Fatty Acid Distribution in Lipids and 32p Incorporation into Phospholipids during Early Amphibian Development C.A. BARASSI 1 and N.G. BAZ/~,N, 2 Instituto de Investigaciones B~oqutrnmas, Universidad Nacional del Sur, Bah(a Blanca, Argentina ABSTRACT

Several aspects of lipid composition and 32p incorporation were studied during early embryogenesis of the toad, Bufo arenarurn, Hensel. The surveyed stages ranged from unfertilized oocyte to neural tube formation. The fatty acid distribution in polar and neutral lipids, as well as in acetone eluate from Unisil columns was similar in unfertilized oocyte and late blastula stage. There was no significant effect of cell cleavage on the fatty acid composition of these lipid fractions. Neutral lipids represent ca. 67% of the total lipids. The main components of the phospholipids were phosphatides of choline and ethanolamine. The total lipid and phospholipid content does n o t change through the studied stage of neurula. However a large increment in the phospholipid's specific radioactivity occurs when 32p is injected along with the hormone to induce ovulation. It is suggested that this may reflect changes in turnover rates rather than net biosynthesis. Since a large amount of cell membranes is being formed during the early development and because the level of phospholipids remains constant, an explanation is offered regarding membranogenesis. Active phospholipid biosynthesis may take place during oogenesis. These lipids may be stored in the yolk platelet, and fertilization may regulate the functioning of a transport mechanism to corresponding membrane sites. The mcreased incorporation of 32p may reflect changes in the activity of new membranes.

exists on the phospholipid composition at one stage of amphibian development (4). However several aspects of lipid biochemistry were studied during the development of invertebrate eggs, such as those of insects (5) and nematodes (6-8). The aim of the present study was three-fold: (a) to examine the effect of cleavage from oocyte until late blastula stage on the fatty acid distribution in lipid fractions; (b) to study the level of total, neutral and polar lipids from unfertilized oocytes until neurula; and (c) to determine the phospholipid turnover during early embryogenesis by following the 32p incorporation. MATERIALS AND METHODS Materials

All solvents used were analytical grade and were distillated before use. Chemicals a n d standards used were purchased as follows; thioglycolic acid from Merck; Unisil from Clarkson Chemical Co.; lipid standards and fatty acid methyl esters from Supelco, Inc., Applied Science Labs. and The Hormel Institute, University of Minnesota; 6% diethylene glycol succinate on diatoport S, from Hewlett Packard. Human chorionic gonadotropin was obtained from E.L.E.A., Argentina, and sterile radioactive orthophosphate (Na2H32PO4, 69 Ci/g of P) from the Comisi6n Nacional de Energi'a Atfmica, Argentina. Ooeytes and Embryos

Adult Bufo arenarum, Hensel toads captured in the surrroundings of the cities of Bahia Blanca and Tucum~n were used. They were kept in a humidified container without feeding for 3-6 weeks prior to the experiments. To induce ovulation adult females were INTRODUCTION injected in the dorsal lymphatic sacs with Biochemical studies on the structure and 1000-1500 IU chorionic gonadotropin or with a metabolism of lipids during the very early freshly prepared suspension of one or two toad stages of vertebrate embryonic development are pituitary glands in amphibian Ringer solution. scarce. Several authors have reported data on The toads began to eliminate oocytes through the lipids of hen egg yolk (1-3), and a report the cloaca 14-18 hr later. At this point they were demedulated, and the abdominal cavity lIn partial fulfilment of the requirements for the was quickly opened; from the ovisacs the Ph.D. in Biochemistry. oocytes were collected in a petri dish contain2Reprint requests should be directed to N.G. ing amphibian Ringer solution. Next the ova Baz~n. were artificially fertilized with a homogenate of 27

28

C.A. BARASSI AND N.G. BAZtkN

toad testes. The medium employed for these and all subsequent procedures was amphibian Ringer soiution (NaC1 0.65 g, KC1 0.01 g and CaC12 0.003 g/liter). Part of the unfertilized ova and the different stages of development was sampled after removal of the jelly coat by brief periods of contact with neutralized 2% thioglycolic acid. Development was allowed to proceed at 20-25 C and was followed by means of a stereoscopic microscope microscope (50x), using as a reference the morphological characteristics described by Del Conte and Sirlin (9).

plished. Then methanolysis was carried out in a boiling water bath with 14% BF 3 in methanol (14,15) for 90, 30 and 90 min for the acetone eluate, neutral lipids and total phospholipids, respectively. After partitioning, the methyl esters of fatty acids contained in the lower phase were separated on a 0.1 mm thick layer of Silica Gel G using toluene as developing solvent. The spots corresponding to the fatty -acid methyl esters were eluted with ether and hexane.

