The correspond-ing genes are expressed in the liver, and the secreted glycoprotein products are transported to the ovary via the bloodstream, where they are assembled into an egg envelop
Trang 1from the egg envelope protein genes of the Japanese eel, Anguilla japonica
Kaori Sano1, Mari Kawaguchi2, Masayuki Yoshikawa3, Ichiro Iuchi4and Shigeki Yasumasu4
1 Graduate Program of Biological Science, Graduate School of Science and Technology, Sophia University, Tokyo, Japan
2 Atmosphere and Ocean Research Institute, The University of Tokyo, Japan
3 Suruga-Bay Deep Sea Water Aquaculture Research Center, Shizuoka Prefectural Research Institute of Fishery, Japan
4 Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Tokyo, Japan
Introduction
Most animal eggs are surrounded by a
glycoproteina-ceous structure, called an egg envelope, which provides
the embryo with physical protection from the
environ-ment [1,2] Although the general term for this structure
is egg envelope, it is specifically named zona pellucida
(ZP) in mammals, perivitelline membrane in birds,
vitel-line envelope in amphibians, or chorion in fishes The
glycoproteins constituting an egg envelope were first iso-lated from Xenopus laevis [3,4] Subsequently, the cDNAs for the three glycoproteins of mouse ZP were cloned They shared a conserved region ( 260 amino acids) called the ZP domain, and were designated as ZP1, ZP2 and ZP3 [5,6] This was the first universal nomenclature proposed for ZP proteins [7] Later, the
Keywords
egg envelope; expression profile; Japanese
eel; molecular evolution; ZP domain
Correspondence
S Yasumasu, Department of Materials and
Life Sciences, Faculty of Science and
Technology, Sophia University, 7-1 Kioi-cho,
Chiyoda-ku, Tokyo 102-8554, Japan
Fax: +81 3 3238 3393
Tel: +81 3 3238 3270
E-mail: s-yasuma@hoffman.cc.sophia.ac.jp
(Received 12 July 2010, revised 30 August
2010, accepted 9 September 2010)
doi:10.1111/j.1742-4658.2010.07874.x
A fish egg envelope is composed of several glycoproteins, called zona pellu-cida (ZP) proteins, which are conserved among vertebrate species Euteleost fishes synthesize ZP proteins in the liver, while otocephalans synthesize them in the growing oocyte We investigated ZP proteins of the Japanese eel, Anguilla japonica, belonging to Elopomorpha, which diverged earlier than Euteleostei and Otocephala Five major components of the egg enve-lope were purified and their partial amino acid sequences were determined
by sequencing cDNA cloning revealed that the eel egg envelope was com-posed of four ZPC homologues and one ZPB homologue Four of the five eel ZP (eZP) proteins possessed a transmembrane domain, which is not found in the ZP proteins of Euteleostei and Otocephala that diverged later, but is found in most other vertebrate ZP proteins This result suggests that fish ZP proteins originally possessed a transmembrane domain and lost it during evolution Northern blotting and RT-PCR revealed that all of the eZP transcripts were present in the ovary, but not in the liver Phylogenetic analyses of fish zp genes showed that ezps formed a group with other fish
zp genes that are expressed in the ovary, and which are distinct from the group of genes expressed in the liver Our results support the hypothesis that fish ZP proteins were originally synthesized in the ovary, and then the site of synthesis was switched to the liver during the evolutionary pathway
to Euteleostei
Abbreviations
CBB, Coomassie Brilliant Blue G; DIG, digoxigenin; eSRS, eel spermatogenesis-related substance; eZP, eel zona pellucida;
TMD, transmembrane domain; ZP, zona pellucida.
Trang 2homologues possessing the conserved sequence of the
ZP domain were identified in amphibians, birds and
fishes The chicken genome contains six zp genes [8,9],
and the Xenopus genome contains five zp genes [2]
Phylogenetic analyses using the sequences of the ZP
domains of various species have suggested that the zp
genes should be classified into several groups However,
the nomenclature of the zp genes is confused because
different names are used for different animal groups
Spargo and Hope [10] classified vertebrate zp genes into
four subfamilies: ZPA, ZPB, ZPC and ZPX We believe
that this nomenclature is preferable to that for fish zp
genes and we employ it in the present study
Fish chorion is made up of a thick inner layer and a
thin outer layer Various studies suggest that the inner
layer of chorion is truly homologous to zona pellucida,
perivitelline membrane and vitelline envelope
Glyco-proteins constituting the inner layer of chorion have
been extensively studied in Medaka (Oryzias latipes),
belonging to the Euteleostei The inner layer is
com-posed of a group of glycoproteins called ZI-1,2 and a
homologous glycoprotein called ZI-3 The
correspond-ing genes are expressed in the liver, and the secreted
glycoprotein products are transported to the ovary via
the bloodstream, where they are assembled into an egg
envelope [11–14] The precursors of ZI-1,2 were named
choriogenin H and choriogenin Hm, classified into
ZPB, and that of ZI-3 was named choriogenin-L,
clas-sified into ZPC The cDNA homologues for
chorioge-nins were cloned from other euteleostean fishes, and
their genes were found to be expressed in the liver
[15,16] An exception, however, was the homologue of
the zpx gene cloned from the euteleostean fish gilthead
seabream, Sparus aurata, which is expressed in the
ovary [17] In addition to choriogenin homologues, the
zpxgene product is a component of the inner layer of
the chorion [18] The chorion of zebrafish (Danio
rerio), carp (Cyprinus carpio) and goldfish
