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Originally published In Press as doi:10.1194/jlr.M800159-JLR200 on June 24, 2008

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Journal of Lipid Research, Vol. 49, 2365-2378, November 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Lipid levels in sperm, eggs, and during fertilization in Xenopus laevis

Douglas W. Petcoff{dagger}, William L. Holland* and Bradley J. Stith1,*

* Department of Biology, University of Colorado Denver, Denver, CO
{dagger} Metropolitan State College, Denver, CO

Published, JLR Papers in Press, June 24, 2008.

This work has been supported by National Science Foundation Grant IBN-0110609, and University of Colorado Denver Downtown Campus Undergraduate Research Opportunity Program grants.

1 To whom correspondence should be addressed. e-mail: brad.stith{at}cudenver.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 LIPID SIGNALING RELATED TO...
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Critical developmental periods, such as fertilization, involve metabolic activation, membrane fusion events such as sperm-egg or plasma membrane-cortical granule merger, and production and hydrolysis of phospholipids. However, there has been no large-scale quantification of phospholipid changes during fertilization. Using an enzymatic assay, traditional FA analysis by TLC and gas chromatography, along with a new method of phospholipid measurement involving HPLC separation and evaporative light-scattering detection, we report lipid levels in eggs, sperm, and during fertilization in Xenopus laevis. Sperm were found to contain different amounts of phospholipids as compared with eggs. During fertilization, total phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, and phosphatidylserine decreased, and ceramide increased, whereas there was no change in phosphatidylcholine, cardiolipin, or phosphatidylethanolamine. FA analysis of phospholipids found numerous changes during fertilization. Because there is an increase in sn-1,2-diacylglycerol at fertilization, the FAs associated with this increase and the source of the increase in this neutral lipid were examined. Finally, activation of phospholipase C, phospholipase D, phospholipase A2, autotoxin, and sphingomyelinase at fertilization is discussed.

Supplementary key words diacylglycerol • phospholipase C • phospholipase D • phospholipase A2 • autotaxin • sphingomyelinase

Abbreviations: CL, cardiolipin; DAG, diacylglycerol; DHA, docosahexaenoic acid; ELSD, evaporative light-scattering detection; EPA, eicosapentaenoic acid; IP3, inositol 1,4,5-trisphosphate; LPC, lysophosphatidylcholine; MBS, modified Barth's medium; MUFA, monounsaturated fatty acid; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLD, phospholipase D; PS, phosphatidylserine; SM, sphingomyelin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
LIPID SIGNALING RELATED TO...
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Major goals of the LIPID Metabolites and Pathways Strategy (LIPID MAPS) are to "separate and detect all of the lipids in a specific cell," "to quantitate each of the lipid metabolites present and determine the changes in their levels," and to study lipid metabolic pathways during biological events (1). Characterizing changes in the lipid composition during defined biological events is part of the concept of "functional lipidomics" (2). These fundamental, descriptive studies will facilitate our understanding of how lipids regulate membrane fluidity and fusion, lipid rafts, the cytoskeleton, kinases, and protein binding to membranes (e.g., 3–5) to induce pleiotropic cellular effects.

About 15 years ago, quantification of lipid mass changes during maturation of the oocyte to the fertilizable egg, and during fertilization of the egg by sperm, was begun in Xenopus laevis (6–9). Critical developmental events can be isolated in vitro with this model system, and large numbers of gametes can be obtained to provide sufficient amounts of second messengers and lipids. During fertilization, membrane bending and fusion are associated with the acrosome reaction of sperm (wherein the large sperm acrosome undergoes exocytosis upon interaction with layers that cover the egg), with sperm-egg merger, and with subsequent fertilization events such as cortical granule exocytosis (10, 11). Furthermore, both Xenopus and human eggs are arrested in meiosis II (12).

Compared with oocytes, one disadvantage of studying eggs is that they have a short life, much less than the estimated time for lipid precursor incorporation to near equilibrium levels (6, 7). Thus, lipid analysis based on label incorporation or turnover is not reliable, and methods that directly measure lipid mass are needed for fertilization studies.


   LIPID SIGNALING RELATED TO FERTILIZATION
TOP
 ABSTRACT
 INTRODUCTION
 LIPID SIGNALING RELATED TO...
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In terms of lipids in sperm, capacitation (13), alcoholism (14), storage (15), and passage through the epididymis (16) or cervical mucus (17) have been found to alter lipid composition. Although there are many studies on lipids in sperm, there has been no comprehensive, comparative study on the lipid composition of eggs versus sperm, or their changes during fertilization. Thus, the following lipids have been examined during fertilization in Xenopus laevis.

Lysophosphatidylcholine (LPC) has been suggested to induce membrane fusion, and it can induce the acrosome reaction of sperm (18). LPC can stimulate tyrosine kinases (18), phospholipase C (PLC), and map kinases (19). Phospholipase A2 (PLA2) breaks down phosphatidylcholine (PC) to LPC, and it is interesting to note that a PLA2 inhibitor inhibited fertilization envelope formation during sea urchin fertilization but did not block the increase in [Ca++]in or acid efflux/zygote alkalinization (20). Furthermore, Riffo and Parraga (21) suggest that PLA2 activation is important in sperm-egg fusion, inasmuch as LPC addition induced the interspecies fusion of human sperm and the hamster egg and anti-PLA2 antibodies were inhibitory.

Plasmalogens, in which instead of an ester bond, a vinyl ether bond is located at the sn-1 position of the glycerol backbone, have been suggested to protect sperm from oxidation and to play a role in sperm-egg fusion (22, 23).

Cardiolipin (CL) is found predominantly (80%) in mitochondria, whereas about 16% is in the nuclear envelope and 4% in the plasma membrane (24). CL binds calcium with higher affinity than PC, phosphatidylethanolamine (PE), or phosphatidylserine (PS) (24), and this binding is associated with stabilization of the hexagonal phase of the membrane and destabilization of the lamellar phase. Thus, CL may alter membrane properties to facilitate membrane bending and fusion during sperm union with the egg (25). In fact, CL has been found to enhance sperm-vesicle fusion (26), antibodies to sperm surface CL may decrease fertility (27, 28), and the lipid may be concentrated at the surface of sperm in the region of fusion (29).

The presence of certain lipids affects the stability of membrane rafts (detergent-insensitive density membrane fractions with high levels of saturated phospholipids) (30). For example, sphingomyelin (SM) stabilizes rafts and may play a role in membrane fusion (31). Membrane rafts have been described in Xenopus laevis eggs (32), and it is believed that sperm-egg interaction occurs through these rafts (33–35).

