DEVELOPMENTAL
BIOLOGY
Phospholipid
46, 309-m316t 1975)
Metabolism
following
Fertilization
in Sea Urchin
Eggs and Embryos E. WILLIAM Marine
Biology
Research Division.
BYRD.
JR.
Scripps Institution of Oceanography. La Jolla. California 9203i U.S.A. Accepted
Unioersity
of California,
San Diego.
May 20, 1975
Phospholipid metabolism during early development was examined in the sea urchins Stronglyocentrotus purpuratus and Lytechinus pictus. Transport of 3H-choline was stimulated fivefold following fertilization in both species. However. the actual percent incorporation of labeled precursors into phospholipids from the TCA soluble pool did not change at fertilization. There was a slight increase in transport of “C-ethanolamine at fertilization but again there was no change in its percent incorporation into phospholipids. When eggs were preloaded with 3H-choline or “C-ethanolamine and fertilized. the eggs or embryos showed similar patterns of incorporation into phospholipids. There was no significant change in the percent phosphorylation of choline in fertilized or unfertilized eggs. An investigation was made of the activity of choline kinase, the first enzyme in the biosynthesis of phosphatidylcholine. This enzyme was found to have similar activities in fertilized and unfertilized eggs using a variety of homogenization media. The activity of choline kinase was found to decrease slightly in activity at fertilization and reach a maximum activity by gastrula. These results indicate that there is no activation of phospholipid synthesis at fertilization of sea urchin eggs. Apparent increased incorporation actually reflects increased transport of precursors and not de noL)o synthesis. INTRODUCTION
Fertilization of the sea urchin egg results in an extensive metabolic activation or derepression (Epel et al., 1969, 1974; Giudice, 1973). Metabolic changes include increased respiration rate, protein synthesis, transport, activation of enzyme systems and nucleic acid synthesis. Some of these events are closely linked to alterations that occur in the plasma membrane at fertilization, including activation of a Na+dependent amino acid transport system, a change in membrane potential, development of K+ -conductance (Giudice, 1973) and a release of bound Ca+ (Mazia, 1937). Although some of the met.abolic event,s associated with the plasma membrane and fertilization have been studied, little information is available on membrane assembly during cleavage. Is phospholipid synthesis also activated with these other membrane related events? If so, could such synthesis be related to these changes? The status of lipid metabolism at fertili309 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
zation is ambiguous. Pasternak (1973) has studied in uivo incorporation of a phospholipid precursor, choline into Stronglyocentrotus purpuratus and Lytechinus pictus eggs and embryos. His results showed increased transport and phosphorylation of choline although incorporation was minimal immediately following exposure to isotope. These results were interpreted as indicat.ing that phospholipid synthesis was stimulat,ed at fertilization. Schmell and Lennarz (1971) found that although there is increased transport of labeled choline into eggs and embryos of Arbacia punctulata, there was no incorporation into phospholipids. Both eggs and embryos transported and incorporated ethanolamine into phosphatidylethanolamine. However, incorporation by fertilized eggs was three to four times smaller than in embryos which actually reflected a decreased rate of transport after fertilization. Also arguing against increased phospholipid synthesis at fertilization is the obser-
310
DEVELOPMENTAL BIOLOGY
vation (Ewing, 1973) that the activity of cholinephosphotransferase (E.C. 2.7.8.2) decreases during early embryonic development of A. punctulata. To resolve whether lipid metabolism is activated at fertilization I investigated the uptake and conversion of the two major phospholipid precursors, choline and ethanolamine, into the eggs and embryos of L. pictus and S. purpuratus. The activity of one enzyme, choline kinase (E.C. 2.7.1.32), was also studied. The results of this study suggest that phospholipid synthesis is not stimulated at fertilization. Rat.her, the observed increase in incorporation of phospholipids at fertilization actually reflects a change in plasma membrane permeability and transport and not an increased rate of synthesis. This suggests that new membranes made during early cleavage may be formed from preexisting phospholipid precursors made during oogenesis. METHODS
AND
MATERIALS
Isolation of gametes. Shedding of gametes of S. purpuratus and L. pictus was induced by intracoelomic injection of 0.5 M KCl. Eggs were then examined microscopically for the presence of germinal vesicles and discarded if oocytes were present. Eggs were dejellied prior to use by adding 0.1 N HCl to pH 5.2 with gentle swirling and left for 2 min before adjusting the pH to 8.0 with 1 M Tris-HCl (pH 8.0). The dejellied eggs were then washed several times to remove jelly coat material. Egg or embryo concentrations were determined by sedimentation in Bauer-Schenck tubes in a hand centrifuge. In all experiments with fertilized eggs at least 95% fertilization was achieved. Uptake and incorporation of isotope. Two different labeling procedures were used. 1. Pulse experiment. Concentrated eggs or embryos (2 ml of 10% egg suspension) were pulsed at indicated times for 5 min with a total of 10 &i of 3H-choline chloride (16 Ci/mmole) or 2 PCi of “C-ethanola-
VOLUME 46, 1975
mine (5 mCi/mmole). Cells were pulsed in 12 ml conical glass centrifuge tubes and kept suspended by gentle bubbling with a Pasteur pipet every 30 sec. Sampling methods were those of Epel (1972). After exposure to isotope, the cells were washed three times with 8 ml of ice cold seawater containing either 20 mM cold choline or ethanolamine carrier to minimize nonspecific absorption. This procedure also washed away exogenous sperm. After the final seawater wash the pellet was extracted with 2 ml of 10% trichloroacetic acid (TCA) at 4°C for 24 hr. The acid-soluble material was removed for determination of radioactivity and acid-insoluble material was incubated with 2 ml of chloroform-methanol (1:3) for 60 min at 37°C to extract lipid-soluble material. The cell residue was collected by centrifugation at top speed in a clinical centrifuge for 10 min and re-extracted with 2 ml of chloroform-methanol. The lipid extracts were pooled and radioactivity determined by liquid scintillation counting in “Aquasol” (New England Nuclear Corp.). Radioactivity in the remaining pellet. was det.ermined following solubilization in “NCS” (AmershamSearle) and counting in a toluene-based scintillation fluid. 2. Preload experiment. Egg suspensions (10%) were incubated with either 2 @Cilml of 3H-choline or 0.3 rCilm1 of “C-ethanolamine for 90 min at 16°C with gentle bubbling every 30 sec. Two ml samples were withdrawn at indicated times to determine uptake as described above. After 90 min the eggs were washed with seawater containing either 20 mM choline or 20 mM ethanolamine. The eggs were then split into two groups, one group was fertilized (38 ml eggs and 2 ml dilute sperm suspension, optical density of sperm suspension was 2.0 at 600 nm) and the final concentration of eggs or embryos was adjusted to 10%. Egg suspensions were bubbled gently every 30 set and a 2 ml aliquot was taken at indicated times and processed as above. Assay of enzyme activity. Choline kinase
