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Properties of chromatin isolated from bull spermatozoa
498
Biochimica et Biophysica Acta, 340 (1974) 498--508
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 97952 PROPERTIES OF CHROMATIN ISOLATED FROM BULL SPERMATOZOA
YASUKO MARUSHIGE and KEIJI MARUSHIGE Laboratories for Reporoductive Biology and Department of Biochemistry, University of North Carolina, Chapel Hill, N.C. 27514 (U.S.A.)
(Received October 29th, 1973)
Summary Isolated bull sperm chromatin consists principally of DNA and a single class of arginine- and cysteine-rich protein, sperm histone, the ratio of sperm histone to DNA by weight being approx. 0.7. In the presence of sufficient concentrations of urea and 2-mercaptoethanol, bull sperm histone can be totally dissociated from DNA at 0.6 M of NaC1. Incomplete dissociation conditions have yielded sperm histone fractions o f various sizes, suggesting t h a t bull sperm histone molecules are present on the DNA in a highly polymerized form as a result of the formation of intermolecular disulfide linkages. A labeling of sulfhydryl groups released by a thiol during extraction of sperm histone has further shown that essentially all the cysteine sulfhydryls of bull sperm histone are present in the chromatin as disulfides, and t h a t these disulfides are between sperm histone molecules. These results clearly indicate that the DNA in bull sperm chromatin is tightly packaged by sperm histone molecules which are cross-linked by disulfide linkages to form a tight network.
Introduction Chromosomal DNA is tightly packaged in the head of spermatozoa. This packaging is achieved by replacement of somatic histones by a new class of basic proteins at the terminal stages of spermatogenesis. In contrast to the remarkable similarity of somatic histones among various organisms [1], basic proteins found in spermatozoa are known to vary widely among different species and have been collectively called sperm histone [2]. Properties of sperm chromatins thus differ markedly depending upon the type of basic proteins with which DNA is complexed. Sea urchin sperm chromatin, whose basic proteins are similar to those of somatic chromatins, can be dispersed in water or in a buffer of low ionic strength [3l, while nucleoprotamine, the sperm chromatin of the salmonid fish, remains undispersed under these conditions [4]. In both cases, sperm chromatins consits principally of DNA and sperm-specific basic proteins which are totally extractable by a high salt or a dilute acid solution
499 [3,4]. It is well known that no basic protein can be extracted from the heads of mammalian spermatozoa by the usual method described above and the extraction requires thiol reagents or pretreatment of the sperm heads with performic acid [5,6], suggesting that mammalian sperm proteins may be crosslinked by disulfide linkages to form a tight network closely associated with DNA. A highly basic, arginine- and cysteine-rich protein from bull sperm heads, has recently been purified, and extensively characterized [7,8]. Sperm histones of a similar nature have also been found in spermatozoa of various other mammals [8,9]. However, it has n o t been established as to what is the composition of mammalian sperm chromatin and as to whether a postulated network of disulfides in the chromatin involves sperm histone alone or sperm histone in combination with other components of sperm proteins. In order to elucidate the nature of DNA packaging in mammalian spermatozoa, we have isolated and characterized sperm chromatin from bull spermatozoa. It will be shown that bull sperm chromatin consists principally of DNA and sperm histone, and that it is the cross-linking between sperm histone molecules that is responsible for the tight packaging of DNA. Materials and Methods
Preparation o f sperm chromatin from bull semen Bull sperm heads were isolated from semen of breeding males by the method of Coelingh et al. [7] Sperm chromatin was prepared therefrom by repeated washings with detergents. Bull semen (10 ml) was centrifuged at 1500 X g for 10 min, and the sediment was washed successively thrice with 0.15 M NaC1 and once with 0.01 M Tris buffer (pH 8) b y resuspension and centrifugation. The sediment was resuspended in 12 ml of 0.01 M Tris buffer (pH 8) and sheared with a VirTis-45 homogenizer at 85 V for 15 min. The suspension was then layered on an equal volume of 0.6 M sucrose containing 0.01 M Tris buffer (pH 8) and centrifuged at 1500 X g for 20 min. The supernatant was discarded b y decantation and the loosely packed sperm-tail fraction was washed away by gently swirling with Tris buffer, leaving the tightly packed sperm-head fraction at the bottom. The sperm-head fraction was suspended in Tris buffer (0.01 M, pH 8) and sedimented again through 0.6 M sucrose in the same manner. The sperm heads thus obtained were then resuspended in 30 ml of 0.01 M Tris buffer (pH 8) containing 1% Triton X-100, kept in an ice bath overnight, and centrifuged at 1500 × g for 10 min. The sediment was next resuspended in 30 ml of 0.01 M Tris buffer (pH 8) containing 0.01 M sodium deoxycholate, and incubated at 37°C for 60 min, followed by centrifugation at 1500 X g for 10 min. The sediment, which will be referred to as bull sperm chromatin, was washed four times with 0.01 M Tris buffer (pH 8), and resuspended in the same buffer. Isolation and characterization o f basic proteins of sperm chromatin Sperm chromatin was incubated in 0.01 M Tris buffer (pH 8) containing various combinations of NaC1 {0.6--2 M), 2-mercaptoethanol (0.2 M), and urea (4 M, ultrapure grade; Schwarz--Mann, Orangeburg, N.Y.) at 37°C for 2 h. To the suspension, 4 M HC1 was added dropwise to a final concentration of 0.2 M
500 while stirring and the stirring was continued for 30 min in an ice bath. This was then centrifuged at 17000 X g for 20 min. The extract thus obtained was desalted by chromatography on a Sephadex G-10 column (2 cm X 20 cm or 1.2 cm X 20 cm) using 0.01 M HC1 as eluant, and proteins were then precipitated with 20% trichloroacetic acid. The protein precipitate was collected by centrifugation (17 000 X g, 20 min), washed successively with acidified acetone (0.1 ml concentrated HC1 in 200 ml acetone) and with acetone, and dried in a vacuum. The precipitate was then dissolved in 5 M guanidinium chloride (ultrapure grade; Schwarz--Mann, Orangeburg, N.Y.) containing 0.05 M Tris buffer (pH 8) and 0.01 M iodoacetamide at a protein concentration of 0.5--1 mg/ml, and incubated at r o o m temperature for 60 min in the dark, followed by desalting and precipitation with 20% trichloroacetic acid as described above. When chromatography on a CM-cellulose column followed immediately, the proteins were desalted on a Sephadex G-10 column using 0.05 M lithium acetate buffer (pH 5) as eluant. In some experiments, acid-soluble protein fractions were incubated again with 0.2 M 2-mercaptoethanol in the presence of 8 M urea and 0.02 M Tris buffer (pH 8) at 37°C for 2 h, followed by desalting, precipitation with 20% trichloroacetic acid, and treatment with iodoacetamide as described above. Sperm basic protein was chromatographed on CM-cellulose columns (Cellex-CM; Bio-Rad, Richmond, Calif.), using a linearly increasing concentration of guanidinium chloride buffered with 0.05 M lithium acetate buffer (pH 5). A sample (2--4 mg protein) in 0.05 M lithium acetate buffer (pH 5) was applied to a column (2 cm X 7 cm) equilibrated with the same buffer, and eluted with a guanidinium chloride gradient at a flow rate of 60 ml/hour. Protein concentration was determined by absorbance at 230 nm. The resulting chromatographic fractions were appropriately combined, concentrated with a flash evaporator, desalted and precipitated as described above. The protein fractions thus obtained were further characterized by electrophoresis in polyacrylamide gels. Sperm basic protein was also characterized by chromatography on a Sephadex G-75 column (1.2 cm X 96 cm), or a Sephadex G-200 column (1.2 cm X 193 cm). A protein sample (1--2.5 mg) in 1--1.5 ml of 0.2 M HC1 containing 20% sucrose was applied to a column equilibrated with 0.01 M HC1, and eluted with 0.01 M HC1. Protein concentration was determined by absorbance at 230 nm. The protein fractions were appropriately combined, precipitated with 20% trichloroacetic acid, and further characterized by electrophoresis in polyacrylamide gels. Disc electrophoresis in 15% polyacrylamide gels containing 6 M urea was performed according to Bonner et al. [10]. A sample of 0.01--0.07 ml (3--15 pg of protein) in 0.2 M HC1 containing 20% sucrose was applied to each gel and electrophoresed for 70 min at 5 mA per tube. The gels were stained with 0.1% Buffalo Black in 40% ethanol containing 7% acetic acid for 4--5 h and destained by an exhaustive washing in 40% ethanol containing 7% acetic acid.
