i98

BBAEXP 91960

Bio:'him&:a et Biophv~ica Acta, 1008 09891 198L202 El~vier

Changes in chromatin structure of boar late spermatids to mature spermatozoa by using modification with dansyl chloride

Kuniko Akama ", Shusaku Oka a, Tohru Tobita a and Hiroaki Hayashi b " lhT'artment Of ('!w~iYtC~ t'~wultv ~/7ciowe~ Chiba ~;tffterzet~" Chiba City 260 and ~ lnxtitute of En&~crinologv, Gu~m~a Utffversity,

Maeba~ht 371 (Japan)

(Received 24 Nm,ember 1988) (Revised manuscript received 9 March 1989)

Key words: Chromatin structure: Protamine; Dansyl modification; (Boar swarm nuclei)

Changes in the chromatin structure of boar late spermatids maturing to spermatozoa were studied by chemical m~nlificalion of their nuclei ~ith dansyl (Dns) chloride. Protaminc was isolated from the dansylated boar spermatid and sperm nuclei, and its dansylated sites and degrees of dansylation were determined by sequence analysis. The N-terminal Mad, Tyr-3 and Tyr-42 of the protamine molecule in cauda epididymal sperm nuclei were dansylated 27%, 22% and 40~, respectively, whereas the respective residues in late spermatid nuclei were about 1.5-tlmes as reactive as those in cauda epididymal sperm nuclei. However, the dansyl ratio of Tvr-3. to Tyr-42 remained unchanged from. the. late: spermatid to mature sperm nuclei. SDS treatmen t did not affect the reactivity of cauda epididymal protaminc and: that of Ala~! of caput epididymal protamine, but raised that of Tyr,3 and Tyr-42 of caput epididymal protaminc by a factor of about 1,5, As a result of the SDS treatment, caput epididymai protamine came to have almost the same reactivity :as late spermatid protamine. These facts suggest that the fundamental structure, in terms of DNA.protamine interaction, of:sperm chromatin was already formed at the st:~ge of the late spermatid, and then during epididymal transit the sperm chromafin was more tightly condensed. ~ with increasing disulfide cross-links, thereby acquiring insensitivity towards the SDS-trcatment.

Introduc!ion

The organization and the fundamental structure of sperm chromatin have not been completely understood, as compared with the well-defined structure of somatic Chromatin. Most mammalian sperm nuclei consist of I)NA, very basic protamine and additional minor non- basic proteins [1,2], and they differ from other sperm nuclei, such as thc, se of fish and fowl [3], in the presence of many disulfide bonds, thereb'~: forming a tight net- work su,acture [4,5], These disulfide bonds are progres- sively formed as spermatozoa traverse the epididymis [6,7], and the sperm nuclei become more resistant to detergents [8~9], trypsin [10], and micrococcal nuclease [11 ]. In this worL we have studied the structural changes in the chrornatin of boar late spermatids maturing to Spermatozoa using chemical modification with dansy[ Chloride. The results show that the fundamental struc- ture o f the ~perm chromatin is already formed in the late spermatid nuclek

Corre~pt!ndcncc: K. Ak;mia, l)q~nmctl~ of Chemistrs', Facuhy of ~ience~ Chiba University. Chiba City 260 Japan~

0i:67-4781/89/$0 ~, sO ::~-; t 989 Elsevier Science Publishers B.V. (Biomedical

Materials and Methods

Materials, Dansyl chloride was purchased from Nakarai Chemicals, Twice-crystallized trypsin was from Milipore. Porcine carboxypeptidase B was prepared by our own method [12]. Bio-Rex 70 and Chemcosorb 3C18H were flora Bio-Rad and Chemco, respectively.

Preparation, t f boar epididymal sperm and late spermatid nuclei, Epididymal sperm nuclei were pre- pared by our' own method [11], and late spermatid nuclei by the method of Marushige and Marushige [13] in the presence of 5 mM sodium iodoacetate.