E. WILLIAM BYRD. JR.
Sea Urchin
activity was measured in 10% homogenates of eggs or embryos, using different media as described in the figure legends. The homogenates were always examined microscopically to ensure that there was complete disrupt,ion of embryos or eggs and were then centrifuged for 30 min at 17,000g at 4°C. The supernatant from unfertilized eggs had 67.7% of total activity while 61.1%’ of the fertilized egg activity was soluble. The 170 ~1 assay mixture contained 50 ~1 of homogenate (100-110 pg protein), 100 ~1 of buffer-ATP mix (0.1 M Tris-HCl, pH 8.5, 15 mM ATP-Mg), 10 ~1 of 10 mM choline and 2 &i of 3H-choline in 10 ~1 of distilled water. The assay mixture was incubated for 30 min at room temperature and the reaction stopped by the addition of 0.4 ml of ice-cold citrate buffer (pH 4.0). The amount of phosphocholine formed was then measured by modification of the method of McCaman (1962). A 1.5 ml slurry (resin:water; 1:l) of Dowex resin AG l-X8 in OH- form was washed into 0.5 x 2.0 cm columns. After washing with water, a 0.5 ml sample of buffered assay mixture was applied to the column. The resin and assay mix were bubbled several times during a 30-min incubation period. The resin was then washed with 10 ml of water, 10 ml of 0.01 N HCl and finally with 10 ml of water to remove any adhering nonphosphorylated choline from the column. Adhering phosphocholine was eluted by incubating the resin with 1 ml of 3 N HCl for 10 min, this step was repeat.ed once. The 2 ml of 3 N HCl wash were pooled and counted in Aquasol. Contaminating 3H-choline still adhering to the resin after washing made up less than 1% of the total cpm applied. The column assay method had a variability of * 2.5% with a standard sample. Protein concentration was determined either by biuret (Layne, 1957) or by the procedure of Lowry et al., 1951. RESULTS
Uptake and incorporation of choline and ethanolamine into phospholipids in eggs
Phospholipid
311
Metabolism
and embryos. Phosphatidylcholine and phosphatidylethanolamine are the two major phospholipids in the eggs of several species of sea urchins (Mohri, 1964; Isono, 1965). Transport and metabolism of 3Hcholine and “C-ethanolamine was studied in eggs and embryos of 5’. purpuratus and L. pictus. As seen in Table 1 there is a fivefold increase in 3H-choline uptake into TCA-soluble pools in fertilized embryos versus unfertilized eggs in S. purpuratus and a sixfold increase in L. pictus. However, there is only a slight increase in uptake of “C-ethanolamine into fertilized eggs of either S. purpuratus or L. pictus. Concomitant with this increase in uptake of labeled precursors into TCA soluble pools is an increased incorporation into lipid soluble materials, as seen in Table 2. TABLE
1
UPTAKE OF 3H-C~~~~~ AND “C-ETHANOLAMINE IN FERTILIZED AND UNFERTILIZED EGGS” Time after fertilizatior (5 min pulse)
Fertilized eggs 0 20 40 60 90 120 150 180 Unfertilized eggs 0 20 40 60 90 120 150 180
S. purpuratus
L. pictus
%H-cholineb x lo-‘1 wm
“C-ethanolamine” x 10-Z) wm
3H-choline (x lo-‘) cpm
‘“C-ethanolamine Ix lo- 3) cpm
4.5 11.7 13.5 18.1 17.3 17.3 18.0 19.5
4.1 3.1 4.9 4.1 5.7 6.4 8.1 9.5
2.4 17.0 25.5 20.7 27.6 27.7
1.2 1.6 2.9 2.8 2.2 2.9
4.4 3.9 3.9 3.9 3.7 4.5 4.5 4.2
3.9 4.4 5.3 1.9 7.6 5.2 5.4 4.4
3.3 5.6 4.8 5.2 3.9 5.2
1.3 1.3 1.6 1.‘; 1.7 1.6
n Total cpm incorporation by 2 ml of 10% egg suspension (6 x 1W eggs) b 3H-choline, 10 &i in 2 ml; “C-ethanolamine, 2 PCi in 2 ml.
312
DEVELOPMENTAL
TABLE
2
INCORPORATION OF 3H-C~~~~~~ “C-ETHANOLAMINE INTO THE LIPID
TI- S. purpuratus Time after fertilization (5 min pulse)
3H-cho. lineb (x
lOXI cpm
BIOLOGY
amine0 (x 10-Z cpm
AND FRACTIONO
L. pictus “C-ethanolamine .x 10-Z) wm
3H-choline x 10-3) cpm
:
Fertilized em 0 20 40 60 90 120 150 180 Unfertilized
6.0
7.9 7.6 11.5 10.6 54.7 15.3 18.0 24.5
17.1
22.4 24.1 27.5 28.8 34.9 38.1
3.0 18.0 29.‘; 18.5 25.9 26.7
2.4 3.4 7.0 5.5 5.0 5.0
VOLUME
suggest that there is no increase in phospholipid synthesis at fertilization. Preloading experiments also indicate t.hat there is no difference in phospholipid metabolism between fertilized and unfertilized eggs. Two groups of eggs were incubated with 3H-choline or “C-ethanolamine for 90 min and then washed four times with fresh filtered seawater containing 20 mM choline or ethanolamine. The eggs were then separated and resuspended in seawater, one group was fertilized, and the incorporation of precursor into lipid was followed over the next 90 minutes. As can be seen in Fig. 1, upon removal of the exogenous isotope net incorporation of soluble precursor into phospholipid ceases TABLE
eggs 0
20 40 60 90 120 150 180
46, 1975
I L
6.0 5.7 6.5 6.4 6.3 6.4 6.3 6.4
L
7.3 10.7 8.3 8.1 11.3 6.6 7.2 6.7
2.8 4.8 4.9 5.4 3.6 5.4
2.7 3.4 4.1 3.1 3.3 2.9
DTotal cpm incorporated by 2 ml of 10% egg suspension (6 x 10’ eggs). b 3H-choline, 10 PCi in 2 ml; “C-ethanolamine, 2 PCi in 2 ml.
This increase in uptake and incorporation of choline at fertilization is similar to the results found by Pasternak (1973) in S. purpuratus and L. pictus for later development. However, this increased incorporation of labeled phospholipid precursors into lipid need not necessarily reflect an increased de nouo synthesis of lipids after fertilization. These findings could also ensue from the increased availability of radioactive precursors resulting from the increased transport of phospholipid precursors after fertilization. Table 3 shows that the actual percent incorporation of labeled phospholipid precursors into lipid is the same in fertilized and unfertilized eggs. This would
3
INCORPORATION OF 3H-C~~~~~~ AND “C-ETHANOLAMINE INTO LIPIDS=
Time after fertilization
F
S. purpuratus Phospholipid
(Choline (%J
Fertilized eggs 0 20 40 60 90 120 150 180 Unfertilized eggs 0 20 40 60 90 120 150 180
L. pictus
f
Phospholipid
Ethanol, - (:holine amine (8) (%)
Ethanolamine (%,)
13.3 14.6 16.5 13.3 15.8 16.4 19.3 19.5
19.0 24.5 23.5 26.1 25.9 24.0 22.2 25.9
12.5 10.5 11.6 8.9 9.3 9.6
19.9 21.2 24.3 19.3 22.9 17.3
13.5 14.7 16.7 16.4 17.0 14.3 14.1 15.2
18.5 26.; 15.5 16.5 14.8 12.8 13.4 15.0
8.7 8.5 9.9 10.2 9.3 10.4
21.1 26.5 25.9 17.9 18.9 18.2
1
a Embryos were exposed to pulses of SH-choline or “C-ethanolamine as recorded in Tables 1 and 2 and extracted as described in Methods and Materials. Values are percent of intracellular isotope incorporated into lipid extracted material.