Preparation of Lipid Extract

A Varian Aerograph gas chromatograph, mode/ 1700, equipped with two hydrogen flame ionization detectors was employed. For identification purposes, a polar (diethylene glycol succinate) and a nonpolar (OV-1) column were used. Identification was completed by the use of pure standards and catalytic hydrogenation. Most of the runs were carried out in a stainless steel column coated with 6% diethylene glycol succinate on Diatoport S, 80-100 mesh (ID 2.3 mm, length 2.20 m) with nitrogen as carrier gas (flow rate 20 ml/min) and at an oven temperature of 200 C (injector port 230 C and detector 210 C).

Samples were homogenized with chloroform-methanol 2:1 v/v by means of a PotterElvehjem type homogenizer with a motor driven teflon pestle (10). In order to assure completeness in the lipid extraction, including tightly bound polyphosphoinositides, the remaining residue was reextracted three times with 4 volumes of 0.25% He1 in chloroformmethanol 2:1 v/v (11) and centrifuged. Acidified chloroform extraction was not applied in the experiments reported in Tables III and IV. Then the supernatant was filtered through glass wool, mixed with 0.2 volumes of 1 N He1, and centrifuged. Afterwards the lower phase was combined with the lipid extract obtained by Folch's procedure. The extracts were stored under nitrogen at -20 C until processing. Column Chromatography

The combined neutral and acidified chloroform-methanol extracts were taken to a small volume under a nitrogen stream and applied in 1-2 ml to a Unisil column (ID 2.5 cm, height 9 cm) from which three fractions were eluted: chloroform, 100 ml; acetone, 200 ml; and methanol, 200 ml (12). The flow rate was 3 ml/min. Each fraction was taken to dryness in a rotary vacuum evaporator and resuspended in a small volume. The behavior of the column was monitored by applying pure neutral and polar lipids, and then following their distribution in the eluted fractions by means of thin layer chromatography (TLC). A similar test was conducted with several embryo extracts, determining in addition the lipid phosphorus content in each fraction. The two dimensional TLC procedure of Rouser et al. (13) was employed for the separation of the phospholipids. Methanolysis and Purification of Methyl Esters by TLC

The volume of the eluted fractions was reduced under a nitrogen stream at 45 C and then was placed in Teflon-lined screw cap tubes where evaporation to dryness was accomLIPIDS, VOL. 9, NO. I

Gas Liquid Chromatography

Experimental Design for in Vivo Incorporation of 32p into Phospholipids

In vivo labeling of oocytes was accomplished by the procedure described elsewhere (17). In brief, 200 pCi of 32p per 100 g of body weight were injected into the dorsal lymphatic sac along with the gonadotrophin or with pituitary gland extract in order to incorporate the radioisotope into the maturating oocytes during the action of the hormone. Thus oocytes with their phosphorylated molecules and their inorganic phosphate pool labeled with 32p were obtained. After fertilization development was followed by maintaining the embryos in amphibian RAnger solution without the further addition of 32p. Determination of Specific Activity

Aliquots from a washed total lipid extract made up to a known volume were taken to dryness; one of them, usually one-tenth of the final volume, was placed on an aluminium planchet and the other in a test tube. Aliquots from the latter were digested with 70% perchloric acid, and phosphorus was determined colorimetrically (18). In all instances duplicates were run. Radioactivity was measured in the planchets using a model RM gas flow counter spectrometer (Alfanuclear, S.A.I.C., Argentina). The efficiency of the system was 73% for Na2H32p, and data was n o t corrected for efficiency.

