Seasonal variations on the biochemical composition and lipid classes of the gonadal and storage tissues of Crassostrea gigas (Thunberg, 1794) in relation to the gametogenic cycle.

ABSTRACT Variations in biochemical composition and lipid classes in relations to the gametogenic cycle were studied in digestive gland, labial palps and gonad in the Pacific oyster Crassostrea gigas from the Western Atlantic coast of France over 1 y. Variations in the weight of the soft body parts, gonad development and spawning depended on environmental conditions, mainly on nutrients availability. The main energy reserve is the glycogen, showing a marked seasonal variability in all tissues studied this component increased in the digestive gland during the phytoplankton blooms and was rapidly transferred and accumulated in labial palps and gonad during stages IIID-0; glycogen was then mobilized during the final stage of somatic growth and during the beginning of the gametogenic cycle (stage IA). Glycogen is consumed almost totally at the beginning of spring, coinciding with an important increase of phospholipids in males and triacylglycerols in females. This fact seems to indicate that the glycogen reserves were used in the synthesis of lipids and in supporting the high energy demand existing during the active vitellogenesis and spermatogenesis. The digestive gland, besides playing an intermediary role in transferring the ingested lipids to other organs, seems to constitute an important organ of reserve of lipids, mobilizing phospholipids in winter towards palps and gonad during the somatic growth, but also during the initial gametogenesis and sexual maturation (triacylglicerols). There is depletion in the reserves of glycogen and triacylglicerols of the labial palps during the last stage of gonadal maturation. Simultaneously, an increase in triacylglicerols in gonad occurs, suggesting that the labial palps were an organ of reserve to sustain the most advanced stages of gonadal ripeness. The changes of sterol esters of gonad are discussed with regard to gametogenic cycle of Crassostrea gigas.

KEY WORDS: biochemical composition, Crassostrea gigas, energy reserves, lipid classes, sexual maturation, oyster 

INTRODUCTION

Since its introduction in France in 1969, the Pacific oyster Crassostrea gigas has been widely farmed on the Atlantic and Mediterranean French coasts (Chevalier et al. 1975). Most of the French oyster production is based on the availability of seeds (only 10% is produced in hatcheries), thus oyster production is in constant fluctuation because of the irregularity in the recruitments of the species, as noted in recent years. Among the factors of variability, the quality of the gametes is a significant element.

Numerous studies have focused on the seasonal variations in the biochemical composition of marine bivalves in relation to the reproductive cycle (Mann 1979, Gabbott 1983, Beninger & Lucas 1984, Barber & Blake 1985, Deslous-Paoli & Heral 1988, Martinez 1991, Ruiz et al. 1992, Berthelin et al. 2000, Kang et al. 2000). In general, results indicate that, before gametogenesis, reserves accumulate in the different tissues in the form of glycogen, lipids and proteins, which are used, together with the available food, according to requirements during gametogenic process (Ansell 1974, Barber & Blake 1981, Gabbott 1983, Strohmeier et al. 2000). Lipids play an important role in the physiology of bivalves; in particular, they are a very important food reserve in the oocytes, assuring the viability of the larvae (Holland 1978, Gallager et al. 1986, Le Pennec et al. 1998). Also, it has been observed that lipids provide energy during winter growing, when carbohydrate reserves are depleted (Beninger & Lucas 1984). In common with other animals, the neutral lipids (triacylglycerols) are used as energy reserves, whereas polar lipids (phospholipids) are structural component of cell and membranes; also in some cases they may constitute an energy reserve in oocytes (Beninger 1984). The major function of sterols in tissues is to maintain the structural integrity of cell membranes (Nes 1974) in which they are in free form; however, the physiologic role of sterol esters has barely been investigated, possibly representing storage forms or metabolic pools (Napolitano et al. 1988, Pazos et al. 1996).

Oysters, as do other marine bivalves in temperate latitudes, exhibit cyclic changes both in reproductive and somatic components as a consequence of the seasonality of environmental conditions (Ruiz et al. 1992). Nevertheless, some differences exist depending on the characteristics of the organ of reserve. In some families such as the Mytilidae, the mantle tissue is the principal organ of reserve of the glycogen, supporting the development of the gonad, whereas in Ostreidae the digestive gland is the main organ related to this process (Gabbott & Head 1980), Among the rest of the tissues that constitute the soft parts in oysters, the adductor muscle is not considered to be an organ of reserve that should contribute to supply energy to the reproduction process, as was observed in Crassostrea madrassensis (Stephen 1980), Ostrea puelchana (Fernandez & De Vido de Mattio 1987) and C. gigas (Berthelin et al. 2000). The mantle, although it is considered as an organ of reserve, has always been studied as a whole together with the gonad. Few studies are focused on the contribution of the labial palps to the gametogenic cycle of C. gigas. An important variation in size and weight of this organ was observed during the gametogenic cycle, suggesting some possible role of this organ as a storage compartment during the gametogenic cycle.

Few studies describe in detail the biochemical changes of the somatic tissues that occur during the sexual maturation depending on the sex and the different stages of the gametogenic cycle of C. gigas. This study investigates the contribution of the biochemical components as energy substrates in relation to the gametogenic cycle of this species. This involves the biochemical study of digestive gland, labial palps and gonad in relation to sex and gametogenic stages during the annual reproductive cycle. The study of the biochemical composition of separate organs makes it possible to identify nutrient storage sites and determination of nutrient used during reproduction or during periods of low food availability. This study is part of a wider project for improving the quality and maximizing the efficiency of oyster seed production in breeding systems. Knowledge of the processes of accumulation and mobilization of the energy reserves in the somatic tissues is essential for a better understanding of the gametogenic process in this species.

MATERIALS AND METHODS

Specimens

Commercial size adult specimens of Crassostrea gigas (2.5 years old), were obtained from an oyster farm in Marennes-Oleron, La Tremblade, France. The sampling was carried out fortnightly from March 1997 until February 1998. Sample size was between 7 and 12 oysters. The data on water temperature, chlorophyll a and seston concentration were supplied by the hydrologic station at Auger (La Tremblade, France).

Biometric Measurements and Condition Indices

Each individual was cleaned in the laboratory and total weight, length, height and width were determined. Oysters were then opened and drained for 5 min on filter paper and the wet weight of the soft body parts was determined. The soft tissues were dissected and separated into mantle, adductor muscle, digestive gland, labial palps and gonad, and the wet weight of each was obtained. The tissues were then frozen and stored at -80[degrees]C until analyzed. Seasonal variation in the weight of gonad, digestive gland and labial palps were determined. Condition index of each part was calculated with the following formula: [CI.sub.organ] = 100x (wet weight organ/wet weight of the soft parts).

