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THE JOURNAL OF EXPERIMENTAL ZOOLOGY 276:307-314 (1996)
ABSTRACT The lipid and protein composition of the perivitelline fluid of the eggs of Pomacea
canaliculata was investigated. Two lipoproteins (PV 1 and PV 2) and one lipoprotein fraction (PV
3) were detected for the first time in gastropods. They represent 57.0, 7.5, and 35.5% of the egg
total proteins, respectively.
PV 1is a glyco-carotene-protein complex with characteristics of a very high-density lipoprotein
(VHDL). It has 0.33% lipids, mainly free sterols and phospholipids. The particle has a MW of 300
Kd and is composed of three subunits of 35, 32, and 28 Kd, respectively.
PV 2 particle is a VHDL of 400 Kd and 3.75% lipids. The major lipid classes are free sterols and
phospholipids and also have significant quantities of energy-providing triacylglycerides and free
fatty acids. I t is composed of two apoproteins of 67 and 31 Kd.
PV 3 density corresponds to a high-density lipoprotein (HDL). It was fractionated into two
subfractions “h” and “p”. Fraction “h” contains 5.16% lipids, mainly free sterols, phospholipids,
and free fatty acids, and two particles of 100 and 64 Kd. Dissociating electrophoresis showed two
subunits of 34 and 29 Kd. Fraction “p” is composed of a single particle of 26 Kd th a t contains 9.5%
lipids, which represents 30% of the total egg lipids. It has high levels of a carotenoid pigment.
Besides it contains free fatty acids, hydrocarbons, sterified sterols, and triacylglycerides. These
three fractions are probably the major supply of lipids and amino acids for the developing embryo.
0 1996 Wiley-Liss, Inc.
During yolk synthesis primary oocytes increase Brink et al., ’83). A more detailed analysis of the
their size by accumulating vitellus. This vitellus perivitelline polysaccharides in the snail Pomacea
is composed of proteins, lipids, and carbohydrates canaliculata showed that they are composed of
and represents the energy source for the develop- galactose and fucose units (Raven, ’72). On the
ing embryo. The major egg yolk proteins are ei- other hand, Cheesman (’58)found a carotene-gly-
ther called vitellins or lipovitellins, being the latter coprotein complex with no lipids associated on the
high-density lipoproteins (HDL). Such lipoproteins eggs of this snail.
have been isolated from the eggs of two molluscs: The physiology and metabolism of E! canaliculata
the bivalve Pecten maximus and the cephalopod are currently under active research since it is a
Sepia oficialis (Lee, ’91). In gastropods, Barre et pest for rice crops. Besides, this snail is the host
al. (’91) reported the presence of vitellins in oo- for the nematode causing eosinophilic meningoen-
cytes and gonads of Helix aspersa. Most gastro- cephalitis in humans (Mochida, ’91).
pods have a perivitelline fluid, mainly synthesized We have previously worked on the hemolymph
in the albumen gland, that represents the major lipid transport in this and other species of molluscs
source of nutrients for the embryo. Therefore, pro- where for the first time the circulating lipoproteins
teinaceous yolk granules found in embryos devel- of bivalves, gastropods, and cephalopods were de-
oping from egg cells provided with perivitelline scribed (Pollero, ’87; Pollero and Heras, ’89; Heras
fluid do not serve as nutrient storage, instead they and Pollero, ’90, ’92 and references thereafter). In
function as primary lysosomes in charge of perivi- P. canaliculata we have recently identified two
telline fluid digestion (Raven, ’72; Jong-Brink et hemolymph lipoproteins of low and high densities,
al., ’83;Wourms, ’87). In the pulmonate snail Lym-
naea stagnalis the perivitelline fluid is composed
of calcium, proteins, and galactogen, but no lipid Received January 15, 1996; accepted June 28, 1996.
Address reprint requests to Dr. R.J. Pollero, INIBIOLP, Fac.
was detected (Horstmann, ’56; Raven, ’72; Jong- Medicina, UNLP, Calk 60 y 120, (1900) La Plata, Argentina.
0 1996 WILEY-LISS,JNC.
308 C.F. GARIN ET AL.
respectively (Pollero et al., '92; Garin and Pollero, 280 nm. A size exclusion column (Superdex 200
'95). In the present study, we isolated and char- HR 10/20, Pharmacia, Uppsala, Sweden) using a n
acterized three lipoprotein fractions of the egg isocratic gradient of sodium phosphate buffer 50
perivitelline fluid which are involved in the em- mM, pH 7.6, at 0.4 ml/min was employed. Purity
bryo development. of the peaks was checked by native polyacryla-
mide gel electrophoresis (PAGE).
