Ovigerous-Hair Stripping Substance (OHSS) in an Estuarine Crab: Purification, Preliminary Characterization, and Appearance of the Activity in the Developing Embryos

14 Pages·1999·5.7 MB·English

by  M Saigusa

Reference: Bio/. Bull. 197: 174-187. (October I4W)
Ovigerous-Hair Stripping Substance (OHSS) in an
Estuarine Crab:
Purification, Preliminary
Characterization, and Appearance of the Activity
in the Developing Embryos
MASAYUKI SA1GUSA AND HIROSHI IWASAKI
Department of Biology, Faculty ofScience, Okuyama University, Tsushima 2-1-1
(General Education Buildings), Oka\amu 700-8530, Japan
Abstract. Ovigerous-hair stripping substance (OHSS) is Introduction
an active factor in crab hatch water (i.e., filtered medium
into which zoea larvae have been released). This factor After egg-laying, the embryos of intertidal and estuarine
crabs and indeed most decapod crustaceans are encased
participates in stripping off the egg attachment structures
(i.e., egg case, funiculus, and the coat investing ovigerous in a thick, protective capsule composed of two or three
layers; these capsules are then attached to the female's
hairs) that remain attached to the female's ovigerous hairs
ovigerous hairs through the funiculus and investment coat
after larval release. Thus this activity prepares the hairs for (Yonge, 1937, 1946; Cheung, 1966; Goudeau and Lachaise,
the next clutch of embryos. OHSS activity of an estuarine 1983). The capsule breaks open during or after embryonic
crab. Sesarma haematoc/ieir, eluted as a single peak on development, and hatching occurs (Davis, 1968, 1981;
molecular-sieve chromatography, but this peak still showed Saigusa, 1997).
two protein bands at 32 kDa and 30 kDa on SDS-PAGE. The funiculus and investment coat, as well as the broken
The two protein bands stained with a polyclonal antiserum egg capsule, remain attached to the hairs after hatching (for
raised to the active fractions from molecular-sieve chroma- further details, see Saigusa, 1994). But at the time ofhatch-
tography. Moreover, antibodies purified from this poly- ing, an active factor we call ovigerous-hair stripping sub-
clonal OHSS antiserum also recognized both the 32-kDu stance (OHSS) is released from the embryoandcauses these
and 30-kDa bands. OHSS immunoreactivity and biological remnant structures to slip offthe hairs (Saigusa, 1994). The
activity were associated with the attachment structures that stripping of these remnant structures is very important be-
remained connected to the ovigerous hairs afterhatching. In cause it leaves the hairs clean, unbroken, and thus prepared
developing embryos, both protein bands could be stained to incubate the next clutch of embryos (Saigusa, 1995).
immunochemically at least 10 days before hatching. But The funiculus. the coat that wraps the ovigerous hairs,
OHSS biological activity appeared only 3 days before and the outermost layerofthe egg capsule are all composed
hatching. The immunoreactive protein bands were not ob- of the same material (Saigusa ct <;/.. unpub. data), and
served in the zoea, but OHSS bioreactivity was present, OHSS may be an enzyme that acts on this material. In many
though greatly reduced. The 32-kDa protein, at least, is other animals, a hatching enzyme is released from the
probably an active OHSS, and the 30-kDaprotein band may embryos upon hatching and digests the layers of the egg
also be OHSS-related. The OHSS appears to be produced case (e.g., Yamagami. 1988; Lepage and Cache. 1989; Roe
and stored by the developing embryo. Upon hatching, most and Lennarz, 1990; Helvik et ai, 1991). However, there is
of the material may be trapped by the remnant structures, noevidence, even afterelectron microscopy studies, that the
and the remainder is released into the ambient water. glaeysetresd boyf OthHeSeSgg(Saciagpussualeete<n//c..asuinnpgublc.radbatae)m.bSroyotsheanroetidoin-
that OHSS is a crustacean hatching enzyme remains unsub-
Received 16 January 1997; accepted 16 June 1999. stantiated.
174
ACTIVE SUBSTANCE IN CRAB HATCH WATER 175
Although the cells that produce OHSS have not yet been and the remaining water was then passed again through a
identified, we do know that this substance is secreted by the filterpaper. The resulting hatch waterwas pooled in a 50-ml
-40C
embryo, and not by the female. The molecular mass of plastic bottle and immediately stored at until used.