Gametogenic Activity

The middle part of the soft part containing the gonadal tissues was fixed in Bouin solution (75% picric acid, 20% formaldehyde and 5% acetic acid) for 2 h. The tissues were dehydrated in series of alcohols, embedded in paraffin wax, sectioned (5 [micro]m) and stained with Wheatley trichrome (Wheatley 1951). The gonad in oyster develops around the digestive gland; the growth and development of gonad tissue is closely associated with this organ. Colored characteristics and morphology of both tissues determine the macroscopic differences; a cone-shape is observed in the gonad of oysters as growth occurs. Dissection of the gonad begun at the cone tip, because this part of the tissue is far enough from the parts of the gonad located close to the digestive gland. Once the gonad and the digestive gland were separated, accidental remains of the tissues were carefully eliminated avoiding mutual contamination. Distal part of the gonad was used in histologic analysis. Slides were examined with microscope and each individual was then classified by sex and assigned a stage of gonadal development based on descriptions made by Lubet (1959) for Mytilus edulis and more recent descriptions for Crassostrea gigas (Pardo-Fernandez et al. 2002), summarized as follows: inactive (stage 0), early gametogenesis (stage I), late active (stage II), growing (stage IIIA1), mature (IIIA2), restoration gonad (stage IIIC), spawned (stage IIIB), postspawning and resorbing (stage IIID). The histologic undifferentiated individuals were defined as undetermined animals. We included in the description of the gametogenic stage of the females and males all properly gametogenic stages (II, IIIA1, IIIA2, IIIC and IIIB) and in the case of undetermined individuals, the maturity stages 0, I and IIID.

Biochemical Measurements

Total lipid, total protein, glycogen, triacylglycerols, phospholipids, total sterols, free sterols and sterol ester contents were determined in fresh samples of digestive gland, labial palps and gonad of each individual. The glycogen content was determined in 0.1-0.4 g of wet tissue homogenized in 10 mL of percloric acid, after elimination of proteic compounds. The glycogen was analyzed by the enzymatic method recommended by Keppler and Decker (1984). After enzymatic hydrolysis, the glucose produced was quantified by the colorimetric glucose oxidase method (Bergmeyer & Bernt 1974) using the commercial kit SPINREACT, using glucose for calibration standard. Total protein determination was based on the binding of Coomassie brilliant blue to protein (Bradford 1976), using bovine serum albumin as a standard. Total lipids were extracted from 0.2-0.6 g of wet tissue homogenized with 10 mL chloroform-methanol (2:1 v/v) according to Folch et al. (1957) method, modified by Beninger and Lucas (1984) and quantified by the Marsh and Weinstein (1966) method with triestearine as a reference. Phospholipids content was determined by organic phosphorus content of total lipids according to Raheja et al. (1973), the phosphatidylcholine (Sigma Aldrich) was used as reference standard. Triacylglycerols were determined on an aliquot of the total lipids by the Wahlefeld (1974) method, after enzymatic hydrolysis, the liberated glycerol was determined using a commercial kit Test-Combination triacylglycerols GOP-PAP (Boehringer Mannheim, No. 701,912), and the calibration curve was prepared from standard glycerol (Boehinger Mannheim). Total sterol content was determined on total lipids by the Cho (1983) method and quantified by colorimetric cholesterol oxidase method, using the Monostest Cholesterol kit (Boehringer Mannheim No. 237,574); free sterols were determined with the test Combination Free Cholesterol (Boehringer Mannheim, No. 310,328). Sterol esters were estimated by the difference between total sterols and free sterols. In each assay, calibration curves were prepared from composite standards (Boehringer Mannheim). Analysis was repeated on three different aliquots. Results were expressed in grams per wet weight tissue.

Data Analysis

Results were expressed as Mean [+ or -] SD. Each value is the mean of all individuals in each sampling separated by sex and gametogenic stage. A nonparametric test (Kruskall-Wallis test) followed by a Mann-Whitney test was also applied to determine the significance of differences between samples (Zar 1996). The degree of association between different parameters was assessed by Spearman rank-order correlations (a nonparametric test). Statistic version SPPS 10.0 was used for all statistical analyses.

RESULTS

Environmental Parameters

The environmental conditions of the area are given in Figure 1. The mean monthly temperature showed a seasonal cycle, with maxima of 22.3[degrees]C in August and minima of 8.2[degrees]C in February. Seasonal variations in Chlorophyll a, indicative of phytoplankton availability showed higher levels from late summer until late autumn. Seston fluctuated between 10.72 mg [L.sup.-1] and 85.90 mg [L.sup.-1] in summer and winter respectively.

[FIGURE 1 OMITTED]

Biometric Measurements and Condition Index

The biometric measurements and condition indices are shown in Table 1. The main period of growth either in size (length, height and width) or total weight were measured in the autumn period, however, the fresh weight significantly increased during the maturing period from March until August (P < 0.01). Fresh weight decreased after spawning (P < 0.01), recovering during autumn when the main period of phytoplankton blooms occurs. The values of the gonad condition index (GCI) and labial palps condition index (LPIC) presented opposite variations; the GCI increased during active gametogenic development (March-August), reaching the highest value in July (40.17), decreasing after a massive spawning at the end of the summer (8.65). The values of digestive gland condition index (DGCI) ranged between 8.43 and 16.36, showing large variations over the year and correlating positively with a LPIC and negatively with GCI (P < 0.01).

Spearman correlation showed positive correlation between all size measurements of the oysters and total and fresh weight (P < 0.01), whereas the GCI only correlated significantly with weight measurements (total and fresh) (P < 0.01).

Biochemical Composition

Seasonal changes in the content of biochemical components of undetermined individuals showed significant increase of the glycogen content (P < 0.01) in the three tissues studied from late summer until the beginning of autumn (Fig. 2), coinciding with gametogenic stage 0 and phytoplanktonic blooms. Reduction of this component in digestive gland and gonad was observed at the beginnings of the gametogenesis (stage IA) in autumn and also early in winter when the availability of nutrients was low. In females, the glycogen content of labial palps and digestive gland decreased significantly and gradually from stage II until stage IIIC (P < 0.01), reaching its minimum level (Fig. 3); the content of this component in gonad presented a large and significant reduction in stage IIIA1 (P < 0.01), remaining at low levels until stage IIIB. The males presented a significant reduction in glycogen greater than observed in females (P < 0.01), as was observed in digestive gland and gonad (Fig. 4). Labial palps showed a different trend with a slight increase in the middle of spring, coinciding with stages IIIA1 and II/A2, descending significantly in stage IIIC (P < 0.01); during the stage of active gametogenesis (spring-summer), the labial palps presented higher levels of glycogen than digestive gland and gonad. In females and males, the Spearman correlation showed a direct relationship between glycogen in gonad with GCI and LPCI, whereas that glycogen content in the gland correlated negatively with GCI (P < 0.01). Digestive gland and labial palps presented an increase in protein content in early autumn, then decline gradually until winter; the proteins content in the three tissues analyzed showed continuous increases and reductions throughout the autumn and during sexual maturation in both sexes (Fig. 2, 3, 4).

