Phospholipase Activity of Sea Urchin Sperm: Its Possible Involvement in Membrane Fusion A. F. CONWAY AND C. B. METZ Department of Biology a n d Institute for Molecular a n d Cellular Evolution, University of M i a m i , Coral Gables, Florida 331 34 a n d Marine Biological Laboratory, W o o d s Hole, Massachusetts 02543
ABSTRACT Phospholipase activity of egg-water treated Arbacia punctulata and Lytechinus variegatus sperm was shown to result from the sequential action of phospholipase A and lysophospholipase. A transient burst of phospholipase A activity followed induction of the acrosome reaction with egg water. The time of appearance suggested an acrosomal localization of the enzyme. The peak activity of phospholipase A correlated with initiation of sperm-egg fusion, suggesting a role for sea urchin sperm phospholipase A in membrane fusion and/ or egg activation during fertilization.
Although the morphological aspects of membrane fusion during the acrosome reaction and sperm-egg fusion in sea urchins have been described (Dan et al., '64; Franklin, '65; Anderson, '68), the biochemical mechanism(s) of these events has not been studied. This investigation was designed to evaluate the hypothesis of Lucy ('69, '71) that lysophospholipids are involved in induction of membrane fusion. If lysophospholipids are responsible for membrane fusion, lysophospholipids should be produced in the vicinity of fusing membranes. Therefore sperm andlor eggs should display enzyme activity of the phospholipase A type (EC.3.1.1.4; catalyzing removal of one fatty acyl residue from diacylglycerophospholipids) after appropriate stimulation. Phospholipase has been reported in sea urchin sperm. Intact sperm (Numanoi, '59) and sperm lysates (Mohri, '59) degrade purified phosphatidyl choline and lysophosphatidyl choline to glycerophosphoryl choline. Intact sperm are capable of releasing and breaking down phospholipids from hen egg yolk lipoprotein (Monroy, '53, '56; Maggio and Monroy, '55). Maggio and Monroy ('55) further observed that hemolytic activity appeared transiently during incubation of sea urchin sperm with hen egg yolk lipoprotein. Numanoi ('59) also observed release of lysophosphatidyl choline during fertilization in sea urchins but its source was not identified. The phospholipase activity observed during these studies was apparently due to production of hemolytic J. EXP.ZOOL., 198: 39-48.
lysophospholipids by phospholipase A, followed by their degradation by lysophospholipase (EC.3.1.1.5; catalyzing removal of the fatty acyl residue from monoacylglycerophospholipids). The incubation times used in all previous studies of sea urchin sperm lipolytic enzymes were far in excess of the 30 seconds required for sperm-egg membrane fusion (Franklin, '65) making correlations between enzyme activities and membrane fusion impossible. In the experiments reported here, the sperm acrosomal reaction was induced by egg water treatment (Dan, '52). This treatment caused rapid reaction of the acrosomes (Gregg, '71), resulting in exposure of any acrosomal enzymes to exogenous substrates rather than the surface of the egg. We hoped that this procedure would allow detection of any sperm-associated enzymes which would normally act on the egg plasma membrane. Since preliminary studies (Conway, unpublished) showed that phosphatidyl choline was the major phospholipid in sea urchin 1 This work is contribution No. 299 from the Institute for Molecular and Cellular Evolution. It was supported in part by grants from NIH (5-T01-HD00026-09 and 5-T01-HD00026-10 to the Fertilization and Gamete Physiology Training Program at the Marine Biological Lahoratory, Woods Hole, Massachusetts), NSF (Predoctoral fellowship to A. F. Conway and Grant GB3899 to C. B. Metz) and a Robert Maytag Predoctoral fellowship from the University of Miami. This work is part of a dissertation submitted to the Graduate School of the University of Miami in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Present address: Department of Biology, Virginia Commonwealth University, Richmond, Virginia 23284.
*
39
40
A. F. CONWAY A N D C. B. MET2
sperm and in crude preparations of sea urchin egg plasma membranes, this lipid (purified or in hen egg yolk lipoprotein) was chosen for the substrate in many of our phospholipase A assays.