E. WILLIAM BYRD. JR.
Sea l’rchin
I -90
-60
-30
0
30
60
90
I
INCUBATION(min) FIG. 1. Incorporation of 3H-choline and “Cethanolamine into lipid soluble fractions after preload experiment. Eggs from S. purpuratus or L. pictus (1OC; suspensions) were pulsed for 90 min with 3Hcholine (16 Ci/mmole sp act I or ‘“C-ethanolamine (5 mCi/mmole sp act) for 90 min. Eggs were washed with filtered seawater containing 20 mM choline or ethanolamine and divided into two groups. One group was fertilized 10 time on x-axis) and ‘L ml of samples were taken at indicated times. Mixtures were assayed as described in text. A) Incorporation of 3H-choline into L. pictus. BI Incorporation of Wcholine into S. purpuratus. CJ Incorporation of “C-ethanolamine into 12. pictus. A = unfertilized eggs. 0 = fertilized eggs.
and there is a decrease in the amount of label in the phospholipid. The rate of decrease, which probably represents turnover, is not changed by fertilization. Analysis of radioactivity in the TCA-soluble pool revealed a similar loss of ethanolamine and choline in fertilized and unfertilized eggs, probably resulting from turnover. This data indicates that there is no difference in incorporation of 3H-choline and l*Cethanolamine into lipids before and after fertilization. The possibility that increased transport of 3H-choline at fertilization is linked to phosphorylation of choline was examined. The phosphorylation of choline present in the TCA soluble pools was measured in eggs and embryos of L. pictus that were preloaded or pulsed with 3H-choline as shown in Tables 4 and 5. The average
Phospholipid
313
Metabolism
percent phosphorylation for the preload experiment was 51.6 + 3.8 for fertilized and 51.7 + 4.0 for unfertilized eggs. In the pulse experiments (5 min pulses), the values were 19.5% f 3.7 in fertilized and 27.6% f 4.1 in unfertilized eggs. Although there is some variation in the percent phosphorylation, choline phosphorylation does not appear to change significantly after fertilization. Indeed, the data might suggest a slight decrease in phosphorylation at fertilization. Choline kinase actillity during early development. The above results constitute direct evidence that synthesis of the two major phospholipids is not stimulated during the first 3 hr of development. An independent method of measuring this is to TABLE
4
PHOSPHORYLATIONOF 3H-C~~~~~~ IN L. pictus PRELOAD EXPERIMENT@ Time of incubation after pulse 0 0 30 30 90 90
IN
‘:, phosphorylation
fertilized unfertilized fertilized unfertilized fertilized unfertilized
57.1 18.1 17.2 57.8 .50.2 49.1
“TCA soluble material was washed extensiveI> with diethyl ether to remove excess TCA. The resulting solution was then buffered with 0.1 M citrate buffer (pH 4.0) and assayed by the column method as described in Methods and Materials. TABLE
5
PHOSPHORFLATIONOF 3H-C~~~~~~ IN L. picks PULSE EXPERIMENTS’. b Time of development at pulse (5 min pulse 1 20 40 60 90 120
IN
? Phosphorylation Fertilized 18.2 20.4 “8.0 15.3 15.6
Unfertilized 34.3 32.2 21.6 26.6 21.3
’ Acid-soluble extracts of the embryos exposed to pulses of ‘H-choline in Table 1. b Assay conditions were the same as in Table 4.
314
DEVELOPMENTAL Bro~ocy
measure activity of choline kinase (E.C. 2.7.1.32) during early development. The properties of choline kinase activity in the 17,000g supernatant fraction of unfertilized eggs and fertilized eggs was studied to determine assay conditions. Saturation curves for choline and ATP in unfertilized eggs are shown in Fig. 2. Concentrations of 0.6 mM choline and 15 mA4 ATP-15 mA4Mg+* were chosen to obtain maximal activity in these enzyme preparations for fertilized and unfertilized eggs. Substitution of Mn+2 for Mg+2 resulted in a 81% decrease in activity. These conditions were optimal for both L. pictus and S. purpuratus. Differences in enzyme activity were looked for between fertilized and unfertilized eggs. No differences were found in the initial experiments; therefore, several different homogenization media were used to preclude the possibility that the homogenization might release the enzyme from a bound or inactive state in the unfertilized egg. Fertilized and unfertilized eggs were homogenized in media containing high or low salt concentration. The results indicate that the composition of the homogeniza-
VOLUME 46. 1975
tion media has little differential effect on enzyme activity between fertilized or unfertilized eggs in L. picks (Table 6). Similar results were also seen in S. purpuratus. Enzymatic activity was measured during early development. Table 7 shows that there is little change in enzymatic activity occurring during the first 18 hr of L. pictus development. There is an initial decrease in specific activity at fertilization of 25% and a slight increase by 18 hr. DISCUSSION
My study on S. purpuratus and L. pictus eggs and embryos, in agreement with a similar study on A. punctulata gametes (Schmell and Lennarz, 1974), indicates TABLE
6
EFFECTS OF VARIOUS HOMOGENIZATION MEDIA ON RESULTANT ENZYMATIC ACTIVITY OF CHOLINE KINASE” Medium
used
Choline kinase activity (nmoles/mg/min) Fertilized eggs (30 min)
0.01 M Tris-HCI, pH 8.5, 10 mM MgCI, 0.01 M Tris-HCI, pH 8.5, 0.2 M NaCl 0.01 M Tris-HCI, pH 8.5, 0.4 M NaCl 0.25 M Sucrose, 0.1 M Tris-HCl, pH 8.0 0 Reaction
conditions
Ur&$ eggs
7.8
7.5
10.3
11.2
10.4
10.5
8.0
9.2
were as described
TABLE
in text.
7
CHOLINE KINASE ACTIWIY IN EXTRACTS OF L. pictus EMBRYOS DURING EMBRYOGENESW b Time after fertilization PH
ATP (mm1
FIG. 2. Enzymatic activities at different substrate and pH values. A) Effect of substrate saturation on choline kinase activity. Assays were performed as in Methods and Materials with 15 mM Mg:ATP, pH 8.5. B) Effect of pH on choline kinase activity. The standard assay was used with 0.1 M Tris-buffer, pH 7.0-9.0. C) Effect of ATP concentration on choline kinase activity. Conditions for assay as described in text.
