Unusual properties of sea urchin unfertilized egg chromatin

Cell Biology

International

Unusual

I.

Properties

Albanese,

Institute Palermo,

Reports, Vol. 4, No. 2, February

I.

of

Di

Liegro

of Comparative via Archirafi

Urchin Sea Chromatin and

G.

201

1980

Unfertilized

Egg

Cognetti

Anatomy, The Palermo, 20,

University Italy

of

Abstract not detected by A typical nucleosomal pattern is of sea urchin mature egg electrophoretic analysis chromatin,following digestion with micrococcal nuclease. Moreover, at least 80 % of the egg nuclear DNA is resistant to nuclease attack, These unusual features of unfertilized egg chromatin, not shared by oocytes or sperms, are discussed in view of the unique properties and fate of mature female germ cells. Introduction Nucleosomal from widely particles different organisms and cell types have essentially identical properties (Kornberg, 1977). This has generally been attributed to the fact that histones are among the most highly conserved proteins. However, chromatin retains a fairly typical nucleosomal organization (Spadafora and Geraci, 1975; Keichline and Wassarman, 1977), even in those cases in which at least some of the histones are known to be replaced by other histone - like proteins, i.e., sea urchin sperm (Easton and Chalkley, or specific histone variants, Johnson et e.,1973) 1972; sea urchz embryos (Cohen et al., 1975; Newrock I.e., et al., 1977). Very little information is still available about the proteic composition of female germ cell chromatin. Earlier investigations suggested that typical histones, or at least lysine - rich histones, are not present in sea urchin mature egg nuclei (Hnilica and Johnson,lg70; Evans and Ozaki,1973). More recently, Carroll and Ozaki (1979) reported that in Stron gylocentrotus purpuratus eggs, as well as in zygotes, histones can indeed be found in the chromatin, but H 1, H 2A and H 2B have a different electrophoretic mobility in acid - urea gels, compared to the correspondent sperm or embryonic 0309-1651/60/020201-10/~2.~/0

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Press Inc. (London)

Ltd.

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Since no data are however yet available about histones. the organization of chromatin from either mature eggs considering the unusual physiology of the or oocytes, egg cell, which contains all the machinery for an active macromolecular metabolism which has on the other hand to be kept still after oogenesis and until fertilization,we decided to investigate some of the structural features of oocyte and egg chromatin. Materials

and

Methods

P. lividus eggs and sperms were collected in Trisbuffered, millipore filtered sea water (pH 8.1). Oocytes were isolated from female F. lividus gonads as described by Cognetti et 2. (19777. Only sea urchin batches which showed greater than 90 % fertilization efficiency and normal embryo development were chosen. Isolation of nuclei. Sperm nuclei were prepared accordOocyte germinal vesicles ing to Spadafora --et a1.(1976). were isolated as described by Sconzo et ~1 c . (1972). Unfertilized egg pronuclei were isolated as follows : eggs were washed once in buffer I (0.1 R :'Na phosphate, 0.5 mM phenylmethylsulfonylfluoride (P&ZSF), 0.1 bI LiCl, pH 6.0) supplemented with 0.02IC M ethylenediaminetetraacetate (EDTA), resuspended in buffer I - 0.01 PI Pig acetate and homogenized with one or two strokes of a tight-fitting pestle in a Dounce homogenizer. The absence of intact cells was checked by light microscopy. The homogenate was centrifuged and the crude nuclear sediment was washed twice in buffer I-0.01 ki Mg acetate -0.5 76 NP40 to remove contaminant membranes and pigment for 10 min. by centrifugation at 800xg The nuclear pellet was washed again in buffer I - 0.01 FI P;g acetate to get rid of the detergent and further purified by centrifugation through a 0.5 M sucrose cushion, always in the same buffer, at 12,OOOxg for 10 min. Sucrose was then removed by two additional washes in buffer I0.01 &I ?Ig acetate. The entire procedure was carried out at 2O-4OC. Centrifugations were at 3OOxg, unless otherwise specified. Proteolysis was prevented by addition of KiSF; LiCl and Na phosphate were found necessary to inhibit endogenous nuclease activity (Parisi and De Petrocellis, 1972), which in these cells is presumably localized mainly in the cytoplasm (De Petrocellis and On the average, 100 ug of 1972). Parisi, DNA was obtained from 1 ml packed eggs, and approximately 11 $ of total egg DNA was recovered in the purified nuclear This is in fairly good agreement wit!? earlier pellet. observations that about 70-90 % of sea urchin egg DNA is cytoplasmic (Ciudice, 1973). Iicrococcr:l nuclease Flicrococcal nuclease digestion. (Worthington Riochemical Corp.) incubation of oocyte germinal vesicles and egg and sperm nuclei was perform-