0.01 M with respect to Tris, adjusted to pH 8.0, and filtered through Whatman No. 1 filter paper. Only “egg water” (EW) preparations showing sperm agglutination at greater than 1,000-fold dilution were used. Sperm enzyme assay mixtures contained MATERIALS AND METHODS substrate (phosphatidyl choline, hen egg All chemicals used were reagent grade. yolk, or lysophosphatidyl choline), sperm, Solvents were Chromatoquality (Matheson, and egg water (see figure legends for exact Coleman, and Bell) or reagent grade and composition). Four methods were used for were used without further purification. detection of phospholipase A activity: Lipid standards were obtained from Ana1. Lysophosphatidyl choline production. labs. Phosphatidyl choline for substrate Enzymatic activity was terminated by exwas prepared from hen egg yolk by the traction with four volumes of chloroformprocedure of Wells and Hanahan (‘69). methanol-concentrated hydrochloric acid Lysophosphatidyl choline from Analabs (2:0.9:0.1, vol/vol). Aliquots of lower phase was used without further purification. The were evaporated under nitrogen, redissolved purity of all substrates used was monitored in chloroform-methanol (4: 1, vollvol), and by thin layer chromatography. Marine Bi- spotted on ASL Prekotes After chromaological Laboratory formula artificial sea tography in chloroform-acetone-methanolwater (Cavanaugh, ’56) either complete glacial acetic acid-distilled water (50:20: (MBLSW) or calcium-free (CFSW) contain- 10:10:5 by volume), lysophosphatidyl choing 0.01 M Tris (Trishydroxyaminometh- line spots were located with iodine vapor ane), pH 8.0, was employed as the experi- and aspirated into Pasteur pipettes plugged mental medium except where otherwise with glass wool. Lipids were eluted with specified. Sucrose isotonic with sea water two milliliters of chloroform-methanol (2: 1, (0.81 M, Cavanaugh, ’56) was prepared in vol/vol) followed by two milliliters of meth0.01 M Tris-HC1buffer, pH 8.0. anol. The total eluate was evaporated over Arbacia punctulata were obtained from a hot water bath and analyzed for organic the Marine Biological Laboratory, Woods phosphorus according to Rosenthal and Hole, Massachusetts, and the Florida Ma- Han (‘69). 2. Free fatty acid release. Enzymatic rine Specimen Company, Panama City, Florida. Lytechinus variegatus were col- activity was terminated in 0.4 ml samples lected near Virginia Key, Dade County, by the addition of 0.4 ml of 1 N hydrochloFlorida. Adult animals were maintained in ric acid in methanol (freshly prepared). aerated aquaria equipped with recirculat- Phospholipids and other interfering substances were removed by treatment with ing filters. A. punctulata and L. variegatus were 0.6 g Zeolite (MacKenzie et al., ’67). Free spawned by electric shock (Harvey, ’56) fatty acids were extracted with four ml of and acetylcholine injection (Hinegardner, petroleum ether (pesticide quality). AIiquots ’61) respectively. Semen was collected di- of the petroleum ether extracts were anarectly from the gonopores in glass capillary lyzed for free fatty acids by the method of micropipettes (Drummond Sci. Co., State Anderson and McCarty (‘72) modified by College, Pennsylvania) and stored undiluted making the rhodamine reagent in petroleum in a humid atmosphere until used. Washed ether instead of benzene. This method was sperm were prepared by centrifugation also used to assay for lysophospholipase by (100 X g, 15 minutes) through a step gra- using lysophosphatidyl choline as substrate. dient consisting of a lower layer of buffered 3. Acylester hydrolysis. Enzymatic acisotonic sucrose, a middle layer of sucrose- tivity in one milliliter samples was termiMBLSW (l:l,vollvol), and an upper layer nated with two milliliters of 95% ethanol of MBLSW (containing semen). Sperm were and assayed for acyl ester content by the method of Augustyn and Elliott (‘69). recovered from the middle layer. 4. Decrease in turbidity of substrate Eggs were collected and washed several times in MBLSW. Egg jelly coat solutions solutions. The turbidometric assay of Rowere prepared by washing eggs in pH 5.0 senthal and Han (’70) was used with subMBLSW. The pH 5 supernatant was made stitution of MBLSW for saline and puri@.
SPERM PHOSPHOLIPASE AND MEMBRANE FUSION
I
Za
la
41
88‘
0.
