Purification and Biochemical Characterization of the Nuclear Sperm-Specific Proteins of the Bivalve Mollusks Agriodesma saxicola1 and Mytilimeria nuttalli

10 Pages·1992·3.6 MB·English

by  J Ausio

Reference: Biol. Bull 182: 31-40. (February, 1992)
Purification and Biochemical Characterization
of the Nuclear Sperm-Specific Proteins of
the Bivalve Mollusks Agriodesma saxicola1
and Mytilimeria nuttalli
JUAN AUSIO
Department ofBiochemistry andMicrobiology, University ofVictoria,
W
I'ictoria. British Columbia I'8 3P6. Canada
Abstract. The proteins from the nuclei of the sperm HI, this protein is soluble in diluted perchloric acid, has a
from two different species ofthe subclass Anomalodes- globulartrypsin-resistant core, islysinerich, andyetiscom-
=
mata oftheclass Bivalvia have been analyzed forthe first positionally related tothe protamines(PL protamine-like)
time. In both instances Agriodesma saxicola (Baird, (Subirana et al., 1973). Ofall the different nuclear sperm-
1863) and Mytilimeria nuttalli (Conrad, 1837) the specific proteins found within a given species, PL-I is the
compositional pattern is very similar. The sperm chro- one with the lowest electrophoretic mobility in urea-acetic
matin isorganizedbyamajorprotamine-like PL-I protein. acid gels (Ausio, 1986). The presence ofa protamine-like
Asin all PL-I, this protein has a trypsin-resistant core. In histone HI-like protein in bivalve mollusks may have im-
both speciesanalyzed, PL-I containscysteine residuesthat portantevolutionary implications, notonly within thephy-
account forthe presence ofthe monomer (M) and dimer lum Mollusca (SubiranaandColom, 1987), butalsowithin
(D) forms observed in the total nuclear HC1 extracts. The other taxonomic groups.
molecular mass ofthese proteins is 21,000 Da in A. sax- Aspointed outby Kasinsky(1989), however, onlysome
icola, and 25,000 Da in M. nuttalli. All ofthe specimens ofthe subclasses within the class Bivalvia have been an-
ofA. saxicola analyzed were hermaphrodites. Asa result, alyzedsofar.andonlyafewspecieshavebeenthoroughly
thenuclearsperm-specificproteinsfrom several preparations characterized. Nevertheless, at least one sperm-specific
were readily and extensively degraded by protease activity histone HI (PL-I) protein has been identified: in Mytilus
from the oocytes. Such degradation was always observed californianus (by Jutglar et al.. 1991), Crassostrea gigas
when cross contamination between thetwo gonadal tissues (by Sellos, 1985), and Glycymeris yesonensis (by Odin-
accidentally occurred during protein extraction. tsovaetal, 1989)(subclassPteriomorphia); inEnsisminor
(by Giancotti et al., 1983), Spisula solidissima (by Ausio
Introduction etal., 1987) and Macoma nasuta (Ausio, 1988) (Subclass
The presence of a highly specialized histone HI (PL-I Heterodonta); and in Anodonta piscinallis (by Rozov et
protein)seemstobeacommon featureofthenuclearprotein al., 1984) (Subclass Palaeoheterodonta). In some ofthese
compositionofthesperm ofbivalve mollusks(Jutglaretal.. species, two histone HI-like proteins have been described.
1991). Despite the structural heterogeneity of the sperm Although the subclass Heterodonta has been widely
proteinswithin thistaxonomicgroup(Ausio, 1986;Zalensky studied (Ausio, 1986), three subclasses, according to
and Zalenskaya, 1980), a PL-I protein has been identified Barnes's(1980)classification ofthebivalve mollusks, have
in each ofthe speciesanalyzed in detail so far. Like histone never been characterized: Cryptodonta, Palaeotaxodonta,
and Anomalodesmata. In the present work, we have an-
alyzed and characterized the sperm-specific proteins of
R'eTcheiisvsepdec1i8esJuisnemo1r9e9c1;oamcmcoepntleydr2e5feNrorevdetmobeasrE1n9t9o1d.esma saxicola, twospecieswithinthesubclassAnomalodesmataandhHave
butsee Bernard. 1983. shown thateach containsahighlyspecialized histone 1-
31
32 AUSIO
J.