OHSS was estimated by primary gel filtration to be 15-20 Most females incubate their next clutch of embryos a few
kDa (Saigusa, 1995), but no other chemical characteristics days after larval release. The females were therefore kept in
are known. If we are ever to characterize the physiological the laboratory for a month, and hatch water was obtained
mechanism ofOHSS. or its expression during development from their second larval release.
underthe control ofacircatidal clock (Saigusa. 1992. 1993, Since the OHSS activity of hatch water declines con-
1997), a purified preparation must first be obtained. stantly at ambient temperatures, the second method, in
Here we describe a relatively simple procedure for puri- which the material is frozen immediately after production,
fying OHSS from the hatch water ofan estuarine terrestrial yielded hatch water solutions with higher concentrations of
crab, Sesarma haematocheir, and we also provide a prelim- OHSS. Moreover, since all ofthe female crabs survived and
inary characterization of this material. A polyclonal anti- were able to incubate another clutch of embryos, we are
serum was raised against OHSS and purified so that, by confident that the disinfection in ethanol and the dry trans-
immunochemical staining, we could examine the appear- port were not deleterious to the animals.
ance ofOHSS in the developing embryos and its disappear- Most of the hatch water used in this study was collected
ance after hatching. Furthermore, we assessed whether in 1994. but some experiments were carried out with mate-
OHSS is still associated with the remnants ofthe embryonic rial collected in 1996-1998. The concentrations of OHSS
attachment structures that remain after hatching and larval obtained by the two methods would certainly have been
release. different, but the biochemical properties observed were
virtually identical.
Materials and Methods
OHSS
Larval release and collection ofhatch water Bioassay of activity
Specimens of Sesarma haematocheir, the estuarine ter- Our biological assay of OHSS is based on the ability of
restrial crab used in this study, were collected at Kasaoka, chemically fixed ovigerous setae to respond to OHSS. In
Okayama Prefecture, Japan. Here, the thicket inhabited by brief, an ovigerous seta with its attached embryos, all in
the crabs is separated from the shore of a tidal creek by a early stages of development, were excised from a female
small road (for their habitat, see Saigusa, 1982). Just after crab, fixed in 70% ethanol, and then stored in the refriger-
4C
sunset, between 1900 and 2000 h, for several days around ator at until used. Ethanol-fixed setae with their at-
the time ofthe full or new moon, large numbers ofoviger- tached clumps of embryos respond well to exogenous
4C
ous female appear onto this road on their way to the shore. OHSS. even after several years of cold storage at
Thus exposed, they can easily be captured. (unpub. data).
In 1994, more than 3000 females were captured on the Shortly before a bioassay was to be performed, the fixed
road and placed individually into large plastic containers setae were suspended in distilled water (DW) to wash out
(10 cm in diameter, 15 cm in height) containing 30 ml of the ethanol, and then placed in a glass dish with DW. The
very clean ground water obtained near the collecting site. tip ofeach ovigerous seta was cut away, and the remainder
The females released their larvae into this water. Immedi- was subdivided into four segments under the stereomicro-
ately thereafter, the females were removed from the con- scope (see Saigusa. 1995). Each segment was placed on a
tainers, and the waterwas filtered through a nylon mesh that paper towel to remove attached water, and one or two (or
retained the larvae. The filtered hatch water was accumu- three in some experiments) of these segments were placed
lated in 1-liter bottles, transported 50 km to the laboratory, in each of the wells (each 0.8 cm in diameter, 1.7 cm in
and finally frozen at 20C. height) of a plastic culture dish; the wells also contained
The method of collection was changed in 1996. In the 300-500 /J.1 of a fraction eluted through chromatography.
three years 1996. 1997. and 1998, ovigerous females were The culture dish was shaken slowly on a mechanical shaker
still collected at the same site at Kasaoka. but they were first for 1.0-2.2 h at about 23C.
disinfected in ice-cold 70%-80% ethanol forafew minutes, Recall that each seta is equipped with 10-15 whorls of
then washed with distilled water, and finally placed indi- ovigerous hairs to which the embryos are actually attached,
vidually in the large plastic containers, but without water. and that the number of ovigerous hairs is about 10-20 per
These containers were transferred to the laboratory, where whorl (see Saigusa, 1994, 1995). For example, each of the
each crab was immediately placed in a small, covered three segments ofa seta would contain 2-5 whorls (30-100
plastic cup (5 cm in diameter, 6 or 8 cm in height) contain- ovigerous hairs). Afterthe incubation described above, each
ing 9 ml ofdistilled water. As soon as larvae were released, setal segment with its cluster ofembryos was again placed
the zoeas were removed by filtration through nylon mesh, in a glass dish with DW. This dish was put under a
176 M. SAIGUSA AND H. IWASAKI
stereomicroscope, and tine forceps were used to pull the kDa), aldolase (39.2 kDa), trypsin inhibitor (20.1 kDa)
embryos gently away from the ovigerous hairs. The per- (Sigma Chemical Co.).
