Table Of ContentReference: Bntl. Bull 185: 186-1%. (October. 1993)
Sperm-Specific Basic Proteins in the Holocephalan
Fish Hydrolagus colliei (Chondrichthyes,
Chimaeriformes) and Comparison with Protamines
from an Elasmobranch
N. SAPERAS123 M. CHIVA1 \ N. C. BOLS1 4 D. KULAK1 AND H. E. KASINSKY1 *
, , ,
^Department ofZoology. University ofBritish Columbia. \'ancouver, British Columbia. Canada l'6T
17.4. andBamfieldMarineStation, Bamfield. British Columbia. Canada I'OR lBO;2Institutde
Ciencies Del Mar. C.S.I.C.. OS039 Barcelona, Spain: 3Department d'Enginyeria Qitimica. Universitat
Politecnica de Catahmya. OS02S Barcelona. Spain: and4Department ofBiology, University of
Waterloo, Waterloo, Ontario, Canada N2L 3G1
Abstract. Seven basic proteins can be isolated from Saperas et al.. 1993, a, b); gene (Dixon et al.. 1985); and
sperm nuclei ofthe holocephalan ratfish Hydrolagus col- geneticexpression (latrou and Dixon, 1978; Hecht, 1989;
liei. Two of these proteins (R3 and mO) are devoid of Oliva and Dixon, 1991). The protamines ofother verte-
cysteine, whereas fiveofthem (Rl, R2, ml, m2, and m3) brates, particularly those from amphibians, reptiles, birds,
contain lowlevelsofthisamino acid residue. The proteins and mammals, haveoftenbeenconsideredtobemolecules
Rl, R2, and R3 are major ones in the sperm nuclei ofH. that are related evolutionarily to these protamines (Nak-
colliei. and they are analogous to basic proteins Zl, Z2, znoetal.. 1976;Chivaa/., 1987, 1989;Olivaand Dixon,
and Z3 (scylliorhinines) from the sperm ofthe elasmo- 1991; Takamune et al.. 1991). Among the cartilaginous
branch Scyliorhinus canicu/a. However, taking into ac- fishes, spermatogenesis in a few species has been studied
count the partial sequence ofR3 protein and the number based on cytochemical staining(Bols and Kasinsky, 1974,
ofcysteines in Rl and R2, these proteins do not seem to 1976)and electrophoresis(Oliverasetal., 1990). However,
be homologous to the scylliorhinines. A comparison of at the level of biochemical characterization, knowledge
sperm basic proteins between H. colliei (a holocephalan) of protamines in cartilaginous fish is based entirely on
and 5. canicn/a (an elasmobranch) suggestsa remarkable studies ofa single species, Scyliorhinus canicula (Quero,
divergence of these proteins from a common ancestral 1984), the lesser spotted dogfish (also known as the small
pattern during the evolution ofChondrichthyes. spotted catshark).
The observations on the changes of nuclear proteins
Introduction that occur during the spermiogenesis of5. canicula show
The spermatozoan nucleus usually contains specific evident differences between this species and bony fish.
proteins that condense the DNA. In most animal sper- Gusse andChevaillier( 1978. 1981)observed thattwoba-
matozoa, these proteins replace somatic histones. They sic "intermediate" proteins. SI and S2, appearin theearly
showa great interspecific variability (Bloch, 1976; Kasin- modificationsofchromatin structureduringspermiogen-
sky, 1989). The sperm-specific basic proteins (SBPs) of esis in S. canicu/a. These proteins partially substitute for
somebony fish(called "protamines." "monoprotamines," histones (Chevaillier, 1991 ), although in more advanced
or "true protamines") have been very well studied at the stages they are in turn replaced by other basic proteins
levels ofprotein (Ando et al.. 1973; McKay et al, 1986; (protamines). These authors found four protamines (Zl,
Z2, Z3, and S4) called "scylliorhinines" in the sperma-
tozoan nucleus. Oneofthese protamines(Z3) isaprotein
Received 16 March 1993;accepted 23June 1993.