2b
L IlC 0
I ’
0
t
30
30 60 INCUBATION TIME (rec)
I
I
60
INCUBATION TIME (see) Fig. 1 Lipolytic action on exogenous lipoproteins of A. punctulatn semen treated with egg water. Incubation mixture: 0.3 ml hen egg yolk stock (8.9 pmoles organic phosphoruslml) 1.0 ml egg water, pH 8.0 2.6 ml MBLSW, pH 8.0 0.1 m l 5 % semen i n MBLSW, no buffer Each point represents the mean of three experiments -t one standard error. l a , lysophosphatidyl choline production; lb, acyl ester hydrolysis; l c , turbidity decrease; *, significantly different from 0 time (p < 0.05); * 9 6 , highly significantly different from 0 time (p < 0.01). Fig. 2 Lipolytic action on exogenous phosphatidyl choline of L. uariegatus semen treated with egg water. Incubation mixture: 1 .O ml phosphatidyl choline stock (0.5pmoles organic phosphorus/ml) 2.0 ml egg water, pH 8.0 1.0 ml 4% semen in CFSW, pH 8.0 Each point represents the mean of three experiments -t one standard error. 2a, lysophosphatidyl choline production; 2b, free fatty acid release; *, significantly different from 0 time (p < 0.05); **,highly significantly different from 0 time (p < 0.01).
42
A. F. CONWAY AND C. B. MET2
fied phosphatidyl choline for egg yolk in mixture declined rapidly. Over the period some experiments. The turbidity changes 15 to 30 seconds after semen addition the were recorded, usually at 15-second inter- rate of free fatty acid release, acyl ester vals, and are presented as net change (1 a hydrolysis, and turbidity decrease did not unit = a decrease of 0.001 absorbance differ significantly from the rate of lysounit) or rate of change (1 A unit = a rate phosphatidyl choline production under the of l a unit per minute). Reaction rates were same conditions (compare fig. l a with fig. linear for at least 15 seconds after the on- l b and fig. 2a with fig. 2b). Between 30 set of activity, and were assumed to repre- and 60 seconds after sperm addition free sent the initial rates of reaction. fatty acid release, acyl ester hydrolysis, Colorimetric and turbidometric measure- and turbidity decrease differed from lysoments were made with a Beckman DU spec- phosphatidyl choline content in exhibiting trophotometer (Method 1) or a Baush and a continued increase while the lysophosLomb Spectronic 20 (Methods 2, 3 , and 4) phatidyl choline content of the incubation equipped with voltage stabilizers. In all mixture decreased rapidly. cases control samples (complete except for The quantity of substrate hydrolyzed by substrate) were analyzed and subtracted egg water treated sea urchin semen defrom experimental values to obtain the pended on the type of substrate used (tavalues used in the figures. In none of the ble 1). Hen egg yolk lipoprotein yielded the assays was significant lipolytic activity con- largest quantity of hydrolysis products of sistently observed in the absence of sub- the substrates tested. Egg water treated strate. The experimental results were ana- semen from A. punctulata and L.variegatus lyzed using statistical methods for small showed no quantitative difference in the samples (Arkin and Colton, '70). phospholipase activity when compared under identical conditions (table 1). RESULTS The origin of the lipolytic activity of egg After egg water treatment sea urchin water treated sea urchin semen was invessemen produced and destroyed lysophos- tigated. The turbidity decreasing activity pholipids (figs. 1, 2). The lysophosphatidyl of egg water treated semen was apparently choline content of the assay mixture in- associated with the semen rather than creased rapidly from 15 to 30 seconds after arising from egg water as a result of actisperm addition (figs. l a , 2a). Thirty to vation by semen, since the initial velocity sixty seconds after sperm addition the lyso- of turbidity decrease was proportional to phosphatidyl choline content of the assay the semen concentration (fig. 3). When sea TABLE 1
Phospholipase action of sea urchin semen treated w i t h egg water Chemical assays (nmoles product formedlbl semen) Semen Substrate
A. vunctulata
PC (0.125 Fmoles/ml) PC 0.01% DOC (0.125 r*.moles/ml) Hen egg yolk Preparation I 0.66pmoles Porglml Hen egg yolk Preparation I1 0.45 @molesPorg/ml PC (0.125 umoles/ml)
+
L. variegatus
Net product produced 15-30 sec
Net product produced 15-60 sec
Assay used
1.9 rt 0.8 1
3.6 f 1.0
FFA release
2.0 .r 0.5
5.6 f0.6
AE hydrolysis
110f40
164 f45
AE hydrolysis
43 -e 14
AE hydrolysis
4.0 f 1.0
FFA release L-PC production
1.0 +- 1.5 1.5rt0.7
-
Abbreviations: PC, phosphatidyl choline; GPC, lysophosphatidyl choline; DOC, sodium deoxycholate; FFA, free fatty acids; AE, acyl ester. Figures represent the mean of three experiments & one standard error.