(Caras-sius auratus), which belong to the Otocephala, are also
composed of several glycoproteins homologous to ZPB
and ZPC However, as found in mammalian species,
these glycoproteins are synthesized in the oocyte
[19–21]
In Medaka, seven ZP domain-containing genes,
expressed specifically in oocytes, have been identified
by subtractive hybridization, in addition to the
liver-specific genes (choriogenin) [22,23] However, these
oocyte-specific gene products have not been detected
as inner layer components of chorion by biochemical
analysis such as peptide mapping [24,25] Further
stud-ies, such as localization of the products, have not been
carried out Thus, the function of these gene products
is unclear
In summary, the organ that synthesizes glycopro-teins constituting the inner layer of chorion is the liver
in Euteleostei and the ovary in Otocephala; an excep-tion is the gilthead seabream, where the glycoproteins originate from both the liver and ovary However, the genes encoding the egg envelope protein of fish belong-ing to the Elopomorpha, which branched paraphyleti-cally to the common ancestor to Euteleostei and Otocephala, have not yet been identified
The zp gene homologues of the Japanese eel (Anguilla japonica), which belongs to the Elopomor-pha, were cloned by subtractive hybridization from a cDNA library derived from the testis of a human chorionic gonadotropin-stimulated immature male, as down-regulated genes [26] The genes named eel sper-matogenesis-related substance 3 (eSRS3) and eSRS4 were subsequently found to be expressed also in the ovary [27] However, functional studies at the protein level have not yet been carried out
In the present study, we describe the purification of the egg envelope proteins from unfertilized egg enve-lopes of Japanese eel, cloning of the corresponding cDNAs and analysis of their expression Finally, we discuss the evolution of fish egg envelope genes using phylogenetic analysis
Results
Purification of envelope proteins from Japanese eel
The unfertilized egg envelope proteins of Japanese eel were separated by SDS⁄ PAGE and stained with Coo-massie Brilliant Blue (CBB) The SDS⁄ PAGE profile
of the unfertilized egg envelope of the eel revealed three strongly staining bands (of 37, 48 and 53 kDa), two moderately staining bands (of 71 and 84 kDa) and several weakly staining bands (Fig 1A) We reasoned that the five proteins of 37, 48, 53, 71 and 84 kDa (i.e which stained moderately or strongly with CBB follow-ing SDS⁄ PAGE) would be major constituents of the egg envelope and therefore each of these proteins was purified The unfertilized egg envelopes were denatured and solubilized in guanidine hydrochloride and then subjected to C8 reverse-phase chromatography The component proteins of the unfertilized egg envelope were subsequently fractionated into three peaks (Fig 1B) The shoulder of the first peak (fraction I) contained the 71 and 84 kDa proteins, and main part
of the first peak (fraction II) contained 37 kDa pro-tein, the second and third peaks (fraction III and IV) contained 53 and 48 kDa protein, respectively (Fig 1C) These four fractions were then separately
Trang 3analysed by SDS⁄ PAGE, and each of the five egg
envelope proteins was gel-purified, as described in the
Materials and methods (Fig 1D)
Cloning of full-length cDNAs for the five
egg-envelope proteins
To determine the partial amino acid sequences, the
purified proteins were digested with either
lysyl-endo-peptidase or endolysyl-endo-peptidase Glu-C, and the resulting
peptides were separately subjected to N-terminal
analy-sis N-terminal sequences from more than two digests
of each protein were determined (Table 1) We
com-pared all the sequences with those deduced from
eSRS3 and eSRS4 cDNAs Four sequences from the
digests of the 37 kDa protein (Table 1) were found in
the sequence deduced from eSRS3 cDNA Similarly,
three sequences from the digests of the 53 kDa protein were identified in the sequence from the eSRS4 cDNA These results suggest that eSRS3 and eSRS4 are the components of the egg envelope that correspond to the
37 and 53 kDa proteins, respectively
For the three other proteins (i.e the 48, 71 and
84 kDa proteins), degenerate primers were designed based on the amino acid sequences obtained from each digest (Figs 2 and 3) RNAs extracted from the ovary and the liver were used as templates for RT-PCR All fragments were amplified exclusively from ovarian RNA, and full-length cDNAs were cloned by RACE-PCR For the 48 and 71 kDa proteins, 1.7 and 1.6 kbp cDNAs were cloned, which encoded sev-eral amino acid sequences from the digests of each protein (Table 1) In the procedure for cloning cDNA for the 84 kDa protein, two different-sized fragments were amplified by 5¢-RACE-PCR After 3¢-RACE-PCR, each full-length cDNA was cloned Sequence analysis revealed that one of the cDNAs, 2.6 kbp cDNA, contained three sequences from the digests of the 84 kDa protein The amino acid sequence deduced from another cDNA, 1.7 kbp cDNA, did not include any sequence identical to those of the digests obtained from the five proteins However, several sequences of the digests were closely similar to regions of the amino acid sequence deduced from the 1.7 kbp cDNA Thus, five cDNAs for five major components
of the egg envelope, and one cDNA closely related to them, were cloned
B
Fig 1 Purification of egg envelope proteins (A) SDS ⁄ PAGE
pat-terns of unfertilized egg envelope (B) A C8 reverse-phase column
chromatogram of unfertilized egg envelope protein denatured with
guanidine hydrochloride Solid line, absorbance at 280 nm; dashed
line, a gradient from 0% to 78% acetonitrile (MeCN).
