Lipid biosynthesis in amphibian oocyte and embryonic cell-free preparations

Comp. Biochem. Physiol. Vol. 79B, No. 3, pp. 395-400, 1984

0305-0491/84$3.00+ 0.00 © 1984PergamonPress Ltd

Printed in Great Britain

LIPID BIOSYNTHESIS IN AMPHIBIAN OOCYTE AND EMBRYONIC CELL-FREE PREPARATIONS TELMA S. ALONSO* and IDA C. BONINI DE ROMANELLI Instituto de Investigaciones Bioquimicas, Universidad Nacional del Sur, Consejo Nacional de Investigaciones Cientificas y Trcnicas, Gorriti 43, 8000 Bahia Blanca, Argentina (Tel: 091-313-42) (Received 12 March 1984)

Abstraet--l. The utilization of [2-3H]glycerol-3-phosphate in the synthesis of lipids during early embr.yogenesis was studied in cell-free preparations from oocytes or embryos of Bufo arenarum Hensel. 2. The precursor was incorporated in all stages of development up to gill circulation, which indicates that oocytes and embryos have the enzymatic machinery necessary to synthesizeat least part of their own lipids. 3. A significant decrease in the labeling of most lipids took place after fertilization, especially in gastrulas, but at gill circulation lipid synthesis was highly stimulated. 4. The incorporation pattern is similar in unfertilized oocyte, fertilized oocyte and gastrulas, where phosphatidylglycerol has the highest amount of radioactivity. At gill circulation stage phosphatidylethanolamine and neutral lipid biosynthesis also became significant. 5. The results suggest a different regulation of the biosynthetic lipid routes through the appearance of new enzymes or modulators of preexisting enzymes during amphibian development.

INTRODUCTION An active membrane formation is prominent among the cellular events triggered by fertilization, since thousands of cells proliferate from a single germinal one. It is generally accepted that the ample supply of preformed phospholipids present in the unfertilized egg probably suffices to support the requirements of membranogenesis during early development and that only at later stages does lipid synthesis really start. However, information about the timing of these events is scarce and in some respects contradictory (Pasternak, 1973; Schmell and Lennarz, 1974; Byrd, 1975). The amphibian egg is a useful model for developmental studies since embryogenesis can be conducted in vitro and stages of differentiation easily followed. However, due to the extreme impermeability of the vitelline membrane to most lipid precursors and also to the enormous amount of lipids preexisting in the system, metabolism studies in intact cells pose some difficulties. These can be partly overcome when working with cell-free preparations as in the present case. Previous work demonstrated that an increase in phosphatidic acid (Bonini de Romanelli et al., 1981) and a decrease in diacylglycerol mass (Alonso et al., submitted for publication) occur after fertilization, suggesting that substantial changes take place when the oocyte evolves into an embryo. Active incorporation of 32p (Barassi and Baz~in, 1974; Alonso et al., 1982) as well as of [14C]acetate and fatty acids (Pechrn de D'Angelo et al., 1977; Miceli and Brenner, 1976) have been reported. However, no information is available on the de novo synthesis of glycerophospholipids. Though oocytes and early embryos *To whom correspondence should be addressed at: Instituto de Investigaciones Bioquimicas (UNS-CONICET) Gorriti 43, 8000 Bahia Blanca, Argentina. CBP(B) 79/3 G

were unable to incorporate [a4C]glycerol (Pech6n and Bazfin, 1977), this does not preclude the operation of de novo biosynthetic pathways: it merely suggests the absence of glycerol kinase activity at early stages of development. Glycero-3-phosphate is a major product of the pentose phosphate shunt, which is known to be very active in these cells (Salom6n de Legname et al., 1971). In this paper we show that this precursor is incorporated into lipids in cell-free homogenates obtained at different developmental stages, including the unfertilized oocyte. MATERIALS AND METHODS Materials