43
SPERM PHOSPHOLIPASE AND MEMBRANE FUSION
urchin semen was layered over a sucrose gradient and centrifuged (see technique for preparation of washed sperm) before treatment with egg water, the turbidity reducing activity (after egg water treatment) of the fractions obtained correlated with the presence of intact sperm cells (table 2). In experiments to determine the effect of egg water treatment of semen, no significant turbidity reducing activity was observed in the absence of egg water treatment (fig. 4). In the presence of egg water a typical pattern of turbidity reduction occurred. In both A. punctulata and L. variegatus the lysophosphatidyl choline produced by sperm phospholipase A disappeared rapidly after 30 seconds of incubation (figs. l a , 2a). A preliminary study was made of the mechanism of lysophosphatidyl choline removal. Incubation of semen with lysophosphatidyl choline (fig. 5) failed to result in any significant formation of phosphatidyl choline. However, incubation of semen with lysophosphatidyl choline resulted in release of free fatty acids (fig. 6), indicating that lysophospholipase was present. The lysophospholipase activity differed from sperm phospholipase A in not undergoing inactivation during two minutes of incubation. DISCUSSION
The lipolytic action of sea urchin semen on exogenous phospholipids was complex (figs. 1, 2). The initial “lag phase” prior to the appearance of hydrolysis products (0-15 seconds after semen addition) probably represents the time required for ex-
posure of lipolytic enzymes by the acrosomal reaction since the time of appearance of lipolytic activity (15 seconds after semen addition) is the same as that for the appearance of reacted acrosomes in egg water treated sperm (Gregg, ’71). The initial phospholipase activity (1 5-30 seconds after semen addition) was largely due to phospholipase A action, since acyl ester hydrolysis and free fatty acid release did not significantly exceed the production of lysophosphatidyl choline over the same period (figs. l a , 2a). Any extensive action of lysophospholipase on phosphatidyl choline (as observed by Kates et al., ’65) should have resulted in free fatty acid release andlor acyl ester hydrolysis in excess of lysophosphatidyl choline production during 0 to 30 seconds of incubation. No significant difference (t test, 95% confidence level) between these values was observed. The small nonsignificant differences which were observed may be explained by initiation of hydrolysis of lysophosphatidyl choline by lysophospholipase prior to the cessation of phospholipase A hydrolysis of phosphatidyl choline. The agreement of turbidity decrease hom 15 to 30 seconds after semen addition with lysophosphatidyl choline production and acyl ester hydrolysis over the same period (fig. 1) indicated that turbidity decrease provided a valid assay for sea urchin semen phospholipase A during this period. The validity of the initial rate of turbidity decrease as an assay of semen phospholipase A activity was further confirmed by the observation that a known inhibitor of pancreatic phospholipase A (cadmium chlo-
TABLE 2
Distribution of turbidity reducing act i vi t y a n d phospholipids of A. punctulata s e m e n after centrifugation on a sucrose s te p gradient
Layer
Upper (Sea water)
Contents (light Damaged sperm, microscope) seminal plasma Lipid organic phosphorus (8of total extractable by Folch et al., ’57) 24.9 1 Turbidity reducing activity 0’ % oftotal 1
Mean of two experiments.
Middle (Sea water: isotonic sucrose, 1:1, vol/vol)
Lower (Isotonic sucrose)
Most of sperm
Few sperm
66.6 1
75.5 1
8.5 1 25.0
1
44
A. F. CONWAY AND C . B. METZ
I s
3
0 0.1 0.2 SEMEN CONCENTRATION (X) Fig. 3 Dependence of the initial velocity of turbidity decrease on semen concentration i n A. pzrnctiilntci. Incubntion m i x t u r e : 0.3 ml hen egg yolk stock (8.9 pmoles organic
phosphorus/ml) 1.0 ml egg water, pH 8.0
2.6 ml MBLSW, pH 8.0 0.1 mi semen suspension i n MBLSW, no buffer Each point represents the mean of three experiments -r- one standard error.
