In vivo Incorporation of Labeled Methionine into Proteins, Vitellogenin, and Vitellin in Females of the Penaeid Shrimp Penaeus semisulcatus de Haan

6 Pages·1992·2.7 MB·English

by  S Shafir

Reference: Bio/, Bull 183: 242- 7. iOctober. 1992)
In vivo Incorporation of Labeled Methionine into
Proteins, Vitellogenin, and Vitellin in Females of the
Penaeid Shrimp Penaeus semisulcatus de Haan
SHAI SHAFIR1 2 MICHAEL OVADIA2 AND MOSHE TOM1
. ,
{Israel Oceanographic and LimnologicalResearch. P.O. Box 8030, Haifa. 31080. Israel, and2Tel
Aviv University, G. S. U'ise Faculty ofLife Sciences, Department ofZoology. TelAviv 69978, Israel
Abstract. ["S]-Methionine was injected into 1 1 intact Introduction
vitellogenic females ofthe penaeid shrimp Penaeus sem- Crustacean vitellin (Vt) is a lipoglycoprotein that ac-
isulcatusdeHaan. Levelsofradiolabeled methionine.total cumulates in the oocyte cytoplasm. Vt is used later for
protein and vitellogenin/vitellin (Vg/Vt) were measured the nutrition ofthe non-feedingembryoand larval stages.
in the hemolymph during 24 h following methionine in- Vitellogenin (Vg). a vitellin-immunoidentical protein, is
jections. The same parameters were measured 24 h after a female-specific protein found in crustacean extraoocytic
injection, in the hepatopancreas and ovaries ofsacrificed tissues and hemolymph (Meusy and Payen, 1988). Vg is
females. Proteins were precipitated by trichloroacetic acid produced by the subepidermal adipose tissue (SAT) of
and Vg/Vt was immunoprecipitated by anti-Vt serum. anostracanfVanBeekrtfl/.. 1987). isopod(Picaud, 1980;
Hemolymphaticproteinand Vg/Vt levelswereconstant Souty and Picaud. 1981; Picaud ct al.. 1989), and am-
throughout the 24 h ofthe experiment starting from the phipod (Junera and Croisille, 1980; Croisille and Junera,
firstsamplingofhemolymph. twohafterinjection. Similar 1980) crustaceans before it is transported by the hemo-
amounts of Vg/Vt were found in the hepatopancreas lymph into the developing oocytes.
compared to the ovary 24 h after injection, and 10% of Decapod Vg is present in the SAT (Meusy et al.. 1983;
Tom
the labeled protein in the ovary and 6.4% in the hepato- Vazquez-Boucard. 1985; et al.. 1987), hepatopan-
pancreas were Vg/Vt. Free-labeled methionine was still creas(Paulusand Laufer, 1987; Quackenbush and Keeley.
present in all tissues examined after 24 h. The labeled 1988;Quackenbush. 1989a, b; Fainzilber et al., 1992) and
protein and Vg/Vt in the ovary and the hepatopancreas hemolymph (Kerr. 1969; Cecalldi. 1970; Wolin ct al.,
could not be explained by the hemolymphatic content of 1973; Fyffe and O'Connor, 1974; Caubere et al., 1976;
thetwoorgans. ThehemolymphaticVgis24-fold labeled Dehn ct al.. 1983; Durliat, 1984; Marzari ct al., 1986;
over the ovarian Vt, 24 h after injection. The results in- Tom et t//.. 1987; Yano, 1987; Quackenbush, 1989a;
dicate more intense involvement ofthe hepatopancreas Shafir ct al.. in prep.). A major source ofdecapod yolk is
in the vitellogenic process than can be deduced from ear- Vt synthesis by the ovary itself(Lui ct al., 1974; Lui and
lier in vitro studies. This study confirms the earlier defi- O'Connor, 1976, 1977; Dehn ct al.. 1983; Eastman-Reks
and Fingerman, 1985; Yano and Chinzei, 1987; Quack-
nition ofthe oogenic stage ofrapid Vt accumulation in
theovary (AOD range of 150-250j/m) and also indicates ethnebsuisshi,n P1e9n89aae;usBjraopwodniycuctsawla..s1s9u9g0g)e.stTehdetosibtee tohfeVftolsliycnl-e
a role for the hemolymph in transporting Vg between its cells(YanoandChinzei. 1987). No Vgsynthesisoccurred
processing sites. in //; vitro incubated SAT ofPenaeussemisii/catus(Fain-
zilber ct al.. 1992); however, three studies have reported
Vg synthesis in the decapod hepatopancreas: Quacken-
ARbebcreeivvieatdio4nsF:ebAruOaDry 1A9v9e2r;agacecoeopctyetde2diJaumleyte1r9;92d.pm disintegrations bush and Keeley ( 1988) in L'ca pugi/ator; Quackenbush
perminute; Mw Molecularweight;SAT Subepidermaladiposetissue; ( 1989a) in Penaeus vannameiand Fainzilberctal. (1992)
TCA Trichloroacetic acid;Vg Vitellogenin; Vt Vitellin. in Penaeus semisulcatus.
