Two Kinds of Active Factor in Crab Hatch Water: Ovigerous-Hair Stripping Substance (OHSS) and a Proteinase

7 Pages·1996·2.4 MB·English

by  M Saigusa

Reference: Bid. Bull. 191:234-240.(October, 1996)
Two Kinds of Active Factor in Crab Hatch Water:
Ovigerous-Hair Stripping Substance (OHSS)
and a Proteinase
M. SAIGUSA
Department ojBiology, FacultyofScience, Okayama University, Tsushima2-1-1.
Okayama 700, Japan
Abstract. The embryos of intertidal and estuarine nism underlying the timing of hatching is not known.
crabs are clustered on the ovigerous seta ofthe female, Tosettle this problem, the processofhatchingshould be
where they are ventilated for 2-4 weeks by the female's studied.
abdomen. When the embryonic development is com- A number of investigations have indicated that the
plete, hatching occurs and zoea larvae are released into embryossecreteaproteinase upon hatching(forreviews,
the water. This study indicates that the crab hatch water see Davis, 1981; Yamagami, 1988). These proteinases
(i.e., the filtered medium intowhich zoeaswere released) are thought to dissolve a portion ofthe egg capsule, thus
contains at least two kinds of active substance: OHSS rupturing it. Some ofthese proteinases have been puri-
(ovigerous-hair stripping substance) and a proteolytic fied and characterized: e.g.. in the sea urchin blastula
enzyme. Both factors were separated by gel filtration. (Barrett and Edwards, 1976; Lepage and Cache, 1989;
Powdered fragmentsofeggcapsuleweredigested bypro- Roe and Lennarz, 1990), fishes (Yamagami, 1972; Ha-
teinase, suggesting that this enzyme actually acts on the genmeier, 1974; Yasumasu et ai, 1989a, b), and am-
eggcapsule. But thisactivitywasat avery lowlevelcom- phibians(Carroll and Hedrick, 1974;Katagiri, 1975).
pared with casein digestion. The proteinase might be di- On the other hand, little is known about the hatching
gesting the thin, sticky layer enclosing the embryo and mechanism in Crustacea. Hatching ofcrustaceans usu-
would not act on the thick, tough layer constituting the ally occurs upon the rupture ofthe egg capsule. Many
main component ofthe egg capsule. Therefore, a prote- investigations have suggested that this is due to an in-
olysisofsuch lowactivitycould notbeexpectedtocause crease of internal pressure caused by osmotic effects
theeggcapsule to rupture. (Yonge, 1937, 1946; Burkenroad, 1947; Marshall and
Orr, 1954; Davis, 1959, 1964; Anderson and Rossiter,
1969), ortotheswellingoftheembryos(Saigusa, 1992b).
Introduction Recently, De Vries and Forward (1991) reported that
Embryosofintertidalandestuarinecrabsareclustered the embryosofa few estuarine crabs release a proteinase
on the ovigerous setae ofthe female and incubated there upon hatching. But in other experiments, the egg cap-
for 2-4 weeks. When the embryonic development is sules after hatching showed no sign ofdissolution (Sai-
complete, hatching occurs and zoeas are liberated into gusa, 1992b). So the role of such a proteinase in the
(tShaeigwuastae.r1b9y82t)h.eTshpeecitailmifnagnnoifnhgabtechhaivnigoirs coofntthreolfleemdablye hatAchfiunrgthoefrdqeuceasptoidoncrisusrtealcaetaendstroeamnaoitnhseorbascctuirvee.factor
the circatidal clocks ofboth the female and the embryos found in crab hatch water. Hatch water(i.e., the filtered
(Saigusa, 1992a, b, 1993), but the physiological mecha- medium into which zoeas have been released) contains
an active substance that causes each ovigerous hair to
slipoutoftheinvestmentcoatthatbindsittotheembryo
Received 31 March 1994;accepted2August 1996. through the funiculus. The embryos is thus lost without
234
ACTIVE FACTORS IN CRAB HATCH WATER 235
hatching (for details, see Saigusa, 1994. 1995). A ques- solution, and 0.2 ml ofthe enzyme solution. This mix-
tion iswhetherthisfactor,which I call OHSS(ovigerous- ture was incubated for 30 min at 30C, and was precipi-
hairstrippingsubstance), isthe proteinase. tatedthrough theaddition of 1.2 ml of5% TCA (trichlo-
I also report herethatcrab hatch watercontainsapro- roacetic acid). In control experiments, TCA was added
teinase. With gel filtration chromatography, the proteo- to the 1% casein solution before the enzyme solution.