M
like (PL-I) protein that is the major protein component tion buffer was 6 guanidinium chloride (Gdn-HCl;
mM
ofthe nuclei ofthe sperm ofthat organism. Schwarz/Mann Biotech), 50 Tris-HCl pH 7.6.
Reversephase highpressure liquidchromatographv
Materials and Methods
(HPCL)
Livingspecimens HPLC was carried out on a 5 n (25 X 0.46 cm) Vydac
C4column, with0.1%trifluoroaceticacid(TFA)aseluant
Specimens of Agriodesrna saxicola and Mytilimeria with different acetonitrile gradients.
nuttalliwere collected alongthe west coast ofVancouver
Island. British Columbia, Canada, by SCUBA divers from Determination ofthe molecular mass
the Biology Department at the University ofVictoria.
The molecular mass of each protein was determined
Nucleipreparation andprotein extraction bygelnitration underdenaturingconditiMonsonaSuperose
12HR column in the presence of 6 Gdn-HCl (see
Isolation ofsperm nuclei and HC1 crude extraction of above). Several protamines, protamine-like proteins, and
the nuclear basic proteins was performed as described histonesofknown molecularmasswere usedasstandards:
elsewhere (Ausio, 1986). Briefly, after carefully opening PL-I from Spisula solidissima (Mr: 33,500 Da) (Ausio
the shell, a small incision was made in thegonadal tissue, and Subirana, 1982a); Histone HI from calfthymus(Mr:
and the spontaneously released sperm were resuspended 22,000 Da) (DeLange, 1976); PL-Ill (01) from Mytilus
mM mM
inNaCl 0.15 M, Tris-HCl 20 pH 7.6,0.2 PMSF edulis (Mr: 9600 Da) (Ausio and Subirana, 1982b); and
(Phenylmethylsulphonyl Fluoride) (bufferA). Because,-!. unfractionated salmine from Oncorhynchus sp. (Mr; 4300
saxicola is hermaphroditic, its sperm would sometimes Da) (Ando et al, 1973). Histone HI from calf thymus
be contaminated with oocytes released accidentally from was purchased from Worthington, and salmine (sulfate
theintimatelyassociatedovaries(seebelowfordiscussion). form) was obtained from Sigma. The rest of the prot-
The sperm suspension was centrifuged at 3000 X g for amineswere prepared in my laboratory. Globularproteins
10min inaSS-34 Sorvall rotorat4C. Thepelletobtained werealso usedasa molecularmass markers: bovineserum
was homogenized in buffer A containing 0.5% TritonX- albumin (Mr: 68,000); ovoalbumin (Mr: 46,000); chy-
100. Afterstanding for 10 min on ice, the suspension was motrypsinogen A (Mr: 25,000); and ribonuclease A (Mr:
spundown underthesameconditionsasbefore. Thisstep 13,200). These proteins werepurchased from Pharmacia;
ismeanttosolubilize mostofthecytoplasmic membranes, all of them were subjected to performic acid oxidation
including the sperm flagella and the acrosome. Notice before being applied to the column (see below). Vitamin
that this step will also expose the sperm nuclei to egg B12 (Mr: 1350) was purchased from Sigma. For the es-
lysatesin those samplesofA. saxicolacontaminated with timationofthemolecularmass,thecolumnwascalibrated
oocytes; suchcytoplasmiccontamination mayberespon- with theabove standard proteins, andaplot ofKav versus
sible for the protein degradation observed under these log Mr was constructed (Mr = molecular mass; Kav
circumstances. The detergent-treated pellet was imme- = distribution coefficient).
N
diately homogenized in 0.4 HC1. Solubilization was
continued for 2 h under stirring at 4C. Finally, the sus- - v
pension wascentrifuged at 12,000 X gfor 10 min at 4C, v,
and the acid extract was precipitated with 6 volumes of wherevoandV,arethevoidandtotal volumeofthecolumn,
acetone, overnight, at -20C. and Ve = the elution volume of a given protein. Blue
Dextran and dansyl-L-alanine (Sigma) were used to de-
Gel electrophoresis termine V and V, experimentally. The proteins of un-
known Mr were mixed with the protein standards and
Polyacrylamide gel electrophoresis was carried out on run together through the column. Their molecular masses
urea-acetic acid gels, asdescribed elsewhere (Ausio, 1986). were estimated by interpolation oftheirKavvalueson the
best fitting line ofthe calibration plot.
