Hatching enzyme of the ovoviviparous black rockfish Sebastes schlegelii – environmental adaptation of the hatching enzyme and evolutionary aspects of formation of the pseudogene Mari Kawaguchi 1 , Masahiro Nakagawa 2 , Tsutomu Noda 3 , Norio Yoshizaki 4 , Junya Hiroi 5 , Mutsumi Nishida 6 , Ichiro Iuchi 1 and Shigeki Yasumasu 1 1 Life Science Institute, Sophia University, Tokyo, Japan 2 National Center for Stock Enhancement, Fisheries Research Agency, Goto Station, Nagasaki, Japan 3 National Center for Stock Enhancement, Fisheries Research Agency, Miyako Station, Iwate, Japan 4 Department of Animal Resource Production, United Graduate School of Agricultural Science, Gifu University, Japan 5 Department of Anatomy, St Marianna University School of Medicine, Kawasaki, Japan 6 Ocean Research Institute, University of Tokyo, Japan At the time of hatching of oviparous fish embryos, the hatching enzyme is secreted from hatching gland cells of the embryos to digest the egg envelope (chorion) [1– 3]. The hatching enzyme cDNAs have been cloned from embryos of various oviparous fish species, such as medaka (Oryzias latipes) [4], zebrafish (Danio rerio) [5], masu salmon (Oncorhynchus masou) [5], yellow- tailed damsel (Chrysiptera parasema) [6], Japanese eel Keywords aberrant splicing; adaptation; astacin family metalloprotease; hatching enzyme; pseudogene Correspondence S. Yasumasu, Life Science Institute, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554, Japan Fax: +81 3 3238 3393 Tel: +81 3 3238 4263 E-mail: s-yasuma@hoffman.cc.sophia.ac.jp Database The nucleotide sequence data have been submitted to the DDBJ ⁄ EMBL ⁄ GenBank nucleotide sequence databases under the accession numbers AB353099–AB353111 (Received 17 February 2008, revised 25 March 2008, accepted 1 April 2008) doi:10.1111/j.1742-4658.2008.06427.x The hatching enzyme of oviparous euteleostean fishes consists of two metalloproteases: high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They cooperatively digest the egg envelope (chorion) at the time of embryo hatching. In the present study, we investigated the hatching of embryos of the ovoviviparous black rockfish Sebastes schlegelii. The chorion-swelling activity, HCE-like activity, was found in the ovarian fluid carrying the embryos immediately before the hatching stage. Two kinds of HCE were partially purified from the fluid, and the relative molecular masses of them matched well with those deduced from two HCE cDNAs, respectively, by MALDI-TOF MS analysis. On the other hand, LCE cDNAs were cloned; however, the ORF was not complete. These results suggest that the hatching enzyme is also present in ovoviviparous fish, but is composed of only HCE, which is different from the situation in other oviparous euteleostean fishes. The expression of the HCE gene was quite weak when compared with that of the other teleostean fishes. Considering that the black rockfish chorion is thin and fragile, such a small amount of enzyme would be enough to digest the chorion. The black rockfish hatch- ing enzyme is considered to be well adapted to the natural hatching envi- ronment of black rockfish embryos. In addition, five aberrant spliced LCE cDNAs were cloned. Several nucleotide substitutions were found in the splice site consensus sequences of the LCE gene, suggesting that the prod- ucts alternatively spliced from the LCE gene are generated by the muta- tions in intronic regions responsible for splicing. Abbreviations DIG, digoxigenin; Ga, Gasterosteus aculeatus; HCE, high choriolytic enzyme; Hh, Helicolenus hilgendorfi; LCE, low choriolytic enzyme; MCA, 7-amino-4-methylcoumarin; MYA, million years ago; Sg, Setarches guentheri; Ss, Sebastes schlegelii. 2884 FEBS Journal 275 (2008) 2884–2898 ª 2008 The Authors Journal compilation ª 2008 FEBS (Anguilla japonica) [7], Fundulus heteroclitus [8], ayu (Plecoglossus altivelis altivelis) [9] and fugu (Taki- fugu rubripes) [10]. Among them, the medaka enzymes have been studied comprehensively. The hatching enzyme is composed of two proteases: high choriolytic enzyme (HCE, choriolysin H, EC 3.4.24.67) and low choriolytic enzyme (LCE, choriolysin L, EC 3.4.24.66). They cooperatively digest the chorion; HCE swells the chorion by its limited proteolytic action, and then LCE digests the swollen chorion completely [11–13]. They act at the same time, and efficient, complete digestion was observed at natural hatching. Both enzymes belong to the astacin family of metallo- proteases [14]. Unlike oviparous fish embryos, ovoviviparous fish embryos grow and hatch within the maternal body and are then delivered from the body. At the time of ovoviviparous fish hatching, it has been unclear whether the hatching enzyme is secreted from hatching gland cells to digest the chorion. In this study, we observed the embryo hatching of the