lyticactivity iseluted in fractionsdifferent from thoseof The incubation mixture was then centrifuged at
OHSS. The proteinase certainly dissolves the debris of 10,000 rpm for20 min,andtheabsorbanceofthedepro-
the egg capsule, but the activity is very weak. Conse- teinized supernatent was measured at 280 nm.
quently I have concluded that this proteinase does not
dissolvethe main componentsoftheeggcapsule.
Assaywith debrisoftheisolatedeggcapsule
Materialsand Methods
Clusters of premature embryos (identified by their
Hatch watercollection browncolor;e.g., seeSaigusa, 1993, 1994)weredetached
from the females and folded into a sheet ofnylon mesh.
OvigerousfemalesofSesarmahaematocheirthatwere Theywerecrushedandwashed repeatedlywithtapwater
expected to hatch within a fewdayswere collected from to remove any embryonic tissue and yolk remaining in-
the field at Kasaoka, Okayama Prefecture, and brought side ofthe egg capsule. The coarse fragments ofthe egg
to the laboratory. They were dipped into 50% EtOH for capsule remaining on the mesh were suspended in dis-
a few minutes, for disinfection, and were individually tilled water for one night at 4C. Broken ovigerous setae
put into beakers (8.5 cm in diameter, 12 cm in height) at the bottom of the glass beaker were removed. After
containing 30 ml ofdistilledwater. lyophilization, thedried sampleswere furthercrushed in
Hatchingofestuarine crabs is highly synchronized; in a mortarand stored at 20C.
5. haematocheir, all zoeas hatched within 5-30 min. As The powderofthe eggcapsuledebris(30 mg)wassus-
soonashatchinghadbeencompletedandthefemalehad pended in 20 ml of 100 mA/ Tris-HCl buffer (pH 8.5),
released all herzoea larvae into the medium, shewas re- and this suspension was used as a substrate in the assay
moved, and the medium was filtered through nylon of proteolytic activity. The assay mixture contained
mesh to remove larvae. This filtered medium (i.e.. hatch 0.1 ml of Tris-HCl buffer, 0.2 ml ofthe substrate, and
water)wasaccumulated in the plastic bottlesand centri- 0.1 ml ofthe enzyme solution. This mixture was incu-
fuged at 15,000 rpm for 30 min to remove solid materi- bated for 1.5 h at 30C and precipitated by the addition
als. The hatch water was then lyophilized, and the pow- of5% TCA (0.6 ml). In thecontrolexperiment, 5% TCA
der was suspended in 10 mA/Tris-HCl buffer (pH 8.5). was added to the substrate before the enzyme solution.
These sample solutions (concentrated hatch water)were These mixtures were centrifuged at 10,000 rpm for
centrifuged at 15,000 rpm for60 min,andwerestoredat 30 min, and thesupernatant was measuredat 270 nm.
-20C
until used.
OHSS
Assayof
Gelfiltrationchromatography
The sample solutions were applied to a column of The activity ofOHSS was assayed on clusters ofem-
Sephacryl S-200 (1.3 X 45 cm) previously equilibrated bryos detached from ovigerous females. The ovigerous
with 10 mA/Tris-HCl buffer(pH 8.5). The flow ratewas seta with their premature embryos were separated and
5 ml per hour, and elution ofthe proteinswascalibrated cut into 4-6 pieces. Each cluster ofembryos was placed
with blue dextran and NaCl. The proteins contained in in a well of a plastic culture dish and incubated with
each fraction was measured at 280 nm. These experi- 0.4 ml ofthe solution that had eluted upon gel filtration
mentswereall carried outat4C. chromatography. After 2 h ofincubation at about 25C,
theembryoswere transferred to aglassdish with a small
Caseinassay quantity ofdistilled water, then gently pulled with a fine
forceps underthestereomicroscope.