(C) SDS ⁄ PAGE patterns of fractions I–IV (lanes 1–4, respectively)
obtained by the C8 reverse-phase column chromatography.
(D) SDS ⁄ PAGE patterns of the five purified proteins Lane 1,
84 kDa protein; lane 2, 71 kDa protein; lane 3, 53 kDa protein; lane
4, 48 kDa protein; and lane 5, 37 kDa protein The numbers on the
left of the SDS ⁄ PAGE pattern refer to the sizes of the molecular
mass markers.
Table 1 N-terminal amino acid sequences of the digests from five major components of eel egg envelope The protein names deter-mined after sequence analyses of the cDNAs are indicated in parentheses The numbers in parentheses at the end of each sequence indicate the position of the amino acid residues deduced from each cDNA.
Protein size Amino acid sequence
VNTVPPPLPV(190–199) GANGXAD(220–226) RTDPNLVLLL(257–266)
48 kDa (eZPCa) AHXGESSVQLEVD(172–184)
TELHSXGSVL(220–229)
53 kDa (eZPCb) VDMDLLGIGH(148–158)
LQLQLDAFRF(353–362) AXSFPLG(389–395)
71 kDa (eZPCc) RQPVAPVSR(39–47)
FIHVPM(69–74) ALGSTPIIRTNGA(213–225)
84 kDa (eZPCd) DSPVIRAIVTGQP(52–64)
ALVGTPIVR(561–569) ASVVQANHVP(639–648)
Trang 4Domain structures of egg envelope proteins
Comparison of the amino acid sequences deduced
from the cDNAs with those of other vertebrate ZP
proteins revealed that all included a ZP domain
(Fig 4) The cDNAs were named based on sequence
similarities of the ZP domains according to the nomen-clature of Spargo and Hope [10] The amino acid sequence of the ZP domain of the 37 kDa protein (eSRS3) indicated a higher degree of similarity to those
of ZPBs (49.7% for chick ZPB: NM_204879, and 41.7% for Xenopus ZPB: XLU44950) than those of
233 ADSLVYTFTLNYQPNALGATPIIRTSSAVVGIQCHYMRLHNVSSNALKPTWIPYHSTLSA 292
199 EDSLVYTFAFNYQPSAIGATPIIRTSSAVVGIQCHYLSLHNVSSNALKPTWIPYHSTLSA 258
198 EDSLVYTFGLDYQPKALGSTPIIRTNGAIVGVQCHYMRLHNVSSNALKPTWIPYRSTLSA 257
546 EDSLIYTFSLNYQPKALVGTPIVRSSEAVVLIQCHYPRLHNVSSNALHPTWIPYQSAMSA 605
228 EDTLVYTFTIRYQPKAIGVTPIIRTNDAAVGVQCHYMRLHNVSSNALKSTWIPYYSTLSA 287
293 EDLLVFSLRLMADNWQTERTSAVFFLGDLINIEASVVQANHVPLRVFVDTCIATLDPDMN 352
259 EDLLVFSLRIMADNWQLERTSNVFFLGDLINIEISVVQANHVPLRVFVDTCVATLDPDMN 318
258 EDLLVFSLRLMDDNWQMERTSNVFFLGDLINIEASVVQANHVPLRVFVDSCVATLDPNMN 317
606 EELLVFTLRLMEDDWQQERAPRIFFLGDTLKIEASVVQANHVPLRVFIERCVAYLDPSL- 664
288 EDLLVFSLRLMTNDWRMERESYVFFLGDIINIEASVIQANHVPLRVFMDTCVATLAPNMD 347
353 AVPRYAFIENKGCLMDSKLTNSRSQFLSRVQDDKLQFQLDAFRFAQETRSAIYIFCHLKA 412
319 AVPRYAFIENKGCLMDSKLTNSRSQFLSRVQDDKLQLQLDAFRFAQETRSAIYIFCHLKA 378
318 AVPRYAFVENQGCLMDSKLTNSRSQFLSRVQNDKLQFQLDAFRFAKETRSAIYFFCHLKA 377
665 -APSYAFVKEDGCLMDSQLPGSHSMFLPRLQDDKLRMEVDAFRFAQEDRSSIYFYCHLKA 723
348 SVPRYTFIDNQGCLMDSKLTSSRSKFQSRIKDDLLQVQLDAFRFAAETRSEIYIFCHLRA 407
413 TAALPDSEGKACSFPLGKE 431
379 TAALPDSEGKACSFPLGKE 397
378 TTALS-PEGKACSFSLGTQ 395
724 TAASDPYGGKACSFSPEAG 742
408 TAALPESEGKACSFLPSKH 426
139 HCGESSVQMEVDMDLLGIGHLNQPSDITLGGCGPVAQAKSTRALLFETELHGCGSVLAMT 198
173 HCGESSVQLEVDIDLLGIGHLIQPTDITLGGCGPVDLDGSTQVLLFETELHSCGSVLAMT 232
138 YCGESSVQLDVDMDLLGNNHLIQPSDITLGGCGPVGQDDSAQVLFFATELHGCNSVLMMT 197
486 ICGDSLLQVEVNAILLGIGQLVHPSEITLGGCGPVEQDKSDWMLHFVTELHDCGSTQMMT 545
170 HCGETSVQLEVDVDLFGIGNLIQPSDITLGGCDPIGQDHS WLLFETQLHACGSTLMMT 227
eZPCa eZPCb eZPCc eZPCd eZPCe eZPCa eZPCb eZPCc eZPCd eZPCe eZPCa eZPCb eZPCc eZPCd eZPCe eZPCa eZPCb eZPCc eZPCd eZPCe eZPCa eZPCb eZPCc eZPCd eZPCe
Fig 2 Alignment of amino acid sequences
of the ZP domains of eZPCs Conserved
amino acid residues are boxed Gray boxes
indicate the sequences identical to
N-termi-nal sequences from the digests of egg
envelope proteins obtained following
incuba-tion with lysyl-endopeptidase or
endopepti-dase Glu-C The amino acid sequences used
to design degenerate primers for RT-PCR
are indicated by horizontal arrows The
direction of the arrows indicates an
upstream primer (fi) or a downstream
primer (‹).