Modification ~:f ,~perm and spermatid nuclei with dansvl chloride. Unless otherwise indicated, the nuclei were dansylated rapidly after the isolation and SDS-treat- ment described below: The sperm and spermatid nuclei were suspended in 0 : I M sodium bicarbonate (pH 8.0) (1 mg DNA or protamine/m!). An equal volume of a solution of dansyl chloride (5 mg/ml: a c e t o n e ) w a s added to the suspension. The niixture was stirred at 37°C for 1 h and then centrifuged at 3000 × g for20 min. The precipitated dans~. ~ nuclei were washed with acidified acetone and distilled water. The late spermatid nuclei were dansylated without the SDS-treatment, be- cause the SDS-treatment destroyed them.

Division)

r

Treatment with SDS. Before or after dansylation, the sperm nuclei were treated with 0.25 SDS/5 mM iodoacetic acid/10 mM Tris-HCi (pH 8.0) (1.5 mg protein/ml) for 16 h at 4°C to remove acrosome and nuclear envelope from the nucleus [14]. The SDS-treated nuclei were well washed with 1 mM PMSF/5 mM ~odium iodoacetate/10 mM Tris-HC! (pH 8.0).

lsolation :oJ modified protamine from dansyl nuclei. The modified protamine was isolated by our method [15] with some modifications. The acid-extracted pro- tein from the nuclei was dissolved in 0.2 M HCI (1 mg protein/ml), and pH of the solution was neutralized with 1 M NaOH. The sc !:~,tion was applied to a column o f Bio-Rex 170. The column was washed successively with 0.1 M sodium acetate buffer (pH 60), distilled water and 0.3 M acetic acid. The protamine was eluted with 0.1 M HCI, and lyophilized. The protein was passed through a column of AG-1 X-2 (C! form) in distilled water to remove contaminated dansylic acid, and !yophilized. The purity of the protamine was con- firmed by amino-acid analysis. The yields of the pro- tamine from the dansyl-nuclei per 10 g each of cauda and caput epididymal ducts and testis were 25, 7 and 0.38 mg, respectively, on the basis of amino-acid com- position.

Tryptic digestion and separation of tryptic peptides. The protamine (50 nmol) in 0.3 ml of 5 mM CaCI:/50 mM T~s, HCI (pH 8.0) was digested with trypsin (0.28 nmol) at 25°C for 24h. 3 # lo f 10~ trifluoroacetic acid was added to stop the digestion. The digest was dried in vacuo, redissolved in 0.1~ trifluoroacetic acid, and sub- jected to reverse-phase high-performance liquid chro- matography on Chemcosorb 3C18H. The peptides con- taining tyrosine residue and dansyl-amino-acid residu,~ were selectively detected by measurement of the ab- sorbance at 280 nm, since both the tyrosine and the dansyl group have high: absorbance at 280 nm. The peptides in each peak were lyophilized and redissolved in 0.5 ml of 0.1% trifluoroacetic acid. Aliquots were used for amino-acid and N-temunal analyses.

Amino-acid analysis. Samples were hydrolyzed in vacuo with 6 M HC! at 110 °C for 24 h. Amino acids were determined on a Hitachi 835 automatic amino-acid analyzer.

N-terminal analysis. The N.terminus of the peptides was determip.-d by the dansylation method of Gray [16].

Resuhs and Discussion

Boar protamine has the N-terminal alanine, one histidine, and two tyrosine residues to be modified with dansyl chloride. The amount of dansyl,amino-acid re- sidues of the modified protamine was estimated from the :difference in the amino-acid residues between the unmodified and modified protamines. Upon acid hy-

199

drolysis (6 M HCI, ]10°C, 24 h), Dns-(imid.)-His is completely decomposed to free histidine [16], and Dns- Ala was recovered by 70¢~, and Dns-(O)-Tyr almost completely. When free boar pr\~tamine was labeled with dansyl chloride, the N-terminal Ala was 67% labeled (corrected for the recovery percentage of 70), and tyro- sine residues were labeled almost completely. Since one of the fish protamines, clupeine Y1 (NH,-Ala-Arg-Arg-) [3], was also 64~ labeled (corrected for the recover' percentage) with dansyl ~hloride under the same experi- mental conditions, the somewhat low reactivity of !he N-terminal Ala of these protamines may be a character- istic of these very basic proteins. When we modified the SDS-treated cauda epididymal sperm nuclei with dansyi chloride at 37°C for 1 h and 16 h, 32 and 36~ of the protamine tyrosines were dansylated, respectively. The percentage of Dns-Ala was low in both cases. We therefore used 1 h as the reaction time to dansylate the nuclei in the following experiments.

