Quantitative analysis of DNA in sea urchin eggs and subcellular distribution of DNA in the eggs

ANALTTICAL

58, 439448

BIOCHEMISTRT

Quantitative

Analysis

Subcellular TAKEHISA Zoological

Institute, Received

(1974)

of DNA

Distribution KANEKO

AND

Urchin

Eggs

DNA

in the

Eggs’

HIROSHI

of Science, University

Faculty June

of

in Sea

8. 1973;

accepted

and

TERAYAMA o.f Tokyo,

September

Tokyo,

Japan

21, 1973

An improved procedure for analyzing DNA in animal eggs which contain a great deal of other chromogenic materials has been developed. As a result the following DNA contents were estimated: 3.59 ? 0.39 pg DNA/egg for H. pulcherrimus, 5.06 k 0.30 pg DNA/egg for Ps. depressus, and 3.78 a 0.33 pg DNA/egg for A. crassispina. Egg nuclei contain the haploid level of DNA. Most of the cytoplasmic DNA was found to be associated with the mitochondria (more than 90%) and only a small portion was detected in the yolk granule fraction.

A number of papers (l-7) dealing with the DNA content of unfertilized eggs of various animals have already been published, reporting the presence of a great deal of DNA stored in the cytoplasm. In the opinion of some workers this was supposed to support the vigorous cell proliferation during the course of early development. However, the analytical procedures adopted in the earlier studies on the DNA content of animal eggs have been recognized as inadequate for application to eggs and, in fact, the cytoplasmic DNA content reported so far varied tremendously in t.he different papers. In the present st.udy we have compared various analytical procedures in an attempt to obtain more accurate calorimetric assay of egg DNA. By the combination of milder procedures (the modified Tyner-Heidelberger-LePage method for extracting DNA and the modified CroftcLubran method for calorimetric estimation) and appropriate blank correction it was found possible to perform a more accurate assay of the DNA content in sea urchin eggs. MATERIALS

AND

METHODS

Preparation of Eggs and Sperm. Sea urchins used in the present study were Pseudocentrotus depressus, Hemicentrotus pulcherrimus, and Anthocidaris crassispina. Eggs and sperm were spawned by the conventional 1 The present study was supported the Ministry of Education. Japan. Copyright All rights

@ 1974 by Academic Press. of reproduction in any form

by a Grant-in-Aid 439 Inc. reserved.

for

Scientific

Research

from

440

KANEKO

AND

TERATAMA

KC1 method. The jelly coat of eggs was removed by treating eggs in slightly acidified sea water, pH 5.0. Extraction of DNA. The following four conventional procedures were compared: the Schneider method (8)) the SchmidLThannhauser method (9), the Ogur-Rosen method (lo), and the Tyner-Heidelberger-LePage method (11) with or without modification. Among them the TynerHeidelberger-LePage method was found to be most suitable with a little modification as follows. The eggs (usually 5 to IO-ml packed cell volume or several X lo7 cells) were homogenized with water and 3 vol of ethanol were added to the homogenate or the eggs were directly homogenized in ethanol. The ethanolic homogenates were stored at 0°C for 2 hr, followed by centrifugation at 3000 rpm for 10 min. The pellet was washed four to six t’imes with ethanol, three times with ethanol-ether (3:l v/v) mixture. The residue was washed again with ethanol and then with cold 10% NaCl acidified with perchloric acid (PCA) (pH 2.0). The washed pellet was suspended in 3 vol of 10% NaCl, adjusted to pH 910 by adding NazCOa, heated in a boiling water bath for 60 min, and then centrifuged. This hot extraction procedure was repeated once more for 30 min. The first and the second ext#racts (supernatants) were combined, mixed with 3 vol of cold ethanol, and the mixture was allowed to stand at 0°C overnight. The precipitates were spun down by centrifugation, resuspendedin 1 N KOH, and the alkaline suspension was incubated at 37°C for 20 hr. Ice-cold 20% PCA was added up to 5% concentration. The precipitates containing DNA were collected by centrifugation, washed twice with cold 5% PCA and suspended in 5% PCA. The suspensionwas heated at 90°C for 15 min and then centrifuged. The supernatant thus obtained was used for DNA calorimetry. Calorimetry of DNA. The following three procedures were compared: the Burton method (12) [using diphenylamine at 30°C for 18 hr], the Croft-Lubran method (13) [diphenylamine at 10°C for 72 hr], and the Ceriotti method (14) [indole at 100°C for 10 min] . As will be presented later the Croft-Lubran method with the following blank correction was found to be most suitable. The Croft-Lubran reagent to develop the color was prepared by mixing 1 g diphenylamine, 49 ml glacial acetic acid, 1 ml coned sulfuric acid, and 0.25 ml of 2% acetaldehyde in water. The DNA extract was mixed with an equal volume of the above chromogenic reagent and kept at 10°C for 72 hr to develop the color. In case of samples other than egg materials, the calorimetry of DNA may be done simply by measuring absorbancy at 600 nm using calf thymus DNA as standard. However, in the case of the egg materials, the following control is necessary. The DNA extract was divided into two equal parts. To one part was added an equal volume of the Croft-Lubran reagent and to another part