Chemical analyses RNA was determined by the orcinol reaction [11] after hydrolysis in 0.3 M KOH (37°C, 18 h), using t r o u t ribosomal RNA as a standard. In order to
501 determine DNA and protein, bull spermatozoa, sperm head, or sperm chromatin was treated with 0.5 M HC104 at 100°C for 10 min, and centrifuged in a clinical centrifuge for 10 min. DNA was determined on the supernatant in which no protein was found, by the diphenylamine method [11] calibrated with salmon testis DNA, and protein was determined on the residue by the method by Lowry et al. [ 1 2 ] , using bovine serum albumin as a standard. Results and Discussion
The weight ratio of protein to DNA in saline-washed bull spermatozoa has been found to be 2.7. The ratio decreases down to 1.1 after removal of the tail by shearing and sedimentation through 0.6 M sucrose. The acrosomal cap of the sperm head as well as contaminated middle pieces can then be removed by washings with detergents. Bull sperm chromatin thus prepared retains the oval shape characteristic of the bull sperm head and possesses the ratio of protein to DNA by weight of 0.79 (average of the values obtained from five preparations ranging between 0.76 and 0.82). Little or no RNA (not more than 0.7% of DNA) has been found in these preparations. It has already been reported that bull spermatozoa contain only trace amounts of RNA [13]. Little protein is extractable from bull sperm chromatin with NaC1 (up to 4 M) or urea (4 M) alone, or a combination of these two or a combination of 2-mercaptoethanol (0.2 M) and urea (4 M). Only a partial extraction of sperm chromatin proteins has been achieved with a combination of NaC1 (2 M) and 2-mercaptoethanol (0.2 M). Efficient dissociation of bull sperm chromatin occurs when it is treated with a mixture containing both NaC1 and 2-mercaptoethanol in the presence of urea, as shown in Fig. 1. In this experiment, bull sperm chromatin was treated with various concentrations of NaC1 in the presence of 0.2 M 2-mercaptoethanol and 4 M urea buffered with 0.01 M Tris buffer (pH 8) in an ice bath for 2 h and centrifuged at 1 1 4 0 0 0 × g for 18 h in a Spinco SW-50.1 rotor. The sediments were then assayed for DNA and protein. Data of Fig. 1 show that the majority of sperm chromatin proteins are removed from DNA at 0.6 M NaC1 under these conditions. Proteins extracted from bull
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Fig. 1. D i s s o c i a t i o n o f b u l l s p e r m c h r o m a t i n . Bull s p e r m c h r o m a t i n w a s t r e a t e d w i t h v a r i o u s c o n c e n t r a t i o n s o f NaC1 in t h e p r e s e n c e o f 4 M u r e a a n d 0 . 2 M 2 - m e r c a p t o e t h a n o l .
502 sperm chromatin in this manner have been found to be totally soluble in 0.2 M HC1 and will be, hereafter, referred to as sperm histone. When bull sperm chromatin is treated with a mixture containing NaC1, 2-mercaptoethanol and urea of a sufficient strength (e.g. 1.2 M, 0.2 M and 4 M, respectively), the suspension becomes almost instantaneously transparent and viscous as a result of dissociation of sperm histone from DNA. As an alternative to a high-speed centrifugation, the viscous solution was treated with 0.2 M HC1. Upon this treatment, all the DNA precipitated and approx. 90% of the total sperm chromatin proteins were recovered in the extract. Bull sperm histone fractions thus prepared have been first analyzed by chromatography on a CMcellulose column (Fig. 2A). Fractions eluted at the beginning of the guanidinium chloride gradient (tubes No. 9--13 in Fig. 2A, similarly in Fig. 3A) are diffusible, not precipitable with 20% trichloroacetic acid, and exhibit no absorption at 280 nm. Furthermore, the size of this peak is constant irrespective of the amounts and kinds of protein fractions applied to the columns. This peak appears, therefore, to be due to a contaminated material from, most likely, the lithium acetate buffer. The main peak (Fig. 2A,a) comprises 85% of the total sperm histone fraction. This component is chromatographed as a single peak on a Sepbadex G-75 column (Fig. 2B) and gives a single major band in gel electrophoresis (Fig. 4,a). Approx. 15% of the sperm histone fraction is eluted at higher concentrations of guanidinium chloride (Fig. 2A,b) and gives three electrophoretic bands, as shown in Fig. 4,b. This minor fraction, however, migrates as one major band with an electrophoretic mobility identical to that of the main c o m p o n e n t (Fig. 4,a) after its incubation in 0.2 M 2-mercaptoethanol in the presence of 8 M urea (37°C, 2 h), while the electrophoretic mobility of the main c o m p o n e n t is not changed by such a treatment.
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Fig. 2. C h r o m a t o g r a p h i c p r o f i l e s of bull s p e r m h i s t o n e e x t r a c t e d w i t h 0 . 2 M HC1 f o l l o w i n g i n c u b a t i o n of s p e r m c h r o m a t i n in 0 . 2 M 2 - m e r c a p t o e t h a n o l - - l . 2 M N a C l - - 4 M u r e a . A c i d - s o l u b l e p r o t e i n w a s t r e a t e d w i t h i o d o a c e t a m i d e a n d s u b j e c t e d to c o l u m n c h r o m a t o g r a p h y . ( A ) C M - c e l l u l o s e c o l u m n (2 c m X 7 c m ) . (B) r e c h r o m a t o g r a p h y o f F r a c t i o n a in A o n a S e p h a d e x G - 7 5 c o l u m n (1.2 c m X 96 c m ) . F r a c t i o n s i n d i c a t e d b y a r r o w s w e r e c o m b i n e d a n d f u r t h e r a n a l y s e d b y e l e c t r o p h o r e s i s ( F i g . 4).
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Fig. 3. C h r o m a t o g r a p h i c p r o f i l e s o f b u l l s p e r m h i s t o n e e x t r a c t e d w i t h 0 . 2 M HC1 f o l l o w i n g i n c u b a t i o n o f s p e r m c h r o m a t i n in 0 . 2 M 2 - m e r c a p t o e t h a n o l - - 4 M u r e a . A c i d - s o l u b l e p r o t e i n w a s t r e a t e d w i t h i o d o a c e t a m i d e a n d s u b j e c t e d t o c o l u m n c h r o m a t o g r a p h y . ( A ) C M - c e l l u l o s e c o l u m n (2 c m X 7 c m ) . (B) Sephadex G-200 column (1.2 cm × 192 cm). Fractions indicated by arrows were combined and further a n a l y s e d b y e l e c t r o p h o r e s i s (Fig. 4).
