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Lipoproteins of the egg perivitelline fluid

ofPomacea canaliculata snails (Mollusca:
Gastropoda)

Article in Journal of Experimental Zoology · December 1996

DOI: 10.1002/(SICI)1097-010X(19961201)276:5<307::AID-JEZ1>3.0.CO;2-S · Source: PubMed

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 THE JOURNAL OF EXPERIMENTAL ZOOLOGY 276:307-314 (1996)

Lipoproteins of the Egg Perivitelline Fluid

of Pomacea canaliculata Snails
(Mollusca: Gastropoda)
CLAUDIA F. GARIN, HORACIO HERAS, AND RICARDO J. POLLERO
Instituto de Investigaciones Bioquimicas de la Plata (INIBIOLP),
CONICETUNLP, (1900) La Plata, Argentina

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
their size by accumulating vitellus. This vitellus
is composed of proteins, lipids, and carbohydrates
and represents the energy source for the develop-
ing embryo. The major egg yolk proteins are ei-
ther called vitellins or lipovitellins, being the latter
high-density lipoproteins (HDL). Such lipoproteins
have been isolated from the eggs of two molluscs:
the bivalve Pecten maximus and the cephalopod
Sepia oficialis (Lee, ’91). In gastropods, Barre et
al. (’91) reported the presence of vitellins in oo-
cytes and gonads of Helix aspersa. Most gastro-
pods have a perivitelline fluid, mainly synthesized
in the albumen gland, that represents the major
source of nutrients for the embryo. Therefore, pro-
teinaceous yolk granules found in embryos devel-
oping from egg cells provided with perivitelline
fluid do not serve as nutrient storage, instead they
function as primary lysosomes in charge of perivi-
telline fluid digestion (Raven, ’72; Jong-Brink et
al., ’83;Wourms, ’87). In the pulmonate snail Lym-
naea stagnalis the perivitelline fluid is composed
of calcium, proteins, and galactogen, but no lipid
was detected (Horstmann, ’56; Raven, ’72; Jong-
0 1996 WILEY-LISS,JNC.
Brink et al., ’83). A more detailed analysis of the
perivitelline polysaccharides in the snail Pomacea
canaliculata showed that they are composed of
galactose and fucose units (Raven, ’72). On the
other hand, Cheesman (’58)found a carotene-gly-
coprotein complex with no lipids associated on the
eggs of this snail.
The physiology and metabolism of E! canaliculata
are currently under active research since it is a
pest for rice crops. Besides, this snail is the host
for the nematode causing eosinophilic meningoen-
cephalitis in humans (Mochida, ’91).
We have previously worked on the hemolymph
lipid transport in this and other species of molluscs
where for the first time the circulating lipoproteins
of bivalves, gastropods, and cephalopods were de-
scribed (Pollero, ’87; Pollero and Heras, ’89; Heras
and Pollero, ’90, ’92 and references thereafter). In
P. canaliculata we have recently identified two
hemolymph lipoproteins of low and high densities,
Received January 15, 1996; accepted June 28, 1996.
Address reprint requests to Dr. R.J. Pollero, INIBIOLP, Fac.
Medicina, UNLP, Calk 60 y 120, (1900) La Plata, Argentina.
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
ducted with a sequence of three different solvent
systems. The first development was carried out for
45 min in hexane:diethyl ether:ethyl acetate:formic
acid (91:6:3:1 v/v/v/v). Chromarods were dried, par-
tially scanned, and then developed in acetone for
15 min, and scanned to a point just below the caro-