Protein purification andfractionation
Amino acidanalysis
Ionic exchange chromatography was carried out on a
10 X 100 mm Protein-Pak SP8HRcolumn fromWaters- Amino acid analysis was carried out on an Applied
MilGleilponrietraastidoenscwraisbecdarerliseedwohuetreo(nMaog1e0nXse3n00elma/m.. 1F9P9L1C). hByidorsoylsytseimsswmaosdcealrr4i2ed0Aoudteriinvagtaisz-eprh-aasneal6yzNerHsCys1teamn.dT1h%e
Superose 12HR 10/30column from Pharmacia. Theelu- phenol under an argon atmosphere at 165C, for 1 h, 2
NUCLEAR PROTEINS FROM THE SPERM OF BIVALVE MOLLUSKS 33
h,and4h,thefinalaminoacidcompositionwasobtained Performicacidoxydation. Performicacidwasprepared
byextrapolation ofthedatatozero time. So thatcysteine accordingto Hirs(1967). Fortheoxidation, 1-mgaliquots
could be quantified, all protein samples were pyridyl- of protein were dissolved in 0.5 ml of performic acid,
ethylated before hydrolysis, as described below. which had been previously cooled on ice. The reaction
was allowed to proceed for 4 h in an ice bath in capped
Chemical modification ofproteins tubes. The sample was then resuspended in a 25-fold ex-
Reduction ofSH groups. The SH groups of cysteine cess ofHPLC grade distilled water and lyophilized.
were reduced as describedMby Kuehl (1979). Briefly, the Cysteine pyridylethylation. Proteins were pyridyleth-
proteins, at 1 mg/ml in 6 urea 20 mAf Tris-HCl pH ylated, providing for a quantitative estimate ofcysteine
7.6, were reduced in the presence of8% |8-mercaptoeth- in the amino acid analysis. The procedure used wasas fol-
anol for 3 h at room temperature. lows: proteins (=1 nanomol) were dissolved in 44 ^1 of
mM
OxidationofSHgroups. Oxidationwascarriedout un- 6.8 Murea, 60 Tris-HCl, 1.25 mA/EDTA (pH 7.6),
der the same buffer conditions as above, but in the pres- and 2.3% /3-mercaptoethanol. The solution was incubated
mM
enceof0.72 O-phenanthrolineand0.36 mA/CuSO4. for 3 h at room temperature in the dark. Subsequently, 8
CE AS USA
I II IV
J
PL-
1
HIST
X
T J-
PHI
PL-
1
PL-IV
PR
Figure 1. UreaaceticacidPAGEanalysisofthenuclearsperm-specificproteinsofAgriodesmasaxicola
(AS) and Mytilimeria nuttalli(MN) in comparison to a histone standard from chicken erythrocytes (CE)
and to a protamine from salmon, salmine(SA). The nuclearsperm-specific proteinsofone representative
ofeachofthefivegroups(O, I, II, III.and IV)oftheclassificationofthebivalvemollusks(Ausio. 1986)are
alsoshown.Therepresentativespecieschosen foreachgroupwere:O:Pectenmaximus;I:Spisulasolidissima;
II: Ensis ensis; III: Mamma nasula. and IV: Mytilus edulis. The regions corresponding to the different
protamine-like (PL-I, PL-II, PL-Ill, and PL-IV) proteins defined in Ausio (1986) are also shown. HIST:
histoneregion. PR: protamine. D: dimerform. M: monomer form ofthe majorsperm protein component
ineach species. X2, Y2: possibledimer formsofthe minorsperm protein components X, Y.
34 AUSIO
J.
A.
0.8
ss a b c d
0.6
2.0
1.5 -
-
0.2 1.0 O
2CO
0.5
0.0 0.0
8 16 32 40 64 72 80
TIME min
,
B.