When bull sperm chromatin is incubated (37°C, 2 h) in a mixture containing 2-mercaptoethanol (0.2 M) and urea (4 M) but without NaC1, the sperm chromatin particles become somewhat swollen. Although no protein is dissociated from the DNA under these conditions, 40--65% of the total sperm histone can be extracted by the subsequent treatment of the mixture with 0.2 M HC1. One preparation of bull sperm histone isolated in this manner (yield of sperm histone, 40%) has been analyzed by chromatography on a CM-cellulose column (Fig. 3A) and by electrophoresis in polyacrylamide gels (Fig. 4). In comparison with bull sperm histone isolated under complete-dissociation conditions (Fig. 2A and Fig. 4,T, }, the sperm histone fraction thus obtained is highly enriched with fractions which are eluted at higher concentrations of guanidinium chloride in CM-cellulose column chromatography (Fig. 3A), and shows a number of slow-moving bands in gel electrophoresis (Fig. 4,T2 ). At least seven bands can be clearly identified in this figure and they are numbered in the order of their electrophoretic mobilities. The fraction eluted from the CM-cellulose column at 1.0--1.3 M guanidinium chloride (Peak c in Fig. 3A) gives Band I in electrophoresis (Fig. 4,c), while Peak d in Fig. 3A contains mainly Band 2 (Fig. 4,d), the following broad shoulder (Fig. 3A,e) being composed of several slower-migrating components (Fig. 4,e). Another sperm-histone preparation isolated in the same manner (yield of sperm histone, 65%) has been analyzed on a Sephadex G-200 column (Fig. 3B). Further electrophoretic analyses of chromatographic fractions obtained from this column have revealed
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Fig. 4. E l e c t r o p h o r e t i e p r o f i l e s of bull s p e r m h i s t o n e . U n f r a c t i o n a t e d s p e r m h i s t o n e isolated a f t e r i n c u b a tion of s p e r m c h r o m a t i n in 0.2 M 2 - m e r c a p t o e t h a n o l - - l . 2 M N a C I - - 4 M u r e a ( T 1 ) as well as f r a c t i o n s labeled a a n d b f r o m CM-cellulose c o l u m n in Fig. 2 A , u n f r a e t i o n a t e d s p e r m h i s t o n e i s o l a t e d a f t e r i n c u b a t i o n of s p e r m c h r o m a t i n in 0.2 M 2 - m e r c a p t o e t h a n o l - - 4 M u r e a (T 2) as well as f r a c t i o n s l a b e l e d c, d a n d e f r o m CM-ceUulose c o l u m n in Fig. 3 A , u n f r a c t i o n a t e d s p e r m h i s t o n e isolated a f t e r i n c u b a t i o n of sperm a t o z o a in 0.2 M 2 m e r c a p t o e t h a n o l - - 5 M g u a n i d i n i u m c h l o r i d e for 1 rain at 0 ° C ( T 3 ) . U n f r a c t i o n a t e d s p e r m h i s t o n e T 2 a n d T 3 w e r e again i n c u b a t e d w i t h 0.2 M 2 m e r c a p t o e t h a n o l f o l l o w e d b y the iodoa c e t a m i d e t r e a t m e n t (R 2 a n d R 3, r e s p e c t i v e l y ) . E l e c t r o p h o r e s e s w e r e c a r r i e d o u t in 5 - c m gets at 5 m A p e r t u b e f o r 70 rain.
that Peaks 1, 2 and 3 {Fig. 3B,1, 2 and 3) consist mainly of Bands 1, 2 and 3 (Fig. 4,T2 ), respectively. All the slow-migrating bands disappear essentially completely upon treatment of these preparations with 0.2 M 2-mercaptoethanol in the presence of 8 M urea (pH 8), and are converted into Band 1 (Fig. 4,R2 ), whose mobility is, in turn, identical to that of the major fraction of bull sperm histone isolated under complete-dissociation conditions (Fig. 4,a). These results suggest that Band 1 represents the m o n o m e r of bull sperm histone. As shown in Fig. 5A, a linear relationship is found when relative electrophoretic mobilities of Band 1--7 obtained from Fig. 4 are semilogarithmically plotted against the band numbers, indicating that Bands 1--7 represent respectively the m o n o m e r to the heptamer of bull sperm histone. Similarly, a semilogarithmic plotting of relative elution volumes of each peak obtained from Fig. 3B versus the peak numbers also gives a linear relationship (Fig. 5B). Such oligomers of bull sperm histone have also been obtained by a brief exposure of spermatozoa to a complete-extraction medium. In this experiment, omitting the lengthy chromatin-preparation processes during which reshuffling and oxidation of sulfhydryl groups might occur, bull spermatozoa sedimented from semen were immediately incubated with 5 M guanidinium chloride containing 0.2 M 2-mer-
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Fig. 5. R e l a t i v e e l e c t r o p h o r e t i c m o b i l i t i e s and relative e l u t i o n v o l u m e s in gel e x c l u s i o n c h r o m a t o g r a p h y of bull s p e r m h i s t o n e f r a c t i o n s o b t a i n e d b y acid e x t r a c t i o n a f t e r i n c u b a t i o n o f s p e r m c h r o m a t i n in 0 . 2 M 2 - m e r c a p t o e t h a n o l - - 4 M urea. ( A ) R F values ( m i g r a t i o n distance o f p r o t e i n / m i g r a t i o n d i s t a n c e o f s o l v e n t ) o f e a c h b a n d n u m b e r e d in the order o f e l e c t r o p h o r e t i c m o b i l i t i e s in Fig. 4 , T 2 w e r e p l o t t e d against the l o g a r i t h m s o f the band n u m b e r s . (B) K a y ( V e - - V o / V t - - V o , w h e r e V e, V o and V t r e p r e s e n t e l u t i o n v o l u m e o f p r o t e i n , void v o l u m e , and t o t a l b e d v o l u m e , r e s p e c t i v e l y ) o f e a c h p e a k n u m b e r e d in t h e reverse order o f e l u t i o n v o l u m e s in Fig. 3B w e r e p l o t t e d against the l o g a r i t h m s o f t h e p e a k n u m b e r s .
captoethanol for 1 min at 0°C. Acid-soluble proteins were then isolated as described in Materials and Methods and their released sulfhydryl groups were blocked with iodoacetamide. Under these conditions, approx. 80% of sperm histone was extracted. They were then chromatographed on a CM-cellulose column (2 cm × 7 cm) using a step-wise elution with guanidinium chloride buffered with 0.05 M lithium acetate buffer (pH 5), and the protein fraction eluted between 0.8 M and 3 M guanidinium chloride was analyzed by polyacrylamide gel electrophoresis. As can be seen in Fig. 4,T3 and R3, sperm histone fractions thus obtained contain a number of slow-migrating bands, which are essentially completely converted to a single band corresponding to the m o n o m e r of bull sperm histone after reincubation of the protein fraction with 0.2 M 2-mercaptoethanol in the presence of 5 M guanidinium chloride. These results suggest that in bull sperm chromatin sperm histone molecules are cross-linked by disulfide bonds into a highly polymerized form, and such intermolecular disulfide linkages are partially retained in the sperm histone fraction isolated under the present conditions. A m i n o acid composition of bull sperm histone in the present study has been found to be in complete agreement with that reported by Coelingh et al.