Sperm membranes obtain large amounts of phospholipids with polyunsaturated fatty acids (PUFAs) as they move through the epididymis (16). This decrease in lipid saturation has been suggested to increase membrane fluidity, affect rafts, and provide protection against oxidation (36). One PUFA, 22:6n3 (docosahexaenoic acid or DHA), is prevalent in sperm (16, 36) and has been found to decrease the amount of diacylglycerol (DAG) in rafts, possibly induce raft SM degradation to ceramide, and increase apoptosis in cancer cells (30).

Inasmuch as C2-ceramide or an inhibitor of alkaline ceramidase can enhance calcium ionophore induction of the acrosome reaction in boar sperm, ceramide may be involved in the acrosome reaction of sperm (37). Ceramide can be produced by degradation of SM, and SM inhibits whereas ceramide facilitates sperm capacitation (38).

The short half-life of eggs (39) is due to ceramide that induces apoptosis (40, 41). Ceramide may also form membrane channels for proteins like cytochrome C, and stabilize membrane rafts (42). The early embryo is resistant to apoptosis, owing to low levels of ceramide (43, 44); however, there has been no measurement of ceramide during fertilization.

Activation of PLC has been noted as the central pathway in fertilization. Activation of this enzyme and production of inositol 1,4,5-trisphosphate (IP3) and sn-1,2 diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (PIP2) may be sufficient to induce all events of fertilization [for Xenopus: (7, 8, 45, 46)]. Much work by Sato and associates (46) suggests that Xenopus sperm activate Src tyrosine kinase, which, in turn, activates PLC-{gamma}.

Our prior work showed that sn-1,2-DAG mass increased 48 pmol per cell (from 62 to 110 pmol/cell) and peaked at about 10–15 min after insemination (9). A translocation of DAG-dependent protein kinase C (PKC) {alpha} and β to a membrane fraction was also noted. PKC may be involved in resumption of endocytosis (47, 48), cortical contraction, cortical granule exocytosis, chromosome decondensation, nuclear envelope and Golgi reformation, and cleavage furrow formation in Xenopus laevis (49, 50).

DAG may act independently of PKC; for example, it may play a role in sperm-egg fusion in the sea urchin, because neomycin (which presumably inhibits DAG production by binding to PIP2) inhibits sperm-egg fusion but calcium chelators do not (51). One DAG species, 18:0/20:4 DAG, induces fusion of vesicles to form a nuclear envelope around sperm DNA in the egg cytoplasm (a fertilization event) (52).

By comparing the time course and size of the increase in DAG mass to the increase in IP3 mass at fertilization [the IP3 increase is about 280 times lower and peaks ~5–10 min before that of DAG; (7, 8)], and considering that precursor PIP2 mass is never greater than 2 pmol per Xenopus zygote (45), production of 48 pmol of DAG from PIP2 would be unlikely (9). Because choline mass increases on a magnitude equivalent to the increase in DAG, we suggested that breakdown of PC by phospholipase D (PLD) is the source of DAG (9). PLD would hydrolyze PC to phosphatidic acid (PA), which would be dephosphorylated to DAG (53). There was no detectable change in phosphocholine, and there is no known eukaryotic PC-specific PLC that would produce DAG and phosphocholine from PC. To search for the source of the DAG increase at fertilization, we compare decreases in individual FAs in phospholipids with increases in those in DAG.

In summary, we report the mass of major phospholipid classes and their FAs: CL, PE, PS, phosphatidylinositol (PI), PC, LPC, and SM in Xenopus laevis sperm, eggs, and through fertilization. Lipids are analyzed by three methods: an enzymatic assay of ceramide by DAG kinase, a traditional approach involving TLC followed by gas chromatography, and a new method involving HPLC separation and evaporative light-scattering detection (ELSD) (54).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 LIPID SIGNALING RELATED TO...
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Xenopus gametes and fertilization
Fertilization protocols have been published (7–9), but are briefly described here. To obtain eggs, Xenopus females were primed 3–10 days before use with 100 IU of pregnant mare's serum gonadotrophin (Calbiochem-EMD Biosciences) in 0.1 mg/ml BSA (Sigma Chemical Co.). Then, 8–12 h before use, primed females were injected with 850 IU of human chorionic gonadotrophin (Sigma Chemical Co.) to induce ovulation. Eggs were kept in 100% modified Barth's medium (MBS) (pH 7.5; 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 0.82 mM MgSO4·7H2O, 0.33 mM Ca(NO3)2·4H2O, 0.41 mM CaCl2·2H2O). Just before fertilization, eggs were rinsed twice with and then placed into 1 ml of 10% MBS. To obtain a suspension of sperm, two testes from a Xenopus laevis male were minced in 2.4 ml of 100% MBS. For fertilization, 167 µl of the minced testes solution (~9.47 million sperm) was added to the eggs. The rate and success of insemination were recorded by evaluation of video tapes that record the percentage of cells that undergo gravitational rotation (the dark animal pole rotates upward by 15 min after insemination) and cleavage (at about 100 min), typically a 100% success rate.

Extraction of lipids from Xenopus cells
Depending upon the phospholipid to be measured, different numbers of eggs or zygotes were extracted: for PE and PC, groups consisting of 3 eggs were used. For PI and SM, 15 eggs per group were used, and for PS, PI, and SM, 80 eggs were used. For analysis of sperm, 1 ml of testes solution (~56.7 million sperm) was extracted.

Because our method of lipid extraction for major phospholipid classes has been described (54), we briefly note that 40 s before the desired time point, excess MBS was removed and the eggs or zygotes were washed with 10% MBS (there was no washing for the 1 ml sperm suspension). For the zygote samples, we chose 5 min after insemination because we have found that PLC activation and DAG increased at this time (8, 9). Then, 1 ml of 1:2 chloroform-methanol was added and cells were homogenized with 20 strokes in a Dounce homogenizer (prefilled with N2). The mortar and pestle were then washed with 0.5 ml chloroform. This was followed by two washes with 0.5 ml of 1 M NaCl (our experience suggests that high salt drives anionic lipids into the organic phase, whereas acidic methanol-chloroform can degrade lipids). The extracts were centrifuged (clinical centrifuge; 500 g, 2 min), and the organic layer was transferred to a fresh, chilled test tube and kept under N2.

HPLC separation and ELSD of major phospholipid classes
After drying under N2 (N-Evap; Organomation Associates, Berlin MA), the sample was resolubilized in 80:19:1 chloroform-methanol-water and injected into a normal-phase silica column on a dual pump HPLC (Rainen HPXL) (54). A ternary gradient of chloroform with increasing amounts of methanol and water was used to elute lipids (54). A Sedex 55 evaporative light scattering detector (Richard Scientific) was used to record the mass of phospholipids. At our settings (54), the detector has a practical lower limit of about 1 nmol (or 1 µg; using an average phospholipid of 780 Da and a minimal peak size of about 50,000 µV-sec). We used the mass of standards (Avanti Polar Lipids, Inc., Alabaster AL), the ELSD response, and an average molecular mass for each phospholipid to calculate the amount of the Xenopus phospholipid in nanomoles. Average molecular mass was: for PC, 760 Da; for PE, 757 Da; for PI, 909 Da; for SM, 745 Da; and for PS, 810 Da.