1
0
1 I1
INCUBATION TIME hid Fig. 5 The phosphatidyl choline content of reacted L. vnriegatus semen incubated with lysophosphatidyl choline. Inczrbation m i x t u r e : 1.0 ml 50 mM lysophosphatidyl choline i n MBLSW, pH 8.0 1.0 ml egg water, pH 8.0 0.5 m l 5 % semen in CFSW, pH 8.0 Analyzed by thin layer chromatography. Each point represents the mean of three experiments t one standard error.
1 6
14
I
I.
-
.
.
.
.
.
.
.
60 90 1% INCUBATION TIME (sec) 30
Fig. 4 Comparison of the turbidity reducing action of A. pzrnctzrlatn semen following treatment with egg water or sea water. Incubation m i x t u r e : 0.3 ml hen egg yolk stock (8.9 pmoles organic phosphoruslml) 1.0 ml egg water, pH 8.0 or MBLSW, pH 8.0 2.6 ml MBLSW, pH 8.0 0.1 m l 5 % semen in MBLSW, no buffer Each point represents the mean of four experiments c one standard error. Upper curve treated with egg water; Lower curve treated with sea water.
1 2 INCUBATION TIME ( m i d
0
Fig. 6 Free fatty acid content of reacted L. uariegntzis semen incubated with lysophosphatidyl choline. Incubation m i x t u r e : 1.0 ml -50 mM lysophosphatidyl choline in MBLSW, pH 8.0 1.0 ml egg water, pH 8.0 0.5 ml 5 % semen in CFSW, p H 8.0 Each point represents the mean of three experiments rt one standard error. Note: The substrate concentration used in these experiments was the maximum which did not cause lysis of the sperm cells. Saturation levels of substrate were probably not reached. Therefore these values probably do not represent the maximum activity. *, significantly different from 0 time (p < 0.05); d9, highly significantly different from 0 time (p < 0.01).
SPERM PHOSPHOLIPASE AND MEMBRANE FUSION
ride) also inhibited the initial velocity of turbidity decrease caused by sea urchin semen (Conway, in preparation). Phospholipase A was reported in sea urchin sperm (Maggio and Monroy, '55) and in the mammalian sperm acrosome (Allison and Hartree, '68). However, the sea urchin sperm phospholipase A activity reported here differed from previous reports in that this sea urchin phospholipase A displayed the characteristics expected of an enzyme involved in membrane fusion. The activity appeared following egg water treatment suggesting that the enzyme(s) responsible was associated with the acrosome, which is the site of sperm-egg fusion (Franklin, '65). The activity was present during the period at which Franklin ('65) reported that sperm-egg membrane fusion occurs (15-30 seconds after sperm addition). If membrane fusion required membrane destabilization, excessive action of the fusion enzyme might lyse the newly formed zygote. Therefore the disappearance of the enzyme activity is expected. The time course of the changes in phospholipase A activity following release of the enzyme indicated that the activity was probably involved in sperm-egg fusion rather than the acrosome reaction (figs. l a , 2a). The enzyme(s) responsible for semen phospholipase activity between 30 and 60 seconds after semen addition were less definable. A mechanism for the removal of lysolipids after sperm-egg fusion was expected to prevent lysis of the newly formed zygote. The decrease in lysophosphatidyl choline content from 30 to 60 seconds after semen addition (figs. l a , 2a) indicated that some mechanism of lysophosphatidyl choline removal was present. Continued decrease of acyl ester content (fig. lb) coupled with the lack of increase in phosphatidy1 choline content during incubation of semen with lysophosphatidyl choline (fig. 5 ) indicated that resynthesis and acyl transfer were not of major importance in lysophosphatidyl choline removal. Incubation of semen with lysophosphatidyl choline did result in free fatty acid release (fig. 6). This activity was not due to induction of endogenous phospholipase A activity by lysophosphatidyl choline since in other experiments (Conway, in preparation) lysophosphatidyl choline inhibited sperm phospholipase A.