[FIGURES 2-4 OMITTED]

Lipid Classes

Table 2, Table 3 and Table 4 show the changes in the total lipids and main lipid classes of the gonad, digestive gland and labial palps of Crassostrea gigas throughout the year and gametogenic stages. The highest value was obtained in the mature gonad and coincided with phytoplanktonic blooms. A similar evolution of triacylglicerols (TG) during autumn-winter was detected in undetermined individuals in the three tissues studied. During autumn the levels of this component decreasing rising to 15%, 27% and 28% of the total lipids (TL) in the digestive gland, labial palps and gonad, respectively, coinciding with stages 0 and IA (Tables 3B, 4B and 2B). During the winter, coinciding with stage IB, TG increased to 70% of TL (Table 2B), then decreased again at the end of this period. The phospholipids (PL) content of undetermined individuals in the digestive gland, gonad and labial palps accounted for almost 50% of the total lipids at the beginning of autumn. Late in autumn, this component decreased gradually, mainly in the digestive gland, reaching levels of almost 27% (Table 3A). At the end of the winter, FL presented a slight increase in labial palps and gonad. The total levels of cholesterol in undetermined individuals at the end of summer were low, according to stage IIID. At the beginning of autumn, the levels increased in gonad and palps rising to 6% of TL in stage 0, decreasing during stage IA and remaining almost constant at 5% during the winter (Tables 2B and 4B).

When the reactivation of the gametogenesis in females and males (stage II) occurred at the beginning of spring, the content of TG in gonad decreased (49% and 25% of TL, respectively) (Table 2B). In males this component fell drastically with the advance of the spermatogenesis, whereas TG in females reached its maximum level (62% of TL) during stage IIIA2 (Table 2B). The TG content was significantly lower in the males (P < 0.01) from stage IIIA1 to the IIIB (spawning). The variation in TG of the digestive gland followed a similar trend to that observed in the gonad with males and females having an inverse profile (Table 3A and B). As gametogenesis progresses, the males undergo a gradual reduction in the TG until levels rise close to 6% of TL, whereas in females, an increase was observed during oogenesis, reaching up to 40% of the TL. In labials palps, the TG decreased in both sexes; the levels in females were constant from stage IIIA1 to stage IIIB, whereas in males, levels decreased until stage IIIC (Table 4). The variations of TG in palps in both sexes correlated positively with glycogen of digestive gland, palps and gonad, whereas TG in gonad of females correlated negatively with glycogen in digestive gland and gonad (P < 0.01).

The levels of phospholipids of the gonad in both sexes presented an increase during stage II. This increase was considerably higher in males (35% of TL). As the gametogenesis progressed, the proportion of PL in females ranges between 36% to 53% of TL, whereas males accumulated between 57% and 78%, presenting opposite trends to that observed in the TG (Table 2A). This indicates that the phospholipids were the principal lipidic component of the gonad in males. Unlike that observed in the triacylglicerols, females and males seem to have a similar trend to that measured in the phospholipids in gonad: a gradual increase from the spring to summer, the period in which the maximum levels were observed (Table 2A and B). The phospholipids content in digestive gland and labial palps increased progressively from the spring to summer, reaching the maximum values in stage IIIC, accumulating 73% and 53% of the TL, respectively (Tables 3B and 4B). Unlike what was observed in the gonad tissues, the phospholipids were the main lipid component of the digestive gland and labial palps during the major part of the year. The variation of PL in gonad correlated positively with TL of gonad and digestive gland and negatively with glycogen of palps (P < 0.01). The seasonal fluctuations of PL in gonad correlated positively with GCI, whereas TG in palps correlated negatively with GIC (P < 0.01).

At the beginning of spring, both sexes exhibited maximum levels of total cholesterol in gonad (Table 2A). Females did not show great changes as the oogenesis took place, whereas in the males the levels fell notably in stage IIIA1, with values significantly lower than those of the females (P < 0,01) (Table 2B). Late in March, the total cholesterol content of the digestive gland and palps increased, representing more than 10% of the TL. The content in sterols in the digestive gland in males increased until stage IIIA1, whereas in the palps it decreased (Tables 3 and 4).

Free cholesterol is the main form of the cholesterol in the three tissues studied, representing between 60% and 98% of total cholesterol (TC) (Tables 2, 3 and 4). At the beginning of the spring just before the sexual differentiation, free cholesterol showed a clear reduction. The free cholesterol of labial palps presented an important depletion in females at the beginning of the spring, with values down to 6% of the TC (Table 4A).

The sterol esters always presented low values in the gonad during the gametogenic cycle, except in the undetermined individuals and during stage IIIA1 in females at the middle of spring, rising to values close to 53% and 58% of TC (Table 2A). This component in gonad in undetermined individuals presented momentary increases in the middle of autumn coinciding with stage 0, at the beginning of spring and summer, coinciding with stages II y IIIC of males at the beginning of August coinciding with spawning in females. Sterol esters in the digestive gland presented lower levels than that in gonad, principally during winter. This component showed momentary increases in stage II of males and during females restorations in summer (Table 3B). The levels of sterol esters in labial palps were in general higher than those found in the digestive gland and gonad, especially during the spring-summer, a period in which they accumulate between 14% and 27% of TC (Table 4A). It is also necessary to emphasize the enormous increase of sterol esters observed at the beginning of spring, when the gametogenesis was reactivated (Tables 2 and 4). The sterol esters and free sterols of gonad and labials palps in females correlated negatively (P < 0.01).

DISCUSSION

Conditions indices and the biomass of the different tissues follow an annual variation, closely linked with the changes in the biochemical composition. During the spring, the increase in the total weight of Crassostrea gigas and the intense gametogenic activity are noted in the increased gonad condition index. The labial palps alone presented a marked fall in biomass and a pattern in their condition index inverse to the gonad condition index, which led to a negative correlation between the two. In this same season, the condition index of the digestive gland showed consecutive increases and decreases parallel to the concentration of chlorophyll a in the water column. During the autumn, the increase in the condition index of labial palps indicates its active participation as a reserve organ; changes in the content of glycogen, triacylglycerols, phospholipids and esterified cholesterol suggest that these substrata are used as energy reserves during gametogenic development. Sastry (1979) and Roman et al. (2002) demonstrated that the fall in the digestive gland index of pectinids coincides with gonadal growth as a result of a fast transfer of the energy reserves to this organ.

