Báo cáo khoa học: Evolution of the teleostean zona pellucida gene inferred from the egg envelope protein genes of the Japanese eel, Anguilla japonica potx

Evolution of the teleostean zona pellucida gene inferred
from the egg envelope protein genes of the Japanese eel,
Anguilla japonica
Kaori Sano
1
, Mari Kawaguchi
2
, Masayuki Yoshikawa
3
, Ichiro Iuchi
4
and Shigeki Yasumasu
4
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:
(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.
4674 FEBS Journal 277 (2010) 4674–4684 ª 2010 The Authors Journal compilation ª 2010 FEBS
homologues 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
zpx gene 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
K. Sano et al. Egg envelope of Japanese eel
FEBS Journal 277 (2010) 4674–4684 ª 2010 The Authors Journal compilation ª 2010 FEBS 4675
analysed 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 endopeptidase 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.
AC D
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
37 kDa (eZPB) NKMSSTY(148–154)
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)
Egg envelope of Japanese eel K. Sano et al.
4676 FEBS Journal 277 (2010) 4674–4684 ª 2010 The Authors Journal compilation ª 2010 FEBS
Domain 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
QAPVTPRPTFGRPGFT PVGQPPYQRPAATLA
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
Consensus Consensus
AB
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.
K. Sano et al. Egg envelope of Japanese eel
FEBS Journal 277 (2010) 4674–4684 ª 2010 The Authors Journal compilation ª 2010 FEBS 4677
other 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).
Egg envelope of Japanese eel K. Sano et al.
4678 FEBS Journal 277 (2010) 4674–4684 ª 2010 The Authors Journal compilation ª 2010 FEBS
Expression 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.
K. Sano et al. Egg envelope of Japanese eel
FEBS Journal 277 (2010) 4674–4684 ª 2010 The Authors Journal compilation ª 2010 FEBS 4679
rt vepβ
masu chgHβ
sal λzp19
rt vepa
masu chgHa
Ol chgH

Om chgH
Oj chgH
Fh chgH
wf chgH
La
chgH
sa zpbb
Tn chgH
Fg chgH
sa zpba
Ol chgHm
Fh chgHm
Cv zr-2
carp zp2
gf zp2
zf zp2
e zpb
zf zp2like1
zfz p2like2
Fg zpb
Tn zpb
Ol zpb
Fg zpax1
sa zpx
Fg zpax2
Ol zpx
zf
zpa
zf zp3cv1
zf zp3v2

Fg
zpc1
Ol zpc1
Fg
zpc2
Ol zpc2
Fg
zpc4
Ol
zpc4
zf
zpb
Fg zpc5
Ol zpc5
Fg zpc3
Ol zpc3
e zpca
e zpcb
e zpcc
e zpce
e zpcd
carp zp3
carp zp3.2
gf zp3
zf
zp3
zf
zp3a.1
Cv
zr-3

Fh
chgL
Oj chgL
Om chgL
Ol chgL
Os
chgL
Fg
chgL
Tn
chgL
sa zpc
La chgL
masu chgL
char zp
γ
rt
vep
γ
0.5
rt vepγ
char
zpγ
masu
chgL
Fg
zpc1
Ol zpc1
Fg
zpc2

Ol zpc2
Fg
zpc3
Ol zpc3
zf zp3cv1
zf zp3v2
Fg zpc4
Ol zpc4
zf zpc
Fg zpc5
Ol zpc5
carp
zp3
carp zp3.2
gf zp3
zf zp3
zf zp3a.1
e zpca
e zpcb
e zpcc
e
zpce
e zpcd
La chgL
Sa
zp3
Tn
chgL
Fg chgL
Os chgL

Ol chgL
Om chgL
Oj
chgL
Fh chgL
Cv zr-3
96
99
94
99
89
58
99
99
94
67
92
100
100
100
100
99
99
87
94
53
98
79
72
99

86
99
99
99
92
61
69
masu chgHa
rt
vepα
sal λzp19
Ol chgH
Om chgH
Oj chgH
Fh chgH
wf chgH
La chgH
Sa zp1b
Tn chgH
Fg chgH
Sa zp1a
Ol chgHm
Fh chgHm
Cvzr-2
e zpb
Tn zpb
Fg zpb
Ol zpb
zf zp2
gf zp2

carp zp2
zf zp2like2
zf zp2like1
masu chgHβ
rt vepβ
100
100
98
98
97
99
66
59
100
100
100
100
100
100
100
100
100
99
99
90
84
97
0.1
ZPB
ZPX