SEA

URCHIN

EGG

DNA

ANALYSIS

441

was added an equal volume of the reagent minus diphenylamine. These mixtures were similarly kept at 10°C for 72 hr and a difference of absorbances at 600 nm between the two mixtures was measured to get the DNA content. Subcellular Fractionation. The homogenizing medium contained 0.36 M KCl, 0.3 M sucrose, 0.03 M Tris-HCl (pH 7.6)) and 0.003 M EDTA (15). One volume of packed eggs was homogenized wit’h 4 vol of the homogenizing medium in a motor-driven, Teflon-glass homogenizer at 0°C with six times up-and-down strokes. The homogenate was centrifuged at 6009 for 10 min to separate the nuclear fraction from the supernatant, which was then centrifuged at 18,OOOg for 40 min to sediment the yolk-mitochondrial fraction. Further fractionation of the latter was carried out as follows. The yolk-mitochondrial pellet was suspended in 1.0 &f sucrose-O.01 M Tris-HCl buffer (pH 7.6)-0.001 M EDTA solution, and the suspension was centrifuged at 23,000 rpm for 2 hr in a Spinco SW 25-l rotor. The yolk fraction which was floated up to form the top surface pellet and the mitochondrial fraction which was sedimented at the bottom were collected separately. The subcellular fractions of sea urchin eggs thus obtained were subjected to DNA assay according to procedures described above. Assay of Protein and Biochemicals. Protein was assayed by the method of Lowry et al. (16) with crystalline bovine serum albumin as standard. Biochemicals used in the present study were: DNA (Sigma Chemical Co., St. Louis, MO, calf thymus type IV, highly polymerized), sialic acid (N-acetylneuramic acid, Sigma Chemical Co., St. Louis, MO). RESULTS

When sea urchin eggs were extracted with various methods (Schneider, Ogur-Rosen, SchmidtrThannhauser, and Tyner-Heidelberger-LePage) and the calorimetric assay of DNA in these extracts was carried out either by the Burton method or by the Ceriotti method, rather high DNA contents with tremendous fluctuations depending on the analytical procedures were obtained (18-83 pg DNA per egg cell). Among the extraction methods compared in the above preliminary study the TynerHeidelberger-LePage method gave the lowest DNA content. On the other hand, DNA contents in sea urchin sperm cells were consistently lower and constant (1.1-1.2 pg DNA per sperm cell) without regards to the analytical procedures adopted. Throughout these casesabsorption spectra of the colors developed from the egg DNA extracts by the Burton method or the Ceriotti method were found to be different from those of the colors developed from the sperm materials or from the reference calf thymus DNA, showing an extra absorption maximum. This additional absorption maximum seemsto be partly due to a sialic acid contaminant in the DNA

