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

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].

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-

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

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

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

(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

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

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-

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

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-

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
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 to becoming completely
silent.
Splicing processes are known to be catalyzed by
spliceosomes including small nuclear ribonucleoprotein
particles. These factors are responsible for the accurate
positioning of the intronic sequence elements consist-
ing of the 5¢-splice site, branchpoint sequence, polypy-
rimidine tract, and 3¢-splice site [27–29]. The consensus
sequences at the exon–intron boundaries are essential
for specifying the splicing sites. More than 90% of
abnormal splicing products have been reported to be
due to the mutation(s) of such consensus sequences
[30]; several splicing mutations causing exon skipping

skipping exon 4 containing the pretermination stop
codon and nucleotide deletion. However, neither tran-
script is dominant, and their expression was much
lower than that of wSsLCE1 cDNA. These aberrant
splicings did not produce functional products, and the
black rockfish established a single enzyme hatching
system.
The present study has shown that various types of
alternative splicing could arise due to nucleotide sub-
stitution(s) at the intronic sequence. Alternative splic-
ing is known to play important roles in generating
variations in protein function [33], and at least 35–
59% of human genes are alternatively spliced [34]. The
present investigation 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 Sta-
tion, Japan. As black rockfish females usually fertilize their
eggs from the beginning to the middle of April in the sys-
tem, the developing embryos were ordinarily collected by
canulation into the ovary from the end of April to the mid-
dle of June. Developmental stages of embryos were deter-
mined 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

) of the supernatant was measured.
Hatching enzyme of ovoviviparous black rockfish M. Kawaguchi et al.
2894 FEBS Journal 275 (2008) 2884–2898 ª 2008 The Authors Journal compilation ª 2008 FEBS
Estimation of MCA-peptide-cleaving activity
MCA-peptides, peptidyl-7-amino-4-methylcoumarins (Pep-
tide Institute, Inc., Osaka, Japan), were employed for evalu-
ating the substrate specificity of the enzyme activity in
ovarian fluid. A 250 lL reaction mixture containing 100 lm
Suc-Leu-Leu-Val-Tyr-MCA or Suc-Ala-Pro-Ala-MCA,
50 mm Tris ⁄ HCl buffer (pH 8.0), 0.128 m NaCl and ovar-
ian fluid was incubated at 30 °C for 30 min. After the reac-
tion was stopped by adding 500 lL of 20% acetic acid, the
fluorescence was measured with a Hitachi 204 fluorescence
spectrophotometer at 380 nm excitation and 460 nm
emission.
Estimation of choriolytic activity
The choriolytic activity of ovarian fluid was measured using
a30lL reaction mixture consisting of 0.128 m NaCl,
10 mm Tris ⁄ HCl buffer (pH 8.0), 0.3 lL of ovarian fluid
and three chorions isolated from stage 11 embryos. After
incubation at 30 °C for 60 min, the mixture was spun down
(at 1000 g for 10s), and 2 lL aliquots of the supernatant
were added to 60 lL of Bradford reagent (Sigma, St Louis,
MO, USA). The protein amount was determined by absor-
bance at 595 nm.
Inhibition of enzyme activity
The effect of EDTA on caseinolytic, MCA-peptide-cleaving
or choriolytic activity was examined. Ovarian fluid was
preincubated with 20 mm EDTA at 30 °C for 10 min in the
buffer, and substrates were then added. The enzyme activi-

containing 0.1% trifluoroacetic acid under the HPLC system.
The protein amount was monitored by measuring absor-
bance at 210 nm. The relative molecular masses of proteins
were determined with a Voyager-DESTR (Applied Bio-
systems, Foster City, CA, USA) mass spectrometer.
Cloning of hatching enzyme cDNAs from black
rockfish embryos
For cloning of black rockfish orthologs of HCE (SsHCE1
and SsHCE2), cDNA fragments were obtained by the RT-
PCR method using four forward and one reverse primers
designed from the conserved regions including active site
consensus sequences of astacin family proteases as previ-
ously described [9]. Then, 5¢-RACE and 3¢-RACE PCR and
nested PCR were performed from cDNAs synthesized from
RNAs extracted from prehatching embryos with the
SMART RACE cDNA Amplification Kit (Clontech,
Mountain View, CA, USA). The following primers were
used: 5¢-RACE (for first PCR), 5¢-AAGTTGTAGGCCTTC
TGCGGGTTGATGTTC-3¢;5¢-RACE (for nested PCR),
5¢-GAGCATGGTTGAT CTCGTGCTGGATGATGC-3¢;
3¢-RACE (for first PCR), 5¢-GTACGACTACATCAGCA
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 5¢-RACE PCR product. The following primers were

