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: 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. 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. 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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