506 [7]. Bull sperm histone (molecular weight, 6200) is characterized by its high content of arginine (24 residues) and cysteine (six residues). In order to determine h o w many of these cysteine residues are present in bull perm chromatin as disulfides, the number of sulfhydryl groups of sperm histone released by 2-mercaptoethanol during its extraction has been assayed. In this experiment, bull sperm chromatin was isolated in the presence of 5 mM iodoacetamide throughout the entire processes of chromatin preparation, and was further incubated with 5 M guanidinium chloride contianing 5 mM iodoacetamide and 0.05 M Tris buffer (pH 8) at 37°C for 1 h in the dark in order to block free sulfhydryl groups, if any. The chromatin thus treated was next incubated in a mixture containing 0.2 M 2-mercaptoethanol, 1.2 M NaC1 and 4 M urea (pH 8) at 37°C for 2 h, followed by extraction of sperm histone with 0.2 M HC1. The monomer of sperm histone was next purified by Sephadex G-75 chromatography using 0.01 M HC1 as eluant. The reaction of sperm histone with iodo[ 14C]acetamide was then followed during incubation in a mixture containing 10 mM iodo[ 14 C] acetamide, 5 M guanidinium chloride and 0.05 M Tris buffer (pH 8) at room temperature in the dark (Fig. 6), Data of Fig. 6 show that average 5.3 sulfhydryls per mole of bull sperm histone are released during its extraction in the presence of 2-mercaptoethanol, indicating that the majority of cysteine residues of bull sperm histone are present in the chromatin as disulfides. Since the same value has been obtained when bull sperm chromatin particles are mechanically disrupted before their incubation with non-radioactive iodoacetamide, the high number (average 5.3 sulfhydryls released by a thiol per sperm histone molecule) does not appear to be due to a failure of ]
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Fig. 6. Determination of released sulfhydryl groups of bull sperm histone. Completely reduced bull sperm histone isolated f r o m bull sperm chromatin which was preincubated with non-radioactive iodoacetamide, was incubated at a protein concentration of 8 0 0 pg/ml with 10 m M iodo[14C]acetamide (spec. act., 8.12 • 105 cprn/~zmole) in the presence of 5 M guanidinium chloride buffered with 0.05 M Tris buffer (pH 8) at r o o m temperature in the dark. S p e r m histone was precipitated f r o m aliquots taken at various lengths of incubation time with 5 % trichloroacetic acid after an appropriate dilution. Protein and radioactivity of each sample were determined and moles of iodo[14C]acetamide reacted per m o l e of sperm histone calculated. Iodo[ 14 C] acetamide was purchased from ICN, Irvine, Calif.
507 iodoacetamide in penetrating into the chromatin particles during the preincubation. In this experiment, bull sperm chromatin was suspended in 5 M guanidinium chloride containing 5 mM non-radioactive iodoacetamide, quickly frozen with a large excess of glass beads (Type 100--5005, 3M Company, St. Paul, Minn.), and ground thoroughly using a mortar and pestle. This was then incubated at 37°C for I h, followed by extraction of sperm histone in the presence of 2-mercaptoethanol and reaction with iodo[ ~4 C] acetamide as described above. As has been described earlier in this report, approx. 10% of the total sperm chromatin proteins precipitate with DNA when bull sperm chromatin is treated with acid following incubation in 0.2 M 2-mercaptoethanol--l.2 M NaC1--4 M urea. To test a possibility that disulfide-bond formation between sperm histone and such acid-insoluble proteins might make any significant contribution for the packaging of DNA, bull sperm chromatin preincubated with non-radioactive iodoacetamide as described above, was first incubated (37°C, 2 h) in a mixture containing 0.1 M 2-mercaptoethanol, 1.2 M NaC1, 4 M urea and 0.05 M Tris buffer (pH 8), and i o d o [ 1 4 C ] a c e t a m i d e (spec. act., 1.74 • 104 cpm/pmole) was then added to a final concentration of 0.2 M. After an additional incubation for 1 h (37°C in the dark), sperm histone and acidinsoluble proteins were isolated and their radioactivities determined. Results showed that the radioactivity found in the acid-insoluble protein fractions was only 6% (315 cpm) of those found in the sperm histone fractions (5260 cpm). The number of sulfhydryls released by 2-mercaptoethanol per mole of sperm histone was found to be 5.5 in this experiment. These results indicate clearly that most, if not all, disulfide linkages present in bull sperm chromatin are those between sperm histone molecules. It seems thus clear that, although it is possible that certain minor components of sperm chromatin proteins might play certain roles in the packaging of DNA, the DNA of bull sperm chromatin is packaged by sperm histone molecules which are cross-linked through formation of disulfide linkages to form a tight network. The cross-linking appears to be so complete that essentially all the cysteine residues of sperm histone are present as disulfides. The amino acid sequence of bull sperm histone [8] has revealed that the majority of arginine residues are in the middle of the molecule, and two cysteine residues are distributed in this region, while two residues of cysteine are present in the amino-terminal region and two in the carboxy-terminal region of the molecule. The cysteine residues present in the less basic terminal regions seem likely to be the ones involved in polymerization of bull sperm histone through formation of intermolecular disulfides between neighboring sperm histone molecules on the DNA. The packaging of DNA might be further tightened by intramolecular disulfide-bond formation of cysteine residues present in the arginine-rich middle region or these cysteine residues might be involved in corss-linking of neighboring chromatin strands through formation of interchromatin-strand disulfide linkages. Further chemical analyses of mammalian sperm histone fractions whose disulfides have been partially preserved (cf. Figs 3, 4 and 5), as well as studies of sperm histone during formation and maturation of mammalian spermatozoa would provide the further information concerning the molecular architecture of mammalian sperm chromatin.