Unfertilized egg 15 minutes 45 minutes 90 minutes 150 minutes 18 hours
Enzyme activity (nmoles/mg/min) 6.35 4.84 4.73 5.33 4.85 6.85
DReaction conditions were as described in text. b Homogenization medium used (0.1 M Tris-HCl, pH 8.5, 10 mM MgC13.
E.
U'ILLIAM
BYRD. JR.
Sea Urchin
that there is no change in the incorporation of the major phospholipid precursors, choline and ethanolamine, into phospholipids after fertilization. This would suggest that de nouo phospholipid synthesis is not activated immediately following fertilization in the sea urchin. This observed constancy of phospholipid synthesis is also consistent with studies on the activity of enzymes involved in phosphat.idylcholine synthesis. The activity of choline kinase does not change significantly after fertilization; if anything, there is a decrease in activity during the first 3 hr of development. This lack of alteration of enzymatic activity was also seen when activity was measured under different homogenization conditions, in media attempting to better reflect the in uiuo activity of the enzyme. This lack of change in activity is also consistent in my in uivo studies which showed that there is no increased phosphorylation of choline after fertilization, as opposed to the earlier finding of Pasternak (1973). My interpretation is also consistent with the earlier work of Ewing (1973) that the activity of choline phosphotransferase decreases after fertilization of A. punctulata eggs. This data suggests that significant de nouo synthesis is not activated after fertilization in the sea urchin as observed by Pasternak (1973). This implies that the membrane changes associated with fertilization may be independent of de novo phospholipid synthesis. As new membranes are synthesized later in development there is a marked increase in phospholipid synthesis as observed by Pasternak ( 1973). There are some differences between this data on S. purpuratus and L. pictus and Schmell and Lennarz’s data on A. punctulata, these may reflect species differences. Although we agree that there is no increase in major phospholipid synthesis immediately following fertilization, there were differences in the degree of incorpora-
Phospholipid
Metabolism
315
tion of various precursors. They observed no incorporation of choline into phospholipids of either eggs or embryos, whereas I have observed about 10% incorporation of choline into chloroform-met,hanol soluble material of both eggs and embryos. This may be due to either nonspecific contamination or differences in precursor pools present. in these eggs. Amino acid pools of these two sea urchins differ radically (Fry and Gross, 1970) and there may be similar differences in choline pool size. Another difference between the two species of sea urchin was the rate of incorporation of ethanolamine into phosphatidylethanolamine. I observed a slightly increased uptake and incorporation after fertilization whereas Arbacia embryos showed a decreased incorporation relative to eggs. Schmell and Lennarz suggest that uptake differences may explain the enhanced incorporation of ethanolamine into egg lipid versus embryo lipid. Both this study and that of Schmell and Lennarz indicate a loss of radioactivity from phospholipid, probably turnover, when exogenous isotope is removed (preload experiment of Fig. 1). This is puzzling since there is a large pool of labelled phospholipid in the egg. Possibly exogenous label is preferentially utilized in spite of large endogenous pools of precursor. Mathews (in press) has shown that exogenous nucleoside triphosphates are preferentially utilized in S. purpuratus. If exogenous lipid precursors were utilized one would expect a rapid turnover aft.er exogenous label is removed and labeled phospholipids are metabolized. Since the level of phospholipids remains constant during early development (Mohri, 1964) and because a large amount of cell membrane components are formed during cleavage, this study suggests that the phospholipid reserves laid down during oogenesis are utilized during early development. It is therefore intriguing to propose that the lipids needed for membrane biogenesis are
316
DEVELOPMENTAL BIOLOGY
stored or compartmentalized, as in yolk, during oogenesis. As is the case with utilization of preformed RNA, fertilization of the egg would then activate metabolic pathways that would enable the embryo to utilize phospholipid st.ores. These experiments do not provide an absolute answer to the question of whether the immediate membrane changes of fertilization are independent of de nouo phospholipid synthesis. The percent of the total egg phospholipid in the plasma membrane is small due to the surface to volume ratio in the sea urchin egg. Although there is no major change in rate of precursor incorporation, a significant incorporation could go undetected against the large background of cytoplasmic incorporation. This work was supported in part by a grant from the National Science Foundation to Dr. D. Epel. E.W.B., Jr. is a postdoctoral trainee supported by Public Health Service Grant No. F22 HD02840-01. Drs. D. Epel and E. J. Carroll, Jr. are thanked for critical review and discussion of this manuscript. I thank Elizabeth Baker and Diana Majchrzak for excellent technical assistance. REFERENCES EPEL. D. (1964). A primary metabolic change of fertilization: Interconversion of pyridine nucleotides. Biochem. Biophys. Res. Commun. 17,62-69. EPEL, D., PRESSMAN, B. C., ELSAESSE~ S., and WEAVER. A. M. (1969). The program of structural and metabolic changes following fertilization of sea urchin eggs. In “The Cell Cycle” (G. M. Padilla. G. L. Whitson, and I. L. Cameron, eds.), pp. 280-298. New York. EPEL, D. (1972). Activation of a Na+-dependent
VOLUME 46. 1975 amino acid transport system upon fertilization of sea urchin eggs. Exp. Cell Res. 72. 74-89. EPEL, D. (1974). The program of and mechanisms of fertilization in the echinoderm egg. Amer. Zool. in press. EWING, R. D. (1973). Cholinephosphotransferase activity during early development of the sea urchin, Arbacia punctulata. Develop. Biol. 31. 234241. FRY, B. J.. and GROSS,P. R. (1970). Patterns and rates of protein synthesis in sea urchin embryos. Develop. Biol. 21, 125-116. GKIDICE, G. (1973). Developmental Biology of the sea urchin embryo. Academic Press. New York. ISONO, N. (1963). Carbohydrate metabolism in sea urchin eggs. J. Faculrv Sci. (Tokyo). 10, 37-53. ISONO, N. (19651. Phospholipids of sea urchin eggs. Sci. Papers Co/l. Gen. Educ. Biol. (Tokyo). 15, 87-94. LAYNE, E. (1957). Spectrophotometric and turbidity methods for measuring proteins. Methods Enzymol. 3, 447-454. LOWRY, 0. H., RQSEBROUGH, N. J., FARR, A. L.. RANDALL, J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. MATHEWS, C. (1975). Giant pools of DNA precursors in sea urchin eggs. Exp. Cell Res. 92,47-56. MAZIA, D. (1937). The release of calcium in Arbacin eggs upon fertilization. J. Cell Comp. Physiol. IO. 291-304. MCCAMAN, R. E. (1962). Intermediary metabolism of phospholipids in brain tissue. J. Biol. Chem. 237, 672-6’76. MOHRI, H. (1964). Utilization of Cl’-labeled acetate and glycerol for lipid synthesis during the early development of sea urchin embryos. Biol. Bull. 126, 440-455. PASTERNAK, C. A. (1973). Phospholipid synthesis in cleaving sea urchin eggs. Deuelop. Biol. 30, 403-410. SCHMELL, E., and LENNARZ, W. J. (1974). Phospholipid metabolism in eggs and embryos of the sea urchin. Arbacia punctulafa. Biochemistry 13, 4114-4121.