29

LIPIDS DURING E A R L Y EMBRYOGENESIS

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LIPIDS, VOL. 9, NO. 1

C.A. BARASSI AND N.G. BAZAN

30

TABLE II Ratios of Acyl Content of Neutral Lipids to Phospholipids and of Saturated to Unsaturated Fatty Acids from Oocytes and Blastula a Phospholipidsb Samples

Neutral lipid

Oocytes A B Blastula

0.22 0.29 0.53

Saturated to unsaturated fatty acids Neutral lipids Acetone eluate Phospholipids 0.37 0.41 0.41

1.86 2.35 1.95

0.56 0.75 1.04

aRatios were obtained by dividing total peak areas of saturated by unsaturated methyl esters of fatty acids. Further details as in Table I, bThese ratios represent total acyl content of phospholipids to total acyl content of neutral lipids obtained by gas liquid chromatography as described in Table II. RESULTS

total lipid fraction.

Lipid Fractions and Their Methyl Esters in Unfertilized Oocytes and Embryos

Purified lipid e x t r a c t s o f u n f e r t i l i z e d o o c y t e s and e m b r y o s were f r a c t i o n a t e d o n silicic acid c o l u m n s . The e l u t e d f r a c t i o n s were a n a l y z e d by TLC and c o m p a r e d w i t h pure r e f e r e n c e comp o u n d s . In a d d i t i o n the t o t a l P c o n t e n t was d e t e r m i n e d in each fraction. Only the runs t h a t y i e l d e d c h l o r o f o r m f r a c t i o n s devoid o f P were used. The c h l o r o f o r m eluate was c o m p o s e d o f n e u t r a l lipids a n d o f p i g m e n t ( u n p u b l i s h e d observations), and the m e t h a n o l eluate was c o m p o s e d o f p h o s p h o l i p i d s , p h o s p h a t i d y l choline a n d p h o s p h a t i d y l e t h a n o l a m i n e being the m a j o r c o m p o n e n t s . A detailed s t u d y on the c o m p o s i t i o n o f p h o s p h o l i p i d s during early dev e l o p m e n t is b e i n g p r e p a r e d for p u b l i c a t i o n in this l a b o r a t o r y . The f r a c t i o n e l u t e d w i t h acet o n e has an u n k n o w n c o m p o s i t i o n . The P c o n t e n t per 1000 e m b r y o s in t h e a c e t o n e eluate was 2.89 + 2.13 /umol and in the m e t h a n o l eluate 36.93 _+ 1.61 /amol, thus ca. 7% o f the

P was r e c o v e r e d in the a c e t o n e

Fatty Acid Distribution in Lipid Fractions of Unfertilized O ocytes and Late Blastula Stage

In the o o c y t e , p a l m i t a t e , p a l m i t o l e a t e and oleate c o m p r i s e s ca. 80% o f t h e f a t t y acids o f n e u t r a l lipids and ca. 75% of p h o s p h o l i p i d s . A similar c o m p o s i t i o n was f o u n d in the f a t t y acids derived f r o m the a c e t o n e eluate w i t h the e x c e p t i o n o f a c o m p o n e n t t h a t behaves in the gas c h r o m a t o g r a p h as lignocerate. This c o m p o n e n t e r e p r e s e n t e d b e t w e e n 3 and 8% o f the total f a t t y acids in o o c y t e s and blastula. The level o f each acyl c o m p o n e n t was q u a n t i f i e d b y gas liquid c h r o m a t o g r a p h y using internal standards, and the data is p r e s e n t e d as m i c r o g r a m s f a t t y acid per 10 m g dry weight (Table I). It is i n t e r e s t i n g t h a t in o o c y t e s the t o t a l unsaturation is slightly higher in p o l a r lipids, while in the a c e t o n e eluate a p r e d o m i n a n c e o f s a t u r a t e d c o m p o n e n t s can be seen (Table II). A similar d i s t r i b u t i o n o f t o t a l e t h y l e n i c f a t t y acids was o b s e r v e d a t blastula stage. The acyl t o t a l

TABLE II1 Level of Phospholipids, Neutral and Total Lipids in Developing Toad Embryos a rag/1000 Oocytes or embryos Stage

Total lipid

Phospholipids

Unfertilized oocytes Fifth cle avage (16 cells) Late blastula First invagination of dorsal lip Neural fold Neural tube