442

KANEKO

Development Concn of DNAu (rdml) 0 0 7.7 7.7

7.7 15.4 15.4 15.4 30.8 30.8 38.5 38.5

AND

TERAYAMA

TABLE 1 of Color from Mixtures of N-Acetylneuramic and DNA by the Croft-Lubran Method Concn of NAN& (&ml) 48.0 500 0 38.4 43.2 0 28.8 38.4 0 28.8 0 500

E560 0.004 0.014 0.020 0.020 0.023 0.035 0.036 0.036 0.072 0.074 0.092 0.105

Acid

E 600

0.002 0.007 0.035 0.035 0.035 0.070 0.070 0.070 0.140 0.138 0.175 0.183

a NANA; N-acetylneuramic acid. Solutions of DNA and NANA in 57, PCA heated at 100°C for 20 min were subjected to color development according to the Croft-Lubran method. Absorbancas were measured at 550- and BOO-nm wavelengths.

extracts because mixtures of authentic N-acetylneuramic acid and DNA gave absorption spectra resembling those developed from the egg materials. Attempts to obtain an empirical equation to correct for the sialic acid contribution to DNA absorbancy in the Burton and Ceriotti methods have failed. Next’, t,he adaptability of the Croft-Lubran method in assaying DNA calorimetrically in the presence of N-acetylneuramic acid was examined. Mixtures of DNA hydrolyxate and N-acetylneuramic acid of various ratios were treated with the diphenylamine reagent at 10°C for 72 hr according to the procedure of Croft and Lubran (13) and absorbances at 550 nm and 600 nm were measured. As summarized in Table 1, the absorbancy at 550 nm which is specific t,o N-acetylneuramic acid was negligibly small even in the presence of a large excessof N-acetylneuramic acid. At the same time it was found that the absorbancy at 600 nm which is specific to DNA was proportional to the contents of DNA in spite of the presenceof N-acetylneuramic acid. The absorption spectra of the colors developed from A. crassispina germ cell materials by the combined procedures of the modified TynerHeidelberger-LePage extraction method and the Croft-Lubran colorimetric method are illustrated in Fig. 1. In Fig. 1 the curves A, B, and C correspond to the colors developed from the sperm DNA fraction, the egg DNA fraction, and the reference calf thymus DNA, respectively. Apparently the curve B does not have

SEA

OL 520

URCHIN

EGG

DNA

h .

I

550

600 Wave

length

443

ANALYSIS

650

671

(nm)

FIG. 1. Absorption spectra of colors developed by the Croft-Lubran method from egg DNA fraction extracted by the modified Tyner-Heidelberger-LePage method. Eggs of A. cmssispi~~a (1.52 X 10’ cells) were subjected to DNA extraction according to the modified Tyner-Heidelberger-LePage method. The final hot PCA extract of 3-ml volume was divided into two equal parts. To one was added an equal volume of the complete Croft-Lubran reagent mixture containing 2% diphenylamine (B) and to another was added an equal volume of the reagent mixture without diphenylamine (D). The sperm DNA fraction in 5 ml hot PCA extract was prepared from A. crassispina sperm (1.86 X 10’ cells) by the Schmidt-Thannhauser method and the extract of 1.5 ml (hot PCA) was mixed with an equal volume of the complete reagent mixture (A). The hot PCA-treated calf thymus DSA (15 pg/ml) was also mixed with an equal volume of the complete reagent mixture (C). All the mixtures were kept at 10” for 72 hr to develop the colors. Curves A, B. and C correspond to the absorption spectra of colors developed from the sperm DNA fraction, the egg DNA fraction, and the calf thymus DNA treated with the complete reagent mixture. respectively. Curve D corresponds to the absorption spectrum of color developed from egg DNA fraction treated with the reagent mixture minus diphenylamine. Curve Bo is obtained by subtracting the curve D from curve B.

an extra absorbance maximum or shoulder but has relatively higher absorbances at, lower wave lengths as compared with the curves A and C. Curve D, showing increasing absorbancy with lowering wavelengths, corresponds to the color developed from the egg DNA extract treated with the Croft-Lubran reagent minus diphenylamine and may be ascribed to the nonspecific colors. Curve Bo which is obtained by subtracting curve D from curve B is quite similar to curves A and C, and may be considered to be more accurate representation of DNA in the egg DNA extract. The DNA contents in the unfertilized eggs of three different speciesof

