- Email: [email protected]
Characterization of the nuclear basic proteins specific of spermiogenesis in cuttlefish, Sepia officinalis
86
Biochimica et Biophysica Acta
953 (1988) 86-94 Elsevier
BBA 33077
Characterization of the nuclear basic proteins specific of spermiogenesis in cuttlefish, Sepia officinalis Dani~le Wouters-Tyrou a, Annie Martin-Ponthieu a, Alain Richard b and Pierre Sauti~re a a Units AssociSe 409 du Centre National de la Recherche Scientifique, Institut de Recherches sur le Cancer, Lille and b Station Marine de Wimereux, UniversitS des Sciences et Techniques de Lille Flandres Artois, Wimereux (France)
(Received12 October 1987)
Key words: Nuclear basic protein; Spermiogenesis;Alkalinephosphatase; CarboxypeptidaseB; (S. officinalis)
Two highly basic proteins which appear during cuttlefish spermiogenesis have been isolated from testis chromatin and from sperm nuclei. (1) Protein T is a spermatid-specific protein which is transiently associated to DNA and disappears in spermatozoa. It is rich in arginine (approx. 60%) and is phosphorylated at different levels. (2) Protein Sp is a typical protamine which appears in the late stage of spermiogenesis and constitutes the major basic nuclear component in mature spermatozoa. It is very rich in arginine (approx. 77%) and contains only four different amino acids. Phosphorylated in the testis, it is completely dephosphorylated in the spermatozoa. It is suggested that protein T could be a precursor of protein Sp.
Introduction In mature sperm cells of most animal species, somatic histones are replaced by generally smaller and more basic proteins called protamines. This protein transition is either direct, as in fishes [1-3], or indirect, involving the transitory existence, in mid- or late-step spermatids, of other basic proteins intermediate between somatic histones and protamines, as described in mammals [4-8] and in dogfish [9]. Biochemical evidence for such intermediate proteins has never been obtained for invertebrate species. Most of the studies have been performed on mature sperm cells where a great variability of nuclear basic proteins was found according to the degree of evolution of the concerned species (for a
Correspondence: P. Sauti6re, UA 409 CNRS, Institut de Recherches sur le Cancer, Place de Verdun, F-59045 Lille C~dex, France.
recent review, see Poccia in Ref. 10). But little information is available about proteins associated with DNA during the early stages of spermiogenesis. To study these proteins, the cuttlefish, S e p i a o f f i c i n a l i s (Cephalopoda), appears as a good model, since its testis is well differentiated and contains, when the animal has reached its sexual maturity, all types of germinal cells, from spermatogonia to elongated spermatids. The choice of this model lies also in the fact that cuttlefish exhibits some features of higher vertebrates such as eye organization and internal fertilization in contrast to other marine invertebrates. The work reported here concerns the char° acterization of the specific nuclear basic proteins from the cuttlefish testis (immature germinal cells) and sperm (mature germinal cells). Biochemical evidence is presented for the existence of a transient protein which appears in round spermatids and is replaced by a major typical protamine in elongated spermatids. These proteins were char-
0167-4838/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
87 acterized by their amino-acid composition and their electrophoretic mobility. Materials and Methods
Testes and spermatozoa from adult cuttlefishes were obtained at the Station Marine de Wimereux (France). After excision, tissues were frozen in liquid nitrogen and stored at - 8 0 °C until use.
Extraction of nuclear basic proteins All operations were carried out at + 4°C and in the presence of 0.1 mM diisopropyl fluorophosphate. From testes. Purified nuclei were prepared from testes and chromatin subsequently isolated from nuclei as described by Chauvi&e et al. [9]. Total acid-soluble proteins were obtained from chromatin by overnight extraction with 0.4 M HC1. The residual pellet was then extracted for 18 h with 5 M guanidinium chloride containing 100 mM 2-mercaptoethanol. Both extracts were dialyzed extensively in Spectrapor 3500 dialysis bags against deionized water and lyophilized. From mature spermatozoa. The isolation of sperm nuclei was performed according to the procedure described by Sautirre et al. [11], using 2% Triton X-100 instead of 4% Tween 80. The nuclear acid-soluble proteins were extracted from purified nuclei by stirring overnight with 0.4 M HC1. The residual pellet was then treated for 18 h with 5 M guanidinium chloride containing 100 mM 2mercaptoethanol. Each extract was subsequently fractionated by two successive precipitations, the first with 3.5 Vol. of acetone containing 0.07 M HC1 and the second with an additional 2.5 Vol. of acidified acetone, for 48 h at - 2 0 ° C. The precipitates were recovered by centrifugation at - 2 0 ° C and dissolved in 0.01 M HC1. Protein solutions were then dialyzed in Spectrapor 3500 dialysis bags against distilled water and lyophilized.
Purification of proteins Spermatid-specific protein (protein T) was obtained in pure form from acid extracts of testis chromatin by gel-filtration chromatography on Bio-Gel P-10 (150 X 5 cm), equilibrated and eluted with 0.01 M HCI saturated with chloroform.
The phosphorylated forms of protein T were separated by ion-exchange chromatography on a carboxymethyl cellulose column (25 × 2.5 cm), equilibrated with 0.5 M NaC1 in 0.05 M sodium acetate (pH 6.0). The proteins were dissolved in the same buffer in the presence of 6 M urea and applied to the column. They were eluted with a linear gradient from 0.5 to 1.5 M NaCI in acetate buffer. Protein Sp was purified by high-performance liquid chromatography (HPLC) fractionation of the testis or sperm extracts on a /tBondapak C18 column (300 x 3.9 mm) (Waters Associates), eluted with a linear gradient of acetonitrile from 0 to 50% in 0.05% trifluoroacetic acid.
Analytical gel electrophoresis Protein fractions were analyzed by polyacrylamide gel electrophoresis on 17% acrylamide slab gels at pH 3.2, in the presence of 6.25 M urea [12].
Amino-acid analysis The amino-acid composition of proteins was determined after hydrolysis in 6 M HC1, in vacuum, at 110 o C, for 24 and 72 h. One drop of 1% phenol was added to prevent excessive degradation of tyrosine. Amino-acid analyses were performed on a Beckman 119 CL amino-acid analyzer. Presence of phosphoamino acids was investigated as described in Ref. 13.