50 *****A*******W** 65
66 *G*A*-***I*G**** 80
96 *G*A*-***I****** 110
111 *G*A*-***I****** 125
126 *G*A*-***I****** 140
141 *********L*E*KGS 156
46 ***I*****L***W** 61
62 ***********G**I* 77
78 *S************I* 93
94 **************I* 109
110 *********L*E*TH* 125
56 **R**AH**V*E*E*I 71
72 HV*M*TY****GA*Y* 87
88 ****S******GA*Y* 103
104 ****S******GA*Y* 119
35 **A**T**I****LP* 50
67 **A**T**I****LP* 82
83 **A**T**I****LP* 98
51 **A**A*******LP* 66
99 **A**A*******LP* 114
115 *S**PV*******VA* 130
131 *S**PV*****K*PV* 146
147 *G*******IKE*PQP 162
81 *G***-***I*G**** 95
89 *E***S**K****MV 103
104 ***HS********ME 118
119 *************MI 133
164 *********L***ME 178
179 *****S***L***ME 193
194 *****S**G****** 208
254 *************** 268
269 *************** 283
284 *************** 298
299 *************** 313
314 *************** 328
329 ***L*********** 343
344 ***L*********** 358
359 ***L*********** 373
374 ***L**Y******** 388
389 ***L**Y******** 403
404 ***L**Y******** 418
419 ***L**Y******** 433
434 *****S********* 448
449 *****S********* 463
239 *A***S********* 253
225 *****SIE**VPH-V 238
209 *****SFE**VLPTFT 224
134 *****S*******MV 148
149 *****SFETLVPPRI 163 eZPCa
eZPCb
eZPCc
eZPCd
eZPCe
Fig 3 Repeat sequences in the N-terminal
regions of eZPCs The repeat sequences of
eZPCa, b, c, e (A) and eZPCd (B) are shown.
The consensus sequences are indicated at
the top of each figure The amino acid
resi-dues identical to the consensus sequences
are highlighted by asterisks The numbers
on each side of the panels refer to the
posi-tions of amino acid residues deduced from
cDNAs A gray box and a horizontal arrow
indicate the amino acid sequence identical
to the N-terminal sequence from a digest of
the 74 kDa protein and the position of a
degenerate primer for RT-PCR, respectively.
Trang 5other subfamilies (< 35.2%) Therefore, the 37 kDa
protein was named eZPB The amino acid sequences
of the ZP domains from the remaining five cDNAs
showed 80–85% similarity (Fig 2) Comparison of the
five cDNAs with those from other vertebrate ZPs
revealed the highest similarity to ZPCs [48.3–52.5%
for chick ZPC (NM_204389) and 45.4–49.0% for
Xenopus ZPC (U44952)] among those of the four
sub-families Therefore, the 48-, 53- (eSRS4), 71- and
84 kDa proteins were designated eZPCa (AB571308),
eZPCb, eZPCc (AB571309) and eZPCd (AB571310),
respectively, and the ZP protein-related 1.8 kbp cDNA
was named eZPCe (AB571311) Thus, the five major
components of eel egg envelope comprise four ZPC
homologues and one ZPB homologue
About 20 consecutive residues from the N terminus
of all eZP proteins were rich in hydrophobic amino
acids, which are characteristic of signal peptides The
N-terminal regions following the signal sequences of
all eZPCs were made up of repeat sequences (Fig 3,
4) The sequences of the repeat units of eZPCa,
eZPCb, eZPCc and eZPCe, each of which comprised
16 residues, displayed similarities (Fig 3) There were
seven repeat units for eZPCa, five for eZPCb, four for
eZPCc and eight for eZPCe (Figs 3 and 4) The
N-termi-nal region of eZPCd was also made up of repeat
sequences The sequence of the repeat unit, which was
composed of 15 residues, was quite different from
those of other eZPCs, and the number of repeat units
was much greater (25 times) than those of other eZPC
homologues (Figs 3 and 4) By contrast, no
repeat-sequence region was found in the N-terminal region of
eZPB However, a trefoil domain, which is characteris-tic of a ZPB subfamily, was found in eZPB just preceding the ZP domain (Fig 4) We also analyzed the C-terminal region following the ZP domain The consen-sus C-terminal processing site (Arg-Lys-X-fl-Arg), which
is processed before the formation of the egg envelope, and the transmembrane domain (TMD) were found in all but two of the eZPs (i.e a clear consensus sequence of the C-terminal processing site was absent in eZPB, and there was no TMD in eZPCa) (Fig 4)
Glycosylation of ZP proteins Many of the ZP proteins have been reported to be glycoproteins [2,24] The egg-envelope proteins from unfertilized egg envelopes of the eel were separated by SDS⁄ PAGE and then stained using a glycoprotein-staining method As shown in Fig 5, bands for four components, except for eZPB, were stained The pre-dicted molecular masses deduced from eZP cDNAs were compared with the molecular masses obtained from SDS⁄ PAGE The molecular mass predicted from eZPB cDNA (from the signal peptide cleavage site to the C-terminal processing site) was similar to the value obtained from SDS⁄ PAGE (37976.39 ⁄ 37 kDa for eZPB), while those from eZPC cDNAs were smaller than those obtained from SDS⁄ PAGE (46157.52 ⁄