* Addresscorrespondence to thisauthorat the University ofBritish with 31 amino acid residues, a high percentage ofarginine
Columbia. (64.5 mol%), and nocysteine(Sautiere el al.. 1981:Gusse
186
PROTAM1NES IN A HOLOCEPHALAN FISH 187
ct at.. 1983). This protein isorganized in arginine clusters, homogenatewasfiltered through fourlayersofcheesecloth
and its primary structure is comparable with that ofthe and centrifuged at 4000 X gfor8 min. Theresultingsedi-
protamines from bony fish. Zl, Z2, and S4 are cysteine- ment (1 vol) was homogenized in 15 vol ofbufferA con-
rich protamines and contain 50. 46, and 32 residues, re- taining proteolytic inhibitor and 0.1% (w/v) Triton X-
spectively. The first two are rich in arginine, whereas S4 100 in a Dounce hand homogenizer underthe samecon-
is rich in lysine (Gusse ct ai, 1983). The sequences of ditions. The resultimngMhomogenate was unMderlaid by M15
these three molecules do not show a significant number vol ofbuffer B (20 Tris pH 7.5/0.15 KC1/1.4
ofidentities in the position oftheir residues (Sautiere ct sucrose) containing the proteolytic inhibitor, and centri-
ai, 1984;MartinagetVa/.. 1985; Chevaillierel ai, 1987); fuged for20 minat 30,000 Xg. Thesedimentwaswashed
norare they comparable to cysteine-rich protamines from in distilled water and the nuclei (or chromatin) obtained
mammals. In ageneral analysis from acomparison ofthe again by centrifugation (4000 X g, 8 min). All the pro-
sequences of these proteins (Sautiere ct ai, 1984; Che- cedures were performed in the cold.
vaillier et ai, 1987), these authors suggest two important
points: (1) Z3 and bony fish protamines probably origi- Proteins
nated from the same ancestral DNA sequence, beforethe
N
divergence between cartilaginousand bony fish occurred; Proteins were extracted from nuclei with 0.25 HC1.
(2) Zl, Z2, and S4 probably had an independent origin In thecasesthatwillbedetailed below, nuclearsediments
from different gene families. The studies ofthe sequence were reduced and alkylated before (or between) the ex-
ofintermediate protaminesSI and S2 alsoshowthat there tractions. For H. colliei. we applied the method used by
isnorelationshipbetween theirsequencesand thosefrom Gusse elai (1983)on S. canicula toallowthecomparison
sperm protamines (Chauviere et ai. 1987. 1989). ofsperm protamines between both species. Nuclearsedi-
In spite ofthis work, we cannot extend these results to mentswere reduced with 20 vol ofbufferC (50 mAfTris,
chondrichthyan fish in general because S. canicula is the pH 8.8/10 mAI DTT/2 mAf disodium EDTA) in a 1-h
only species of cartilaginous fish that has been studied incubation at 37C under N2 atmosphere. Aftercentrifu-
biochemically. In this work, we examine the sperm pro- gation at 7700 Xgfor 10min, thesedimentwasalkylated
teins of the ratfish (rabbitfish) Hydrolagus colliei. This with 25 vol of 12.5 mAf iodoacetamide in buffer C for
species is a holocephalan (order Chimaeriformes). It was 1 h at the same conditions and centrifuged.
selected because the holocephalans diverged very early ForFigure 1, basic nuclearproteinswereprepared from
from the rest ofthecartilaginous fish (Nelson, 1984). They testicular zones. In this case, the micromethod ofLouie
are considered to be more primitive than elasmobranchs and Dixon (1972) was used to isolate and extract sperm
(Schaeffer. 1981). Consequently, a comparison between basic proteins from cell suspensions. We have utilized
the protamines of//, collieiand 5. canicula may provide thismethodpreviously, asdescribed indetailbyKasinsky
valuable information about the common characteristics et ai (1985).