ovoviviparous black rockfish Sebastes schlegelii, which is a member of the Scorpaeniformes within the Euteleostei [15]. The hatching enzyme was identified from ovarian fluids of the black rockfish, and the cDNAs and the genes for the hatching enzyme were cloned from the embryos. Results Detection of metalloprotease activity in ovarian fluid We expected that enzymes secreted from ovoviviparous fish embryos (hatching enzymes) would be present in the ovarian fluid after the embryos hatched. Ovarian fluid was collected from the ovarian cavity, and its proteolytic activity was examined using several sub- strates added in isotonic saline (0.128 m NaCl, similar to the natural hatching environment of embryos in the ovarian cavity). The teleostean hatching enzymes are generally known to belong to the astacin family of metalloproteases, and they are inactivated by a chelating reagent such as EDTA. Enzyme activities were determined with or without EDTA. First, the caseinolytic activity of ovarian fluid was examined. The ovarian fluid was prepared from female fish carrying embryos at the following stages: stages of late blastula (stage 11), 22–23 somites (optic cups, stage 20), auditory placodes (stage 21), 26–27 somites (pectoral fins, stage 24), pigmentation of retina (stage 25), openings of mouth and anus (stage 28), pig- mentation of peritoneal wall (stage 29), depletion of yolk (stage 30), immediately before hatching (stage 31), and after embryo delivery [16]. As shown in Fig. 1A, constant activities were observed in the ovarian fluids carrying stage 11 to stage 30 embryos (stage 11 to stage 30 ovarian fluid). The activity was sharply increased in the stage 31 ovarian fluid, and disap- peared from the fluid after embryo delivery. The activi- ties in stage 11 to stage 30 ovarian fluid were not inhibited by EDTA, but the activity in stage 31 ovar- ian fluid dropped to about a half because of EDTA. Although some proteases are present in ovarian fluid carrying embryos throughout all developmental stages, the stage 31 ovarian fluid is suggested to contain metalloprotease(s). Next, the substrate specificity of the enzyme activity was examined using Suc-Leu-Leu-Val-Tyr-7-amino-4- methylcoumarin (MCA) and Suc-Ala-Pro-Ala-MCA as substrates; these are the best substrates for medaka HCE [12] and Fundulus HCE [8], respectively. Fig- ure 1B shows the change in MCA-peptide-cleaving activity of the ovarian fluid towards Suc-Leu-Leu-Val- Tyr-MCA. Little or no activity was observed in stage 11 to stage 30 ovarian fluid. The activity was sharply increased in the stage 31 fluid, and was not detected in the ovarian fluid after embryo delivery. The activity in the stage 31 fluid was strongly inhibited by EDTA. The activity towards Suc-Ala-Pro- Ala-MCA in stage 31 ovarian fluid was about 30 times less than that towards Suc-Leu-Leu-Val-Tyr-MCA. The changes in the activities throughout development were the same as those towards Suc-Leu-Leu-Val- Tyr-MCA. These results suggest that the metallo- Fig. 1. Caseinolytic activity (A) and Suc-Leu-Leu-Val-Tyr-MCA-cleav- ing activity (B) of ovarian fluid carrying embryos at various develop- mental stages (from stage 11 to stage 31) and after embryo delivery, D. Black circles and white squares indicate the activities of the fluid preincubated without and with 20 m M EDTA, respec- tively. Caseinolytic and MCA-cleaving activities are expressed as DA 280 30 min )1 and nmolÆmin )1 , respectively. M. Kawaguchi et al. Hatching enzyme of ovoviviparous black rockfish FEBS Journal 275 (2008) 2884–2898 ª 2008 The Authors Journal compilation ª 2008 FEBS 2885 protease with the substrate specificity similar to that of known HCEs is present specifically in the stage 31 ovarian fluid. Choriolytic activity in stage 31 ovarian fluid and morphological changes of the chorion As stage 31 of black rockfish embryos is the stage immediately before hatching, it is conceivable that metalloprotease(s) present in the stage 31 ovarian fluid are the hatching enzyme(s) of black rockfish. When the stage 31 ovarian fluid was incubated with chorion frag- ments, the amount of liberated peptides was increased up to 30 min and became constant thereafter (Fig. 2A). Most of the peptides were not liberated after the treatment with EDTA, suggesting that metallopro- tease efficiently digesting the chorion is present in the stage 31 ovarian fluid. After 30 min of incubation, the chorion was swollen (Fig. 2D), and the thickness of the chorion was increased about four times when com- pared with that of the control chorion (Fig. 2B,C). Eighty minutes later, the inner layer of the chorion was completely digested, and the thin outer layer remained undigested (Fig. 2E). The fine structure of the black rockfish chorion before or after incubation with ovarian fluid was observed with an electron