centage of hairs in each whorl that were stripped clean but
were still undamaged was calculated. The activity ofOHSS Effects oftemperature andpH
was usually taken as the mean of 3-10 whorls (one or two
segments); the standard deviation was also calculated. The active fractions from molecular-sieve chromatogra-
phy (fractions 6-8; Fig. 2A) were pooled and incubated for
4C
15 min at temperatures from to 100C, and were
Purification procedure immediately returned to 23C. Segments of the ovigerous
Stored, frozen, crude hatch water was thawed and centri- seta with their attached embryos were incubated with these
fuged at 18,000 rpm for 30 min at 4C to remove the solid solutions for 1 h at 23C, and OHSS activity was bioas-
pmaotwedreiral,s.lefTthoevehrnaitgchht,waatnedrcwenatsrisfautguerdataetd18wi.t0h00(iNpHm4f)o-r,S3O04 wsaeyreed.maIninatnaoitnheedraetxp4e'rCimeanntd, 2th3eCpoofolred0-ac1t2i0ve hf.raTcthieosnes
min. The supernatant contained no OHSS activity. The solutions were then bioassayed for 1 h.
precipitate was dissolved in 100 mM Tris-HCl buffer (pH The effects ofpH were examinedmsiMmilarly. Buffers used
9.0) and, at this stage, could also be stored at -20C. This for this studymweMre as follows: 100 Na-acetate (pHm3.M0
material was called "concentrated hatch water." OHSS was and 5.0; 100 Tris-HCl (pH 7.0 and 8.5); and 100
ppurroicfeideudrefsurtwheerreianlltchraereriesdteopsu,t wasithdeascrfaisbtedprobteelionw.liTquhied ganlyecqiunale-qNuaanOtHit(ypHof1e0a.c5)h.bAucffteirv,eafnrdactthieonOsHwSerSeamctiixveitdywwiatsh
chromatography system (FPLC; Pharmacia) in an experi- bioassayed for 1 h.
mental chamber with the temperature controlled at 4"C: In each experiment (A-C), assays were repeated three
protein elution was monitored at 280 rim. OHSS rapidly times with the same OHSS solution. The mean percentage
loses its activity during purification, so the following pro- of stripped hairs in 2-5 whorls of the hair was first esti-
cedures were completed within 12 h. mated, and the mean of three assays was estimated.
Step 1: Hydrophobic chromatography. Concentrated
hatch water (described above) was mixed with an equal Electrophoretic analysis (SDS-PAGE)
mM
qmuaMntity of20 Tris-HCl buffer (pH 9.0) containing 300 SDS (sodium dodecyl sulfate)-polyacrylamide gel elec-
Na,SO4. This medium was applied to a column con- trophoresis (SDS-PAGE) was carried out according to the
taining 10 ml of HiTrap-Octyl-Sepharose m4FMF (prepack method ofLaemmli (1970). Each fraction from gel filtration
cbuoflfuemrn,conPthaairnmiancgia3)00eqmuiMlibNraat2SedO4wi(tphH 290.0). TheTrciosl-uHmCnl wbraasnecon(cCeennttrraitceodnb1y0,paAsmsaigceont)h.roTughheanfilutletrreadfilmtartaetrioinalmewma-s
(wa2s emluMt/emdlwitahndaalinfelaorwgrraatdeienmtaionftaNiane2dSOa4t: 4-.80 mmlM//mimni)n. d5i%ssoSlDvSe,d i5n%an2-emqeuraclaqputaonettihtaynoolf,ly8siMs buurfefae,r 5[commpMosEitDioTnA:
Fractions of 10 ml were collected. (ethylenediaminetetraacetic acid), 5% sucrose, in 125 mM
Step 2: Ion-exchange chromatography. The active frac- Tris-HCl (pH 6.8)], and then kept at room temperature for
tions from step 1 were pooled (60 ml), and this sample was 30 min. The molecular mass markers employed were glu-
applied to an anion-exchange column (MONO-Q HR5/5. tamate dehydrogenase (55.6 kDa). aldolase (39.2 kDa),
prepack, Pharmacia: 0.5mXM5 cm). The column had been triosephosphase isomerase (26.6 kDa), trypsin inhibitor
pre-equilibrated with 20 Tris-HCl buffer (pH 9.0). and (20.1 kDa). and lyso/yme (14.3 kDa) (Sigma Chemical
the sample was eluted with the same solution. The flow rate Co.). The gels (15%) were transblotted onto PVDF (poly-
was 1.0 ml/min. and 2-ml fractions were collected. The vinyliden difluoride) membranes (Clear Blot Membrane P;
fractions from the void volume were pooled, and were Atto, Japan), and were stained with Coomassie brilliant blue
concentrated to 500 ju,l with an ultrafiltration membrane R-250 (CBB).
(YM 10, Amicon).
