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JEZ 797
THE JOURNAL OF EXPERIMENTAL ZOOLOGY 278:339–348 (1997)
Isolation and Characterization of the High-Density
Lipoproteins From the Hemolymph and Ovary of
the Penaeid Shrimp Penaeus semisulcatus (de
Haan): Apoproteins and Lipids
E. LUBZENS,1* T. RAVID,1,2 M. KHAYAT,1,2 N. DAUBE,1 AND A. TIETZ2
1
Israel Oceanographic and Limnological Research, National Institute of
Oceanography, Haifa 31080, Israel
2
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel
Aviv University, Tel Aviv 699878, Israel
ABSTRACT
The high-density lipoproteins (HDLs) found in the male and female hemolymph
of Penaeus semisulcatus de Haan were isolated by NaBr (1.22 g/ml) followed by sucrose gradient
(5–25%) ultracentrifugation. The male HDL contained one protein, lipoprotein 1 (LP1), composed
of one 110-kDa peptide subunit. The female HDL contained two proteins: 1) the LP1 that was
immunoidentical to the male LP1 and was similarly composed of one 110-kDa peptide subunit and
2) vitellogenin (Vg), reacting positively with the rabbit antiserum generated against vitellin (Vt)
that was isolated from vitellogenic ovaries. Both Vg and Vt consisted mainly of three polypeptide
subunits (200, 120, and 80 kDa) as revealed by denatured PAGE and Western blot. The LP1 from
males or females did not react with the Vt rabbit antiserum. Similarly, Vg and Vt did not react
with the rabbit antiserum prepared against LP1.
Phospholipids (PL) constituted 71–76% of the total lipids in the hemolymph and HDLs of both
male and female hemolymph. Cholesterol (Ch) amounted to 17–20% , and small amounts (5%) of
diacylglycerols (DAG) were also carried by these HDLs. Both the PL and DAG contained highly
unsaturated fatty acids (20:5 ω3 and 22:6 ω3) that are transported from the food or hepatopancreas to the tissues, including the vitellogenic ovaries in females. In the present study we show for
the first time the separate lipid composition of female LP1 and Vg and compare them with the
lipids attached to the Vt. Vg had a lower lipid content than LP1 (540 and 1089 mg/g protein,
respectively). Differences were also found in the relative abundance of PL, Ch, and DAG classes in
the LP1 in comparison with Vg. Furthermore, small amounts (~3.8%) of triacylglycerols (TAG)
were found only in the hemolymph of vitellogenic females, and they were associated with the Vg.
Although Vg and Vt were composed of similar polypeptides, their lipid composition was different.
Vt, in contrast to Vg, carried considerable amounts of TAG (~22%) and only trace amounts of
DAG. The significance of the TAG in the hemolymph of vitellogenic females is not known, and the
functional relationship between Vg and Vt requires future extensive studies. Lipids were not detected in hemocyanin that was purified from clotted hemolymph. J. Exp. Zool. 278:339–348, 1997.
© 1997 Wiley-Liss, Inc.
Yolk is the nutritive material accumulating in
substantial quantities in the ooplasm of developing
oocytes and is intended to meet the basic requirements of embryonic development, independent of
the maternal organism. It is accepted that yolk proteins provide the basic structural material needed
for tissue buildup, whereas lipids, especially the
neutral lipids, serve as the major fuel and a source
of essential fatty acids. The main protein fraction
of the crustacean egg proteins is a high-density
lipoprotein/lipoglycoprotein frequently associated
with carotenoid pigments and usually referred to
© 1997 WILEY-LISS, INC.
as lipovitellin. In addition to lipovitellin, lipid
spheres or globuli have been observed within the
developing oocytes (Adiyodi and Subramonian, ’83;
Charniaux-Cotton and Payen, ’88).
The ovary increases in weight during oocyte development, reaching 7–15% of the total fresh
weight in females of Penaeus semisulcatus de
*Correspondence to: E. Lubzens, Israel Oceanographic and Limnological Research, National Institute of Oceanography, P.O. Box 8030,
Haifa 31080, Israel.
Received 10 June 1996; accepted 30 January 1997
340
E. LUBZENS ET AL.
Haan (Shlagman et al., ’86). This increase is associated with the accumulation of proteins and lipids. Protein content in the ovary of Penaeus
semisulcatus increases from 1 to 12 mg/g fresh
body weight (Shafir et al., ’92). Lipids constitute between 18–41% of the dry weight of the
mature ovary in various crustaceans (Teshima
and Kanazawa, ’83; Castille and Lawrence, ’88;
Lautier and Lagarrigue, ’88; Harrison, ’90). In
most species, phospholipids (PL), triacylglycerols
(TAG), and sterols are the most abundant lipid
classes in the ovaries. The fatty acid composition
of PL and TAG showed an abundance of highly
unsaturated fatty acids (HUFA) including 20:5 ω3
(EPA) and 22:6 ω3 (DHA), supporting the assumption that HUFA are necessary for vitellogenesis
in penaeid shrimp (Middleditch et al., ’80; Teshima
et al., ’89). Furthermore, the so-called “quality” of
penaeid shrimp eggs is usually correlated with
their ω3 HUFA content (Middleditch et al., ’80;
Cahu et al., ’86, ’94; Lytle et al., ’90).