182 184

59 60

Nonpolar lipids l 23 l 24

173 174 172 181

56 57 57 59

117 l 17 115 122

aThree samples of 250 oocytes or embryos of each stage were used. Figures represent average values, and results agreed within 6% for determinations in different samples. Total liplds Were determined by drying aliquots of washed chloroform-methanol extracts to constant weight at 110 C. Phospholipid levels were obtained from colorimetric measurements of lipid P (16) using a conversion factor of 25. Nonpolar lipid values were obtained by difference. Other details are given in Materials and Methods. LIPIDS, VOL. 9, NO. 1

LIPIDS DURING EARLY EMBRYOGENESIS

31

TABLE IV Incorporation of 32p and Specific Activity of Phospholipids during Early Stages of Toad D e v e l o p m e n t a Stage Unfertilized oocytes Fifth cleavage (16 cells) Blastula

Late blastula First invagin ation of dorsal lip

Neural fold Neural tube

Specific activity, cpm/#mol of P Incorporation, cpm/1000 oocytes or embryos 559 729

42,492 56,168

915 998 1075 2210 2405

67,748 71,912 75,580 163,140 182,848

aln vivo incorporation of 32p was performed as described elsewhere (15) and in Materials and Methods. The samples were three of 250 oocytes or embryos as in Table III, with the e x c e p t i o n o f blastula that were two samples of 500 embryos each. The figures represent mean values of three determinations and in all cases t h e y agreed within 7%. Other details as in Material and Methods. content of neutral lipids slightly decreased from fertilization until blastula (Table II). Lipids during Early Development and 32p Incorporation into Total Phospholipids

No significant changes were detected in the level of total phospholipids and lipids from unfertilized oocytes until the stage of neural tube (Table III). Phosphatidyl choline and phosphatidyl ethanolamine were the most abundant constituents of the phospholipids in unfertilized oocyte and throughout the early stages of development until the surveyed stage of dorsal lip. Phosphatidyl choline and phosphatidyl ethanolamine comprise 44.9 + 5.6 and 18.0 + 114% of the total phospholipids, respectively, and their level is n o t modified during the studied period of embryogenesis. The average level from oocyte to neural tube was 57.7 + 1.3 mg phospholipid per 1000 oocytes or embryos. A small decrease was observed in neutral lipids at the stage of blastula, dorsal lip and neural fold (Table III). Until the neural tube stage, ca. 67% of the total lipids was nonpolar. 32p injected i n t o . t h e dorsal lymphatic sac along with the hormone to induce ovulation was used to label the phospholipids. In Table IV d a t a on the incorporation of 32p and specific activity from oocyte to the stage of neural tube are shown. A steady rise in incorporation can be seen. Since the total phospholipid content remains constant, there is an increase in specific activity. The experimental design utilized by us for in vivo radiophosphoms labeling of the cells enabled assessment of phospholipid turnover. Since it is known that some tightly bound lipids, mainly polyphosphoinositides (11), remain in tissue residues after the Folch extraction, a further experiment (Table V) was devised to assure the completeness of the

phospholipid extraction. Most of the lipid P was found to be recovered in the neutral chloroform-methanol extracts of unfertilized oocytes, blastula, dorsal lip and tail bud. Only a small amount of lipid P was found when extracts were made with acidified solvents, representing ca. 2% of the total lipid P until the stage of first invagination of dorsal lip. The most striking finding about the acidified chloroform-methanol extract is the high specific activity of the incorporated 32p. Fertilization promotes an increase in the specific activity in this fraction, while during the early development changes were not seen (Table V). The lipids that comprise the fraction extracted by acidified chloroform-methanol (Table V) are not well known as yet. The results presented in Tables IV and V show different incorporation of 32p into the phospholipids extracted with neutral solvents; this is mainly due to differences in the amounts of 32p injected in the two sets of experiments (Table V: 200 /JCi and 400 /ICi/100 g). However, in spite of the large differences in incorporation, the rise in specific activity was similar in both experiments. The per cent increases in specific activities, as compared to unfertilized oocytes found at the blastula stages, were 50 and 65% and at the first invagination of dorsal lip, 87 and 94%. DI SC USSI ON