microscope. The control chorion was com- posed of a thick inner layer and a thin outer layer. The inner layer seems to be composed of two layers, which are morphologically distinct (Fig. 3A). No significant change of the chorion was observed after the incubation with stage 24 ovarian fluid (data not shown). On the other hand, stage 31 ovarian fluid swelled both of the inner layers of the isolated chorion (Fig. 3B), and fine fibrillar structures were observed in the outer region of the inner layer (Fig. 3C). This structural change was similar to that of the chorion isolated from stage 31 embryos (Fig. 3D). The chorion-digesting property of the stage 31 ovarian fluid was similar to that of HCEs that have been previously reported in medaka and Fund- ulus [8,13]. This observation suggests that an HCE-like activity, rather than an LCE-like activity, exists in stage 31 ovarian fluid. Identification of HCE from stage 31 ovarian fluid The protease(s) in stage 31 ovarian fluid was par- tially purified by successive HPLC steps through a gel Fig. 2. (A) Time course of chorion solubilization by stage 31 ovarian fluid. Black circles and white squares indicate the activities of the fluid preincubated without and with 20 m M EDTA, respectively. The activity is expressed as the value of DA 595 . Black rockfish chorion isolated from stage 11 embryos was incubated for 0 min (B, C), 30 min (D) and 80 min (E). Scale bars: 100 lm. Arrows indicate thickness of chorion. Fig. 3. Electron microscopic observation of morphological change of the chorion by stage 31 ovarian fluid. The chorion isolated from stage 11 embryos was incubated with only the buffer (A) and with stage 31 ovarian fluid (B). (C) High magnification of the part shown in the box in (B). The bar indicates the outer layer. (D) The chorion isolated from a stage 31 embryo. Scale bars: 1 lm (A, B, D) and 0.5 lm (C). Hatching enzyme of ovoviviparous black rockfish M. Kawaguchi et al. 2886 FEBS Journal 275 (2008) 2884–2898 ª 2008 The Authors Journal compilation ª 2008 FEBS filtration column, S-Sepharose column and Source 15S column. Figure 4 shows the chromatogram of the Source 15S column. Most of the proteins were adsorbed to the column, and the proteolytic activity was eluted as two peaks just after a large protein peak. Then, the fraction containing the two peaks was sub- jected to reversed-phase column chromatography. The five protein peaks thus obtained were analyzed by SDS ⁄ PAGE. The major peak, containing a 23 kDa protein, the molecular mass of which was anticipated to be the molecular mass of other euteleostean HCEs, was subjected to MALDI-TOF MS analysis (Fig. 4). The values (m ⁄ z 22 789.68 and 23 075.27) were almost identical to the relative molecular masses calculated from two black rockfish HCE cDNAs (SsHCE1, M r = 22 584; SsHCE2, M r = 23 056) cloned in the present study (described later). These results strongly suggest that the chorion-swelling activity in the stage 31 ovarian fluid is responsible for the action of HCEs, the genes of which are orthologous to those of other euteleostean HCEs. Cloning of black rockfish hatching enzyme cDNAs It has been suggested that both HCE and LCE genes are present in euteleostean fishes [10]. However, only HCE was identified in stage 31 ovarian fluid. Whether black rockfish possess both the HCE and LCE genes or not remains unclear. First, we performed cloning of hatching enzyme cDNAs by RT-PCR and RACE PCR from the RNA of black rockfish embryos. As a result, the 1009 bp and 1088 bp cDNAs were cloned from black rockfish embryos. Figure 5 shows the phyloge- netic tree constructed from the previously cloned hatching enzyme cDNAs of fishes belonging to the Elopomorpha (Japanese eel) and the Euteleostei (medaka, Fundulus, fugu, and Tetraodon), together with the cDNAs cloned in the present study. The tree clearly shows that euteleostean hatching enzymes are divided into HCE and LCE clades with high probabil- ity (92% for the maximum likelihood tree, 100% for the neighbor-joining tree, and 100% for the Bayesian tree). On the basis of the tree, the two cloned cDNAs were named black rockfish Seb. schlegelii HCEs, SsHCE1 and SsHCE2. Fig. 4. Elution pattern of cation exchange Source 15S chromatogra- phy with a linear gradient from 0 to 1 M NaCl. Solid line, absor- bance at 280 nm; dashed line, Suc-Leu-Leu-Val-Tyr-MCA-cleaving activity shown as nmolÆmin )1 . The inset shows the MALDI-TOF MS spectrum obtained from the major peak by RP-HPLC with the range of m ⁄ z values from 21 716 to 24 768. Ions at m ⁄ z 22 789.68 and 23 075.27 were identified as the black rockfish HCE. Fig. 5. A 55% majority rule consensus phylogenetic tree con- structed by the maximum likelihood method. The tree was con- structed using nucleotide sequences at the mature enzyme portion of hatching enzymes of arowana (AwHE, AB276000), bony tongue (BtHE, AB360712), Japanese eel (EHE, AB071423–9), Fundulus (FHCE, AB210813; and FLCE, AB210814), medaka (MHCE, M96170; and MLCE, M96169), Tetraodon (TnHCE, AB246043; and TnLCE, AB246044), fugu (FgHCE, AB246041; and FgLCE, AB246042), stickleback (GaHCE, AB353108–9; and GaLCE, AB353110), Set. guentheri (SgHCE, AB353105–6; and SgLCE, AB353107), H. hilgendorfi (HhHCE, AB353102–3; and HhLCE, AB353104), and black rockfish (SsHCE, AB353099–100; and wSsLCE, AB353101). Numbers at the nodes indicate bootstrap val- ues for the maximum likelihood tree and neighbor-joining tree, and Bayesian posterior probabilities, shown as percentages. M. Kawaguchi et al. Hatching enzyme of ovoviviparous black rockfish FEBS Journal 275 (2008) 2884–2898 ª 2008 The Authors Journal compilation ª 2008 FEBS 2887 To obtain evolutionary information, we amplified HCE genes from genomic DNAs of Helicolenus hil- gendorfi and Setarches guentheri, which belong to the same subfamily (Sebastinae) as that of black rockfish [15]. From both the species, SsHCE1 and SsHCE2 or- thologs (HhHCE1 and HhHCE2 for H. hilgendorfi, and SgHCE1 and SgHCE2 for Set. guentheri) were cloned (Fig. 5). HCE (GaHCE1 and GaHCE2) cDNAs were also cloned from the stickleback Gaster- osteus aculeatus, belonging to the Gasterosteiformes [15], which is an order different from the Scorpaenifor- mes. Both the orders belong to the same series, the Percomorpha. The amino acid sequences of HCEs deduced from the newly cloned cDNAs are shown in Fig. 6A. All of them possessed two active site consensus sequences of the astacin family proteases: HExxHxx- GFxHExxRxDR (zinc-binding site) and SxMHY (methionine turn) [17–19]. In addition, six cysteines, which are present in all of the previously cloned fish hatching enzymes [9], were conserved among them. Fig. 6. (A) A multiple alignment of amino acid sequences of hatching enzymes. White and black triangles indicate putative signal sequence cleavage sites and N-terminals of mature enzymes, respectively. Arrows indicate intron insertion sites of LCE genes. Identical residues are boxed. Dashes represent gaps. Two active site consensus sequences of the astacin family protease are given in dark (zinc-binding site) and light (methionine turn) gray boxes, and conserved cysteine residues are in black boxes. (B) Exon–intron structures of black rockfish (wSsLCE), H. hilgendorfi (HhLCE), Set. guentheri (SgLCE) and stickleback (GaLCE) LCE and HCE genes. The exons and introns are indicated by boxes and solid lines, respectively. Numbers in parentheses indicate intron phases. Hatching enzyme of ovoviviparous black rockfish M. Kawaguchi et al. 2888 FEBS Journal 275 (2008) 2884–2898 ª 2008 The Authors Journal compilation ª 2008 FEBS The gene structures of all the HCE genes were deter- mined to be intron-less (Fig. 6B), which is characteris- tic of HCE genes [10]. Southern blot analysis showed that the SsHCE1 probe hybridized with at least four EcoRI fragments of 4.4, 3.8, 3.4 and 3.2 kbp of black rockfish genomic DNA (Fig. 7A), indicating that the black rockfish HCE gene is a multicopy gene, like other euteleostean HCE genes examined so far [10]. As no LCE cDNA fragments were obtained from the black rockfish by the above strategy, we employed another strategy: that is, primers were generated from the sequence of stickleback LCE (GaLCE) cDNA. Six different-size cDNAs (600–2 kbp) were cloned from black rockfish embryos, and five of the six were the transcripts that would be formed by abnormal splicing (see later). The other one (929 bp, SsLCE1) was well aligned with other known LCE cDNAs, but its ORF was incomplete. Thus, the black rockfish LCE gene is transcribed, but the gene is not translated into a func- tional protein. The LCE gene is predicted to be a pseudogene. We named it black rockfish pseudo-LCE gene (wSsLCE). These results support the finding from the protein level experiment that only HCE activity, not the cooperative activity of HCE and LCE, is pres- ent in stage 31 ovarian fluid. LCE genes were cloned from H. hilgendorfi (HhLCE) and Set. guentheri (SgLCE). Their ORFs were predicted to be complete. Figure 8 shows nucle- otide and deduced amino acid sequences of wSsLCE1 and HhLCE cDNAs. The identity of the nucleotide sequences of the ORF between them was 95%. When compared with HhLCE cDNA, wSsLCE1 cDNA possessed a pretermination stop codon due to nucleotide substitution of 262G to 262T, and a frameshift mutation due to one nucleo- tide deletion (288delA) (Fig. 8). The gene structure of wSsLCE was determined using the nucleotide sequence of wSsLCE1 cDNA. The wSsLCE gene was composed of eight exons and seven introns; its structure, including the positions of exon– intron boundaries and intron phases, was the same as that of other euteleostean LCE genes (Fig. 