FA analysis of lipids
For analysis of FAs connected to the various lipids, Xenopus eggs or zygotes (5 min after insemination) were extracted for lipids (see above) and lipid classes were separated by TLC (55). Individual lipids were then transesterified (3 N methanolic-HCl, sealed vials, under nitrogen, at 100°C for 45 min), and the resulting FA methyl esters were extracted (hexane, 0.05% butylated hydroxytoluene), separated, and quantified by capillary gas chromatography (Hewlett-Packard model 6890, Wilmington, DE), using a 30 m DB-225MS capillary column (J and W Scientific, Folsom, CA) and a flame-ionization detector (55). Comparisons with known FA standards identified peaks, and standards for recovery efficiency were included. The data for the mole percent of each FA represent the amount of a FA multiplied by 100 and divided by the total FA in that particular lipid class. Data for the mole percent of a class of phospholipid are the total amount in the lipid class multiplied by 100 and divided by the total of all major phospholipid classes.

For sperm DAG FA analysis, two testes were minced in 2.4 ml of 100% MBS, and 0.5 ml of this sperm suspension was extracted for lipids with 2:1 chloroform-methanol (followed by phase separation and chloroform isolation as noted above). This amount of sperm should produce amounts of lipid similar to the egg sample. For eggs, 10 cells were placed in 1 ml of 100% MBS and extracted as with sperm. Zygotes were obtained at 12 min after insemination (when maximal levels of DAG are achieved) (9), and lipids were extracted as noted above. For zygotes, fertilization was achieved by addition of 50 µl of sperm suspension in 100% MBS to groups of 10 eggs in 300 µl of 20% MBS. Before extraction, excess sperm were washed away. FA species of DAG were determined by gas chromatography as noted above.

Ceramide mass measurement
The amount of ceramide was determined by use of a DAG kinase assay developed by Bell and associates [(56); for a detailed procedure using Xenopus oocytes, eggs, and zygotes, see Refs. 9, 57)]. Lipids were extracted as noted above and were phosphorylated with [32P]ATP (ICN, Costa Mesa, CA) and DAG kinase (EMD/Calbiochem, Inc., La Jolla, CA). The labeled ceramide phosphate was purified by TLC, and the radioactivity was quantified by liquid scintillation counting. Standards (Avanti Polar Lipids) were used to calculate the amount of ceramide per cell.

Statistics
Data were analyzed by two-sided Student's t-test, which was calculated by Sigmaplot 2001 (version 7.101). The average ± the standard error, along with the sample size (e.g., n = 3), are stated in the text and shown in the figures.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 LIPID SIGNALING RELATED TO...
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lipids in Xenopus sperm and eggs
Using our new method of phospholipid analysis by HPLC and ELSD, sperm were found to contain a very different ratio of phospholipids than that found in eggs (Fig. 1 ). Approximately equal volumes of cells were used for comparison. In a second comparison, data from sperm and eggs were transformed into mole percent and compared directly (Fig. 2 ).


Figure 1
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  		Fig. 1. Xenopus sperm and egg phospholipids. Cells were extracted, separated by HPLC, and mass amounts were recorded by evaporative light-scattering detection (ELSD) and use of a standard line for each lipid. The values for 1 million sperm (B), the approximate volume of one Xenopus egg, were used to facilitate comparison with the eggs (A). PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine; SM, sphingomyelin. Error bars indicate SEM.

 

Figure 2
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  		Fig. 2. A comparison of Xenopus egg and sperm phospholipids. Based on HPLC/ELSD methodology, the mole percent of the major classes of phospholipids from Xenopus eggs (open bar) and from sperm (hatched bar) were compared. The amount of each phospholipid was multiplied by 100 and divided by the total mass of all major lipids. When sperm were compared with eggs, significant differences were noted with an asterisk (P < 0.05).

 
Sperm have ~40% higher levels of PE than eggs, but sperm have much less PC (only 25% of the PC found in eggs). There were ~7-fold higher amounts of PS and ~3-fold greater amounts of SM in sperm than in eggs. There was no obvious difference in the relative amounts of PI in eggs or sperm.

Because DAG is an important second messenger, the total amount of DAG (all diesters of glycerol, e.g., enantiomeric sn-1,2/2,3 and isomeric sn-1,3) determined by TLC/gas chromatography was 531 pmol in Xenopus eggs, 571 pmol in zygotes (12 min after insemination), and 1,238 pmol in sperm (a sperm sample estimated to be the same volume as one Xenopus egg) (Fig. 3A ). This increase in total DAG (40 pmol) is similar to that we reported earlier for sn-1,2 DAG (48 pmol), which was measured by the DAG kinase assay (9). If basal levels of the sn-1,2 form of DAG are about 62 pmol (9), and total DAG is 531 pmol, then the sn-1,2 form of DAG makes up only ~12% of all DAG forms.


Figure 3
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  		Fig. 3. A comparison of Xenopus egg and sperm sn-1,2-diacylglycerol FAs. In an analysis by gas chromatography, the different diacylglycerol (DAG) FAs are shown on the abscissa based on the numbering system noted in Table 1. The amount of each DAG FA in egg (first bar) or sperm (second bar of the pair) is shown (A). To be able to compare relative levels of FA in the sperm and egg, the amount of 18:1n9 DAG in sperm was made equivalent to the amount in one egg. Thus, the values for the different molecules of sperm DAG were divided by 109 (this final amount is equivalent to 0.00458 ml). In another method of comparison (B), the mole percents of each DAG FA in either egg or sperm are shown grouped together.

 

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  		TABLE 1. Numbers on the X axis of Figs. 3, 5, and 8–10 representing different FAs of the phospholipid or DAG and corresponding to the following list

 
The different FAs of DAG in both sperm and eggs were compared (Fig. 3). Table 1 has a guide to the 40 FAs shown in the figures. As compared with eggs, sperm contain more 16:0 (peak 5), t16:1n7 (peak 6), 18:0 (peak 11), and 20:4n6 (peak 27). Those FAs of DAG that differ in eggs and sperm are presented in Table 2 .