A lkaline phosphatase treatment Protein T and protein Sp were hydrolyzed with alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1.) as described in Ref. 13. The proteins were recovered by precipitation with 6 Vol. of cold acetone and dried before electrophoretic analysis.
End-group determination The amino-terminal sequence was established by manual Edman degradation performed as in Ref. 14, and phenylthiohydantoin derivatives of amino acids were identified by high-pressure liquid chromatography on a column of #Bondapak C18 as in Ref. 15. For the carboxy-terminal sequence determination, proteins (50 nmol) were hydrolyzed in 1 ml
88
0
-I.-
H 4 J,,--
•
-qT
~Sp
1
2
3
4
5
6
7
8
9
10 11
5
+
s sU
C
m
Fig. 1. (a) Electrophoretic analysis of nuclear basic proteins specific of spermiogenesis from cuttlefish Sepia officinalis. Electrophoresis was performed on slab gel (160x180×0.75 mm) at pH 3.2, in 0.9 M acetic acid/6.25 M urea, using a 17% acrylamide concentration. Samples (3 ~tg), dissolved in 0.01 M HC1/8 M urea/0.5 M 2-mercaptoethanol, were run at 22 m_A for 2.5 h, after a pre-electrophoresis of 1.5 h. The gel was stained for 60 rain with 0.04% Coomassie blue R-250, in the mixture 0.08 M sodium picrate/2% acetic acid/2.5% ethanol, as described in Ref. 16. The gel was destained by diffusion in acetic acid/ethanol/water (7 : 20 : 73, v/v). Lane 1, whole histone from calf thymus, as reference; lane 2, 0.4 M HC1 extract from cuttlefish testis chromatin; lane 3, 5 M guanidinium chloride extract from cuttlefish testis chromatin; lane 4, ram protamine; lanes 5 and 6, 0.4 M HC1 extract from cuttlefish sperm nuclei, precipitated with 3.5 and 6 Voi. of acidified acetone, respectively; lanes 7 and 8, 5 M guanidinium chloride extract from cuttlefish sperm nuclei, precipitated with 3.5 and 6 Vol. of acidified acetone, respectively; lane 9, cuttlefish protein T; lane 10, cuttlefish protein Sp isolated from testis chromatin; lane 11, cuttlefish protein Sp isolated from sperm nuclei. (b and c) Densitometric scannings of 0.4 M HCI extract and 5 M guanidinium chloride extract from cuttlefish testis chromatin. Lanes 2 and 3 of (a) were scanned at a wavelength of 540 nm in a Vernon model PHI-6 spectrophotometer fitted with a linear transport assembly.
89
Protein T is only partially extracted from chromatin with 0.4 M HC1. Its complete extraction is ensured by treatment of the residual chromatin with 5 M guanldinium chloride (lane 3). In addition to protein T, guanidinium chloride releases a smaller and more basic protein, called protein Sp, the mobility of which is similar to that of ram protamine (lane 4). In testis extracts, protein T and protein Sp migrate as at least four bands. This has to be related to the existence of structural variants and post-translational modifications of the proteins, and will be discussed later. Somatic-type histones, protein T and protein Sp represent, respectively, 58, 28 and 14% of the total nuclear basic proteins of cuttlefish testis chromatin, as calculated from densitometric scanning of electrophoretic analyses of these proteins (Fig. lb and c).
of 0.2 M N-methylmorpholine acetate (pH 8.0), at 37°C, with carboxypeptidase B (peptidyl-Llysine(L-arginine) hydrolase, EC 3.4.17.2), using an enzyme/substrate ratio of 1:5000 by weight. Aliquots were taken off at time points, freezedried, and analyzed on the amino-acid analyzer. Results and Discussion As shown in Fig. la, the major nuclear basic proteins extracted from cuttlefish testis chromatin with 0.4 M HC1 (lane 2) have an electrophoretic migration very similar to that of somatic-type histones. The 0.4 M HC1 extract contains also another protein called protein T, of electrophoretic mobility intermediate between that of histone H4 (M r = 11 300) and that of ram protamine (M r -- 6 700, lane 4) [17].
b
a 1.0
+
6
50 °/, Acetonitrile
//- . . . . . . . . . . . . . /
I
/ /
E e/
¢0 0
t
// ,/
¢
t
/ / /
C.E
1
2
3 .J
0.5
/ / / / / i / /
/
//
2'5
Effluent (ml)
50
Fig. 2. (a) Fractionation of the 5 M guanidinium chloride extract from cuttlefish testis chromatin by reverse-phase high-pressure liquid chromatography. The extract, dissolved in 0.01 M HCI was loaded on a/~Bondapak C18 column (3.9 x 300 mm), equilibrated with 0.05% trifluoroacetic acid. Elution was performed with a linear gradient of acetonitrile ( . . . . . . ) from 0 to 50% for 30 min. Fractions of 0.5 ml were collected at a flow rate of 1 m l / m i n . The ehition of fractions was monitored at 206 nm. Fractions were pooled as indicated by solid bars. (b) Polyacrylamide gel electrophoresis of the fractions 1, 2 and 3 of (a). Electrophoresis was performed as described in the legend to Fig. 1. C.F., crude fraction, whole 5 M guanidinium chloride extract.