Cell Biology international

Reports, Vol. 4, No. 2, February 1980

ed as described by Hewish and fiurgoyne (1!'73),in zuffer Chick embryo erythroA supplemented with 1 mM CaCl?. cyte nuclei were isolated and-digested with micrococcal nuclease as described by Riley and Weintraub (1978). For further details on the incubation conditions, see the text. The digestion reaction was stopped by chilling and adding EDTA to 2 mM. The nuclei were pelleted at 300xg and lysed in 0.2 mM EDTA.Soon after the lysis, the suspension was made 5 mM EDTA to solubilize the chromatin fragments. Gel electrophoresis. For electrophoretic analysis of the nucleoprotein particles, aliquots of the nuclear lysate were directly diluted in the electrophoresis buffer and loaded onto 5% polyacrylamide gel(Weintraub, Total or 0.25N H2S04-soluble nuclear proteins 1976). were analyzed on sodiumdodecylsulfate (SDS) - 15% polyacrylamide gels prepared as described by Weintraub et Calf thymus histones (Sigma) were used z a1.(1975). markers. Diphenylamine assay. The amount of DNA was determined by the diphenylamine(DFA) reaction (Dische,lF55) on the samples, after extraction according to the procedure described by Sconzo and Giudice (1971). Results

and

Discussion

We first tried structure conld be egg chromatin.

to determine detected in

whether sea urchin

a nucleosomal unfertilized

gel Fig. 1. Polyacrylamide electrophoresis of chromatin A) unfertilized egg from: nuclei digested with (from left to right): O;5;lO;2O; micrococcal nu600 U/ml at 37°C for 10 min. clease, (following incubation, EDTA was added to 2mK and nuclei were incubated with 50 ug/ml pancreatic RNAse (Sigma) at 37°C for 30 min.) B) Chick embryo erythrocyte nuclei digested with 30 U/ml micrococcal nuclease, at 37” c for 10 min.

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Nucleosomes can phoresis, following chromatin or intact

be directly micrococcal nuclei.

2.

visualized nuclease lividus

by electrodigestion of mature egg

nuclei were therefore isolated and incubated in the presence Of micrococcal nuclease, as described. Unexpectedly, electrophoretic analysis of the nuclear 1Ysate (Fig. IA) showed that no material migrates onto the gel under digestion conditions that produce a typiCal nucleosomal Pattern with chick embryo erythrocyte nuclei (Fig. 1B). It is only by using much higher nuclease concentrations that a single band appears in the mononucleosome position, but a regular series of bands, corresponding to dimers, trimers and higher oligomers was never observed, although DNA and enzyme concentrations were widely varied in a series of experiments. A brightly stained band was instead reproducibly present at the top of the gel. This result cannot be explained with a higher nuclear resistance because it is also obtained when micrococcal nucletise digestion is carried out on purified egg chromatin, rather than on intact nuclei. of the egg chromatin to This unexpected resista!?ce prodlIce a nucleosomal pattern prompted us to analyze the extent of enzymatic hydrolysis to acid - soluble incubation with fragments of egg nuclear DNA, following increasing amounts of micrococcal nuclease. The results obtained in such experiments indicate is dethat no more than 20 5; of the egg nuclear DNA even under the more fragments, graded to acid - soluble U / ml for drastic incubation conditions tested (1000 60 min.). In contrast, up to 90 :ti DNA hydrolysis could be obtained in parallel experiments with hatching blastula incubation in the presence of as after 15 min. nuclei, nuclease; under even little as 100 U / ml micrococcal milder incubation conditions a typical Plateau was observed when 50 76 of the DNA had been degraded. The different susceptibility of egg and embryo nuclear DNA to degradation could not be explained bY the presence of a nuclease inhibitor in our egg nuclei since blastulae DNA was hydrolyzed to the preparations, incubated alone or same extent when their nuclei were mixed in various ratios with egg nuclei.

The fact that nucleoprotein particles with the electrophoretic mobility t$pical of nucleosomes are not nuclease digestion of egg chroreleased by micrococcal matin is not a general property of sea urchin germ typical nucleosomal patAS shown in Fig. 2a,b, cells. terns can in fact be generated by nuclease digestion of sperm nuclei and oocyte germinal vesicles.