Figure 5
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  		Fig. 5. Changes in FA content of major phospholipids during fertilization. Data are expressed as a mole percent of each lipid class (n = 3 for all determinations). Mole percent was expressed as the percent of each species in each major class of lipid. The different FAs along the abscissa are identified in Table 1. * P < 0.05. CL, cardiolipin; LPC, lysophosphatidylcholine. Error bars indicate SEM.

 

Figure 8
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  		Fig. 8. Changes in SM FAs during fertilization. The first bar of the pair is the egg data, and the second is the zygote data. As noted for prior figures, the FAs are identified in Table 1. Asterisks denote significance at P < 0.05. Error bars indicate SEM.

 

Figure 9
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  		Fig. 9. The change in FAs of DAG during fertilization. The first bar of the pair is the egg data, and the second is the zygote data. Mole percent of each FA (identified in Table 1) refers to the amount of a FA in DAG, multiplied by 100 and divided by the total amount of all FAs in DAG.

 

Figure 10
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  		Fig. 10. Comparison of FAs from DAG and major phospholipids. To search for the source of the DAG increase at fertilization, the FAs from the elevated DAG and major phospholipid classes are compared. The top panel shows the change in FAs in DAG through fertilization and is the difference between the amount in the egg versus the zygote. An upward bar represents an increase in a DAG FA during fertilization. The lower three panels represent a comparison of the change in the phospholipid with the change in DAG during fertilization. The change in the amount of each FA in PC, PE, or PI/PS in the zygote was determined, and then the change in the amount of DAG (top panel) was subtracted. In the lower three panels, the panel showing the smallest bars would show which phospholipid has the fewest differences with the DAG increase and would represent a possible source for the DAG increase at fertilization.

 

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  		TABLE 2. Comparison of DAG data expressed in mole percent

 
There also appears to be more plasmalogen dm18:1n9 in DAG (peak 9; which is not detectable in egg; Fig. 3) in sperm.

Changes in major phospholipid classes through fertilization
After fertilization, PI decreased by 1.17 nmol per cell (Fig. 4 ; Table 3 ), whereas total PE and PC did not change at fertilization. The level of PS dropped 234 pmol at 3 min (0.916 ± 0.057 nmol/egg to 0.682 ± 0.037 nmol/cell, n = 5, P < 0.01) but returned to basal levels by 5 min.


Figure 4
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  		Fig. 4. The mass of major phospholipids during Xenopus fertilization. As measured by HPLC/ELSD, samples were obtained after insemination. We previously estimated (7, 9) that sperm-egg contact occurs at ~1 min after insemination. A calcium/surface contraction wave traverses the zygote at ~3–6 min to induce cortical granule exocytosis. Asterisks denote significance at * P < 0.05. Error bars indicate SEM.

 

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  		TABLE 3. Summary of lipid changes at fertilization

 
The measurement of individual FAs of phospholipids by gas chromatography showed that there was a significant decrease in 18:1n9 PC (peak 13) from 12.665 ± 0.0531 mol% in the egg to 12.355 ± 0.074 mol% in the zygote (n = 3 each; P < 0.027) (top panel, Fig. 5 ).

In analysis of a combined PI/PS fraction during Xenopus fertilization, the largest change was a drop in 18:1n9 (11.383 ± 0.270 mol% to 6.691 ± 0.265 mol%; n = 3 each, P < 0.00024), and there were additional decreases in 16:0 (peak 5), 16:1n7 (peak 7), and 18:0 (peak 11) (Fig. 5). There was an increase in 20:5n3 [eicosapentaenoic acid (EPA), peak 30].

The largely mitochondrial lipid CL made up only 0.75 mol% of all major phospholipids in the Xenopus egg. There was no significant change in total CL during fertilization (Table 3), but there was a significant decrease in 16:1n7 CL (from 8.287 ± 0.296 mol% to 6.286 ± 0.599 mol%; n = 3 each, P < 0.04) and trans-18:1n9 increased (from 0.987 ± 0.391 mol% to 2.247 ± 0.054 mol%; P < 0.03), whereas 20:0 doubled (0.483 ± 0.122 mol% to 0.948 ± 0.123 mol%; n = 3 each, P < 0.05)(Fig. 5).

These FAs in PE changed during fertilization: 18:1n9 decreased (8.410 ± 0.0183 mol% to 6.629 ± 0.047 mol%; n = 3 each, P < 0.000003), and both arachidonate 20:4n6 (14.596 ± 0.272 mol% to 15.311 ± 0.079 mol%; n = 3 each, P < 0.06), and 20:5n3 EPA increased (9.402 ± 0.241 mol% to 10.493 ± 0.270 mol%; n = 3 each, P < 0.04) (Fig. 5).

The total amount of LPC made up 1.78 mol% of all major phospholipids. During fertilization, LPC mass declined from 1.671 ± 0.234 to 0.869 ± 0.146 nmol per cell (n = 3 each, P < 0.043) (Table 3). As shown in Fig. 5, the 18:1n9 LPC decreased from 20.923 ± 0.660 mol% to 13.988 ± 1.183 mol% (n = 3 each, P < 0.007), whereas there was an increase in 22:6n3 (peak 37; DHA) from 1.998 ± 0.343 mol% to 4.809 ± 0.978 mol% (n = 3 each, P < 0.05). The apparent increase in peak 5 (16:0) was not significant (24.75 ± 1.36 mol% to 29.9.975 ± 2.71 mol%; P < 0.16; n = 3 each). Similarly, the apparent decrease in peak 6 (t16:1n7) was not significant (11.947 ± 1.12 mol% to 8.93 ± 1.02 mol%, P < 0.11; n = 3 each).

Saturation changes through fertilization
Increasing degrees of saturation can lower membrane fluidity and affect other membrane properties. During Xenopus fertilization, the mole percent of saturated FAs decreased in PI/PS (43.22 ± 0.73 mol% to 39.42 ± 0.61 mol%; P < 0.016; n = 3 each) and increased in SM (50.94 ± 0.60 mol% to 59.45 ± 0.39 mol%; P < 0.0002; n = 3 each) (Fig. 6 ).


Figure 6
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  		Fig. 6. Saturation changes in fertilization. Within each of the phospholipid classes, the mole percent of saturated (top), monounsaturated (middle), or polyunsaturated (bottom) FAs were determined and graphed. The first vertical bar of each pair represents the egg and the second bar is the zygote. Asterisks denote significance at P < 0.05. Error bars indicate SEM.

 
Monounsaturated fatty acids (MUFAs) decreased in LPC, PC, PE, PI/PS and SM but not in CL (Fig. 6). The largest decreases were in LPC, PI/PS, and SM. MUFA in LPC declined by 29% (26.9 ± 0.5 mol% to 19.1 ± 2.1 mol%; P < 0.001; n = 3 each), whereas MUFA in SM decreased 30% (from 35.5 ± 1.4 mol% to 24.9 ± 1.4 mol%; P < 0.001; n = 3 each), and in PI/PS declined 35% (17.1 ± 0.6 mol% to 11.2 ± 1.1 mol%; P < 0.01; n = 3 each).