MATERIALS PLND METHODS Absorbance spectra of each purified fraction
Sample collection were obtained using a DW-2000 UV-VIS Spectro-
photometer (SLM Instruments Inc. Aminco, Ur-
Eggs from I? canaZicLdata were collected from bana, IL). Samples dissolved i n water were
females raised in our la'boratory between Decem- scanned from 250 to 600 nm and spectra were
ber and April 1994. Spscimens were collected in plotted in a Color Pro Plotter (Hewlett-Packard,
the Zapata stream, Buerios Aires Province, Argen- San Diego, CAI. The presence of hemocyanin was
tina. All egg masses used had embryos developed checked in each fraction by monitoring copper ab-
t o no more than the moi-ula stage. Embryo devel- sorption at 340 nm before and after the addition
opment was checked in each egg mass microscopi- of KCN 0.2 M (Nickerson and Van Holde, '71).
cally. Wet and dry weights were obtained from ten
clutch aliquots. Gel electrophoresis
Total proteins of each fraction were measured by
Preparation aizd fi-actionation the method of Lowry et al. ('51).Fractions obtained
of soluble t?ggproteins by HPLC or ultracentrifugation were dialyzed
Eggs from three to five clutches were pooled, against 20 mM Tris-HC1, pH 7.0, and concentrated
weighed, cooled on ice, amd homogenized in a Pot- either by lyophilization on a Freezmobile 5SL lyo-
ter type homogenizer (Thomas Sci., Swedesvoro, philizer (Virtis, New York) or by Centripep mem-
NJ) using Tris-HC1 buffer 0.02 M, pH 7.5, contain- brane concentrators with a MW 10,000 cut-off
ing 2 pg/ml aprotinine (Trasylol, Mobay Chemical (Amicon, Beverly, MA). Nondissociating electro-
Co., New York). The relation buffer:sample was kept phoresis analysis was done by polyacrylamide gra-
5:l vlv. Homogenate was; centrifuged sequentially dient gel electrophoresis (PAGGE) using a 4-20%
at 10,OOOg for 30 min arid at 100,OOOg for 60 min. acrylarnide gradient (acrylamide-bis acrylamide
Both pellets were discarded and the second su- ratio 30:0.8 w/w) (Davis, '64; Felgenhauer, '74;
pernatant was dialyzed for 24 h against NaBr 6 = Margolis and Wrigley, '75).Apoproteins from each
1.017 g/ml. The dialyzed sample was layered over fraction were analyzed by sodium dodecylsulfate
NaBr 1.26 g/ml and ultracentrifuged at 207,OOOg (SDS)-PAGGE using a gradient of 4-23% acryla-
for 22 h, at 10°C on a 13eckman L8M (Beckman, mide (Laemmli, '70). Proteins were visualized by
Palo Alto, CA). A tube layered with NaBr 6 = 1.07 Coomassie Brilliant Blue R-250 staining (Sigma
g/ml in lieu of the sample was used as a blank for Chemical Co.) or silver staining (Merril, '90). Mo-
density calculations. After the run, 19 aliquots of lecular weight standards (Pharmacia) were run
200 p1 were collected sequentially down the top in the same gels. Carbohydrates attached to the
of the tubes. Absorbance at 280 nm was performed purified proteins, determined in native gel elec-
on each aliquot to obtain the protein profile. Re- trophoresis, were revealed with a thymol-sulfuric
fractive index of the blank tube aliquots was deter- reagent (Gander, '84). Protein subunit ratios were
mined with a refractometer (Bausch & Lomb, New calculated by densitometric analysis of the gels
York) and converted to g/ml using tabulated data at 550 nm on a scanning photodensitometer (Zeiss,
(Lindgren, '75). In another experiment, samples Oberkschen, Germany).
prestained with sudan black B (Sigma Chemical
Co., Saint Louis, MO) 0.01% in ethyleneglycol Lipid analysis
(Bauer, '91) were ultraclantrifuged under the same Lipids were extracted with a chlorofonn-metha-
conditions as above. no1 mixture following the method of Bligh and Dyer
('59). Lipid classes were analyzed by thin layer chro-
Purification of lipoproteins matography (TLC) on silica gel (chromarods type
Purification was done using a Merck-Hitachi S-111)with quantitation by flame ionization detec-
high-performance liquid chromatograph (HPLC) tion (FID) using a Iatroscan TH-10, Mark 111(Iatron
(Hitachi Ltd., Tokyo, Japan) with a L-6200 Intel- Laboratories, Inc., Tokyo, Japan) as described by
ligent Pump and a n L,-4200 W detector set at Parrish and Ackman ('85). The separation was con-
SNAIL EGG LIPOPROTEINS 309
ducted with a sequence of three different solvent profiles of the aliquots obtained from the centri-
systems. The first development was carried out for fuge tube with the location of the three fractions.