ofthese molecules in the early stages ofthe evolution of
fish. We should take into account that the long period of
Chromatography
separate evolution between these two groups could result
in important differences between their sperm proteins. Chromatographic separation of proteins by ion ex-
change columns was performed on CM-52 cellulose
Materials and Methods (Whatman). Proteins were dissolved in 50 mAf acetate
MNaCl
buffer(pH 6.0)containing0.2 (Chivaet ai, 1987,
Animals 1988). In some cases, the proteins extracted from sperm
H. colliei testes were obtained in April at the Friday nuclei were reduced and alkylated again before chro-
Harbor Laboratories. San Juan Island. Washington, USA; matographic separation. MReduction Mwas performed in
in May atComox, British Columbia, Canada; and inJune buffer D (20 mAf DTT/8 urea/0.5 NaCl) for 1 h at
at the Bamfield Marine Station. B.C., Canada. 37C under N2 atmosphere. Alkylation with 50 mAf io-
doacetamide in buffer D was done under the same con-
ditions(Gusse etai, 1983). Afterwards, the proteinswere
Nuclei
dialyzed against acetate buffer/NaCl and loaded onto the
Nuclei were prepared separately from wholetestis. epi- columns.
didymis, and ampulla ductus deferentis after Gusse and When necessary, fractionscollected fromCM-cellulose
Chevaillier (1978) as follows. The tissues were dissected columns were applied on a 5 ^m Spherisorb column and
on ice with scissorsand homogenized for30 sin a Sorvall purified by reverse-phase high-performance liquid chro-
Omnimixerin bufferA(20 rrLl/TrispH 7.5/0.15 A/KC1/ matography (HPLC). The column was equilibrated with
M
0.34 sucrose) containing either 50 mAf benzamidine 0.05% trifluoroacetic acid (TFA) and proteins separated
chloride or0.5 m.M PMSFasa proteolytic inhibitor. The with a gradient ofacetonitrile in 0.05% TFA.
188 N. SAPERAS ET AL.
Aniino acidanalysis Results
Amino acid analyses were performed as described by Nuclearproteins in testicular andsperm cells
CdehtievramainnedtMheezmqouliatrac(o19m8p3o)s.itTihoensseoafnaprloytseeisnsw.erTeheusneudmt-o durFionrgtthheeecloecutrrsoephoofrteetsitcicaunlaalryssipseromfabtaosgicenneuscilse,aronpreotteesitniss
ber ofamino acid residues in each protein was inferred was dissected into concentric zones (A to E). A portion
from the compositional values and from the electropho- of each zone was examined cytochemically to establish
retic mobility in acetic-urea polyacrylamide gels (Daban the stages ofspermiogenesis present in the tissuethat had
etai, 1991). tobeexamined by electrophoresis. Spermiogenesis in the
Inaddition, we usedthe methoddescribedbyCreighton ratfish has been divided into seven stages on the basis of
(1980) and Hollecker (1989) to determine the integral nuclearproteincytochemistry(Bolsand Kasinsky, 1976).
cnhaousmmmbbeeerercnioafalpcpyslstitaeeniddnaetrorde/s/(.iBdPcuToelIsl,ioefBiotpehrehormtioanlmgeeicrnuelMesasn.asnThhweeiilslmm)ae.stTthohodea cInh19e7Fm6ii)gc,uarlaensdt1atglheefesty,1czoaonnntedasi2nAirdaeonnutdnidfBiesdpaerbreymeaBqtouilidsvsaal(neAdn)taKnatosdicsnypstekory--
psurrooedtaie/ui1nm0wEmaDsMTdeADnTaftTour/r13e00d ammnidnMraeTtdru3ics7e-CdH.CblyAfpitnHecruw8ba.ar0t/di1,ngamliAinqlu8odAtist- Z(maotniedsCju(ssttabgeegsin3n-i4n)gitsohuentdeerroggoenneuoculsearanedlosnghaotwisonsp(eBr).-
M matidsstartingthe processofspiralization, aswell asthose
ofthe sampleMwere alkylated by adding0.25 iodoacet- still in the midst ofnuclear elongation. In zone D (stages
amide, 0.25 iodoacetate, and varying ratios of iodo-
arcoeotmamtiedmepetroatiuordeo,actehteatseo,lurteisopnesctwiveerley.plAafcteedr o1n5 imcienanadt zi5n-ot6no)e,tiEsghp(telsrytmapagaetci7kd)esdtahcreyesttsec.sotmNipcoulnleeatrionsfgptehserpsmierazcelolinlzesastaiirosenc,oomrwpghlaienltieezleiydn
adequate vMolumes analyzed in polyacrylamide gels con- homogeneous, but each zone (except for C) is enriched
taining 8 urea, following the low pH discontinuous for cells at a particular stage ofspermiogenesis.