PUFA in the PI/PS fraction increased 24% during fertilization (37.2 ± 1.2 mol% to 46.2 ± 1.3 mol%; P < 0.01; n = 3 each), whereas that in PE increased, from 46.65 ± 0.49 mol% to 49.08 ± 0.15 mol% (P < 0.009; n = 3 each) (Fig. 6). There were no detectable changes in the mole percents of PUFA in CL, LPC, PC, and SM.

Plasmalogens in phospholipids
Plasmalogens are detected after acid hydrolysis and subsequent dimethyl (dm) acetyl production. There were no detectable changes in the level of plasmalogens (dm16:0, dm18:0, dm18:1n9, dm18:1n7) during fertilization.

As is typical (23), small amounts of plasmalogens were present in the PC and PE family from the Xenopus egg or zygote, and those from other major lipid classes were negligible. The largest plasmalogen PC was dm16:0 PC (0.16 ± 0.003 mol%; n = 6), and dm18:1n7 PC (0.09 ± 0.002 mol%; n = 5), whereas dm18:0 and dm18:1n9 PC were undetectable.

PE plasmalogens were more common than those from PC in the Xenopus egg and zygote: dm16:0 PE was 2.0 ± 0.16 mol%, dm18:0 PE was 0.40 ± 0.013 mol%, dm18:1n9 PE was 0.96 ± 0.012 mol%, and dm18:1n7 PE was 1.26 ± 0.063 mol% (n = 6 for each).

Sphingomyelinase activation at fertilization
SM can be hydrolyzed by sphingomyelinase to produce ceramide, and there is evidence that this enzyme is activated at fertilization. With the HPLC/ELSD method, a decrease of 230 pmol/cell in SM was found (Fig. 7A ; Table 3) and, as measured by DAG kinase assay [for Xenopus, see (9)], ceramide levels increased 208 pmol/cell (Fig. 7B). Thus, we find a similar decrease of the substrate and increase in the product of sphingomyelinase and time course for these changes.


Figure 7
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  		Fig. 7. Comparison of sphingomyelin (SM) and ceramide during fertilization. A: As measured by our HPLC method, SM decreased at fertilization. B: Ceramide, as measured with the enzymatic assay with DAG kinase, increased at fertilization. Asterisks denote significance at P < 0.05.

 
During fertilization, FA analysis finds a very large (53%) decrease in 18:1n9 SM (20.2 ± 1.0 mol% to 9.4 ± 0.2 mol%; P < 0.02; n = 3 each) (peak 13 in Fig. 8 ) and a small decrease in 22:1n9 (peak 32).

There were also significant increases in the SM species 16:0 (from 34.112 ± 0.798 mol% in the egg to 40.478 ± 0.478 mol% in the zygote; n = 3 each, P < 0.009) and two longer chain species: 24:0 (peak 38; 1.258 ± 0.111 mol% to 2.497 ± 0.210 mol%, n = 3 each, P < 0.006) and 24:1n9 (peak 39; 5.204 ± 0.429 mol% to 6.594 ± 0.278 mol%; n = 3 each, P < 0.05)(there was no significant change in peak 25, 20:2n6 SM; Fig. 8).

DAG levels during fertilization
In comparison with the differences in DAG noted between egg and sperm (Fig. 3), the egg and zygote values were similar (Table 2; Fig. 9 ). The major DAG species in the egg and the zygote are: 16:0 (peak 5), 18:1n9 (peak 13), and 22:1n9 (32). Reporting the difference in mass of DAG species (top panel, Fig. 10 ), there was an increase of 35 pmol/cell in 18:1n9 DAG and of 8 pmol/cell of 22:1n9 DAG during fertilization.

One way of searching for the source of DAG produced at fertilization (9) has been to compare changes in various phospholipid FAs with the change of those in DAG (58). Thus, the changes in the amount of DAG FAs during fertilization (top panel, Fig. 10) were compared with the changes in those of major phospholipids (bottom three panels of Fig. 10). In these three bottom panels, the change in the amount of each FA in the phospholipid classes (PC, PE, PI/PS) during fertilization was first calculated by subtracting the amount of each FA in the egg from that in the zygote. From this change in FAs during fertilization, the change in each FA in DAG was subtracted. When one looks at the lower panels in Fig. 10 for the shortest bars (which represent the least differences from the DAG increase), PC appears to be the closest, and thus PC may be the source of the DAG. More specifically, one can compare the 18:1n9 increase in DAG during fertilization (see top panel of Fig. 10) with the same peak in each of the lower three panels. For PC, there was only a small difference (0.021 nmol) from that for PE (0.907 nmol) or PI/PS (0.488 nmol). However, these data are open to other interpretations and DAG can be produced through multiple pathways (59, 60).


   DISCUSSION
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 ABSTRACT
 INTRODUCTION
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 MATERIALS AND METHODS
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We have now examined the following lipids during fertilization: IP3 (7, 8), DAG, choline, phosphocholine (9), and, from this report, DAG FA analysis, ceramide, SM, PC, LPC, PI, PE, PS, CL, and plasmalogens at fertilization (for summary, see Table 3).

Xenopus sperm and egg had very different lipid profiles, but they both undergo [Ca2+]in-induced exocytotic events and membrane fusion. We note that sperm had much higher levels of PE, PS, and SM, with much lower amounts of PC (PI was about the same; Figs. 1, 2). Similar to our high values of sperm SM (12 mol%; Fig. 2), Furland et al. (61) report higher than typical levels of SM (13–15 mol%) in bull and ram sperm and that SM may be required for a large production of ceramide during sperm capacitation. The high levels of SM may also reflect higher levels of rafts in sperm membranes.

When compared with Xenopus sperm (hatched bars, Fig. 2), Buhr, Curtis, and Somnapan Kakuda (62) found that boar sperm had lower PE (23 mol%), PI (3 mol%), PS (5 mol%), and higher PC (40 mol%) and SM (20 mol%).

In a pioneering lipid analysis of a plasma membrane-enriched fraction from sea urchin eggs by Kinsey, Decker, and Lennarz (63), unusually high levels of PI and PE were noted. In comparison, our analysis showed that PE (30 vs. 56 mol%), and PI (11 vs. 23 mol%) were lower in the Xenopus egg, whereas Xenopus PC was higher (55 vs. 16 mol%). No CL was detected in the sea urchin egg. Because the authors used purified KI-extracted cells and TLC methodology to analyze an enriched fraction of this invertebrate egg, many factors could explain the differences.