ZPC
A
B
C
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).
Egg envelope of Japanese eel K. Sano et al.
4680 FEBS Journal 277 (2010) 4674–4684 ª 2010 The Authors Journal compilation ª 2010 FEBS
encode 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 choriogenin 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 necessary 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
5mm iodoacetic acid. After centrifugation (2000 g, for 30 s
at 4 °C), the supernatant was decanted. This procedure was
K. Sano et al. Egg envelope of Japanese eel
FEBS Journal 277 (2010) 4674–4684 ª 2010 The Authors Journal compilation ª 2010 FEBS 4681
repeated 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
)1
of endopep-
tidase Glu-C (Roche, Indianapolis, IN, USA), at 37 °C
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 37 °C for 1 h, the total
RNA on the membrane was hybridized at 37 °C 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 60 °C, 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. 1
3, 7
]
decan}-4-yl) phenyl phosphate, 0.1% diethanolamine and
Table 2. Primer sequences specific for each eZP gene.
Gene Primer (5¢-to3¢)
ezpb Forward: GCAAAGAAGGTCAATTGCTCC
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
Egg envelope of Japanese eel K. Sano et al.
4682 FEBS Journal 277 (2010) 4674–4684 ª 2010 The Authors Journal compilation ª 2010 FEBS
1mm MgCl
2
for 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 94 °C, 30 s at
56 °C 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.
References
1 Yamagami K, Hamazaki TS, Yasumasu S, Masuda K
& Iuchi I (1992) Molecular and cellular basis of

formation, hardening, and breakdown of the egg
envelope in fish. Int Rev Cytol 136, 694–705.
2 Hedrick JL (2008) Anuran and pig egg zona pellucida
glycoproteins in fertilization and early development.
Int J Dev Biol 52, 683–701.
3 Wyrick RE, Nishihara T & Hedrick JL (1974) Fertiliza-
tion envelope formation by glycoprotein agglutination
and block to polyspermy in xenopuslaevis. Fed Proc 33,
1453–1453.
4 Wolf DP, Nishihara T, West DM, Wyrick RE &
Hedrick JL (1976) Isolation, physicochemical properties,
and macromolecular-composition of vitelline and
fertilization envelopes from xenopus-laevis eggs.
Biochemistry 15, 3671–3678.
5 Bleil JD & Wassarman PM (1980) Structure and func-
tion of the zona pellucida: identification and character-
ization of the proteins of the mouse oocyte’s zona
pellucida. Dev Biol 76, 185–202.
6 Epifano O, Liang LF, Familari M, Moos MC Jr &
Dean J (1995) Coordinate expression of the three zona
pellucida genes during mouse oogenesis. Development
121, 1947–1956.
7 Harris JD, Hibler DW, Fontenot GK, Hsu KT,
Yurewicz EC & Sacco AG (1994) Cloning and charac-
terization of zona-pellucida genes and cDNAs from a
variety of mammalian-species – the ZPA, ZPB and ZPC
gene families. DNA Seq 4, 361–393.
8 Bausek N, Waclawek M, Schneider WJ & Wohlrab F
(2000) The major chicken egg envelope protein ZP1 is
different from ZPB and is synthesized in the liver.

J Biol Chem 275, 28866–28872.
9 Smith J, Paton IR, Hughes DC & Burt DW (2005)
Isolation and mapping the chicken zona pellucida genes:
an insight into the evolution of orthologous genes in
different species. Mol Reprod Dev 70, 133–145.
10 Spargo SC & Hope RM (2003) Evolution and nomen-
clature of the zona pellucida gene family. Biol Reprod
68, 358–362.
11 Hamazaki TS, Ichiro I & Yamagami K (1987) Isolation
and partial characterization of a ‘‘spawning female-
specific substance’’ in the teleost, Oryzias latipes. J Exp
Zool 242, 343–349.
12 Hamazaki TS, Ichiro I & Yamagami K (1987) Produc-
tion of a ‘‘spawning female-specific substance’’ in hepa-
tic cells and its accumulation in the ascites of the
estrogen-treated adult fish, Oryzias latipes. J Exp Zool
242, 325–332.
13 Hamazaki TS, Nagahama Y, Iuchi I & Yamagami K
(1989) A glycoprotein from the liver constitutes the
inner layer of the egg envelope (zona pellucid interna) of
the fish, Olyzias latipes. Dev Biol 133, 101–110.
14 LaFleur GJ Jr, Raldu´ a D, Fabra M, Carnevali O,
Denslow N, Wallace RA & Cerda
`
J (2005) Derivation
of major yolk proteins from parental vitellogenins and
alternative processing during oocyte maturation in
Fundulus heteroclitus. Biol Reprod 73, 815–824.
15 Berg AH, Westerlund L & Olsson PE (2004) Regulation
of Arctic char (Salvelinus alpinus) egg shell proteins