The protamine isolated from the modified nuclei was digested with trypsin, and the resulting tryptic peptides were separated by reverse-phase high-performance liquid chromatography on Chemcosorb 3C18H. Fig. 1 shows a typical elution pattern of the tryptic peptides of the protamine isolated from the intact dansylated-caput epididymal nuclei. Table 1 shows amino-acid composi- tions, N,terminal amino acids and sequences of the peptides in peaks PI-P6. The amino-acid sequence of boar protamine [17] is also shown at the top of Table 1. The peptides in peak P1 were rechromatographed on Chemcosorb 3C18H (Fig. 2). Amino-acid comp~:~sitio~= of the peptides in each peak are shown in Table II. The

t 1p I

i J ~ 4JO O 20

i P 4 / 40

i l z u. ~s

| ! |

6O Fraction Number

Fig. I. High-performance liquid chromatography (HPLC) of the t~ptic pcptidrs of the protamin¢ from the intact dansylatcd caput ¢pididy- ma! nuclei on Chemcosorh 3C18H. The digest of the protamine (56 nmol) was chromatographer on a column (6×100 ram) of Chem- cosorb 3C18H in 0.I~ trifluoroac©tic acid with an acctonitrile (McCN) linear gradient of 0-80q[ at 40 o C. Fractions (I m~) were collected at a

flow rate of I ml/mim

2 0 0

TABLE l

Complet e amino.a~.id sequence of I~mr protamine, and amino -acid compositions. N-terminal amino acids and sequences of tO'pric peptides of the modified protamim, isolated from the intact damylated caput epidi,,~rrnal nuclei 1 10 20 30 4 0 50

A R Y R C C R S H S R S R C R P R R R R C R R R R R R C C P R R R R R A V C C R R Y T V I R C R R C

The values of amino-acid residues are normalized to 100 nmol of the protamine.

Residue PI E? P3 P4 P5 P6

CM-Cys 140 Thr 1.9.~ 43.8 1.94 28.6

Ala a a Val 2.58 42.2 3.02 26.8 lie Z08 38.8 228 24.6 Tyr 66.2 1.4.8 45.4 b b b Arg 149 l'i.J 55.2 15.9 4.88 32.6

N-terminal amino acids Tyr Ala ~ Tyr Tyr c Arg Tyr " CM-Cys Tyr Ala c

S~-n:luences of peptides a 3 -4 D n s - l - 2 42 - 46 3 -4 41-42 - 46 42 ~ 46

I 1 I 42 - 46 Dns-1-2 Dns Dns Dns

and e, after acid hydrolysis, (a) and (b) were identified as Dns-Aia and Dns-(O)-Tyr, respectively. Derived from DNS-Ala and Dns-(O)-Tyr of original peptides, respectively.

a Residue numbers in boar protamine; Dns-1 indicates Dn.,;-Ala; I indicates Dns-(O)-Tyr. Ons

o

/ -

"7"

"/" J ~'i Plc " - I ~ ~ z / /

t. 1 , I l I I l ~

0 20 4 0 F r o c f l o n N u m b e r

Fig. 2. HPLC of the peptides in peak P1 in Fig~ 1 on Chemcosorb 3Ct8H. The peptides in peak PI in Fig. I from the protamine (39 nmol) ~ere chromatographed on a column (6× 100 ram) of Chem- co,orb 3C18H in 0.tq~ trifluoroacetic acid with an acetonitrile linear gradient of 0-3% at 40 o C, Fractions (0.5 mt) were collected at a flow

rate a l l ml /min .