Proteolytic activity was assayed with casein (Zwilling Theratioofthe numberofovigeroushairsthatslipped
and Neurath, 1981). Thesubstratewas 1% casein (Ishizu from the investment coat without breaking, to the total
SeiyakuCo.,Japan)thatwassuspended in 100 mA/Tris- numberofhairs on the seta wasestimated foreach clus-
HCl buffer(pH 8.5) and heated for 10-15 min in a boil- ter of embryos. The activity of OHSS was determined
ing wmatMer bath. The assay mixture contained 0.2 ml of with respect to the 45% response level (ED50): i.e.. the
100 Tris-HCl buffer (pH 8.5), 0.4 ml of 1% casein valueat which thedose-response curve intersectsthede-
236 M. SA1GUSA
fined 45% line (for further details ofthe assay, see Sai- minedwithgel filtrationchromatography. Butthisactive
gusa, 1995). factor could be a product ofthe enzyme-substrate reac-
tion, since hatch wateristhe medium into which the zo-
eas were hatched and released. The proteinase could be
Results
combining with substrate in the egg capsule (or on the
Elution ofproteolyticactivity surface ofthe embryos) to form conjugated proteins. So
further investigations will be required to determine the
Concentrated hatch water(1.5 ml; about five females) molecularsizeofthisenzyme.
was fractionated on the Sephacryl S-200 column with Hatch water was also collected from other species (S.
mM
10 Tris-HCl buffer, and the proteolytic activity of pictiini. S. dehaani, and Hemigrapsitssanguineus), sub-
each fraction was examined. As shown in Figure 1, the jectedtothesamegel nitration protocol aswasusedwith
proteolytic activity eluted near the void volume of the S. hacnialochcir, and assayed for proteolytic activity in
column. Fractions in the latter halfshowed noactivity. the same way. Proteolytic activities were detected in the
The molecular size ofthis proteinase should be deter- hatch water from all ofthe species, suggesting that the
2.0
-i
1.5-
ACTIVE FACTORS IN CRAB HATCH WATER 237
2.0n
,5J 150n
O! i.o-
00
CNI
--*
ft
0.5
in
.Q
0-
10 20 30
Fraction number
Figure2. ElutionpatternofOHSSactivityongelfiltrationchromatography.Concentratedhatchwater
(1.5 ml;5females)wassubjectedtothesamegelfiltrationprotocolshowninFigure 1.Clustersofembryos,
freshlydetachedfromthreefemales,wereincubatedwitheachfractionfor2hatabout25C.OHSSactivity
( ):Absorbanceofprotein(O O).
proteinaseoccurswidelyinintertidalandestuarinecrabs fore, at least two kinds ofactive factors are contained in
(data not shown). crab hatch water, and both factorsoccurwidely in inter-
tidal andestuarinecrabs.
OHSS
Elution oj activity
Assaywith egg-capsuledebris
Concentrated hatch water(1.5 ml; about five females)
was subjected to the same gel filtration protocol used in I next asked whether the hatch water would dissolve
the experiment ofFigure 1. The activity ofeach fraction the eggcapsule, and ifso, whetherOHSS and proteinase
was assayed with unhatched embryos of Se.sanna couldberesponsiblefortheactivity.Theassaywasthere-
haenwtocheir for 2 hours (Fig. 2).The activity ofOHSS fore performed with the powdered debris ofthe egg cap-
extends over a wide range of fractions. As shown else- sule as substrate. Hatch water (1.5 ml; about five fe-
where (Saigusa. 1995), the molecular size ofOHSS was males) that had been lyophilized and suspended in
estimatedtobe 15-20 kDabyacomparison ofitselution lOmA/Tris-HCl buffer (pH 8.5) was subjected to the
volumewith those ofthe standard proteins. same gel filtration protocol used in the experiments
The pattern ofOHSS activity on gel filtration (Fig. 2) shown in Figures 1 and 2.