tenoid peak. Finally, the Chromarods were devel-
oped in ch1oroform:methanol:formic acid:water
(50:30:4:2 v/v/v/v) for 60 min and completely
scanned to reveal the different phospholipids.
Tetracosanol was used as a n internal standard
and quantitation was performed by obtaining cali-
bration curves of authentic standards r u n under
the same conditions. Lipids were also identified
on HP-TLC plates (Merck, Darmstadt, Germany)
developed with hexane: diethy1 ether:acetic acid
(80:20:1.5 v/v/v) for neutral lipids and chloro-
form:methanol:acetic acid:water (65:25:4:4 v/v/v/v)
for polar lipids. Standard lipids, iodine vapors, and
specific reagents were used to identify lipid classes.
RESULTS
Separation of perivitelline
fluid lipoprotein fractions
After ultracentrifugation in a NaBr gradient of
the soluble proteins from just-laid eggs, three li-
poprotein fractions were obtained. An orange frac-
tion in the upper part of the ultracentrifuge tube
(henceforth called PV 3 fraction) with a relative
density of 1.19-1.21 g/ml; a red fraction at the
bottom (PV 1)with a relative density of 1.26-1.28
g/ml; and between them, a third colorless frac-
tion, were detected (PV 2) that were nevertheless
intensely stained by the lipophilic dye, Sudan
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
Fig. 1. Protein and density profiles of fractions obtained
from the ultracentrifugation gradient of the soluble fraction of
I! canaliculutu eggs. Egg cytosol was layered on NaBr 6 = 1.26
g/ml and centrifuged at 207,OOOg for 22 h. AU, arbitrary units.
309
profiles of the aliquots obtained from the centri-
fuge tube with the location of the three fractions.
To learn about the protein composition of the
peaks and to check their degree of homogeneity,
we performed a native gel electrophoresis of each
aliquot obtained from ultracentrifugation (Fig. 2).
Results showed that PV 1 had only one protein
band and that the proteins of the other two frac-
tions were cross-contaminated. To further sepa-
rate them, a pool of the aliquots of each fraction
was dialyzed, concentrated, and analyzed by size
exclusion HPLC (Fig. 3).
Characteristics of the red particle PV 1
It was found that PV 1 had a high chromato-
graphic and electrophoretic purity (Figs. 3A, 4A).
On native PAGGE, only one protein band was ob-
served. By comparison with molecular weight
standards it was calculated that PV 1has a n ap-
proximate M W of 300 Kd (Fig. 4A). PV 1 protein
concentration is 7.99 mg/g egg (wet weight) or
3.27% dry weight, and represents 57% of the to-
tal proteins of the perivitellus.
By subjecting this fraction to SDS-PAGGE,
three bands of 35, 32, and 28 Kd were observed
(Fig. 4B). I n order to check the presence of carbo-
hydrates attached t o these apoproteins, the gel
was treated with thymol-sulfuric reagent giving
a positive stain in the three subunits.
Lipids from the whole fraction were separated,
identified, and quantified by TLC-FID and HP-
TLC. Table 1summarizes the results of the quali-
tative a n d quantitative lipid classes i n this
lipoprotein. Lipids account for 0.33% (w/w) of the
u
PV2 + PV3
-
PV1
Fig. 2. PAGE of the aliquots from the ultracentrifuge gra-
dient region containing fractions PV 3, PV 2, and PV 1. Na-
tive PAGE was done using 46 (w/v)acnilamide pels. Proteins
I
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 ~