444

KANEKO

AND

TERAYAMA

sea urchins thus obtained from the maximal absorbancy of curve Bo at 590-600 nm are summarized in Table 2 in comparison to the sperm DNA contents which may be determined without any trouble by any conventional method. Apparently, the DNA content in the eggs thus estimated by our procedures is much smaller than the values obtained by the other conventional procedures, showing only 3.S4.5 times the haploid level of DNA (sperm was assumed to have the haploid level of DNA). In order to obtain additional confirmation that the modified TynerHeidelberger-LePage method can extract DNA quantitatively, A. crassispina egg homogenate was mixed with different amounts of reference calf thymus DNA or with different amounts of isolated sea urchin nuclear fraction, and the mixtures were subjected to DNA assay according to our procedures described in the present paper. It was found that calf thymus DNA or DNA in the nuclei added to the egg homogenate could be extracted and assayed almost quantitatively with 91 + 4% recoveries in spite of very small amounts of total DNA (16-37 pg) used in the present tests. If the nucleus of unfertilized eggs of sea urchins is assumed to have a haploid DNA content similar to the sperm, 2.0-3.5 X haploid DNA may be considered to be present in the cytoplasm. DNA contents of the subcellular fractions of sea urchin eggs determined by our method are summarized in Table 3. It is clear that the yolk-mitochondrial fraction and the nuclear fraction are the main DNA-containing organelles in the unfertilized sea urchin eggs. Almost 95% of the total cellular DNA was recovered in these fractions. The ratio of DNA in the yolk-mitochondrial fraction to that in the nuclear fraction was about 2.5 (Ps. depressus) or 3.9 (H. pulcherrimus). The yolk-mitochondrial fraction of H. pulcherrimus was further fractionated into the yolk and mitochondrial fractions and DNA in these fractions was assayed by our method. It was found that more than 90% of DNA in the yolk-mitochondrial fraction is recovered in the mitochondria, leaving only a small portion of DNA in the yolk granules, suggesting that the cytoplasmic DNA in the unfertilized sea urchin eggs is mainly associated with the mitochondria. The content of DNA recovered in the nuclear fraction was 1.16 pg (Ps. depressus) or 0.91 pg (H. pulcherrimus) per egg cell. The values may be considered to be very close to the DNA content in the sperm cell (1.13-1.20 pg/sperm) if we take the possible loss of a small portion of nuclei in the fractionation course into consideration. In our calorimetric procedures the blank correction (control lacking diphenylamine) has been introduced. The correction appears to correspond to 2040% of the apparent estimate of DNA in the whole egg homogenate. When the yolk and mitochondrial fractions were separated

Contenk

pul. pul. cm. era. dep. dep.

Egg Sperm Egg Sperm

Egg Sperm

Germ cell

in the Germ E&action

method

Modified Tyner-Heidelberger-LePage Schmidt-Thannhauserc Modified Tyner-Heidelberger-LePage Schmidt-Thannhauser Modified Tyner-Heidelberger-LePage Schmidt-Thannhauser

Extraction

serious

Pseudocentrotus

trouble.

Ps. dep.;

Croft-Lubran Croft-Lubran Croft-Lubran Croft-Lubran Crof t-Lubran Croft-Lubran

Colorimetry

TABLE 2 Cells of Various Species of Sea Urchins Estimated by the Modified Method Combined with the Modified Croft,-Lubran Calorimetric

n H. pul.; Hemicentrotus pulcherrinlus, A. cm.; Anthocidaris crassispina, * Mean + average deviation (number of determinations). c Sperm DNA may be extracted by any convent,ional method without

H. H. A. A. Ps. Ps.

Speciesa

DNA

depressus.