[2-3H]Glycerol-3-phosphate(7.1 Ci/mmole)was from New England Nuclear (Boston, MA, USA). L-Phosphatidyl-DLglycerol, phospholipase D, thioglycolic acid, myoinositol and HEPES were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All solvents used were of analytical grade. Methods

Ovulation was induced in mature toads (Bufo arenarum Hensel) by injecting the dorsal lymphatic sacs with a homologous pituitary extract. Glands were stored and used as described by Pisan6 (1956). Oviposition began 16-20 hr thereafter. The animals were killed and the ovisacs excised to obtain the oocytes which were subsequently fertilized with a homologous testicular homogenate. Spermatozoid motility and mobility were previously checked. Development was allowed to take place in a diluted Ringer solution (0.65g/1 NaC1; 0.01 g/1 KCI; 0.003g/l CaCl2) at 22-25°C. The developmental stages were identified by morphological criteria (Del Conte and Sirlin, 1952). Unfertilized oocytes, oocytes 1.5 hr after fertilization and embryos at late gastrula and gill circulation stages were taken and dejelliedby brief exposure to 2~ thioglycolicacid previously neutralized with NaOH. The incorporation of [2-3H]glycerol-3-phosphate (5 #Ci/ 250 units) was studied using 10% (w/v) oocyte or embryo

395

396

TELMA S. ALONSO and IDA C. BONINI DE ROMANELLI extracts were taken to dryness and washed according to Folch et al. (1957). Phospholipids were isolated according to Rouser et al. (1970). The procedure described by Poorthuis et al. (1976) was also used to resolve phosphatidylglycerol from phosphatidylethanolamine. Neutral lipids were eluted from the two dimensional plates using the solvents described by Arvidson (1968) and resolved with hexane:ether: acetic acid (80:20:2.3, per vol) on silica gel G. Lipid spots were viewed with iodine vapours. Radioactivity was determined using a Beckman L-S-250 liquid scintillation counter. Lipid phosphorus was measured as described by Rouser et al. (1970). Proteins from the residues after lipid extraction were dissolved with 1N NaOH and quantitated according to the procedure of Lowry et ell. (1951). using bovine serum albumin as standard.

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The time course of [2-3H]glycerol-3-phosphate incorporation into total lipids of oocyte and embryonic cell-free preparations is shown in Fig. 1. During 120 rain incubations the precursor was incorporated into lipids in all four developmental stages investigated. In unfertilized oocytes the precursor was incorporated at a nearly linear rate. Fertilized oocytes (1.5 hr later) showed a 25% higher labeling than unfertilized oocytes for the earliest incubation times (5-15 min). Similar incorporation levels were attained at 30 min. Thereafter the incorporation of the fertilized oocyte reached constant levels amounting after 120min incubation to 60% that of the unfertilized oocyte. The lowest level of incorporation was observed for the gastrula stage (34th hr of development). When the embryo reached the gill circulation stage (132ndhr of development) the labeling pattern was quantitatively similar to that of" the unfertilized oocyte though the quantitative lipid synthetic activity was several-fold higher (Fig. 1). In order to investigate whether these changes affected equally all the metabolic pathways or whether they were due to different enzymatic activities, individual labeling of the lipids was carried out. As shown in Fig. 2, synthesis of different lipids

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INCUBATt0N TIME(minutes) Fig. 1. Time-course for [2-3H]gtycerol-3-phosphate incorporation in total lipids during early amphibian development. Cell-free oocyte and embryo preparations in HEPES buffer 50 mM, pH 7.4, were incubated at 2 3 C with the labeled precursor (701zCi/3500units). At the specific intervals aliquots corresponding to 250 units were taken and lipids were extracted. Each point is the mean + SD from three independent samples with the exception of 120 rain (two samples). cell-free preparations in 50mM HEPES buffer, pH 7.4. Homogenates were incubated in a shaking bath at 230C and aliquots corresponding to about 250units were taken at different intervals. When analyzing the effect of myoinositol, this was added to the incubation medium at a concentration of 2.5 mM. Lipids were extracted according to Bligh and Dyer (1959). The proteins at the interphase were removed and washed twice with chloroform-methanol (2: 1, v/v). The combined

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PC 20

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incubation time (minutes) Fig. 2. Individual lipid labeling by [2-3H]glycerol-3-phosphate in oocyte and embryo homogenates. Details as in Fig. 1.