PLC activation at fertilzation
The major reaction of fertilization is considered to be the sperm-induced breakdown of PIP2 to IP3 and DAG through activation of PLC (46, 64–67). Although one would expect a decrease in PIP2, two reports show an increase in PIP2 during fertilization. First, PIP2 increases from 0.8 to 1.8 pmol of PIP2 per Xenopus egg/zygote during the first 10 min of fertilization (45). Second, presumably due to stimulation of PI 4 kinase by elevated calcium, there is an increase in PIP2 (as detected by an increased signal from a fluorescent PIP2 binding protein) during mouse fertilization (68). Kinsey and coworkers (64) report a 50% decline in PI in sea urchin fertilization and suggest that it is due to a replenishment of PIP2. A similar suggestion has been made (69) in regard to nuclear envelop reformation.

Consistent with prior work, we report a 32% decrease in total PI during Xenopus fertilization (1,172 pmol/cell; Fig. 4, Table 3). Thus, during Xenopus fertilization and activation of PLC, there might be a decrease in PI as PIP2 is regenerated through phosphorylation of PI and phosphatidylinositol 4-phosphate. Note that this PI decrease is 1,172-fold greater than the increase in PIP2, and this suggests that PI metabolism largely produces products other than PIP2 (perhaps phosphatidylinositol 3- or 4-phosphate). In addition to serving as a substrate for PLC, PIP2 may play many other roles, and the decrease in PI levels may produce active lipids other than PIP2. Finally, related to this discussion, if one assumes PIP2 levels of about 1 pmol per cell, and production of 0.173 pmol of IP3 (8) (Table 3), we estimate that only ~17% of PIP2 may be hydrolyzed at fertilization.

There were decreases in various FAs in the PI/PS fraction (e.g., 16:0, 16:1n7), but the largest declines were in 18:0 (~167 pmol/cell) and 18:1n9 (~352 pmol/cell). The latter FA, 18:1n9, decreased in LPC, PE, PI/PS, PC, and SM but increased in DAG (Fig. 5).

Both PI and, for the 3 min time point only, PS (234 pmol/cell) decrease (Fig. 4). Although PI and PS were examined together for FA analysis, the large change in total PI and the small, brief change in PS suggest that 18:0 and 18:1n9 may be the major species that decline in PI.

PLC prefers 18:0 at the sn-1 position and arachidonate 20:4n6 (and to a lesser extent, 22:5) in the sn-2 position of PIP2 (59, 69). These FAs would correspond to peaks 11, 27, 35, and 36 (the latter two for 22:5) in Fig. 5 for the PI/PS fraction. As noted above, there was a decrease in 18:0 in the PI/PS fraction, but the other FAs are either not detectable or do not decrease in this combined Xenopus phospholipid fraction.

Membrane phospholipid saturation and unsaturation
Wolf et al. (70) report both increases and decreases in membrane fluidity during fertilization and suggest that the results are due to different probes that lie in different membrane microdomains. Increasing saturation of phospholipids typically reduces membrane fluidity, but there were no detectable changes in saturation in CL, LPC, PC, or PE during fertilization (Fig. 6). The relative levels of saturation in the phospholipids were 1.0 CL, 2.15 LPC, 1.95 PC, 1.65 PE, 2.0 PS/PI, and 2.5 SM (Fig. 6). Thus, CL has the lowest level of saturation and SM has the highest.

In Xenopus eggs, the FA profile of LPC and SM were different from other major phospholipids in that they were predominately made up of saturated 16:0 and MUFA 18:1n9 with very low levels of PUFAs arachidonate 20:4n6, 20:5n3 EPA, and 22:6n3 DHA (Figs. 5, 8).

All phospholipids except CL showed a decline in the levels of MUFA during fertilization. In PI/PS, the levels of saturated and MUFA decreased by 10% and 17%, respectively, whereas PUFA increased 24%. Xenopus PLC is active at fertilization (7, 8, 46), and because PLC prefers tetraunsaturated PIP2 (59) and PIP2 is regenerated during fertilization, one might expect a decrease in PUFA in the combined PI/PS fraction.

PUFA levels did not change in CL, LPC, PC, or SM, but there was a small (7%) increase in PUFA in PE. Sperm, melanocytes, and nerve cells have high levels of PUFA (16, 36). The Xenopus egg averages ~40 mol% PUFA in the phospholipids CL, PC, PE, and PI/PS, whereas there is only ~13 mol% in LPC and SM.

The PUFA 22:6n3 (DHA) has been suggested to be a major regulator of membrane fluidity and raft structure, whereas high levels offer oxidation protection and can lead to apoptosis (16, 30, 36). In a comparison with other FAs in all phospholipids, Xenopus eggs have a lower value of 9.4% of PUFA as 22:6n3, whereas silurid fish eggs contain 15–18% (71) and red blood cells contain 4% (16). Considering the amount of 22:6n3 in Xenopus egg CL, LPC, PC, PE, PS/PI, and SM, about 93% of 22:6n3 is found in PC (65%) and PE (27%), whereas all of the other phospholipids contain very small amounts. There were no changes in 22:6n3 PC or 22:6n3 PE during fertilization; however, 22:6n3 LPC increased over 2-fold (Fig. 5).

The long, saturated FA tail of SM stabilizes rafts. Thus, the increase in saturation (19%) and the decrease in MUFA (30%) SM could represent a stabilization of rafts during fertilization. However, as noted, there is also a decrease in the amount of SM to produce ceramide, and this could represent a destabilization of rafts (although ceramide can stabilize rafts). Perhaps the increase in SM saturation is a method to stabilize rafts in the presence of decreased SM.

LPC changes in fertilization
There are relatively few major species of Xenopus egg LPC and most are less than 18 carbons long or less: 16:0, trans-16:1n7, 18:0, and 18:1n9 (Fig. 5).

The 50% decrease in total LPC during fertilization might reflect the activation of an enzyme such as autotaxin. Autotaxin is a lysophospholipase D that catalyzes the hydrolysis of LPC to lysophosphatidic acid (74). Lysophosphatidic acid is a well-known signaling lipid that binds to G protein-coupled receptors to release calcium but also may have intracellular action (19).

As noted earlier, PLA2 inhibition blocks some fertilization events, and there is evidence that LPC, produced through the action of PLA2 on PC, plays a role in membrane fusion (20, 21). Although this should be examined directly, the ~50% drop in total amount LPC during fertilization suggests that high levels of LPC do not play a role in sperm-egg fusion or subsequent cortical granule exocytosis.