6B) [10]. Southern blot analysis was performed using genomic DNA digested with BamHI, HindIII, ScaI and BglII. The wSsLCE1 DNA probe hybridized with a single fragment in each digest (Fig. 7B), suggesting that the wSsLCE gene is a single-copy gene, like other euteleos- tean LCE genes examined so far [10]. As described above, in addition to wSsLCE1 cDNA, five different-size cDNAs were cloned from black rock- fish embryos using primers designed from the 5¢-UTR and 3¢-UTR for wSsLCE1 cDNA. The wSsLCE2 (724 bp) and wSsLCE3 (606 bp) cDNAs were shorter than wSsLCE1 cDNA (870 bp), whereas wSsLCE4 (1033 bp), wSsLCE5 (2036 bp) and wSsLCE6 (1852 bp) cDNAs were longer than wSsLCE1 cDNA (Fig. 9A). wSsLCE2 and wSsLCE3 cDNAs lacked the entire region of exon 4 (146 bp) and exon 4⁄ 5 (264 bp) of the wSsLCE gene, respectively. Considering that the wSsLCE gene is a single-copy gene, wSsLCE2 and wSsLCE3 cDNAs are predicted to be the products resulting from exon skipping by aberrant splicing. As the pretermination stop codon and the nucleotide dele- tion are present in exon 4, wSsLCE2 and w SsLCE3 cDNAs have complete ORFs. However, their trans- lated products lack the N-terminal region of the mature enzyme encoded by exon 4, and are considered to be nonfunctional. On the other hand, wSsLCE4 and wSsLCE5 cDNAs possessed the entire intron 1 (163 bp) and intron 5 (1166 bp) sequences, respec- tively, showing cancellation of splicing of intron 1 and intron 5, respectively. wSsLCE6 cDNA was 184 bp shorter than wSsLCE5 cDNA, due to partial deletion of exon 5 and partial inclusion of intron 5. wSsLCE6 cDNA is considered to be the transcript that appears as a result of imprecise splicing. As shown in Fig. 9B, intron regions including the 5¢-splicing boundary of intron 5 also showed the simi- larity among the black rockfish, H. hilgendorfi and Set. guentheri. When we focused on the 5¢-splicing con- sensus sequence (gtragt) [20], we found a G to A sub- stitution in the +5 site of the wSsLCE gene (gtra gt to gtga at), whereas those of the HhLCE and SgLCE genes were well conserved. An experiment has demon- strated that +5 site mutation causes the exon skipping [21]. These results suggest that the mutation found in the wSsLCE gene probably results in intron 5 being Fig. 7. Southern blot analysis of SsHCE1 (A) and wSsLCE (B) genes. The restriction enzymes are shown at the top. Numbers on the left refer to the positions of size markers. M. Kawaguchi et al. Hatching enzyme of ovoviviparous black rockfish FEBS Journal 275 (2008) 2884–2898 ª 2008 The Authors Journal compilation ª 2008 FEBS 2889 retained by the cancellation of splicing, as seen in wSsLCE5 cDNA, and in the exon deletion, as seen in wSsLCE3 cDNA (Fig. 9A). Half of the wSsLCE cDNAs cloned in the present study had one nucleotide deletion (73delG) located at the 5¢-end of exon 2 (Fig. 8). The region including the exon–intron boundary between intron 1 and exon 2 was amplified by PCR from the genomic DNA. Sequence analysis revealed that the gene is heterozy- gous, and that a nucleotide substitution-destroying splicing acceptor consensus sequence (A GtoAA; Fig. 9B) is present in one of the alleleic wSsLCE genes. One of the alleles used the original AG acceptor sequence, and the other mutated allele used a pseudo- AG acceptor sequence by shifting one nucleotide to the 3¢-site; that is, )1A in the intronic sequence and 73G in the exonic sequence were used as the acceptor sites. The occurrence of 73delG in wSsLCE cDNA can be explained if the 73G was spliced out for use as a pseudo-AG acceptor sequence (Fig. 9B). The substi- tution might also cause the intron 1 retention, as seen in wSsLCE4 cDNA (Fig. 9A). Expression of black rockfish hatching enzyme genes First, the gene expression of SsHCE and wSsLCE was analyzed by northern blot analysis. An SsHCE1 DNA probe was used for detecting the HCE transcript. This probe probably detects both the SsHCE1 and SsHCE2 transcripts, because of their high level of similarity (88%). The hybridization of this probe with 10 lgof total RNA did not show any signal. This amount of RNA, 10 lg, is known to be enough for detecting the HCE transcripts of medaka and Fundulus [8,22]. The result suggests that the expression of SsHCE genes is much weaker than that in other fish species, and there- fore, poly(A)-rich RNA purified from 100 lg of total Fig. 8. Nucleotide and predicted amino acid sequences of wSsLCE1 and HhLCE. Arrows indicate intron insertion sites with intron numbers. Boxes indicate mutation sites found in the wSsLCE gene as described in the text. Hatching enzyme of ovoviviparous black rockfish M. Kawaguchi et al. 2890 FEBS Journal 275 (2008) 2884–2898 ª 2008 The Authors Journal compilation ª 2008 FEBS RNA was employed. The SsHCE1 probe hybridized with about 1 kb of transcript; this size was consistent with