242
SYNTHESIS OF YOLK. IN SHRIMPS 243
Vtendocytosisbytheovariesofseveraldecapod species, Materials and Methods
including penaeids has been reported by Beams and Kes-
sel. 1963; Hinsch and Cone. 1969; Wolin ct ai. 1973;
Duronslet el ai. 1975; Zerbib, 1979; Schade and Shivers, AdultsofPelicanssemisulcaluswerecollected in Haifa
1980; Zerbib and Mustel. 1984; and Jugan and Soyez, Bay, Israel. Thirty shrimp were held in a 3000-liter sea-
1985. Vg receptors were found and isolated from the oo- watertank. Watertemperature ranged from 18C(winter)
cyticcytoplasm membrane oftwodecapod species(Jugan. to 27C (summer). Water was replaced at a rate of300%-
1985; Laverdure and Soyez. 1988; Jugan and Van Herp. per day. Animals were fed daily with defrosted Anemia
1989). and a mixture ofdefrosted shrimp, fish, and squid. Fe-
The penaeid ovary isawell established majorsynthetic maleswere individually tagged byclipping their uropods.
siteofVtcompared totheminorquantitativecontribution Theiroogenic stage was monitored periodically by visual
ofthe penaeid hepatopancreas to Vg production, as was examination of the ovaries according to the method of
revealedby in vitroincubationsofpenaeid hepatopancreas Browdy and Samocha (1985). Ovarian developmental
and ovariesin the presence oflabeled amino acids. Yano stages were determined more accurately after sacrificing
and Chinzei (1987) found no Vg synthesis at all in P. the females, by measuring the average oocyte diameter
japonicushepatopancreasincontrasttoahigh Vtsynthesis (AOD) as described by Shlagman et al. (1986). Molting
in the ovary. Two additional studies, carried out in P. ofindividual femaleswasrecorded dailyby identification
vannamei (Quackenbush. 1989a) and in P. semisulcatus oftheclippedcodsonthe uropodsofthecollectedexuviae.
(Browdyetal., 1990; Fainzilberric?/.. 1992)revealedlower
rates ofyolk synthesis in the hepatopancreas compared Chemicals
totherespectiveovarian rates. TherateofVgproduction,
normalized per mg protein, in the hepatopancreas ofP. [35S]-Methionine, 300 mCi/mMol was purchased from
vannamei was around 10% ofthe ovarian value at early Amersham, U.K. All other reagents were of analytical
and mid vitellogenesis. An apparent increase ofthis per- grade.
centageto 50% at late vitellogenesis ofP. vannainci. isan
artifact caused by the normalization method, as the ac- [JfS]-methionine labeling andsampling
cumulation ofyolk in the ovary duringvitellogenesis adds oflabeledtissues
protein that does not contribute to Vt synthesis but re- Labeled methionine was injected through the thin cu-
duces the rate ofsynthesis per mg protein. A further re- ticle connecting the thorax and the abdomen using 1 ml
duction ofthe contribution ofthe whole hepatopancreas disposable syringes. Samples ofhemolymph from live in-
versusthewholeovaryisduetoitssmallerproteincontent. dividuals were collected from the same site with 1 ml
The Vg rate ofsynthesis in the whole hepatopancreas of syringes containing a premeasured volume of the anti-
P. semisulcatus reached 4.35% ofthe Vt rate ofsynthesis coagulant 10% tri-sodium citrate. Hemolymph from sac-
in the whole ovary. The percentages of synthesized Vg rificed females wascollected in a premeasured volume of
from total protein synthesis in the hepatopancreas were 10% tri-sodium citrate through an excision cut in the an-
also low in both P. vannamei and P. semisulcatus (1 and teriorpartofthecephalothorax, atthebaseoftheeyestalk.
14.4%. respectively) compared to similar percentages in Theovaryandhepatopancreasweredissectedfrom each
the ovary (8 and 40%, respectively). ofthe sacrificed females and weighed before a portion of
The small contribution of the //; vitro incubated he- the ovary was fixed in 4% formaldehyde in seawater for
patopancreasto Vgsynthesisshould be furtherestablished themeasurement ofoocytediameters. The remainingpart
by complementary //; vivo methods, having better resem- ofthe ovarywasthenweighed again. The hepatopancreas
blanceto the natural vitellogenic process. An appropriate and ovary from each of the females were homogenized
M
method for examination ofthe hepatopancreas share in separatelyin0.1 phosphatebuffer. pH 7.4. Hemolymph
yolk synthesis is the identification of Vg-mRNA in the samples and homogenized tissues were centrifuged and
penaeid hepatopancreasand ovary, astudyyettobedone. thesupernatantswereused fortheTCA precipitation and
Anotherapproach, theone taken by thepresent research, radioimmunoprecipitation (RIP).
includes determination of the presence of radioactively
labeled Vg and Vt in the hemolymph. hepatopancreas. TCA precipitation and measurement ojtotal
and the ovary of vitellogenic penaeid females following labeled methionine
injection oflabeled amino acid. It is aimed at validating
the overall involvement of the hepatopancreas and the Four 50 n\ aliquots taken from each supernatant were
ovary (not distinguishing between production and pro- pipetted onto Whatman 3 mm filterpaperdiscsanddried.