The origin of the lipids reaching the crustacean
ovary and the mode of their transport in the
hemolymph are not fully known. The hepatopancreas (HEP) was suggested to operate as a metabolic juncture for lipids coming in from the gut
(Teshima and Kanazawa, ’80a, b; Lee and Puppione, ’88). In several cases, it was shown that
these lipids are stored in the HEP and transported
to the ovary during egg development (Castille and
Lawrence, ’88; and a review on this topic by
Harrison, ’90). Teshima et al. (’88) showed that
intensive labeling of the HEP lipids occurred after female shrimp were fed a diet supplemented
with [9,10-3H]palmitatic acid and [1-14C]linolenic
acid. These labeled lipids were transported to the
ovaries in the group of females that were subjected
to eyestalk ablation, which led to acceleration of
ovarian development. Teshima et al. (’86a, b) also
showed that female shrimp doubled their food consumption, further supporting that the lipids accumulating in the ovary originate in the food.
Lipids, being insoluble in water, are transported
in the hemolymph as lipoproteins (LPs). Three
LPs were isolated from crustacean hemolymph;
all belong to the high-density lipoprotein (HDL)
and very high density lipoprotein (VHDL) classes
(Lee and Puppione, ’78, ’88; Teshima and Kanazawa, ’80a, b; Puppione et al., ’86; Spaziani et al.,
’86; Lee, ’90; Komatsu and Ando, ’92; Komatsu et
al., ’93; Chen and Chen, ’94; Hall et al., ’95). One
of them is the female-specific vitellogenin (Vg),
the presence of which is correlated with ovarian
development. The other LPs, lipoprotein 1 (LP1)
and VHDL, are common to both sexes. Spaziani
et al. (’86) found two LP1s, one belonging to the
HDL2 and the other to the HDL3 density class.
Both had similar subunit profiles on SDS-PAGE,
suggesting that these two proteins serve as a reusable lipid shuttle transporting lipid from the
HEP to other tissues (Lee and Puppione, ’88) with
the HDL2 being loaded and HDL3 being the unloaded phase of the same LP, in a manner similar
to that found in insects. Komatsu et al. (’93) suggested that VHDL was involved in lipid transfer
reaction between lipoproteins in crustaceans. Hall
et al. (’95) showed that HDL and VHDL were identical to two proteins previously studied for their
role in immune recognition and clotting in the
crayfish Pacifastacus leniusculus. Recently, Spaziani et al. (’95) argued that there are no femalespecific proteins in crustaceans.
In the present work we isolated the high-density lipoprotein (LP1) found in both the male and
the female hemolymph of adult Penaeus semisulcatus (de Haan) and the Vg specific to females.
We also isolated the vitellin (Vt) from vitellogenic
ovaries. We determined for the first time, for each
of the lipoproteins, their apoprotein profiles, lipid
classes, and fatty acid composition. The results
presented here and in Ravid (’94) and Ravid et
al. (in preparation) will permit the construction
of a model explaining the transport mechanism
of lipids into maturing ovaries in this commercially important species. Furthermore, this information will be relevant in future determinations
of evolution and comparative trends with other
invertebrate groups, including insects, which are
also arthropods.
MATERIALS AND METHODS
Animals
Marine shrimp (Penaeus semisulcatus de Haan)
caught in Haifa Bay, Israel, were kept in 3-m3
tanks in running sea water (salinity 400/00; three
exchanges per day) and fed each morning with
food pellets (prepared by Dr. George Kissil, National Center for Mariculture, Israel Oceanographic and Limnological Research, Eilat, Israel)
and in the afternoon with bait shrimp and squid.
Water temperatures ranged between 19–21°C
year-round. Under these conditions, females produced vitellogenic ovaries all year round.
Determination of oocyte diameter
The stage of oocyte maturation was determined
in all females used. A piece of tissue was removed
HDL APOPROTEINS, LIPIDS, SHRIMP
from the ovary of each female and fixed in 4%
formalin in seawater, and the average oocyte diameter (AOD) was determined (Shlagman et al.,
’86). Previtellogenic females were those with oocytes of 100 µm or less in diameter.