Step 3: Molecular-sieve chromatography. A sample of
Electrophoresis ofthe activefractions without
these concentrated active fractions (500 was fraction-
/u,l) denaturation {native SDS-PAGE) anil biotissuy with the
pateerddebxy7m5olHecRul1a0r/-3s0i,epvreecpharcko.maPthoagrrmaapchiay)(.geTlhfeilctroaltiuomn)n(hSaud- crushed gel
mM
been equilibrated previously with 20 Tris-HCl buffer The active fractions from gel filtration were pooled (2 ml)
mM
containing 150 NaCl (pH 9.0). The sample was eluted and concentrated to less than 200 jid by passage through the
with the same buffer at a flow rate of0.25 ml/min, and I-ml ultrafiltration membrane. This material (80 /xl) was dis-
fractions were collected. The molecular mass ofOHSS was solved in a solution containing 45 mg of saccharose added
M
determined by comparison with the elution volume of the in 100 n\ of 0.5 Tris-HCl (pH 6.8), and was electropho-
following marker proteins: glutamate dehydrogenase (55.6 resed on an SDS-polyacrylamide gel (15%). After electro-
ACTIVE SUBSTANCE IN CRAB HATCH WATER 177
phoresis, a narrow strip of this gel was cut parallel to the glycine-HCl (pH 2.0) for 10 min, on ice, to elute bound
direction of migration, and then stained with CBB. This antibody. The eluate was adjusted to pH 7.4-7.5 by the
M
strip was used to indicate the position ofthe protein bands. addition of 30-32 jul of I Tris.
The remainder of the gel was cut into four equal segments For staining, a 1:1000 dilution of the antibody that was
perpendicular to the direction of migration. Each of these eluted from either the 32-kDa or 30-kDa band was used as
gel segments was crushed with a pestle, 500 jul of DW was the primary; dilution was with 0.5% nonfat milk in T-TBS.
added, and the OHSS activity was bioassayed. The secondary antibody was peroxidase-conjugated goat-
rabbit immunoglobulin (Cappel) diluted 1:5000 with 0.5%
Preparation ofpolyclonal antiserum, electrophoresis, mul nonfat milk in T-TBS.
mm
western blotting ofOHSS Because the two bands were at most 2 apart on the
gel and were not stained with CBB, we could not be sure
onInSDprSe-lPimAiGnaEryweaxspecruitmefnrtosm, ath3e2g-eklDawiptrhotaeinshbarapndknsiefeen, fthoarte,thceotncweontprraotteeidnsachtaidvebefernacctoimopnlsetferloymsempoalreactueld.arT-hseireev-e
mixed with Freund's complete adjuvant, and injected into a chromatography (6 and 7 in Fig. 2A) were electrophoresed
c2omwmeeerkcsiaalpawrhtiteyriaeblbdite.dTnhoeseanttriesaetrmuemntswhefnourasisnjaeycetdionbsy, otrnan1sf5e%rreSdDtSo agePlVfDorFamleomnbgrearnpee.riTohdis(9prho)c,eadnurdewperroedutcheedn
westernblotting. Therefore,the pooledactive fractions from a gap of5-6 mm between the upper and lower bands. The
gel filtration (2 ml; 6 and 7 in Fig. 2A) were mixed with the whole membrane was stained with 0.01% Ponceau S
adjuvant, and two additional injections, 2 weeks apart, were (Sigma) in 5% acetic acid for 10 min. The protein bands, 2
givIenn wtoesttheernsabmleotrtaibnbgi,t.the electrophoresed sample was wmimde)wiwdaes,ewxecrleudceudt. Touhteabnoduntdheanmtiidbdoldeiesstwriepre(atbhoeunte2lutmedm,
transblotted onto PVDF membrane, and immunoreactivity
as described above.
was detected by chemobioluminescence with ECL western
blotting reagents (Amersham). For the primary antibody, Immunochemical detection ofOHSS during
the polyclonal antiserum was diluted 1:1000 with 0.5%
mM mM embryonic development
nonfat milk inT-TBS ( 10 Tris-HCl, 150 NaCl, and
0.05% Tween 20). The PVDF membrane was incubated At various times before hatching, an embryo cluster with
with the primary antibody for 1 h at room temperature, then its attached hairs and seta (i.e., one-third of an ovigerous
washed five times with T-TBS. The secondary antibody was seta altogether) was detached from a single female, crushed
peroxidase-conjugated goat anti-rabbit immunoglobulin with a pestle in distilled water (300 ;ul) for 5 min. and
(Cappel) diluted 1:5000 with 0.5% nonfat milk in T-TBS. denatured by the addition of 300 jid of the lysis buffer (pH
After a 1 h incubation with the secondary antibody at room 6.8). The solutionwascentrifuged at 15.000rpmfor20min.
temperature, the membrane was washed five times with Fifty microliters ofthe supernatant was pipetted, and 100 ^\
DW
T-TBS. and was further incubated (for 1 min at room of and 100 /ul of the lysis buffer were added to this
temperature) with ECL western blotting reagents. This supernatant. Thirty microliters of this solution was sub-
membrane was exposed to Hyperfilm ECL (Amersham). jected to electrophoresis, and OHSS was examined by the
immunochemical staining of western blots. The purified
Affinitv purification ofthe OHSS antiserum antibody was used in the experiments.