Our results show that during the autumn phytoplankton bloom, the digestive gland accumulates glycogen and lipids. The parallel increase of glycogen in labial palps and gonad appears to indicate a transfer from the digestive gland to these tissues. The importance of the digestive gland in the relationship between food availability and the accumulation of energy reserves in the different somatic tissues has been studied in diverse species of bivalves (Vassallo 1973, Barber & Blake 1985, Berthelin et al. 2000) and its role as a reserve organ for lipids and glycogen (Barber & Blake 1981, Barber & Blake 1985, Pazos et al. 1996, Strohmeier et al. 2000, Le Pennec et al. 2001, Roman et al. 2002, Pazos et al. 2003b). The accumulation of glycogen in the digestive gland not only appears to be linked to the availability of nutrients, but also to the quality of the same during the phytoplankton blooms (Deslous-Paoli & Heral 1988). In winter, the fall in glycogen, lipids and proteins in the digestive gland may be related to the stress involved in individuals with a scarcity of food and the drastic decrease in temperature at this time of the year (Beukema & De Bruin 1979, Beninger & Lucas 1984, Whyte et al. 1990, Ruiz et al. 1992).

The sexual differentiation in males at the outset of spring leads to decrease of over 70% in glycogen reserves, mainly in the digestive gland and gonad; in females, this decrease only involved 30%. These results indicate the use of glycogen in the synthesis of lipids, as suggested by the negative correlation between the gonad phospholipids and the glycogen in palps, as noted in other species of bivalves (Ansell 1974, Taylor & Venn 1979, Barber & Blake 1981, Martinez 1991, Frolov & Pankov 1992, Paez-Osuna et al. 1993, Strohmeier et al. 2000). The decrease in glycogen levels in males coincides with an increase of phospholipids in gonad significantly greater than that noted in females. This appears to indicate a greater energy and lipid requirement in males in view of the higher number of germinal cells formed (spermatogonias and spermatocytes I and II). In females, the greater decrease of glycogen in gonad and somatic tissues occurs in stage IIIA1, suggesting a high energy consumption in forming large amounts of lipid and protein reserves in the vitellogenic oocytes. These lipidic reserves ensure that embryonic and larval development concludes successfully in diverse species of bivalves (Holland 1978, Gallager et al. 1986, Lee & Heffernan 1991). As noted in numerous works on ostreidae, glycogen is mobilized during the fast proliferation of the sexual cells and also in periods when food concentration in the medium is low (Mann 1979, Maurer & Borel 1986, Deslous-Paoli & Heral 1988, Ruiz et al. 1992, Berthelin et al. 2000). The high reproductive effort in Crassostrea gigas entails a considerable consumption of energy, of which the glycogen appears to be the main contributor. A deficient accumulation of these substrata has been directly related to a low production of gametes (Kang et al. 2000). Mobilization of glycogen in females appears to be more important as from stage IIIA1, whereas in males this mainly occurs in stages II and IIIC.

During the periods of minimum gametogenic activity, triacylglycerols are the main class of lipids in gonad and labial palps; this occurs during the autumn-winter months, except in October and November when the triacylglycerols are exceeded by the phospholipids. The accumulation of triacylglycerols in the three tissues studied coincides with the second phytoplanktonic bloom, as noted in the digestive gland in different species of bivalve (Napolitano & Ackman 1992, Le Pennec et al. 2001, Pazos et al. 2003b). The triacylglycerols accumulated in the digestive gland during the winter appear to be transferred to the labial palps in early spring and, to a lesser extent, to the gonad. Because the gametogenic cycle progresses and the females reach stage IIIA1 (onset of vitellogenesis), the accumulation of triacylglycerols in the gonad coincides with a marked fall in palp triacylglycerols and, to a lesser extent, in the digestive gland; this situation points to a possible mobilization of triacylglycerols from the somatic tissues to the gonad during the vitellogenesis process. The molecules from the catabolism of triacylglycerols (fatty acids and glycerol) may be transferred to the gonad to form part of the more complex lipidic structures. Triacylglycerols seem to have a 2-fold function: they may store fatty saturated acids for energy purposes and/or serve as a temporary reservoir for polyunsaturated fatty acids used in the synthesis of structural lipids or on specific metabolic routes, as noted by Napolitano et al. (1988) and Pazos et al. (2003b). The accumulation of triacylglycerols in the gonad of female C. gigas during oogenesis is inversely related to the glycogen content in the different tissues analyzed. Whereas the triacylglycerols increase to maximum levels in the advanced stages of maturity (IIIA2 and IIIC), the glycogen of gonad, digestive gland and labial palps decreases to its minimum levels. This suggests that the mobilization of glycogen is necessary for forming triacylglycerols during vitellogenesis in females, as occurs in other bivalves (Barber & Blake 1985, Maurer & Borel 1986), possibly because of its participation in the synthesis de novo of saturated and monosaturated fatty acids (Waldock & Holland 1976, Kluytmans et al. 1985).

The increase in phospholipids in female gonad during gonadal maturation (spring-summer) may be related to its participation as structural membrane lipids and as part of the lipovitellines that accumulate oocytes as reserve for the cellular dividing process following fertilization (Frolov & Pankov 1992, Napolitano & Ackman 1992, Li et al. 1998). Unlike the triacylglycerols, it seems unlikely that the accumulation of phospholipids in the female gonad should derive from the mobilization of these components from the somatic tissues (digestive gland and palps), because during this period a simultaneous increase in phospholipids is noted in the three tissues analyzed. The origin of the gonadal phospholipids in female C. gigas could be in the mobilization of triacylglycerols and glycogen in the three somatic tissues analyzed. This mobilization of triacylglycerols in the somatic and gonadal tissue occurs as the maximum levels of glycogen are reached in gonad (stages IIIA2 and IIIC), which may be related to the energy requirement of the spermatozoids to maintain their mobility and functionality. The scarcity of food during spring may also induce mobilization of the triacylglycerols in the digestive gland, palps and gonad to act as an energy substratum and to form structural lipids.

Although triacylglycerols are described as the main lipidic form in the digestive gland of bivalves (Pollero et al. 1979, Lubet et al. 1985, Le Pennec et al. 2001, Pazos et al. 2003b), the results of this work show that, at certain points in time, phospholipids are considerably greater in proportion than triacylglycerols, as occurs in P. magellanicus (Napolitano & Ackman 1992) and in C. gigas (Allen & Conley 1982). These results reinforce the notion that the digestive gland in C. gigas is an essential organ for lipidic reserves, not only for triacylglycerols but also for phospholipids, which are to be used in various ways depending on sex, gametogenic stage, the nutritional and physiologic condition of the organism.