and vitellogenin during reproduction and in response to
17beta-estradiol and cortisol. Gen Comp Endocrinol 135,
276–285.
16 Fujita T, Fukada H, Shimizu M, Hiramatsu N &
Hara A (2008) Molecular cloning and characterization
of three distinct choriogenins in masu salmon,
Oncorhynchus masou. Mol Reprod Dev 75,
1217–1228.
K. Sano et al. Egg envelope of Japanese eel
FEBS Journal 277 (2010) 4674–4684 ª 2010 The Authors Journal compilation ª 2010 FEBS 4683
17 Modig C, Modesto T, Canario A, Cerda
`
J, von Hofsten J
& Olsson PE (2006) Molecular characterization and
expression pattern of zona pellucida proteins in
gilthead seabream (Sparus aurata). Biol Reprod 75,
717–725.
18 Modig C, Raldu´ a D, Cerda
`
J & Olsson PE (2008)
Analysis of vitelline envelope synthesis and composition
during early oocyte development in gilthead seabream
(Sparus aurata). Mol Reprod Dev 75, 1351–1360.
19 Chang YS, Wang SC, Tsao CC & Huang FL (1996)
Molecular cloning, structural analysis, and expression
of carp ZP3 gene. Mol Reprod Dev 44, 295–304.
20 Chang YS, Hsu CC, Wang SC, Tsao CC & Huang FL
(1997) Molecular cloning, structural analysis, and
expression of carp ZP2 gene. Mol Reprod Dev 46, 258–
267.

21 Wang H & Gong Z (1999) Characterization of two
zebrafish cDNA clones encoding egg envelope proteins
ZP2 and ZP3. Biochim Biophys Acta 1446, 156–160.
22 Kanamori A (2000) Systematic identification of genes
expressed during early oogenesis in medaka. Mol
Reprod Dev 55, 31–36.
23 Kanamori A, Naruse K, Mitani H, Shima A & Hori H
(2003) Genomic organization of ZP domain containing
egg envelope genes in medaka (Oryzias latipes). Gene
305, 35–45.
24 Murata K, Sasaki T, Yasumasu S, Iuchi I, Enami J,
Yasumasu I & Yamagami K (1995) Cloning of cDNAs
for the precursor protein of a low-molecular-weight
subunit of the inner layer of the egg envelope (chorion)
of the fish Oryzias latipes. Dev Biol 167, 9–17.
25 Sugiyama H, Yasumasu S, Murata K, Iuchi I & Yama-
gami K (1996) The third egg envelope subunit in fish:
cDNA cloning and analysis, and gene expression. Dev
Growth Differ 40, 35–45.
26 Miura T, Yamauchi K, Nagahama Y & Takahashi H
(1991) Induction of spermatogenesis in male Japanese
eel, Anguilla japonica, by a single injection of human
chorionic gonadotropin. Zool Sci 8, 63–73.
27 Miura T, Kudo N, Miura C, Yamauchi K & Nagahama Y
(1998) Two testicular cDNA clones suppressed by
gonadotropin stimulation exhibit ZP2- and ZP3-like
structures in Japanese eel. Mol Reprod Dev 51, 235–242.
28 Sano K, Inohaya K, Kawaguchi M, Yoshizaki N,
Iuchi I & Yasumasu S (2008) Purification and
characterization of zebrafish hatching enzyme: an evolu-

tionary aspect of the mechanism of egg envelope diges-
tion. FEBS J 275, 5934–5946.
29 Liu X, Wang H & Gong Z (2006) Tandem-repeated
zebrafish zp3 genes possess oocyte-specific promoters
and are insensitive to estrogen induction. Biol Reprod
74, 1016–1025.
30 Jovine L, Qi H, Williams Z, Litscher E & Wassarman PM
(2002) The ZP domain is a conserved module for
polymerization of extracellular proteins. Nat Cell Biol 4,
457–461.
31 Conner SJ & Hughes DC (2003) Analysis of fish
ZP1 ⁄ ZPB homologous genes – evidence for both
genome duplication and species-specific amplification
models of evolution. Reproduction 126, 347–352.
32 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F
& Higgins DG (1997) The CLUSTAL_X windows
interface: flexible strategies for multiple sequence
alignment aided by quality analysis tools. Nucleic
Acid Res 25, 4876–4882.
33 Hall BG (2005) Phylogenetic Trees Made Easy, 2nd edn.
Massachusetts, Sinauer Associates, Inc.
34 Tanabe AS (2007) KAKUSAN: a computer program
to automate the selection of a nucleotide substitution
model and the configuration of a mixed model on
multilocus data. Mol Ecol Notes 7, 962–964.
35 Jobb G (2008) TREEFINDER version of October
2008. www.treefinder.de
Egg envelope of Japanese eel K. Sano et al.
4684 FEBS Journal 277 (2010) 4674–4684 ª 2010 The Authors Journal compilation ª 2010 FEBS