Time [miri] Time [min]

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

particle and represent only 6.72% of the total lip-

A B ids present in the perivitelline fluid, although this
is the main particle in terms of perivitellus pro-
tein concentration. Free sterols account for half
of the total lipids of PV 1, while phospholipids
(phosphatidylcholine and phosphatidylethanola-
mine) account for approximately 30%.The remain-
669 67 ing lipid classes were a carotenoid pigment and
60 free fatty acids.
440
36
232 Fig. 4. Native (A) and dissociating gel electrophoresis (B)
140 18,s of PV 1 particle. Both PAGGEs were done using acrylamide
gradients of 4-20% wlv. Proteins were revealed by Coomassie
blue staining. STD, high molecular weight standards (Phar-
macia). Native proteins: thyroglobulin (MW 669,000), ferritin
( M W 440,000), catalase (MW 232,0001, lactate dehydrogenase
(MW 140,000), and albumin (MW 67,000). Standards under
dissociating conditions: ferritin (MW 18,5001, lactate dehy-
Kd drogenase (MW 36,000), catalase (MW 60,000), and albumin
kd (MW 67,000).
 SNAIL EGG LIPOPROTEINS 311

TABLE 1. Lipid class composition of lipoprotein fractions from perivitelline fluid of P. canaliculata’

pg lipidlg egg, wet wt

(9%w/w)
Lipid classes PV1 PV2 PV 3h PV 3p
Hydrocarbons + tr 3.4 2 1.0 tr 33.3 f 5.4
Sterified sterols - (8.5) - (28.1)
TriacylgIycerides 0.3 k 0.0 7.0 2 2.9 17.0 f 1.5 13.9 f 6.5
(1.0) (17.5) (8.1) (11.8)
Free fatty acids 1.4 2 0.7 6.4 0.5 38.5 f 0.5 19.9 f 6.3
(5.5) (16.0) (18.4) (16.8)
Free sterols 13.7 2 2.1 8.9 2 2.8 86.7 2 3.5 7.5 f 1.0
(51.7) (22.3) (41.5) (6.3)
Pigment 2.6 2 0.0 1.8 2 0.2 14.7 1.8 26.5 * 5.3
(9.9) (4.5) (7.0) (22.4)
Phosphatidylethanolamine 3.8 f 1.5 6.4 f 2.0 25.4 f 8.4 6.5 f 1.2
(14.4) (16.0) (12.1) (5.5)
Phosphatidylcholine 4.6 2 0.9 3.4 2 0.3 27.4 6.4 5.3 f 1.0
(17.6) (8.5) (13.1) (4.5)
Sphingomyelin nd 2.7 2 0.5 tr 5.4 f 0.9
- (6.7) - (4.6)
Total 26.4 39.9 209.1 118.2
(100) (100) (100) (100)
‘Values are the mean of triplicate analysis 2 1SD. Values in parentheses correspond to the percentage of each lipid class w/w; tr, traces; nd,
not detectable; -, not determined.

Characteristics of the particle PV 2 phoresis the presence of two bands of 67 and 31

The fraction PV 2 was purified by HPLC, cor- Kd was observed (Fig. 5). Densitometry of the gels
responding t o the first eluting peak of the chro- allowed us t o calculate the relative proportion of
matogram (Fig. 3). By native electrophoresis we the 67 and 31 Kd apolipoproteins which was 7:3.
confirmed the high degree of purity of this par- Table 1shows the lipid class composition of PV 2.
ticle, even after silver staining the gels. The mo- This lipoprotein particle contained a higher lipid
lecular weight of the only band present was 400 content than PV 1 (3.75%) which accounted for
Kd (Fig. 5A). 10.14% of the total lipids of the perivitellus. PV
This particle was present at a concentration of 2 was mainly composed of free sterols, similarly
1.02 mg/g egg (wet weight) and 0.42% egg dry to PV 1, but it also had important amounts of
weight. It represents 7.32% of the total proteins triacylglycerides, free fatty acids, and phosphati-
of the perivitelline fluid. By dissociating electro- dylethanolamine. This particle was the only one
with significant amounts of triacylglycerides (18%).
A B Characteristics of the orange fraction PV 3
669 Fraction PV 3 was found to be more heteroge-
440 neous than the other ones. By HPLC we found
three major peaks, the two first ones termed frac-
232 tion “h,” and a third peak called fraction “p” (Fig.
3B). The PV 3 fraction had a perivitelline fluid
140 protein concentration of 4.97 mg/g egg (wet weight)
or 2.03% dry weight. This protein value represents
35.52% of the total proteins of perivitellus. The lipid
67
and protein compositions of subfractions “h” and
“p” were studied separately. Fraction “h” is the
major one with a concentration of 3.84 mg/g egg
Kd while fraction “p”is only 1.13 mg/g egg wet weight.
Fig. 5. Native PAGGE (A) and SDS-PAGGE (B) of PV This represents 27% and 8.08% by weight of the
2 particle. Electrophoresis conditions were the same as in total perivitellus proteins, respectively. Fraction
Figure 4. “h” is composed of two major proteins of 100 and
312 C.F. GARIN ET AL.