90
Protein T, separated from somatic-type histones by fractionation of the 0.4 M HC1 extract from cuttlefish testis chromatin on a column of Bio-Gel P-10, was obtained in pure form as shown in Fig. la, lane 9. Testis protein Sp was purified to homogeneity from the guanidinium chloride extract by reverse-phase HPLC (Fig. 2a). Three protein fractions were isolated which correspond, respectively, to protein Sp (peak 1), protein T (peak 2), and residual somatic-type histones, mostly H3 and H4 (peak 3), as shown by analytical polyacrylamide gel electrophoresis (Fig. 2b). On the other hand, the 0.4 M HC1 and guanidinium chloride extracts from purified sperm nuclei contain a major protein, migrating as two bands, whose electrophoretic mobility is very close to that of testis protein Sp. Histones and protein T are absent from these extracts (Fig. la, lanes 5-8). The purification of the sperm nuclear protein can
be performed in a similar manner to that of testis protein Sp (Fig. 3), although pure protein can be obtained more easily in large quantity simply by fractional precipitation with 6 Vol. of acetone from the guanidinium chloride extract from sperm nuclei (Fig. la, lane 8). The amino-acid compositions of proteins T and Sp from testis and sperm are presented in Table I. These proteins are characterized by a very large content of arginine (59, 77 and 77%, respectively) and significantly high amounts of serine and tyrosine. Testicular protein Sp and major sperm nuclear protein are very similar. They are constituted of only four different amino acids: arginine, serine, tyrosine and proline present in the same ratio. Therefore, they appear to be the same protein, which presents typical features of fish protamines: very high content of arginine (60-70%), low num-
b
+
a
50 °/~ Acetonitrile
1.0
/ . . . . . . .
/ /
//
E ¢-
/
/
/
//
£O
o
/
O4
//
< /
/
/
/
// /
0.5
/
C.F. l a l b lc 2 3 4 5 / / / / /
// /
/
/
2
// // /
/
//
0 °/oAcetonitrile 20
Effluent ( ml )
40
Fig. 3. (a) Fractionation of the 0.4 M HCI extract of sperm nuclei, precipitated with 3.5 Vol. of acidified acetone, by reverse-phase high-pressure liquid chromatography. See legend to Fig. 2a. (b) Polyacrylamide gel electrophoresis of the fractions 1-5 of (a) Electrophoresis was performed as described in the legend to Fig. 1. C.F., crude fraction, whole 0.4 M HC1 extract, precipitated with 3.5 Vol. of acidified acetone. Lanes la, l b and lc, peak 1 (ascending slope, top and descending slope). Lanes 2-5: peaks 2 to 5.
91 TABLE I
-I-
AMINO-ACID COMPOSITIONS OF NUCLEAR BASIC PROTEINS SPECIFIC OF SPERMIOGENESIS IN CUTTLEFISH SEPIA O F F I C I N A L I S Results are expressed as moi per 100 mol. The values for threonine and serine were obtained by linear extrapolation to zero hydrolysis time. Values in parentheses represent the number of residues per molecule of protein. Testis protein T
Sperm protein Sp
protein Sp
11.4 (6)
11.3 (6)
2.3 (1)
2.2 (1)
Asp Thr Ser Glu Pro Gly Ala Val Met Leu Tyr Lys Arg
2.1 (1-2) 0.6 (1) 9.6 (7) 2.6 (1-2) 1.4 (1) 3.1 (2) 3.3 (2-3) 1.7 (1-2) 1.0 (3) a 4.3 (3) 6.5 (5) 5.0 (4) 58.7 (42)
9.1 (5)
9.0 (5)
77.1 (42)
77.4 (42)
%Basic N-terminal C-terminal
63.7 Met Arg
77.1 Arg Arg
77.4 Arg Arg
a From preliminary structural data.
ber of constituent amino acids, absence of cysteine or acidic residues [2,11]. However, the presence of tyrosine is rather characteristic of mammalian protamines, although this amino acid was found in two fish species: tunafish [18] and dogfish [19]. The amino-acid composition of cuttlefish protamine is very close to that of calamar protamine, but quite different from that of octopus protamine [20]. Phosphoserine was identified only in testis protein Sp after limited acid hydrolysis and thin-layer electrophoresis in the presence of phosphoserine and phosphotyrosine. This can explain the difference between the electrophoretic patterns of testis and sperm proteins Sp. After treatment with alkaline phosphatase, the two proteins are resolved as the same two bands on polyacrylamide gel electrophoresis (Fig. 4). From these results, we can conclude that protein Sp in testis is a family of molecules phosphorylated at different levels, whereas protein Sp, in mature sperm, is not found
12
34
Fig. 4. Effects of alkaline phosphatase on the electrophoretic mobility of protein Sp. Lanes 1 and 2, testicular protein Sp after (1) and before (2) incubation with the enzyme. Lanes 3 and 4, sperm protein Sp after (3) and before (4) incubation with the enzyme.
phosphorylated. Nevertheless, the electrophoretic heterogeneity of protein Sp, even after treatment with alkaline phosphatase, strongly suggests that it could be constituted of at least two structural variants. Protein T differs from protein Sp by its molecular size and a greater amino-acid diversity. Acidic and hydrophobic residues are little represented and cysteine is absent. The features of this composition exclude the possibility that protein T results of a proteolytic degradation of an histone. The amino-acid diversity as well as the electrophoretic mobility and the size of protein T are typical of spermatid-specific proteins which, in
92
mammals and dogfish [5,7-9], appear transiently between removal of histones and deposition of protamines on DNA. However, cuttlefish protein T differs markedly from intermediate proteins identified in mammals and dogfish by a higher content in arginine which is close to that found in fish protamines [2,19]. When submitted to ion-exchange chromatography on carboxymethyl cellulose, protein T was resolved into five fractions of increasing electrophoretic mobility (Fig. 5). The amino-acid compositions of these fractions were identical. Presence of phosphoserine was detected in all fractions after acid hydrolysis for 2 h at l l 0 ° C , and the level of phosphorylation was found to decrease from the first eluted fraction to the last
one: thus, fraction 1 was found to be 3-times more phosphorylated than fraction 5. Moreover, after hydrolysis with alkaline phosphatase, all fractions exhibited the same electrophoretic mobility, on acid-urea gel (Fig. 6). Therefore, the apparent heterogeneity of protein T has to be related to different levels of phosphorylation of the protein. In conclusion, cuttlefish spermiogenesis is characterized by protein transitions similar to that observed during dogfish and mammalian spermiogenesis [9,21]. Two specific proteins appear sequentially to replace somatic-type histones in testis chromatin. Protein T appears in round spermatids is abundant in elongating spermatids and is no longer detected in elongated spermatids, as shown by immunocytochemical studies [22].
fl Turbidity 400 nm
b -IiN iiii
o.1
. 1 . 5 M NaCI
e
.