Isolation and purification of proteins
Isolation of high-density lipoproteins (HDL)
Hemolymph was collected, after cutting off the
anterior part of the cephalothorax, near the base
of the eyestalks, into a 10% sodium citrate solution or alternatively was allowed to clot at 4°C
for 1–2 hr. Protease inhibitors were added immediately after collection of hemolymph to a final
concentration of 1 mM phenylmethylsulphonylfluoride (PMSF) and 1 µg/ml aprotinin. Hemolymph samples from several individuals were
pooled (separately for males or females) and centrifuged (10 min at 9,000 × g in a Sorval 5-CB,
Dupont, USA) to remove cells and the clot. To isolate lipoproteins, NaBr was added to the supernatant at a final density of 1.22 g/ml and subjected
to 48-hr density ultracentrifugation (125,000 × g
and 12°C, in a T-875 fixed angle rotor, Sorval,
Dupont). The upper yellow colored fraction containing the HDL was collected. The HDL samples
were dialyzed for 24 hr at 4°C to remove the NaBr,
in two changes of 1 L dialysis buffer (10 mM phosphate buffer solution containing 150 mM NaCl
and 0.01% EDTA at pH 7.4 and protease inhibitors as described above). Samples were stored in
sterile tubes in the dark at 4°C and used within
1–2 mo after isolation.
Vg and LP1 from female hemolymph HDL and
LP1 from male hemolymph HDL were obtained
from hemolymph samples that were allowed to
clot. The HDL (1–1.5 mg) was incubated at room
temperature for 45 min in a final concentration
of 0.1% acytelated Sudan black B (Ribero and
McDonald, ’63) and loaded on a stepwise sucrose
gradient of 5, 10, 15, 20, and 25% (w/w prepared
in phosphate buffer saline). After a 24-hr centrifugation (swinging bucket T-641 rotor, at 180,000 ×
g and 6°C; Sorval, Dupont), one stained band was
observed in the tube containing the male HDL
samples. The female HDL showed two stained
bands. One was running in the same position as
the male LP1 and was presumed to be the female
LP1. The second lower band was assumed to be
Vg, because its mobility was similar to that of Vt
that was run on an identical gradient. The purified bands were collected from the side of the tube
by piercing and suction into a 5-ml syringe and
341
stored at 4°C in the dark until used in electrophoresis, Western blot, and lipid determinations.
Apoprotein and lipid determinations of LP1 (from
males and females) and Vg were performed on sucrose gradient purified proteins.
Isolation of vitellin (Vt)
Vt was isolated and purified from the ovaries
of vitellogenic females by two methods giving similar results: 1) as described by Browdy et al. (’90)
and Tom et al. (’92), except that 1 mM PMSF, 10
µg/ml leupeptine, and 1 µg/ml aprotinin were
added during the first homogenation step and
PMSF and aprotinin were included in the buffers
used during the gel and ion exchange chromatography and 2) through high-density ultracentrifugation, using the same method described above
for isolation of hemolymph HDL. Briefly, ovaries
were homogenized in phosphate buffer containing protease inhibitors (as mentioned earlier) and
centrifuged (10 min at 10,000 × g and 4°C; Sorval
5-CB, Dupont) to remove cell debris. The supernatant was subjected to NaBr and sucrose gradient ultracentrifugation as described above for
HDL. After sucrose gradient ultracentrifugation,
only one green band was observed and collected
for further lipid and protein analysis.
Protein determinations
Protein concentrations were determined by a
modification of the Lowry procedure (Markwell et
al., ’78).
Characterization of apoproteins
Male and female HDL and sucrose isolated fractions were characterized by electrophoresis on native and denatured polyacrylamide gels (PAGE)
as described previously (Khayat et al., ’94a). Protein bands were visualized after staining with
Coomassie blue. Immunoidentity of proteins was
established after the transfer of proteins to nitrocellulose in Western blot and was visualized as
described by Khayat et al., ’94b.
Preparation of rabbit-specific antisera
Specific polyclonal antibodies against purified
Vt were generated by Dr. M. Tom (Browdy et al.,
’90; Tom et al., ’92). An LP1 polyclonal antibody
was generated in rabbits that were injected with
LP1 from males. The HDL prepared from male
hemolymph was run on a native PAGE, and the
Coomassie blue stained band was cut and injected
into rabbits (Yeda, Weizmann Institute, Rehovot,
Israel). Preimmunized and immunized samples
342
E. LUBZENS ET AL.
were tested by dot blot and PAGE followed by
Western blot, as described above.