cessing ofVg/Vt) and at examining the dynamics ofcir- The proteins were precipitated onto two of the discs in
culating Vg. 10% TCA according to Mans and Novelli (1961), and all
244 S. SHAFIR ET AL.
fourdiscswere radioactivelycountedin scintillation vials Table I
with4 ml Aquasol-2 (NEN)inaKontron Betamaticliquid Incorporation of[35S]-methionineni/o TCAprecipitableproteinsand
scintillation counter. ]g/Vt in litehepatopancreasamiovary<>tPenaeussemisulcatus
24 halterinjection
Radioimmunoprecipitation (RIP)
Radioimmunoprecipitation wascarried out by mixing Parameters Ovary Hepatopancreas
25 ,ul samplesofeach supernatantwith unlabeled purified Total ['5S]-methionine
vitellin (15 /ug in 25 /il) and 200 /ul ofanti-Vt serum. The TCA precipitahle protein
anti-Vt serum was prepared and examined for Vt speci- tmmunoprecipitable Vt
ficity according to Browdy el al. ( 1990) and Tom el al.
(1992). Control samples contained normal rabbit serum
(NRS) instead ofanti-Vt serum. The total volume in the
M
reaction tube was adjusted to 0.5 ml with 0.4 NaCl
and was mixed thoroughly before overnight incubation
at 4C. Following incubation, samples wereMcentrifuged
and the pellets washed three times with 0.4 NaCl. Fi-
nally, each pellet was dissolved in 75 jul of0.5 A/ NaOH
by vigorous shaking for '/: h at rMoom temperature and
was neutralized with 25 ^1 of 2 HC1 before it was
counted.
Complete immunoprecipitation ofthe labeled antigen
was achieved with an adequate antigen-antibody ratio.
The determination ofsuitable amounts ofreactants was
carriedoutbyreactingfixedamountsofunlabeled Vtand
labeled sample with increasing amounts ofanti-Vt serum,
followed by washing and counting the pellets.
25
Figure 1. Twenty-tourhtimecoursepresentationof(A)hemolymph
total-labeled methionine, (B| labeled protein, and (C) labeled Vg nor-
malized bythecountsoftotal-labeled methioninein thehemolymph 2
h after injection (TC2h) in Penaem semisulcatiis. Vertical lines show
standarddeviations.
SYNTHESIS OF YOLK IN SHRIMPS 245
Table II
.Iirrdv'""'"I" =H) oflabeledproteins inthehepatopancreas i
ovarv10labeledproteinsfoundinan identicalhemolymph volume
in Penaeussemisulcatus
246 S. SHAFIR ET AL.
thedataofShafirc: i'ep.)whoshowedthatoocytes should have been seen in both the ovary and the hemo-
ofthissizeare > -nulating Vt. The reduced per- lymph.
m
centage ofIn1- the labeled protein found in In conclusion, evidence from this and previous studies
thepreset e vitellogenesis(4.4%, Fig. 2)when supports the hypothesis that the hepatopancreas has a
combin slow increase ofaccumulated Vt in moreintenseinvolvement inthevitellogenicprocessthan
AOD
ovaries ime range (Shafir et a/., in prep.) could be deduced from earlier in vitro studies, but is not
indii 'iion of Vt accumulation in the ovary of necessarily the only site of Vg synthesis. The rapid ac-
the intact female at AOD > 250 /urn. In contrast, ovaries cumulation ofVtthatoccursearly in vitellogenesis(AOD
incubated in vitro showed no inhibition of Vt synthesis range of 150-250 /urn) and the arrest ofVt accumulation
at any stage of vitellogenesis although a decrease in the later (AOD > 250 urn), first found by Shafir et al. (in
percentageofsynthesized Vtwasobservedat AOD> 350 prep.), isconfirmed by the present findings. Furthermore,
^m, presumably due to increased synthesis ofother pro- the present study shows that circulating Vg is not a de-
teins (Browdy el a!., 1990). graded Vt which issecreted unselectively from the ovary.
The amount of labeled Vg and its percentage of the
total labeled protein in the hepatopancreas were similar Acknowledgments
to the corresponding values of labeled Vt in the ovary
after the 24 h experiment (Table I and Fig. 2). These la- Wethank Ms. Alisa Hadani forherdedicatedtechnical
beled Vg/Vt amounts can not be explained by the hem- assistance. This study was supported by the Israeli Min-
olymphaticVgcomponentofthetwoorgansalone(Table istry ofEnergy and Infrastructure through their support
II). Theyareprobablytheresultofanequilibrium between ofresearch projects in mariculture at IOLR and also by
theinflux ofcirculating Vgand thecontribution ofinsitu grant No. 1-1435-88 to Dr. M. Tom from the U. S.
produced Vg and Vg secretion during the 24 h of the Israel Binational Agricultural Research and Development
experiment. These findings strongly suggest that the he- Fund (BARD).
patopancreashasa moreprominentroleinthevitellogenic
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