However, the increase in DHA 22:6n3 LPC during fertilization (Fig. 5) could facilitate membrane fusion and represent an activation of PLA2. There was no detectable decrease in 22:6n3 PC (10.523 ± 0.150 mol% to 10.036 ± 0.274 mol%; P < 0.19; peak 37 in Fig. 5), although the large amount of this species could obscure small changes.

Mammalian PLA2 is believed to act through release of arachidonic acid (20:4n6) from PC, and arachidonate can have effects on Xenopus cells (72). During fertilization, there was no detectable change in the 20:4n6 arachidonate species of CL, LPC, PC, PS/PI, or SM (arachidonate PE actually increased; Fig. 5). PLA2 may be able to break down the plasmalogen form of PC (e.g., 73), but there was no detectable change in this species during fertilization. Further work is needed to directly examine whether decreased LPC, lysophosphatidic acid, arachidonate release, PLA2, or autotaxin plays a role in fertilization.

Phospholipid changes
Although the total level of PC did not significantly change during fertilization, there was a decrease in 18:1n9 species of PC from 12.7 to 12.3 mol% (estimated as an increase of ~430 pmol/cell) (Fig. 5). In addition to any PLA2 activation (see discussion above), PLD activation at fertilization would degrade PC to PA and choline, and any PA produced at fertilization should reflect the FA species of PC. To lend support for an activation of PLD during fertilization, we have reported a 134 pmol/cell increase in choline mass during Xenopus fertilization (Table 3) (9).

At fertilization, there was no detectable change in total PE mass, but there was a decrease in 18:1n9 and an increase in the arachidonate and EPA species in PE [DHA PE showed a trend toward an increase, P < 0.08; (Fig. 5)]. As noted for phosphoinositides, there may be a hydrolysis and subsequent synthesis of PE during fertilization.

Note that addition of 20:5n3 EPA to cancer cells has been found to remove SM and cholesterol from rafts, increase ceramide in rafts, and induce apoptosis (30). However, there were no decreases in any phospholipid EPA, so there is no support for a release of this FA at fertilization (peak 30; Fig. 5). As noted, EPA increased during fertilization in PE and PS/PI.

Because CL may play a role in membrane fusion (25–29), we note that total CL did not change during fertilization, so this does not provide support for elevated CL producing sperm-egg fusion or cortical granule exocytosis. However, there was a decrease in 16:1n7, and increases in trans-18:1n9 and 20:0 (Fig. 5) that may relate to a role for CL in fertilization. CL was very unusual in that it contained very large amounts (~22 mol%) of 22:2n6 (peak 33; Fig. 5) and low levels of arachidonate, EPA, and DHA. All other phospholipids contained only trace amounts, up to ~2 mol% 22:2N6.

Plasmalogens
Plasmalogens have been implicated as crucial to survival of sperm owing to their antioxidant properties (22, 23). Furthermore, plasmalogens may raise membrane fluidity and play a role in membrane fusion (69). Not surprisingly, sperm have relatively high levels of plasmalogens (22, 23). As compared with Xenopus eggs, there appears to be more sperm plasmalogen dm16:0 (peak 4, Fig. 3) and dm18:1n9 DAG (peak 9), because these plasmalogens were not detectable in eggs. There were no detectable changes in plasmalogen species during fertilization.

In comparison to other animal tissues, the relative amounts of plasmalogens found in the Xenopus egg were typical, in that they were present at low levels, predominately in PE, with lower levels in PC (little or none found in PI or PS) (22). Total Xenopus PE plasmalogens were 4.7-fold greater than those from PC. The relative amount of PC plasmalogens was dm16:0 (0.16 mol%) > dm18:1n7 (0.08 mol%) >> dm18:0 and dm18:1n9, and for those in PE, dm16:0 (~2 mol%) > dm18:1n7 (~1.3 mol%) > dm18:1n9 (0.98 mol%) > dm18:0 (0.4 mol%).

Highly fusogenic vesicles from sea urchin egg cytoplasm contain PC that is almost exclusively plasmalogen, with especially high levels of dm18:1/20:5 (12 mol%), dm18:0/20:5 (20 mol%), and dm18:0/22:6 PC (15 mol%) (69). Xenopus egg PC also has a relatively high amount of dm18:1n7 and 20:5n3 EPA (7.72 mol%; compare peak 30 with others in Fig. 5, top panel). Perhaps of interest, Xenopus egg PE has a relatively high mole percent of these two species.

SM and ceramide
We find evidence that sphingomyelinase is activated at fertilization, because there was an equivalent decrease in total SM and increase in ceramide (~220 pmol/cell) (Table 3) and similar time course for change (Fig. 7). Because 18:1n9 SM declined by 53% (Fig. 8), any activation of a Xenopus sphingomyelinase at fertilization might prefer this substrate, and the ceramide produced would reflect this species. As noted, SM shows the highest level of saturation and the lowest (3-fold lower) percentage of PUFAs of the major phospholipids. During fertilization, there was an increase in saturated FAs and a decrease in MUFAs in SM.

During fertilization, ceramide increased from 392 to 600 pmol per egg or zygote (Fig. 7; Table 3). Similarly, Coll et al. (75) report the basal level of ceramide in Xenopus oocytes as ~200 pmol/oocyte, and artificial production of ceramide maximizes at ~800 pmol/oocyte.

One might expect that the SM decrease in fertilization would lead to the breakdown of membrane rafts, increased membrane disorder (76–78), and inhibition of secretory vesicle fusion (31). However, functional rafts are present during fertilization (46), and cortical granule exocytosis proceeds during this decrease in SM. Perhaps the increase in saturation and decrease in MUFA SM ameliorate any negative effects of a decrease in total SM.

The increase in ceramide during fertilization is surprising, inasmuch as the lipid is typically associated with increased apoptosis in eggs (41). The early embryo is resistant to ceramide-induced apoptosis (42), so ceramide presumably would have functions other than induction of apoptosis. Ceramide stabilizes membrane rafts (79) and alters PKC activity (59, 80). Ceramide could be metabolized to sphingosine and sphingosine-1-phosphate, and both have been found to release [Ca++]in (81). Addition of ceramide to mammalian sperm enhanced exocytosis (e.g., the acrosome reaction) (37, 38, 82), but we were unable to find any references linking ceramide (or sphingomyelinase or SM) and fertilization. Preliminary experiments with a neutral sphingomyelinase inhibitor (GW4869) found that the drug did not inhibit fertilization.

It is interesting that the addition of ceramide to Xenopus oocytes induces meiosis (75) and that one event of fertilization is the completion of meiosis. Furthermore, ceramide addition to Xenopus oocytes released [Ca2+]in through Gq, PLCβ, and IP3 receptor activation (83). Because this G protein pathway is not thought to be involved in [Ca2+]in release as initiated by sperm (84), ceramide may not play a role in the calcium release at fertilization. However, understanding sphingomyelinase activation and the role of ceramide during fertilization requires further study.