Extraction and lipid determinations
Lipids from male and female HDL samples obtained after high-density NaBr ultracentrifugation
and LP1 and Vg samples obtained by sucrose
stepwise gradient centrifugation were extracted
in methanol:chloroform (2:1 v/v) as described by
Bligh and Dyer (’59). Lipids were separated by
thin layer chromatography (TLC; silica gel 60,
Merck, Darmstadt, Germany) employing benzene:
ethylether; ethylacetate:acetic acid (80:10:10:0.2 by
volume) for separation of neutral lipids and
chloroform:methanol:acetic acid:water (100:20:
12:5 by volume) for the separation of PL. Lipid
spots were detected after spraying with 2,7
dichlorofluorescine (0.2% in ethanol). Lipid spots
were collected, saponified with 0.5 M methanolicKOH at 50°C, and the fatty acids were recovered
after acidification by hexane extraction. The fatty
acids were methylated with diazomethane (Schlenck
and Gellerman, ’60) and separated by gas liquid
chromatography on a 25-M fused silica column
cross-linked with methylsilicone (0.52-m film
thickness) at a temperature range of 185–230°C
employing a Hewlett-Packard 5990A gas chromatograph equipped with a flame ionization detector. The relative composition of a fatty acid
mixture was calculated from the areas under the
peaks employing a Hewlett-Packard 3390A integrator. For quantitative analysis, heptadecanoic
acid was added as the internal standard, assuming that the response of the detector for all methylfatty esters was identical. Ch was eluted from silica
gel with chloroform-methanol (1:2, by volume) and
quantitated by using Boehringer’s cholesterol-oxidase kit (Boehringer, Mannheim, Germany). The efficiency of the procedure was estimated by adding
trace amounts of 14C-cholesterol prior to TLC, and
the results were corrected for losses.
RESULTS
Male and female lipoproteins
The hemolymph lipoproteins isolated from the
top fraction after ultracentrifugation in NaBr at
a density of 1.22 gm/ml fall in the class of HDL.
No lipoproteins were found after ultracentrifugation in NaBr at a density of 1.063 gm/ml. Density
sucrose ultracentrifugation revealed one Sudan
black stained protein in the HDL of males and
two in that of adult vitellogenic females, and one
band in lipoproteins isolated from the ovary (Fig.
1). The differences in the staining intensity of the
Fig. 1. Purification of high-density lipoproteins (HDL) of
male and female hemolymph and vitellin (Vt) by density sucrose ultracentrifugation. The HDLs and Vt were obtained
by NaBr density ultracentrifugation, stained with Sudan black
B and loaded on a stepwise sucrose gradient as described in
Materials and Methods. Vt is shown in lane 1, and female
and male HDL separations are shown in lanes 2 and 3, respectively. The lower band in lane 2 (indicated by an arrow)
correlates with Vt.
two lipoproteins in female HDL after sucrose ultracentrifugation indicate the lower concentration
of Vg (lower band) in comparison with LP1 (upper band). An excess stain of Sudan black precipitated after ultracentrifugation to the bottom
of the tube holding the male HDL (Fig. 1., lane
3). One Coomassie blue stained band was revealed
after gel electrophoresis under native conditions
in each of the following preparations obtained after sucrose gradient ultracentrifugation of HDL:
male LP1 (Fig. 2, lane 1) from the hemolymph of
males, the upper band and lower bands of female
HDL (Fig. 2; lanes 2 and 3, respectively) from the
hemolymph of females, and Vt (Fig. 2, lane 4) from
the ovarian homogenates. The upper Sudan black
stained band in the female HDL subjected to sucrose gradient ultracentrifugation showed similar
mobility on PAGE to that of LP1 of males and
reacted positively with the polyclonal antibody
prepared against male LP1 in Western blot analysis (Fig. 3, lanes 1 and 2). Vg and Vt run on the
same gel did not react with the LP1 antibody. Furthermore, both the LP1 of males and the LP1 of
females were found to be composed of one 110-kDa
polypeptide, as revealed by gel electrophoresis under denaturing and reduced conditions (Fig. 4a) and
HDL APOPROTEINS, LIPIDS, SHRIMP
Fig. 2. Polyacrylamide gel (5%) electrophoretic analysis
(PAGE) under native conditions of lipoproteins collected by
sucrose gradient ultracentrifugation (shown in Fig. 1). The
gel was stained with Coomassie blue. Lane 1: male lipoprotein (m-LP1; see Fig. 1, lane 3); lane 2: upper fraction of
female lipoproteins (f-LP1; see Fig. 1, lane 2); lane 3: lower
fraction of female lipoproteins (vitellogenin [Vg]; see Fig. 1,
lane 2); lane 4: vitellin isolated from vitellogenic ovaries (Vt;
see Fig. 1, lane 1).