508
Acknowledgments We are grateful to Drs H. Stanley Bennett and J. Logan Irvin for their stimulating discussion, and to Dr Edward Ezrailson for his helpful information. We would like to thank Dr Lester Ulberg and his staff, Reproductive Physiology Research Laboratories, North Carolina State University for their kind supply of bull semen, and Dr J. Logan Irvin and Mr Jack Faircloth for amino acid analysis. This work was supported by a grant from the Rockefeller Foundation to the Laboratories for Reproductive Biology, University of North Carolina. References 1 2 3 4 5 6 7 8 9 10
11 12 13
D e L a n g e , R . J . a n d S m i t h , E . L . ( 1 9 7 1 ) A n n u ° B,ev. B i o c h e m . 4 0 , 2 7 9 - - 3 1 4 B l o c h , D.P. ( 1 9 6 9 ) G e n e t i c s 6 1 , S u p p l . I~ 9 3 - - 1 1 1 P a o l e t t i , R . A . a n d H u a n g , R , C . C . ( 1 9 6 9 ) B i o c h e m i s t r y S, 1 6 1 5 - - 1 6 2 5 M a r u s h i g e , K. a n d D i x o n , G . H . ( 1 9 6 9 ) Dev. Biol. 1 9 , 3 9 7 - - - 4 1 4 B r i l - P e t e r s e n , E. a n d W e s t e n b r i n k , H . G . K . ( 1 9 6 3 ) B i o c h i m . B i o p h y s . A c t a 7 6 , 1 5 2 - - 1 5 4 H e n d r i c k s , D.M. a n d M a y e r , D . T . ( 1 9 6 5 ) E x p . Cell Res. 4 0 , 4 0 2 - - 4 1 2 C o e l i n g h , J.P., R o z i j n , T . H , a n d M o n f o o r t , C . H . ( 1 9 6 9 ) B i o c h i m . B i o p h y s . A c t a 1 8 8 , 3 5 3 - - 3 5 6 C o e l i n g h , J.P., M o n f o o r t , C . H . , R o z i j n , T . H . , L e u v e n , J . A . G . , S c h i p h o f , R., S t e y n - P a r v ~ , E.P., B r a u n i t z e r , G . , S c h r a n k , B. a n d R u h f u s , A . ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 8 5 , 1 - - 1 4 K i s t l e r , W . S . , G e r o c h , M.E. a n d W i l l i a m s - A s h m a n , H , G . ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 4 5 3 2 - - 4 5 4 3 B o n n e r , J . , C h a l k l e y , G . R . , D a h m u s , M,, F a m b r o u g h , D., F u j i m u r a , F., H u a n g , R . C . C . , H u b e r m a n , J., Jensen~ R., M a r u s h i g e , K . , O h l e n b u s e h , H., O l i v e r a , B. a n d W i d h o l m , J. ( 1 9 6 8 ) in M e t h o d s in E n z y m o l o g y ( G r o s s m a n , L. a n d M o l d a v e , K., eds), Vol. XII, P a r t B, p p . 3 - - 6 5 , A c a d e m i c Press, N e w York D i s c h e , Z. ( 1 9 5 5 ) in T h e N u c l e i c A c i d ( C h a r g a f f , E. a n d D a v i d s o n , J . N . , e d s ) , Vol. I, p p . 2 8 5 - - 3 0 5 , A c a d e m i c Press, N e w Y o r k L o w r y , O . H . , R o s e b r o u g h , N . J . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 B h a r g a v a , P.M., B i s h o p , M . W . H . a n d W o r k , T.S. ( 1 9 5 9 ) B i o c h e m . J. 7 3 , 2 4 2 2 4 7
Biochimica et Biophysica Acta, 340 (1974) 498--508
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 97952 PROPERTIES OF CHROMATIN ISOLATED FROM BULL SPERMATOZOA
YASUKO MARUSHIGE and KEIJI MARUSHIGE Laboratories for Reporoductive Biology and Department of Biochemistry, University of North Carolina, Chapel Hill, N.C. 27514 (U.S.A.)
(Received October 29th, 1973)
Summary Isolated bull sperm chromatin consists principally of DNA and a single class of arginine- and cysteine-rich protein, sperm histone, the ratio of sperm histone to DNA by weight being approx. 0.7. In the presence of sufficient concentrations of urea and 2-mercaptoethanol, bull sperm histone can be totally dissociated from DNA at 0.6 M of NaC1. Incomplete dissociation conditions have yielded sperm histone fractions o f various sizes, suggesting t h a t bull sperm histone molecules are present on the DNA in a highly polymerized form as a result of the formation of intermolecular disulfide linkages. A labeling of sulfhydryl groups released by a thiol during extraction of sperm histone has further shown that essentially all the cysteine sulfhydryls of bull sperm histone are present in the chromatin as disulfides, and t h a t these disulfides are between sperm histone molecules. These results clearly indicate that the DNA in bull sperm chromatin is tightly packaged by sperm histone molecules which are cross-linked by disulfide linkages to form a tight network.
Introduction Chromosomal DNA is tightly packaged in the head of spermatozoa. This packaging is achieved by replacement of somatic histones by a new class of basic proteins at the terminal stages of spermatogenesis. In contrast to the remarkable similarity of somatic histones among various organisms [1], basic proteins found in spermatozoa are known to vary widely among different species and have been collectively called sperm histone [2]. Properties of sperm chromatins thus differ markedly depending upon the type of basic proteins with which DNA is complexed. Sea urchin sperm chromatin, whose basic proteins are similar to those of somatic chromatins, can be dispersed in water or in a buffer of low ionic strength [3l, while nucleoprotamine, the sperm chromatin of the salmonid fish, remains undispersed under these conditions [4]. In both cases, sperm chromatins consits principally of DNA and sperm-specific basic proteins which are totally extractable by a high salt or a dilute acid solution
499 [3,4]. It is well known that no basic protein can be extracted from the heads of mammalian spermatozoa by the usual method described above and the extraction requires thiol reagents or pretreatment of the sperm heads with performic acid [5,6], suggesting that mammalian sperm proteins may be crosslinked by disulfide linkages to form a tight network closely associated with DNA. A highly basic, arginine- and cysteine-rich protein from bull sperm heads, has recently been purified, and extensively characterized [7,8]. Sperm histones of a similar nature have also been found in spermatozoa of various other mammals [8,9]. However, it has n o t been established as to what is the composition of mammalian sperm chromatin and as to whether a postulated network of disulfides in the chromatin involves sperm histone alone or sperm histone in combination with other components of sperm proteins. In order to elucidate the nature of DNA packaging in mammalian spermatozoa, we have isolated and characterized sperm chromatin from bull spermatozoa. It will be shown that bull sperm chromatin consists principally of DNA and sperm histone, and that it is the cross-linking between sperm histone molecules that is responsible for the tight packaging of DNA. Materials and Methods
Preparation o f sperm chromatin from bull semen Bull sperm heads were isolated from semen of breeding males by the method of Coelingh et al. [7] Sperm chromatin was prepared therefrom by repeated washings with detergents. Bull semen (10 ml) was centrifuged at 1500 X g for 10 min, and the sediment was washed successively thrice with 0.15 M NaC1 and once with 0.01 M Tris buffer (pH 8) b y resuspension and centrifugation. The sediment was resuspended in 12 ml of 0.01 M Tris buffer (pH 8) and sheared with a VirTis-45 homogenizer at 85 V for 15 min. The suspension was then layered on an equal volume of 0.6 M sucrose containing 0.01 M Tris buffer (pH 8) and centrifuged at 1500 X g for 20 min. The supernatant was discarded b y decantation and the loosely packed sperm-tail fraction was washed away by gently swirling with Tris buffer, leaving the tightly packed sperm-head fraction at the bottom. The sperm-head fraction was suspended in Tris buffer (0.01 M, pH 8) and sedimented again through 0.6 M sucrose in the same manner. The sperm heads thus obtained were then resuspended in 30 ml of 0.01 M Tris buffer (pH 8) containing 1% Triton X-100, kept in an ice bath overnight, and centrifuged at 1500 × g for 10 min. The sediment was next resuspended in 30 ml of 0.01 M Tris buffer (pH 8) containing 0.01 M sodium deoxycholate, and incubated at 37°C for 60 min, followed by centrifugation at 1500 X g for 10 min. The sediment, which will be referred to as bull sperm chromatin, was washed four times with 0.01 M Tris buffer (pH 8), and resuspended in the same buffer. Isolation and characterization o f basic proteins of sperm chromatin Sperm chromatin was incubated in 0.01 M Tris buffer (pH 8) containing various combinations of NaC1 {0.6--2 M), 2-mercaptoethanol (0.2 M), and urea (4 M, ultrapure grade; Schwarz--Mann, Orangeburg, N.Y.) at 37°C for 2 h. To the suspension, 4 M HC1 was added dropwise to a final concentration of 0.2 M