Tyr-Arg peptide (residues 3-4) was found in peak Plc. Siac¢ P2 and P3 were not completely separated, the az:,o~,j,~ of Dns-Ala-Arg (residues 1-2) was estimated

Amin:~ aci~ , :mpositions of peaks in Fig. 2

The ~atues ~: ! anfino-acid residues are normalized to 1(~ nmol of the protaminc CM-Cys, Carboxymethyi cysteine.

Residue PI a P lb Plc P ld

CM-Cys T~F

Arg

38.4 53.3 19 1 49.5

94.5 40.9 65.1 4L6

from the difference between the total amount of arginine and the amount of arginine derived from Tyr-Thr-Val- Ile-Arg (residues 42-46) in each of P2 and P3. Dns-Ala- Arg in peak P2 was ascertained as follows. The digest of the peptides in peak P2 with carboxypeptidase B was chromatographed on Chemcosorb 3C18H (Fig. 3). When the effluent was detected by measurement of the fluo-

!j° t . , ¢ / f

/ / ~

O

~ t I i i 0

b

0o

U

0 2 0 40 T i m e ( m l n )

Fig. 3. HPLC of Dns-alanine and the digest of the peptides in peak P2 in Fig. i with carboxypeptidase B. (a)Dns-alanine (9.8 nmol); (b) t h e digest of t he peptides in peak P2 in Fig. 1 (2.5 nn~l ) with porcine carboxypeptidase B (12.5 pmo!) at 37 ° C for 4 h i Each sampl e was chromatographed on a co lumn ( 4 x 150 mm) of Chemcosorb 3C18H in 0.1% trifluoroacetic acid with an acetonitrile l inear gradient o f

0-40% at a flow rate of 1 m l / m i n at 40°C .

rescence at 547 nm upon excitation at 322 nm on a Hitachi F1000 fluorescence spectrometer, a major peak had the same retention time as a standard Dns-Aia. and its amount was almost the same as that estimated from the amino-acid analysis described above. The peptides containing a tyrosine residue were completely separated from those containing a Dns-(O)-Tyr residue. Thus, each peak in Fig. 1 was identified as follows: peak P1, Tyr-Arg (residues 3-4) and some other peptides; peak /'2, a 1:0.13 mixture of Dns-Ala-Arg (residues 1-2) and Tyr,'i hr-Val-lle-Arg (residues 42-46); peak P3, a 1:0.30 mixtme o f Tyr,Thr-Val-lle-Arg (residues 42-46) and Dns-Ala,Arg (residues 1-2); peak P4, Dns-(O)-Tyr-Arg (residues 3-4); peak P5, Arg-Dns-(O)-Tyr-Thr-Val-lle- Arg (residues 41-46); peak P6, Dns-(O)-Tyr-Thr-Val- lle-Arg (residues 42-46).

The dansylation percentage of Tyr-Arg peptide was calculated from the molar ratio: [Dns-(O)-Tyr-Arg]/ ([Tyr-Arg] + [Dns-(O)-Tyr-Arg]); likewise for the dansy- lation percentage of the peptides containing Tyr-42. The dansylation percentage of Ala-Arg peptide was calcu- lated from the molar ratio of Dns-Ala-Arg whose amount was corrected for the chromatographic recovery (82%) to the protamine used for digestion. The average percentage of Dns-Tyr,3 and Dns-Tyr-42, and the per- centage of: Dns'AIa,1 were almost equal to those esti- mated from the results o f amino-acid analysis of the modified protamine without tryptic digestion. These results showed that 32~ of Ala-1, 20~ Of tyr-3, and 41% of Tyr-42 of the protamine were dansylated in the intact caput epididymal sperm nuclei.