343
Fig. 4. Electrophoretic and Western blot analysis of male
and female LP1 proteins, under denatured and reduced conditions (7.5–17% gel). Coomassie blue stain of molecular
weight standards (left lane), male (m-LP1), and female (fLP1) LP1 are shown in (a). Proteins were transferred to a
nitrocellulose membrane and incubated with LP1 antiserum
(b). The color was developed as described in Figure 3.
by Western blot analysis (Fig. 4b). On several occasions, the female LP1 migrated slightly differently
from the male LP1 on a native gel (not shown).
The higher density protein (lower stained band
in Fig. 1) found in sucrose gradient ultracentrifugation of the female HDL is Vg because it reacted
positively with the polyclonal antibody prepared
against purified Vt in Western blot analysis (Fig.
5). Coomassie blue staining and Western blot
analysis revealed three stained bands at 200, 120,
and 80 kDa in both Vg and Vt (Fig. 6a, b). The
relative abundance of the polypeptide species of
Vg was similar to that of Vt.
Fatty acid and lipid composition
Fig. 3. Western blot analysis of high-density lipoproteins
(HDL) showing immunoidentity with LP1. Lipoproteins were
collected after sucrose gradient ultracentrifugation (see Fig.
1) and were subjected to polyacrylamide gel electrophoresis
(5%) under native conditions. Proteins were transferred to a
nitrocellulose membrane and incubated with LP1 antiserum,
followed by GAR-HRP. The color was developed after incubation with 3,3´-diaminobenzidine and hydrogen peroxide. Lane
1: male lipoprotein (m-LP1; see Fig. 1, lane 3); lane 2: the
upper fraction in sucrose gradient (f-LP1; see Fig. 1, lane 2);
lane 3: the lower fraction in sucrose gradient (vitellogenin [Vg];
see Fig. 1, lane 2); lane 4: vitellin (Vt; see Fig. 1, lane 1).
Table 1 shows the lipid composition of male and
female hemolymph and the HDLs isolated from
them. Because the HDL of females contains two
lipoproteins, LP1 and Vg, the lipid composition of
each lipoprotein was determined separately (Table
2). Table 2 also presents the lipid composition of
ovarian Vt. Considerable amounts of lipids were
detected in the hemolymph of male and female
shrimp, most of which were concentrated in the
HDL upon centrifugation, after addition of NaBr.
PL constituted 71–76% of the total lipid in the
hemolymph and the HDL isolated from it. Ch
amounted to 17–20%, yielding a PL:Ch ratio of
3.0:5.3. Only relatively small amounts of diacylglycerol (DAG) were detected. TAGs were detected
only in the hemolymph and HDL of vitellogenic
females and were carried by Vg (Table 2). The average lipid concentrations of 94 mg% (mg/100 ml),
344
E. LUBZENS ET AL.
Fig. 5. Western blot analysis of high-density lipoproteins
(HDLs) showing immunoidentity with vitellin (Vt). Lipoproteins were collected after sucrose gradient ultracentrifugation (see Fig. 1) and were subjected to polyacrylamide gel
electrophoresis (5%) under native conditions. Proteins were
transferred to a nitrocellulose membrane and incubated with
Vt antiserum, followed by GAR-HRP. The color was developed after incubation with 3,3´-diaminobenzidine and hydrogen peroxide. Lane 1: male lipoproteins (m-LP1); lane
2: the upper fraction in sucrose gradient (f-LP1; see Fig. 1,
lane 2); lane 3: the lower fraction in sucrose gradient
(vitellogenin [Vg]; see Fig. 1, lane 1); lane 4: vitellin (Vt)
isolated from vitellogenic ovaries.
136 mg%, and 113 mg% was found in males,
vitellogenic, nonvitellogenic and young females,
respectively. A ratio of 1.01, 0.78, and 0.94 lipids
per 1 g protein was found for the HDL of males,
vitellogenic, and nonvitellogenic females, respectively (Table 1).
Table 2 shows the lipid composition of female
LP1, Vg, and ovarian Vt. The lipid content of Vg
is much smaller than that of LP1 (0.54 and 1.09
mg/g protein, respectively). This difference can
explain the higher lipid content of male HDL compared with female HDL (Table 1). The lipid composition of female LP1 is very similar to that of
male HDL. In contrast, Vg carries relatively less
Ch (12%) than LP1, yielding a PL:Ch ratio of 7.1
DAG are carried by both Vg and LP1, but TAG
are carried only by Vg. Vt, in contrast to Vg, carries considerable amounts of TAG (22.1 and 7.6%,
respectively) and negligible amounts of DAG.
Table 3 shows the fatty acid (FA) composition
of LP1, Vg, and Vt. Because the FA composition
of lipids isolated from male and female hemolymph and of male and female HDL are very similar to that of female LP1, these results are not
described in detail in this table. All the lipoproteins carried EPA and DHA. Generally, the HUFA
content of PL is higher than that of DAG and TAG
(Table 3), and the ratio of ω3:ω6 fatty acids is also
higher (1.71–1.72) in the PL than in the DAG
(0.95–1.02) of the hemolymph lipoproteins. Vt
showed a relatively high ω3:ω6 ratio in both the
PL and TAG (2.00 and 2.30, respectively).