DAG levels
Relative to eggs, DAG levels in sperm appeared to contain more 16:0, trans-16:1n7, 18:0, arachidonate 20:4n6, and plasmalogen dm18:1n9 (Fig. 3). Because the primary functions of sperm are to move and fuse with the egg, these lipid levels may relate to these or other functions (e.g., enzyme activation, protection against lipid oxidation).

DAG 18:0/20:4n6 is very high during fusion of vesicles around sperm DNA that has just entered the egg/zygote cytoplasm after fertilization, and has been implicated in membrane fusion in other cells (52, 69, 85). As noted above, higher levels of these FAs in DAG are present in Xenopus sperm, so they may play a role in the acrosome reaction (membrane fusion of the acrosome) or in sperm-egg fusion.

Source of DAG increase during fertilization
Based on our prior measurement of IP3, there should be a small (173 fmol/cell), rapid (5 min or less after insemination) increase in DAG that derives from PIP2 hydrolysis by PLC (7–9). The molecular species of this DAG increase should reflect that of PIP2 and PI (59). We report that sn-1,2-DAG activated PKC {alpha} and β translocated to the plasma membrane by 7–10 min after insemination (9). However, with basal sn-1,2-DAG in Xenopus eggs at 62 pmol, it is difficult to detect the small DAG increase.

Elevated levels of arachidonate 20:4n6 DAG during membrane fusion are believed to be due to production by PLC{gamma} in the fusogenic vesicles (69, 85). That is, 20:4n6 DAG derives from PIP2 (see earlier discussion of PLC specificity), not PC. Because PI molecular species reflect PIP2 species, note that the two highest levels of FAs in Xenopus egg PI/PS are 18:0 and 20:4n6 (Fig. 5). The mole percent of 18:0 or 20:4n6 in PI/PS is higher than that in PC, CL, PE, or LPC (Fig. 5). Thus, although we were unable to detect any change in the small amount of arachidonate DAG during fertilization, DAG containing 20:4n6 may be produced from PIP2 hydrolysis (as noted, we would not expect to see a change of 0.173 fmol/cell based on the change in IP3 mass).

The late (12 min), larger 48 pmol/cell increase in DAG at fertilization is easily measurable, inasmuch as it is ~300-fold larger than the IP3 increase at fertilization (9). Based on data presented in Fig. 10, this larger DAG increase may be derived from PC hydrolysis. Some 80% of the increase in the large, late DAG during fertilization was due to 18:1n9, whereas 22:1n9 contributed another ~17% (Fig. 10, top panel). These data suggest that the DAG increase of 48 pmol/cell during fertilization (which was measured with a DAG kinase assay; Table 3) (9) is due to increases in these two species of DAG (measured with gas chromatography). There is support for this suggestion because the size of the increase of 18:1n9 was 35 pmol and 22:1n9 was 8 pmol, for a total of 43 pmol.

The molecular species of DAG derived from PIP2 is largely made up of highly polyunsaturated FAs (e.g., 20:4 and 22:6), whereas that from PC is made up of saturated and monounsatured FAs (3, 59, 85). Thus, because the large, late DAG increase at fertilization is due to monounsaturated DAG (e.g., 18:1n9 and 22:1n9), there is further evidence that it derives from PC.

If this large 48 pmol increase in DAG was produced through PLC hydrolysis of PIP2, one might expect more PIP2 present during Xenopus fertilization (~1 pmol/cell) (45). Further support for the large DAG increase deriving from PC, not PIP2, hydrolysis is the timing: we found that the IP3 (and thus, PIP2-derived DAG) production peak was much earlier (5 min) than that of the larger DAG (~12 min). Many other cellular systems have a DAG production from the breakdown of PIP2 that is much faster and smaller than that of DAG production from PC (60).

The 20,000 pmol of PC present in a Xenopus egg (Fig. 4) is obviously sufficient to produce the late, large DAG increase. More specifically, we showed that the only detectable change in PC FAs at fertilization was a decrease in the amount of 18:1n9 (peak 13; Fig. 5), the same FA that increases in DAG (peak 13; Figs. 9, 10, top panel). The decrease in 18:1n9 PC was estimated as ~430 pmol/cell, and this is ~9-fold larger than the 48 pmol increase in DAG. This difference may mean that DAG is not from this species of PC, that data are inaccurate, or that other lipases (e.g., PLD) also degrade this species of PC. 18:1n9 PI/PS, PE, and LPC also show a decrease of about 220–310 pmol/cell and could be sources of the late DAG increase at fertilization. However, Wakelam (59) suggests that reports on PE or PI hydrolysis to DAG are unconfirmed, or if real, represent unusual circumstances.

PKC is believed to be important in fertilization (47–50), but the type of DAG is important for PKC activation. DAG that derives from PC hydrolysis may not stimulate PKC, because this DAG is typically made up of saturated fatty acids or MUFAs, and DAGs made up of these species are unable to stimulate PKC (58, 59, 86, 87; however, see 88). Thus, the late, large monounsaturated DAG fertilization increase may not stimulate Xenopus PKC but may play other roles (perhaps aiding membrane fusion in the massive cortical granule exocytosis that occurs at ~7–13 min postfertilization).

In summary, this examination of the total amount and FA composition of the major lipid classes closes gaps that exist in the fertilization literature. These lipidomic data provide baseline values that will aid in developing a more comprehensive model of fertilization. Relative amounts of lipids in sperm, eggs, and zygotes are important for understanding gamete physiology, the role of rafts, and the events of fertilization. We report levels of species of DAG, the major classes of phospholipids, CL, LPC, plasmalogens, and sphingolipids (including SM and ceramide). We found a decrease in total LPC, SM, and PI and suggest that phosphorylation of PI leads to higher levels of PIP2 during fertilization. There were changes in MUFA and PUFA associated with fertilization. We report an increase in ceramide and the FA species of DAG during fertilization. With the data presented here, further studies on PLA2, PLD, autotoxin, and sphingomyelinase are warranted.


   ACKNOWLEDGMENTS
 
The authors thank students Thomas E. Morrison, Cheney Lupe, Patricia Medina, Pablo Joucovsky, Carolyn Anello, Erinn Stauter, Liliana Cabrera, David Carujo, Stephen Sharp, Crystal Szczesny, Cassandra Shum, Elizabeth Lampert, Ingar Krebs, and Ryan Koonze.

Submitted on March 27, 2008
Revised on June 23, 2008


   REFERENCES
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 ABSTRACT
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 LIPID SIGNALING RELATED TO...
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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