I

120

I0

397

Lipid biosynthesis in amphibian oocytes and embryos Table 1. Phospholipid content in oocytes and developingembryos Phospholipid

Unfertilized

Fertilized

Late

Gill

Oocyte

Oocyte

Gastrula

Circulation

mol/100 Phosphatidic

mg protein

Acid

0.03

± 0.004

0.03

+ 0.001

Phosphatidylserine

0.11

+ 0.01

0.18

± 0.03

Phosphatidylinositol

1.17

4 0.20

1.19

± 0.04

1.26 + 0 . 0 5

Sphingomyelin

0.56

÷ 0.14

0.88

+ 0.03

0.92

± 0.07

11.20

+ 1.07

11.61

+ 1.57

12.82

± 0.68

Phosphatidylcholine

*

0.03

+ 0.001

0.17

+ 0.02

0.05

± 0.004

*

*

0.22

± 0.04

*

1.30

± 0.13

*

0.97

+ 0.08

*

13.89

+ 0.73

* *

Phosphatidylethanolamine

A

4.81

+ 0.40

4.16

± 0.10

5.15

+ 0.62

5.70

± 0.38

Phosphatidylglycerol

A

0.01

+ 0.002

0.01

± 0.001

0.01

± 0.001

0.01

+ 0.001

Diphosphatidylglycerol

A

0.10

± 0.03

0.09

± 0.03

0.10

± 0.02

0.14

+ 0.03

Mean values + SD from three independent samples. *Significant differences with respect to unfertilized oocyte (P < 0.05). Phospholipids were separated according to Rouser et al. (1970), except for A, which were resolved as described by Poorthuis et al. (1976).

have not as yet fully materialized. In contrast, at gill circulation stage the expression of phosphatidate phosphohydrolase is evidenced by the active consumption of phosphatidate and the concomitant increase in diacylglycerols (Fig. 2). The time-course of the labeling of these two lipids is consistent with a precursor-product relationship. In turn, the subsequent decrease in DG* and the increase in PC, PE and TG labeling indicate that cells at this stage contain the enzymes that utilize DG. An unexpected finding from these in vitro experiments was the massive labeling of PG that occurred at all stages investigated. The high labeling of PG represented, however, only 0.05~o of the total phospholipid content (Table 1). PA, PS, SpH, PC and PE underwent significant changes between unfertilized oocyte and gill circulation stage. Despite these changes in their absolute content the percenage distribution followed

proceeded at different rates in each developmental stage. This suggests that the triggering of enzymatic activities during differentiation follows a concerted pattern. After fertilization phosphatidic acid was most actively synthesized, in coincidence with the reported accumulation of phosphatidate at this stage (Bonini de Romanelli et al., 1981, Table 1). However, the newly formed phosphatidate was not efficiently used to synthesize other lipids, which suggests that enzymes from lipid synthesis are either inactive or *Abbreviations: PA, phosphatidic acid; PS, phosphatidylserine; PI, phosphatidylinositol; SpH, sphingomyelin; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; DG, diacylglycerol; TG, triacylglycerol; CDP-DG, cytidine diphosphate diacylglyceride. Table

2. Specific

activity of phospholipids labeled with phosphate during early developmental stages Incubation

PG

[2-3H]glycerol-3 -

PA

Ratio

lipid

PG/PA

Stage time

(min)

5

pmol

644 ±

x I0-2/~ mol

47

114 ±

46

5.6

822

273 ±

18

53.3

Unfertilized 60

14543

+

oocyte 120

5

19865

595 ±

332

83

432 ±

59.8

38

1.4

1048 ± 128

7.1

Fertilized 60

7 4 0 9 ± 427

oocyte 120

8134

5

347 ±

825

83

53 ±

9.9

11

6.5

Late 60

4667

± 850

41

±

7

113.8

gastrula 120

9692

36

269.2

Figures are mean values + SD from three independent samples except for 120 min (mean of two samples).