1.5
SS
L/L/UUUUUUU
60
1.0 50
o UJ
fO
CM 405!
300
0.5
r-
LU
20 <
0.0 10
10 20 30 40 50 60
TIME min
,
Figure 2. Fractionation ofa crude 0.4 A' HCl extract from the nuclei of the sperm ofAgnodesma
saxicola. (A) Ionicexchangechromatographyona(10 X 100 mm) Protein-Pak SP 8HR column. Proteins
wereeluted with a linear(0-2 A/) NaCl gradient in 50 m.1/ Na-phosphate buffer(pH 6.8) at a flow rate of
1 ml/min. Theinsetshowstheurea-aceticacid PAGE analysisofthe fractionsindicated. (B) Reverse-phase
HPLConaVydacC4column. Elutionwascarriedwithanacetonitrilegradient in0.1%trifluoroaceticacid
ataflowrateof1 ml/min.Theinsetshowstheelectrophoreticanalysisofthefractionation.Thelanesshown
intheinset, and thechromatogram hasbeen aligned to match the fraction analyzed with itscorresponding
position inthechromatogram. SS: startingsample.
NUCLEAR PROTEINS FROM THE SPERM OF BIVALVE MOLLUSK.S 35
A. B.
1.2 mn a b c d e f 9
_
I
0.8
?v5SHHBI^HBM^H
as a b c d e f 9
_
0.4
IL
0.2
0.0 16 24 32 48
TIME,min.
M
Figure 3. (A) Gel filtration FPLC on a Superose 12 HR 10/30 column. The elution buffer was 6
Gdn-HCI in 50 mA/Tris-HCl pH 7.6. The flow rate was 0.4 ml/min. Theelution profiles ofHC1 nuclear
extractsfromthesperm(ofM)ytilimerianuttalli( )andAgriodesmasaxicola( )areshowntogether
withtheelutionprofile ofsomeofthestandardsusedtocalibratethiscolumn: I: PL-IfromSpisula
solidissima; II: Histone HI from calfthymus; III: PL-HI from Mytilus edulis; IV: protamine salmine; V:
vitamin B 12; VI: dansyl-L-alanine. (B) Electrophoreticanalysison urea-aceticacidgelsofthe fractionsa,
b. c. d. e, f, g ofthe elution profiles ofAt. nuttalli and A. saxicola. mn: starting sample ofA/ nuttalli. as:
startingsampleofA. saxicola.
H\ of4-vinylpyridine was added, and the reaction was al- established fortheclassification ofthe nuclearsperm-spe-
lowedtoproceed for2 hatroom temperature. Thesample cific proteins of the bivalve mollusks (Ausio, 1986). In
wasthen immediately desalted in an HPLCreverse phase each ofthe two speciesanalyzed, two majorprotein bands
C8Vydac column, which was eluted for 5 min with 0.1% run in the region ofthe PL-I proteins (Ausio, 1986). In
TFA (trifluoroacetic acid), and for 20 min with a 0-70% addition tothese proteins, 10-20% ofminorprotein frac-
acetonitrile gradient in 0.1%- TFA. /3-lactoglobulin from tions X and Y, which run in the histone region, are also
Applied Biosystems Inc. was used as a standard for this observed (see Fig. 1-AS and 1-MN). This protein com-
procedure. position was sufficiently novel that the two organisms
TrypsMindigestion. Trypsin digestion ofproteinsin high could not, at first, beassigned toany oftheproteingroups
salt 2 NaCl, 50 mA/Na-phosphate buffer(pH 6.8) previously established in my classification ofthe bivalves
wascarried outasdescribed elsewhere (Ausio el ai, \987). (Ausio, 1986). Thatwasnotsurprisingbecausetheybelong
to a subclass (Anomalodesmata) that had not been ana-
Results lyzed before. I thereforedecided topurify andcharacterize
each ofthe major protein components ofthese organisms.
sCpherromm-aspteocgirfaipchniucclaenaarlypsriosteainnsdfpurroimfiAc.atsiaoxnicoofltaheand M. FPTLhCeufnidrsetrantotne-mdpetnaatturfirnagctcioonndaittiioonnsbyissihonoiwcneixncFhiagnugree
nuttalli
2A. Most ofthe protein components coeluted in a single
M
Figure 1 showsthe0.4NHC1 protein extracts from the multiphasic peak at around 2 NaCl, but some protein
nuclei ofthe sperm ofA. saxicola and Al. nuttalli. They separation was clearly achieved as is shown in the inset
are shown in comparison to the five groups previously ofthe same figure.