3.59 1.20 3.78 1.19 5.06 1.13

+ + f f k *

0.39 0.06 0.33 0.07 0.30 0.04

DNA content (pdcell) (4)b (3) (4) (3) (4) (3)

Tyner-Heidelberger-LePage Met)hod

5 fs 2 E

%

3.2 4.5

B

s E s

E 9

3.0

Ratio of DNA (egg/ sperm)

dep.)

0 Abbreviation

(H. pd.)

VII

(H. pd.)

VI

(H. pd.)

V

(H. pd.)

IV

(Ps. dep.)

III

&.

(Ps. dep.)

I

Exp no. (Speciesp

of species

2.7

3.5

3.0

is the same

Yolk Mitoch.

Yolk Mitoch.

as in Table

2.

0.062 0.280

0.102 0.400

0.146 0.482 0.032

0.150 0.504 0.068

Nuclei Yolk-mitoch. Supn.

3.0

Nuclei Yolk-mitoch. Supn.

0.200 0.430 0.050

Nuclei Yolk-mitoch. Supn.

3.0

0.190 0.398 0.134

Nuclei Yolk-mitoch. Supn.

3.0

0.276 0.822 0.105

B

0.040 0.000

0.066 0.000

0.050 0.080 0.014

0.044 0.120 0.040

0.052 0.120 0.050

0.044 0.080 0.050

0.090 0.126 0.045

D

0.022 0.280

0.036 0.400

0.096 0.402 0.018

0.106 0.384 0.028

0.148 0.310 0.000

0.146 0.318 0.084

0.186 0.696 0.060

Bo

at 600 nm

TABLE 3 in Subcellular Fractions

Absorbance

Nuclei Yolk-mitoch. Supn.

Subcellular fraction

of DNA

6.0

Amount of eggs (X10’ cells )

Assay

5.9 75.6

9.7 108.0

25.9 108.5 4.9

28.6 103.7 7.6

40.0 83.7 0.0

39.4 85.8 22.7

50.2 188 16.2

Pf3

DNA

of Sea Urchin

0.22 0.280

0.27 3.08

0.86 3.62 0.16

0.95 3.46 0.25

1.33 2.79 0.0

1.31 2.86 0.76

0.84 3.13 0.27

pg/egg

content

Eggs

0.25 f 0.03 Mitoch. 2.94 + 0.14

Yolk

Nuclei 0.91 * 0.05 Yolk-mitoch. 3.54 f 0.08 Supn. 0.21 + 0.05

Nuclei 1.16 + 0.21 Yolk-mitoch. 2.93 f 0.14 Supn. 0.34 & 0.28

m DNA/egg

Average

(4.5)

(75.9)

(19.5)

(7.6)

(66.1)

(26.4)

(o/o)

2 5

5 c;l

P 3 g

WA

URCHIN

EGG

DNA

ANALYSIS

447

and assayed individually, this nonspecific colors necessitating the blank correction was found to be predominantly ascribable to the yolk fraction. DISCUSSION

In spite of the recognition that the falsely high estimates of DNA in the cytoplasm of unfertilized eggs may at least partly be ascribed to analytical errors due to some other chromogenic contaminants (7,15,17~, the quantitative estimation of DNA in animal eggs has yet not been fully investigated and perfected. Such techniques as the thymidine microbioassay method by Hoff-Jorgensen (2) and the diaminobenzoic acid (DABA) -fluorometric method by Kissane and Robins (18) have been considered t.o be more reliable because they give lower DNA estimates compared with the calorimetric methods (1,2,6,7,19-22). The analytical procedure described in the present paper represents further improvement of the egg DNA assay by introducing additional mildness into both the chemical extraction and the calorimetric assay of DNA, selectively suppressing both extraction and calorimetric interference of chromogenic storage materials. The improved method still requires a blank correction for interfering color, which can not be neglected when DNA in the whole eggs or the subcellular fractions except isolated mitochondria is assayed. As to the DNA content of sea urchin eggs, the following values have so far been reported: 20-30 X haploid DNA for Paracentrotus lividus (thymidine microbioassay) (I), 37 X haploid DNA for Hemicentrotws lividus (DABA-fluoromctry) (21) and 180X haploid DNA for Strongylocentrotus purpuratus (DABA-fluorometry) (22). Hovvever, Piko et al. (15) have reported much smaller DNA contents of seaurchin eggs by the CsCl density gradient centrifugation technique: i.e., 4.3 X haploid DNA for Strongylocentrotus purpuratus and 9.5 X haploid DNA for Lytechinus pi&us. The latter values seem to be closer to the DNA contents of sea urchin eggs obtained by our calorimetric method: i.e., 3.0-3.2X haploid DNA for Hemicentrotus pulcherrimus and Anthocidaris crassispina and 4.5 X haploid DNA for Pseudocentrotus depressus, indicating that the cytoplasmic DNA content is not as large as considered in the past. Unfertilized sea urchin egg nuclei have been reported to contain the haploid level of DNA (23). The results of our present study on the subcellular distribution of DNA have also indicated the presence of the haploid level DNA in the nuclei. Most of the cytoplasmic DNA seemsto be associated with the mitochondria rather than with the yolk granules. The possibility of nuclear contamination in the mitochondrial fraction is considered very small. The ratio of DNA to protein in our mitochondrial fraction was 0.73-0.83 pg DNA/mg protein. The value is similar to the