Sterols may be present in a free form or sterified with fatty acids. Free sterols form a part of membranes and play an important role in regulating their viscosity and permeability (Nes 1974). Our results show that the gonad in both sexes presents an increase in free cholesterol from stage IIIA1, decreasing following spawning, which suggests their inclusion in the gametes formed. Cholesterol seems to be the main sterol required for forming new cellular membranes during the proliferation of gametes and has an important function during embryogenesis and early phases of larval development (Krishnamoorthy et al. 1980, Pollero et al. 1983, Piretti et al. 1989, Pazos et al. 2003a). The decrease in sterified cholesterol in female gonad is probably caused by its inclusion in the oocyte membranes in the form of free cholesterol (Napolitano et al. 1988, Frolov & Pankov 1992, Pazos et al. 1996), as suggested by the negative correlation between both components. The fall in sterified cholesterol in labial palps parallel to the increase in female gonad during stage IIIA1, suggests that this component may be mobilized from these somatic tissues to the gonad. In Placopecten magellanicus, the loss of sterols in the adductor muscle, in the summer months, seems to be because they are transferred to the developing gonad (Idler et al. 1964). The negative correlation between sterified cholesterol in the female gonad with the free cholesterol in the labial palps and gonad may be interpreted as a possible interconversion between these two components.

Apart from participating in the formation of new cellular membranes during the gamete proliferation process and during somatic growth, sterols are precursor of all the steroid hormones that also control reproduction in marine invertebrates (Goad 1976, Krishnamoorthy et al. 1980, Piretti et al. 1989). Thus the variation in sterified cholesterol in gonad, at certain points in the gametogenic cycle, may indicate a possible hormonal relationship. Therefore, during the proliferation of gonias during stage I there is a marked fall of this component in undetermined individuals, a time when production, proliferation and propagation of gonias occur. Also in the course of the sexual differentiation of males (stage II), in early spring, the sterified cholesterol content in undetermined individuals is almost double the number found in the sexually differentiated individuals. At the onset of vitellogenesis (stage IIIA1), a notable increase of this component occurs, following which the gonad reaches stage IIIA2. Studies on steroids in female gonad of Patinopecten yessoensis and C. gigas show that the formation of vitelline seems to be regulated by the action of certain estrogens such as estradiol-17[beta] ([E.sub.2]), whose variation is synchronous with gonadal maturity. The [E.sub.2] seems to be one of the factors participating in the control of vitellogenesis in these two species of bivalves (Matsumoto et al. 1997, Osada et al. 2003); thus, the variation in cholesterol observed in our study may indicate its use as a precursor of an inducer of vitellogenesis.

ACKNOWLEDGMENTS

The author thanks Ian Emmett for the revision and translation into English and anonymous referees for their critical comments and suggestions on the manuscript. This project was carried out by EC research contract (FAIR IV). Project title: Breeding improvement of Crassostrea gigas by nutritional and gametogenesis control (GIGANUGA). A. Matus de la Parra was supported by a ICI fellowship from the AECI (Agencia Espanola de Cooperacion Internacional).

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ANA MATUS DE LA PARRA, (1) * OSCAR GARCIA (2) AND FUENCISLA SAN JUAN (3)

(1) Department of Ecology and Animal Biology, Faculty of Science, University of Vigo. Lagoas Marcosende, 36200, Vigo, Pontevedra, Spain; (2) Department of Biochemistry and Molecular Biology, Faculty of Pharmacia. University of Santiago de Compostela, Campo Sur, A Coruha, Spain; (3) Department of Biochemistry and Molecular Biology, Faculty of Science, University of Vigo, Pontevedra, Spain

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Biometric measurement of the samples individuals of Crassostrea
gigas: length, height, width, total weight (TW), fresh weight (FW)
and condition index of gonad (GCI), digestive gland (DGCI) and
labial palps (LPCI).

                 Length (mm)     Height (mm)     Width (mm)

    Date       Mean      SD     Mean     SD    Mean     SD

Mar 26, 1997    81.36    8.33   25.25   2.95   43.05    6.68
Apr 10, 1997    88.68   13.49   24.71   3.68   42.95    3.92
May 14, 1997    87.9     7.38   26.10   3.75   41.21    2.76
May 22, 1997    85.86    9.80   25.61   4.19   43.97    3.39
Jun 5, 1997     85.07    7.59   24.99   2.84   43.92    4.35
Jun 26, 1997    91.46   11.78   26.48   5.16   40.61    3.66
Jul 4, 1997     84.01    5.57   24.66   2.77   42.66    4.48
Jul 10. 1997    82.5     7.78   26.82   3.23   43.31    2.91
Jul 24, 1997    80.75   16.28   27.77   3.34   43.86    3.09
Aug 7, 1997     88.59   13.38   26.47   2.37   46.07    3.85
Aug 21, 1997    87.75    8.67   26.37   2.44   48.64    4.10
Sep 8, 1997     91.85    6.41   28.33   4.28   49.40    5.89
Sep 19, 1997    89.9     6.89   27.59   4.37   47.58    7.11
Oct 6, 1997     93.66   11.21   30.10   1.83   51.37    9.42
Oct 17, 1997    94.61    9.11   31.16   4.79   48.16   18.84
Nov 21, 1997    97.97    4.83   31.08   3.73   53.24    3.86
Dec 5, 1997     99.68   19.88   28.51   4.43   50.17    4.56
Dec 19, 1997   105.70   15.33   32.13   5.16   51.86    5.77
Jan 16, 1998    88.62    9.21   33.27   3.26   49.06    5.37
Jan 30, 1998   102.21    9.39   32.70   2.79   52.21    3.86
Feb 13, 1998    93.71   10.19   28.05   4.64   46.61    5.51

                   TW (g)          FW (g)

    Date        Mean     SD     Mean     SD     GCI    DGCI    LPCI

Mar 26, 1997    47.16    6.30    4.93   0.92    8.84   12.52   6.96
Apr 10, 1997    46.27    4.42    5.44   0.58    8.17   14.19   4.73
May 14, 1997    47.54    5.24    6.47   1.24   17.42   11.58   4.29
May 22, 1997    49.37    5.58    6.83   1.62   20.67   10.20   4.42
Jun 5, 1997     48.73    5.05    8.20   1.94   32.65    8.43   4.01
Jun 26, 1997    53.98    6.89    8.42   1.99   26.67   13.09   3.61
Jul 4, 1997     49.73    3.57    8.14   1.38   30.21   12.31   2.81
Jul 10. 1997    51.66    6.53    8.90   2.42   39.96   12.06   3.63
Jul 24, 1997    60.51    9.17   11.40   2.19   40.17   10.76   4.76
Aug 7, 1997     61.49    8.08   11.49   2.11   38.46   10.99   5.34
Aug 21, 1997    58.83    7.42    8.25   1.27    8.65   16.36   7.49
Sep 8, 1997     68.10    10.6    9.52   2.99    8.97   14.83   7.55
Sep 19, 1997    63.90   12.13    9.64   2.30    8.28   13.37   7.48
Oct 6, 1997     76.87   15.07   10.90   3.27   10.98   13.41   7.30
Oct 17, 1997    94.05   16.54   13.86   2.11   11.13    8.47   6.86
Nov 21, 1997   104.03   12.93   14.61   1.72   15.15   10.32   9.28
Dec 5, 1997     87.01   10.70   12.09   3.48    9.10   12.94   8.01
Dec 19, 1997   107.13   11.79   13.45   1.15   10.44   13.13   9.27
Jan 16, 1998    90.55   12.80   11.51   2.33   13.73   15.41   7.25
Jan 30, 1998   120.08   17.03   13.57   2.69   15.96   10.33   7.99
Feb 13, 1998    84.05   19.64    9.24   3.53   10.86   10.28   6.54

TABLE 2.
A. Seasonal variation of the major lipid classes (% of total lipids)
and total lipids content (mg/g wet weight) from gonad. B. Variation
by maturity stage of the major classes (% total lipids)
from gonad.

