EJCB
European Journal of Cell Biology 81, 341 ± 349 (2002, June) ¥ ¹ Urban & Fischer Verlag ¥ Jena
http://www.urbanfischer.de/journals/ejcb
341
Chromatin condensation, cysteine-rich protamine,
and establishment of disulphide interprotamine
bonds during spermiogenesis of Eledone cirrhosa
(Cephalopoda)
Pepita Gimenez-Bonafe¬2)a, Enric Ribes2)b, Pierre Sautie¡rec, Angel Gonzalezd, Harold Kasinskye, Mustafa
Kouachc, Pierre-Eric Sautie¡ref, Juan Ausio¬g, Manel Chiva1)d
a
b
c
d
e
f
g
The Prostate Center, Vancouver General Hospital, Vancouver, BC/Canada
Dept. Biologia Cel.lular. Fac. Biologia, Univ. Barcelona, Barcelona/Spain
URA, CNRS 1309, Chimie des Biomol., Inst. Pasteur, Lille/France
Dept. Ciencies Fisiolo¡giques II, Fac. Medicina, Univ. Barcelona, Campus Bellvitge, Barcelona/Spain
Dept. of Zoology, Univ. of British Columbia, Vancouver, BC/Canada
Endocrinologie des Annelides, UPRESA, CNRS 8017 SN3-USTL, Villeneuve d−Asq/France
Dept. Biochem. Microbiol., Univ. of Victoria, Victoria, BC/Canada
Received December 10, 2001
Received in revised version March 7, 2002
Accepted March 8, 2002
Eledone ± spermiogenesis ± chromatin ± cysteine-rich
protamine ± disulphide interprotamine bonds
During spermiogenesis in Eledone cirrhosa a single protamine
substitutes for histones in nuclei of developing spermatids. This
protein displays a peculiar primary structure. It contains
22.6 mol% cysteine residues (19 cysteines in 84 residues).
This makes it the most cysteine-rich protamine known. The
proportion of basic residues is relatively low (arginine
36.9 mol%, lysine 19.0 mol%). The protamine of E. cirrhosa
condenses spermiogenic chromatin in a pattern which comprises fibres with a progressively larger diameter and lamellae
that finally undergo definitive coalescence. We have also
performed a study that estimates the number of interprotamine
disulphide bonds formed during the process of spermiogenic
chromatin condensation by means of sequential disappearance
of MMNA (monomaleimido-nanogold) labelling. During the
first step of spermiogenesis, protamines are found spread over
very slightly condensed chromatin with their cysteines in a
reactive state (protamine-cys-SH). From this stage the interprotamine disulphide bonds are established in a progressive
way. First they are formed inside the chromatin fibres.
Subsequently, they participate in the mechanism of fibre
coalescence and finally, in the last step of spermiogenesis, the
1)
Prof. Manel Chiva, Dept. Ciencies Fisiolo¡giques II, Fac. Medicina,
Univ. Barcelona, Feixa Llarga s/n. L−Hospitalet de Llobregat,
E-08071 Barcelona/Spain, e-mail: [email protected], Fax:
‡ 34 93 402 4213.
2)
P. Gimenez-Bonafe¬ and E. Ribes contributed equally to this work.
remaining free reactive -SH groups of cysteine form disulphide
bonds, thus promoting a definitive stabilization of the nucleoprotein complex in the ripe sperm nucleus.
Abbreviations. BSA Bovine serum albumin. ± CAM S-Carboxyamidomethylcysteine. ± Da Daltons. ± DTT Dithiothreitol. ± EDTA Ethylenediamine
tetraacetic acid. ± HPLC High performance liquid chromatography. ± ISMS
Ion spray mass spectrometry. ± MEA Mercaptoethylamine hydrochloride.
± MMNA Monomaleimido-nanogold. ± PBS Phosphate-buffered saline. ±
SNBPs Sperm nuclear basic proteins.
Introduction
The packaging of DNA, as well as the architecture of the
genome in ripe sperm nuclei are the result of the pathway
followed in the process of chromatin condensation. The
essential fact of this process is an organized substitution of
structural proteins (mainly histones) in nuclei of early spermatids by more basic proteins (SNBPs, protamines) in ripe sperm
nuclei, that are rich in arginine and/or lysine residues (Poccia,
1986; Kasinsky, 1989). SNBPs, which are an extremely heterogeneous group of proteins, were originally placed into five