OHSS was also examined as follows. Embryo clusters
mtPraVotTDpohhgFeorrapemposeheomydlberd(oa6nnaaecnt1.id5v%Ae7fiSnrnaaDcrFStriigoo.gwnmesl2m,sAftr)raiopnwmdeorfmteohtlehceniocsnutclmreaaenrntmsrbfsaeirtreaervdnee,decehtlworeaocs--a dwattreeeonrlpaeyhtouccrrreeeunsdsethdrie.indfautgwhneeiddtlhtyhfsaeoirspOebH2su0tSflfSeemriiwnnda.e5ss0Tc0erhxie/abLnLme!diotnfaheebDdoWsvbue,ypeftarohnnredat1iiamhnm,mtmueenlwdeoaic--s-
then cut on both edges (5 in width), parallel to the chemical staining of western blots. The sediment was de-
dCiBreBctaionndowfermiegruasteidont.o Tinhdeisceatefilttheer sptorsiiptsiowneroefesatacihnepdrowtietihn Tnahteurseodlusteipoanrawtealsy bfuyrtthheeradcdeinttiroinfuogfedthefolrys2is0bmuifnf,erafnord 1thhe.
band; the membrane was notstainedwithCBB. except these supernatant was subjected to electrophoresis followed by
strips. immunochemical detection.
Two protein bands located with the CBB-stained tiller
strips, an upper (32 kDa) and lower (30 kDa), were cut Appearance ofOHSS activity in developing embryos:
separately from the membrane. Each band was further cut bioassa\ with crushed emhryos
into pieces and was incubated for 24 h in 1 ml antiserum at
4C. The pieces ofmembrane were then rinsed in PBS (pH One-third ofthe embryoclusters attached to an ovigerous
7.4) for 10 min, and this operation was repeated 5-6 times. seta were detached from a single female every day until
The membrane pieces were overlain with 200 jul of 0.2 M hatching, crushed in 500 /ul of DW, and then centrifuged at
178 M. SAIGUSA AND H. IWASAKI
15.000 rpm. The OHSS activity contained in these samples extracted with the detergent (Triton-X). Remnant matter
(i.e., the supernatant and sediment ofcrushed embryos) was (500 mg wet weight) was thawed and washed repeatedly
bioassayed. with DW. The remnants were DthWen crushed in 600 ju.1 of
Triton-X solution dissolved in (5%, 10%. and 20%),
Bioassa\ ofOHSS activity in broken egg cases and were held for 30 min at room temperature (23 C). Each
suspension was centrifuged for 20 min (15,000 rpm), the
eulmi,bArsaynoddnesitcchreiabtcetodaactehlmisenenvwethsestriynesgt(eSomavii(gig.uees.r,ao,busr1o9hk9ae4in)r,se)rgegrmecnmaaasnietnss,ofonufnittchh-ee ds1iu.s5phear(nneadatca2hn.t23hw0.a0s/xdl)i,viadneddOinHtSoStwaoctwieviltlyswoafsabpiloasatsiscayceuldtufroer
ovigerous hairs after hatching and larval release. These
remnants were removed with tine forceps after hatching,
and stored at -20C until used. Ovigerous hairs and setae Results
were notcontained in these remnants. So the possibility that Chromatography and estimation ofthe molecular mass
the OHSS activity is present in the ovigerous hairs and setae
of the female was excluded. Crude hatch water from about 90 females was saturated
Five hundred milligrams of the remnant matter (wet with (NH4)2SO4 overnight; no OHSS activity was found in
weight) was thawed and washed repeatedly with DW, and the supernatant. The precipitate was dissolved in 100 mM
centrifuged for 20 min (15,000 rpm). The sediment was Tris-HCl buffer. This solution (concentrated hatch water)
crushed in 600 of DW; the suspension was divided into was subjected to hydrophobic chromatography, and each
/n.1
two wells ofaplastic dish (each 300 /il), and OHSS activity fraction (10 ml) was bioassayed for 1.5 h.
was bioassayed for 1.5 h and 2.0 h. As shown in Figure 1, most proteins were removed by
We also determined whether the OHSS activity could be this fractionation. The peak of OHSS activity was very
2.0r
0.3
M
0.2
0.1
J
30 40 50
Fraction number
Figure 1. HlutinnofOHSSactivityfromahydrophobiccolumn(HiTrap-Octyl-Sepharose4FF;mPMharmacia).
Concentratedhatchwaterfrom90femaleswasappliedtoacolumnpreviouslyequilibratedwith20 Tris-HCl
buffer containing 300 m/M Na2SO4, and the proteins were eluted with a lineargradient: -8 mM Na:SO4/min
(broken line). Open circles (O): 280 nm absorption indicating protein concentration in each fraction (10 ml).
Bioassay: incubation time, 1.3 h; OHSS activity, percentage ofstripped, unbroken ovigerous hairs per whorl:
datapointsarcthe meanpercentagesof3-10whorls(Asolidtriangles);verticalbarsindicatestandarddeviation;
conltol assay(Abottom right),incubationwithbuffer(detailsofassay in Methods). Verticalarrowsindicatethe
6 active tractions (60 ml) applied to anion exchange chromatography.