A

             Total lipids (mg/g wet weight)

             Females   Males   Undetermined

03/26/1997    59.58    81.04      62.96
04/10/1997    55.44    31.42
05/14/1997    45.82    34.39
05/22/1997    36.68    25.99
06/05/1997    50.64    34.14
06/26/1997    52.99    36.78
07/04/1997    59.63    59.79
07/10/1997    54.38    38.22
07/24/1997    57.62    50.15
08/07/1997    54.68    49.98
08/21/1997                        33.64
09/08/1997                        38.24
09/19/1997                        39.28
10/06/1997                        35.84
10/17/1997                        49.68
11/21/1997                        38.73
12/05/1997                        35.86
12/19/1997    46.86               39.73
01/16/1998    42.23               49.14
01/30/1999    44.68               40.79
02/13/1998    37.14               37.47

             Triacylglicerols (% total lipids)

             Females   Males   Undetermined

03/26/1997    22.82    25.01      28.90
04/10/1997    36.05    30.45
05/14/1997    43.37     9.42
05/22/1997    47.44     6.13
06/05/1997    52.54     7.94
06/26/1997    53.14     8.74
07/04/1997    49.58     5.00
07/10/1997    51.08     7.70
07/24/1997    34.57     4.44
08/07/1997    43.47     6.49
08/21/1997                        38.14
09/08/1997                        52.80
09/19/1997                        52.67
10/06/1997                         5.62
10/17/1997                         7.25
11/21/1997                         6.91
12/05/1997                        53.45
12/19/1997    60.62               63.90
01/16/1998    73.68               69.11
01/30/1999    61.27               64.69
02/13/1998    57.80               38.62

             Phospholipids (% total lipids)

             Females   Males   Undetermined

03/26/1997    22.82    35.02      45.12
04/10/1997    36.05    64.66
05/14/1997    43.37    57.79
05/22/1997    47.44    76.85
06/05/1997    52.54    67.27
06/26/1997    53.14    73.56
07/04/1997    49.58    57.87
07/10/1997    51.08    78.13
07/24/1997    34.57    76.05
08/07/1997    43.47    65.72
08/21/1997                        52.32
09/08/1997                        32.88
09/19/1997                        41.79
10/06/1997                        40.68
10/17/1997                        35.64
11/21/1997                        26.89
12/05/1997                        27.76
12/19/1997    60.62               23.21
01/16/1998    73.68               14.29
01/30/1999    61.27               22.89
02/13/1998    57.80               39.19

                Sterols (% total lipids)

             Females   Males   Undetermined

03/26/1997    3.55     4.69        7.87
04/10/1997    2.42     2.69
05/14/1997    3.14     3.10
05/22/1997    4.88     5.41
06/05/1997    3.29     4.28
06/26/1997    3.40     4.76
07/04/1997    2.16     2.72
07/10/1997    2.29     4.31
07/24/1997    2.98     2.63
08/07/1997    3.28     3.92
08/21/1997                         3.98
09/08/1997                         4.11
09/19/1997                         3.02
10/06/1997                         9.26
10/17/1997                         4.96
11/21/1997                         4.74
12/05/1997                         5.92
12/19/1997    4.62                 4.76
01/16/1998    3.86                 3.32
01/30/1999    4.99                 4.85
02/13/1998    6.33                 7.79

             Free Sterols (% total sterols)

             Females   Males   Undetermined

03/26/1997    53.85    59.17      46.99
04/10/1997    87.31    78.10
05/14/1997    41.97    79.33
05/22/1997    89.18    93.47
06/05/1997    77.40    91.19
06/26/1997    90.24    91.35
07/04/1997    76.18    78.46
07/10/1997    76.27    95.32
07/24/1997    89.74    92.98
08/07/1997    65.72    89.19
08/21/1997                        84.25
09/08/1997                        88.79
09/19/1997                        81.42
10/06/1997                        86.71
10/17/1997                        60.34
11/21/1997                        96.61
12/05/1997                        84.92
12/19/1997    92.83               95.75
01/16/1998    90.53               88.08
01/30/1999    88.23               96.85
02/13/1998    91.99               85.80

             Sterol Esters (% total sterols)

             Females   Males   Undetermined

03/26/1997    46.15    40.83      53.00
04/10/1997    12.22    21.90
05/14/1997    58.03    20.68
05/22/1997    10.84     6.47
06/05/1997     7.73    25.80
06/26/1997     9.75     8.65
07/04/1997    23.85    21.54
07/10/1997    23.74     4.68
07/24/1997    12.92     6.98
08/07/1997    34.28    10.80
08/21/1997                        15.81
09/08/1997                        11.24
09/19/1997                        18.53
10/06/1997                        13.28
10/17/1997                        39.66
11/21/1997                         3.38
12/05/1997                        15.08
12/19/1997     7.16                4.23
01/16/1998     9.47               11.90
01/30/1999    11.78                3.13
02/13/1998     8.02               14.20

B

             Triacylglicerols (% total lipids)

             Females   Males   Undetermined

IIID                              41.17
O                                 33.00
IA                                28.70
IB                                46.22
II            49.16    25.01
IIIA1         49.93    25.14
IIIA2         62.18     7.48
IIIC          50.15    10.14
IIIB          44.04     5.67

             Phospholipids (% total lipids)

             Females   Males   Undetermined

IIID                              56.65
O                                 58.47
IA                                36.02
IB                                29.94
II            28.63    33.26
IIIA1         43.64    57.44
IIIA2         45.42    68.68
IIIC          40.35    49.08
IIIB          48.74    71.58

                Sterols (% total lipids)

             Females   Males   Undetermined

IIID                               4.52
O                                  8.05
IA                                 5.05
IB                                 5.36
II            3.97     3.43
IIIA1         4.34     2.88
IIIA2         3.94     4.28
IIIC          2.74     2.80
IIIB          3.03     3.59

             Free Sterols (% total sterols)

             Females   Males   Undetermined

IIID                              84.80
O                                 70.22
IA                                78.73
IB                                90.29
II            78.60    59.17
IIIA1         70.77    86.96
IIIA2         82.36    84.18
IIIC          84.05    82.52
IIIB          76.65    88.46

             Sterol Esters (% total sterols)

             Females   Males   Undetermined

IIID                              15.20
O                                 29.78
IA                                21.27
IB                                 9.71
II            21.40    40.83
IIIA1         29.23    13.04
IIIA2         17.64    15.82
IIIC          15.95    17.48
IIIB          23.35    11.54

TABLE 3.
A. Seasonal variation of the major lipid classes (% of total lipids)
and total lipids content (mg/g wet weight) from digestive gland.
B. Variation by maturity stage of the major classes (% total
lipids) from digestive gland.