categories by Bloch (1969, 1976). One of the types in Bloch−s
classification is the ∫cysteine-containing protamines∫, mainly
described and studied in sperm nuclei of mammals (reviewed
by Oliva (1995)) and chondrichthyan fish (reviewed by Chiva
0171-9335/02/81/06-341 $15.00/0
EJCB
342 P. Gimenez-Bonafe¬ et al.
et al. (1995)). In a classical work, Bedford and Calvin (1974)
showed that cysteine-rich mammalian protamines established
intermolecular disulphide bonds leading to a high physical and
chemical stability of the nucleoprotamine complex in mature
sperm nuclei. More recently, Balhorn×s group has presented a
model in which, mammalian protamines can also form some
intra-protamine disulphide bridges, thus lending a specific
three-dimensional structure to these molecules (Balhorn, 1982;
Balhorn et al., 1999). In fact, knowledge of compositional and
structural changes in the nucleus during mammalian spermiogenesis progressed during the last decade (Haaf and Ward,
1995; Oko et al., 1996; Baarends et al., 1999; Sotolongo and
Ward, 2000). Nonetheless, both the process by which spermiogenic chromatin condenses in a definite pattern, and the precise
functional role of specific amino acid types of protamines in
chromatin condensation remain poorly understood.
In a previous work (Gimenez-Bonafe¬ et al., 1999) we found
that the DNA-condensing protein in sperm nuclei of the
octopod E. cirrhosa contained a high proportion of cysteine
residues (> 22 mol%). This protein offers an interesting model
in which to analyze the participation of cysteines in sperm
chromatin condensation. In the present work, we first describe
the pathway of chromatin condensation during E. cirrhosa
spermiogenesis; secondly we study the primary structure of the
DNA-interacting protein (™protamine™); and, finally, we analyze in situ the development of disulphide bond formation
during nuclear spermiogenesis, as it correlates with chromatin
condensation.
Materials and methods
Organisms
The gonads and adjacent structures of several individuals of the octopod
E. cirrhosa were dissected and analyzed for this work. Sexually mature
specimens were collected from the Mediterranean sea (Costa Brava,
Spain) during the reproductive season (July-August).
Extraction and purification of nuclear proteins
Spermatid nuclei were prepared from cells obtained from whole gonads
while mature sperm nuclei were obtained from sperm packaged into the
spermatophores. In both instances, nuclear purification was performed
as described in (Gimenez-Bonafe¬ et al., 1999).
Nuclear proteins were extracted by solubilization in 0.4 N HCl after
chemical reduction with DTT and iodoacetamide alkylation of chromatin (Gusse et al., 1983). This process reduces disulphide covalent
bridges between cysteine-containing protamines yielding S-carboxyamidomethylcysteine (CAM). Due to the large amounts of disulphide
interprotamine bonds present in spermiogenic chromatin of E. cirrhosa,
it was not possible to solubilize the protamine without this previous
chemical reduction. Upon extraction, the proteins were extensively
dialyzed against distilled water and lyophilized.
In order to convert all cysteines to the CAM form, the lyophilized
proteins were subjected to a second and more drastic process of
reduction-alkylation. This chemical treatment was performed by
incubating the protein in 2 mM EDTA, 50 mM Tris-HCl, pH 8.8,
10 mM DTT, 40 mM iodoacetamide at 37 8C for 2 hours under a
nitrogen atmosphere. The contribution of the mass of the carboxyamidomethyl group (57.04 Da) was taken into consideration in interpreting
the mass spectrometry results obtained with alkylated protamine.
E. cirrhosa protamine was purified from spermatid nuclei by reversephase HPLC (Ca¡ceres et al., 1999).
Protein sequence and molecular mass
determination
The purified protamine was hydrolyzed with thermolysin as described
by Daban et al. (1995). Under these conditions three peptides were
obtained and fractionated by reverse-phase HPLC. Both the intact
protamine and each peptide were subjected to automated Edman
degradation on a Procise Sequenator/ABI-492 (Perkin Elmer) using the
Pulse Liquid 42L program. Molecular masses of proteins were
determined by ion spray mass spectrometry (ISMS) (Ca¡ceres et al.,
1999). Amino acid compositional analyses were carried out as in (Chiva
and Mezquita, 1983). Comparative sequence analyses were performed
using the BLAST program (Altschul et al., 1990).
Gel electrophoresis was done according to (Panyim and Chalkley,
1969), with the modifications described by Hurley (1977).
Microscopy
For the analysis of the samples by conventional transmission electron
microscopy (TEM), gonads or sperm were fixed in 2.5% (v/v)
glutaraldehyde, 0.1 M cacodylate buffer, pH 7.3, and postfixed in
0.1% (w/v) osmium tetroxide. Following this, the samples were
dehydrated, soaked in Spurr−s resin and stained with uranyl acetate.
Specific labelling of free thiol groups was achieved by fixing pieces of
gonads or sperm pellets in 4% (v/v) paraformaldehyde/0.2% (v/v)
glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, and embedding in
Lowicryl resin. No staining was performed at this point. The grids were
incubated in 12 nmol/ml of monomaleimido-nanogold (MMNA)/PBS for
30 minutes and rinsed several times with deionized water. The gold label
was developed with HQ silver reagent (Lin et al., 1995). In order to label
the thiol groups of mature sperm chromatin, ripe sperm nuclei were first
reduced with 1-mercaptoethylamine hydrochloride (MEA) and treated
as in (Gimenez-Bonafe¬ et al., 1999). Quantitation of MMNA labelling
was performed using the image-analysis OPTIMASTM 6.0 program.