ACTIVE SUBSTANCE IN CRAB HATCH WATER 179
broad, and only halfofit coincided with a protein peak that 20.1 kDa
eluted after mM Na2SO4 was reached. A bioassay with an
incubation of 1.0 h was also attempted; the results were 0.3 r
similar to those shown in Figure 1, although the peak of
activity was narrower than that with the longer incubation
(not shown).
The pooled active fractions from hydrophobic chroma-
jteocgtreadphtyo a(n6i0onml;exscihxanvgereticchalroamrartoowgsraipnhyF.ig.Th1e) OweHrSeSsuabc-- 0.2 - -1100 %
tivity appeared in the pass-through fractions (not shown).
These active fractions (60 ml) were concentrated to 500 4
by ultrafiltration and were then subjected to molecular-
/Lil >,
sieve chromatography. Each fraction was bioassayed for
1.5 h. As shown in Figure 2A, the activity appeared as a 0.1 50
single peak in fractions 6-8. The molecular mass of the
X
eluted protein peak was estimated at 35 kDa by comparison o
with standard proteins. Similar results were also obtained
with the solutions bioassayed for 1.0 h (not shown).
01 23456 78910
SDS-PAGE Conlro1
Fraction number
Each fraction eluted in molecular-sieve chromatography
(Fig. 2A) was concentrated, and the proteins were analyzed B 23Frac4tion5num6ber7
by SDS-PAGE. As shown in Figure 2B. two common 89
protein bands appeared in fractions 6 and 7, both of which
had high OHSS activity (Fig. 2A). Fraction 8. which also
had high OHSS activity, had little or no staining. The 55.6
molecular masses of these bands were estimated to be 32 39.2
kDa and 30 kDa by comparison with the marker proteins.
An additional band (22 kDa) appeared in fractions 5 and 6;
26.6
itis veryweak in Figure 2B, but isclearin Figure 5A. Itwas
clearthat the OHSS activity bioassayed with ovigerous setal
segments does not correspond to the 22-kDa band in frac-
tions 5 and 6 (compare Fig. 2A with Fig. 2B). 14.3
The concentrated active fractions (6 and 7) from molec-
kDa
ular-sieve chromatography were also electrophoresed with-
out denaturation; the proteins were clearly separated (Fig.
3A). The gel was cut into quarters, and each segment ofthe Figure 2. Purification ofOHSS activity by molecular-sieve chroma-
gel was bioassayed for 1.5 h and 2.0 h. The OHSS activity tography with protein analysis on SDS-PAGE. (A) The pass-through
ualpapreamraesdsibnetthweeesnec4o0ndkDsaegamnedn2t3(kb)Dao)f,twhheigcehlcsotrnitpai(nmeodletch-e bfryacutlitornasfil(t6r0atmioln),farnodmaanniaolniq-ueoxtch(a5n0g0e/cilh)rowmaastoagprplaipehdytwoermoelceocnucleanrt-rsaiteevde
chromatography; fractions are I ml. Open circles (O): protein concentra-
32-kDa and 30-kDa protein bands (Fig. 3B). tion in each fraction (280 nm absorption). Solid triangles (A): the OHSS
activityofeach fraction; bioassayscarriedout for 1.5 h withone (ortwo)
Characterization ovigerous setal segments perfraction; errorbars: standarddeviation. Con-
trol assay (A bottom right). Downward-pointing arrows indicate the mo-
Active fractions 6-8 from molecular-sieve chromatogra- lecular masses of marker proteins: glutamate dehydrogenase (55.6 kDa).
phy (Fig. 2A) were pooled, and the thermostability of the aldolase (39.2 kDa). and trypsin inhibitor (20.1 kDa). (B) Analysis, by
OHSS solution was examined. Active solutions that were SDS-PAGE. ofthe proteins ineach fraction (1-9)eluted from molecular-
exposed for 15 min to temperatures between 4C and 80C smieemvebrcahnroemawthoigcrhapwhays.TsthaeinpeodlywaictrhylCaomoimdaessgeilewbarsilltiraanntsbblloutet.edThtoeamPaVrkDeFr
showed virtually no decrease in OHSS activity. In contrast, proteins were glutamate dehydrogenase (55.6 kDa), aldolase (39.2 kDa),
solutions exposed to 100C lost activity (Fig. 4A). triosephosphate isomerase (26.6 kDa), and lysozyme (14.3 kDa). The two
Least-square regression lines fitted to the data (Fig. 4B) bandsthat appearin fractions6and7 (arrowstotheright) have molecular
indicated that active solutions incubated at either 4C or masses of32 kDa and 30 kDa.