45
Therefore release of free fatty acids during incubation of sea urchin sperm with lysophosphatidyl choline must be due to the action of a lysophospholipase. Sea urchin sperm therefore differ from other membrane systems in which resynthesis (Oliveira and Vaughan, '62; Mulder and Van Deenen, '65; Stahl and Trams, '68; Redman, '71) and acyl transfer (asback et al., '65) were demonstrated. Whether these processes occur in the newly formed zygote membrane is not clear, since one or both of these processes may be present in the egg plasma membrane and participate in lysolipid removal during fertilization. Under the conditions used in this study, the predominant mode of lysophosphatidyl choline removal was lysophospholipase. The continued turbidity decrease during the decrease in lysophosphatidyl choline content (compare figs. l a and l c from 30-60 seconds after semen addition) was surprising, since this indicated continued release of surface active products. Lysophosphatidyl choline was believed to be the major cause of turbidity decrease in systems in which the turbidity assay has been used previously (Marinetti, '65). The correspondence of the pattern of turbidity decrease with the pattern of acyl ester hydrolysis from 30 to 60 seconds after semen addition suggested that the continued turbidity decrease over this period might be due to release of surface active fatty acids by lysophospholipase action. A scheme for the mechanism of membrane fusion based on these experiments is presented in figure 7. In the model phospholipids contribute to a stable membrane structure. Conversion of membrane phospholipids to lysophospholipids results in an
Fig. 7 Diagrammatic representation of the role of sea urchin sperm enzymes in membrane fusion. Heavy arrows indicate reactions which have demonstrated. Thin arrows indicate reactions which were not detected. Tapered arrows represent postulated effects on cell membranes.
46
A. F. CONWAY AND C. B. METZ
unstable membrane structure, possibly by local conversion of a predominantly bilayer structure to a micellar structure as proposed by Lucy (‘69, ’70). In this form the membrane is capable of fusion with another membrane in a similar condition if they are brought into close apposition. Once fusion has occurred the lysolipids are converted to water soluble glycero-phosphoryl esters by lysophospholipase. Alternatives to lysophospholipase action include resynthesis and acyl ester transfer, but no evidence for these processes was found in sperm. The relevance of this membrane fusion model to membrane fusion systems other than during sperm-egg fusion is uncertain. The demonstration by Uvnas (‘63) that phospholipase A treatment resulted in degranulation of mast cells in vitro indicates that a Lucy-type mechanism is capable of causing fusion of secretory granules with the plasma membrane, but whether this mechanism normally operates in vivo is unknown. A further attractive feature of this model is that it might also explain egg activation during sperm-egg fusion. The transient existence of lysophospholipids in the egg membrane should result in a transient breakdown in the permeability barrier at the egg surface. The ion flux(es) resulting from such temporary permeability changes of the egg plasma membrane might serve as a trigger for many of the biochemical events resulting from egg activation. The results of these experiments are consistent with the hypothesis of Monroy (‘53, ’56) and Maggio and Monroy (‘55) that the lipolytic activity which they observed in sea urchin sperm functioned in egg activation. ACKNOWLEDGMENTS
The guidance, advice and assistance of Drs. Kurt Koehler, Marco Crippa, and Carolyn Conway is gratefully acknowledged. LITERATURE CITED Allison, A. C., and E. F. Hartree 1968 Lysosomd nature of the acrosomes of ram spermatozoa. Biochem. J., 1 I I : 35. Anderson, E. 1968 Oocyte differentiation i n the sea urchin, Arbncia punctulata, with particular reference to the origin of cortical granules and their participation i n the corticaI reaction. J. Cell Biol.. 37: 514-539.
