a function of time after injection. 7SSe-labeled cytosol obtained from rat testis 3 h after injection was applied to a 10--35% sucrose density gradient for 16 h at 369 000 × g [6]. A single peak migrating at approximately 7.0 S obtained. The sedimentation was not altered by having 0.4 KC1 in the prepared gradients. Chromatography of that

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testis cytosol on Blue-Sepharose showed an almost quantitative recovery (95%) of radioactivity in the first fraction. Results indicate that the selenoprotein in rat testis cytosol is not associated with albumin. Strong 7SSe binding to protein in the cytosol fraction was demonstrated by overnight dialysis at 4°C (Table I) against water (91% retention of label), 2 M NaC1 (93%), 0.5 mM mercaptoethanol (76%), 8 M urea {75%) and 0.1 M selenite (87%). However, 85% of the 7SSe was removed by dialysis against 0.5 M NaOH. This stability to disulfide reagents contrasts with the lability of selenite-protein complexes formed in TABLE I STABILITY OF TESTES CYTOSOL AGENTS AND DISULFIDE REAGENTS

SELENOPROTEiN

TO

DENATURANTS,

HYDROLYTIC

I rrfl testis c y t o s o l f r o m rats t r e a t e d w i t h [ 7 $ S e ] - s e l e n t t e f o r 4 h was d i a l y z e d in w a s h e d Visking t u b i n g [ 9 ] against 1 1 o f t h e i n d i c a t e d s o l u t i o n s o v e r n i g h t at 4°C. T h e c o n t e n t s o f t h e bags w e r e r e m o v e d and counted. P e r c e n t of 7SSe a f t e r dialysis for:

Qontrois water 2 M NaC1 Denaturants 8 M urea 1 M a c e t i c acid Hydrolytic agents 0.1 M HC1 0.5 M N a O H Disulfide r e a g e n t s fl-mercaptoethanol 0.1 M s o d i u m sulfite 0.1 M s o d i u m selenite 8 M urea + 0.5 M ~-mercaptoethanol

24 h

48 h

84 85

84 87

45 84

33 84

82 17

81 10

72 65 84 77

71 58 74 77

118

TABLE II SELENIUM

CONTENT

OF TISSUES

Values are based on neutron are i n d i c a t e d in p a r e n t h e s e s .

Liver (14) Kidney (14) Testes (14 pairs) E p i d i d y m i s ( 1 3 pairs)

a c t i v a t i o n a n a l y s i s [ 1 1 ] a n d a r e m e a n s +-S.E. N u m b e r s o f s a m p l e s a v e r a g e d

#g Se/g dry wt.

#g Se/organ

2.46 3.75 3.68 0.74

6.23 1.62 1.14 0.14

-+ 0 . 8 + 0.09 + 0.21 -+ 0 . 2 2

+ 0.44 +- 0 . 0 6 + 0.05 _+ 0 . 0 5

vitro [10]. Selenium content of some organs of interest was established by neutron activation analysis (Table II). Expressed per unit weight, the selenium of the testis is as concentrated as in the kidney and more so than in the liver and epididymis. On the basis of pg Se/per organ, the liver and kidney contained more than the testis. Epididymides from rats 4 weeks after injection of labeled selenite were found to possess as much radioactivity as the testes, in agreement with studies of Gunn [2]. About one-third of that radioactivity could be recovered in the form of a suspension o f spermatozoa which was obtained by slitting the intact organs and expressing epididymal ducts with tweezers. SDS-polyacrylamide gel electrophoresis of 4-week epididymal cytosol shows a preponderance of the 15 000 molecular weight species, as seen in the testis cytosol at one week (Fig. 3). Discussion