Immunostaining analysis was carried out as follows. The grids with the
samples were washed in PBS (0.1 M, pH 8.0) containing 1% (w/v) BSA
and 20 mM glycine for 30 minutes. They were then incubated for
2 hours, with anti-a-tubulin antibodies (Amersham N356) diluted 1 : 50
in PBS, 1% (w/v) BSA, 0.01% (w/v) Tween-20. Next, they were washed
three times in PBS-BSA-Tween (5 minutes each), and incubated for
1 hour with the secondary antibody goat anti-mouse IgG-IgM-10 nm
gold (Biocell) diluted 1 : 25 in PBS-5%(w/v) BSA. After rinsing in PBS
and deionized water (three times), the grids were finally stained with
uranyl acetate (15 minutes) and lead citrate (5 minutes).
Results
Chromatin condensation in the spermiogenesis
of E. cirrhosa
Some aspects related to nuclear development during E. cirrhosa
spermiogenesis have been described by Maxwell (1974). This
section presents a selection of the most significant morphological
changes undergone by chromatin during its condensation, many
of which have not been previously reported.
By conventional TEM the chromatin of early haploid
spermatids appears to be similar to that of somatic nuclei.
However the proportion of heterochromatin becomes progressively lower and tends to accumulate at the nuclear periphery
(Figure 1A). The next step of nuclear development is an
elongation of the organelle in which heterochromatin has
totally disappeared, except for a peripheral sheet (Figure 1B).
Once the first part of nuclear elongation is established,
chromatin starts to condense, forming fibres. The diameter of
the fibres is relatively constant at the beginning of condensation
(Figure 1C) but increases progressively. This occurs concomitantly with a transient loss of nuclear membrane. It is
noteworthy that, in contrast with other cephalopods (Maxwell,
EJCB
Cysteine-rich protamine and chromatin condensation 343
Fig. 1. First stages of spermiogenic chromatin condensation in E.
cirrhosa. A. Early spermatid nucleus. In these phases, heterochromatin
accumulates at the nuclear periphery, whereas euchromatin prevails in
the inner zone of nuclei. B. Spermatid undergoing nuclear elongation.
Heterochromatin has practically disappeared, except for a peripheral
layer. C. At the initial stages of condensation, chromatin forms fibres
with a relatively constant diameter. D. Transversal view of a spermatid
nucleus showing the coalescence of fibres and their supercoiled
arrangement. E. In the next step all the nuclei appear supercoiled.
Bars: 1 mm (A ± C, E), 0.4 mm (D). m mitochondrion; n nucleus; v
acrosomal vesicle.
1975), chromatin fibres in E. cirrhosa are supercoiled (Figure 1D) and grow by coalescence. It seems to be that supercoiled growth of chromatin fibres generates a three-dimensional coiling of the whole spermatid nuclei at these stages
(Figure 1E). The subsequent coalescence of supercoiled fibres,
producing a network of lamellae and their ordered joining,
constitutes the advanced step of spermiogenic chromatin
condensation. Figure 2 (A, B) shows the morphological pattern
344 P. Gimenez-Bonafe¬ et al.
Fig. 2. Advanced stages of E. cirrhosa spermiogenic chromatin
condensation. A, B. The coalescence of chromatin fibres forms lamellae
(shown in transversal section in (A)), and subsequently a network of
condensed chromatin (B). C, D. At the last steps, the cylindrical nucleus
acquires a helical shape. During this nuclear transformation, the
chromatin is not completely condensed (see white areas of nonelectrondense nucleoplasm inside nuclei in (C and D)). E. Scanning
microscopy of a E. cirrhosa ripe spermatozoal nucleus showing its
helical shape. In this final stage the chromatin is wholly condensed.
Bars: 0.2 mm (A), 0.25 mm (B), 0.5 mm (C), 0.6 mm (D). In (E), the pitch
of nuclear helix has a length of 1 mm.
by which the chromatin condenses during these phases.
Interestingly, these kinds of patterns appear to be almost
identical in the spermiogenesis of other unrelated species (see,
for instance, (Watson and Rohde, 1994)).
At more advanced steps, the spermatid nucleus becomes a
cylinder, and the chromatin is found to be highly, but not
completely, packed (Figure 2C). From these stages, perinuclear
microtubules undergo a progressive contraction, and the
cylindrical nucleus will transform into a helical structure
(Figure 2D). Only after that, in the last step of spermiogenesis
leading towards ripe spermatozoa (Figure 2E), chromatin will
complete its definitive packing (Gimenez-Bonafe¬ et al., 1999).