500 while stirring and the stirring was continued for 30 min in an ice bath. This was then centrifuged at 17000 X g for 20 min. The extract thus obtained was desalted by chromatography on a Sephadex G-10 column (2 cm X 20 cm or 1.2 cm X 20 cm) using 0.01 M HC1 as eluant, and proteins were then precipitated with 20% trichloroacetic acid. The protein precipitate was collected by centrifugation (17 000 X g, 20 min), washed successively with acidified acetone (0.1 ml concentrated HC1 in 200 ml acetone) and with acetone, and dried in a vacuum. The precipitate was then dissolved in 5 M guanidinium chloride (ultrapure grade; Schwarz--Mann, Orangeburg, N.Y.) containing 0.05 M Tris buffer (pH 8) and 0.01 M iodoacetamide at a protein concentration of 0.5--1 mg/ml, and incubated at r o o m temperature for 60 min in the dark, followed by desalting and precipitation with 20% trichloroacetic acid as described above. When chromatography on a CM-cellulose column followed immediately, the proteins were desalted on a Sephadex G-10 column using 0.05 M lithium acetate buffer (pH 5) as eluant. In some experiments, acid-soluble protein fractions were incubated again with 0.2 M 2-mercaptoethanol in the presence of 8 M urea and 0.02 M Tris buffer (pH 8) at 37°C for 2 h, followed by desalting, precipitation with 20% trichloroacetic acid, and treatment with iodoacetamide as described above. Sperm basic protein was chromatographed on CM-cellulose columns (Cellex-CM; Bio-Rad, Richmond, Calif.), using a linearly increasing concentration of guanidinium chloride buffered with 0.05 M lithium acetate buffer (pH 5). A sample (2--4 mg protein) in 0.05 M lithium acetate buffer (pH 5) was applied to a column (2 cm X 7 cm) equilibrated with the same buffer, and eluted with a guanidinium chloride gradient at a flow rate of 60 ml/hour. Protein concentration was determined by absorbance at 230 nm. The resulting chromatographic fractions were appropriately combined, concentrated with a flash evaporator, desalted and precipitated as described above. The protein fractions thus obtained were further characterized by electrophoresis in polyacrylamide gels. Sperm basic protein was also characterized by chromatography on a Sephadex G-75 column (1.2 cm X 96 cm), or a Sephadex G-200 column (1.2 cm X 193 cm). A protein sample (1--2.5 mg) in 1--1.5 ml of 0.2 M HC1 containing 20% sucrose was applied to a column equilibrated with 0.01 M HC1, and eluted with 0.01 M HC1. Protein concentration was determined by absorbance at 230 nm. The protein fractions were appropriately combined, precipitated with 20% trichloroacetic acid, and further characterized by electrophoresis in polyacrylamide gels. Disc electrophoresis in 15% polyacrylamide gels containing 6 M urea was performed according to Bonner et al. [10]. A sample of 0.01--0.07 ml (3--15 pg of protein) in 0.2 M HC1 containing 20% sucrose was applied to each gel and electrophoresed for 70 min at 5 mA per tube. The gels were stained with 0.1% Buffalo Black in 40% ethanol containing 7% acetic acid for 4--5 h and destained by an exhaustive washing in 40% ethanol containing 7% acetic acid.
Chemical analyses RNA was determined by the orcinol reaction [11] after hydrolysis in 0.3 M KOH (37°C, 18 h), using t r o u t ribosomal RNA as a standard. In order to
501 determine DNA and protein, bull spermatozoa, sperm head, or sperm chromatin was treated with 0.5 M HC104 at 100°C for 10 min, and centrifuged in a clinical centrifuge for 10 min. DNA was determined on the supernatant in which no protein was found, by the diphenylamine method [11] calibrated with salmon testis DNA, and protein was determined on the residue by the method by Lowry et al. [ 1 2 ] , using bovine serum albumin as a standard. Results and Discussion
The weight ratio of protein to DNA in saline-washed bull spermatozoa has been found to be 2.7. The ratio decreases down to 1.1 after removal of the tail by shearing and sedimentation through 0.6 M sucrose. The acrosomal cap of the sperm head as well as contaminated middle pieces can then be removed by washings with detergents. Bull sperm chromatin thus prepared retains the oval shape characteristic of the bull sperm head and possesses the ratio of protein to DNA by weight of 0.79 (average of the values obtained from five preparations ranging between 0.76 and 0.82). Little or no RNA (not more than 0.7% of DNA) has been found in these preparations. It has already been reported that bull spermatozoa contain only trace amounts of RNA [13]. Little protein is extractable from bull sperm chromatin with NaC1 (up to 4 M) or urea (4 M) alone, or a combination of these two or a combination of 2-mercaptoethanol (0.2 M) and urea (4 M). Only a partial extraction of sperm chromatin proteins has been achieved with a combination of NaC1 (2 M) and 2-mercaptoethanol (0.2 M). Efficient dissociation of bull sperm chromatin occurs when it is treated with a mixture containing both NaC1 and 2-mercaptoethanol in the presence of urea, as shown in Fig. 1. In this experiment, bull sperm chromatin was treated with various concentrations of NaC1 in the presence of 0.2 M 2-mercaptoethanol and 4 M urea buffered with 0.01 M Tris buffer (pH 8) in an ice bath for 2 h and centrifuged at 1 1 4 0 0 0 × g for 18 h in a Spinco SW-50.1 rotor. The sediments were then assayed for DNA and protein. Data of Fig. 1 show that the majority of sperm chromatin proteins are removed from DNA at 0.6 M NaC1 under these conditions. Proteins extracted from bull
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502 sperm chromatin in this manner have been found to be totally soluble in 0.2 M HC1 and will be, hereafter, referred to as sperm histone. When bull sperm chromatin is treated with a mixture containing NaC1, 2-mercaptoethanol and urea of a sufficient strength (e.g. 1.2 M, 0.2 M and 4 M, respectively), the suspension becomes almost instantaneously transparent and viscous as a result of dissociation of sperm histone from DNA. As an alternative to a high-speed centrifugation, the viscous solution was treated with 0.2 M HC1. Upon this treatment, all the DNA precipitated and approx. 90% of the total sperm chromatin proteins were recovered in the extract. Bull sperm histone fractions thus prepared have been first analyzed by chromatography on a CMcellulose column (Fig. 2A). Fractions eluted at the beginning of the guanidinium chloride gradient (tubes No. 9--13 in Fig. 2A, similarly in Fig. 3A) are diffusible, not precipitable with 20% trichloroacetic acid, and exhibit no absorption at 280 nm. Furthermore, the size of this peak is constant irrespective of the amounts and kinds of protein fractions applied to the columns. This peak appears, therefore, to be due to a contaminated material from, most likely, the lithium acetate buffer. The main peak (Fig. 2A,a) comprises 85% of the total sperm histone fraction. This component is chromatographed as a single peak on a Sepbadex G-75 column (Fig. 2B) and gives a single major band in gel electrophoresis (Fig. 4,a). Approx. 15% of the sperm histone fraction is eluted at higher concentrations of guanidinium chloride (Fig. 2A,b) and gives three electrophoretic bands, as shown in Fig. 4,b. This minor fraction, however, migrates as one major band with an electrophoretic mobility identical to that of the main c o m p o n e n t (Fig. 4,a) after its incubation in 0.2 M 2-mercaptoethanol in the presence of 8 M urea (37°C, 2 h), while the electrophoretic mobility of the main c o m p o n e n t is not changed by such a treatment.
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Fig. 3. C h r o m a t o g r a p h i c p r o f i l e s o f b u l l s p e r m h i s t o n e e x t r a c t e d w i t h 0 . 2 M HC1 f o l l o w i n g i n c u b a t i o n o f s p e r m c h r o m a t i n in 0 . 2 M 2 - m e r c a p t o e t h a n o l - - 4 M u r e a . A c i d - s o l u b l e p r o t e i n w a s t r e a t e d w i t h i o d o a c e t a m i d e a n d s u b j e c t e d t o c o l u m n c h r o m a t o g r a p h y . ( A ) C M - c e l l u l o s e c o l u m n (2 c m X 7 c m ) . (B) Sephadex G-200 column (1.2 cm × 192 cm). Fractions indicated by arrows were combined and further a n a l y s e d b y e l e c t r o p h o r e s i s (Fig. 4).