system ofReisfeld el ul. (1962). The electrophoretic analysis ofbasic nuclear proteins
from each ofthese zones is shown in Figure 1, right. This
N-tenninal sequence analysisrevealsthatsomatichistonesarethemain proteins
The amino-terminal sequence ofratfish protamine R3 contained in nuclei from early spermiogenic stages, but
was determined by automated Edman degradation with in the mostadvanced stages, thespermatid nuclei contain
an Applied Biosystems 470A Protein Sequencer, and the a collection ofproteins with higher mobility than histone
phenylthiohydantoin derivatives ofamino acid residues H4. Threeoftheseproteinsappeartobe major;they have
were analyzed on an Applied Biosystems 120A on-line been designated as Rl, R2, R3. The proteins appearing
HPLC system using a microbore C18 Brownlee column in Figure 1 have been extracted from nuclei with 0.25 A'
(2.1 X 220 mm) (Saperas et a!., 1993b). HC1 withoutpreviousreduction/alkylation ofthese nuclei.
A subsequent reduction/alkylation and extraction ofthe
Electrophoresis chromatin did not solubilize any additional proteins.
Nuclei ofsperm cellshavebeen isolated separately from
forTrhoeutpirnoeceedluercetroofphPoarneytiicmaannadlyCshisalokflepyro(t1e9i6ns9.)wTahseugseelds aepmipduildlyamidsucatnuds tdheeferaenntteirsi.orFiagnudrepo2stserhioowrsrtehgeiopnrootfeitnhse
waneorle/ascteatiincedacwiidt/hH02O.2(55%:1(:1w/bvy) vCooloummaes)siaendbldueestianinmeedthi-n cexotnrdaicttieodnsfrfoormchthreosmeatniunc.leWihweinthnaucnldeiwiatrehoruetdurceedducainndg
tt2hh0ee-cscmyasmtgeeeilmnsiexwtreuerrseei.duusGeesedlosftwotehiremeppr1r0ootvceeimnrlemosonolgl,eutcbiuoulnte.ss,Tomoweetcioumusenestd tawlhikrtyehlea0mt.ea2dj5,orNabcaHonCmd1pslceeoxxitnrcsaeictdteioowfnipt(rhFoitgR.eli,2n,sR2lc,aananensdbeb,R3sco,loubdb)s.ielriTvzheeedd
the methodology descriMbed by Reisfeld et al. (1962), but in spermiogenic testicular nuclei, but some minor bands
using gels containing 8 urea.
also appear in the electrophoretic pattern (arrows in Fig.
2). When nuclei are extracted with 0.25 A' HC1 without
Histology previous reduction, only the protein R3 (Fig. 2, lane e)
In two specimens, midsagittal sections were dissected plus one minor band (named mO in lane e) can be solu-
from concentric zones ofampullae in the testis (Stanley, bilized. ProteinsRl andR2canbesolubilized from sperm
1963). Starting from the ampullogenic zone, a portion of nuclei only after reduction ofchromatin (Fig. 2, lane g).
eachzonewasfixedin 10% neutral-buffered formalin and In this'case, some minor bands also appear in the elec-
used forcytochemical analyses(Bolsand Kasinsky, 1974, trophoretic pattern (designated as ml, m2, and m3 in
1976). The remainder of each zone was used to isolate lane g). We have found these electrophoretic patterns to
and extract sperm basic proteins from nuclei by the mi- be highly reproducible with many sperm-nuclei prepa-
cromethod ofLouie and Dixon (1972). rations using different proteolytic inhibitors. We conclude
I'ROTAMINES IN A HOLOCEPHALAN FISH 189
-""' :''.:--V '.* -.