36 AUSIO
J.
1.0 Characterization ofthe sperm-specific nuclearproteins
from A. saxicola andM. nuttalli
The fractionation problemsdescribed in the preceding
0.8 section began to be elucidated when the molecular mass
ofthese proteins was analyzed. Figure 4 shows the cali-
bration plot used to estimate the molecular mass ofpro-
0.6 teins from gel filtration analysis. The molecular mass of
the two major protein components of the sperm nuclei
in A. saxicola were: 21,000 Da for the fastest protein
component and 43,000 Da for the slowest moving frac-
0.4
tion. The values were 25,000 Da and 49,000 Da for M.
nuttalli. These results suggested a monomer-dimer rela-
tionship between the slow and the fast moving protein
0.2
fractions present in each species. To analyze this rela-
tionship, and to examine the nature of the association
phenomenon, 1 incubated the crude HC1 extracts in the
0.0103 10" 105 presence of either /3-mercaptoethanol or copper phen-
Mr anthroline. Figure 5 shows the results ofthese treatments
in the case ofA. saxicola, and identical results were ob-
Figure4. Calibration plot usedtodeterminethe molecularmassof
thespermproteinsdeterminedonasuperose 12HR 10/30columnunder tained with M. nuttalli (results not shown). The slower
theelutionconditionsdescribedinFigure3A.GlobularOandnonglob- moving band completely disappears underreducingcon-
ular proteinswereusedasstandards. 1: Vitamin B 12(Mr: 1350Da); ditions for cysteine. Under oxidizing conditions (in the
2: ribonuclease A (Mr: 13.200 Da); 3: chymotrypsingen A (Mr: 25,000 presence ofcopper phenanthroline) the relative intensity
6Da8),:0040: Doav)o:al1b*:umpirnot(aMmri:ne46s,a0l0m0inDea)(:Mr5:: b4o,v3i0n0eDsa)e;ru2m*:alprboutmeiinn (PMLr-I: ofthe faster moving band (see Fig. 5b) slightly decreases,
from Myliluseditlis (Mr: 9,600 Da); 3*: histone HI from calfthymus and higher association complexes are formed (see arrow
We
(Mr: 22,000Da);4*: ProteinPL-IfromSpisulasolidissima(Mr: 33,000 in Fig. 5b). are therefore dealing with the association
Da). M = monomer form and D = dimer form ofthe major sperm ofthe faster moving protein components. Although the
protein components of: Mytillineria mtnalli , and Agriodesma association seems to involve primarily the formation of
saxicola O. dimers, the number ofcysteines present in the monomer
To increase the resolution in the separation, the 0.4 N
SS A B
HC1 protein extracts were fractionated by reverse-phase U
HPLC;theelution profile isshown in Figure 2B. Although . .
two peaks corresponding to each ofthe two major com-
^^M
ponentscouldbeclearlyseparated, both ofthem exhibited
different amounts of what appeared to be overlapping d
cross-contamination. PL-I x
2
Size fractionation of the starting HC1 extracts by gel m
filtration under denaturing conditions in the presence of
6 A/guanidinium chloride (Gdn-HCl) isshown in Figure
3. Although the peaks could not be completely resolved,
the sample was partially fractionated as shown in Figure
3B. Indeed, when some ofthe eluting fractions from the
different regions corresponding to the two major protein
components were pooled together and rerun under the
same conditions, two distinct peaks could then be clearly
resolved. This was used as a basis for estimating the
molecular mass ofeach ofthese protein components. Figure5. Urea-aceticacidpolyacrylamidegelelectrophoresisofthe
Nevertheless, when the fraction under each separate nuclearsperm specific proteins fromAgriodesmasaxicola underA: re-
peak was analyzed by urea acetic acid PAGE, the same ducing(6%/3-mercaptoethanol)orB:oxidizing(copper-phenanthroline)
cross-contamination observed in Figure 2B was csopnedrimtiopnrso.teSinSc=osmtpaortnienngtsa(mPpLl-Ie),.mX,,dX=2 =momnoonmoemrearndanddimdeirmeorfmofajtohre
again observed, although to a lesser extent (results not minorsperm proteincomponent. Thearrowheadindicatesthepresence
shown). ofhigherassociation oligomersobtained upon oxidativetreatment.