398

TELMA S. ALONSO a n d IDA C. BONINI DE ROMANELLI Table 3. Time course of phospholipid labeling by [2-3H]glycerol-3-phosphate in cell free preparation Irom embryoa at ~'tJl circulation stage Incubation

PA

PG time

PG/PA

PC

PE

?S

PI

DPG

(min)

p mol

x

I0-2/%Jmoi

lipid

5

9471

+ 1178

1299

~ 168

7.3

1.9

+ 0.4

0.2

L 0.06

0.5

t 0.02

23

+ 9

0.8

÷ 0.1

15

18852

± 3696

2721

+

6.9

12.4

~ 0.3

0.2

+ 0.06

2.4

+ 0.6

30

± 7

2.0

~ 0.1

30

26527

+

1083

1905

60

33230

+ 2793

591

120

87661

321

129

+ 209

13.9

17.7

! 2.2

0.7

! 0.06

2.6

+ 0.7

31

± 5

4.9

~ 0.9

±

56.2

36.2

+ 3.1

0.9

~ 0.06

3.6

4 0.2

17

*

8.9

- 0.5

273.1

162.3

6

1.7

7.9

23

~

55.1

Conditions as in Table 2.

the same pattern through the different stages; PC (60°/) and PE (25%) constituted the major lipids followed by PI, SpH and PS. Specific activities of PG and PA in the first three developmental stages are shown in Table 2. For the sake of simplicity negligible levels of incorporation were omitted. At 5min incubation in unfertilized oocyte the amount of labeled PG was 3-fold higher than that of PA. At the maximal incubation time analyzed (120 min) there was a large increase in the specific activity of PG (30-fold) and a smaller increase in PA (3-fold). After fertilization the specific activities of PA reached higher values than those of unfertilized oocytes. PG showed similar specific activities at 5 min, being about 50% lower after 60 and 120 min of incubation. The differences between the specific activities of PG and PA observed in unfertilized oocyte were therefore eliminated upon fertilization. At the late gastrula stage, specific activities were lower for both phospholipids, but more dramatic for PA which decreased concomitantly with an increase in PG. The highest values for the PG/PA ratio were observed at 120 min. The specific activities of the different phospholipids were analyzed in more detail for the gill circulation stage. In accordance with the results obtained for earlier development stages (Table 2) PG and PA constituted very active pools (Table 3). Although specific activities were several-fold higher than in late gastrula (Table 2) a similar PG increase/PA decrease as a function of incubation time was observed. These two phospholipids represented the most active pools at this developmental stage, other phospholipids attaining much lower incorporation (Table 3). Among them the value of PE was the highest, showing also the largest increase throughout incubation. PS was the only phospholipid whose specific activity remained unchanged up to 120 min. Since the specific activity of PG was much higher than that of PA at all stages and incubation times analyzed (Tables 2 and 3) the results suggest that most of the label in PG originated from the active turnover of the polar glycerol moiety rather than from de novo synthesis of the phosphatidyl moiety. This conclusion is supported by the fact that phospholipase D hydrolysis of a preparation of PG from gill circulation homogenates yielded only 12~, of the label as PA, the remainder being recovered in the