Table 4 compares the fatty acid composition of
phosphatidylcholine (PC) and phosphatidylethanolamine (PE) isolated from female LP1, Vg, and
Vt. The results shown for female LP1 are very
similar to those determined for male and female
hemolymph and HDL and are not presented in
this table. PE constituted about 10% of the PL of
all lipoproteins. As indicated by the SAT:HUFA
fatty acid ratio, PE contained more HUFA than
PC and less 16:0 and 18:1.
The hemocyanin isolated from clotted hemolymph contained only trace amounts of lipids
whereas that isolated from citrated (nonclotted)
hemolymph contained 2.5–3.7 mg/g hemocyanin.
DISCUSSION
Fig. 6. Electrophoretic and Western blot analysis of vitellin (Vt) and vitellogenin (Vg) under denatured and reduced
conditions (7.5–17% gel). Coomassie blue stain of molecular
weight standards (left lane), Vt, and Vg are shown in (a).
Proteins were transferred to a nitrocellulose membrane and
incubated with Vt antiserum (b). The color was developed as
described above in Figure 3.
One lipoprotein was found in the hemolymph
of Penaeus semisulcatus males that consisted of
one 110-kDa peptide unit, supporting results reported previously (Khayat et al., ’94a; Lubzens et
al., ’95). This lipoprotein, known as LP1, was
found in several species of crustaceans, including
HDL APOPROTEINS, LIPIDS, SHRIMP
345
TABLE 1. Lipid content of hemolymph (HL) and high-density lipoproteins (HDL)
PL
Male HL
Female HL
Non-vit female HL
Male HDL
Female HDL
Non-vit female HDL
DAG
68.1 ± 7.3
100.5 ± 17.5
81.3 ± 20.4
775 ± 361
559 ± 251
674
TAG
7.1 ± 1.7
11.9 ± 4.9
11.9 ± 4.9
50 ± 11
43 ± 15
40
Trace
4.5
Trace
Trace
36 ± 24
Trace
Ch
PL/Ch
Total
19.0 ± 0.9
19.0 ± 4.1
19.2 ± 3.9
185 ± 50
146 ± 41
225
3.6
5.3
4.2
4.2
3.8
3.0
94
136
113
1,012
784
939
Hemolymph was collected with citrate (10%), and HDL was prepared as described in Materials and Methods. The lipid content of hemolymph
is expressed as mg100 ml hemolymph. The lipid content of HDL is expressed as mg/g protein. The results for HDL of nonvitellogenic (non-vit)
females were performed only once. All other results represent averages and SD for three different preparations.
other penaeid species (Lee and Puppione, ’88; Lee,
’90; Komatsu et al., ’93; Hall et al., ’95; see also
Table 1 in Yepiz-Plascencia et al., ’95). A protein
immunologically identical to male LP1 and similarly possessing one 110-kDa peptide was also
found in the hemolymph of females. The site of
synthesis of LP1 is not known. Although Lee and
Puppione (’88) suggested that LP1 was synthesized in the HEP, no direct evidence has been presented. Direct evidence for cell-free translation of
LP1 mRNA extracted from the male HEP failed,
because of the relatively greater abundance of
mRNA encoding hemocyanin in this organ (Khayat et al., ’95). The turnover rate of LP1 in the P.
semisulcatus hemolymph was found to be significantly lower than that of Vg (Khayat et al., ’94a).
This may be attributed to a relatively higher concentration of LP1 or higher removal rate of Vg
from the hemolymph. Recently, Hall et al. (’95)
have shown that major lipoproteins in crayfish
hemolymph are also involved in immune recognition and clotting in addition to their role of lipid
transport and suggested that the LP1 is identical
to the β-1,3-glucan binding protein.
The hemolymph of vitellogenic females contained a second lipoprotein, not found in males.
It was found to react with the Vt antiserum, the
main lipoprotein found in mature ovaries. By definition, this protein is termed Vg. In previous publications we showed that Vt is synthesized within
the ovary of P. semisulcatus (Browdy et al., ’90;
Khayat et al., ’94b). However, the HEP was also
found to synthesize a protein that was immunologically identical to Vt and was therefore termed
Vg (Fainzilber et al., ’92). The exact relationship
between Vg and Vt in penaeid shrimp is not fully
established. In vertebrates and most insects
extraovarian synthesis of Vg has been shown. The
Vg was found to enter the ovary through receptor
mediated endocytosis and, following some modifications, forms the Vt (Byrne et al., ’89; Sappington
et al., ’95; Schneider, ’95).