When bull sperm chromatin is incubated (37°C, 2 h) in a mixture containing 2-mercaptoethanol (0.2 M) and urea (4 M) but without NaC1, the sperm chromatin particles become somewhat swollen. Although no protein is dissociated from the DNA under these conditions, 40--65% of the total sperm histone can be extracted by the subsequent treatment of the mixture with 0.2 M HC1. One preparation of bull sperm histone isolated in this manner (yield of sperm histone, 40%) has been analyzed by chromatography on a CM-cellulose column (Fig. 3A) and by electrophoresis in polyacrylamide gels (Fig. 4). In comparison with bull sperm histone isolated under complete-dissociation conditions (Fig. 2A and Fig. 4,T, }, the sperm histone fraction thus obtained is highly enriched with fractions which are eluted at higher concentrations of guanidinium chloride in CM-cellulose column chromatography (Fig. 3A), and shows a number of slow-moving bands in gel electrophoresis (Fig. 4,T2 ). At least seven bands can be clearly identified in this figure and they are numbered in the order of their electrophoretic mobilities. The fraction eluted from the CM-cellulose column at 1.0--1.3 M guanidinium chloride (Peak c in Fig. 3A) gives Band I in electrophoresis (Fig. 4,c), while Peak d in Fig. 3A contains mainly Band 2 (Fig. 4,d), the following broad shoulder (Fig. 3A,e) being composed of several slower-migrating components (Fig. 4,e). Another sperm-histone preparation isolated in the same manner (yield of sperm histone, 65%) has been analyzed on a Sephadex G-200 column (Fig. 3B). Further electrophoretic analyses of chromatographic fractions obtained from this column have revealed
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that Peaks 1, 2 and 3 {Fig. 3B,1, 2 and 3) consist mainly of Bands 1, 2 and 3 (Fig. 4,T2 ), respectively. All the slow-migrating bands disappear essentially completely upon treatment of these preparations with 0.2 M 2-mercaptoethanol in the presence of 8 M urea (pH 8), and are converted into Band 1 (Fig. 4,R2 ), whose mobility is, in turn, identical to that of the major fraction of bull sperm histone isolated under complete-dissociation conditions (Fig. 4,a). These results suggest that Band 1 represents the m o n o m e r of bull sperm histone. As shown in Fig. 5A, a linear relationship is found when relative electrophoretic mobilities of Band 1--7 obtained from Fig. 4 are semilogarithmically plotted against the band numbers, indicating that Bands 1--7 represent respectively the m o n o m e r to the heptamer of bull sperm histone. Similarly, a semilogarithmic plotting of relative elution volumes of each peak obtained from Fig. 3B versus the peak numbers also gives a linear relationship (Fig. 5B). Such oligomers of bull sperm histone have also been obtained by a brief exposure of spermatozoa to a complete-extraction medium. In this experiment, omitting the lengthy chromatin-preparation processes during which reshuffling and oxidation of sulfhydryl groups might occur, bull spermatozoa sedimented from semen were immediately incubated with 5 M guanidinium chloride containing 0.2 M 2-mer-
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Fig. 5. R e l a t i v e e l e c t r o p h o r e t i c m o b i l i t i e s and relative e l u t i o n v o l u m e s in gel e x c l u s i o n c h r o m a t o g r a p h y of bull s p e r m h i s t o n e f r a c t i o n s o b t a i n e d b y acid e x t r a c t i o n a f t e r i n c u b a t i o n o f s p e r m c h r o m a t i n in 0 . 2 M 2 - m e r c a p t o e t h a n o l - - 4 M urea. ( A ) R F values ( m i g r a t i o n distance o f p r o t e i n / m i g r a t i o n d i s t a n c e o f s o l v e n t ) o f e a c h b a n d n u m b e r e d in the order o f e l e c t r o p h o r e t i c m o b i l i t i e s in Fig. 4 , T 2 w e r e p l o t t e d against the l o g a r i t h m s o f the band n u m b e r s . (B) K a y ( V e - - V o / V t - - V o , w h e r e V e, V o and V t r e p r e s e n t e l u t i o n v o l u m e o f p r o t e i n , void v o l u m e , and t o t a l b e d v o l u m e , r e s p e c t i v e l y ) o f e a c h p e a k n u m b e r e d in t h e reverse order o f e l u t i o n v o l u m e s in Fig. 3B w e r e p l o t t e d against the l o g a r i t h m s o f t h e p e a k n u m b e r s .
captoethanol for 1 min at 0°C. Acid-soluble proteins were then isolated as described in Materials and Methods and their released sulfhydryl groups were blocked with iodoacetamide. Under these conditions, approx. 80% of sperm histone was extracted. They were then chromatographed on a CM-cellulose column (2 cm × 7 cm) using a step-wise elution with guanidinium chloride buffered with 0.05 M lithium acetate buffer (pH 5), and the protein fraction eluted between 0.8 M and 3 M guanidinium chloride was analyzed by polyacrylamide gel electrophoresis. As can be seen in Fig. 4,T3 and R3, sperm histone fractions thus obtained contain a number of slow-migrating bands, which are essentially completely converted to a single band corresponding to the m o n o m e r of bull sperm histone after reincubation of the protein fraction with 0.2 M 2-mercaptoethanol in the presence of 5 M guanidinium chloride. These results suggest that in bull sperm chromatin sperm histone molecules are cross-linked by disulfide bonds into a highly polymerized form, and such intermolecular disulfide linkages are partially retained in the sperm histone fraction isolated under the present conditions. A m i n o acid composition of bull sperm histone in the present study has been found to be in complete agreement with that reported by Coelingh et al.
506 [7]. Bull sperm histone (molecular weight, 6200) is characterized by its high content of arginine (24 residues) and cysteine (six residues). In order to determine h o w many of these cysteine residues are present in bull perm chromatin as disulfides, the number of sulfhydryl groups of sperm histone released by 2-mercaptoethanol during its extraction has been assayed. In this experiment, bull sperm chromatin was isolated in the presence of 5 mM iodoacetamide throughout the entire processes of chromatin preparation, and was further incubated with 5 M guanidinium chloride contianing 5 mM iodoacetamide and 0.05 M Tris buffer (pH 8) at 37°C for 1 h in the dark in order to block free sulfhydryl groups, if any. The chromatin thus treated was next incubated in a mixture containing 0.2 M 2-mercaptoethanol, 1.2 M NaC1 and 4 M urea (pH 8) at 37°C for 2 h, followed by extraction of sperm histone with 0.2 M HC1. The monomer of sperm histone was next purified by Sephadex G-75 chromatography using 0.01 M HC1 as eluant. The reaction of sperm histone with iodo[ 14C]acetamide was then followed during incubation in a mixture containing 10 mM iodo[ 14 C] acetamide, 5 M guanidinium chloride and 0.05 M Tris buffer (pH 8) at room temperature in the dark (Fig. 6), Data of Fig. 6 show that average 5.3 sulfhydryls per mole of bull sperm histone are released during its extraction in the presence of 2-mercaptoethanol, indicating that the majority of cysteine residues of bull sperm histone are present in the chromatin as disulfides. Since the same value has been obtained when bull sperm chromatin particles are mechanically disrupted before their incubation with non-radioactive iodoacetamide, the high number (average 5.3 sulfhydryls released by a thiol per sperm histone molecule) does not appear to be due to a failure of ]
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Fig. 6. Determination of released sulfhydryl groups of bull sperm histone. Completely reduced bull sperm histone isolated f r o m bull sperm chromatin which was preincubated with non-radioactive iodoacetamide, was incubated at a protein concentration of 8 0 0 pg/ml with 10 m M iodo[14C]acetamide (spec. act., 8.12 • 105 cprn/~zmole) in the presence of 5 M guanidinium chloride buffered with 0.05 M Tris buffer (pH 8) at r o o m temperature in the dark. S p e r m histone was precipitated f r o m aliquots taken at various lengths of incubation time with 5 % trichloroacetic acid after an appropriate dilution. Protein and radioactivity of each sample were determined and moles of iodo[14C]acetamide reacted per m o l e of sperm histone calculated. Iodo[ 14 C] acetamide was purchased from ICN, Irvine, Calif.