Anderson, M. M., and R. E. McCarty 1972 Rapid and sensitive assay for free fatty acids using rhodamine 6G. Anal. Biochem., 45: 260-270. Arkin, H., and R. R. Colton 1970 Statistical Methods. Fifth ed. Barnes & Noble, Inc., N. Y. Augustyn, J. M., and W. B. Elliott 1969 A modified hydroxamate assay of phospholipase A activity. Anal. Biochem., 31 : 246-250. Cavanaugh, G. M. 1956 Formulae and Methods V of the Manne Biological Laboratory. Marine Biological Laboratory, Woods Hole, Mass. Dan, J. C. 1952 Studies on the acrosome. I. Reaction to egg-water and other stimuli. Biol. Bull., 103:54-66. Dan, J. C., Y. Ohori and A. Kushida 1964 Studies on the acrosome. VII. Formation of the acrosoma1 process in sea urchin spermatozoa. J. U1trastruct. Res., 11: 508-524. Elsbach, P., J. W. 0. Van den Berg, H. Van den Bosch and L. L. M. Van Deenen 1965 Metabolism of phospholipids by polymorphonuclear leukocytes. Biochim. Biophys. Acta, 106: 338-
347. Franklin, L. E. 1965 Morphology of gamete membrane fusion and of sperm entry into oocytes of the sea urchin. J. Cell Biol., 25: 81-100. Gregg, K. R. 1971 A Study of Cortical Response Antigens, Egg Jelly and the Induction of the Acrosome Reaction Using Gametes of the Sea Urchin, Arbacia punctulata. Doctoral dissertation, University of Miami, Coral Gables, Fla. Harvey, E. B. 1956 The American Arbacia and Other Sea Urchins. Princeton University Press, Princeton, N. J. Hinegardner, R. T. 1961 The DNA content of isolated sea urchin egg nuclei. Exp. Cell Res., 2 5 : 231-247. Kates, M., J. R. Madeley and J. L. Beare 1965 Action of phospholipase B on ultrasonically dispersed lecithin. Biochem. Biophys. Acta, 106:
630-634. Lucy, J. A. 1969 Lysosomal membranes. In: Lysosomes in Biology and Medicine. Vol. 11. J. T. Dingle and H. B. Fell, eds. North-Holland Publishing Co., Amsterdam, pp. 313-341. Lucy, J. A. 1971 The fusion of biological membranes. Nature, 227: 814-817. MacKenzie, R. D., T. R. Blohm, E. M. Auxier and A. C. Luther 1967 Rapid colorimetric micromethod for free fatty acids. J. Lipid Res., 8: 589-
597. Maggio, R., and A. Monroy 1955 Some experiments pertaining to the chemical mechanisms of the cortical reaction in fertilization of sea urchin eggs. Exp. Cell Res., 8: 240-244. Marinetti, G. V. 1965 The action of phospholipase A on lipoproteins. Biochim. Biophys. Acta, 98:554565. Mohri, H. 1959 Enzymic hydrolysis of phospholipids i n sea urchin spermatozoa. Sci. Pap. Coll. Gen. Educ. Univ. (Tokyo), 9:270-278. Monroy, A. 1953 A model for the cortical reaction of fertilization in the sea urchin egg. Experientia, 9:424-425. 1956 Some experiments concerning the chemical mechanisms of the activation of the sea urchin egg. Exp. Cell Res., 10: 320-323. Mulder, E., and L. L. M. Van Deenen 1965 Metabolism of red-cell lipids. I. Incorporation in uitro of fatty acids into phospholipids from ma-
SPERM PHOSPHOLIPASE AND MEMBRANE FUSION ture erythrocytes. Biochim. Biophys. Acta, 106:
106-1 17. Numanoi, H. 1959 Studies on the fertilization substance. 8. Enzymic degradation of lecithin during development of sea urchin eggs. Sci. Pap. Coll. Gen. Educ. Univ. (Tokyo), 9:285-296. Oliveira, M. M., and M. Vaughan 1962 Incorporation of fatty acids into phospholipids of red blood cell membrane. Fed. Proc., 21 ; 296. Redman, C. M. 1971 Phospholipid metabolism in intact and modified erythrocyte membranes. J. Cell Biol., 49: 35-49. Rosenthal, A. F.,and S . C.-H. Han 1969 Phosphorus determination i n phosphoglycerides from thin-layer chromatograms. J. Lipid Res., 1 0 : 243-245.
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1970 A study of phospholipase A inhibition by glycerophosphatide analogs in various systems. Biochim. Biophys. Acta, 218: 213-220. Stahl, W. L., and E. G. Trams 1968 Synthesis of lipids by liver plasma membranes. Incorporation of acyl-coenzyme A derivatives into membrane lipids in uitro. Biochim. Biophys. Acta, 163: 459-471. Uvnas, B. 1963 Mechanism of histamine release i n mast cells. Ann. N. Y. Acad. Sci., 103: 278-284. Wells, M. A,, and D. J. Hanahan 1969 Phospholipase A from Crotnlus ndnmenteus venom. In: Methods in Enzymology. Vol. 14. S. P. Colowick and N. 0. Kaplan, eds. Academic Prees, New York, pp. 178-184.