48 kDa for eZPCa; 43207.54⁄ 53 kDa for eZPCb; 43411.50⁄ 71 kDa for eZPCc; and 79578.11 ⁄ 84 kDa for eZPCd) These results indicate that eZPCa, eZPCb, eZPCc and eZPCd are glycoproteins In particular, the predicted mass from eZPCc cDNA was much smaller than the corresponding value obtained from SDS⁄ PAGE Such a large discrepancy is caused by a highly glycosylated state of eZPCc or for some other reason (see below)
Fig 4 Schematic representations of the structures of eZPs The
ZP domains are shown in the light gray box The repeat units and a
trefoil domain in the N-terminal regions are in dark and meshed
boxes, respectively Transmembrane domains are indicated by
diag-onal boxes White and black triangles indicate the putative cleavage
sites of signal sequence and the deduced C-terminal processing
sites, respectively.
Fig 5 Glycosylation of egg envelope proteins SDS ⁄ PAGE pat-terns of unfertilized egg envelope stained by CBB (lane 1) or by the glycoprotein staining kit (lane 2).
Trang 6Expression of ezp genes analyzed by northern
blotting and RT-PCR
For northern blotting, digoxigenin (DIG)-labelled
DNA probes were synthesized from the cDNAs for
the ZP domain of eZPB and from the repeat sequence
regions for eZPCa, eZPCc and eZPCd All four
tran-scripts of eZPs were detected exclusively in the ovary
(Fig 6A) Each probe for eZPB, eZPCa and eZPCd
specifically hybridized with a single transcript, the sizes
of which were 1.75, 1.9 and 3.0 kb, respectively The
size of each transcript corresponded to those of the
respective cDNAs (1385, 1690 and 2655 bp) However,
the probe for eZPCc hybridized to two different-sized
transcripts, 1.6 and 2.7 kb The 1.6 kb transcript was
consistent with the size of the corresponding cDNA
(1585 bp) When a sequence for the ZP domain of
eZPCc was employed as a probe, the same pattern was
obtained (data not shown) These results suggest the
presence of a longer transcript whose sequence is
highly similar to that of eZPCc cDNA This transcript would have an extended 5¢ and ⁄ or 3¢ noncoding region and⁄ or a longer coding sequence (e.g the repeat sequence in the N-terminal region might be longer than that of cloned eZPCc)
Expression of the six ezp genes was analyzed semi-quantitatively by RT-PCR using RNA extracted from the ovary or liver (Fig 6B) In the ‘RT-PCR, all frag-ments for eZP transcripts were exclusively amplified by RNA derived from the ovary A faint band corre-sponding to eZPCa was observed after 30 cycles of amplification using liver RNA, but this result was not reproducible The bands amplified from ovarian RNA were visualized after 21 cycles for eZPB, eZPCa and eZPCb, after 24 cycles for eZPCc and after 30 cycles for eZPCd These results suggest that the transcripts for eZPB, eZPCa and eZPCb are more abundant than those for eZPCc and eZPCd Moreover, this result supports the relative band intensity of ZP glyco-proteins obtained from SDS⁄ PAGE pattern of the unfertilized egg envelope (Fig 1A) Unexpectedly, the RT-PCR analysis detected a considerable amount of the eZPCe transcript in the ovary, despite a lack of the corresponding protein In summary, all genes for the major glycoproteins constituting the eel egg envelope were found to be expressed exclusively in the ovary
Phylogenetic analysis First, we made a phylogenetic tree using the sequences
of ZP domains from various teleostean fishes Accord-ing to the tree, all fishes possessed two subfamilies of
zp genes: ZPB and ZPC groups Several fishes had additional zp gene(s) called ZPX (Fig 7A) According
to such analyses using vertebrate zp genes, Spargo and Hope [10] proposed that ZPC divided earlier from other subfamilies Indeed, the branch length between the ZPC group and the ZPB group is long We sepa-rately made the trees of zpb and zpc genes In both trees, zp genes could be classified into two groups in terms of their expression profiles: ovary-specific and liver-specific genes The ezpb gene and five ezpc genes belonged to the ovary-specific gene groups of ZPB and
of ZPC, respectively (Fig 7B, C)
In the ovary-specific gene group of the ZPC tree, zpc genes were separated into three groups Two of the three groups were eel zpc genes and otocephalan zpc genes In zebrafish, the egg envelope is known to
be composed of two glycoproteins, which are encoded by zfzp3 and zfzp2 [21,28] The otocephalan zpc gene group contained zfzp3 and its orthologues
of carp and goldfish zpc genes (carpzp3, carpzp3.2, gfzp3) [19,20] Thus, these two groups of zpc genes
A
B
Fig 6 Expression analyses of ezp genes (A) Northern blot of
ezpb, ezpca, ezpcc and ezpcd Five micrograms of RNA extracted
from ovary (O) or liver (L) was loaded onto each lane The numbers
on the left indicate the sizes of the RNA size markers (B)
Semi-quantitative analysis of expression of ezp genes by RT-PCR The
numbers of the PCR cycle are indicated at the bottom of each
panel b-actin was used as the control.