507 iodoacetamide in penetrating into the chromatin particles during the preincubation. In this experiment, bull sperm chromatin was suspended in 5 M guanidinium chloride containing 5 mM non-radioactive iodoacetamide, quickly frozen with a large excess of glass beads (Type 100--5005, 3M Company, St. Paul, Minn.), and ground thoroughly using a mortar and pestle. This was then incubated at 37°C for I h, followed by extraction of sperm histone in the presence of 2-mercaptoethanol and reaction with iodo[ ~4 C] acetamide as described above. As has been described earlier in this report, approx. 10% of the total sperm chromatin proteins precipitate with DNA when bull sperm chromatin is treated with acid following incubation in 0.2 M 2-mercaptoethanol--l.2 M NaC1--4 M urea. To test a possibility that disulfide-bond formation between sperm histone and such acid-insoluble proteins might make any significant contribution for the packaging of DNA, bull sperm chromatin preincubated with non-radioactive iodoacetamide as described above, was first incubated (37°C, 2 h) in a mixture containing 0.1 M 2-mercaptoethanol, 1.2 M NaC1, 4 M urea and 0.05 M Tris buffer (pH 8), and i o d o [ 1 4 C ] a c e t a m i d e (spec. act., 1.74 • 104 cpm/pmole) was then added to a final concentration of 0.2 M. After an additional incubation for 1 h (37°C in the dark), sperm histone and acidinsoluble proteins were isolated and their radioactivities determined. Results showed that the radioactivity found in the acid-insoluble protein fractions was only 6% (315 cpm) of those found in the sperm histone fractions (5260 cpm). The number of sulfhydryls released by 2-mercaptoethanol per mole of sperm histone was found to be 5.5 in this experiment. These results indicate clearly that most, if not all, disulfide linkages present in bull sperm chromatin are those between sperm histone molecules. It seems thus clear that, although it is possible that certain minor components of sperm chromatin proteins might play certain roles in the packaging of DNA, the DNA of bull sperm chromatin is packaged by sperm histone molecules which are cross-linked through formation of disulfide linkages to form a tight network. The cross-linking appears to be so complete that essentially all the cysteine residues of sperm histone are present as disulfides. The amino acid sequence of bull sperm histone [8] has revealed that the majority of arginine residues are in the middle of the molecule, and two cysteine residues are distributed in this region, while two residues of cysteine are present in the amino-terminal region and two in the carboxy-terminal region of the molecule. The cysteine residues present in the less basic terminal regions seem likely to be the ones involved in polymerization of bull sperm histone through formation of intermolecular disulfides between neighboring sperm histone molecules on the DNA. The packaging of DNA might be further tightened by intramolecular disulfide-bond formation of cysteine residues present in the arginine-rich middle region or these cysteine residues might be involved in corss-linking of neighboring chromatin strands through formation of interchromatin-strand disulfide linkages. Further chemical analyses of mammalian sperm histone fractions whose disulfides have been partially preserved (cf. Figs 3, 4 and 5), as well as studies of sperm histone during formation and maturation of mammalian spermatozoa would provide the further information concerning the molecular architecture of mammalian sperm chromatin.
508
Acknowledgments We are grateful to Drs H. Stanley Bennett and J. Logan Irvin for their stimulating discussion, and to Dr Edward Ezrailson for his helpful information. We would like to thank Dr Lester Ulberg and his staff, Reproductive Physiology Research Laboratories, North Carolina State University for their kind supply of bull semen, and Dr J. Logan Irvin and Mr Jack Faircloth for amino acid analysis. This work was supported by a grant from the Rockefeller Foundation to the Laboratories for Reproductive Biology, University of North Carolina. References 1 2 3 4 5 6 7 8 9 10
11 12 13
D e L a n g e , R . J . a n d S m i t h , E . L . ( 1 9 7 1 ) A n n u ° B,ev. B i o c h e m . 4 0 , 2 7 9 - - 3 1 4 B l o c h , D.P. ( 1 9 6 9 ) G e n e t i c s 6 1 , S u p p l . I~ 9 3 - - 1 1 1 P a o l e t t i , R . A . a n d H u a n g , R , C . C . ( 1 9 6 9 ) B i o c h e m i s t r y S, 1 6 1 5 - - 1 6 2 5 M a r u s h i g e , K. a n d D i x o n , G . H . ( 1 9 6 9 ) Dev. Biol. 1 9 , 3 9 7 - - - 4 1 4 B r i l - P e t e r s e n , E. a n d W e s t e n b r i n k , H . G . K . ( 1 9 6 3 ) B i o c h i m . B i o p h y s . A c t a 7 6 , 1 5 2 - - 1 5 4 H e n d r i c k s , D.M. a n d M a y e r , D . T . ( 1 9 6 5 ) E x p . Cell Res. 4 0 , 4 0 2 - - 4 1 2 C o e l i n g h , J.P., R o z i j n , T . H , a n d M o n f o o r t , C . H . ( 1 9 6 9 ) B i o c h i m . B i o p h y s . A c t a 1 8 8 , 3 5 3 - - 3 5 6 C o e l i n g h , J.P., M o n f o o r t , C . H . , R o z i j n , T . H . , L e u v e n , J . A . G . , S c h i p h o f , R., S t e y n - P a r v ~ , E.P., B r a u n i t z e r , G . , S c h r a n k , B. a n d R u h f u s , A . ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 8 5 , 1 - - 1 4 K i s t l e r , W . S . , G e r o c h , M.E. a n d W i l l i a m s - A s h m a n , H , G . ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 4 5 3 2 - - 4 5 4 3 B o n n e r , J . , C h a l k l e y , G . R . , D a h m u s , M,, F a m b r o u g h , D., F u j i m u r a , F., H u a n g , R . C . C . , H u b e r m a n , J., Jensen~ R., M a r u s h i g e , K . , O h l e n b u s e h , H., O l i v e r a , B. a n d W i d h o l m , J. ( 1 9 6 8 ) in M e t h o d s in E n z y m o l o g y ( G r o s s m a n , L. a n d M o l d a v e , K., eds), Vol. XII, P a r t B, p p . 3 - - 6 5 , A c a d e m i c Press, N e w York D i s c h e , Z. ( 1 9 5 5 ) in T h e N u c l e i c A c i d ( C h a r g a f f , E. a n d D a v i d s o n , J . N . , e d s ) , Vol. I, p p . 2 8 5 - - 3 0 5 , A c a d e m i c Press, N e w Y o r k L o w r y , O . H . , R o s e b r o u g h , N . J . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 B h a r g a v a , P.M., B i s h o p , M . W . H . a n d W o r k , T.S. ( 1 9 5 9 ) B i o c h e m . J. 7 3 , 2 4 2 2 4 7