The tryptic peptides of the protamines from other dansylated nuclei were also analyzed as described above. These results are shown in Table III. The N-terminal Ala-l, Tyr-3, and Tyr-42 of the protamine in the cauda epididymal sperm nuclei were dansylated 27%, 22% and 40%, respectively. The respective residues of the pro- tamine were about 1.5-times more reactive in the l.'.te spermatid nuclei than in the cauda epididymal sperm nuclei. However, the dansyl ratio of Tyr-3 to Tyr-42 remained unchanged from the late spermatid to mature

TABLE ill

Percentage of Dns.alanine-l, Dns-(O).tyrosine.3, and Dns-(O).tyrosine. 42 o[ the modified protamine [rom the dansylated eauda and caput epididymal sperm and late spermatid nuclei

Treatment Dansylated Cauda Caput Late resir~ue sperm sperm spermatid

Intact Ala-1 27 32 37 Tyr-3 22 20 35 Tyr-42 40 41 60

SDS-trcated Ala-1 22 33 Tyr-3 22 36 Tyr-42 40 56

20!1

sperm nuclei. The SDS treatment did not change the reactivity of cauda epididymal protamir~e, but enhanced that of Tyr-3 and Tyr-42 of caput epidk~ymal protamine about 1.5-times. However, the reactivity of Ala-I of intact caput epididymai protamine wa:~ unaffected by the SDS treatment. The reactivity of the SDS-treated caput epididymal protamine was almost the same as that of the late spermatid protamine. The~ results showed that the fundamental structure, in terms of DNA-orotamine interaction~ of the sperm cm~omatin was already formed in the late spermatid nuclei, and that the caput sperm chromatin was mostly conden~d, but sensitive to the SDS treatment, whereas the cauda sperm chromatin was rigidly condensed and locked by disulfide bonds, being insensitive to the SDS treatment.

The o:ganization of mammalian sperm nuclei has been studied by electron microscopy [18-22], micrococ- cai nuclease digestion [22-24], X-ray di, ffraction [22] and fluorescence anisotropy [25], giving ,conflicting re- sults such as a beaded chromatin [~8,24], a sm~x~th chromatin [20-22], or both [19]. The reconstitution ex- pefimems from dansylated protamine and DNA pr;,w:~ that boar protamines bind to DNA through two iunc- tional domains of a central arginine-clustering basic region and N- and C, terminal less basic regions (arm region) [15:], supporting Balhom's proposal [26]. The results of differential cleavages of disulfide cross-links by limiting reduction of boar sperm nuclei irMicated that the cross-linkings of the protamine are formed in the order cysteine-47, cysteine-50, cysteine-38-cYsteine- 39, cysteine-14, cysteine-21, cysteine-5-cysteine-6, and cysteine-28-cysteine-29 [27]. Considering the Balhorn model [26] together with our results, the formation of the sperm chromatin is thought to be as follows. The central region of boar protamine predominantly binds DNA, forming a core complex. The N-terminal arm bends back, up and over the DNA-bound a;ginine cluster. The C-terminal region of an adjacent protamine molecule containing several cysteine and tyrosine re- sidues overlaps with the N-terminal region. Tyr-42 in the C-terminal region was more reactive to dansyl chlo- ride than Tyr-3 and Ala-1 in the N-terminal region among the late spermatid and epididymal sperm nuclei. Although the cysteine residues in the C-terminal region have been crosslinked at the early stage of chromatin packing [27], the side-chain of Tyr,42 would be oriented outwards to be more accessible to the reagent; On the other hand, the side'chain of Tyr,3 as well as the a-amino group of Ala-1 in the N-terminal region, where the disulfide bonds are formed later than those in the C-terminal region [27], probably interacts with some other sites o f the chromatin through hydrogen bonds and/or hydrophobic bonds, thereby being less accessi- ble to the reagenL: The caput sperm chromatin that is about 20~ stabilized by disulfide bonds from th,; results of nuclease digestion [11] is mostly condensed, bu:t

2 0 2

sensit ive to the SDS- t r ea tmen t . The re fo r e , h y d r o g e n b o n d s a n d / o r h ) d r o p h o b i c in te rac t ion will be necessa ry

in ~,he organiza t ion of the capu t spe rm ch roma t in . T h e cauda spe rm c h r o m a t i n is ful ly c o n d e n s e d and s tabi-

l ized by disui~Me bonds, and a lmos t total ly res is tant to

trypsi:~, nuclease and de te rgen ts such as S D S [8-11].

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