The results reported here for P. semisulcatus
show that all three Coomassie blue stained
polypeptide bands of Vg were also found in the
Vt, and the antiserum prepared against Vt reacted
with all three bands of Vg. In P. monodon the isolated Vg consisted of 82- and 170-kDa subunits
and was very different from the purified Vt that
contained eight subunits, with molecular weights
ranging from 35 to 91 kDa (Chang et al., ’94). The
Vt of several penaeid species had molecular
weights ranging between 283–540 kDa, with two
to eight subunits in denatured gels (see comparisons in Lubzens et al., ’95). Because in most cases,
Vt was isolated in the absence of protease inhibitors, the subunits reported in the various publications could represent products of enzymatic
digestion. Surprisingly, Chen and Chen (’93), did
not find cross-immunoreactivity between three of
TABLE 2. Lipid content of LP1 and vitellogenin (Vg) isolated from feamle HDL, and vitellin (Vt) isolated from ovaries
LP1
Vg
Vt
PL
DAG
TAG
Ch
PL/Ch
Total
847 ± 119.9
(77.8)
382.9 ± 20.5
(70.9)
324.4 ± 114.4
(67.0)
54.0 ± 10.8
(5.0)
51.1 ± 17.4
(9.5)
—
—
220.9 ± 24.2
(20.3)
64.9 ± 37.1
(12.0)
38.8 ± 5.0
(8.6)
3.9 ± 0.95
1,088.9 ± 114.6
7.1 ± 3.2
539.8 ± 50.2
8.7 ± 4.3
320.0 ± 248.2
40.9 ± 10.2
(7.6)
99.0 ± 13.6
(22.1)
HDL was obtained from clotted hemolymph. Vg and LP1 were isolated on a sucrose gradient as described in Materials and Methods. Results
were expressed as mg lipid/g proteins. The results represent the average and SD for three different preparations.
346
E. LUBZENS ET AL.
TABLE 3. The fatty acid composition (in % of total) of vitellogenin and LP1 isolated from vitellogenic female high-density
lipoproteins (HDL) and vitellin that was isolated from the ovaries
Phospholipids
Fatty acids
16:0
16:1 ω7
18:0
18:1
18:2 ω6
20:1 ω9
20:4 ω6
20:5 ω3
22:6 ω3
ω3/ω6
1
2
Diacyglycerols
Triacylglycerols
LP1
Vg
Vt
LP1
Vg
Vg
Vt
18.5 ± 3.8
5.2 ± 1.3
7.8 ± 0.4
18.1 ± 2.0
8.0 ± 3.5
2.11
3.9 ± 0.3
10.7 ± 0.4
9.8 ± 2.9
1.72
18.9 ± 3.3
5.6 ± 1.4
8.1 ± 3.2
19.1 ± 2.7
8.6 ± 3.1
192
4.4 ± 0.4
11.9 ± 1.7
9.8 ± 2.2
1.71
12.9 ± 3.0
4.0 ± 1.2
7.7 ± 0.6
17.9 ± 2.1
6.0 ± 0.9
3.7 ± 1.4
2.9 ± 0.7
8.1 ± 1.4
9.8 ± 1.3
2.00
26.2 ± 2.1
5.7 ± 2.0
8.5 ± 3.6
20.0 ± 2.7
8.3 ± 3.4
4.21
4.9 ± 1.2
8.4 ± 0.8
4.2 ± 0.8
0.95
23.2 ± 5.9
5.2 ± 1.5
8.4 ± 2.6
19.2 ± 1.6
7.7 ± 3.6
2.01
5.8 ± 0.7
9.4 ± 0.8
4.4 ± 1.0
1.02
29.7 ± 2.7
4.6 ± 1.1
7.5 ± 2.9
22.7 ± 2.2
6.6 ± 2.6
4.51
2.1 ± 0.1
5.3 ± 1.8
4.2 ± 0.1
1.09
15.2 ± 1.9
3.5 ± 0.9
3.3 ± 0.6
11.0 ± 4.8
3.8 ± 0.7
4.3 ± 1.1
0.9 ± 0.1
4.0 ± 1.3
5.1 ± 1.5
2.30
Found only in two replicate samples.
Found only in one replicate sample.
the four polypeptides of Vt of P. monodon. On the
other hand, immunocross-reactivity was reported
between the antiserum prepared against one
penaeid species with the Vt of other species. For
example, the antiserum raised against Vt of P.
monodon cross-reacted in immunodiffusion with
the ovarian extracts from mature P. indicus, P.
merguiensis, and P. semisulcatus (Quinitio et al.,
90). Similarly, the antiserum raised against P.
semisulcatus was found to react with the Vt of P.
vannamei (Tom et al., ’92).
