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Nucleotides and sugar nucleotides in early development of Bufo arenarum
ARCHIVES
OF BIOCHEMISTRY
Nucleotides
AND
BIOPHYSICS
and Sugar
269-275
180,
Nucleotides in Early Development Bufo arenarum
Coelomic
MARIA
Embriologia Facultad
LEONOR
Animal y Biologh de Ciencias Exactas
(1977)
CANTORE, EDUARDO Molecular y Naturales,
Oocytes’,
Received
2
MARTA E. F. DE RECONDO, F. RECOND03
Departamentos Universidad September
of
AND
de Ciencias Bioldgicas y Q&mica Bioldgica, de Buenos Aires, Buenos Aires, Argentina 13, 1976
Nucleotides and sugar nucleotides from coelomic oocytes of Bufo arenarum were extracted with trichloroacetic acid and analyzed by ion-exchange chromatography. The hypoxanthine and guanine were sequentially eluted from the column with water. Nucleotides and sugar nucleotides were eluted with a linear gradient of ammonium chloride. The first peak of ultraviolet adsorption eluted from the resin was a complex mixture of at least three substances. The main component was identified as cytidine diphosphocholine by chemical, enzymatic, and chromatographic analyses. Preliminary experiments suggest a possible role for this compound during oogenesis, since immature oocytes incubated in vitro with [L4Clcholine showed an active metabolism of this substance with rapid incorporation in choline phosphate, cytidine diphosphocholine, and lecithin.
In amphibians, oogenesis begins immediately after the breeding season and continues until fall, by which time eggs to be ovulated the following year are in the prophase of the first maturation division and their metabolic activity becomes quiescent. Increase of gonadotrophic activities breaks the equilibrium state. The giant nucleus (germinal vesicle) of the ovarian oocyte bursts, and chromosomes restart their first meiotic division. Oocytes are released from the ovaries over a period of a few hours and they fall into the peritoneal cavity, where cillia propel them toward the end of the adjacent oviduct. Each oocyte completes the first maturation divi-
sion and, simultaneously, a secretory lining deposits several layers of jelly coat around them (1). Oocytes from the body cavity are interesting because of their high metabolic activity and of their transition stage between a differentiated cell, the full-grown oocyte of the ovary, and a totipotent cell, the female gamete able to produce a new multicellular organism after fertilization. In this paper, research is described in which nucleotides and sugar nucleotides from coelomic oocytes were analyzed by ion-exchange chromatography. CDP-choline was found to be a normal component of the acid-soluble extract. Its amount decreased slowly during early development (2) and increased little after gastrulation. It was not detected in a somatic tissue like liver (3). A possible function of this nucleotide during oogenesis is being investigated. Preliminary results indicate a rapid incorporation of choline to phospholipid in immature oocytes via choline phosphate and CDP-choline.
1 Dedicated to Professor Luis F. Leloir on the occasion of his seventieth birthday. 2 This investigation was supported in part by a research grant from the Consejo National de Investigaciones Cientificas y Tecnicas (Argentina) and from the Universidad de Buenos Aires. This paper is third in a series. 3 Career Investigator of the Consejo National de Investigaciones Cientificas y Tecnicas (Argentina). 269 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
270
CANTORE, MATERIALS
AND
DE
RECONDO,
METHODS
Materials. n-Glucose oxidase (a-n-glucose: oxygen oxidoreductase, EC 1.1.3.41, alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) protease type V (P5005), collagenase type I (C-0130), choline phosphate, and lecithin were obtained from Sigma Chemical Co. (St. Louis, Missouri). Purine and pyrimidine bases, nucleosides, nucleotides, sugar nucleotides, and sugars were commercial preparations. lnC]Choline (413 mCi/ mmol) was obtained from New England Nuclear. Animals were purchased from a local supplier. Obtention of coelomic oocytes. Mature females from Bufo arenarum, carefully selected, were anesthetized. The upper ends of their oviducts were tied to force oocytes to remain in the coelomic cavity. After surgery, toads were kept in cold running water for recuperation; 24 h later, ovulation was induced by injecting an aqueous extract of homologous hypophyses. Females were kept approximately 16 h at about 25°C. Jelly-free oocytes which had accumulated in the coelomic cavity were removed through two cuts made at both sides of the media ventral line. Extraction procedure. Oocytes were washed with Holfreter solution, suspended in 2 vol of 10% trichloroacetic acid and homogenized with a blender for 1 min. The suspension was centrifuged at 5000 rpm for 30 min. The supernatant solution was extracted three times with cold ether to remove most of the acid and finally neutralized with NH,OH solution to pH 7. The precipitate was reextracted with 10% trichloroacetic acid and removed by centrifugation, and the superantant solution was extracted with ether and neutralized as described. Both extracts were pooled, the organic solvent was removed by evaporation at room temperature in uacuo, and the solution was stored frozen at -20°C until used (acidsoluble extract). As was reported in preceding papers (2-4), extreme care was taken to preserve labile structures, and a great number of oocytes were used to ensure the detection of compounds present in very small quantities. Three samples of oocytes, each coming from five to six females, were analyzed. Each acid-soluble extract came from approximately 90,000 oocytes (500 oocytes/ml) and contained from 500 to 600 pmol of total nucleotides. Zon-exchange column chromatography. Acid-soluble extracts were chromatographed on Dowex 1 x 4 (Cl-) resin as previously described (4). The bases were eluted with water, and the nucleotides were eluted with a linear gradient of ammonium chloride (O-O.9 M). Fractions were pooled and processed as before (4). Analytical methods. The same analytical methods described in preceding papers (2-4) were used. The choline content of CDP-choline was measured