that of the cDNAs. The transcripts were detected in stage 17 ⁄ 18 embryos, decreased in amount towards stage 25, and disappeared thereafter (Fig. 10A). We failed to detect the positive signal of the wSsLCE gene transcript by northern blot analysis. Next, gene expression was determined by RT-PCR (Fig. 10B). After 28 cycles of PCR, sufficient expres- sion of the SsHCE1 and SsHCE2 genes was detected, and the band intensity of SsHCE2 tran- scripts was about half that of SsHCE1. For the wSsLCE gene, the 33 cycles of RT-PCR gave these bands at about 700 bp, 800 bp, 1 kbp, and 1.2 kbp, corresponding to wSsLCE3, wSsLCE2, wSsLCE1 and wSsLCE4 cDNAs, respectively. The expression pat- tern of the wSsLCE gene through the developmental stages was similar to that of the SsHCE genes, but the expression was much weaker than that of the SsHCE genes. As shown in Fig. 11, whole-mount in situ hybrid- ization using an antisense RNA probe for the SsHCE1 gene revealed a distribution of cells express- ing SsHCE transcripts in developing black rockfish embryos. It is well known that the fish hatching gland cells differentiate at the anterior end of the hypoblast layer, called the pillow, in the late gastrula embryos, and until hatching, the gland cells migrate to the final destination in a species-dependent man- ner [5,22]. In stage 17 embryos of the black rockfish, positive cells were first observed along the edge of the anterior head. These cells seem to make a start in migration from the pillow (Fig. 11A). From stage 18 to stage 22, the cells migrated posteriorly (Fig. 11B), and they were finally distributed widely in the epidermis of both lateral sides of the head Fig. 9. (A) A schematic representation of the splicing variants of the wSsLCE gene. The black triangle indicates putative N-terminals of mature enzymes. The structures of the normally spliced form (w SsLCE1) and the alternatively spliced forms (wSsLCE2–6) are shown. wSsLCE2, wSsLCE3, wSsLCE4, wSsLCE5 and wSsLCE6 have an exon 4 deletion, an exon 4 and 5 deletion, an intron 1 inclusion, an intron 5 inclusion, and partial deletion of exon 5 and partial inclusion of intron 5, respectively. (B) Nucleotide mutations found on the splice site con- sensus sequence at intron 5 and intron 1. The upper part gives a comparison of the exon–intron boundary between exon 5 and intron 5 among the wSsLCE, HhLCE and SgLCE genes. The consensus sequence of splicing donor site is shown at the top. The lower part is an electropherogram of the PCR product around the boundary between intron 1 and exon 2. The splicing acceptor consensus sequence and pseudo-AG consensus sequence are indicated by red boxes on the upper and lower lines, respectively, together with each cDNA product. The regions of the exon and intron are indicated by upper-case and lower-case letters, respectively. M. Kawaguchi et al. Hatching enzyme of ovoviviparous black rockfish FEBS Journal 275 (2008) 2884–2898 ª 2008 The Authors Journal compilation ª 2008 FEBS 2891 (Fig. 11C,D). In stage 24 and stage 25 embryos, the signals in positive cells became weak and their num- bers were decreased. No signals were observed in stage 29 and stage 31 embryos and posthatching fry, and nor were signals from sense RNA observed in any embryos. Fig. 10. Expression analysis of the SsHCE1, SsHCE2 and wSsLCE genes. (A) Northern blot analysis of expression of the SsHCE gene during development. Arrowheads indi- cate the positions of 28S and 18S rRNA. (B) RT-PCR analysis of SsHCE1, SsHCE2 and wSsLCE during development. b-Actin was used as a control. PCR cycles were 28 for SsHCE1 and SsHCE2, 33 for wSsLCE, and 24 for b-actin. Developmental stages are shown at the top. Fry, posthatching embryos. The 200 bp (SsHCE1, SsHCE2, and wSsLCE) and 100 bp (b-actin) ladder markers are shown in the left lane. Fig. 11. Whole-mount in situ hybridization of SsHCE gene during the development of black rockfish embryos. The SsHCE1 RNA probe was hybridized with stage 17 (A), stage 18 (B), stage 22 (C, D), stage 24 (E) and stage 25 (F) embryos. (A, B) Dorsal views of head regions. Upper, the anterior- most. (C, E, F) Lateral views. Upper, dorsal. (D) Dorsal view of the head region. Right, the anterior-most. Yolk was removed from stage 22 embryos (C, D). Scale bars: 200 lm. (G) Average number of hatching gland cells per embryo. The values are expressed as the mean of five embryos. Error bars indicate the standard deviation. Hatching enzyme of ovoviviparous black rockfish M. Kawaguchi et al. 2892 FEBS Journal 275 (2008) 2884–2898 ª 2008 The Authors Journal compilation ª 2008 FEBS Throughout the developmental stages, the total number of SsHCE-expressing cells per embryo seemed to be less than in other fishes. The number of hatching gland cells in hybridized embryos was counted, and the average number per embryo was determined