room temperature (about 23C) for up to 70 h showed no
significant decrease in activity, and that there was no sig-
180 M. SAIGUSA AND H IWASAKI
B
1.5 h 2.0 h
100 r 100 r
55.6 I
80 - 80 -
39.2 I 40
26.6 60 -
23
40 40
14.3
--
kDa 12 kDa
20 20
abed abed
Figure 3. Distribution of OHSS activity in a polyacrylamide gel divided into four equal segments after
SDS-PAGE. (A)Thegel. Numbersattheleftofthesmall arrowsindicatethemolecularmassesofthestandards
(see Fig. 2B). The molecular mass limits ofeach gel segment are indicated by the large arrows. (B) Bioassay
with twoorthree ovigerous setal segments. Open bars indicate OHSS activity ofeach gel segment. Incubation
periods for the bioassay: 1.5 h and 2.0 h. Stippled bars: controls; the ovigerous setal segments were incubated
DW
in for 1.5 h and 2.0 h. Error bars: standard deviation.
nificant difference between the two experiments. Solutions bands on SDS-PAGE, and they appeared in fractions 6-8
maintained for longer times (100 and 120 h) decreased in (Fig. 5). These immunostained protein bands clearly corre-
activity in both experiments. sponded to the peak of OHSS activity in Figure 2A.
Figure 4C shows the pH dependency of OHSS activity. The antibodies that had stained the 32-kDa and 30-kDa
Afteran assay incubation of 1 h, the pH optimum was quite protein bands were purified by immunochemical affinity to
broad, about 7.0-11.0. determine the specificity of binding. As shown in Figure
6A, the antibodies eluted from the 32-kDa protein band on
Specificity ofantibodies and affinity purification SDS-PAGE stained the 30-kDa protein as well; and the
An antiserum raised from molecular-sieve chromatogra- antibodies eluted from the 30-kDa band similarly stained
phy (fractions 6 and 7) detected only two strong protein both the 30-kDa and 32-kDa proteins.
B
100- 100- 100-i
80- 80- 80-
S 60^ 60- 60-
w
w f
O 40- 40 -i 40-
20- 20- 20-
*
i i i i i i i 2i 4i 81012
20 40 60 80 100 20 40 60 8800 100 120 6
Temperature ( C) Time of incubation ( h ) pH
Figure4. Characteristics ofOHSS activity. (A) Heat stability. Active solutions were incubated for 15 mm
at each temperature, and OHSS activity was bioassayed for 1 h. (B) Effects ofprolonged incubation (up to 5
days). For each time, active solutions were maintained at either 4C (O) or 23C (A), and the activity was
bioassayedfor 1 h. (C) Dependenceofactivityon pH. Buffers: 100mMNa-acetate(): 100mMTns-HCl(A);
100 mM Gly-NaOH (O). The activity was bioassayed for 1 h. Broken line in (C) indicates the least-square
regression curve fitted to the data. Error bars: standard deviation ofthree experiments.
ACTIVE SUBSTANCE IN CRAB HATCH WATER 181
234Frac5tion6num7ber89 B 34Fra5ctio6n nu7mber
2 8 9
556
392
*
266
143-
kDa
Figure 5. Specificity of the polyclonal antiserum raised against OHSS. (A) SDS-PAGE of fractions
eluted from molecular-sieve chromatography. The polyacrylamide gel was transblotted onto a PVDF
membrane and stained with Coomassie brilliant blue. (B) Immunostaining ofthe PVDF membrane with a
polyclonal antiserum raised to OHSS. Numbers to the left show the molecular masses of the standards
showninFigure 2B. The intensely immunostainedbandsin fractions6-8havemassesofabout32 kDaand
30 kDa (arrows to the right).
In this experiment (Fig. 6A), however, the 32-kDa band ular sieve chromatography (6 and 7 in Fig. 2A) were elec-
was at most 2 mm apart from the 30 kDa band on the gel. trophoresed for a longerperiod (9 h), which produced a gap
In addition, the PVDF membrane was not stained with of5-6 mm between the upper and lower bands. The whole
CBB, except the narrow stripthat had beencut on both sides membrane was stained by Ponceau S, and each protein band
mm mm
(5 in width), because the antibodies were not bound to (2 in width) was subjected to affinity purification.
CBB-stained proteins. So these protein bands could have Again, the antibody raised from the 32-kDa band bound to
been incompletely separated. the 30-kDa band as well; and the antibody produced from
In Figure 6B, concentrated active fractions from molec- the 30-kDaband alsorecognizedthe 32-kDaband (Fig. 6B).
B
55.6
55.6-
39.2- 39.2 i a
26.6 26.6
14.3- 14.3
kDa kDa
Figure6. ImmunostainingwithantibodiesaffinitypurifiedfromSDS-PAGEbandsthathadOHSSactivity.