BIOLOGY
Phospholipid
46, 309-m316t 1975)
Metabolism
following
Fertilization
in Sea Urchin
Eggs and Embryos E. WILLIAM Marine
Biology
Research Division.
BYRD.
JR.
Scripps Institution of Oceanography. La Jolla. California 9203i U.S.A. Accepted
Unioersity
of California,
San Diego.
May 20, 1975
Phospholipid metabolism during early development was examined in the sea urchins Stronglyocentrotus purpuratus and Lytechinus pictus. Transport of 3H-choline was stimulated fivefold following fertilization in both species. However. the actual percent incorporation of labeled precursors into phospholipids from the TCA soluble pool did not change at fertilization. There was a slight increase in transport of “C-ethanolamine at fertilization but again there was no change in its percent incorporation into phospholipids. When eggs were preloaded with 3H-choline or “C-ethanolamine and fertilized. the eggs or embryos showed similar patterns of incorporation into phospholipids. There was no significant change in the percent phosphorylation of choline in fertilized or unfertilized eggs. An investigation was made of the activity of choline kinase, the first enzyme in the biosynthesis of phosphatidylcholine. This enzyme was found to have similar activities in fertilized and unfertilized eggs using a variety of homogenization media. The activity of choline kinase was found to decrease slightly in activity at fertilization and reach a maximum activity by gastrula. These results indicate that there is no activation of phospholipid synthesis at fertilization of sea urchin eggs. Apparent increased incorporation actually reflects increased transport of precursors and not de noL)o synthesis. INTRODUCTION
Fertilization of the sea urchin egg results in an extensive metabolic activation or derepression (Epel et al., 1969, 1974; Giudice, 1973). Metabolic changes include increased respiration rate, protein synthesis, transport, activation of enzyme systems and nucleic acid synthesis. Some of these events are closely linked to alterations that occur in the plasma membrane at fertilization, including activation of a Na+dependent amino acid transport system, a change in membrane potential, development of K+ -conductance (Giudice, 1973) and a release of bound Ca+ (Mazia, 1937). Although some of the met.abolic event,s associated with the plasma membrane and fertilization have been studied, little information is available on membrane assembly during cleavage. Is phospholipid synthesis also activated with these other membrane related events? If so, could such synthesis be related to these changes? The status of lipid metabolism at fertili309 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
zation is ambiguous. Pasternak (1973) has studied in uivo incorporation of a phospholipid precursor, choline into Stronglyocentrotus purpuratus and Lytechinus pictus eggs and embryos. His results showed increased transport and phosphorylation of choline although incorporation was minimal immediately following exposure to isotope. These results were interpreted as indicat.ing that phospholipid synthesis was stimulat,ed at fertilization. Schmell and Lennarz (1971) found that although there is increased transport of labeled choline into eggs and embryos of Arbacia punctulata, there was no incorporation into phospholipids. Both eggs and embryos transported and incorporated ethanolamine into phosphatidylethanolamine. However, incorporation by fertilized eggs was three to four times smaller than in embryos which actually reflected a decreased rate of transport after fertilization. Also arguing against increased phospholipid synthesis at fertilization is the obser-
310
DEVELOPMENTAL BIOLOGY
vation (Ewing, 1973) that the activity of cholinephosphotransferase (E.C. 2.7.8.2) decreases during early embryonic development of A. punctulata. To resolve whether lipid metabolism is activated at fertilization I investigated the uptake and conversion of the two major phospholipid precursors, choline and ethanolamine, into the eggs and embryos of L. pictus and S. purpuratus. The activity of one enzyme, choline kinase (E.C. 2.7.1.32), was also studied. The results of this study suggest that phospholipid synthesis is not stimulated at fertilization. Rat.her, the observed increase in incorporation of phospholipids at fertilization actually reflects a change in plasma membrane permeability and transport and not an increased rate of synthesis. This suggests that new membranes made during early cleavage may be formed from preexisting phospholipid precursors made during oogenesis. METHODS
AND
MATERIALS
Isolation of gametes. Shedding of gametes of S. purpuratus and L. pictus was induced by intracoelomic injection of 0.5 M KCl. Eggs were then examined microscopically for the presence of germinal vesicles and discarded if oocytes were present. Eggs were dejellied prior to use by adding 0.1 N HCl to pH 5.2 with gentle swirling and left for 2 min before adjusting the pH to 8.0 with 1 M Tris-HCl (pH 8.0). The dejellied eggs were then washed several times to remove jelly coat material. Egg or embryo concentrations were determined by sedimentation in Bauer-Schenck tubes in a hand centrifuge. In all experiments with fertilized eggs at least 95% fertilization was achieved. Uptake and incorporation of isotope. Two different labeling procedures were used. 1. Pulse experiment. Concentrated eggs or embryos (2 ml of 10% egg suspension) were pulsed at indicated times for 5 min with a total of 10 &i of 3H-choline chloride (16 Ci/mmole) or 2 PCi of “C-ethanola-
VOLUME 46, 1975
mine (5 mCi/mmole). Cells were pulsed in 12 ml conical glass centrifuge tubes and kept suspended by gentle bubbling with a Pasteur pipet every 30 sec. Sampling methods were those of Epel (1972). After exposure to isotope, the cells were washed three times with 8 ml of ice cold seawater containing either 20 mM cold choline or ethanolamine carrier to minimize nonspecific absorption. This procedure also washed away exogenous sperm. After the final seawater wash the pellet was extracted with 2 ml of 10% trichloroacetic acid (TCA) at 4°C for 24 hr. The acid-soluble material was removed for determination of radioactivity and acid-insoluble material was incubated with 2 ml of chloroform-methanol (1:3) for 60 min at 37°C to extract lipid-soluble material. The cell residue was collected by centrifugation at top speed in a clinical centrifuge for 10 min and re-extracted with 2 ml of chloroform-methanol. The lipid extracts were pooled and radioactivity determined by liquid scintillation counting in “Aquasol” (New England Nuclear Corp.). Radioactivity in the remaining pellet. was det.ermined following solubilization in “NCS” (AmershamSearle) and counting in a toluene-based scintillation fluid. 2. Preload experiment. Egg suspensions (10%) were incubated with either 2 @Cilml of 3H-choline or 0.3 rCilm1 of “C-ethanolamine for 90 min at 16°C with gentle bubbling every 30 sec. Two ml samples were withdrawn at indicated times to determine uptake as described above. After 90 min the eggs were washed with seawater containing either 20 mM choline or 20 mM ethanolamine. The eggs were then split into two groups, one group was fertilized (38 ml eggs and 2 ml dilute sperm suspension, optical density of sperm suspension was 2.0 at 600 nm) and the final concentration of eggs or embryos was adjusted to 10%. Egg suspensions were bubbled gently every 30 set and a 2 ml aliquot was taken at indicated times and processed as above. Assay of enzyme activity. Choline kinase