64 Kd (Fig. 6A), which analyzed by SDS-PAGGE DISCUSSION

showed the presence of two apoproteins of 34 and
29 Kd (Fig. 6B). On the other hand, fraction “p”
was composed of only m e protein of 26 Kd under
native electrophoresis and 21 Kd under dissociat-
ing conditions (Fig. 6A,B, respectively).
The lipid content of the two fractions is depicted
in Table 1. Each subfraction of PV 3 had a dis-
tinct lipid composition. Fraction “h” had 5.16% (w/
w) of total lipids which represents 53.10% of the
perivitellus total lipids, The most abundant lipid
class was found to be fiee sterols, as in PV 1and
PV 2, followed by free Estty acids and phospholip-
ids. Pigment content was only 7% of the total lip-
ids. Fraction “p” showe cl the highest 1ipid:protein
ratio of all fractions, reaching 9.47% (w/w) of the
lipoprotein. This small fraction contains more than
30% of the total lipids present in the perivitelline
fluid. The lipid composition showed t h a t it is
mainly composed of hydrocarbons, sterified ste-
rols, and pigments, thle latter giving the whole
fraction a strong yellowish color.
Absorption spectra hetween 250 and 600 nm
were obtained for the t ~ PV o 3 fractions and the
other two lipoproteins (results not shown). We ob-
served a n absorption maximum at 420 nm in PV
3p (Fig. 7) that was present neither in particles
PV 1, PV 2, nor in fraction PV 3h. This result
was coincident with the fact that PV 3p contains
more than 60% of the total pigments of the perivi-
telline fluid, probably a carotenoid.
P canaliculata belongs to the invertebrate group
where the yolk is a minor source of nutrients to
the embryo. Nutrients are provided by the extra-
cellular perivitelline fluid and yolk granules in
these species contain mainly zymogens whose
function would be the hydrolysis of the perivi-
telline fluid nutrients (Jong-Brink e t al., ’83;
Bretting et al., ’91). Perivitelline fluid is synthe-
sized by accessory glands of the female tract,
called albumen glands in P. canaliculata.
Based on density differences, we were able to iso-
late and characterize two high-molecular weight li-
poprotein particles and one lipoprotein fraction. PV
1 is a red lipoprotein particle of high molecular
weight. This particle was first described by
Cheesman (’58) in the same snail, and it was
called ovorubin. Our results show that this caro-
tene-glycoprotein complex also has lipids attached
(0.33%), which account for 6.7% of the total lipids
of the perivitelline fluid. The presence of lipids
has not been reported before for the ovorubin and
it would allow the inclusion of PV 1 as a lipo-
glyco-caroteneprotein, similar to other inverte-
brate lipovitellins (Zagalsky, ’85; Cheesman et al.,
’67). Nevertheless, it cannot be considered a true
lipovitellin because it is not transported by
hemolymph to the vitellogenic oocytes, b u t it
would be incorporated to the fertilized oocyte as
a secretion of the albumen gland instead. This dis-
tinction between ovorubin and true lipovitellins