SDS-PAGEoftheproteinsfrom molecular-sievefractions6and7(seeFig. 2A) wascarriedoutforeither3.5h
(A) or9h (B), the longerelectrophoresis producing the greater separation between the active bands at 32 kDa
and 30kDa. Thebands werecutoutofthe PVDFmembranes, incubated with apolyclonal antiserum raisedto
OHSS,andtheboundantibodieswereeluted(seeMethods).Theseantibodies,obtainedfromthe shortandlong
electrophoresis,wereappliedtotwonewSDS-PAGEruns,bothof3.5h;thesefinalrunsareshowninthisfigure.
Immunostaining was effected by antibodies bound (a) to protein in the upper band (32 kDa), and (b) to the
proteininthelowerband(30kDa).ThestripstotheleftofthetwopanelswerestainedwithCoomassiebrilliant
blue (A) and Ponceau S (B); the numbers (with arrow) are the molecular masses of the standard molecules
(identified in Fig. 2B).
182 M. SAIGUSA AND H. IWASAKI
OHSS activity in the post-hatching remnants ofthe during development, these activities would appear. Immu-
embryo attachment system nochemical and biological observations were made.
After hatching, the broken egg cases, funiculus, and in- Immunochemistry. Embryo clusters (one-third of an
vestment coat remain attached to the ovigerous hairs. The ovigerous seta) were taken at successive times from a single
female picks these remnant attachment structures off the female and crushed; the suspensions were denatured with
ovigeroushairs, but this must occurafterthe OHSS released the lysis bufferbefore centrifugation. As shown in Figure 8,
with the hatch water has been greatly diluted in the estuary. the 32- and 30-kDa proteins became noticeable at least 2
We therefore examined the possibility that at least some weeks before hatching. The 30-kDa band was the stronger
OHSS is present in the remnant attachment structures them- of the two bands at 10 and 6 days before hatching; but its
selves. intensity declined and became very faint in embryos 4 h
The remnants were collected and stained with FITC- before hatching; and it disappeared completely in post-
conjugated, OHSS antiserum (Fig. 7). OHSS was clearly hatched zoeas. In contrast, the 32-kDa band appeared later
detected all over the remnants, including the prezoeal cuti- than the 30-kDa band, but it was still quite clear in embryos
cles. In contrast, the egg capsule, funiculus, and investment 4 h before hatching, and again was not detected in the
coat did not react to the FITC-conjugated OHSS antiserum post-hatched zoeas. In addition to the two bands of OHSS-
when the embryos were squeezed in the egg cases (not related protein, an immunoreactive band appeared at about
shown). 55 kDa, from 14 to about 2 days before hatching (Fig. 8).
When samples for electrophoresis and immunoblotting In another female, the supernatant and the sediment were
were prepared from remnants that had been crushed and denatured separately by the lysis buffer after the embryo
denatured, only the 32-kDa band was detected (Fig. 7, clusters had been crushed and centrifuged. In the superna-
right). tant (Fig. 9A). OHSS first appeared as a weak 32-kDa band
Remnants storedat 20C were thawed andcrushed, and 14 days before hatching. The 30-kDa band was noticeable
suspensions of this material (not centrifuged) were bioas- 10 days before hatching, and the amount increased abruptly
sayedfor 1.5 h and 2.0h. As shown inTable I, strong OHSS in the embryos collected 6 days before. This protein de-
was detected in this solution. In another experiment, the creased markedly just before hatching, but was still visible
remnants were thawed and crushed, and then treated with 4 h before hatching.
5%, 10%, and 20% Triton-X. After centrifugation for 20 On the other hand, the sediment contained broken egg
min (15,000 rpm), the supernatant was bioassayed for 1.5 h cases, funiculi, the investment coat, ovigerous hairs,
and 2.2 h and showed strong OHSS activity, particularly at prezoeal cuticles, and probably only a portion of the em-
the longer incubation (Table II). bryos. When this material was centrifuged and the superna-
Appearance ofOHSS in the developing embryos tkaDnta wbaasndexaamppienaerdedonwtehaeklsyameinftehmealelan(eFisg.de9rBi)v,edthefr3o0m-
Because OHSS immunoreactivity and biological activity embryos collected 10, 6, and 4 days before hatching; but it
occur in the remnants after hatching, we determined when. was absent at other times. In contrast, the 32-kDa band
55.6
39.2
32 kDa
26.6
Figure7. Immunochemica] stainingofthestructuresremaining attached toafemale'sovigeroushairsafter
hatching. Left: the iminunoblot of an extract of the remnants subjected to SDS-PAGE. Arrowhead: 32-kDa
protein band. Numbers to the left show the molecular masses ofthe standard molecules (as in Fig. 2B). Right:
the remnants stained with polyclonal FITC-conjugated OHSS antiserum. ci1: broken eggcase:/: funiculus;pc:
prezoeal cuticle; oh: female ovigerous hair (see fig. 2 in Saigusa. 1994). Scale: 100 /nl.
ACTIVE SUBSTANCE IN CRAB HATCH WATER 183
Table I
iatissiiv iffsuspensions ofcrushedattachment slniciu
ificr hatching
Incubation*