• ee
•
_
0.0
j j"
2 I
0.SM NaCI
650
3
4
13bd
Effluent (ml)
Fig. 5. (a) Separation of phosphorylated forms of spermatid-specific protein T from cuttlefish testis by chromatography on carboxymethyl cellulose. Five fractions (1-5) were eluted successively with a linear gradient of NaCI from 0.5 to 1.5 M in 0.05 M sodium acetate pH 6.0. Elntion of protein was monitored by turbidimetry at 400 nm. Fractions of 6.5 ml were collected at a flow rate of 20 m l / h . (b) Polyacrylamide gel electrophoresis of the fractions recovered from chromatography on carboxymethyl cellulose. Eleetrophoresis was performed as described in the legend to Fig. 1. C.F., crude fraction of protein T. A and B: peaks A and B from Fig. 5a. Lanes 1-5: fractions 1-5 from Fig. 5a, phosphorylated forms of protein T.
93
+
precursor molecule (in this case, protein T) like mouse protamine 2, as shown recently by Yelick et al. [23]. The complete determination of the amino-acid sequence of proteins T and Sp would provide reliable arguments in favor of this hypothesis.
Acknowledgements
D
mmb
The authors are grateful to Professor S. Frontier (Station Marine de Wimereux) for providing the cuttlefishes. They acknowledge with pleasure the skilful technical assistance of A. H6mez, F. Boutteau, M.J. Dupire and T. Ernout. They also thank T. Ernout for editorial assistance. This work was supported by grants from the Centre National de la Recherche Scientifique (RCP 0680), from Universit6 de Lille II, from the Minist6re de l'Industrie et de la Recherche (Contrat 84 C 0942) and from the Fondation h la Recherche M6dicale.
References
123456 Fig. 6. Effects of alkaline phosphatase on the electrophoretic mobility of protein T. Lanes 1-3: fractious 1, 3 and 5 from the carboxymethyl cellulose fractionation of protein T; before incubation with the enzyme. Lanes 4-6: the same fractions after incubation with the enzyme.
Protein T is a transitional protein phosphorylated in the testis and absent from the spermatozoa. Protein Sp appears in elongated spermatids and is a typical protamine, phosphorylated in the testis and nonphosphorylated in the mature sperm cells where it constitutes the major basic protein associated to DNA. On the other hand, the fact that protein T and protein Sp contain similar amounts of serine, proline, tyrosine and arginine strongly suggests that a close structural relationship exists between the two proteins. Protein Sp could be initially synthesized as a
1 Dixon, G.H. (1972) in Karohnska Symposium on Research Methods in Reproduction and Endocrinology (Diczfalusy, E., ed.), pp. 130-154, Karolinska Institute, Stockholm. 2 Ando, T., Yamasaki, M. and Suzuki, K. (1973) in Molecular Biology Biochemistry and Biophysics, Vol. 12, pp. 1-114, Springer-Verlag, Berlin. 3 Louie, A.J., Candido, E.M.P. and Dixon, G.H. (1973) Cold Spring Harbor Symp. Quant. Biol. 38, 803-819. 4 K.istler, W.S., Noyes, C., Hsu, R. and Heinrikson, R.L. (1975) J. Biol. Chem. 250, 1847-1853. 5 Grimes, S.R., Meistrich, M.L., Platz, R.D. and Hnilica, L.S. (1977) Exp. Cell Res. 110, 31-39. 6 Balhorn, R., Weston, S., Thomas, C. and Wyrobek, A.J. (1984) Exp. Cell Res. 150, 298-308. 7 Dupressoir, T., Sauti6re, P., Lanneau, M. and Loir, M. (1985) Exp. Cell Res. 161, 63-74. 8 Gusse, M., Sauti&e, P., B61aiche, D., Martinage, A., Roux, C., Dadoune, J.P. and Chevailher, P. (1986) Biochim. Biophys. Acta 884, 124-134. 9 Chauvi~re, M., Laine, B., Sauti~re, P. and Chevaillier, P. (1983) FEBS Lett. 152, 231-235. 10 Poccia, D. (1986) Intern. Rev. Cytol. 105, 1-65. 11 Sauti6re, P., Briand, G., Gusse, M. and Chevaillier, P. (1981) Eur. J. Biochem. 119, 251-255. 12 Panyim, S. and Chalkley, R. (1969) Arch. Biochem. Biophys. 130, 337-346. 13 Martinage, A., Gusse, M., B61ai'che, D., Sauti6re, P. and Chevailfier, P. (1985) Biochim. Biophys. Acta 831, 172-178. 14 Edman, P. and Henschen, A. (1975) in Protein Sequence Determination (Needleman, S.B., ed.), pp. 232-279, Springer-Verlag, Berlin.
94 15 Hermann, J., Titani, K., Ericsson, L.H., Wade, R.D., Neurath, H. and Walsh, K.A. (1978) Biochemistry 17, 5672-5679. 16 Stephano, J.L., Gould, M. and Rojas-Galicia, L. (1986) Anal. Biochem. 152, 308-313. 17 Sauti&e, P., Brlaiche, D., Martinage, A. and Loir, M. (1984) Eur. J. Biochem. 144, 121-125. 18 Bretzel, G. (1973) Hoppe Seyler's Z. Physiol. Chem. 354, 543-549. 19 Gusse, M., Sautirre, P., Chauvirre, M. and Chevaillier, P. (1983) Biochim. Biophys. Acta 748, 93-98.
20 Subirana, J.A., Cozcolluela, C., Palau, J. and Unzeta, M. (1973) Biochim. Biophys. Acta 317, 364-379. 21 Grimes, S.R., Jr. (1986) Comp. Biochem. Physiol. 83B, 495-500. 22 Rousseaux-Prrvost, R., Engelhardt, R.P., Rousseaux, J., Wouters-Tyrou, D. and Sauti~re, P. (1988) Gamete Res. in press. 23 Yelick, P.C., Balhorn, R., Johnson, P.A., Corzett, M., Mazrimas, J.A., Kleene, K.C. and Hecht, N.B. (1987) Mol. Cell. Biol. 7(6), 2173-2179.