The primary structure of E. cirrhosa protamine
As has been explained before, during spermiogenesis in E.
cirrhosa, all the histones are replaced by a protamine that
appears to be the only nuclear basic protein present in mature
EJCB
Fig. 3. Purification and molecular mass of E. cirrhosa protamine. A.
Electrophoretic analysis of E. cirrhosa protamine. Lane w: Whole
complement of basic proteins contained in gonadal nuclei. Proteins
were extracted with 0.4 N HCl after chemical reduction and alkylation
of chromatin; lane p: E. cirrhosa protamine purified from whole
proteins. H Histones, P protamine. B. Chromatogram of purified
protamine analyzed by HPLC. Three different components can clearly
be observed (arrows). C. ISMS of protamine. The protein also shows
three components with masses of 11696 Da, (11696 ‡ 80) Da, and
(11696 ‡ 160) Da.
sperm nuclei. In order to study its primary structure, we have
purified this protein from whole gonadal nuclei (Figure 3A).
The mature gonads contained spermatids in different steps of
development (mainly advanced spermatids). Because of this
mixed-cell nature of the tissue, both histones and protamine
were present in the extract (H and P, respectively, in Figure 3A).
Although the protamine purified from gonads behaves as a
single electrophoretic band, HPLC purification indicates the
presence of three components (Figure 3B) that can also be
detected by mass spectrometry (Figure 3C). The molecular
EJCB
Cysteine-rich protamine and chromatin condensation 345
Tab. I. Molecular masses of protamine peptides Th-1, Th-2 and
Th-3 resulting from the digestion of E. cirrhosa protamine with
thermolysin.
Th-1 (1 ± 33)
Th-2 (34 ± 60)
Th-3 (61 ± 84)
STh-2H2O
Protamine
Estimated from sequence
Determined by ISMS
(Da)
4526.3
3776.5
3429.2
11694.0
11696.1
(Da)
ND
3777
3430
ND
11696
Cysteines were chemically modified to CAM (Mr ˆ 160.18) (see Materials
and methods). ND ± Not determined.
Fig. 4. Determination of E. cirrhosa protamine sequence. A. ISMS of
peptide Th-2 (residues 34 ± 60) obtained by thermolysin cleavage and
purified by HPLC. A minor peak of (3777 ‡ 81) Da represents a monophosphorylated form of this peptide. B. ISMS of peptide Th-3
generated by thermolysin cleavage and purified as Th-2. (Molecular
mass ˆ 3430 Da). C. Complete sequence of E. cirrhosa protamine
inferred from amino acid sequence and mass spectrometry analyses.
Partial sequences have been determined from intact protamine (a) and
Th-1, Th-2, Th-3 thermolysin peptides (b, c and d, respectively). See
also Table I.
masses of these protein fractions (11696 Da, 11776 Da and
11856 Da) indicate that they correspond to non-, mono- and diphosphorylated forms of protamine. Thus, as has been observed
with other protamines, postranslational modification (phosphorylation/dephosphorylation) may play an important role in
the modulation of the interaction between DNA and the
protamine of E. cirrhosa (see (Oliva and Dixon, 1991) for a
review on protamine phosphorylation).
The terminal amino acid sequence of E. cirrhosa protamine
was determined up to residue 43 by automated Edman
degradation of the intact protein (Figure 4C). Cleavage of the
protamine with thermolysin resulted in three major peptides:
Th-1 (residues 1 ± 33), Th-2 (residues 34 ± 60) and Th-3 (residues 61 ± 84) (Figure 4C). The amino-terminal sequence of the
intact protein overlaps with Th-1 and Th-2. Mass spectrometry
of these peptides (Figure 4A, B and Table I), in conjunction
with their amino acid analyses (not shown), indicated that these
peptides comprise the complete sequence of the protamine in
which Th-3 is at the carboxy-terminal end.
The protamine of E. cirrhosa contains 84 amino acid residues,
31 of which are arginines (36.9 mol%) and 16 are lysines
(19.0 mol%). Except for the amino-terminal region, the basic
residues tend to be clustered, as in other protamines. These
groups of basic residues are responsible for the cooperative
interaction of protamine with DNA (Willmitzer and Wagner,
1980). The neutralization of the negative charge of the DNA
phosphates by basic amino acid residues of protamine induces
the condensation of sperm chromatin. However, the most
striking characteristic of E. cirrhosa protamine lies in its
cysteine-rich composition (19 cysteines in 84 residues;
22.6 mol%). In this respect, it represents the most cysteinerich protamine known. Cysteine residues are also present in
mammalian and chondrichthyan protamines, where the main
role of this residue appears to be in the formation of interprotamine disulphide bonds that physically and chemically
contribute to the stability of the chromatin structure of sperm
(Bedford and Calvin, 1974; Balhorn, 1982).