E. WILLIAM BYRD. JR.
Sea Urchin
activity was measured in 10% homogenates of eggs or embryos, using different media as described in the figure legends. The homogenates were always examined microscopically to ensure that there was complete disrupt,ion of embryos or eggs and were then centrifuged for 30 min at 17,000g at 4°C. The supernatant from unfertilized eggs had 67.7% of total activity while 61.1%’ of the fertilized egg activity was soluble. The 170 ~1 assay mixture contained 50 ~1 of homogenate (100-110 pg protein), 100 ~1 of buffer-ATP mix (0.1 M Tris-HCl, pH 8.5, 15 mM ATP-Mg), 10 ~1 of 10 mM choline and 2 &i of 3H-choline in 10 ~1 of distilled water. The assay mixture was incubated for 30 min at room temperature and the reaction stopped by the addition of 0.4 ml of ice-cold citrate buffer (pH 4.0). The amount of phosphocholine formed was then measured by modification of the method of McCaman (1962). A 1.5 ml slurry (resin:water; 1:l) of Dowex resin AG l-X8 in OH- form was washed into 0.5 x 2.0 cm columns. After washing with water, a 0.5 ml sample of buffered assay mixture was applied to the column. The resin and assay mix were bubbled several times during a 30-min incubation period. The resin was then washed with 10 ml of water, 10 ml of 0.01 N HCl and finally with 10 ml of water to remove any adhering nonphosphorylated choline from the column. Adhering phosphocholine was eluted by incubating the resin with 1 ml of 3 N HCl for 10 min, this step was repeat.ed once. The 2 ml of 3 N HCl wash were pooled and counted in Aquasol. Contaminating 3H-choline still adhering to the resin after washing made up less than 1% of the total cpm applied. The column assay method had a variability of * 2.5% with a standard sample. Protein concentration was determined either by biuret (Layne, 1957) or by the procedure of Lowry et al., 1951. RESULTS
Uptake and incorporation of choline and ethanolamine into phospholipids in eggs
Phospholipid
311
Metabolism
and embryos. Phosphatidylcholine and phosphatidylethanolamine are the two major phospholipids in the eggs of several species of sea urchins (Mohri, 1964; Isono, 1965). Transport and metabolism of 3Hcholine and “C-ethanolamine was studied in eggs and embryos of 5’. purpuratus and L. pictus. As seen in Table 1 there is a fivefold increase in 3H-choline uptake into TCA-soluble pools in fertilized embryos versus unfertilized eggs in S. purpuratus and a sixfold increase in L. pictus. However, there is only a slight increase in uptake of “C-ethanolamine into fertilized eggs of either S. purpuratus or L. pictus. Concomitant with this increase in uptake of labeled precursors into TCA soluble pools is an increased incorporation into lipid soluble materials, as seen in Table 2. TABLE
1
UPTAKE OF 3H-C~~~~~ AND “C-ETHANOLAMINE IN FERTILIZED AND UNFERTILIZED EGGS” Time after fertilizatior (5 min pulse)
Fertilized eggs 0 20 40 60 90 120 150 180 Unfertilized eggs 0 20 40 60 90 120 150 180
S. purpuratus
L. pictus
%H-cholineb x lo-‘1 wm
“C-ethanolamine” x 10-Z) wm
3H-choline (x lo-‘) cpm
‘“C-ethanolamine Ix lo- 3) cpm
4.5 11.7 13.5 18.1 17.3 17.3 18.0 19.5
4.1 3.1 4.9 4.1 5.7 6.4 8.1 9.5
2.4 17.0 25.5 20.7 27.6 27.7
1.2 1.6 2.9 2.8 2.2 2.9
4.4 3.9 3.9 3.9 3.7 4.5 4.5 4.2
3.9 4.4 5.3 1.9 7.6 5.2 5.4 4.4
3.3 5.6 4.8 5.2 3.9 5.2
1.3 1.3 1.6 1.‘; 1.7 1.6
n Total cpm incorporation by 2 ml of 10% egg suspension (6 x 1W eggs) b 3H-choline, 10 &i in 2 ml; “C-ethanolamine, 2 PCi in 2 ml.
312
DEVELOPMENTAL
TABLE
2
INCORPORATION OF 3H-C~~~~~~ “C-ETHANOLAMINE INTO THE LIPID
TI- S. purpuratus Time after fertilization (5 min pulse)
3H-cho. lineb (x
lOXI cpm
BIOLOGY
amine0 (x 10-Z cpm
AND FRACTIONO
L. pictus “C-ethanolamine .x 10-Z) wm
3H-choline x 10-3) cpm
:
Fertilized em 0 20 40 60 90 120 150 180 Unfertilized
6.0
7.9 7.6 11.5 10.6 54.7 15.3 18.0 24.5
17.1
22.4 24.1 27.5 28.8 34.9 38.1
3.0 18.0 29.‘; 18.5 25.9 26.7
2.4 3.4 7.0 5.5 5.0 5.0
VOLUME
suggest that there is no increase in phospholipid synthesis at fertilization. Preloading experiments also indicate t.hat there is no difference in phospholipid metabolism between fertilized and unfertilized eggs. Two groups of eggs were incubated with 3H-choline or “C-ethanolamine for 90 min and then washed four times with fresh filtered seawater containing 20 mM choline or ethanolamine. The eggs were then separated and resuspended in seawater, one group was fertilized, and the incorporation of precursor into lipid was followed over the next 90 minutes. As can be seen in Fig. 1, upon removal of the exogenous isotope net incorporation of soluble precursor into phospholipid ceases TABLE
eggs 0
20 40 60 90 120 150 180
46, 1975
I L
6.0 5.7 6.5 6.4 6.3 6.4 6.3 6.4
L
7.3 10.7 8.3 8.1 11.3 6.6 7.2 6.7
2.8 4.8 4.9 5.4 3.6 5.4
2.7 3.4 4.1 3.1 3.3 2.9
DTotal cpm incorporated by 2 ml of 10% egg suspension (6 x 10’ eggs). b 3H-choline, 10 PCi in 2 ml; “C-ethanolamine, 2 PCi in 2 ml.
This increase in uptake and incorporation of choline at fertilization is similar to the results found by Pasternak (1973) in S. purpuratus and L. pictus for later development. However, this increased incorporation of labeled phospholipid precursors into lipid need not necessarily reflect an increased de nouo synthesis of lipids after fertilization. These findings could also ensue from the increased availability of radioactive precursors resulting from the increased transport of phospholipid precursors after fertilization. Table 3 shows that the actual percent incorporation of labeled phospholipid precursors into lipid is the same in fertilized and unfertilized eggs. This would
3
INCORPORATION OF 3H-C~~~~~~ AND “C-ETHANOLAMINE INTO LIPIDS=
Time after fertilization
F
S. purpuratus Phospholipid
(Choline (%J
Fertilized eggs 0 20 40 60 90 120 150 180 Unfertilized eggs 0 20 40 60 90 120 150 180
L. pictus
f
Phospholipid
Ethanol, - (:holine amine (8) (%)
Ethanolamine (%,)
13.3 14.6 16.5 13.3 15.8 16.4 19.3 19.5
19.0 24.5 23.5 26.1 25.9 24.0 22.2 25.9
12.5 10.5 11.6 8.9 9.3 9.6
19.9 21.2 24.3 19.3 22.9 17.3
13.5 14.7 16.7 16.4 17.0 14.3 14.1 15.2
18.5 26.; 15.5 16.5 14.8 12.8 13.4 15.0
8.7 8.5 9.9 10.2 9.3 10.4
21.1 26.5 25.9 17.9 18.9 18.2
1
a Embryos were exposed to pulses of SH-choline or “C-ethanolamine as recorded in Tables 1 and 2 and extracted as described in Methods and Materials. Values are percent of intracellular isotope incorporated into lipid extracted material.