NUCLEAR PROTEINS FROM THE SPERM OF BIVALVE MOLLUSKS 37
cannot be clearlyascertained from theabove experiments. Table I
Thus, although the strong tendency toward dimer for- Aminoacidanalysis(mol%)ofthenuclearsperm-specificPL-I
mation would suggest the presence only ofone cysteine proteinso/'Agriodesmasaxicola PL-I(AS) andMytilimeria nuttalli
permolecule, thelackofcompletedimerization observed PL-I(MN) incomparison tothoseofSpisulasolidissimaPL-I(SS)
in Figure 5b, and the presence ofassociation complexes (AusioandSubirana. I982a) andMacoma nasutaPL-I(MC)
higher than dimers. would strongly suggest the presence (Ausio. 1988)
ofmore than one cysteine. The presence oftwo cysteine Pl-I(AS) PL-I(MN) PL-I(SS) PL-I(MC) X (AS)
residuespermolecule, which couldeasily form an internal
disulfide bond, would explain the incomplete polymer- Lys
ization of the monomer, otherwise expected under the
oxidizing conditions used here (Fig. 5b).
To determine the number of cysteines, as well as to
establish theaminoacid composition ofthe majornuclear
protein component ofthe sperm ofA. saxicola and M.
mittalli, protein fractionssuch asthoseshown in the insets
ofFigure 2A and B were pyridylethylated before amino
acid analysis. A /5-lactoglobulin sample was simulta-
neously treated andanalyzed tocheck forthecompletion
ofthe reaction. Theaminoacidanalysesclearlyshow(see
Table I) that the proteins ofbothA. saxicola and M. nut-
talli contain two cysteine residues per molecule. Com-
parison with the amino acid analysesofotherPLproteins,
revealsthe PL-I natureofthe major nuclearprotein com-
ponent of the sperm of the two species analyzed. Like
other PL-I proteins (Ausio, 1986; Ausio, 1988; Jutglar et
al., 1991), these have an internal trypsin resistant core
(Fig. 6).
M
Besides the major protein components and D, we
have also characterized the minor component X ofA.
saxicola. Thisprotein exhibitsan amino acidcomposition
that is almost identical to PL-I (see Table I). Although
38
D
Figure7. Microscopicanalysiswithphasecontrastofpuresperm(A),andofspermpreparationscontaining
an increasing amount ofcontamination by eggs (B) and (C). The samples were obtained from different
specimensofAgrtiidesmasaxicola. a,b,andc:electrophoreticanalysis,inurea-aceticacidPAGE,ofsperm
preparations containing an increasing amount ofcontamination by eggs. As contamination increases, an
extensivedegradation ofboth the monomer(M)and dimer(D) formsofthe majornuclearsperm-specific
PL-Icomponentisobserved.Arelativelyresistantpeptide r isgeneratedduringthisdegradationprocess.
Thewhitebarcorresponds to50pm. X and X2 areas in Figure 5.