A

             Total lipids (mg/g wet weight)

             Females   Males   Undetermined

03/26/1997    47.02    53.79      57.37
04/10/1997    48.49    42.33
05/14/1997    34.45    34.77
05/22/1997    27.82    29.64
06/05/1997    39.10    32.48
06/26/1997    44.21    47.09
07/04/1997    44.29    34.47
07/10/1997    35.80    52.38
07/24/1997    48.61    63.86
08/07/1997    46.64    45.64
08/21/1997                        36.50
09/08/1997                        40.53
09/19/1997                        45.63
10/06/1997                        30.96
10/17/1997                        40.40
11/21/1997                        37.91
12/05/1997                        35.43
12/19/1997    40.53               34.47
01/16/1998    36.22               35.95
01/30/1998    36.82               34.56
02/13/1998    38.76               30.67

             Triacylglicerols (% total lipids)

             Females   Males   Undetermined

03/26/1997    18.41     9.49       5.82
04/10/1997    21.54    25.25
05/14/1997    30.40    18.83
05/22/1997    26.18    12.39
06/05/1997    21.26    12.57
06/26/1997    32.61     6.06
07/04/1997    39.72    11.36
07/10/1997    30.35    10.78
07/24/1997    26.89     8.22
08/07/1997    26.96    12.18
08/21/1997                        34.93
09/08/1997                        34.20
09/19/1997                        42.94
10/06/1997                         5.32
10/17/1997                         5.97
11/21/1997                         4.63
12/05/1997                        43.63
12/19/1997    58.29               55.46
01/16/1998    53.31               62.71
01/30/1998    58.60               54.03
02/13/1998    47.71               34.71

             Phospholipids (% total lipids)

             Females   Males   Undetermined

03/26/1997    58.64    51.59      51.47
04/10/1997    52.28    54.96
05/14/1997    48.53    55.60
05/22/1997    55.84    70.26
06/05/1997    54.23    62.95
06/26/1997    45.83    51.07
07/04/1997    52.21    78.85
07/10/1997    53.85    49.26
07/24/1997    64.24    53.11
08/07/1997    58.36    66.28
08/21/1997                        55.06
09/08/1997                        50.18
09/19/1997                        44.97
10/06/1997                        56.78
10/17/1997                        37.45
11/21/1997                        46.07
12/05/1997                        36.44
12/19/1997    24.13               27.25
01/16/1998    22.69               20.93
01/30/1998    28.33               28.19
02/13/1998    21.71               33.77

                Sterols (% total lipids)

             Females   Males   Undetermined

03/26/1997     7.67     4.66       5.58
04/10/1997    10.08    10.70
05/14/1997     5.59     4.67
05/22/1997     8.36     5.04
06/05/1997     6.21     8.19
06/26/1997     5.89     4.62
07/04/1997     4.86     5.14
07/10/1997     5.99     3.85
07/24/1997     3.28     1.87
08/07/1997     4.53     5.51
08/21/1997                         7.29
09/08/1997                         5.21
09/19/1997                         5.95
10/06/1997                         9.97
10/17/1997                         5.39
11/21/1997                         5.85
12/05/1997                         6.63
12/19/1997     5.74                5.86
01/16/1998     5.16                5.51
01/30/1998     6.09                6.03
02/13/1998     6.11               10.68

             Free Sterols (% total sterols)

             Females   Males   Undetermined

03/26/1997    86.01    94.13      74.67
04/10/1997    81.15    88.19
05/14/1997    80.95    80.32
05/22/1997    97.85    92.98
06/05/1997    92.87    96.32
06/26/1997    94.80    95.06
07/04/1997    63.66    83.12
07/10/1997    89.82    92.44
07/24/1997    93.11    77.80
08/07/1997    93.47    95.19
08/21/1997                        86.36
09/08/1997                        95.46
09/19/1997                        95.93
10/06/1997                        97.73
10/17/1997                        89.27
11/21/1997                        89.53
12/05/1997                        94.67
12/19/1997    93.11               94.37
01/16/1998    97.13               95.50
01/30/1998    94.27               92.23
02/13/1998    95.14               95.63

             Sterol Esters (% total sterols)

             Females   Males   Undetermined

03/26/1997    14.00     5.87      25.33
04/10/1997    18.85    11.81
05/14/1997    19.04    19.68
05/22/1997     2.15     7.05
06/05/1997     7.13     3.68
06/26/1997     5.20     4.92
07/04/1997    36.34    16.88
07/10/1997    10.18     7.56
07/24/1997     6.89    22.20
08/07/1997     6.51     4.81
08/21/1997                        13.73
09/08/1997                         4.50
09/19/1997                         4.06
10/06/1997                         2.27
10/17/1997                        10.73
11/21/1997                        10.49
12/05/1997                         5.32
12/19/1997     6.91                5.62
01/16/1998     2.68                3.99
01/30/1998     5.72                7.71
02/13/1998     4.83                4.37

B

             Triacylglicerols (% total lipids)

             Females   Males   Undetermined

IIID                              30.37
O                                 15.68
IA                                23.90
IB                                39.10
IIID          39.90     9.49
IIIA1         25.35    22.83
IIIA2         26.40    14.31
IIIC          44.29    16.30
IIIB          28.36     9.59

             Phospholipids (% total lipids)

             Females   Males   Undetermined

IIID                              53.20
O                                 47.59
IA                                47.07
IB                                38.43
IIID          38.90    51.59
IIIA1         52.94    55.63
IIIA2         52.11    64.00
IIIC          51.70    72.90
IIIB          62.21    62.66

                Sterols (% total lipids)

             Females   Males   Undetermined

IIID                               6.40
O                                  6.65
IA                                 6.82
IB                                 6.83
IIID          6.98      6.92
IIIA1         7.10     10.20
IIIA2         6.38      5.70
IIIC          6.12      4.93
IIIB          4.52      4.46

             Free Sterols (% total sterols)

             Females   Males   Undetermined

IIID                              87.75
O                                 92.53
IA                                92.93
IB                                94.34
IIID          91.43    78.38
IIIA1         91.44    94.77
IIIA2         91.34    88.27
IIIC          84.80    91.23
IIIB          91.89    88.84

             Sterol Esters (% total sterols)

             Females   Males   Undetermined

IIID                              12.25
O                                  7.47
IA                                 7.07
IB                                 5.66
IIID           8.57    21.62
IIIA1          8.56     5.23
IIIA2          8.66    11.73
IIIC          15.20     8.77
IIIB           8.11    11.16

TABLE 4.
A. Seasonal variation of the major lipid classes (% of total lipids)
and total lipids content (mg/g wet weight) from labial palps. B.
Variation by maturity stage of the major classes (% total lipids)
from labial palps.