aqueous phase after partition of the reaction products by established procedures (Kates and Sastry, 1969; Chalifour et al., 1980). This indicates, however, that even if 90~ of the radioactivity incorporated in PG came from turnover of its polar moiety the remaining 10% is still higher than the label in other membrane lipids such as PI. The latter phospholipid is believed to originate in the same de novo metabolic pathway as PG, entailing a common C D P - D G intermediate (Bleasdale and Johnston, 1982). In order to investigate whether the negligible synthesis of PI as opposed to that of PG arose from a dilution of myoinositol produced by homogenization, 120min incubations were carried out in the presence and absence of 2.5 mM myoinositol (Table 4). The results clearly indicate that the synthesis of PI was not affected by the presence of inositol even when PG synthesis was decreased. The strong stimulation of D G and T G labeling by inositol at gill circulation stage may be secondary to this PA stimulation. DISCUSSION

The results presented here show that both the unfertilized oocyte and the embryo at early stages of development possess enzymatic activities conducive to the synthesis of membrane lipids, and suggest that at least part of these enzymes are operative right from oviposition. The qualitative and quantitative changes in lipid labeling observed after fertilization suggest that the synthesis of lipids is a closely regulated event, synchronized to meet the requirements of a rapidly dividing cell system. The exact stage in the development process when a cleidoic growing system such as the amphibian embryo starts to synthesize its own lipids has been a matter of some controversy (Pasternak, 1973; Schmell and Lennarz, 1974), but there is general consensus that the massive deposits of lipids with which the egg is released would suffice to support membrane formation in the embryo up to the particular stage in the process at which the synthesis of lipids is somehow "switched on". Our results suggest that the synthesis of membrane lipids is not an all-or-none process but rather a series of concerted events which evolve, like the cell itself, to increased levels of complexity. Thus, from phosphatidate being virtually the only lipid synthesized de novo after fertilization, the cells reach a stage in which all of

399

Lipid biosynthesis in amphibian oocytes and embryos Table 4. Myoinositol effect on [2-3H]glycerol-3-phospbateincorporation into lipids of oocytes and developingembryos Unfertilized Oocytes

Fertilized

Late

Gill

Oocytes

Gastrula

Circulation

Myoinositol (2.5 mM)

-

+

+

Lipid

+

-

+

dpm/mg protein

PG

561

204

299

229

932

708

PE PA

22

4

22

17

1034

540

70

104

4

16

6

23

PC

3

2

2

4

28

59

Pl

11

11

PS

6

3

DG TG

262

382

53

15

24

44

6

4

11

11

13

20

18

196

578

-

-

35

103

504

Cell-free oocyteand embryopreparations in HEPES buffer 50mM pH 7.4 were incubated at 23°C during 2 hours with the labeled precursor (70#Ci/3500units). Values are the mean of three independent samples with SD lower than 10yo.Control values were obtained from two samples.

their glycerolipids are labeled, even in the unfavorable experimental situation of a tissue that has lost its integrity and compartmentalization. In addition to de novo synthesis, additional enzymatic activities required to build up or chemically modify a phospholipid remain in the embryo, such as the ability to de novo synthesize fatty acids from acetate (Miceli and Brenner, 1976), to incorporate this label into lipids (Miceli and Brenner, 1976; Pechrn de D'Angelo et al., 1977) and to produce the turnover of preformed fatty acids (Miceli and Brenner, 1976) and the polar moiety (Barassi and Bazfin, 1974; Alonso et al., 1982). The net decrease in lipid labeling seen after fertilization and gastrula with respect to oocyte and later developmental stages could be the expression of mechanisms regulating lipid synthesis in the embryo. Thus, an active ionic mobilization takes place in the egg after fertilization (Epel, 1980), which could conceivably modulate the activity of enzymes like acyltransferases. Moreover, most of the energetic demands during this period are known to be supplied by the pentose phosphate shunt (Salom6n de Legname et al., 1971). Dilution of labeled glycerol-3phosphate by an increased production of the endogenous metabolite could partially explain its decreased utilization for lipid synthesis under these conditions. However, dilution of the precursor is not likely to be the only operating mechanism since phosphatidate synthesis after fertilization is stimulated rather than inhibited with respect to the oocyte (Fig. 2). Hence, most of the decrease in lipid labeling comes from the decreased activity of enzymes not directly utilizing glycerophosphate but lipid intermediates like PA and DG. On the other hand, the progressive decrease of PG labeling from oocyte to gastrula (Fig. 2; Tables 2 and 4) could be more affected by its dilution, since most of the label arises from the turnover of the polar glycerol moiety.