decrease, and the proteins finally disappear completely. clearly fulfill the compositional definition ofprotamines
Thisisaccompanied by theappearance ofacomplex pat- (Subirana, 1983): (Lys + Arg) = 45-80%, (Ser + Thr)
ternofnewbandswith fasterelectrophoretic mobility(Fig. = 10-25%. Indeed, of all the PL proteins characterized
7b,c). Such protein pattern transition isclearlyindicative so far, the onesanalyzed here exhibit the highest arginine
ofadegradation processelicited by specific proteases from content within the PL classification (Ausio, 1986). The
the contaminating eggs. A similar in vitro degradation of presence ofa trypsin-resistant core (see Fig. 6) indicates
sperm histones by the cytoplasm ofsea urchin eggs has that these proteins are also related to the proteins ofthe
also been reported (Betzalel and Moav, 1987). A quite histone H1 family. Therefore, the PL majorcomponents
resistant degradation peptide. designated r, is produced ofbothA. saxicolaand M. nuttallishould be undoubtedly
duringthis process. Although the composition and nature classified withinthe PL-I class(Ausio, 1986). The presence
ofpeptide rarecompletely unknown, itiscertainly much ofcysteine in these proteins does not seem to be an un-
smallerthan the trypsin-resistant peptide obtained under usual feature; indeed, two cysteines also occur in the PL-
in vitro conditions (see Fig. 6). A nuclear HC1 protein I component ofMacoma nasuta (Ausio, 1988).
extract from pure oocytes contained none ofthe proteins Becausethe minorprotein component XofA. saxicola
observed in Figure 7 (results not shown). has an amino acid composition indistinguishable from
PL-I, these two proteins must be closely related, and X:
Discussion (see Fig. 1-AS) may represent a dimer form of X. The
In this work I have analyzed the protein composition same observations apply to the X and Y components of
ofthe nuclei ofthe sperm of two representatives ofthe M. nuttalli (results not shown). The structural relation-
subclass Anomalodesmata within the class Bivalvia. In ships among PL-I and the X and Y fractions remain ob-
both ofthe species analyzed Agriodesma saxicola and scure, but the similarity oftheir amino acid analyses to
Mytilimeria nuttalli 80-90% ofthe nuclear sperm-spe- that ofthe major protein component suggests that X and
cificproteinsconsistofamixtureofdimer(D)and mono- Y maybeproteolytic peptidesfrom PL-I. Theycouldarise
mer (M) forms of a protamine-like (PL-I) protein. The fromtheactivityofeitheranuclearoranacrosomal sperm
remaining 10-20% includes the minor protein fractions protease (Miiller-Esterl and Fritz, 1981) during protein
X and Y. extraction. However, they do not seem to be related to
The protamine-like nature ofthe major proteins is re- any of the protein fragments produced by the protease
vealedbytheiraminoacidcomposition(seeTableI).They digestion resulting from oocytic contamination, because
NUCLEAR PROTEINS FROM THE SPERM OF BIVALVE MOLLUSKS 39
Table II
Classification oftheclassBivalviaaccordingtollieirprotamine-like
group(Ansid. 1986)
Subclass(a)
40 AUSIO
J.
Kmhi.J.1979. Synthesisofhighmobilitygroupproteinsinregenerating Sellos, D. 1985. The histones isolated from the sperm ofthe oyster
ratliver./ Biol. Chem. 254: 7276-7281. Crassostreagigas. CellDiffer. 17: 183-192.
Mogensen,C.,S.Carlos,andJ.Ausio. 1991. Microheterogeneityand Subirana,J. A.,C. Cozcolluela, J. Palau, and M. Unzeta. 1973. Pro-
interspecific variabilityofthe nuclearsperm proteinsfrom Mytilus. tamines and other basic proteins from spermatozoa of molluscs.
FEESLett. 282: 273-276. Biochim. Biophys. Ada317: 364-379.
Miiller-Esterl, W., and H. Fritz. 1981. Sperm acrosin. Methods En- Subirana,J.A. 1983. NuclearProteinsinSpermatozoaandTheirIn-
:ymol. 80: 621-632. teractions with DNA. Pp. 197-214in TheSperm Cell, J.Andre,ed.
Odintsova,N.A.,S.M.Rozov,and I.A.Zalenskaya. 1989. Thechro- Martinus NijhoffB. V., The Hague.
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Rozov, S. M., V. A. Brednikov, F. K. Corel, M. V. Lavrenteva, and Zalensky,A.O.,andI.A.Zalenskaya. 1980. Basicchromosomalpro-
L.P.Solonenko.1984. Structureoflysine-nchhistonefromsperm teinsofmarineinvertebratesII.TheproteinsfromtheBivalviamol-
ofAnodontapiscinalis. Mol. Biol. (transl)(USSR) 18: 1497-1508. luscs. Comp. Biochem. Physiol. 66B: 415-419.