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chgH
Fg chgH
Sa zp1aOl chgHm
Fh chgHm
Cvzr-2
e zpb
Tn zpb
Fg
zpb
Ol
zpb
gf
zp2
carp
zp2
zf zp2like2
zf zp2like1 masu
chgH
β
rt vepβ 100
100 98
98 97
99 59 100 100 100
100 100 100
100
99 99 90 97
0.1
ZPB
A
Ovary
Ovary
Liver
Liver
Fig 7 Maximum-likelihood (ML) trees of the nucleotide sequences of the zp genes of Teleostei (A) Phylogenetic tree of teleostean zp genes The subfamilies of zp genes are labelled (ZPB, ZPC and ZPX) (B) A tree of teleostean zpb genes, or (C) zpc genes The groups of ovary-specific and liver-specific genes are labelled in pink and in light blue, respectively The group of the ‘unknown-function zpc genes’ is labelled in dark blue
in the ZPC tree Numbers at the nodes indicate bootstrap values estimated by ML, which are shown as percentages Accession numbers: masu (Oncorhynchus masou) chgHa (EU042124); masu chgHb (EU042125); masu chgL (EU042126); rt (rainbow trout) vepa (AF231706); rt vepb (AF231707); rt vepc (AF231708); sal (Salmo salar) kzp19 (AJ000664); char (Arctic char) zpc (AY426717); Ol (Oryzias latipes) chgH (D89609); Ol chgHm (AB025967); Ol zpb (AF128808); Ol chgL (D38630); Ol zpc1 (AF128809); Ol zpc2 (AF128810); Ol zpc3 (AF128811); Ol zpc4 (AF128812); Ol zpc5 (AF128813); Ol zpx (AF128807); Oj (Oryzias javanicus) chgH (AY913759); Oj chgL (AY913760); Om (Oryzias melastigma) chgH (EF392363); Om chgL (EF392364); Os (Oryzias sinensis) chgL (AY758411); Fh (Fundulus heteroclitus) chgH (AB533328); Fh chgHm (AB533329); Fh chgL (AB533330); Cv (Cyprinodon variegatus) zr-2 (AY598615); Cv zr-3 (AY598616); La (Liparis atlanticus) chgH (AY547502); La chgL (AY547503); Sa (Sparus aurata) zp1a (AY928800); Sa zp1b (AY928798); Sa zp3 (X93306); Sa zpx (AY928799); Tn (Tetraodon nigroviridis) chgH (CR665164); Tn chgL (CR639306); wf (winter flounder) chgH (U03674); gf (goldfish) zp2 (Z72495); gf zp3 (Z48974); carp zp2 (Z72491); carp zp3 (L41639); carp zp3.2 (L41638); zf (zebrafish) zp2 (AF095456); zf zp2like1 (NM_001105104); zf zp2like2 (NM_001089502); zf zp3 (AF095457); zf zp3a.1 (NM_001013271); zf zpc (NM_131696); zf zp3cv1 (XM_680521); zf zp3v2 (NM_001162847); zf zpa (NM_212718); Fg (Takifugu rubripes) chgH, zpb, chgL, zpc1, zpc2, zpc3, zpc4 and zpc5 (in silico cloning from Fugu Genome Project); and Tn zpb (in silico cloning from the Tetraodon Genome Project).
Trang 8encode major components of the inner layer (i.e.