AND
RECONDO
spectrophotometrically by the method of Appleton (5) and Krisman (6) after acid and enzymatic hydrolyses (see Results). Chromatography and electrophoresis. Paper chromatography was performed on Whatman No. 1 paper with the following solvents (v/v): (I) ethanol-l M ammonium acetate (7:3.5) at pH 7.5 (7); (II) ethanol1 M ammonium acetate (7:3.5) at pH 3.8 (71; (III) butyl alcohol-pyridine-water (6:4: 3) (8); (IV) ethanol-ammonia (7:3) (7); (V) hydrochloric acid-2propanol-water (41:170:39) (9); (VI) butyl alcohol (saturated with water at about 23”C)-ammonia (100: 1) (10); (VII) 2-propanol-acetic acid-water (27:4:9) (11); (VIII) 4.5 M ammonium sulfate in 0.1 M phosphate buffer at pH 6.8-n-propanol (5O:l) (12); (IX) butyl alcohol-acetic acid-water (5:2:3) (13); (X) chloroform-methanol-acetic acid-water (25:15:4:2) (14). Paper electrophoresis was performed on Whatman No. 4 paper as described by Markham and Smith (15) with the following buffers: (Al 0.05 M potassium tetraborate buffer, pH 9.2; (B) sodium phosphate buffer, pH 7.5; (C) sodium carbonatesodium hydrogen carbonate buffer, pH 9.2 (16); (Dl sodium acetate buffer, pH 3.8; (El pyridine acetate buffer, pH 6.5. Thin-layer chromatography was carried out on glass chromatoplates prepared with silica gel G (according to Stahl, E. Merck AG, Darmstadt, Germany). The plates were prepared by making a slurry of 30 g of silica gel G with 65 ml of distilled water and applying it with a Desaga applicator (250 pm thickness). Plates were allowed to dry at room temperature and were stored in a cabinet. Just prior to application of samples, chromatoplates were activated at 80-100°C for 60 min. Obtention of immature oocytes. After ovulation was induced as described before, females were killed. Small residual ovaries were quickly removed and carefully washed with 0.02 M NaCl-0.08 M KC1 (salt solution). Tissue was cut in small pieces and incubated for 2 h at 37°C in an enzymatic mixture (protease, 35 mg; collagenase, 3.5 mg; salt solution, 70 ml). Free oocytes were separated by filtration through gauze and exhaustively washed with the salt solution. Incubation and extraction procedure. Isolated immature oocytes (0.2 ml) were suspended in 0.02 M NaCl-0.08 M KC1 (7 ml) containing [i4Clcholine (1 pCi) and incubated at 30°C for different periods. At the end of the incubation period, residual radioactivity was washed out with the salt solution. Oocytes were homogenized in 10% trichloroacetic acid and centrifuged off, the pellet was successively extracted with 5% trichloroacetic acid and water. The trichloroacetic extracts (washed with ether as described before) and aqueous extracts were pooled, adjusted to pH 6, and used as the “trichloroacetic acid-soluble fraction.” The pellet was extracted
NUCLEOTIDES
IN
EARLY
twice with a chloroform-methanol (2:l) mixture to yield the “lipid fraction.” The trichloroacetic acid-soluble fraction was analyzed by paper chromatography in solvent IX. The lipid fraction was analyzed by thin-layer chromatography in solvent X. Radioactivity on the paper strips or thin-layer plates was located with a Packard radiochromatograph. RESULTS
The ultraviolet-absorbing material eluted from the column with water consists of two main fractions named C and D. Fraction C and fraction D were identified as hypoxanthine and guanine by ultraviolet absorption spectra, behavior in paper chromatography with solvents V, VI, and VII, and paper electrophoresis with buffer A. Elution of the nucleotides and sugar nucleotides was obtained with a linear gradient of 0 to 0.9 M ammonium chloride (Fig. 1). The ultraviolet-absorbing fractions were analyzed as follows.
271
DEVELOPMENT
Fraction I. This fraction usually contained cytosine derivatives and traces of guanine and consisted of a complex mixture of compounds. Although it behaved as a single compound on paper chromatography in solvents I and II and on paper electrophoresis with buffer B, it gave four or five ultraviolet-absorbing bands, with solvent IV, one of which was identified as cytidine diphosphate choline; the others were not further studied. The component with a mobility similar to that of standard CDP-choline was eluted from the paper and exhaustively analyzed. The ultraviolet absorption spectra (neutral and acid) was identical to that of cytosine compounds. Extensive hydrolysis (3 N HCl, 1 h, 1OoOC)followed by paper chromatography of the hydrolysate on solvent V showed that the only base present was cytosine. Pentose determination showed the pres-
1.8
0.9
E =:= w s
1.4
0.7
Y f
1.0
0.5 -5 0 I‘ r 0.3
-
s =: 0.6 2 a2
0.1 0
1.0
2.0
3.0
4.0
5.0
VOLUME
FIG.
COMPARATIVE
1. Elution
ANALYTICAL
Sample
to 1000 kmol
obtained
DATA Base
Coelomic oocytes Mature oocytes Fertilized eggs Morula Gastrula Neurula a Refers
pattern
with
ZO
8.0
9.0
a linear
gradient
TABLE
I
of ammonium
chloride
OF THE MAIN COMPONENT OF PEAK I OBTAINED DIFFERENT STAGES OF DEVELOPMENT Pentose
Cytosine Cytosine Cytosine Cytosine Cytosine Cytosine of bases,
6.0
(liters)
0.9 0.9 1.0 1.0 1.0 0.8 nucleotides,
Total phosphorus
Labile phosphol7lS
2.4 1.8 1.8 1.7 1.8 2.1 and sugar
nucleotides
Inorganic phosphorus
0 0
0 0
0 -
0 0 eluted
from
(O-O.9
FROM Choline
1.0 1.1 1.0 1.0 0.9 0.9 the column.