Biochimica et Biophysica Acta
953 (1988) 86-94 Elsevier
BBA 33077
Characterization of the nuclear basic proteins specific of spermiogenesis in cuttlefish, Sepia officinalis Dani~le Wouters-Tyrou a, Annie Martin-Ponthieu a, Alain Richard b and Pierre Sauti~re a a Units AssociSe 409 du Centre National de la Recherche Scientifique, Institut de Recherches sur le Cancer, Lille and b Station Marine de Wimereux, UniversitS des Sciences et Techniques de Lille Flandres Artois, Wimereux (France)
(Received12 October 1987)
Key words: Nuclear basic protein; Spermiogenesis;Alkalinephosphatase; CarboxypeptidaseB; (S. officinalis)
Two highly basic proteins which appear during cuttlefish spermiogenesis have been isolated from testis chromatin and from sperm nuclei. (1) Protein T is a spermatid-specific protein which is transiently associated to DNA and disappears in spermatozoa. It is rich in arginine (approx. 60%) and is phosphorylated at different levels. (2) Protein Sp is a typical protamine which appears in the late stage of spermiogenesis and constitutes the major basic nuclear component in mature spermatozoa. It is very rich in arginine (approx. 77%) and contains only four different amino acids. Phosphorylated in the testis, it is completely dephosphorylated in the spermatozoa. It is suggested that protein T could be a precursor of protein Sp.
Introduction In mature sperm cells of most animal species, somatic histones are replaced by generally smaller and more basic proteins called protamines. This protein transition is either direct, as in fishes [1-3], or indirect, involving the transitory existence, in mid- or late-step spermatids, of other basic proteins intermediate between somatic histones and protamines, as described in mammals [4-8] and in dogfish [9]. Biochemical evidence for such intermediate proteins has never been obtained for invertebrate species. Most of the studies have been performed on mature sperm cells where a great variability of nuclear basic proteins was found according to the degree of evolution of the concerned species (for a
Correspondence: P. Sauti6re, UA 409 CNRS, Institut de Recherches sur le Cancer, Place de Verdun, F-59045 Lille C~dex, France.
recent review, see Poccia in Ref. 10). But little information is available about proteins associated with DNA during the early stages of spermiogenesis. To study these proteins, the cuttlefish, S e p i a o f f i c i n a l i s (Cephalopoda), appears as a good model, since its testis is well differentiated and contains, when the animal has reached its sexual maturity, all types of germinal cells, from spermatogonia to elongated spermatids. The choice of this model lies also in the fact that cuttlefish exhibits some features of higher vertebrates such as eye organization and internal fertilization in contrast to other marine invertebrates. The work reported here concerns the char° acterization of the specific nuclear basic proteins from the cuttlefish testis (immature germinal cells) and sperm (mature germinal cells). Biochemical evidence is presented for the existence of a transient protein which appears in round spermatids and is replaced by a major typical protamine in elongated spermatids. These proteins were char-
0167-4838/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
87 acterized by their amino-acid composition and their electrophoretic mobility. Materials and Methods
Testes and spermatozoa from adult cuttlefishes were obtained at the Station Marine de Wimereux (France). After excision, tissues were frozen in liquid nitrogen and stored at - 8 0 °C until use.
Extraction of nuclear basic proteins All operations were carried out at + 4°C and in the presence of 0.1 mM diisopropyl fluorophosphate. From testes. Purified nuclei were prepared from testes and chromatin subsequently isolated from nuclei as described by Chauvi&e et al. [9]. Total acid-soluble proteins were obtained from chromatin by overnight extraction with 0.4 M HC1. The residual pellet was then extracted for 18 h with 5 M guanidinium chloride containing 100 mM 2-mercaptoethanol. Both extracts were dialyzed extensively in Spectrapor 3500 dialysis bags against deionized water and lyophilized. From mature spermatozoa. The isolation of sperm nuclei was performed according to the procedure described by Sautirre et al. [11], using 2% Triton X-100 instead of 4% Tween 80. The nuclear acid-soluble proteins were extracted from purified nuclei by stirring overnight with 0.4 M HC1. The residual pellet was then treated for 18 h with 5 M guanidinium chloride containing 100 mM 2mercaptoethanol. Each extract was subsequently fractionated by two successive precipitations, the first with 3.5 Vol. of acetone containing 0.07 M HC1 and the second with an additional 2.5 Vol. of acidified acetone, for 48 h at - 2 0 ° C. The precipitates were recovered by centrifugation at - 2 0 ° C and dissolved in 0.01 M HC1. Protein solutions were then dialyzed in Spectrapor 3500 dialysis bags against distilled water and lyophilized.
Purification of proteins Spermatid-specific protein (protein T) was obtained in pure form from acid extracts of testis chromatin by gel-filtration chromatography on Bio-Gel P-10 (150 X 5 cm), equilibrated and eluted with 0.01 M HCI saturated with chloroform.
The phosphorylated forms of protein T were separated by ion-exchange chromatography on a carboxymethyl cellulose column (25 × 2.5 cm), equilibrated with 0.5 M NaC1 in 0.05 M sodium acetate (pH 6.0). The proteins were dissolved in the same buffer in the presence of 6 M urea and applied to the column. They were eluted with a linear gradient from 0.5 to 1.5 M NaCI in acetate buffer. Protein Sp was purified by high-performance liquid chromatography (HPLC) fractionation of the testis or sperm extracts on a /tBondapak C18 column (300 x 3.9 mm) (Waters Associates), eluted with a linear gradient of acetonitrile from 0 to 50% in 0.05% trifluoroacetic acid.
Analytical gel electrophoresis Protein fractions were analyzed by polyacrylamide gel electrophoresis on 17% acrylamide slab gels at pH 3.2, in the presence of 6.25 M urea [12].
Amino-acid analysis The amino-acid composition of proteins was determined after hydrolysis in 6 M HC1, in vacuum, at 110 o C, for 24 and 72 h. One drop of 1% phenol was added to prevent excessive degradation of tyrosine. Amino-acid analyses were performed on a Beckman 119 CL amino-acid analyzer. Presence of phosphoamino acids was investigated as described in Ref. 13.