45 min in hexane:diethyl ether:ethyl acetate:formic To learn about the protein composition of the
acid (91:6:3:1 v/v/v/v). Chromarods were dried, par- peaks and to check their degree of homogeneity,
tially scanned, and then developed in acetone for we performed a native gel electrophoresis of each
15 min, and scanned to a point just below the caro- aliquot obtained from ultracentrifugation (Fig. 2).
tenoid peak. Finally, the Chromarods were devel- Results showed that PV 1 had only one protein
oped in ch1oroform:methanol:formic acid:water band and that the proteins of the other two frac-
(50:30:4:2 v/v/v/v) for 60 min and completely tions were cross-contaminated. To further sepa-
scanned to reveal the different phospholipids. rate them, a pool of the aliquots of each fraction
Tetracosanol was used as a n internal standard was dialyzed, concentrated, and analyzed by size
and quantitation was performed by obtaining cali- exclusion HPLC (Fig. 3).
bration curves of authentic standards r u n under
the same conditions. Lipids were also identified Characteristics of the red particle PV 1
on HP-TLC plates (Merck, Darmstadt, Germany) It was found that PV 1 had a high chromato-
developed with hexane: diethy1 ether:acetic acid graphic and electrophoretic purity (Figs. 3A, 4A).
(80:20:1.5 v/v/v) for neutral lipids and chloro- On native PAGGE, only one protein band was ob-
form:methanol:acetic acid:water (65:25:4:4 v/v/v/v) served. By comparison with molecular weight
for polar lipids. Standard lipids, iodine vapors, and standards it was calculated that PV 1has a n ap-
specific reagents were used to identify lipid classes. proximate M W of 300 Kd (Fig. 4A). PV 1 protein
concentration is 7.99 mg/g egg (wet weight) or
RESULTS 3.27% dry weight, and represents 57% of the to-
Separation of perivitelline tal proteins of the perivitellus.
fluid lipoprotein fractions By subjecting this fraction to SDS-PAGGE,
After ultracentrifugation in a NaBr gradient of three bands of 35, 32, and 28 Kd were observed
the soluble proteins from just-laid eggs, three li- (Fig. 4B). I n order to check the presence of carbo-
poprotein fractions were obtained. An orange frac- hydrates attached t o these apoproteins, the gel
tion in the upper part of the ultracentrifuge tube was treated with thymol-sulfuric reagent giving
(henceforth called PV 3 fraction) with a relative a positive stain in the three subunits.
density of 1.19-1.21 g/ml; a red fraction at the Lipids from the whole fraction were separated,
bottom (PV 1)with a relative density of 1.26-1.28 identified, and quantified by TLC-FID and HP-
g/ml; and between them, a third colorless frac- TLC. Table 1summarizes the results of the quali-
tion, were detected (PV 2) that were nevertheless tative a n d quantitative lipid classes i n this
intensely stained by the lipophilic dye, Sudan lipoprotein. Lipids account for 0.33% (w/w) of the
Black B. This latter fraction had a density of 1.22-
1.25 g/ml. Figure 1shows the density and protein
1 2
Volume [mi]
3 4 u
PV2 + PV3
-
PV1
Fig. 1. Protein and density profiles of fractions obtained Fig. 2. PAGE of the aliquots from the ultracentrifuge gra-
from the ultracentrifugation gradient of the soluble fraction of dient region containing fractions PV 3, PV 2, and PV 1. Na-
I! canaliculutu eggs. Egg cytosol was layered on NaBr 6 = 1.26 tive PAGE was done using 46 (w/v)acnilamide pels. Proteins
I
g/ml and centrifuged at 207,OOOg for 22 h. AU, arbitrary units. were revealed by silver staining.
C.F. GARIN ET AL.
PV1
A B
PV3p
L --
I l l ~ l I l l ~ l l l l ~ l l l l ~ I I I I ~ I I I1I1~
1 (I1I1 1 1 ~ 1 1 1 1 ~ 1 1 1 1 [ 1 1 1 1 ~ 1 1 1 1 ~ 1 1 1 1 ~ 1 1 1 1 ~ 1 1 1 1 ~ 1 1 1 1 ~ 1 1 1 1 ~ 1 I I I I I I I I ~ I I I I ~
Fig. 3. HPLC elution profile of the lipoprotein fractions buffer 50 mM, pH 7.6, on a Superdex 200 column and visual-
obtained by density gradient ultracentrifugation. Purification ized by monitoring elution at 280 nm. AU, arbitrary units.
was done using an isocratic gradient of sodium phosphate
TABLE 1. Lipid class composition of lipoprotein fractions from perivitelline fluid of P. canaliculata’
A B
232 - 67
60
140 - 36
18.5
67 -