M).
SAMPLES
IN
Total amount” (pm011 63.6 14.0 16 19.5 26
Uracil
Adenine Guanine
Adenine Guanine
VII
VIII
IX
ATP GTP
ADP GDP
-
-
-
-
-
-
+
TABLE
II
-
-
0.96
0.91
-
0.96
0.85
0
-
1.72
1.72
1.73
1.75
__ Total Labile
FROM COE~MIC Phosphorus”
-
-
-
1.08
0. 91
0.65
Pent.oses”
NUCLEOTIDES Sugar
~-Glucose “-Galactose
B.
2-Acetamido-2deoxyglucw? 2-Acetamido-2deoxygalactose@
-
ChromatograPW
OOCYTES
” Identification by paper chromatography in solvent V after hydrolysis with 3 &J HCI, 1 h, 100°C. b Identification by paper chromatography in solvents I and II and electrophoresis with buffers A and c Increase of absorbance at 327 “m in the presence of potassium cyanide (17). * Mole per mole of base. s Paper chromatography in solvent III. ‘Identification by paper chromatography in solvent III after degradation with ninhydrin (16). * Detected with the reagent of Trevelyan et al. (20). h Paucity of the material prevented further identification. 1 Proportion was determined by spectrophotometric method (19).
(65%)’ (35%)’
(40%)’ (6O%Y
UDP-glucuronic
Uracil (75%)’ Cytosine (25%)
VI
acid
UTP CTP
Adenine” Cytosine” GuanineO Uracil”
Vb
NADP UDP
Adenine” Uracil”
Va
-
UDP-Glc UDP-Gal
Uracil
IVb
(73%) (27%)
-
(78%) (22%)
UDP-GlcNAc UDP-GalNAc
Uracil
IVa
~___~
CW nide compit%’
AND SUGAR
+
Adenine
II
CDP-choline
Nucleotidesb
-~
NAD
Cytosine
BEWe”
I
Peak
NUCLEOTIDES after
-
-
-
-
-
2-Acetamido-2. deoxyglucose 2-Acetsmido-%deoxygalactose
-
Electrophoresis
hydrolysis
-
+
acid
-
-
Arabinose xylose
-
Degradation with ninhydrid
component
NUCLEOTIDES
IN
EARLY
ence of 1 mol of pentose/mol of base, and analysis of the total and acid-labile phosphorous gave two and zero phosphate groups per mole of base, respectively. Acid hydrolysis with 1 N hydrochloric acid for 1 h at 100°C gave an ultravioletabsorbing compound that behaved as authentic CMP on chromatography and electrophoresis. The hydrolysate previously neutralized was treated with alkaline phosphatase, and choline and inorganic phosphate were measured (5, 6); 1 mol of choline/2 mol of inorganic phosphate was found. It was concluded that acid hydrolysis splits CDP-choline into cytidine monophosphate and choline phosphate. Comparative analytical data obtained from samples of coelomic oocytes, mature oocytes, fertilized eggs, and embryos in morula, gastrula, and neurula stages are summarized in Table I. Fractions II through IX. As in samples from other stages of development (2-41, the four ribonucleotide triphosphates (UTP, CTP, GTP, and ATP), NADP, NAD, ADP, GDP, and six sugar nucleotides with uracyl as the base moiety were isolated. Analytical data are summarized in Table II. Table III shows the amount of the different nucleotides and sugar nucleotides found in oocytes from the coelomic cavity. The averages of three samples from five to six females are shown. Values are expressed as micromoles of each compound per 1000 pmol of total bases, nucleotides, and sugar nucleotides eluted from the column. Immature oocytes isolated from the ovaries as described under Materials and Methods were incubated with [14Clcholine for different periods, and the fate of the radioactivity was followed in the trichloracetic acid-soluble fraction and in the lipid fraction material. Paper chromatography in solvent IX of the trichloroacetic acid-soluble extract showed five peaks of radioactivity which had the mobilities of CDP-choline, choline phosphate, betain, choline, and acetyl choline, respectively (Fig. 2). Thin-layer chromatography of the lipid fraction showed two areas of radioactivity. The main one had the same mobility as phosphatidyl choline and the small one
273
DEVELOPMENT TABLE
III
QUANTITATIVE DATA FOR THE PEAKS OBTAINED BY ION-EXCHANGE CHROMATOGRAPHY OF EXTRACTS FROM OOCYTES AT BODY CAVITY Peak
Constituent
Amount”
(pmol,
mean
i SD)
C
Hypoxanthine
314.7
D
Guanine
60.2 t 6
I
CDP-choline
63.6 + 1.8
II
NAD
19.2 rf. 0.9
IVa
UDP-N-acetylglucosamine UDP-N-acetylgalactosamine
IVb
UDP-glucose UDP-galactose
17 c 1.2 6.4 + 0.5
VI
UTP CTP
160 ? 10 53 2 3.6
VII
UDP-glucuronic
VIII
ADP GDP
9.3 2 1.8 14 t 2.7
IX
ATP GTP
84 + 9.6 45 + 5.2
acid
t 17
103.3 2 6.2 26 -+ 1.5
26.2 r 2.5
0 Micromoles of each compound refers to 1000 pmol of bases, nucleotides, and sugar nucleotides eluted from the column.
had the mobility of lysophosphatidyl line.