A lkaline phosphatase treatment Protein T and protein Sp were hydrolyzed with alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1.) as described in Ref. 13. The proteins were recovered by precipitation with 6 Vol. of cold acetone and dried before electrophoretic analysis.
End-group determination The amino-terminal sequence was established by manual Edman degradation performed as in Ref. 14, and phenylthiohydantoin derivatives of amino acids were identified by high-pressure liquid chromatography on a column of #Bondapak C18 as in Ref. 15. For the carboxy-terminal sequence determination, proteins (50 nmol) were hydrolyzed in 1 ml
88
0
-I.-
H 4 J,,--
•
-qT
~Sp
1
2
3
4
5
6
7
8
9
10 11
5
+
s sU
C
m
Fig. 1. (a) Electrophoretic analysis of nuclear basic proteins specific of spermiogenesis from cuttlefish Sepia officinalis. Electrophoresis was performed on slab gel (160x180×0.75 mm) at pH 3.2, in 0.9 M acetic acid/6.25 M urea, using a 17% acrylamide concentration. Samples (3 ~tg), dissolved in 0.01 M HC1/8 M urea/0.5 M 2-mercaptoethanol, were run at 22 m_A for 2.5 h, after a pre-electrophoresis of 1.5 h. The gel was stained for 60 rain with 0.04% Coomassie blue R-250, in the mixture 0.08 M sodium picrate/2% acetic acid/2.5% ethanol, as described in Ref. 16. The gel was destained by diffusion in acetic acid/ethanol/water (7 : 20 : 73, v/v). Lane 1, whole histone from calf thymus, as reference; lane 2, 0.4 M HC1 extract from cuttlefish testis chromatin; lane 3, 5 M guanidinium chloride extract from cuttlefish testis chromatin; lane 4, ram protamine; lanes 5 and 6, 0.4 M HC1 extract from cuttlefish sperm nuclei, precipitated with 3.5 and 6 Voi. of acidified acetone, respectively; lanes 7 and 8, 5 M guanidinium chloride extract from cuttlefish sperm nuclei, precipitated with 3.5 and 6 Vol. of acidified acetone, respectively; lane 9, cuttlefish protein T; lane 10, cuttlefish protein Sp isolated from testis chromatin; lane 11, cuttlefish protein Sp isolated from sperm nuclei. (b and c) Densitometric scannings of 0.4 M HCI extract and 5 M guanidinium chloride extract from cuttlefish testis chromatin. Lanes 2 and 3 of (a) were scanned at a wavelength of 540 nm in a Vernon model PHI-6 spectrophotometer fitted with a linear transport assembly.
89
Protein T is only partially extracted from chromatin with 0.4 M HC1. Its complete extraction is ensured by treatment of the residual chromatin with 5 M guanldinium chloride (lane 3). In addition to protein T, guanidinium chloride releases a smaller and more basic protein, called protein Sp, the mobility of which is similar to that of ram protamine (lane 4). In testis extracts, protein T and protein Sp migrate as at least four bands. This has to be related to the existence of structural variants and post-translational modifications of the proteins, and will be discussed later. Somatic-type histones, protein T and protein Sp represent, respectively, 58, 28 and 14% of the total nuclear basic proteins of cuttlefish testis chromatin, as calculated from densitometric scanning of electrophoretic analyses of these proteins (Fig. lb and c).
of 0.2 M N-methylmorpholine acetate (pH 8.0), at 37°C, with carboxypeptidase B (peptidyl-Llysine(L-arginine) hydrolase, EC 3.4.17.2), using an enzyme/substrate ratio of 1:5000 by weight. Aliquots were taken off at time points, freezedried, and analyzed on the amino-acid analyzer. Results and Discussion As shown in Fig. la, the major nuclear basic proteins extracted from cuttlefish testis chromatin with 0.4 M HC1 (lane 2) have an electrophoretic migration very similar to that of somatic-type histones. The 0.4 M HC1 extract contains also another protein called protein T, of electrophoretic mobility intermediate between that of histone H4 (M r = 11 300) and that of ram protamine (M r -- 6 700, lane 4) [17].
b
a 1.0
+
6
50 °/, Acetonitrile
//- . . . . . . . . . . . . . /
I
/ /
E e/
¢0 0
t
// ,/
¢
t
/ / /
C.E
1
2
3 .J
0.5
/ / / / / i / /
/
//
2'5
Effluent (ml)
50
Fig. 2. (a) Fractionation of the 5 M guanidinium chloride extract from cuttlefish testis chromatin by reverse-phase high-pressure liquid chromatography. The extract, dissolved in 0.01 M HCI was loaded on a/~Bondapak C18 column (3.9 x 300 mm), equilibrated with 0.05% trifluoroacetic acid. Elution was performed with a linear gradient of acetonitrile ( . . . . . . ) from 0 to 50% for 30 min. Fractions of 0.5 ml were collected at a flow rate of 1 m l / m i n . The ehition of fractions was monitored at 206 nm. Fractions were pooled as indicated by solid bars. (b) Polyacrylamide gel electrophoresis of the fractions 1, 2 and 3 of (a). Electrophoresis was performed as described in the legend to Fig. 1. C.F., crude fraction, whole 5 M guanidinium chloride extract.