The present work provides evidence that the total lipid and phospholipid level remains unchanged from unfertilized oocyte through the early stages of amphibian embryogenesis until neurula formation. These findings are surprising, because at that stage a very large number of cells comprises the embryo. In fact, late neurulas of other amphibians, Rana pipiens and Xenopus laevis contain 172,000 and 180,000 LIPIDS, VOL. 9, NO. 1

32

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LIPIDS DURING EARLY EMBRYOGENESIS

33

cells (19). Consequently a great amount of cell These processes may be efficient and under membranes must be formed and a large require- genetic control. Second, another possible mechment of phospholipids must be met. In addition anism may be that yolk plateleL as well as our data show that cell cleavage up to the other organelles, contributes to maintain lipid blastula stage, 2500 to 3100 cells (19), does not precursors for the biosynthesis of phosphoalter the fatty acid composition in polar and lipids. Results presented herein support the neutral lipids. These findings are of interest, former path, because the structure and level of because the pathways involved in membrane phospholipids do not change during cell cleavbiosynthesis during early embryogenesis are not age. Furthermore the phospholipid classes, as known. Moreover many important functions well as the molecular species of their major are related to membranes, such as control of classes-phosphatidyl choline and phosphatidyl growth (20), initiation of DNA biosynthesis e t h a n o l a m i n e - d u r i n g early cleavage are also and cell division (21). Although little is known unchanged (unpublished observations); alof the biochemistry of cell membranes during though the presented data shows a steady early embryogenesis, important processes are increase in specific radioactivity when 32p is related to it. In the sea urchin egg, ultrastruc- injected along with the hormone to induce tural studies have shown that the surface coat is ovulation. At late blastula stage an 80% rise of complex and that fertilization causes very rapid specific radioactivity was observed, while at and extensive changes. These events are the neural fold and neural tube stages the incresplitting of the surface membrane into the ment amounted to 336%. vitelline and plasma membrane, promoting the In other systems where increased cell divirelease of cortical granules and leaving a highly sion occurs, phospholipids vary rapidly, such as convoluted plasma membrane coated by a in cultured 3T3 mouse cells (27), regenerating mucoprotein-like hyaline layer. When cleavage rat liver (28,29) and rat hepatoma cells grown starts, tubular formation and a dense layer in suspension culture (30,31), and in neoplastic develop beneath the outer cell membrane (22). mast cells (32). Moreover, at early cleavage stages in a wide The present results show that during the variety of embryos, begins the development of early embryogenesis of the toad an increased tight junctions that lead to electrical coupling incorporation of 32p into phospholipids takes between cells (23). Trelstad et al. (24) have place. This may reflect metabolic changes asproposed that these specialized cell contacts sociated with the activity of the newly formed may participate in cell adhesion and also in cell membranes or rapid exchange reactions of the movements. Close membrane appositions con- polar moiety or both. Rapid exchange reactions stitute areas of low electrical resistence, allow- may be taking place upon the arrival of the ing relatively free movement of ions (23). Also, phospholipid carrier complex to the membrane these electrical pathways may be involved in vicinity in order to unload the polar molecule the intercellular transmission of substances re- or to make the appropriate assemblage of the lated to the coordination of growth and differ- phospholipid into the membrane. These procentiation. Besides, membranes form transitory esses may generate a transitory diacylglycerol syncytia allowing cytoplasmic connections be- and may depend on the presence of an active tween blastomeres of early developing embryos phosphoryl choline and phosphory ethanol(25). amine pool. The increased specific radioactivity It is evident from all the functions of in phospholipids may also reflect, in part, membranes during early embryogenesis that the breakdown and synthesis. If such metabolic events underlying their biosynthesis may be changes also contribute to the rise in incorporavery precisely regulated. The polar lipids re- tion they may be very precisely regulated, quired for membrane biogenesis may evolve because the resulting level and composition of through one of the following paths. First, yolk the phospholipids do n o t change. In so far as platelets of amphibians are known to store the origin of the membrane protein is conmolecules to be used during early development. cerned, our hyphothesis does n o t exclude the In Rana pipiens 14% of yolk platelet dry weight possibility that the carrier phospholipid comis lipids (26). Phospholipid biosynthesis may plex is comprised of membrane subunits. Curtake place concurrently with yolk platelet rent work in our laboratory is testing the formation. Consequently most polar lipids of proposed ideas. the unfertilized oocyte may be compartmentalized in the yolk platelet. Fertilization may REFERENCES trigger a transport mechanism for carrying the phospholipids from the storage location, yolk 1. Noble, R.C., and J.H. Moore, Can J. Biochem. 45:1125 (1967). platelet, to corresponding membrane sites. LIPIDS, VOL. 9, NO. 1