E. WILLIAM BYRD. JR.
Sea l’rchin
I -90
-60
-30
0
30
60
90
I
INCUBATION(min) FIG. 1. Incorporation of 3H-choline and “Cethanolamine into lipid soluble fractions after preload experiment. Eggs from S. purpuratus or L. pictus (1OC; suspensions) were pulsed for 90 min with 3Hcholine (16 Ci/mmole sp act I or ‘“C-ethanolamine (5 mCi/mmole sp act) for 90 min. Eggs were washed with filtered seawater containing 20 mM choline or ethanolamine and divided into two groups. One group was fertilized 10 time on x-axis) and ‘L ml of samples were taken at indicated times. Mixtures were assayed as described in text. A) Incorporation of 3H-choline into L. pictus. BI Incorporation of Wcholine into S. purpuratus. CJ Incorporation of “C-ethanolamine into 12. pictus. A = unfertilized eggs. 0 = fertilized eggs.
and there is a decrease in the amount of label in the phospholipid. The rate of decrease, which probably represents turnover, is not changed by fertilization. Analysis of radioactivity in the TCA-soluble pool revealed a similar loss of ethanolamine and choline in fertilized and unfertilized eggs, probably resulting from turnover. This data indicates that there is no difference in incorporation of 3H-choline and l*Cethanolamine into lipids before and after fertilization. The possibility that increased transport of 3H-choline at fertilization is linked to phosphorylation of choline was examined. The phosphorylation of choline present in the TCA soluble pools was measured in eggs and embryos of L. pictus that were preloaded or pulsed with 3H-choline as shown in Tables 4 and 5. The average
Phospholipid
313
Metabolism
percent phosphorylation for the preload experiment was 51.6 + 3.8 for fertilized and 51.7 + 4.0 for unfertilized eggs. In the pulse experiments (5 min pulses), the values were 19.5% f 3.7 in fertilized and 27.6% f 4.1 in unfertilized eggs. Although there is some variation in the percent phosphorylation, choline phosphorylation does not appear to change significantly after fertilization. Indeed, the data might suggest a slight decrease in phosphorylation at fertilization. Choline kinase actillity during early development. The above results constitute direct evidence that synthesis of the two major phospholipids is not stimulated during the first 3 hr of development. An independent method of measuring this is to TABLE
4
PHOSPHORYLATIONOF 3H-C~~~~~~ IN L. pictus PRELOAD EXPERIMENT@ Time of incubation after pulse 0 0 30 30 90 90
IN
‘:, phosphorylation
fertilized unfertilized fertilized unfertilized fertilized unfertilized
57.1 18.1 17.2 57.8 .50.2 49.1
“TCA soluble material was washed extensiveI> with diethyl ether to remove excess TCA. The resulting solution was then buffered with 0.1 M citrate buffer (pH 4.0) and assayed by the column method as described in Methods and Materials. TABLE
5
PHOSPHORFLATIONOF 3H-C~~~~~~ IN L. picks PULSE EXPERIMENTS’. b Time of development at pulse (5 min pulse 1 20 40 60 90 120
IN
? Phosphorylation Fertilized 18.2 20.4 “8.0 15.3 15.6
Unfertilized 34.3 32.2 21.6 26.6 21.3
’ Acid-soluble extracts of the embryos exposed to pulses of ‘H-choline in Table 1. b Assay conditions were the same as in Table 4.
314
DEVELOPMENTAL Bro~ocy
measure activity of choline kinase (E.C. 2.7.1.32) during early development. The properties of choline kinase activity in the 17,000g supernatant fraction of unfertilized eggs and fertilized eggs was studied to determine assay conditions. Saturation curves for choline and ATP in unfertilized eggs are shown in Fig. 2. Concentrations of 0.6 mM choline and 15 mA4 ATP-15 mA4Mg+* were chosen to obtain maximal activity in these enzyme preparations for fertilized and unfertilized eggs. Substitution of Mn+2 for Mg+2 resulted in a 81% decrease in activity. These conditions were optimal for both L. pictus and S. purpuratus. Differences in enzyme activity were looked for between fertilized and unfertilized eggs. No differences were found in the initial experiments; therefore, several different homogenization media were used to preclude the possibility that the homogenization might release the enzyme from a bound or inactive state in the unfertilized egg. Fertilized and unfertilized eggs were homogenized in media containing high or low salt concentration. The results indicate that the composition of the homogeniza-
VOLUME 46. 1975
tion media has little differential effect on enzyme activity between fertilized or unfertilized eggs in L. picks (Table 6). Similar results were also seen in S. purpuratus. Enzymatic activity was measured during early development. Table 7 shows that there is little change in enzymatic activity occurring during the first 18 hr of L. pictus development. There is an initial decrease in specific activity at fertilization of 25% and a slight increase by 18 hr. DISCUSSION
My study on S. purpuratus and L. pictus eggs and embryos, in agreement with a similar study on A. punctulata gametes (Schmell and Lennarz, 1974), indicates TABLE
6
EFFECTS OF VARIOUS HOMOGENIZATION MEDIA ON RESULTANT ENZYMATIC ACTIVITY OF CHOLINE KINASE” Medium
used
Choline kinase activity (nmoles/mg/min) Fertilized eggs (30 min)
0.01 M Tris-HCI, pH 8.5, 10 mM MgCI, 0.01 M Tris-HCI, pH 8.5, 0.2 M NaCl 0.01 M Tris-HCI, pH 8.5, 0.4 M NaCl 0.25 M Sucrose, 0.1 M Tris-HCl, pH 8.0 0 Reaction
conditions
Ur&$ eggs
7.8
7.5
10.3
11.2
10.4
10.5
8.0
9.2
were as described
TABLE
in text.
7
CHOLINE KINASE ACTIWIY IN EXTRACTS OF L. pictus EMBRYOS DURING EMBRYOGENESW b Time after fertilization PH
ATP (mm1
FIG. 2. Enzymatic activities at different substrate and pH values. A) Effect of substrate saturation on choline kinase activity. Assays were performed as in Methods and Materials with 15 mM Mg:ATP, pH 8.5. B) Effect of pH on choline kinase activity. The standard assay was used with 0.1 M Tris-buffer, pH 7.0-9.0. C) Effect of ATP concentration on choline kinase activity. Conditions for assay as described in text.