448

KANEKO

AND

TERBYAMA

one reported for the purely isolated amphibian mitochondria (0.51-0.53 pLg DNA/mg protein) (24) and is much smaller than the ratio reported for the isolated sea urchin nuclear fraction (43.5 pg DNA/mg protein) (25). Predominant mitochondrial localization of the cyt,oplasmic DNA has been reported by Dawid (24) on the eggs of Xenopus laevzls as well as Rana pipiens and also by Piko et al. (15) on the eggs of Lytechinus pictw. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23, 24. 25.

ELSON, D. T., AND CHARGAFF, E. (1952) Experientia 8, 143. HOFF-JORGENSEN, E. S., AND ZEUTHEN, E. (1952) Nature (London) 169, 245. SOLOMON, J. B. (1957) Biochim. Biophys. Acta 24, 584. CHEN, P. S. (1960) Exp. Cell Res. 21, 523. COLLIER, J. R., AND MCCANN-COLLIER, M. (1962) Exp. Cell Res. 27, 553. BALTUS, E., AND BRACHET, J. (1962) B&him. Biophys. Acta 61,157. HAGGIS, A. J. (1964) Develop. Biol. 10, 358. SCHNEIDER, W. C. (1945). J. Biol. Chem. 161, 293. SCHNEIDER, W. C. (1946) J. Biol. Chem. 164, 747. OGUR, M., AND ROSEN, G. (1950) Arch. Biochem. 25,262. TYNER, E. P., HEIDELBERGER, C., AND LEPAGE, G. A. (1953) Cancer Res. 13, 186. BURTON, K. (1956) Biochem. J. 62,315. CROFT, D. N., AND LUBRAN, M. (1965) Biochem. J. 95, 612. CERIOTTI, G. (1955) J. Biol. Chem. 214, 59. PIKO, L., ?~NER, A., AND VINOGRAD, J. (1967) Biol. Bull. 132, 68. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. DAWID, I. B. (1965) J. Mol. Biol. 12, 581. KISS~NE, J. M., AND ROBINS, E. (1958) J. Bid. Chem. 233, 184. GRANT, P. (1958) J. Cell. Comp. Physiol. 52, 227. SUGINO, Y., SIJGINO, N., OKASAKI, R., AND OKASAKI, Y. (1960) Biochim. Biophys. Acta 40, 417. BALTUS, E., QUERTIER, J., FICQ, A., AND BRACHET, J. (1965) Biochim. Biophys. Acta 95, 408. EBERHARD, A., AND MAZIA, D. (1965) Biochem. Biophys. Res. Commun. 21, 460. HINEGARDNER, R. T. (1961) Exp. Cell Res. 25,341. DAWID, I. B. (1966) Proc. Nat. Acad. Sci. USA 56, 269. HNILICA, L. S. (1970) Biochim. Biophys. Acta 224, 518.