isclearlydifferentfromthatofproteinase(Fig. 1). There- The debris of the egg capsule was dissolved by the
238 M. SAIGUSA
2.0-,
1.5-
E
c
O
0.6-
E
c
O
OO
.o
1O1 0)
o
0.5- -0.05
t/i
O)
O)
0-
10 20 30 50
Fraction number
Figure 3. Dissolution ofegg capsule debris by the proteinase. Concentrated hatch water (1.5 ml; 5
females)wassubjectedtothesamegelfiltrationchromatographyshowninFigure 1.Thesubstratesolutions
containingeggcapsuledebrisor 1%caseinwereassayedwitheach fraction. Bothsolutionswereincubated
for 1.5 hat 30C. Absorbanceofprotein (O. . . .O);proteolyticactivity(A A);digestionofegg-cap-
suledebris(D D).
hatch water(Fig. 3). The activity eluted asa single peak, troanilide (BValGlyArgNA); the method was that of
and its pattern clearly agrees with that ofthe proteolytic Grant et at. (1981). BValGlyArgNA was dissolved in
activity (Fig. 1). and not with OHSS (Fig. 2). Thus the 50 mAl Tris-HCl buffer (pH 8.5) to a concentration of
eggcapsuledebrisisprobablydigested bytheproteinase, 1 mg/ml ( 1.7 mAm/)M. The solution was further diluted 20
but notbyOHSS. timeswith 100 Tris-HCl buffer(pH 8.5). The assay
mixture contained 200 ^1 ofthis diluted substrate solu-
Someotherexperiments tion and 200 ^' ofthe enzyme solution. The amount of
/>-nitroanilide liberated was monitored continuously at
To determine whether a chitinase is contained in the 385 nm for4-5 min. Concentrated hatch water (1.5 ml;
hatchwater,degradationofcolloidalchitin(i.e., ahomo- five females) wassubjectedtothe samegel filtration pro-
polymer of ,8-linked TV-acetylglucosamine) was exam- tocol used in Figures 1 and 2.
ined. The assay method followed was that of Bade and This substrate (i.e.. BValGlyArgNA) was also decom-
Stinson (1979). In addition, the release of/7-nitrophenol posed by hatch water. The pattern ofthisactivity on gel
from 0.1 mjU/7-nitrophenyl J?-acetoamide-2-deoxy-/3-D- filtrationwasthesameasthatshowninFigure2,suggest-
glucopyranoside (pNP-0-GlcNAc) solution. The assay ing that BValGlyArgNA is decomposed by the protein-
methodwasthatofDziadik-Turneretat. (1981). Neither ase, not by OHSS(data not shown).
7V-acetylglucosamine nor ;>nitrophenol was released
from these substrates, suggesting that a chitinase is not Discussion
contained in crab hatch water(data notshown).
Proteolytic activity was also assayed with an amidase This study indicates that the hatch water ofthe estua-
substrate, i.e., jV-benzoyl-L-valylglycyl-L-arginine-/>-ni- rine terrestrial crab Sesarma haematocheir contains a
ACTIVE FACTORS IN CRAB HATCH WATER 239
proteolytic enzyme (Fig. 1) in addition to OHSS (i.e., Grant-in-Aid forScientific Research (C) from the Minis-
ovigerous-hairstrippingsubstance) (Fig. 2). Both factors try of Education, Science and Culture, No. 06839017
wereseparated bygel filtration. The proteinasedissolved and No. 08833009 (Marine Biology).
theeggcapsuledebris, buttheabsorbanceat 270 nmwas
very low, even at itspeak, comparedwith thedissolution
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