The biochemical role of PG is not as yet completely understood. In bacteria it may function as precursor of some membrane oligosaccharides (Jackson and Kennedy, 1983) and it plays an important role in the stabilization of the lung surfactant (Bleasdale and Johnston, 1982). Even when there is no net increase in the mitochondrial mass from the 2nd to 3rd day after fertilization (Chase and Dawid, 1972) structural alterations and changes in mitochondrial enzymatic activity (Nelson and Lgvtrup-Rein, 1983) do take place during amphibian development. The active PG formation described in this paper could be linked to this mitochondrial differentiation, since PG is a direct precursor of DPG, a typical component of the inner mitochondrial membrane. The relative rate of PI and PG synthesis may be regulated by the availability of myoinositol through a redirecting of the common intermediate CDPdiacylglycerol. The addition of myoinositol to the incubation medium has been reported to stimulate the incorporation of [14C]glycerol into PI with a concomitant decrease in PG in lung (Bleasdale et al., 1983). In our preparation, the addition of myoinositol did not affect PI labeling from glycerophosphate. A dilution of cofactors necessary for PI synthesis by homogenate preparations could partially explain this phenomenon. It must be borne in mind that the levels of cytidine nucleotides may also play a regulatory role (Bleasdale and Johnston, 1982). Since in Bufo arenarum the levels of most nucleotides vary during development (Cantore et al., 1977) it should be interesting to explore the role of cytidine nucleotides on lipid synthesis during these stages. On the other hand, myoinositoi stimulates labeling of PA, DG and TG while decreasing PG and PE incorporation particularly at the gill circulation stage. This may suggest that inositol stimulates the phosphatidate phosphohydrolase favoring in turn TG biosynthesis (Jamdar and Fallon, 1973), and in-

400

TELMA S. ALONSO and IDA C. BONINI I)E ROMANELLI

hibiting the PE and C D P - D G f o r m a t i o n with a c o n c o m i t a n t decrease in the de novo P G biosynthesis. U n d e r o u r conditions PC was not highly labeled despite being the m a j o r lipid c o m p o n e n t of embryos (Table 1). Sea urchin oocytes a n d e m b r y o s are also inactive in i n c o r p o r a t i n g [3H]choline into PC (Schmell a n d Lennarz, 1974). The p o o r synthesis of PC with respect to t h a t of PE is s o m e w h a t surprising if b o t h lipids are synthesized by the K e n n e d y pathway. However, this p h e n o m e n o n m i g h t well be more closely related to a higher sensitivity o f the enzymes synthesizing PC to the mechanical disruption of c o m p a r t m e n t a l i z a t i o n p r o d u c e d by h o m o g e n i z a t i o n (Ilincheta de Boschero a n d Baz/m, 1982) than to the relative rates of synthesis in rh, o. Given the vectorial topology o f PC a n d PE as well as of the enzymes involved in their synthesis in the endoplasmic reticulum m e m b r a n e ( H u t s o n and Higgins, 1982), it is conceivable t h a t cell disruption dilutes or detaches peripheral enzymes or cofactors necessary for the synthesis of PC in the resulting microsomes, while those for the synthesis of PE remain protected in the vesicle interior. Acknowledgements--The authors are grateful to Drs M. 1. Aveldafio, F. J. Barrantes, N. M. Giusto and E. B. Rodriguez de Turco for critical reading of the manuscript and for helpful criticism. REFERENCES

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