Most of the lipids in the hemolymph of both
males and females are carried by HDL:LP1 in
males, LP1 and Vg in females. PL and Ch are the
major lipid components (71–77% and 18–20%, respectively; Tables 1 and 2) of LP1. DAG constitute only approximately 5%. Vg in comparison
with LP1 has a lower lipid content (540 and 1,089
mg/g protein, respectively); it carries much less
Ch (12%) and slightly more DAG (9.5%). This may
contribute to the differences observed between
male and female HDL (Table 1) and to the lower
intensity in the Vg stained with Sudan black and
subjected to ultracentrifugation (Fig. 1). Vg also
carries TAG, which are not found in male or female LP1 and in the hemolymph of nonvitellogenic
females. Although Vg and Vt are composed of similar polypeptides, their lipid composition is different. Vt, in contrast to Vg, carries considerable
amounts of TAG (22%) and only trace amounts of
DAG. The relatively high amounts of TAG are
reminiscent of the lipid composition of intact ovaries at the last stages of oocyte development in P.
semisulcatus (Ravid et al., in preparation). All lipoproteins contain EPA and DHA. Generally, the
HUFA content of PL is higher than that of DAG
or TAG (Table 3), and the ratio ω3/ω6 fatty acids
was higher in the PL than in the DAG of the
hemolymph lipoproteins. The higher ω3/ω6 ratio
in both the PL and TAG (2.0 and 2.3, respectively)
TABLE 4. The fatty acid composition (in % of total) of the phosphatidylcholine (PC) and phosphoethanolamine (PE) from the
phospholipids (PL) of the LP1, vitellogenin (Vg), and vitellin (Vt)1
Fatty acid
Vg
PL
PC
PE
PL1
PL
PC
PE
Vt
PL
PC
PE
1
16:0
18:0
18:1
18:2
20:5
20:6
SAT/HUFA
23.5
24.5
18.9
8.5
10.0
13.2
15.5
16.6
6.8
12.7
13.2
6.8
9.7
10.0
16.3
12.9
13.9
15.3
1.4
1.4
1.0
23.5
23.4
8.2
7.3
7.2
11.5
15.3
16.3
8.2
12.8
14.3
6.6
14.1
10.2
24.6
10.2
14.2
21.3
1.3
1.3
0.4
17.5
18.6
10.0
8.2
7.6
8.8
19.9
19.3
9.6
8.8
9.0
5.6
12.2
10.4
20.6
16.0
13.9
20.7
0.9
1.1
0.5
The ratio of saturated to highly unsaturated fatty acids (SAT/HUFA) is shown in the last column.
HDL APOPROTEINS, LIPIDS, SHRIMP
was found in Vt. Although Vt and several of its
lipids are synthesized within the ovary (Browdy
et al., ’90; Fainzilber et al., ’92; Shenker et al.,
’93), the HUFA originate from the food.
Lipid fatty acid compositions were similar
among the HDLs from male, female, and vitellogenic female blue crabs (Lee and Puppione, ’88),
with phosphatidylcholine as the predominant lipid
(80–85%). Phosphatidylethanolamine (3%), lysophosphatidylcholine (~1%), TGs (5–8%), cholesterol (Ch) (3–4%), and sphigomyelin (2–3%) were
also carried by these HDLs.
The functional relationship between Vg and Vt
has not been established so far. One possibility,
that Vg is associated with lipid transport to the
ovary, as previously suggested by Quackenbush
(’89), has yet to be verified. Lipids are carried from
the HEP by the hemolymph lipoproteins, and we
have not established the role of each lipoprotein
in this process. The presence of an ovarian receptor for Vg has been demonstrated in a lobster
(Lavedure and Soyez, ’88). It is not known whether
this receptor is specific only to Vg or could serve
for receptor-mediated transport of lipids also for
LP1, as has been shown for the Vg/VLDL ovarian
receptor in the chicken (Schneider, ’92). Recently,
we have preliminary results showing the presence
of a Vg/Vt receptor in ovarian membranes of P.
semisulcatus (Tietz et al., ’96). These membranes
also bind LP1, however, at a lower affinity.
In recent years it has been claimed that hemocyanin functions as a lipid carrier (Hall et al., ’95).
We did not detect any lipids on hemocyanin that
was isolated from clotted hemolymph. However,
small amounts of lipids, with a composition identical to that of HDL, were detected in hemocyanin that was isolated from citrated hemolymph
(unpublished results). Whether these lipids are
associated with the hemocyanin, the shrimp
VHDL (Komatsu et al., ’93; Hall et al., ’95), or
constitute a contamination of LP1 is currently being investigated.
ACKNOWLEDGMENTS
This work was supported by grants from the
National Academy of Sciences and Humanities
(231/91) and the Binational Science Foundation
(93-00083).
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