Eledone protamine has two additional relevant features. The
protein does not appear to be homologous to any known
protein although it shares some identities with the sequence of
mammalian P1 protamines. Also, the sequence between
residues 4 to 21 consists of nine alternating groups of nonbasic/basic residues. These sequences (particularly the SR
motifs) are widespread in the N-terminal region of molluscan
SNBP and vertebrate protamines (Carlos et al., 1993; Daban
et al., 1995; Bandiera et al., 1995; Hunt et al., 1996), and they
are shared also with other proteins, such as the family of SR-rich
splicing factors (Tacke and Manley, 1999). Unfortunately, the
functional role of alternating residues in protamines has not yet
been characterized.
346 P. Gimenez-Bonafe¬ et al.
Fig. 5. Establishment of interprotamine disulphide bonds. A. Early
stage of chromatin condensation in spermiogenesis of E. cirrhosa.
MMNA labelling is observed over the non-condensed chromatin. B.
Middle stage. Chromatin is arranged in fibres, some of which appear to
coalesce. MMNA labelling appears to be located preferentially at the
periphery of fibres. C. Late stage. Two advanced spermatid nuclei
EJCB
undergoing helicoidization. Note that MMNA labelling is low inside
the nucleus, but that it accumulates in delimited areas. Compare these
areas with non-electrondense nucleoplasm in Figure 2C, D. Arrowheads show the boundaries of nuclei, where unorganized (A, B) and
organized (C) tubulins are located (see also Figure 6). Bars: 0.2 mm.
EJCB
Cysteine-rich protamine and chromatin condensation 347
Establishment of disulphide bonds and its
relation with condensing chromatin
Monomaleimide-nanogold (MMNA) is a specific reagent for
free -SH groups of proteins, that can be detected by electron
microscopy. Due to the high content of cysteines present in E.
cirrhosa protamine, we have used MMNA to study the
protamine distribution in developing spermiogenic nuclei, as
well as to monitor the establishment of inter-protamine
disulphide bonds by sequential disappearance of MMNA
labelling during chromatin condensation.
As shown in Figure 1 (A, B), during the first stages of
spermiogenesis the spermatidyl nucleus elongates without any
concomitant condensation of its chromatin. The MMNA
labelling of nuclei belonging to these steps is very low and
cannot be distinguished from the background labelling.
The chromatin begins to condense only when the nucleus has
acquired an elongate shape. Figure 5 shows the MMNA
labelling of nuclei at three significantly different stages of
spermiogenic chromatin condensation. In these images, the
strong reaction seen in the peripheral boundaries of the nucleus
(arrowheads in Figure 5) is not due to the presence of
protamine in these regions but to an important set of microtubules that develop around the nucleus simultaneously with
the ongoing process of chromatin condensation. The main
constitutive proteins of microtubules (tubulins) also exhibit a
cysteine-rich composition, with free thiol groups that react with
MMNA. In Figure 6A we discriminate between tubulins and
protamine using anti-a-tubulin antibodies.
The earliest stage of chromatin condensation is shown in
Figure 5A. At this stage, the labelling with MMNA is very
intense (see also the quantification presented in Figure 6B) and
homogeneously spread over a very low condensed chromatin.
This is a significant result for three main reasons. First it shows
that E. cirrhosa protamine enters the spermatid nuclei with free
cysteine-SH groups; second, it indicates that chromatin condensation does not start at a preferential specific site or nuclear
region; and third, and most important, demonstrates that in
early spermiogenic stages, DNA and protamine are already in
contact with only a low degree of chromatin condensation.
From this stage, the nuclear labelling of MMNA progressively decreases (see Figure 6B represented as a number of
points/m2). This fact is concomitant both with the establishment
of interprotamine disulfide bonds and with the formation of
fibres and their coalescence which takes place in the next stages
of spermiogenesis. As can be seen in Figure 5B the first interprotamine disulphide bridges are mainly established within the
fibres, while the formation of larger fibres and lamellae is driven
by the protamine-S-S-protamine interactions among different
fibres. Figure 5B distinctively shows that the MMNA labelling
prevails on the periphery of the fibres. At the stages where the
nucleus acquires its characteristic shape, MMNA labelling is
very low and confined to the non-electrodense nucleoplasm
(see Figure 5C and compare with Figure 2C, D). This implies
that the major part, but not all, of inter-protamine disulphide
bonds are already established. The presence of remnant areas
still containing protamine with free -SH groups agrees with the
concept of chromatin plasticity necessary to allow the helical
shaping of the nucleus by microtubules (Maxwell, 1974) (note
also that the nucleus shown in Figure 5C is in process to
acquiring the helical shape). In contrast, the MMNA labelling is
practically non-existent in mature sperm nuclei ((GimenezBonafe¬ et al., 1999) and Figure 6B) indicating that at this point,
all chromatin is tightly linked by disulphide bridges. Figure 6B
Fig. 6. Anti-tubulin and MMNA labelling. A. Anti-a-tubulin antibody labelling of a section of a spermatid, showing the nucleus with
chromatin in a fibrillar state, the material surrounding the nucleus, and
several flagella. a-Tubulin is found associated to microtubules of
flagella and to the surrounding nuclear material (arrows). This material
constitutes a perinuclear complex of microtubules in later stages (some
of them can be seen in Figure 2C, D). Bar: 0.1 mm. B. Concentration of
MMNA labelling in chromatin of different spermiogenic phases.