Unfertilized egg 15 minutes 45 minutes 90 minutes 150 minutes 18 hours
Enzyme activity (nmoles/mg/min) 6.35 4.84 4.73 5.33 4.85 6.85
DReaction conditions were as described in text. b Homogenization medium used (0.1 M Tris-HCl, pH 8.5, 10 mM MgC13.
E.
U'ILLIAM
BYRD. JR.
Sea Urchin
that there is no change in the incorporation of the major phospholipid precursors, choline and ethanolamine, into phospholipids after fertilization. This would suggest that de nouo phospholipid synthesis is not activated immediately following fertilization in the sea urchin. This observed constancy of phospholipid synthesis is also consistent with studies on the activity of enzymes involved in phosphat.idylcholine synthesis. The activity of choline kinase does not change significantly after fertilization; if anything, there is a decrease in activity during the first 3 hr of development. This lack of alteration of enzymatic activity was also seen when activity was measured under different homogenization conditions, in media attempting to better reflect the in uiuo activity of the enzyme. This lack of change in activity is also consistent in my in uivo studies which showed that there is no increased phosphorylation of choline after fertilization, as opposed to the earlier finding of Pasternak (1973). My interpretation is also consistent with the earlier work of Ewing (1973) that the activity of choline phosphotransferase decreases after fertilization of A. punctulata eggs. This data suggests that significant de nouo synthesis is not activated after fertilization in the sea urchin as observed by Pasternak (1973). This implies that the membrane changes associated with fertilization may be independent of de novo phospholipid synthesis. As new membranes are synthesized later in development there is a marked increase in phospholipid synthesis as observed by Pasternak ( 1973). There are some differences between this data on S. purpuratus and L. pictus and Schmell and Lennarz’s data on A. punctulata, these may reflect species differences. Although we agree that there is no increase in major phospholipid synthesis immediately following fertilization, there were differences in the degree of incorpora-
Phospholipid
Metabolism
315
tion of various precursors. They observed no incorporation of choline into phospholipids of either eggs or embryos, whereas I have observed about 10% incorporation of choline into chloroform-met,hanol soluble material of both eggs and embryos. This may be due to either nonspecific contamination or differences in precursor pools present. in these eggs. Amino acid pools of these two sea urchins differ radically (Fry and Gross, 1970) and there may be similar differences in choline pool size. Another difference between the two species of sea urchin was the rate of incorporation of ethanolamine into phosphatidylethanolamine. I observed a slightly increased uptake and incorporation after fertilization whereas Arbacia embryos showed a decreased incorporation relative to eggs. Schmell and Lennarz suggest that uptake differences may explain the enhanced incorporation of ethanolamine into egg lipid versus embryo lipid. Both this study and that of Schmell and Lennarz indicate a loss of radioactivity from phospholipid, probably turnover, when exogenous isotope is removed (preload experiment of Fig. 1). This is puzzling since there is a large pool of labelled phospholipid in the egg. Possibly exogenous label is preferentially utilized in spite of large endogenous pools of precursor. Mathews (in press) has shown that exogenous nucleoside triphosphates are preferentially utilized in S. purpuratus. If exogenous lipid precursors were utilized one would expect a rapid turnover aft.er exogenous label is removed and labeled phospholipids are metabolized. Since the level of phospholipids remains constant during early development (Mohri, 1964) and because a large amount of cell membrane components are formed during cleavage, this study suggests that the phospholipid reserves laid down during oogenesis are utilized during early development. It is therefore intriguing to propose that the lipids needed for membrane biogenesis are
316
DEVELOPMENTAL BIOLOGY
stored or compartmentalized, as in yolk, during oogenesis. As is the case with utilization of preformed RNA, fertilization of the egg would then activate metabolic pathways that would enable the embryo to utilize phospholipid st.ores. These experiments do not provide an absolute answer to the question of whether the immediate membrane changes of fertilization are independent of de nouo phospholipid synthesis. The percent of the total egg phospholipid in the plasma membrane is small due to the surface to volume ratio in the sea urchin egg. Although there is no major change in rate of precursor incorporation, a significant incorporation could go undetected against the large background of cytoplasmic incorporation. This work was supported in part by a grant from the National Science Foundation to Dr. D. Epel. E.W.B., Jr. is a postdoctoral trainee supported by Public Health Service Grant No. F22 HD02840-01. Drs. D. Epel and E. J. Carroll, Jr. are thanked for critical review and discussion of this manuscript. I thank Elizabeth Baker and Diana Majchrzak for excellent technical assistance. REFERENCES EPEL. D. (1964). A primary metabolic change of fertilization: Interconversion of pyridine nucleotides. Biochem. Biophys. Res. Commun. 17,62-69. EPEL, D., PRESSMAN, B. C., ELSAESSE~ S., and WEAVER. A. M. (1969). The program of structural and metabolic changes following fertilization of sea urchin eggs. In “The Cell Cycle” (G. M. Padilla. G. L. Whitson, and I. L. Cameron, eds.), pp. 280-298. New York. EPEL, D. (1972). Activation of a Na+-dependent
VOLUME 46. 1975 amino acid transport system upon fertilization of sea urchin eggs. Exp. Cell Res. 72. 74-89. EPEL, D. (1974). The program of and mechanisms of fertilization in the echinoderm egg. Amer. Zool. in press. EWING, R. D. (1973). Cholinephosphotransferase activity during early development of the sea urchin, Arbacia punctulata. Develop. Biol. 31. 234241. FRY, B. J.. and GROSS,P. R. (1970). Patterns and rates of protein synthesis in sea urchin embryos. Develop. Biol. 21, 125-116. GKIDICE, G. (1973). Developmental Biology of the sea urchin embryo. Academic Press. New York. ISONO, N. (1963). Carbohydrate metabolism in sea urchin eggs. J. Faculrv Sci. (Tokyo). 10, 37-53. ISONO, N. (19651. Phospholipids of sea urchin eggs. Sci. Papers Co/l. Gen. Educ. Biol. (Tokyo). 15, 87-94. LAYNE, E. (1957). Spectrophotometric and turbidity methods for measuring proteins. Methods Enzymol. 3, 447-454. LOWRY, 0. H., RQSEBROUGH, N. J., FARR, A. L.. RANDALL, J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. MATHEWS, C. (1975). Giant pools of DNA precursors in sea urchin eggs. Exp. Cell Res. 92,47-56. MAZIA, D. (1937). The release of calcium in Arbacin eggs upon fertilization. J. Cell Comp. Physiol. IO. 291-304. MCCAMAN, R. E. (1962). Intermediary metabolism of phospholipids in brain tissue. J. Biol. Chem. 237, 672-6’76. MOHRI, H. (1964). Utilization of Cl’-labeled acetate and glycerol for lipid synthesis during the early development of sea urchin embryos. Biol. Bull. 126, 440-455. PASTERNAK, C. A. (1973). Phospholipid synthesis in cleaving sea urchin eggs. Deuelop. Biol. 30, 403-410. SCHMELL, E., and LENNARZ, W. J. (1974). Phospholipid metabolism in eggs and embryos of the sea urchin. Arbacia punctulafa. Biochemistry 13, 4114-4121.