GTGTGACAAGTCTCAGAAGTGAC-3¢; and reverse, 5¢-
GTCATACTGTCACATTGAAACATACAGTAATAC-3¢.
In this procedure, three cDNAs that were regarded as
the products of aberrant splicing were newly obtained in
addition to the former three. Finally, six LCE cDNAs
(wSsLCE1–6) were cloned.
Stickleback hatching enzyme genes were cloned in silico
using the Ensembl genome database (embl.
org/Gasterosteus_aculeatus/index.html), and then full-
length cDNAs for HCEs and LCE were cloned by RT-PCR
from RNA extracted from prehatching embryos.
Phylogeny
A multiple sequence alignment of amino acid sequences of
mature enzyme portions was performed using the clus-
tal x program [35], and the codon-based alignment of their
nucleotide sequences was done using the codonalign 2.0
program. Trees were constructed according to the maxi-
mum likelihood method in the program phyml [36], the
Bayesian inference in the program mrbayes 3.1.2 [37,38]
with the HKY [39] +I+G model, and the neighbor-joining
method with the distance matrix calculated using the HKY
[39] model in the program paup* 4.0b [40]. The arowana
hatching enzyme (AwHE) gene was used as an outgroup.
The reliability of the tree was assessed by bootstrap values
obtained with 2000 pseudoreplicates for maximum likeli-
hood and neighbor-joining trees.
Gene amplification
Genomic DNAs were obtained by proteinase K digestion
followed by phenol ⁄ chloroform extraction and ethanol pre-
cipitation. The black rockfish hatching enzyme genes were

Tween-20 for 30 min at room temperature, and with
1 : 5000-diluted alkaline phosphatase-conjugated antibodies
to digoxigenin in the same buffer for 1 h. After three 5 min
washes with NaCl ⁄ P
i
containing 0.3% Tween-20, the mem-
brane was incubated in a reaction buffer consisting of 0.1%
diethanolamine and 1 mm MgCl
2
for 5 min at room
temperature. The membrane was incubated with 1% 3-[4-
methoxyspiro{1,2-dioxetane-3,2¢-(5¢-chloro)tricyclo[3.3.1.1
3,7
]
decan}-4-yl]phenyl phosphate in the buffer and exposed to
scientific imaging film (Kodak, Rochester, NY, USA) in the
dark.
Northern blot analysis
Poly(A)-rich RNA was extracted from total RNA
(100 lg) using a PolyATract mRNA Isolation System
(Promega, Madison, WI, USA), electrophoresed on 1%
formaldehyde–agarose gel, and transferred to a nylon
membrane (Hybond N; Amersham). Hybridization was
performed using the same protocol as for the Southern
blot analysis.
Semiquantitative estimation of expression of
hatching enzyme genes by RT-PCR
RT-PCR was performed using 0.1 lg of RNA with a One-
Step RT-PCR kit (Qiagen, Valencia, CA, USA), according
to the manufacturer’s instructions. b-Actin was used as a

Cit (pH 6.0) containing 0.1% Tween-20 (SSCT) at 68 °C,
the embryos were incubated for 4 · 15 min in 2 · SSCT
at 68 °C, washed for 3 · 20 min in 0.2 · SSCT at 68 °C,
transferred to NaCl ⁄ P
i
containing 0.1% Tween-20, and
washed for 3 · 5 min at room temperature. The embryos
were incubated for 90 min with 1% blocking reagent in
NaCl ⁄ P
i
containing 0.1% Tween-20, and then with
1 : 8000-diluted alkaline phosphatase-conjugated antibod-
ies to digoxigenin in NaCl ⁄ P
i
containing 0.1% Tween-20
at 4 °C overnight. After eight 30 min washes in NaCl ⁄ P
i
containing 0.1% Tween-20, the embryos were incubated
in a staining buffer consisting of 100 mm Tris ⁄ HCl
(pH 9.5), 50 mm MgCl
2
, 100 mm NaCl and 0.1% Tween-
20 for 2 · 5 min, and stained with 1 : 50 (v ⁄ v) Nitro Blue
tetrazolium ⁄ 5-bromo-4-chloroindol-2-yl phosphate in the
staining buffer. After the reaction, the embryos were
washed with NaCl ⁄ P
i
.
Acknowledgements
We express our thanks to Professor F. S. Howell,

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