'
V -H4
B
Rl tf
-SP3-5
R2
-SP6
' &s" R3
-3
-^ft-
D '**$*
-P
A x
l
A B C D E
v<
Figure 1. Left: Midsagittal sections ofHydrolagus collici testis showing follicles containing different
stagesofspermiogenesis. Feulgen staining, zones A-D; hematoxylin-eosin staining. Zone E. Zones A and
B,sperniatidsbeginningtheprocessofnuclearelongation;ZoneC, mixtureofspermatidsundergoingnuclear
elongationandspermatidsbeginningtheprocessofspiralization;ZoneD,spermatidsundergoingspiralization;
Zone E, sperm organized into tightly packedcysts. Scale(in A)denotes 10/jm foreach zone. Right: Elec-
trophoreticprofilesofbasicproteinsextractedwith0.25NHC1(withoutreduction)fromdissectedtesticular
zones.Lane 1,//.colliciwholetestis;lanes2-6,//.collieitesticularzonesA-E.respectively;lane7,standards
offrog(XeniipM /i/cv/.v) sperm basic proteins(histonesand proteins SP 3-5, SP6)and herring protamine
(P). H4 = evolutionaryconservative histone H4. Electrophoresis in thisand subsequent figuresisfrom top
(+)tobottom (-).
N
that sperm-specific basic protamines of//. collici consist were extracted directly with 0.25 HC1, cleared by cen-
of three major (Rl, R2, and R3) and four minor (mO, trifugation; and reduced, alkylated, and re-extracted with
N
ml, m2, and m3) proteins. Only two ofthem (R3 and 0.25 HC1. The fractionscontainingproteinssolubilized
mO)can beextracted from sperm nuclei without previous without reduction (R3 and mO) and proteins extracted
reduction. In addition, Rl and R2 reproducibly have a after reduction (Rl, R2, ml, m2, and m3) were precipi-
slightly lower mobility when extracted without reduction tated with the addition of six volumes of cold acetone
from testis (Fig. 2, lane f) than when extracted with re- ( 20C overnight), rinsed with acetone, and dried.
duction from epididymal sperm (Fig. 2, lane g). The former fraction (R3 and mO) wasplaced on aCM-
cellulose column and proteins were collected in two
Purification amianalysis ofproteins "peaks"(I and II, and Fig. 3A). Whereaspeak II contained
the purified protein R3 (Fig. 3, lane II), peak I contained
Weused sperm cellsfrom epididymis,aswellasanterior a mixture ofR3 and mO (Fig. 3A, lane I). Proteins from
and posterior parts ofthe ampulla ductus deferentis, for this peak were separated by reverse-phase HPLC. In this
the purification ofratfish nuclear sperm proteins. Nuclei system we obtained a series ofpeaks, some ofthem con-
190 N. SAPERAS ET AL
\
****
Rl R1
R2
R2
-mi
-m2
-ma
' R3
**-
f 9
Figure 2. Effectofreductionandextractionofsperm basicproteinsofHydrolaguscalliei. Left: Proteins
extractedwith0.25 NHCI afterreduction andalkylation ofsperm nuclei. Lane b. sperm fromepididymis;
lanec,sperm fromtheanteriorregionofampullaductusdeferentis;laned.sperm fromtheposteriorregion
oftheampullaductusdeferentis.Laneashowsastandardofhistonesfromunnpetestesof// colliei.Arrows
nexttolanedindicateminorbandsml,m2.m3. Right:Proteinsextractedwith0.25A'HCIwithoutprevious
reduction.Lanee,epididymalspermnuclei:lanef.testicularnuclei. Lanegshowsthepatternwhenepididymal
sperm basic proteins remaining in nuclei after removal ofR3 and mO (with 0.25 .VHCI, lane e) are re-
extracted with0.25 A'HCI afterreduction/alkylation.