authentic zp genes) The third group was that of
‘unknown-function zpc genes’ which were first
reported as Medaka ovary-specific zp genes (see the
Introduction) [22] This group also included Medaka
orthologues of fugu zp genes and three zebrafish zpc
genes (zfzp3v1, zfzp3v2, zfzp3a.1) [29] whose products
are not considered to be major components of egg
envelope Hence, the authentic zp genes for egg
enve-lope proteins were clearly discriminated from the
‘unknown-function zpc genes’ in the phylogenetic
analysis of zpc genes The zebrafish genome contains
two zpb genes (zfzp2like1 and zfzp2like2) in addition
to an authentic zpb gene (zfzp2), all of which are
located in the ovary-specific zpb gene group [29]
Therefore, the ‘unknown-function zpb and c genes’
were found in both euteleostean and otocephalan
fishes, suggesting that these genes have been
con-served during teleostean evolution
Discussion
The ZP proteins, which constitute the egg envelope of
mammals, birds and amphibians, are synthesized in
the oocyte, except for chicken ZP1 and ZP3, which are
synthesized in the liver and in the granulosa cells of the
ovary, respectively [9] The C-terminal regions of the
ZP proteins contain a TMD The TMD is thought to
anchor the ZP proteins into the plasma membrane of
the oocyte after secretion, but is removed by C-terminal
processing before the formation of the egg envelope In
mouse, the TMD is not required for secretion, but is
essential for assembly of the ZP proteins [30] All fish
ZP proteins previously reported lack a TMD [22] The
TMD is presumably unnecessary for the ZP proteins of
euteleostean fishes because the corresponding genes are
expressed in the liver and the secreted proteins are
transported to the ovary However, the otocephalan ZP
proteins synthesized in the ovary also lack a TMD [22]
In the present study, five of the six eZPs possessed a
TMD, suggesting that the fish zp genes originally
encoded a TMD, like those of other vertebrates
Fur-thermore, ezpca, one of the high-expression genes, does
not possess a TMD It is possible that the TMD is
dis-pensable for egg-envelope formation in teleosts and
thus disappeared during evolution In the present
study, all genes encoding the major components of eel
egg-envelope were found to be expressed in the ovary,
as is the case for other vertebrate zp genes Our results
suggest that eel zp genes have retained the ancestral
form of the teleostean zp gene
Some ZP proteins are reported to possess a repeat
sequence in their N-terminal regions, while others do
not Here, we found that the N-terminal regions of all eZPCs were composed of a repeat sequence, with each repeat unit being made up of 15 or 16 amino acid residues The ZPBs of carp (carpzp) and goldfish (gfzp) belonging to Otocephala also possess repeat sequences whose units are 14–16 amino acids in length [19] However, there is no obvious sequence similarity in repeat units between eZPCs and otocephalan ZPBs Thus, the N-terminal regions of the ovary-specific ZP proteins are highly variable in terms of both amino acid sequence and length Nonetheless, the N-terminal regions of many euteleostean liver-specific ZPB (chor-iogenin H and chor(chor-iogenin Hm) glycoproteins are composed of a characteristic three-amino-acid motif called the Pro-Xaa-Yaa repeat sequence This result suggested that euteleostean zpb genes have acquired Pro-Xaa-Yaa repeat sequences in the evolutionary pathway to Euteleostei Therefore, the liver-specific zpb genes can be distinguished from the ovary-specific zp genes by phylogenetic analysis as well as by the char-acteristics of the repeat sequences Our results support the hypothesis that gene duplication of zp genes occurred at an early phase of teleostean evolution, and then one of the duplicates changed its site of expres-sion from the ovary to the liver [23,31]
The present phylogenetic analysis suggests that the additional ovary-specific zp genes, other than the zp genes encoding major components of the egg envelope, are present in several fish species The ‘unknown-func-tion zp genes’ were first identified in Medaka, and then homologous genes were identified from the genome sequences of zebrafish and fugu These results suggest that these genes are widely distributed in both euteleostean and otocephalan fishes Therefore, the
‘unknown-function zp genes’ may play an essential role
in the ovary To fully understand the evolutionary pro-cess of the fish zp genes, it is nepro-cessary to clarify a bio-logical role of the ‘unknown-function zp genes’, and also to clone more cDNAs for ZP proteins from a wider variety of fish species
Materials and methods
Materials
The females of the Japanese eel, A japonica, sexually matured by hormonal injection, were supplied from Hama-nako Branch, Shizuoka Prefectural Research Institute of Fishery of Japan Unfertilized eggs were squeezed out from spawning female fish and homogenized in 0.13 m NaCl,
20 mm Tris⁄ HCl (pH 8.0) containing 5 mm EDTA and
5 mm iodoacetic acid After centrifugation (2000 g, for 30 s
at 4C), the supernatant was decanted This procedure was
Trang 9repeated several times to completely remove yolk proteins
and cell debris The isolated unfertilized egg envelopes were
stored at)20 C The RNA was extracted from ovary and
liver tissue using RNAiso (Takara Bio Inc., Tokyo, Japan),
following the manufacturer’s recommendations
Purification of egg envelope proteins
Unfertilized egg envelopes were completely dissolved in 6 m
guanidine hydrochloride and then diluted six-fold with 0.1%
trifluoroacetic acid After centrifugation (12 000 g, 5 min at
room temperature), the supernatant was injected onto a C8