Cell Biology

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pattern Fig. -2. Klectrophoretic of nucleosomes prepared from : sperm nuclei digested with (4 100 U/ml micrococcal nuclease, at 37°C for 5 min. (b) oocyte germinal vesicles, incubated with 20 U/ml micrococcal nuclease, at 37°C for 10 min.

a

b

Analysis of the proteins bound to DNA in egg nuclei may be helpful in understanding why typical nucleosomes cannot be detected in chromatin from these cells. Since well the proteic composition of egg chromatin is not has and even the presence of histones in it known, often been questioned in the past, we performed an electrophoretic analysis of -the egg nuclear proteins 3A). Despite the high extractable in 0.2 5 N H~SOI, (Fig. four bands which comigrate complexity of this pattern, with calf thymus H 3, II 213, H 2A and H 4 (Fig. 3Ll) are The reduced intensity of the band corclearly visible. responding to H 4 must be attributed to a partial loss of this histone during the H2SOI, extraction and the subsequent acetone washes of the acid-soluble protein This band is in fact reproduci31y present with pellet. relative to the other three, when the same intensity, total egg nuclear proteins are analyzed on similar gels. Proteins with the same electrophoretic mobility in and therefore with the same SDS of typical histones, are thus present in sea urchin egg molecular weight, Presumably, these proteins, chromatin. or at least II 2A and II 33, are specific histone variants, given their altered mobility in any gel system in the absence of SDS (Carroll and Ozaki, 1375; our unp. ohs.). The presence of atypical histones alone however may not be sufficient to account for the unusual properties of egg chromatin. First of all, egg histones are not significantly different from the calf thymus ones, at

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1;:”

A Fig. 3. Electrophoretic on SDS - polyacrylamide proteins from unfertilized thymus histones. C) The four main histones

6 pattern of nuclear proteins gel. A) Acid - soluble egg nuclei. B) calf Nuclear proteins from sperm. are indicated.

least in terms of molecular weight. Moreover, typical nucleosome particles are present in sperms (Fig. Za), even though sperm II 2E and probably H 3A are different from the somatic ones, not only in primary structure but also in molecular weight (Fig. 3C) (Easton and 1972; Johnson et al., 1973). Similarly, nuChalkley, cleosomes with normal electrophoretic behaviour can be prepared from sea urchin embryos at very early stages (our unp. obs.), when the so called C S (2 - 1( cells)

Cell Biology

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proteins replace the histones in the chromatin(Newrock 1977).Fi&A shows however a highly complex patet al., ternThe very large number of basic, non-histone nuclear proteins in mature eggs is rather surprising, given the very low metabolic activity of these cells. Whether they play any structural role in egg chromatin is still totally unknown. However, it is not unreasonable to suppose that they may be somehow responsible for the great resistance of egg chromatin DNA to enzymatic degradation and our failure to detect typical nucleosomal particles. Conclusion Two main features characterize the mature sea urespecially reDNA is chin egg chromatin, i.e., its sistant to nuclease attack and nucleoprotein particles typical of nucleowith the electrophoretic mobility following micrococcal nuclease somes are not released, digestion. Preliminary experiments indicate that at least part of the DNA of the unfertilized egg chromatin is cut into typical fragments, although no nucleosomal pattern is generated upon electrophoresis of the digested chrothus suggesting that some change in the egg matin, chromatin structure holds the DNA fragments together after nuclease treatment of the chromatin. These unusual properties of mature egg chromatin are not observed in either sperms or growing oocytes, before meiotic division, and may be related to the unique differentiation process which leads to egg maturation. Although DNA replication and transcription are repressed in eggs, these cells retain in fact the potentiality to resume full metabolic activity within a very short period of time. Furthemore, no external factors are really needed by the egg, which can be activated not only by fertilization but also by a variety of chemical or mechanical stimulations. It is then conceivable that some sort of remodeling of the oocyte genome takes place at the end of the maturation process such that DNA replication and transcription are prevented in the absence of proper stimulation. Although chromosomal proteins are known to be synthesized by growing oocytes (Adamson and Woodland,l97&; Cognetti et al., 1974; 1977), no investigation has yet been performed to examine the possibility that these proteins are directly incorporated into the oocyte Experiments are currently in progress in our chromatin. aimed to better characterize the chromatin laboratory, composition and organization in mature eggs, as compared to oocytes, in the hope to understand whether genome remodeling is indeed a relevant aspect of the more

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general phenomenon of oocyte inactivation and subseFurther investigations will quent egg reactivation. also be devoted to establish whether the unusual properties of egg chromatin, here described for sea urchin, are also shared by the mature egg chromatin from other organisms. Acknowledgements We wish to thank Prof. G. Giudice for continuous and helpful discussions and for critically reading the manuscript. We thank Mr. D. Cascino for his excellent technical assistance. This work was supported in part by the Italian Research Council (CNR) (R esearch Project on Biology of Reproduction, contract no. 79.01155.85). References E.D. and Woodland, H.R. Adamson, sis in early amphibian development: syntheses are not co-ordinated. Biology, 88, 263 - 285.