Phases 1, 2 and 3 correspond to stages A, B and C of Figure 5. Phases 4
and 5 represent the MMNA labelling of ripe sperm nuclei before (4),
and after (5) chemical reduction. For each stage we have measured the
extranuclear background without considering the labelling on nuclear
boundaries. Total area ˆ 1 mm2.
also indicates that after chemical reduction it is possible to
revert the labelling inability of mature sperm nuclei.
Discussion
The interaction of protamine and DNA has been studied by
several methods, mainly from complexes reconstituted in vitro
and in some cases using mature sperm nuclei. All these studies
348 P. Gimenez-Bonafe¬ et al.
show that in spite of the remarkable differences in the primary
structure of protamines, they share the same kind of local
interaction with DNA. Protamines are positioned inside the
major groove of DNA, with their arginine or lysine residues
establishing hydrogen bonds with DNA phosphate groups, thus
stabilizing the B form of the nucleic acid (Mirzabekow et al.,
1977; Suau and Subirana, 1977; Fita et al., 1983; Gatewood
et al., 1987; Suzuki and Wakabayashi, 1988). The strong
electrostatic interaction between protamines and DNA seems
to be the most important cause of the high degree of chromatin
packing in ripe sperm nuclei.
Nevertheless, as has been noted in the Introduction, the
process driving towards mature sperm chromatin is poorly
understood; that is, the way in which protamine and DNA
progressively interact in order to produce the packing and
organization of sperm genome.
The present work shows for the first time the distribution of
protamines and the disappearance of the free cysteine-SH
groups in situ during spermiogenesis of E. cirrhosa. The most
significant facts stated here are: a) In the initial stages of
spermiogenesis protamines are found throughout the nucleus,
but chromatin does not display a significant condensed state.
During this stage the major part of cysteine-SH groups are in
the reactive form. We conjecture that at this point, the
phosphorylated protamines may establish relatively weak and
reversible interactions in order to replace histones and find a
correct positioning on the DNA molecule. These suggestions
must, however, be examined more carefully. b) The chromatin
fibres (that is, the nucleoprotamine complexes) are formed
individually from the earliest stages. They grow by coalescence
and produce a network of lamellae. c) The interprotamine
disulphide bonds are first established inside chromatin fibres
and subsequently stabilize the coalesced structures: larger
fibres, lamellae and finally the whole mature sperm chromatin.
Finally, it is necessary to point out that the condensation of
chromatin during spermiogenesis does not solely depend on
protamine interacting with DNA, but appears to be a highly
complex process including, at least in some species, a partial
reorganization of the genome architecture (Joffe et al., 1998;
Hazzouri et al., 2000), the presence of a spermiogenic nuclear
matrix (Ward and Zalensky, 1996; Kramer and Krawetz, 1996;
Alsheimer and Benavente, 1996), and some additional nonprotamine protein components (Zalensky et al., 1997; Gineitis
et al., 2000).
Acknowledgements. We are very indebted to Drs. N. Cortadellas, J.
Serratosa and J. Subirana for their scientific assistance. This work has
been supported by grant PB98 ± 1191 (Spain) (M. Chiva, E. Ribes),
NSERC grant 585401 (Canada) (H. Kasinsky), NSERC grant OGP
82368 (J. Ausio¬ the NATO Collaborative Linkage Grant LST.CLG.
976607 (M. Chiva, J. Ausio¬).
References
Alsheimer, M., Benavente, R. (1996): Change of karyoskeleton during
mammalian spermatogenesis: Expression pattern of nuclear lamin
C2 and its regulation. Exp. Cell Res. 228, 181 ± 188.
Altschul, S. F., Gish, W., Miller, W. Myers, E. W., Lipman, D. J. (1990):
Basic local alignment search tool. J. Mol. Biol. 215, 403 ± 410.
Baarends, W. M., Hoogerbrugge, J. W., Roest, H. P., Ooms, M., Vreeburg, J., Hoeijmakers, J. H. J., Grootegoed, J. A. (1999): Histone
ubiquitination and chromatin remodeling in mouse spermatogenesis.
Dev. Biol. 207, 322 ± 333.
EJCB
Balhorn, R. (1982): A model for the structure of chromatin in
mammalian sperm. J. Cell Biol. 93, 298 ± 305.