tainingthepurified proteins R3 and mO(Fig. 3B). Proteins amino acid analysis data that there is a large difference
R3 and mO from those columns were analyzed for their between ratfish R3 and dogfish Z3.
amino acid content. Compositional values are shown in Theproteinsextracted with0.25 TVHCI afterreduction
Table I togetherwith theamino acid composition ofnon- of sperm nuclei were purified using the same types of
keratinous protamine Z3 from S. canicula. The amino chromatography as before. First we separated three frac-
acid content of//, collieiR3 isremarkablydifferent from tions (I, II, and III in Fig. 5A) by ion exchange on CM-
that ofS. canicula Z3 (see Discussion). cellulose. Fraction IIcontained purified Rl, and Fraction
tenAsinveallyiqaugoatinosftt5h0empuMrifNieHd 4pHroCteOin3 aRn3dwlayosphdiilailzyezde.dFeoxr- tIIhIecroensttaoifneRdlR2plu(Fsigt.he5Cm,ilnaonrespcr,odt)e.inFsramclti,onm2I,coanntadinme3d
sequencing purposes, the protein was further purified by (not shown). Proteins from fraction I were purified in re-
reverse-phase HPLC (see Materials and Methods). The verse-phase HPLC (Fig. 5, B and C) and analyzed com-
16 N-terminal amino acid sequence for protein R3 ob- positionally. Table II shows theiramino acid composition
tained by automated Edman degradation (Fig. 4) shows compared with scylliorhinines Zl, Z2, and S4. Although
two heterogeneous clusters ofbasic amino acid residues the compositional valuesare partially similaramongst the
(RRRH) and (KKKRK). The comparison ofthis part of ratfish sperm basic proteins and scylliorhinines Zl and
the R3 molecule with the complete sequence ofZ3 prot- Z2, this does not mean that they all belong to the same
amine ofS. caniculasupportsthe idea emerging from the family ofproteins; i.e.. ratfish and dogfish proteins need
PROTAMINHS IN A HOLOCFPHALAN FISH 191
A225
5
192 N. SAPERAS ET AL.
Table I
Aminolicit/computation (mol%)olrattuli sperm basicproteins>
andmilcomparedwith 7-3proiamine(S. canicuia)andsalmint
Aminoacid R3 mO Z3a Salmineh
Lys
PROTAMINES IN A HOLOCEPHALAN FISH 193
O -
08
(NaCl)
^m B1
R2
m?
I mi
02
40 80 12O 10 20 30 4O b c d e f g h
fraction number
B c
Figure5. ChromatographicpurificationofRI, R2. mI, m2,and m3proteins.A.CM-celluloseseparation
ofthe complete set ofproteins; B. HPLC purification ofproteins eluted in fraction I from panel A; C.
Electrophoreticcontrol ofproteins punned: lanec, protein Rl from fraction 11 ofpanel A; laned. protein
R2 from fraction III ofpanel A; lane f, protein ml from peak 3 ofpanel B: lane g. protein m2 from peak
4 ofpanel B; lane h. protein m3 from peak I ofpanel B. Lanes b and e are the complete set ofproteins
alter0.25 .VHC1extraction and the reduction/alkylation process. Laneashowsastandard ofhistonesand
protein R3 from Hydrolagtiscollie/.