at each developmental stage (Fig. 11G). In stage 17 and stage 22 embryos, about 100 cells were observed, and the number was decreased to about one-half at stage 24, to about one-quarter at stage 25, and to zero at stage 29. These results were consistent with the developmental expression profile obtained by northern blot analysis. In comparison, we counted the numbers of hatching gland cells of rainbow trout, ayu or loach embryos at the middle to late stages of somitogenesis. There were about 3000 (loach), 2000 (rainbow trout) and 1000 (ayu) per embryo. Thus, black rockfish hatching gland cells were about 10–30 times fewer in number than those of other fish species. Summing up the results, the black rockfish hatching enzyme gene is actively expressed, but its expression stops at the ear- lier stages. In addition, the expression level is consid- ered to be suppressed to a greater extent than in other fishes. Discussion We investigated the hatching of an ovoviviparous black rockfish. The EDTA-sensitive protease activity with a substrate specificity similar to that of known HCEs was detected in the ovarian fluid carrying embryos immediately before hatching stage (stage 31). Furthermore, the protease was found to swell the inner layer of the egg envelope (chorion) and to release some water-soluble peptides from the chorion. HCE, one of the euteleostean hatching enzymes, is well known to swell the chorion by its proteolytic action. The prote- ases in the stage 31 ovarian fluid were partially puri- fied, and a proteolytically active fraction containing proteins had a molecular mass corresponding to the cloned SsHCE1 and SsHCE2 cDNAs according to MALDI-TOF MS analysis. Therefore, these results strongly suggest that HCEs are secreted from black rockfish embryos immediately before the hatching stage. This is the first demonstration of hatching enzymes in ovoviviparous fish. At the natural hatching of medaka and Fundulus embryos, the chorion is efficiently solubilized, and no swelling of the chorion has been observed, due to the concurrent and cooperative action of LCE and HCE [8,13]. The morphological change of the chorion observed in black rockfish embryos implies that its chorion digestion mechanism is different from that of other euteleostean fishes. In addition, the present study revealed that HCE cDNAs were cloned and their gene expression was observed specifically in the hatching gland cells of embryos, whereas the LCE gene was pseudogenized. These results suggest that the chorion digestion at black rockfish hatching is performed by HCE alone. The intact chorion of the black rockfish was thin and fragile when compared with the medaka and Fundulus chorions (Fig. 2B), and had about one- fourth the thickness of the medaka chorion [23]. According to in vitro experiments, the chorion was completely digested by a long period of incubation (80 min) with stage 31 ovarian fluid. Considering that the hatching enzyme stays with the chorion for a long time in the ovarian cavity, HCE alone would be suffi- cient for chorion digestion. The northern blot analysis and in situ hybridization experiment showed that expression of the HCE gene was suppressed to a very low extent when compared with that of other euteleostean HCE genes. In addi- tion, the hatching enzyme synthesis of the black rock- fish ceased around the middle of somitogenesis, whereas that of other teleostean fishes, such as medaka, zebrafish, Japanese eel and ayu, could be detected at stages from the beginning of its expression to immediately before hatching [5,7,9,22]. These results imply that the black rockfish embryo synthesizes an amount sufficient for, but limited to, chorion digestion. Such an amount would not be harmful for embryos, as embryos might be damaged by a long period of incubation with a high concentration of the protease. Thus, the hatching enzyme system in oviparous fish embryos is conserved in the ovoviviparous black rockfish, with adaptations to their specific hatching environment. According to the teleostean phylogenetic tree pro- posed by Nelson, the ovoviviparous black rockfish and oviparous H. hilgendorfi belong to the same tribe (Sebastinae) but different genera, and oviparous Set. guentheri belongs to the same subfamily (Sebasti- nae) but a different tribe [15]. The mitochondrial DNA-based phylogenetic tree indicates that the genus Helicolenus is sister to Sebastes, which includes the black rockfish [24]. The nucleotide sequences of black rockfish hatching enzyme cDNAs indicated high simi- larity (93% and 97% for HCE1 and HCE2, respec- tively, and 95% for LCE) to those of H. hilgendorfi, and the phylogenetic analysis (Fig. 5) agreed well with the mitochondrial phylogenetic tree. Despite this phy- logenetically close relationship, the LCE genes of H. hilgendorfi and Set. guentheri