The results of this investigation are part of a growing body of evidence on a biological role for selenoproteins in the testis. In particular, our data confirm work of Gunn et al. [ 2] showing the kinetics of uptake and retention of selenium to parallel spermatogenesis. Accordingly, we have found epididymal cytosol 7SSe levels rise after the 4th week whereas testicular levels are falling. Spermatozoa recovered from the epididymis and purified by low speed centrifugation contained about a third of total epididymal radioactivity. Conceivably all epididymal radioactivity may be present in spermatozoa. Our own experiments with gel filtration and dodecyl sulfate gel electrophoresis of the cytosol preparation from testes obtained at 4 h to 4 weeks after injection of radioselenium show that higher molecular weight forms appear early, whereas there is a gradual increase of 15 000 molecular weight selenoprotein during sperm maturation. The results suggest that the different size selenoproteins appearing in the testis may represent more than one type of active molecule. Work by Lawerence and Burk [12] and Prohaska et al. [5] has shown the glutathione-cumene peroxidase activity of testis to reside primarily in nonselenium-containing glutathione transferases and giutathione peroxidase(s). The latter workers [ 5] have gel filtered rat testis cytosol and shown clear absence of glutathione peroxidase activity in the 15 000 molecular weight region. We sug-

119

gest that other activities for the approximately 15 000 molecular weight testicular selenoprotein must be found. This protein is synthesized in testis and appears in spermatozoa of the epididymis [Ref. 1 and this work]. Brown and Burk's data showing sperm immotility in severely selenium-deprived mice [13] would suggest a vital function for this spermatic selenoprotein. Their data indicated an association of a selenoprotein with keratinous structures of the sperm midpiece. Calvin [14] as well as Pallini and Bacci [15] have reported a selenoprotein associated with the spermatic mitochondria in rodents and bull, respectively. It remains for further work to purify and characterize these intriguing selenoproteins and to elucidate the biological roles. Black et al. [16] have shown sheep liver, heart and kidney to contain a major selenoprotein in the 10 000 molecular weight range. The relationship to the 15 000 molecular weight selenoprotein of rat spermatozoa remains to be clarified.

Acknowledgements This work received principal support from the Medical Research Service of the Veterans Administration, as well as from American Cancer Society Grant In l l l C . Neutron activation analyses of selenium were carried out by Alan Blotcky, General Medical Research, Veterans Administration Hospital, Omaha, NE. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Rosenfield, I. and Beath, O.A. (1964) Selenium, pp. 91--140, Academic Press, New York Gunn, S.A., Gould, T.C. and Anderson, W.A.D. (1967) Proc. Soc. Exp. Biol. Med. 124, 1260--1263 Burk, R.F., Brown, D.G., Seely, R.J. and Scaief, R.C. (1972) J. Nutr. 102, 1049--1056 Chen, R.W., Whanger, P.D. and Weswig, P.H. (1975) Bioinorg. Chem. 4, 125--133 Prohaska, J.R., Mowafy, M. and Ganther, H,E. (1977) Chem.-Biol. Interact. 18, 253--265 Kute, T.E., Hcidemann, P. and Wittliff, J.L. (1978) Cancer Res. 38, 4307--4313 Maurer, H.R. (1971) Disc Electrophorests and Related Techniques of Polyacrylamide Gel Elcctrophores/s, pp. 44--45, Walter de Gruyter, New York Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, J. (1951) J. Biol. Chem. 193, 265--275 Westphal, U. (1961) Methods Enzymol. 15, 761--769 Burton, R.M., Higgins, P.J. and McConnell, K.P. (1977) Biochim. Biophys. Acta 493,323--331 Blotcky, A.L., Arsenwault, L.J. and Rack, E.P. (1973) Anal. Chem, 45, 1056--1060 Lawerence, R.A. and Burk, R.F. (1978) J. Nutr. 108, 211--215 Brown, D.G. and Burk, R.F. (1973) J. Nutr. 103, 102--108 Calvin, H.T. (1978) J. Exp. Zool. 204,445---452 Pallini, V. and Bacci, E. (1979) J. Submicr. Cytol. 11,165--170 Black, R.S., Tripp, M.J., Whanger, P.D. and Weswig, P.H. (1978) Bioinorg. Chem. 8,161--172