and S2) that are present in the nuclei of differentiating isa protamine structurally very similartothe typical true
spermatids but not in nuclei ofspermatozoa. The follow- protamines of bony fish (Sautiere et ai, 1981). These
ing evolutionary generalizations can be made. First, Z3 authors suggest that "scylliorhinine Z3 and teleost
Table II
.liniiuiacidcomposition !mol ~e)ojnitfisli Rl, R2. nil. in2. andin3sperm basicproteinscompared it/ikeralinousprutaminesZl, 7.2. andS4of
S. canicula
Aminoacid
194 N. SAPERAS ET AL
BPTI
2345
1
R1 i
R2 *
R3
12345
R3 W
C
W
2 3
1
Figure 6. Determination ofthe number of cysteine residues according to the method ofCreighton
(1980).A.StandardofBPTI(BoehringerMannheim);B. Rl from llydmlaxnscolliei:C. R2fromH colliei.
iRoedaocatcieotnatweit(h5).loWdac=etwahmoildeeb(aIs);iciopdrooatceeitnapmaitdtee/rinodforaocmet/a/.tecoilnlireaititeosstiosf. l:l (2). 1:3(3).and 1:9(4);andwith
protamines originated very probably from the same an- ferent types), contrasted with only five types of residues
DNA
cestral sequence before the divergence of Chon- that constitute Z3. In addition, ratfish is relatively poor
drichthyes and Osteichthyesduring the Devonian period" in arginine (27.6 mol % versus 64.5 mol % in dogfish),
(Chevaillieret ai. 1987). Second, there are very few iden- and its N-terminal sequence showsthe presenceofclusters
tities in the sequencesofprotamines Zl, Z2, and S4. Pos- ofheterogeneous basic residues (RRRH, KKKRK) that
sibly, these basic proteins have evolved neither from the are not present in Z3, nor in the protamines ofbony fish.
same singleancestral polypeptide norfrom the samegene The minor protamine mt) presents compositional char-
family. Third, the intermediate basic proteins SI and S2 acteristics similar to R3 (Table I).
are not homologous to the sperm proteins. These data The comparison between the amino acid composition
mean thatthesperm (and spermiogenic) nuclearproteins of Rl and R2 and the scylliorhinines Zl and Z2 (Table
from 5. caniculaarecoded byaseriesofgenesthatexpress II) shows that, globally, all these proteins possess a com-
coordinately during spermiogenesis but do not possess a parable composition. However, this similarity does not
close evolutionary relationship. necessarily mean that there isa homology between them.
The comparison ofratfish protamines with those from Two points are inconsistent with such a homology: ( 1)
5. canicula brings out yet agreater variability in this sys- scylliorhininesZ1 andZ2 arecompositionallysimilarbut
tem of molecules. Ratfish R3 protamine differs greatly theirsequences present hardlyany identities; (2) thereare
from S. canicula Z3 protamine (Table I). R3 contains a important deviations in the compositions of Rl and R2
relatively great diversity of amino acid residues (14 dif- with respect to Z1 and Z2, as can be seen by the larger
PROTAMINES IN A HOLOCEPHALAN FISH 195
M. MacKay, as well as E. Rosenberg. A number ofstu-
dents and assistants deserve thanks for their efforts in
R1
various aspects of this study: these include the late J.
Boyko. D. Braille, L. Gutovich, J. Ip, S. Jessa, H. Lau, L.
R2 Johnstone, E. Soorany, M. Tsui, and I. Uzuraga. This
work was supported in part by a Travel Fellowship from
the Ministerio de Educacion y Ciencia, Spain (N.S.);
CICYT. Spain (M.C.); and NSERC, Canada (H.E.K.).
Z1
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Figure 7. Comparison of electrophoretic patterns of sperm basic 1987. Nuclearbasicprotein transitionduringsperm differentiation.
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and M. Gusse kindly provided us with purified samples w1i9t9h1.diffPerroenttammiondeessionfprreopsroobdruacnticohni.anJ.gEaxspt.roZpooodls.(2M5o7l:lu2s6c5a-)28v3a.ry
ofS. canicit/a sperm basic proteins and assisted us with Dixon,G.H.,J. M.Aiken,J. M.Jankowsky, D.I.McKenzie, R. Moir,
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