90
Protein T, separated from somatic-type histones by fractionation of the 0.4 M HC1 extract from cuttlefish testis chromatin on a column of Bio-Gel P-10, was obtained in pure form as shown in Fig. la, lane 9. Testis protein Sp was purified to homogeneity from the guanidinium chloride extract by reverse-phase HPLC (Fig. 2a). Three protein fractions were isolated which correspond, respectively, to protein Sp (peak 1), protein T (peak 2), and residual somatic-type histones, mostly H3 and H4 (peak 3), as shown by analytical polyacrylamide gel electrophoresis (Fig. 2b). On the other hand, the 0.4 M HC1 and guanidinium chloride extracts from purified sperm nuclei contain a major protein, migrating as two bands, whose electrophoretic mobility is very close to that of testis protein Sp. Histones and protein T are absent from these extracts (Fig. la, lanes 5-8). The purification of the sperm nuclear protein can
be performed in a similar manner to that of testis protein Sp (Fig. 3), although pure protein can be obtained more easily in large quantity simply by fractional precipitation with 6 Vol. of acetone from the guanidinium chloride extract from sperm nuclei (Fig. la, lane 8). The amino-acid compositions of proteins T and Sp from testis and sperm are presented in Table I. These proteins are characterized by a very large content of arginine (59, 77 and 77%, respectively) and significantly high amounts of serine and tyrosine. Testicular protein Sp and major sperm nuclear protein are very similar. They are constituted of only four different amino acids: arginine, serine, tyrosine and proline present in the same ratio. Therefore, they appear to be the same protein, which presents typical features of fish protamines: very high content of arginine (60-70%), low num-
b
+
a
50 °/~ Acetonitrile
1.0
/ . . . . . . .
/ /
//
E ¢-
/
/
/
//
£O
o
/
O4
//
< /
/
/
/
// /
0.5
/
C.F. l a l b lc 2 3 4 5 / / / / /
// /
/
/
2
// // /
/
//
0 °/oAcetonitrile 20
Effluent ( ml )
40
Fig. 3. (a) Fractionation of the 0.4 M HCI extract of sperm nuclei, precipitated with 3.5 Vol. of acidified acetone, by reverse-phase high-pressure liquid chromatography. See legend to Fig. 2a. (b) Polyacrylamide gel electrophoresis of the fractions 1-5 of (a) Electrophoresis was performed as described in the legend to Fig. 1. C.F., crude fraction, whole 0.4 M HC1 extract, precipitated with 3.5 Vol. of acidified acetone. Lanes la, l b and lc, peak 1 (ascending slope, top and descending slope). Lanes 2-5: peaks 2 to 5.
91 TABLE I
-I-
AMINO-ACID COMPOSITIONS OF NUCLEAR BASIC PROTEINS SPECIFIC OF SPERMIOGENESIS IN CUTTLEFISH SEPIA O F F I C I N A L I S Results are expressed as moi per 100 mol. The values for threonine and serine were obtained by linear extrapolation to zero hydrolysis time. Values in parentheses represent the number of residues per molecule of protein. Testis protein T
Sperm protein Sp
protein Sp
11.4 (6)
11.3 (6)
2.3 (1)
2.2 (1)
Asp Thr Ser Glu Pro Gly Ala Val Met Leu Tyr Lys Arg
2.1 (1-2) 0.6 (1) 9.6 (7) 2.6 (1-2) 1.4 (1) 3.1 (2) 3.3 (2-3) 1.7 (1-2) 1.0 (3) a 4.3 (3) 6.5 (5) 5.0 (4) 58.7 (42)
9.1 (5)
9.0 (5)
77.1 (42)
77.4 (42)
%Basic N-terminal C-terminal
63.7 Met Arg
77.1 Arg Arg
77.4 Arg Arg
a From preliminary structural data.
ber of constituent amino acids, absence of cysteine or acidic residues [2,11]. However, the presence of tyrosine is rather characteristic of mammalian protamines, although this amino acid was found in two fish species: tunafish [18] and dogfish [19]. The amino-acid composition of cuttlefish protamine is very close to that of calamar protamine, but quite different from that of octopus protamine [20]. Phosphoserine was identified only in testis protein Sp after limited acid hydrolysis and thin-layer electrophoresis in the presence of phosphoserine and phosphotyrosine. This can explain the difference between the electrophoretic patterns of testis and sperm proteins Sp. After treatment with alkaline phosphatase, the two proteins are resolved as the same two bands on polyacrylamide gel electrophoresis (Fig. 4). From these results, we can conclude that protein Sp in testis is a family of molecules phosphorylated at different levels, whereas protein Sp, in mature sperm, is not found
12
34
Fig. 4. Effects of alkaline phosphatase on the electrophoretic mobility of protein Sp. Lanes 1 and 2, testicular protein Sp after (1) and before (2) incubation with the enzyme. Lanes 3 and 4, sperm protein Sp after (3) and before (4) incubation with the enzyme.
phosphorylated. Nevertheless, the electrophoretic heterogeneity of protein Sp, even after treatment with alkaline phosphatase, strongly suggests that it could be constituted of at least two structural variants. Protein T differs from protein Sp by its molecular size and a greater amino-acid diversity. Acidic and hydrophobic residues are little represented and cysteine is absent. The features of this composition exclude the possibility that protein T results of a proteolytic degradation of an histone. The amino-acid diversity as well as the electrophoretic mobility and the size of protein T are typical of spermatid-specific proteins which, in
92
mammals and dogfish [5,7-9], appear transiently between removal of histones and deposition of protamines on DNA. However, cuttlefish protein T differs markedly from intermediate proteins identified in mammals and dogfish by a higher content in arginine which is close to that found in fish protamines [2,19]. When submitted to ion-exchange chromatography on carboxymethyl cellulose, protein T was resolved into five fractions of increasing electrophoretic mobility (Fig. 5). The amino-acid compositions of these fractions were identical. Presence of phosphoserine was detected in all fractions after acid hydrolysis for 2 h at l l 0 ° C , and the level of phosphorylation was found to decrease from the first eluted fraction to the last
one: thus, fraction 1 was found to be 3-times more phosphorylated than fraction 5. Moreover, after hydrolysis with alkaline phosphatase, all fractions exhibited the same electrophoretic mobility, on acid-urea gel (Fig. 6). Therefore, the apparent heterogeneity of protein T has to be related to different levels of phosphorylation of the protein. In conclusion, cuttlefish spermiogenesis is characterized by protein transitions similar to that observed during dogfish and mammalian spermiogenesis [9,21]. Two specific proteins appear sequentially to replace somatic-type histones in testis chromatin. Protein T appears in round spermatids is abundant in elongating spermatids and is no longer detected in elongated spermatids, as shown by immunocytochemical studies [22].
fl Turbidity 400 nm
b -IiN iiii
o.1
. 1 . 5 M NaCI
e
.