A

             Total lipids (mg/g wet weight)

             Females   Males   Undetermined

03/26/1997    47.23    65.31      57.76
04/10/1997    81.59    76.23
05/14/1997    58.59    45.28
05/22/1997    28.22    37.41
06/05/1997    42.61    38.01
06/26/1997    33.57    39.77
07/04/1997    45.61    44.00
07/10/1997    36.12    35.92
07/24/1997    32.67    42.79
08/07/1997    42.43    53.14
08/21/1997                        40.60
09/08/1997                        38.78
09/19/1997                        41.09
10/06/1997                        37.30
10/17/1997                        42.06
11/21/1997                        43.51
12/05/1997                        36.29
12/19/1997    43.66               43.13
01/16/1998    46.44               47.71
01/30/1998    44.04               38.86
02/13/1998    49.92               43.75

             Triacylglicerols (% total lipids)

             Females   Males   Undetermined

03/26/1997    39.83    31.30      14.99
04/10/1997    28.71    31.48
05/14/1997    25.68    20.94
05/22/1997    28.87    31.27
06/05/1997    21.52    15.67
06/26/1997    33.64    13.66
07/04/1997    24.35    16.85
07/10/1997    19.10    12.57
07/24/1997    23.76    17.39
08/07/1997    31.11     9.02
08/21/1997                        52.78
09/08/1997                        53.41
09/19/1997                        56.55
10/06/1997                         5.76
10/17/1997                         6.43
11/21/1997                         5.82
12/05/1997                        48.45
12/19/1997    66.65               61.06
01/16/1998    57.24               67.06
01/30/1998    67.40               58.54
02/13/1998    70.22               42.43

             Phospholipids (% total lipids)

             Females   Males   Undetermined

03/26/1997    33.88    37.88      36.17
04/10/1997    19.96    21.45
05/14/1997    18.40    32.21
05/22/1997    52.11    35.78
06/05/1997    25.55    39.07
06/26/1997    48.21    45.43
07/04/1997    44.15    53.47
07/10/1997    60.85    65.25
07/24/1997    63.43    58.57
08/07/1997    44.54    33.93
08/21/1997                        37.36
09/08/1997                        38.08
09/19/1997                        38.82
10/06/1997                        37.22
10/17/1997                        29.01
11/21/1997                        25.01
12/05/1997                        34.24
12/19/1997    24.26               21.06
01/16/1998    18.38               17.73
01/30/1998    20.43               24.56
02/13/1998    17.15               27.39

                Sterols (% total lipids)

             Females   Males   Undetermined

03/26/1997    12.41    14.54       6.49
04/10/1997     1.80     1.52
05/14/1997     2.36     2.15
05/22/1997     4.98     3.70
06/05/1997     4.76     5.16
06/26/1997     5.29     4.96
07/04/1997     2.35     3.15
07/10/1997     2.42     3.32
07/24/1997     5.48     3.32
08/07/1997     3.25     0.71
08/21/1997                         3.92
09/08/1997                         4.41
09/19/1997                         2.48
10/06/1997                         5.68
10/17/1997                         3.44
11/21/1997                         3.34
12/05/1997                         5.94
12/19/1997     4.56                3.83
01/16/1998     4.05                3.08
01/30/1998     4.47                4.86
02/13/1998     4.76                6.72

             Free Sterols (% total sterols)

             Females   Males   Undetermined

03/26/1997     6.49    44.50      60.73
04/10/1997    75.87    75.70
05/14/1997    76.11    74.22
05/22/1997    82.60    74.87
06/05/1997    73.15    84.14
06/26/1997    75.71    72.83
07/04/1997    83.27    75.87
07/10/1997    80.12    85.52
07/24/1997    80.23    78.85
08/07/1997    77.68    71.26
08/21/1997                        79.85
09/08/1997                        91.36
09/19/1997                        79.35
10/06/1997                        97.94
10/17/1997                        93.94
11/21/1997                        92.94
12/05/1997                        81.68
12/19/1997    83.29               85.85
01/16/1998    81.50               94.76
01/30/1998    89.13               95.97
02/13/1998    92.60               83.33

             Sterol Esters (% total sterols)

             Females   Males   Undetermined

03/26/1997    93.53    55.50      39.27
04/10/1997    24.13    24.30
05/14/1997    23.88    25.78
05/22/1997    17.40    25.13
06/05/1997    26.86    15.86
06/26/1997    24.29    27.17
07/04/1997    16.71    24.14
07/10/1997    19.88    14.48
07/24/1997    19.77    21.18
08/07/1997    22.30    13.09
08/21/1997                        20.39
09/08/1997                         8.67
09/19/1997                        20.65
10/06/1997                         2.03
10/17/1997                         6.02
11/21/1997                         7.11
12/05/1997                        18.33
12/19/1997    16.71               14.17
01/16/1998    18.49                5.24
01/30/1998    10.86                4.03
02/13/1998     7.40               17.50

B

             Triacylglicerols (% total lipids)

             Females   Males   Undetermined

IIID                              50.19
O                                 27.59
IA                                33.37
IB                                42.49
II            52.13    32.36
IIIA1         24.12    30.19
IIIA2         35.45    24.23
IIIC          21.99     9.94
IIIB          24.62    13.48

             Phospholipids (% total lipids)

             Females   Males   Undetermined

IIID                              47.36
O                                 25.79
IA                                35.11
IB                                27.25
II            23.55    37.88
IIIA1         37.98    21.22
IIIA2         41.12    38.91
IIIC          48.32    53.22
IIIB          55.60    53.95

                Sterols (% total lipids)

             Females   Males   Undetermined

IIID                               4.52
O                                  8.50
IA                                 5.05
IB                                 5.36
II            6.37     14.54
IIIA1         4.25      1.60
IIIA2         4.92      3.54
IIIC          3.63      3.61
IIIB          3.86      3.05

             Free Sterols (% total sterols)

             Females   Males   Undetermined

IIID                              83.19
O                                 87.43
IA                                85.60
IB                                89.03
II            73.76    44.50
IIIA1         79.82    73.17
IIIA2         80.39    74.92
IIIC          78.19    70.43
IIIB          79.68    82.25

             Sterol Esters (% total sterols)

             Females   Males   Undetermined

IIID                              16.81
O                                 12.57
IA                                14.40
IB                                10.97
II            26.24    55.50
IIIA1         20.18    26.83
IIIA2         19.61    25.08
IIIC          21.81    29.57
IIIB          20.32    17.75