Balhorn, R., Cosman, M., Thornton, K., Krishnan, V. V., Corzett, M.,
Bench, G., Kramer, C., Lee, J., Hud, N. V., Allen, M., Prieto, M.,
Meyer-Ilse, W., Brown, J. T., Kirz, J., Zhang, X., Bradbury, E. M.,
Maki, G., Braun, R. E., Breed, W. (1999): Protamine mediated
condensation of DNA in mammalian sperm. In: C. Gagnon (ed.): The
Male Gamete. From Basic Science to Clinical Applications. Cache
River Press, McGill University, pp. 56 ± 70.
Bandiera, A., Patel, U. A., Manfioletti, G., Rustighi, A., Giancotti, V.,
Crane Robinson, C. (1995): A precursor product relationship in
molluscan sperm proteins from Ensis minor. Eur. J. Biochem. 185,
1 ± 6.
Bedford, J. M., Calvin, H. I. (1974): The occurrence and possible
functional significance of -S-S- crosslinks in sperm heads, with
particular reference to eutherian mammals. J. Exp. Zool. 188, 137 ±
156.
Bloch, D. P. (1969): A catalog of sperm histones. Genetics 61 (Suppl.),
93 ± 111.
Bloch, D. P. (1976): Histones of sperm. In: R. C. King Jr. (ed.):
Handbook of Genetics. Plenum Press, pp. 136 ± 167.
Ca¡ceres, C., Gimenez-Bonafe¬, P., Ribes, E., Wouters-Tyrou, D., Martinage, A., Kouach, M., Sautie¡re, P., Muller, S., Palau, J., Subirana, J. A.,
Cornudella, L., Chiva, M. (1999): DNA-interacting proteins in the
spermiogenesis of the mollusc Murex brandaris. J. Biol. Chem. 247,
649 ± 656.
Carlos, S., Jutglar, L., Borrell, I., Hunt, D. F., Ausio¬, J. (1993): Sequence
and characterization of a sperm-specific histone H1-like protein of
Mytilus californianus. J. Biol. Chem. 268, 185 ± 194.
Chiva, M., Mezquita, C. (1983): Quantitative changes of high mobility
group non-histone chromosomal proteins HMG 1 and HMG 2 during
rooster spermatogenesis. FEBS Lett. 162, 324 ± 328.
Chiva, M., Saperas, N., Ca¡ceres, C., Ausio¬, J. (1995): Nuclear basic
proteins from the sperm of tunicates, cephalochordates, agnathans
and fish. In: B. G. M. Jamieson, J. Ausio¬, J. L. Justine (eds.): Advances
in Spermatozoal Phylogeny and Taxonomy. Mem. Mus. natn. Hist.
nat. Paris Vol. 166, pp. 501 ± 514.
Daban, M., Martinage, A., Kouach, M., Chiva, M., Subirana, J. A.,
Sautie¡re, P. (1995): Sequence analysis and structural features of the
largest known protamine isolated from the sperm of the archaeogastropod Monodonta turbinata. J. Mol. Evol. 40, 663 ± 670.
Fita, I., Campos, J. L., Puigjaner, L., Subirana, J. A. (1983): X-ray
diffraction study of DNA complexes with arginine peptides and their
relation to nucleoprotamine structure. J. Mol. Biol. 167, 157 ± 177.
Gatewood, J. M., Cook, G. R., Balhorn, R., Bradbury, E. M., Schmid,
C. M. (1987): Sequence specific packaging of DNA in human sperm
chromatin. Science 263, 962 ± 964.
Gimenez-Bonafe¬, P., Ribes, E., Kasinsky, H. E., Subirana, J. A., Chiva,
M. (1999): Keratinous state of Eledone cirrhosa sperm cells and their
special nuclear protein. J. Exp. Zool. 283, 580 ± 589.
Gineitis, A. A., Zalenskaya, I. A., Yan, P. M., Bradbury, E. M., Zalensky, A. O. (2000): Human sperm telomere-binding complex involves
histone H2B and secures telomere membrane attachment. J. Cell
Biol. 151, 1591 ± 1598.
Gusse, M., Sautie¡re, P., Chauvie¡re, M., Chevaillier, P. (1983): Extraction,
purification and characterization of the sperm protamines of the
dog-fish Scylliorhinus caniculus. Biochim. Biophys. Acta 748, 93 ±
98.
Haaf, T., Ward, D. (1995): Higher order nuclear structure in mammalian
sperm revealed by in situ hybridization and extended chromatin
fibers. Exp. Cell Res. 219, 604 ± 611.
Hazzouri, M., Rousseaux, S., Mongelard, F., Usson, Y., Pelletier, R.,
Faure, A. K., Vourc−h, C., Se¡le, B. (2000): Genome organization in the
human sperm nucleus studied by FISH and confocal microscopy. Mol.
Reprod. Dev. 55, 307 ± 315.
Hunt, J. G., Kasinsky, H. E., Elsie, R., Wright, C., Rice, P., Bell, J., Sharp,
D., Kiss, A., Hunt, D., Arnott, D., Russ, M., Shabonowitz, J., Ausio¡, J.
(1996): Protamines of reptiles. J. Biol. Chem. 271, 23547 ± 23557.