had complete ORFs, whereas that of the black rockfish was incomplete. The Sebastes fossils can be traced back to the late Miocene (about 6–10 million years ago, MYA) [25]. This time M. Kawaguchi et al. Hatching enzyme of ovoviviparous black rockfish FEBS Journal 275 (2008) 2884–2898 ª 2008 The Authors Journal compilation ª 2008 FEBS 2893 [...].. .Hatching enzyme of ovoviviparous black rockfish M Kawaguchi et al agrees well with the divergence time of Sebastes, about 8 MYA, obtained by molecular clock estimation [26] These results suggest that the pseudogenization occurred within about 8 MYA of the evolutionary pathway to Sebastes Considering that the expression of the wSsLCE gene was very low, the wSsLCE gene is presumed to be on the way... Structure and developmental expression of hatching enzyme genes of the Japanese eel Anguilla japonica: an aspect of the evolution of fish hatching enzyme gene Dev Genes Evol 214, 17 6–1 84 8 Kawaguchi M, Yasumasu S, Shimizu A, Hiroi J, Yoshizaki N, Nagata K, Tanokura M & Iuchi I (2005) Purification and gene cloning of Fundulus heteroclitus hatching enzyme A hatching enzyme system composed of high choriolytic enzyme. .. exon–intron structures of fish, amphibian, bird and mammalian hatching enzyme genes, with special reference to the intron loss evolution of hatching enzyme genes in Teleostei Gene 392, 7 7–8 8 FEBS Journal 275 (2008) 288 4–2 898 ª 2008 The Authors Journal compilation ª 2008 FEBS 2897 Hatching enzyme of ovoviviparous black rockfish M Kawaguchi et al 11 Yasumasu S, Iuchi I & Yamagami K (1988) Medaka hatching enzyme. .. from the beginning to the middle of April in the system, the developing embryos were ordinarily collected by canulation into the ovary from the end of April to the middle of June Developmental stages of embryos were determined according to the criteria proposed by Kusakari [16], and eggs and ovarian fluid were collected separately Stage 17, 18, 21, 22, 24, 25, 29 and 31 prehatching embryos, and posthatching... consists of two kinds of proteases which act cooperatively Zool Sci 5, 19 1–1 95 12 Yasumasu S, Iuchi I & Yamagami K (1989) Purification and partial characterization of high choriolytic enzyme (HCE), a component of the hatching enzyme of the teleost, Oryzias latipes J Biochem 105, 20 4–2 11 13 Yasumasu S, Iuchi I & Yamagami K (1989) Isolation and some properties of low choriolytic enzyme (LCE), a component of the. .. rockfish hatching enzyme genes were amplified from the genomic DNA using primers designed from nucleotide sequences of the 5¢- and 3¢-ends of each full-length cDNA 2896 Hatching enzyme genes for H hilgendorfi and Set guentheri were cloned by PCR from the genomic DNA of each species, using primers generated from nucleotide sequences of the 5¢-UTR and 3¢-UTR for SsHCE1, SsHCE2 and wSsLCE cDNAs Southern blot analysis... Molecular and cellular basis of formation, hardening, and breakdown of the egg envelope in fish Int Rev Cytol 136, 5 1–9 2 4 Yasumasu S, Yamada K, Akasaka K, Mitsunaga K, Iuchi I, Shimada H & Yamagami K (1992a) Isolation of cDNAs for LCE and HCE, two constituent proteases of the hatching enzyme of Oryzias latipes, and concurrent expression of their mRNAs during development Dev Biol 153, 25 0–2 58 5 Inohaya K, Yasumasu... on the pseudogenized LCE gene gives us an idea of the evolutionary process generating alternative splicing, i.e the mutations of the intronic sequences of the genes and their subsequent natural selection Experimental procedures Fish Black rockfish (Seb schlegelii) were maintained in an indoor culturing system at Miyako Fisheries Research Station, Japan As black rockfish females usually fertilize their... have never been cloned from other fish species [4,5, 8–1 0], suggesting that the aberrant splicing of the wSsLCE gene occurred only in the black rockfish lineage We found some nucleotide substitutions in the splice site consensus sequences of the wSsLCE gene, as shown in Fig 9B One possible evolutionary pathway to the occurrence of aberrant splicing is as follows After the black rockfish LCE gene had became... TCGAGAACAGAGC-3¢; and 3¢-RACE (for nested PCR), 5¢-ATGTTTCTCCTCTCTGGGCAGAACTGGAGG-3¢ Two and one fragments were obtained by 5¢-RACE and 3¢-RACE PCR, respectively The nucleotide sequences of overlapping regions of one of the 5¢-RACE fragments were identical to the 3¢-RACE PCR product, whereas those of the other were not The 3¢-RACE PCR and its nested PCR were performed to obtain the full-length cDNAs for the other . Hatching enzyme of the ovoviviparous black rockfish Sebastes schlegelii – environmental adaptation of the hatching enzyme and evolutionary aspects of formation. the time of embryo hatching. In the present study, we investigated the hatching of embryos of the ovoviviparous black rockfish Sebastes schlegelii. The chorion-swelling