reverse-phase column (YMC Co., Ltd., Tokyo, Japan),
equil-ibrated in 0.1% trifluoroacetic acid, using an HPLC system
(Gilson Inc., Middleton, WI, USA) Bound proteins were
then eluted with a linear gradient of 0–78% acetonitrile
(MeCN) Peak fractions were concentrated using a
centrifu-gal vaporizer CVE-100D (Tokyo Rikakikai Co Ltd., Tokyo,
Japan), and analyzed by SDS⁄ PAGE After staining with
CBB, each band was cut out, and the gel pieces thus obtained
were crushed in 5 mL of 0.1% SDS, 20 mm Tris⁄ HCl (pH
8.0) After incubation with shaking, overnight at room
tem-perature, the supernatant was collected by centrifugation and
then concentrated to 250 lL using a centrifugal vaporizer
CVE-100D (Tokyo Rikakikai Co) Then, 1 mL of ice-cold
acetone was added to the mixture, which was incubated at
)80 C for 1 h After centrifugation at 12 000 g for 5 min at
room temperature, the precipitate was evaporated to dryness
and dissolved in 0.1% SDS
Determination of partial amino acid sequences of
egg envelope proteins
The purified egg envelope proteins were digested in a
mix-ture containing 50 mm Tris⁄ HCl (pH 9.0), 0.05% SDS and
20 lgÆmL)1 of lysyl-endopeptidase (Wako Pure Chemical
Industries, Ltd., Osaka, Japan), or in a mixture of 50 mm
Tris⁄ HCl (pH 8.0) 0.05% SDS and 10 lgÆmL)1of
endopep-tidase Glu-C (Roche, Indianapolis, IN, USA), at 37C
overnight The digests were analyzed by SDS⁄ PAGE and
electroblotted onto polyvinylidene difluoride membrane
(Hybond-P; GE Healthcare UK Ltd., Little Chalfont, UK)
After staining with CBB, the protein bands were cut out
and subjected to sequencing using a Procise 491HT
sequen-cer (Applied Biosystems, Foster City, CA, USA)
Cloning of cDNAs for egg envelope proteins
RT-PCR was carried out using a OneStep RT-PCR kit
(Qiagen, Valencia, CA, USA) according to the manufacturer’s
instructions PCR amplification was performed using
degenerate primers and RNA extracted from the ovary or
the liver as a template The cloning of full-length cDNA
was performed by the 5¢- and 3¢-RACE-PCR methods
using a SMART RACE cDNA Amplification kit (Clon-tech, Mountain View, CA, USA)
Staining of sugar chain
Unfertilized egg envelopes were subjected to SDS⁄ PAGE, and components of egg envelope containing sugar chain were stained using the GelCode Glycoprotein Staining kit (Thermo Scientific Inc., Rockford, IL, USA), according to the manufacturer’s instructions
Northern blot
Five micrograms of total RNA extracted from the ovary or liver of a female eel was electrophoresed on a 1% agarose gel containing 18% formaldehyde, and transferred to nylon membrane (Hybond N+; GE Healthcare UK Ltd.) Digoxi-genin-labelled DNA probes were synthesized with a PCR Probe Synthesis kit (Roche), using cloned eZPB, eZPCa, eZ-PCc and eZPCd cDNAs as templates After prehybridiza-tion in DIG Easy Hyb (Roche) at 37C for 1 h, the total RNA on the membrane was hybridized at 37C overnight with the DNA probe in DIG Easy Hyb The membrane was washed twice with 2· NaCl ⁄ Cit containing 0.1% SDS for
5 min at room temperature, once with 1· NaCl ⁄ Cit con-taining 0.1% SDS for 15 min at 60C, and twice with 0.2· NaCl ⁄ Cit containing 0.1% SDS for 15 min at 60 C The membrane was incubated with a 0.2% blocking reagent
in phosphate buffered saline with Tween-20 (PBST, 20 mm phosphate buffer, 0.13 m NaCl, 0.1% Tween) PBST for
30 min at room temperature, and with 5000-fold diluted alkaline phosphatase-conjugated anti-DIG Ig in the same buffer for 1 h After washing three times with PBST, for
5 min each wash, the membrane was incubated in a sub-strate solution consisting of 1% Disodium 3-(4-methoxy-spiro {1, 2-dioxetane-3, 2¢-(5¢-chloro)tricyclo [3 3 1 13, 7
] decan}-4-yl) phenyl phosphate, 0.1% diethanolamine and
Table 2 Primer sequences specific for each eZP gene.
Reverse: TACGACAGCCAATGCCAGGAT ezpca Forward: GGAAAGGAACAGTGGGTTAGT
Reverse: ATCAGCCGCCAAAGTGCCAGG ezpcb Forward: GGGAAGGAACGGTGGATTGAG
Reverse: CTGCATTCAGAGGGCTAATGG ezpcc Forward: GGAACTCAACGGTGGATTAGT
Reverse: CTCTACCACCAAGTGTTGGCT ezpcd Forward: TTCCTACCTTCAAAGCATGGG
Reverse: GTGCTCAACTCAGGCATGTCA ezpce Forward: CTCATTCTCTCCAGAAGCTGG
Reverse: GCTCCTAGACTCTGACACCAG
Trang 101 mm MgCl2for 5 min, and then exposed to scientific
imag-ing film (Kodak Japan Ltd., Tokyo, Japan) in the dark
Semiquantitative estimation of expression of
ezp genes by RT-PCR
The PCR amplification cycle was 30 s at 94C, 30 s at
56C and 1 min at 72 C The primers specific for each ezp
gene are presented in Table 2
Aliquots of the PCR cocktail were loaded onto 1.8%
aga-rose gels containing 0.1 lg⁄ mL ethidium bromide The
desired amplified products were confirmed by DNA
sequencing
Phylogenetic analysis
A codon-based alignment of nucleotide sequences of the ZP
domain was made using the Clustal X2 program [32] and
the CodonAlign 2.0 program [33] Data were partitioned
into first, second and third positions The best-fitting
mod-els for each position were selected using Kakusan4 [34], as
follows: SYM+G+I, TVMef+I and HKY85+G for the
data set of all zp genes; J2+G, GTR+G and J2+G+I for
the data set of zpb genes; and J1+G, TVM+G and
TVM+G for the data set of zpc genes Using the models,
maximum-likelihood analysis was employed in the program
Treefinder [35] The best-scoring tree was obtained and then
bootstrap values were generated from 1000 replicates
Acknowledgements
We express our cordial thanks to Professor F S
Howell, Department of Materials and Life Sciences,
Faculty of Science and Technology, Sophia University,
Tokyo, for reading the manuscript The present study
was supported, in part, by Grants-in-Aid for Scientific
Research (C) from J S P S to S Y
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