34

C.A. BARASSI AND N.G. BAZAN

2. Noble, R.C., and J.H. Moore, Ibid. 43:1677 (1965). 3. Christie, W.W., and J.H. Moore, Comp. Biochem. Physiol. 41 B:297 (1972). 4. Morril, G.A., and A.B. Kostellow, J. Cell Biol. 25:21 (1965). 5. Fast, P.G., in "Progress in the chemistry of Fats and other lipids," Vol. 11, Edited by R.T. Holman, Pergamon Press, Oxford, 1970, p. 226. 6. Theise, H., and G. Scharte, Acta Biol. Med. GeL 21:755 (1968). 7. Fairbairn, D., Can. J. Biochem. Physiol. 33:122 (1965). 8. Ward, C.W., and D. Fairbairn, Develop. Biol. 22:366 (1970). 9. Del Conte, E . , a n d J.L. Sirlin, Anat. Rec. 112:125 (1952). 10. Folch, J., M. Lees and G.H. Sloane-Stanley, J. Biol. Chem. 226:497 (1957). 11. Dawson, R.M.C., and J. Eichberg, Biochem. J. 96:634 (1965). 12. Rouser G., G. Kritchevsky and A. Yamamoto, in "Lipid Chromatographic Analysis," Vol. 1, Edited by G.V. Marinetti, Marcel Dekker, Inc., N e w York, 1967, p. 118. 13. Rouser G., S. Fleischer and A. Yamamoto, Lipids 5:494 (1970). 14. Morrison, W.R., and L.M. Smith, J. Lipid Res. 5:600 (1964). 15. Baz~n, N.G., Biochim. Biophys. Acta 218:1 (1970). 16. Avelda'~o, M.I., and N.G. Baz~n, Ibid., 296:1 (1973). 17. Crupkin M., C.A. Barassi and N.G. Bazdn, Comp. Biochem. Physiol, 45 : 523 (1973).

LIPIDS, VOL. 9, NO. 1

18. Chen, P.S., T.Y. Toribara and H. Warner, Anal. Chem. 28:1756 (1956). 19. Deuchar, E.M., "Biochemical Aspects of Amphibian Development," Methuen & Co. Ltd., London, 1966, p. 163. 20. Pardee, A.B., Nat. Cancer Inst. Monogr. 14:7 (1964). 21. Shapiro, B.M., A.G. Siccardi, Y. Hirota and F. Jacob, J. Mol. Biol. 52:75 (1970). 22. Wolpert, L., in "Cell Growth and Cell Division," Edited by R.J.C. Harris, Academic Press, New York, 1963, p. 277. 23. Furshpan, E., and D. Potter, in "Current Topics in Developmental Biology," Edited by A. Moscona and A. Monroy, Academic Press, New York, 1968, p. 95. 24. Trelstad, R.L., E.D. Hay and J.P. Revel, Develop. Biol. 16:78 (1967). 25. Hagstrom, B.E., and S. Lonning, Protoplasma 68:271 (1969). 26. Wallace, R.A., Biochim. Biophys. Acta 74:495 (1963). 27. Cunningham, D.D., J. Biol. Chem. 247:2464 (1972). 28. Fex, G., Biochem. J. 119:743 (1970). 29. Fex, G., Biochim. Biophys. Acta 231:161 (1971). 30. Plagemann, P.G.W., Arch. Biochem. Biophys. 128:70 (1969). 31. Plagemann, P.G.W., J. Cell Biol. 42:766 (1969). 32. Pasternak, C.A., and J.J.M. Bergeron, Biochem J. 119:473 (1970). [ Revised m a n u s c r i p t r e c e i v e d F e b r u a r y 5, 1 9 7 3 ]