• ee
•
_
0.0
j j"
2 I
0.SM NaCI
650
3
4
13bd
Effluent (ml)
Fig. 5. (a) Separation of phosphorylated forms of spermatid-specific protein T from cuttlefish testis by chromatography on carboxymethyl cellulose. Five fractions (1-5) were eluted successively with a linear gradient of NaCI from 0.5 to 1.5 M in 0.05 M sodium acetate pH 6.0. Elntion of protein was monitored by turbidimetry at 400 nm. Fractions of 6.5 ml were collected at a flow rate of 20 m l / h . (b) Polyacrylamide gel electrophoresis of the fractions recovered from chromatography on carboxymethyl cellulose. Eleetrophoresis was performed as described in the legend to Fig. 1. C.F., crude fraction of protein T. A and B: peaks A and B from Fig. 5a. Lanes 1-5: fractions 1-5 from Fig. 5a, phosphorylated forms of protein T.
93
+
precursor molecule (in this case, protein T) like mouse protamine 2, as shown recently by Yelick et al. [23]. The complete determination of the amino-acid sequence of proteins T and Sp would provide reliable arguments in favor of this hypothesis.
Acknowledgements
D
mmb
The authors are grateful to Professor S. Frontier (Station Marine de Wimereux) for providing the cuttlefishes. They acknowledge with pleasure the skilful technical assistance of A. H6mez, F. Boutteau, M.J. Dupire and T. Ernout. They also thank T. Ernout for editorial assistance. This work was supported by grants from the Centre National de la Recherche Scientifique (RCP 0680), from Universit6 de Lille II, from the Minist6re de l'Industrie et de la Recherche (Contrat 84 C 0942) and from the Fondation h la Recherche M6dicale.
References
123456 Fig. 6. Effects of alkaline phosphatase on the electrophoretic mobility of protein T. Lanes 1-3: fractious 1, 3 and 5 from the carboxymethyl cellulose fractionation of protein T; before incubation with the enzyme. Lanes 4-6: the same fractions after incubation with the enzyme.
Protein T is a transitional protein phosphorylated in the testis and absent from the spermatozoa. Protein Sp appears in elongated spermatids and is a typical protamine, phosphorylated in the testis and nonphosphorylated in the mature sperm cells where it constitutes the major basic protein associated to DNA. On the other hand, the fact that protein T and protein Sp contain similar amounts of serine, proline, tyrosine and arginine strongly suggests that a close structural relationship exists between the two proteins. Protein Sp could be initially synthesized as a
1 Dixon, G.H. (1972) in Karohnska Symposium on Research Methods in Reproduction and Endocrinology (Diczfalusy, E., ed.), pp. 130-154, Karolinska Institute, Stockholm. 2 Ando, T., Yamasaki, M. and Suzuki, K. (1973) in Molecular Biology Biochemistry and Biophysics, Vol. 12, pp. 1-114, Springer-Verlag, Berlin. 3 Louie, A.J., Candido, E.M.P. and Dixon, G.H. (1973) Cold Spring Harbor Symp. Quant. Biol. 38, 803-819. 4 K.istler, W.S., Noyes, C., Hsu, R. and Heinrikson, R.L. (1975) J. Biol. Chem. 250, 1847-1853. 5 Grimes, S.R., Meistrich, M.L., Platz, R.D. and Hnilica, L.S. (1977) Exp. Cell Res. 110, 31-39. 6 Balhorn, R., Weston, S., Thomas, C. and Wyrobek, A.J. (1984) Exp. Cell Res. 150, 298-308. 7 Dupressoir, T., Sauti6re, P., Lanneau, M. and Loir, M. (1985) Exp. Cell Res. 161, 63-74. 8 Gusse, M., Sauti&e, P., B61aiche, D., Martinage, A., Roux, C., Dadoune, J.P. and Chevailher, P. (1986) Biochim. Biophys. Acta 884, 124-134. 9 Chauvi~re, M., Laine, B., Sauti~re, P. and Chevaillier, P. (1983) FEBS Lett. 152, 231-235. 10 Poccia, D. (1986) Intern. Rev. Cytol. 105, 1-65. 11 Sauti6re, P., Briand, G., Gusse, M. and Chevaillier, P. (1981) Eur. J. Biochem. 119, 251-255. 12 Panyim, S. and Chalkley, R. (1969) Arch. Biochem. Biophys. 130, 337-346. 13 Martinage, A., Gusse, M., B61ai'che, D., Sauti6re, P. and Chevailfier, P. (1985) Biochim. Biophys. Acta 831, 172-178. 14 Edman, P. and Henschen, A. (1975) in Protein Sequence Determination (Needleman, S.B., ed.), pp. 232-279, Springer-Verlag, Berlin.
94 15 Hermann, J., Titani, K., Ericsson, L.H., Wade, R.D., Neurath, H. and Walsh, K.A. (1978) Biochemistry 17, 5672-5679. 16 Stephano, J.L., Gould, M. and Rojas-Galicia, L. (1986) Anal. Biochem. 152, 308-313. 17 Sauti&e, P., Brlaiche, D., Martinage, A. and Loir, M. (1984) Eur. J. Biochem. 144, 121-125. 18 Bretzel, G. (1973) Hoppe Seyler's Z. Physiol. Chem. 354, 543-549. 19 Gusse, M., Sautirre, P., Chauvirre, M. and Chevaillier, P. (1983) Biochim. Biophys. Acta 748, 93-98.
20 Subirana, J.A., Cozcolluela, C., Palau, J. and Unzeta, M. (1973) Biochim. Biophys. Acta 317, 364-379. 21 Grimes, S.R., Jr. (1986) Comp. Biochem. Physiol. 83B, 495-500. 22 Rousseaux-Prrvost, R., Engelhardt, R.P., Rousseaux, J., Wouters-Tyrou, D. and Sauti~re, P. (1988) Gamete Res. in press. 23 Yelick, P.C., Balhorn, R., Johnson, P.A., Corzett, M., Mazrimas, J.A., Kleene, K.C. and Hecht, N.B. (1987) Mol. Cell. Biol. 7(6), 2173-2179.