1996 Garin Jez Pomacea Egg Perivitellins.pdf

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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 developing embryo. The major egg yolk proteins are either 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 oocytes and gonads of Helix aspersa. Most gastropods have a perivitelline fluid, mainly synthesized in the albumen gland, that represents the major source of nutrients for the embryo. Therefore, proteinaceous yolk granules found in embryos developing from egg cells provided with perivitelline fluid do not serve as nutrient storage, instead they function as primary lysosomes in charge of perivitelline fluid digestion (Raven, ’72; Jong-Brink et al., ’83;Wourms, ’87). In the pulmonate snail Lymnaea stagnalis the perivitelline fluid is composed of calcium, proteins, and galactogen, but no lipid was detected (Horstmann, ’56; Raven, ’72; Jong0 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-glycoprotein 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 meningoencephalitis 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 described (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 polyacrylamide gel electrophoresis (PAGE). MATERIALS PLND METHODS Absorbance spectra of each purified fraction were obtained using a DW-2000 UV-VIS SpectroSample collection photometer (SLM Instruments Inc. Aminco, UrEggs 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 abt 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 lyoter type homogenizer (Thomas Sci., Swedesvoro, philizer (Virtis, New York) or by Centripep memNJ) 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 electroCo., New York). The relation buffer:sample was kept phoresis analysis was done by polyacrylamide gra5: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% acrylafor 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). Modensity 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 elecon 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 Lipid analysis Co., Saint Louis, MO) 0.01% in ethyleneglycol Lipids were extracted with a chlorofonn-metha(Bauer, '91) were ultraclantrifuged under the same no1 mixture following the method of Bligh and Dyer conditions as above. ('59). Lipid classes were analyzed by thin layer chroPurification of lipoproteins matography (TLC) on silica gel (chromarods type Purification was done using a Merck-Hitachi S-111)with quantitation by flame ionization detechigh-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, partially scanned, and then developed in acetone for 15 min, and scanned to a point just below the carotenoid peak. Finally, the Chromarods were developed 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 calibration 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 chloroform: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 lipoprotein fractions were obtained. An orange fraction 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 fraction, were detected (PV 2) that were nevertheless intensely stained by the lipophilic dye, Sudan Black B. This latter fraction had a density of 1.221.25 g/ml. Figure 1shows the density and protein

1

2

3

4

Volume [mi]

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 centrifuge 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 fractions were cross-contaminated. To further separate 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 chromatographic and electrophoretic purity (Figs. 3A, 4A). On native PAGGE, only one protein band was observed. By comparison with molecular weight standards it was calculated that PV 1has a n approximate 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 total 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 carbohydrates 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 HPTLC. Table 1summarizes the results of the qualitative 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 gradient region containing fractions PV 3, PV 2, and PV 1. Native PAGE was done using 46 (w/v)acnilamide pels. Proteins were revealed by silver staining. I

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 obtained by density gradient ultracentrifugation. Purification was done using an isocratic gradient of sodium phosphate

B

A

669 440

67

60 36

232 140

kd

18,s

Kd

buffer 50 mM, pH 7.6, on a Superdex 200 column and visualized by monitoring elution at 280 nm. AU, arbitrary units.

particle and represent only 6.72% of the total lipids present in the perivitelline fluid, although this is the main particle in terms of perivitellus protein concentration. Free sterols account for half of the total lipids of PV 1, while phospholipids (phosphatidylcholine and phosphatidylethanolamine) account for approximately 30%.The remaining lipid classes were a carotenoid pigment and free fatty acids. Fig. 4. Native (A) and dissociating gel electrophoresis (B) 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 (Pharmacia). 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 dehydrogenase (MW 36,000), catalase (MW 60,000), and albumin (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 Hydrocarbons + Sterified sterols TriacylgIycerides

PV1

PV2

PV 3h

PV 3p

tr

3.4 2 1.0 (8.5) 7.0 2 2.9 (17.5) 6.4 0.5 (16.0) 8.9 2 2.8 (22.3) 1.8 2 0.2 (4.5) 6.4 f 2.0 (16.0) 3.4 2 0.3 (8.5) 2.7 2 0.5 (6.7) 39.9 (100)

tr

33.3 f 5.4 (28.1) 13.9 f 6.5 (11.8) 19.9 f 6.3 (16.8) 7.5 f 1.0 (6.3) 26.5 * 5.3 (22.4) 6.5 f 1.2 (5.5) 5.3 f 1.0 (4.5) 5.4 f 0.9 (4.6) 118.2 (100)

-

0.3 k 0.0 (1.0) 1.4 2 0.7 (5.5) 13.7 2 2.1 (51.7) 2.6 2 0.0 (9.9) 3.8 f 1.5 (14.4) 4.6 2 0.9 (17.6) nd

Free fatty acids Free sterols Pigment

Phosphatidylethanolamine Phosphatidylcholine Sphingomyelin

-

26.4 (100)

Total ‘Values are the mean of triplicate analysis not detectable; -, not determined.