Hurley, C. K. (1977): Electrophoresis of histones: a modified Panyim
and Chalkley system for slab gels. Anal. Biochem. 80, 624 ± 626.
EJCB
Joffe, B. I., Solovei, I. V., Macgregor, H. C. (1998): Ordered arrangement and rearrangement of chromosomes during spermatogenesis in
two species of planarians (Plathelminthes). Chromosoma 107, 173 ±
183.
Kasinsky, H. E. (1989): Specificity and distribution of sperm basic
proteins. In: L. S. Hnilica, G. S. Stein, J. L. Stein (eds.): Histones and
Other Basic Nuclear Proteins. CRC Press, Boca Raton, Florida,
pp. 73 ± 163.
Kramer, J. A, Krawetz, S. A. (1996): Nuclear matrix interactions within
the sperm genome. J. Biol. Chem. 271, 11619 ± 11622.
Lin, M., Sistina, Y., Rodger, J. C. (1995): Electron-microscopic localization of thiol and disulfide groups by direct monomaleimido-nanogold
labelling in the spermatozoa of a marsupial, the tammar Wallaby
(Macropus eugenii). Cell Tissue Res. 282, 291 ± 296.
Maxwell, W. L. (1974): Spermiogenesis of Eledone cirrhosa Lamarck
(Cephalopoda, Octopoda). Proc. R. Soc. Lond. B186, 181 ± 190.
Maxwell, W. L. (1975): Spermiogenesis of Eusepia officinalis (L), Loligo
forbesi (Streenstrup) and Alloteuthis subulata (L) (Cephalopoda,
Decapoda). Proc. R. Soc. Lond. B191, 527 ± 535.
Mirzabekow, A. D., Sanko, D. F., Kolchinsky, A. M., Melnikova, A. F.
(1977): Protein arrangement in the DNA grooves in chromatin and
nucleoprotamine in vitro and in vivo revealed by methylation. Eur. J.
Biochem. 75, 379 ± 390.
Oko, R. J., Jando, V., Wagner, C. L., Kistler, W. S., Hermo, L. S. (1996):
Chromatin reorganization in rat spermatids during the disappearance
of testis-specific histone, H1t, and the appearence of transition
proteins TP1 and TP2. Biol. Reprod. 54, 1141 ± 1157.
Oliva, R., Dixon, G. R. (1991): Vertebrate protamine genes and the
histone-to-protamine replacement reaction. Prog. Nucleic Acid Res.
Mol. Biol. 40, 25 ± 94.
Cysteine-rich protamine and chromatin condensation 349
Oliva, R. (1995): Sequence, evolution and transcriptional regulation of
avian-mammalian P1 type protamines. In: B. J. M. Jamieson, J. Ausio¬,
J. L. Justine (eds.): Advances in Spermatozoal Phylogeny and
Taxonomy. Mem. Mus. natn. Hist. nat. Paris, vol 166, pp. 537 ± 548.
Panyim, S., Chalkley, R. (1969): High resolution acrylamide gel
electrophoresis of histones. Arch. Biochem. Biophys. 130, 337 ± 346.
Poccia, D. (1986): Remodelling of nucleoproteins during gametogenesis,
fertilization and early development. Int. Rev. Cytol. 105, 1 ± 65.
Sotolongo, B., Ward, W. S. (2000): DNA loop domain organization: The
three-dimensional genomic code. J. Cell. Biochem. 80, 23 ± 26.
Suau, P., Subirana, J. A. (1977): X-ray diffraction studies of nucleoprotamine structure. J. Mol. Biol. 117, 909 ± 926.
Suzuki, M., Wakabayashi, T. (1988): Packaging of DNA in cricket sperm.
A compact mode of DNA packaging. J. Mol. Biol. 204, 653 ± 661.
Tacke, R., Manley, J. L. (1999): Determinants of SR protein specificity.
Curr. Opin. Cell Biol. 11, 358 ± 362.
Ward, W. S., Zalensky, A. O. (1996): The unique, complex organization
of the transcriptionally silent sperm chromatin. Crit. Rev. Eukaryot.
Gene Expr. 6, 139 ± 147.
Watson, N. A., Rohde, K. (1994): Ultrastructure of spermiogenesis and
spermatozoa in Phaenocora anomalocoela (Platyhelminthes,
Typhloplanida, Phaenocorinae). Inv. Reprod. Dev. 25, 237 ± 246.
Willmitzer, L., R. G. Wagner (1980): The binding of protamine to DNA;
role of protamine phosphorylation. Biophys. Struct. Mech. 6, 95 ± 110.
Zalensky, A. O., Tomilin, N. V., Zalenskaya, I. A., Teplitz, R. L., Bradbury, E. M. (1997): Telomere-telomere interactions and candidate
telomere binding protein(s) in mammalian sperm cells. Exp. Cell Res.
232, 29 ± 41.