cho-
DISCUSSION
The importance of the metabolic processesoccurring during oogenesis has been emphasized in the last few years. All of the experimental evidence accumulated points to the conclusion that differentiation actually starts at oogenesis. rRNA and mRNA, macromolecules of great importance during development, are synthesized in the oocytes at the ovaries and they are not used until early embryogenesis (21). Simultaneously, reserve materials such as yolk are also synthesized during the long prophase of the first meiotic division, when oocytes pass from the pachytene to the diplotene stages. Yolk platelets are built in these stages, and, again, this reserve material is stored and used only at
274
CANTORE,
DE RECONDO,
CPM
COP-choline
FIG. 2. Radioactivity pattern of the trichloroacetic acid-soluble extracts after paper chromatography in solvent IX.
the beginning of development. During these events, oocytes exhibit an enormous increase in volume, so that a remarkable amount of nucleic acid and protein is packaged within a single cell over a relatively short time. A systematic study of the levels of the precursors of these macromalecules during the early development of our common toad B. arenarum should be interesting. We found that oocytes from the coelomic cavity have the same nucleotides and sugar nucleotides as in other stages of development, but differences were detected in their levels. As compared with oocytes at morula stages, CDP-choline was four times higher in coelomic oocytes. The levels of UDP-galactose were also high: 20 times higher than in the morula. There were less di- and triphosphate nucleotides of purine base in coelomic oocytes than in embryos at the morula stage (Table III) cl).
The presence of CDP-choline at high levels in oocytes from the coelomic cavity could be related to the synthesis of lipopro-
AND RECONDO
teins at earlier stages of development during oogenesis in the ovaries. Yolk platelets, which are the main components in the cytoplasm of oocytes from the ovary, are formed by a homogenous central body surrounded by a superficial layer, both enclosed within a simple membrane. The crystalline main body consists of 2 mol of a highly phosphorylated protein, called phosvitin, for every mole of a lipoprotein called lipovitellin. Wallace and Dumont (22) have followed the fate of radioactive precursors injected into amphibians and demonstrated that at least the protein moiety of phosvitin and lipovitellin is synthesized in the liver and passes to the blood as a vitellogenin complex. Bergink and Wallace (23) later showed that this complex consists of the precursor molecules for lipovitellin and phosvitin and that, once the complex is transported to the ovary, it is transformed into the two proteins of the yolk platelets. The origin of the lipid moiety of lipovitellin is not well understood. This lipoprotein contains phosphatidyl choline (lecithin), and the metabolic pathway of choline to lecithin through choline phosphate and CDP-choline is well known. All of these facts suggest that CDP-choline could be an intermediate in the biosynthesis of the lipovitellin of yolk during oogenesis. Immature oocytes were incubated with [14C]choline and the fate of the radioactivity was followed in the acid-soluble fraction and in the lipid fraction. Our preliminary results showed that choline is rapidly metabolized by the oocyte and incorporated in choline phosphate, CDP-choline, and, finally, in lecithin. It remains to be elucidated which is the distribution of lecithin: the lipids or the lipoproteins of the oocyte organelles. REFERENCES 1. RUGH, R. (1951) The Frog, pp. 31-71, Blakiston, Philadelphia. 2. MORENO, S., CUKIER, M., GUERRERO, G., FERNANDEZ DE RECONDO, M. E., AND RECONDO, E. F. (1976) Carbohydrate Res. 47, 275-284. 3. MAGGESE, M. C., SPAIZMAN, R. C., AND FERNANDEZ DE RECONDO, M. E. (1976) Acta Physiol. Latinoamer., in press. 4. FERNANDEZ DE RECONDO, M. E., MAGGESE, M.
NUCLEOTIDES
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
IN
EARLY
C., GUERRERO, G., SPAIZMAN, R. C., AND RECONDO, E. F. (1973) Carbohydrate Res. 26, 365-375. APPLETON, H. D. (1950) Fed. Proc. 9, 146. KRISMAN, C. R. (1962)Anal. Biochem. 4, 17-23. PALADINI, A. C., AND LELOIR, L. F. (1952) Biothem. J. 51, 426-430. JEANES, A., WISE, C. S., AND DIMLER, R. J. (1951) Anal. Chem. 23, 415-420. WYATT, G. R. (1951) Biochem. J. 48, 584-590. MACNUTT, J. (1952) Biochem. J. 50, 384-396. TUNG, K. K., AND NORDIN, J. M. (1968)Biochim. Biophys. Acta 158, 154-156. MARKHAM, R., AND SMITH, J. D. (1951) Biochem. J. 49, 401-406. PASTERNAK, C. (1973)Deuelop. Biol. 30,403-410. SKIPSKI, V. P., PETERSON, R. F., AND BARCLAY, M. (1964) Biochem. J. 90, 374-377. MARKHAM, R., AND SMITH, J. D. (1952) Biochem.
DEVELOPMENT
275
J. 52, 552-557. 16. RECONDO, E. F., GONCALVES, R. J., AND DANKERT, M. (1964) J. Chromatogr. 16, 415-416. 17. Pabst Laboratories (1965) Circular OR-lo. 18. GARDELL, S., HEIJKENSKJOLD, F., AND ROCHNORLUND, A. (1950) Acta Chem. Sand. 4, 970. 19. LORING, H. S. (1955) in The Nucleic Acids (Chargaff, E., and Davidson, J. N., eds.), Vol. 1, pp. 199-204, Academic Press, New York. 20. TREVELYAN, W. E., PROCTER, D. P., AND HARRISON, J. S. (1950) Nature (London) 166, 444445. 21. DAVIDSON, E. H. (1971) Gene Activity in Early Development, 1st ed., pp. 202-216, Academic Press, New York. 22. WALLACE, R. A., AND DUMONT, J. W. (1968) J. Cell Physiol. 72, 73-89. 23. BERGINK, W., AND WALLACE, R. A. (1974) J. Biol. Chem. 249, 2897-2903.