2

-

17.0 f 1.5 (8.1) 38.5 f 0.5 (18.4) 86.7 2 3.5 (41.5) 14.7 1.8 (7.0) 25.4 f 8.4 (12.1) 27.4 6.4 (13.1) tr -

209.1 (100)

1SD. Values in parentheses correspond to the percentage of each lipid class w/w; tr, traces; nd,

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 10.14% of the total lipids of the perivitellus. PV Kd (Fig. 5A). 2 was mainly composed of free sterols, similarly This particle was present at a concentration of to PV 1, but it also had important amounts of 1.02 mg/g egg (wet weight) and 0.42% egg dry triacylglycerides, free fatty acids, and phosphatiweight. It represents 7.32% of the total proteins dylethanolamine. This particle was the only one of the perivitelline fluid. By dissociating electrowith significant amounts of triacylglycerides (18%). A

B

669 440

232

140

67 Kd Fig. 5. Native PAGGE (A) and SDS-PAGGE (B) of PV 2 particle. Electrophoresis conditions were the same as in Figure 4.

Characteristics of the orange fraction PV 3 Fraction PV 3 was found to be more heterogeneous than the other ones. By HPLC we found three major peaks, the two first ones termed fraction “h,” and a third peak called fraction “p” (Fig. 3B). The PV 3 fraction had a perivitelline fluid 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 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 while fraction “p”is only 1.13 mg/g egg wet weight. This represents 27% and 8.08% by weight of the total perivitellus proteins, respectively. Fraction “h” is composed of two major proteins of 100 and

C.F. GARIN ET AL.

312

64 Kd (Fig. 6A), which analyzed by SDS-PAGGE 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 dissociating conditions (Fig. 6A,B, respectively). The lipid content of the two fractions is depicted in Table 1. Each subfraction of PV 3 had a distinct 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 phospholipids. Pigment content was only 7% of the total lipids. 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 sterols, 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 observed 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 perivitelline fluid, probably a carotenoid.

B

A

232 140

DISCUSSION P canaliculata belongs to the invertebrate group where the yolk is a minor source of nutrients to the embryo. Nutrients are provided by the extracellular perivitelline fluid and yolk granules in these species contain mainly zymogens whose function would be the hydrolysis of the perivitelline fluid nutrients (Jong-Brink e t al., ’83; Bretting et al., ’91). Perivitelline fluid is synthesized by accessory glands of the female tract, called albumen glands in P. canaliculata. Based on density differences, we were able to isolate and characterize two high-molecular weight lipoprotein 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 carotene-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 lipoglyco-caroteneprotein, similar to other invertebrate 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 distinction between ovorubin and true lipovitellins

-

67 60 36

18.5 67

Kd

STD

PV2

+

PV3h PV3p

PV3

STD Kd

PV3p STD

PV3 Fig. 6. Native PAGGE (A) and SDS-PAGGE (B) of PV 3 fraction. Electrophoresis conditions 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.”

Kd

SNAIL EGG LIPOPROTEINS

250

100

350

450 500 Wavelength (nm)

400

550

600

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 embryo by pinocytosis (Elbers and Bluemink, ’60; Rigby, ’79; Raven, ’72) as the embryo synthesizes its structural components. The fact that the predominant lipid classes of ovorubin are biomembrane components (free sterols and phospholipids) would suggest that the major function of these lipids during 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 methodology. 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 perivitelline fluid was unexpected, since in previous descriptions 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 hydration 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 composition 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 proteins of great evolutive interest. Fraction PV 3 is composed of at least three lipoprotein particles. Hydration density of this fraction corresponds to t h e upper limit of HDLs. Analysis of this fraction allowed us to study a 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 ultracentrifuge gradient. This absorption maximum was already reported by Cheesman (’58)for the whole homogenate of the eggs during ovorubin purification. 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 particle 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 perjvitelline fluid. The major lipid classes are free sterols 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 possible to consider homologies between t h e s e perivitelline lipoproteins and yolk lipovitellins based on their functional analogy. The composition and amount of lipids suggest these particles would play a role in providing structural components and metabolic precursors for the developing 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 laboratory on the energetics of the development will probably clarify this matter.

C.F. GARIN ET AL.

314

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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