A B

232 - 67
60
140 - 36
18.5
67 -

Kd STD PV2 PV3h PV3p PV3 STD Kd PV3p STD Kd

+
PV3
Fig. 6. Native PAGGE (A) and SDS-PAGGE (B) of PV 3 fraction. Electrophoresis condi-
tions were the same as in Figure 4 except protein bands in (A) were revealed by silver
staining. P V 3 includes PV 3 “p” + PV 3 “h.”
 SNAIL EGG LIPOPROTEINS
250 100 350 400 450 500 550
Wavelength (nm)
Fig. 7. Absorption spectra of PV 3p subfraction. The scan
was measured in the range of 250-600 nm. Lipoprotein was
prepared in sodium phosphate buffer, 50 mM, pH 7.6.
agrees with some of the differences reported by
Zagalsky (’71) on the amino acid composition.
PV 1 and other components of the perivitelline
fluid would be taken up by the developing em-
bryo by pinocytosis (Elbers and Bluemink, ’60;
Rigby, ’79; Raven, ’72) as the embryo synthesizes
its structural components. The fact that the pre-
dominant lipid classes of ovorubin are biomembrane
components (free sterols and phospholipids) would
suggest that the major function of these lipids dur-
ing embryogenesis would be structural, while
galactogen and apoproteins would provide for the
energy and precursor molecules.
The hydration density of PV 1 corresponds to a
very high-density lipoprotein (VHDL), and the
molecular weight of 300 Kd agrees with the one
found by Cheesman (’58) using another method-
ology. I n his original work, Cheesman did not
study the subunits of ovorubin. According to our
results, the molecular weight of the three subunits
found was 35,32, and 28 Kd.
The presence of the PV 2 particle in the perivi-
telline fluid was unexpected, since in previous de-
scriptions of gastropod perivitellus proteins (Morrill
et al., ’64;Cheesman, ’58)there was no mention of
a particle comparable to PV 2. This particle of 400
Kd is bigger than PV 1and it is composed of two
subunits as revealed by SDS-PAGE, one of them
67 Kd and the other 31 Kd. Considering its hy-
dration density, this particle also falls into the
VHDL range. It is the least concentrated of the
three lipoprotein fractions of perivitellus (7.3%)
and holds 3.75%lipids which corresponds to 10.1%
of the total lipids for the egg. The lipid class com-
position of this particle suggested it may play a n
important role as both a structural and an en-
313
ergy source because it had significant amounts of
triacylglycerides and free fatty acids a s well as
free sterols and phospholipids. A detailed study
on the structure, amino acid composition, and
lipid-binding properties of this lipoprotein would
establish patterns and homologies with other pro-
teins of great evolutive interest.
Fraction PV 3 is composed of at least three li-
poprotein particles. Hydration density of this frac-
tion corresponds to t h e upper limit of HDLs.
Analysis of this fraction allowed us to study a
600
separate particle “p” which is a carotenoprotein.
This particle has a n absorption maximum at 420
nm and is responsible for the strong yellowish
color of the fraction obtained from the ultracen-
trifuge gradient. This absorption maximum was
already reported by Cheesman (’58)for the whole
homogenate of the eggs during ovorubin purifica-
tion. This author suggested that the chromoprotein
responsible for that peak would be helicorubin, a
chromoprotein found in the digestive tract of Helix
pomatia. Particle “p” is the most lipid-rich par-
ticle with 9.5% lipids t h a t corresponds to 30%
of the total perivitelline lipids. It is the fraction
with the least concentration of proteins (8%)and
it contains most of the carotenoid pigment as
found by comparison of t h e absorption spectra
at 420 n m of all fractions a n d also by t h e
amount of the acetone-mobile peak i n TLC-FID.
The other fraction of PV 3 (fraction “h”) is more
heterogeneous, it represents 27% of the total
protein, a n d contains 5.16% lipids. The lipids
account for 53.2%of the total lipids of the perjvi-
telline fluid. The major lipid classes are free ste-
rols a n d phospholipids, as i n t h e other two
fractions, b u t in this case PV 3 also contains
free fatty acids.
In conclusion, all lipoprotein fractions of the
perivitelline fluid of this freshwater prosobranch
fall into the VHDL and HDL categories. This is
basically the only feature i n common with other
invertebrate and vertebrate lipovitellins (Lee,
,911 because, as we already stated, it is not pos-
sible to consider homologies between t h e s e
perivitelline lipoproteins and yolk lipovitellins
based on their functional analogy. The composi-
tion and amount of lipids suggest these particles
would play a role in providing structural com-
ponents and metabolic precursors for the devel-
oping embryo, a n d t h a t t h e y would not be
considered as energy sources for t h e embryo as
lipovitellins are. Ongoing research i n our labo-
ratory on the energetics of the development will
probably clarify this matter.
 314 C.F. GARIN ET AL.
ACKNOWLEDGMENTS
This work was partially supported by grants from
CONICET, Fundacih Antorchas, Argentina, and
Efamol Res. Institute, Canada. R.J.P. is a member of
Carrera del Investigador, U C (Bs. As.), Argentina.
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