OF BIOCHEMISTRY
Nucleotides
AND
BIOPHYSICS
and Sugar
269-275
180,
Nucleotides in Early Development Bufo arenarum
Coelomic
MARIA
Embriologia Facultad
LEONOR
Animal y Biologh de Ciencias Exactas
(1977)
CANTORE, EDUARDO Molecular y Naturales,
Oocytes’,
Received
2
MARTA E. F. DE RECONDO, F. RECOND03
Departamentos Universidad September
of
AND
de Ciencias Bioldgicas y Q&mica Bioldgica, de Buenos Aires, Buenos Aires, Argentina 13, 1976
Nucleotides and sugar nucleotides from coelomic oocytes of Bufo arenarum were extracted with trichloroacetic acid and analyzed by ion-exchange chromatography. The hypoxanthine and guanine were sequentially eluted from the column with water. Nucleotides and sugar nucleotides were eluted with a linear gradient of ammonium chloride. The first peak of ultraviolet adsorption eluted from the resin was a complex mixture of at least three substances. The main component was identified as cytidine diphosphocholine by chemical, enzymatic, and chromatographic analyses. Preliminary experiments suggest a possible role for this compound during oogenesis, since immature oocytes incubated in vitro with [L4Clcholine showed an active metabolism of this substance with rapid incorporation in choline phosphate, cytidine diphosphocholine, and lecithin.
In amphibians, oogenesis begins immediately after the breeding season and continues until fall, by which time eggs to be ovulated the following year are in the prophase of the first maturation division and their metabolic activity becomes quiescent. Increase of gonadotrophic activities breaks the equilibrium state. The giant nucleus (germinal vesicle) of the ovarian oocyte bursts, and chromosomes restart their first meiotic division. Oocytes are released from the ovaries over a period of a few hours and they fall into the peritoneal cavity, where cillia propel them toward the end of the adjacent oviduct. Each oocyte completes the first maturation divi-
sion and, simultaneously, a secretory lining deposits several layers of jelly coat around them (1). Oocytes from the body cavity are interesting because of their high metabolic activity and of their transition stage between a differentiated cell, the full-grown oocyte of the ovary, and a totipotent cell, the female gamete able to produce a new multicellular organism after fertilization. In this paper, research is described in which nucleotides and sugar nucleotides from coelomic oocytes were analyzed by ion-exchange chromatography. CDP-choline was found to be a normal component of the acid-soluble extract. Its amount decreased slowly during early development (2) and increased little after gastrulation. It was not detected in a somatic tissue like liver (3). A possible function of this nucleotide during oogenesis is being investigated. Preliminary results indicate a rapid incorporation of choline to phospholipid in immature oocytes via choline phosphate and CDP-choline.
1 Dedicated to Professor Luis F. Leloir on the occasion of his seventieth birthday. 2 This investigation was supported in part by a research grant from the Consejo National de Investigaciones Cientificas y Tecnicas (Argentina) and from the Universidad de Buenos Aires. This paper is third in a series. 3 Career Investigator of the Consejo National de Investigaciones Cientificas y Tecnicas (Argentina). 269 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
270
CANTORE, MATERIALS
AND
DE
RECONDO,
METHODS
Materials. n-Glucose oxidase (a-n-glucose: oxygen oxidoreductase, EC 1.1.3.41, alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) protease type V (P5005), collagenase type I (C-0130), choline phosphate, and lecithin were obtained from Sigma Chemical Co. (St. Louis, Missouri). Purine and pyrimidine bases, nucleosides, nucleotides, sugar nucleotides, and sugars were commercial preparations. lnC]Choline (413 mCi/ mmol) was obtained from New England Nuclear. Animals were purchased from a local supplier. Obtention of coelomic oocytes. Mature females from Bufo arenarum, carefully selected, were anesthetized. The upper ends of their oviducts were tied to force oocytes to remain in the coelomic cavity. After surgery, toads were kept in cold running water for recuperation; 24 h later, ovulation was induced by injecting an aqueous extract of homologous hypophyses. Females were kept approximately 16 h at about 25°C. Jelly-free oocytes which had accumulated in the coelomic cavity were removed through two cuts made at both sides of the media ventral line. Extraction procedure. Oocytes were washed with Holfreter solution, suspended in 2 vol of 10% trichloroacetic acid and homogenized with a blender for 1 min. The suspension was centrifuged at 5000 rpm for 30 min. The supernatant solution was extracted three times with cold ether to remove most of the acid and finally neutralized with NH,OH solution to pH 7. The precipitate was reextracted with 10% trichloroacetic acid and removed by centrifugation, and the superantant solution was extracted with ether and neutralized as described. Both extracts were pooled, the organic solvent was removed by evaporation at room temperature in uacuo, and the solution was stored frozen at -20°C until used (acidsoluble extract). As was reported in preceding papers (2-4), extreme care was taken to preserve labile structures, and a great number of oocytes were used to ensure the detection of compounds present in very small quantities. Three samples of oocytes, each coming from five to six females, were analyzed. Each acid-soluble extract came from approximately 90,000 oocytes (500 oocytes/ml) and contained from 500 to 600 pmol of total nucleotides. Zon-exchange column chromatography. Acid-soluble extracts were chromatographed on Dowex 1 x 4 (Cl-) resin as previously described (4). The bases were eluted with water, and the nucleotides were eluted with a linear gradient of ammonium chloride (O-O.9 M). Fractions were pooled and processed as before (4). Analytical methods. The same analytical methods described in preceding papers (2-4) were used. The choline content of CDP-choline was measured
AND
RECONDO
spectrophotometrically by the method of Appleton (5) and Krisman (6) after acid and enzymatic hydrolyses (see Results). Chromatography and electrophoresis. Paper chromatography was performed on Whatman No. 1 paper with the following solvents (v/v): (I) ethanol-l M ammonium acetate (7:3.5) at pH 7.5 (7); (II) ethanol1 M ammonium acetate (7:3.5) at pH 3.8 (71; (III) butyl alcohol-pyridine-water (6:4: 3) (8); (IV) ethanol-ammonia (7:3) (7); (V) hydrochloric acid-2propanol-water (41:170:39) (9); (VI) butyl alcohol (saturated with water at about 23”C)-ammonia (100: 1) (10); (VII) 2-propanol-acetic acid-water (27:4:9) (11); (VIII) 4.5 M ammonium sulfate in 0.1 M phosphate buffer at pH 6.8-n-propanol (5O:l) (12); (IX) butyl alcohol-acetic acid-water (5:2:3) (13); (X) chloroform-methanol-acetic acid-water (25:15:4:2) (14). Paper electrophoresis was performed on Whatman No. 4 paper as described by Markham and Smith (15) with the following buffers: (Al 0.05 M potassium tetraborate buffer, pH 9.2; (B) sodium phosphate buffer, pH 7.5; (C) sodium carbonatesodium hydrogen carbonate buffer, pH 9.2 (16); (Dl sodium acetate buffer, pH 3.8; (El pyridine acetate buffer, pH 6.5. Thin-layer chromatography was carried out on glass chromatoplates prepared with silica gel G (according to Stahl, E. Merck AG, Darmstadt, Germany). The plates were prepared by making a slurry of 30 g of silica gel G with 65 ml of distilled water and applying it with a Desaga applicator (250 pm thickness). Plates were allowed to dry at room temperature and were stored in a cabinet. Just prior to application of samples, chromatoplates were activated at 80-100°C for 60 min. Obtention of immature oocytes. After ovulation was induced as described before, females were killed. Small residual ovaries were quickly removed and carefully washed with 0.02 M NaCl-0.08 M KC1 (salt solution). Tissue was cut in small pieces and incubated for 2 h at 37°C in an enzymatic mixture (protease, 35 mg; collagenase, 3.5 mg; salt solution, 70 ml). Free oocytes were separated by filtration through gauze and exhaustively washed with the salt solution. Incubation and extraction procedure. Isolated immature oocytes (0.2 ml) were suspended in 0.02 M NaCl-0.08 M KC1 (7 ml) containing [i4Clcholine (1 pCi) and incubated at 30°C for different periods. At the end of the incubation period, residual radioactivity was washed out with the salt solution. Oocytes were homogenized in 10% trichloroacetic acid and centrifuged off, the pellet was successively extracted with 5% trichloroacetic acid and water. The trichloroacetic extracts (washed with ether as described before) and aqueous extracts were pooled, adjusted to pH 6, and used as the “trichloroacetic acid-soluble fraction.” The pellet was extracted
NUCLEOTIDES
IN
EARLY
twice with a chloroform-methanol (2:l) mixture to yield the “lipid fraction.” The trichloroacetic acid-soluble fraction was analyzed by paper chromatography in solvent IX. The lipid fraction was analyzed by thin-layer chromatography in solvent X. Radioactivity on the paper strips or thin-layer plates was located with a Packard radiochromatograph. RESULTS
The ultraviolet-absorbing material eluted from the column with water consists of two main fractions named C and D. Fraction C and fraction D were identified as hypoxanthine and guanine by ultraviolet absorption spectra, behavior in paper chromatography with solvents V, VI, and VII, and paper electrophoresis with buffer A. Elution of the nucleotides and sugar nucleotides was obtained with a linear gradient of 0 to 0.9 M ammonium chloride (Fig. 1). The ultraviolet-absorbing fractions were analyzed as follows.
271
DEVELOPMENT
Fraction I. This fraction usually contained cytosine derivatives and traces of guanine and consisted of a complex mixture of compounds. Although it behaved as a single compound on paper chromatography in solvents I and II and on paper electrophoresis with buffer B, it gave four or five ultraviolet-absorbing bands, with solvent IV, one of which was identified as cytidine diphosphate choline; the others were not further studied. The component with a mobility similar to that of standard CDP-choline was eluted from the paper and exhaustively analyzed. The ultraviolet absorption spectra (neutral and acid) was identical to that of cytosine compounds. Extensive hydrolysis (3 N HCl, 1 h, 1OoOC)followed by paper chromatography of the hydrolysate on solvent V showed that the only base present was cytosine. Pentose determination showed the pres-
1.8
0.9
E =:= w s
1.4
0.7
Y f
1.0
0.5 -5 0 I‘ r 0.3
-
s =: 0.6 2 a2
0.1 0
1.0
2.0
3.0
4.0
5.0
VOLUME
FIG.
COMPARATIVE
1. Elution
ANALYTICAL
Sample
to 1000 kmol
obtained
DATA Base
Coelomic oocytes Mature oocytes Fertilized eggs Morula Gastrula Neurula a Refers
pattern
with
ZO
8.0
9.0
a linear
gradient
TABLE
I
of ammonium
chloride
OF THE MAIN COMPONENT OF PEAK I OBTAINED DIFFERENT STAGES OF DEVELOPMENT Pentose
Cytosine Cytosine Cytosine Cytosine Cytosine Cytosine of bases,
6.0
(liters)
0.9 0.9 1.0 1.0 1.0 0.8 nucleotides,
Total phosphorus
Labile phosphol7lS
2.4 1.8 1.8 1.7 1.8 2.1 and sugar
nucleotides
Inorganic phosphorus
0 0
0 0
0 -
0 0 eluted
from
(O-O.9
FROM Choline
1.0 1.1 1.0 1.0 0.9 0.9 the column.
M).
SAMPLES
IN
Total amount” (pm011 63.6 14.0 16 19.5 26
Uracil
Adenine Guanine
Adenine Guanine
VII
VIII
IX
ATP GTP
ADP GDP
-
-
-
-
-
-
+
TABLE
II
-
-
0.96
0.91
-
0.96
0.85
0
-
1.72
1.72
1.73
1.75
__ Total Labile
FROM COE~MIC Phosphorus”
-
-
-
1.08
0. 91
0.65
Pent.oses”
NUCLEOTIDES Sugar
~-Glucose “-Galactose
B.
2-Acetamido-2deoxyglucw? 2-Acetamido-2deoxygalactose@
-
ChromatograPW
OOCYTES
” Identification by paper chromatography in solvent V after hydrolysis with 3 &J HCI, 1 h, 100°C. b Identification by paper chromatography in solvents I and II and electrophoresis with buffers A and c Increase of absorbance at 327 “m in the presence of potassium cyanide (17). * Mole per mole of base. s Paper chromatography in solvent III. ‘Identification by paper chromatography in solvent III after degradation with ninhydrin (16). * Detected with the reagent of Trevelyan et al. (20). h Paucity of the material prevented further identification. 1 Proportion was determined by spectrophotometric method (19).
(65%)’ (35%)’
(40%)’ (6O%Y
UDP-glucuronic
Uracil (75%)’ Cytosine (25%)
VI
acid
UTP CTP
Adenine” Cytosine” GuanineO Uracil”
Vb
NADP UDP
Adenine” Uracil”
Va
-
UDP-Glc UDP-Gal
Uracil
IVb
(73%) (27%)
-
(78%) (22%)
UDP-GlcNAc UDP-GalNAc
Uracil
IVa
~___~
CW nide compit%’
AND SUGAR
+
Adenine
II
CDP-choline
Nucleotidesb
-~
NAD
Cytosine
BEWe”
I
Peak
NUCLEOTIDES after
-
-
-
-
-
2-Acetamido-2. deoxyglucose 2-Acetsmido-%deoxygalactose
-
Electrophoresis
hydrolysis
-
+
acid
-
-
Arabinose xylose
-
Degradation with ninhydrid
component
NUCLEOTIDES
IN
EARLY
ence of 1 mol of pentose/mol of base, and analysis of the total and acid-labile phosphorous gave two and zero phosphate groups per mole of base, respectively. Acid hydrolysis with 1 N hydrochloric acid for 1 h at 100°C gave an ultravioletabsorbing compound that behaved as authentic CMP on chromatography and electrophoresis. The hydrolysate previously neutralized was treated with alkaline phosphatase, and choline and inorganic phosphate were measured (5, 6); 1 mol of choline/2 mol of inorganic phosphate was found. It was concluded that acid hydrolysis splits CDP-choline into cytidine monophosphate and choline phosphate. Comparative analytical data obtained from samples of coelomic oocytes, mature oocytes, fertilized eggs, and embryos in morula, gastrula, and neurula stages are summarized in Table I. Fractions II through IX. As in samples from other stages of development (2-41, the four ribonucleotide triphosphates (UTP, CTP, GTP, and ATP), NADP, NAD, ADP, GDP, and six sugar nucleotides with uracyl as the base moiety were isolated. Analytical data are summarized in Table II. Table III shows the amount of the different nucleotides and sugar nucleotides found in oocytes from the coelomic cavity. The averages of three samples from five to six females are shown. Values are expressed as micromoles of each compound per 1000 pmol of total bases, nucleotides, and sugar nucleotides eluted from the column. Immature oocytes isolated from the ovaries as described under Materials and Methods were incubated with [14Clcholine for different periods, and the fate of the radioactivity was followed in the trichloracetic acid-soluble fraction and in the lipid fraction material. Paper chromatography in solvent IX of the trichloroacetic acid-soluble extract showed five peaks of radioactivity which had the mobilities of CDP-choline, choline phosphate, betain, choline, and acetyl choline, respectively (Fig. 2). Thin-layer chromatography of the lipid fraction showed two areas of radioactivity. The main one had the same mobility as phosphatidyl choline and the small one
273
DEVELOPMENT TABLE
III
QUANTITATIVE DATA FOR THE PEAKS OBTAINED BY ION-EXCHANGE CHROMATOGRAPHY OF EXTRACTS FROM OOCYTES AT BODY CAVITY Peak
Constituent
Amount”
(pmol,
mean
i SD)
C
Hypoxanthine
314.7
D
Guanine
60.2 t 6
I
CDP-choline
63.6 + 1.8
II
NAD
19.2 rf. 0.9
IVa
UDP-N-acetylglucosamine UDP-N-acetylgalactosamine
IVb
UDP-glucose UDP-galactose
17 c 1.2 6.4 + 0.5
VI
UTP CTP
160 ? 10 53 2 3.6
VII
UDP-glucuronic
VIII
ADP GDP
9.3 2 1.8 14 t 2.7
IX
ATP GTP
84 + 9.6 45 + 5.2
acid
t 17
103.3 2 6.2 26 -+ 1.5
26.2 r 2.5
0 Micromoles of each compound refers to 1000 pmol of bases, nucleotides, and sugar nucleotides eluted from the column.
had the mobility of lysophosphatidyl line.
cho-
DISCUSSION
The importance of the metabolic processesoccurring during oogenesis has been emphasized in the last few years. All of the experimental evidence accumulated points to the conclusion that differentiation actually starts at oogenesis. rRNA and mRNA, macromolecules of great importance during development, are synthesized in the oocytes at the ovaries and they are not used until early embryogenesis (21). Simultaneously, reserve materials such as yolk are also synthesized during the long prophase of the first meiotic division, when oocytes pass from the pachytene to the diplotene stages. Yolk platelets are built in these stages, and, again, this reserve material is stored and used only at
274
CANTORE,
DE RECONDO,
CPM
COP-choline
FIG. 2. Radioactivity pattern of the trichloroacetic acid-soluble extracts after paper chromatography in solvent IX.
the beginning of development. During these events, oocytes exhibit an enormous increase in volume, so that a remarkable amount of nucleic acid and protein is packaged within a single cell over a relatively short time. A systematic study of the levels of the precursors of these macromalecules during the early development of our common toad B. arenarum should be interesting. We found that oocytes from the coelomic cavity have the same nucleotides and sugar nucleotides as in other stages of development, but differences were detected in their levels. As compared with oocytes at morula stages, CDP-choline was four times higher in coelomic oocytes. The levels of UDP-galactose were also high: 20 times higher than in the morula. There were less di- and triphosphate nucleotides of purine base in coelomic oocytes than in embryos at the morula stage (Table III) cl).
The presence of CDP-choline at high levels in oocytes from the coelomic cavity could be related to the synthesis of lipopro-
AND RECONDO
teins at earlier stages of development during oogenesis in the ovaries. Yolk platelets, which are the main components in the cytoplasm of oocytes from the ovary, are formed by a homogenous central body surrounded by a superficial layer, both enclosed within a simple membrane. The crystalline main body consists of 2 mol of a highly phosphorylated protein, called phosvitin, for every mole of a lipoprotein called lipovitellin. Wallace and Dumont (22) have followed the fate of radioactive precursors injected into amphibians and demonstrated that at least the protein moiety of phosvitin and lipovitellin is synthesized in the liver and passes to the blood as a vitellogenin complex. Bergink and Wallace (23) later showed that this complex consists of the precursor molecules for lipovitellin and phosvitin and that, once the complex is transported to the ovary, it is transformed into the two proteins of the yolk platelets. The origin of the lipid moiety of lipovitellin is not well understood. This lipoprotein contains phosphatidyl choline (lecithin), and the metabolic pathway of choline to lecithin through choline phosphate and CDP-choline is well known. All of these facts suggest that CDP-choline could be an intermediate in the biosynthesis of the lipovitellin of yolk during oogenesis. Immature oocytes were incubated with [14C]choline and the fate of the radioactivity was followed in the acid-soluble fraction and in the lipid fraction. Our preliminary results showed that choline is rapidly metabolized by the oocyte and incorporated in choline phosphate, CDP-choline, and, finally, in lecithin. It remains to be elucidated which is the distribution of lecithin: the lipids or the lipoproteins of the oocyte organelles. REFERENCES 1. RUGH, R. (1951) The Frog, pp. 31-71, Blakiston, Philadelphia. 2. MORENO, S., CUKIER, M., GUERRERO, G., FERNANDEZ DE RECONDO, M. E., AND RECONDO, E. F. (1976) Carbohydrate Res. 47, 275-284. 3. MAGGESE, M. C., SPAIZMAN, R. C., AND FERNANDEZ DE RECONDO, M. E. (1976) Acta Physiol. Latinoamer., in press. 4. FERNANDEZ DE RECONDO, M. E., MAGGESE, M.
NUCLEOTIDES
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
IN
EARLY
C., GUERRERO, G., SPAIZMAN, R. C., AND RECONDO, E. F. (1973) Carbohydrate Res. 26, 365-375. APPLETON, H. D. (1950) Fed. Proc. 9, 146. KRISMAN, C. R. (1962)Anal. Biochem. 4, 17-23. PALADINI, A. C., AND LELOIR, L. F. (1952) Biothem. J. 51, 426-430. JEANES, A., WISE, C. S., AND DIMLER, R. J. (1951) Anal. Chem. 23, 415-420. WYATT, G. R. (1951) Biochem. J. 48, 584-590. MACNUTT, J. (1952) Biochem. J. 50, 384-396. TUNG, K. K., AND NORDIN, J. M. (1968)Biochim. Biophys. Acta 158, 154-156. MARKHAM, R., AND SMITH, J. D. (1951) Biochem. J. 49, 401-406. PASTERNAK, C. (1973)Deuelop. Biol. 30,403-410. SKIPSKI, V. P., PETERSON, R. F., AND BARCLAY, M. (1964) Biochem. J. 90, 374-377. MARKHAM, R., AND SMITH, J. D. (1952) Biochem.
DEVELOPMENT
275
J. 52, 552-557. 16. RECONDO, E. F., GONCALVES, R. J., AND DANKERT, M. (1964) J. Chromatogr. 16, 415-416. 17. Pabst Laboratories (1965) Circular OR-lo. 18. GARDELL, S., HEIJKENSKJOLD, F., AND ROCHNORLUND, A. (1950) Acta Chem. Sand. 4, 970. 19. LORING, H. S. (1955) in The Nucleic Acids (Chargaff, E., and Davidson, J. N., eds.), Vol. 1, pp. 199-204, Academic Press, New York. 20. TREVELYAN, W. E., PROCTER, D. P., AND HARRISON, J. S. (1950) Nature (London) 166, 444445. 21. DAVIDSON, E. H. (1971) Gene Activity in Early Development, 1st ed., pp. 202-216, Academic Press, New York. 22. WALLACE, R. A., AND DUMONT, J. W. (1968) J. Cell Physiol. 72, 73-89. 23. BERGINK, W., AND WALLACE, R. A. (1974) J. Biol. Chem. 249, 2897-2903.