(1974) Histone synthehistone and DNA Journal of Molecular

Carroll, A.G. and Ozaki, H. (1979) Changes in histones of the sea urchin Strongylocentrotus at fertilization. Experimental Cell Research, 307 - 315. Cognetti, thesis of Biochimica

G., Spinelli, histones during et Biophysics

G.

and Vivoli, sea urchin Acta, 349,

the purpuratus 119,

A. (1974) oogenesis. 447 - ltfi5.

Syn-

Cognetti, G., Platz, R.D., Iieistrich, (1977) Studies on protein Liegro, I. I-Synthesis of sea urchin oogenesis. Cell Differentiation, 5, 283 - 291.

14.L. and Di synthesis during histone I" 2b.

Cohen, specific genesis

A. (1975) Stage during embryo190, 794 - 997.

L.H., Newrock, K.M., switches in histone of the sea urchin.

Zweidler, synthesis Science,

De Petrocellis, B. and Parisi, E. (1972) Changes alkaline deoxyribonuclease activity in sea urchin dxperimental Cell during embryonic development. Research, 73, 496 - 500.

in

Color reactions of nucleic acid comDische, 2. (1955) and J.N. Davidson (eds.). The ponents. In: E. Chargaff Nucleic Acids. Ch. 9, pp. 285 - 305. Academic Press New York. Inc.,

Cell Biology

Zaston,

International

II.

electrophoretic and sperm itesearch,

Reports, Vol. 4, No. 2, February

1980

209

and of 72,

Chalkley, R. (1972) High-resolution analysis of the histones from Arbacia punctulata. Experimental 5CZ

-

embryos Cell

508.

L.3. and Ozaki, II. (1973) Wans, unfertilized sea urchin eggs. Research, 79, 228 - 230.

Nuclear Xxperimental

histones Cell

of

Deoxyribonucleic acid. In: DevelopGiudice, G. (lT73) mental Biology of the Sea Urchin 3mbryo. Ch. 9, New York and London. press, PP. 200 - ,204. .Icademic (1973) Chromatin subL.B. of chromatin DNA at regularly deox)7ribonuclease. Research Communications,

Hewish, D.11. and Burgoyne, The digestion structure. spaced sites by a nuclear I3iochemical and 5iophysical 52, rob - 510. Hnilica, L.S. and analysis Experimental Johnson, (13711)

histones.

and Johnson, A.1IT. of nuclear proteins Cell Zesearch, 63,

A.iJ., .iilhelm, The composition 3iochimica

(1970)

in 261

J.A., !iiard, of sea urchin et niophysica

Fractionation sea urchin embryos. - 270. D.N. and Hni1ica;L.S sperm and embryo 1~.IO-147. Acta, 2yjfj,

L.D. and Ifassarman, P.bi. (l('77) DevelopmentKeichline, al study of the structure of sea urchin embryo and sperm chromatin using micrococcal nuclease. niochimica et Eiophysica hcta, (775, 13? 151. Kornberg, Review of

!c.D. (1<‘$7) Eiochemi~str;~,

Structure k6,

of ij31

-

chromatin.

-4nnual

yTj/~.

Newrock, ‘-:.I,;., hlfzgeme, C.D., Nardi, 1z.V. and (17!77) ITistone changes during chromatin Cohen, L.H. remodeling in embryogenesis. Cold Spring Harbor Symposia on !)uantitative Hiology, 'C2, I*? 1 - 931. Parisi, 2:. and De I)etrocellis, 5. (1972) properties a deoxyribonuclease from a nuclear extract of Paracentrotus lividus embryos. Biochemical and 5iophysical Research Communications, Ilo, 706 - 71:~.

Riley , D. and Weintraub, digested to repeats of Cell, 113, 281 - 233. Sconzo, somal

G. and Ciudice, RNA in sea urchin

10

H. ( 1978) bases by

G. (1971) embryos.

Xucleosomal

Sxonuclease

synthesis V.Further

DNA is iIT.

of riboevidence

of

Cell Biology International Reports, Vol. 4, No. 2, February 1980

210

for an stage.

activation Biochimica

following the et Biophysics

Sconzo, G., Bono, A., (1972) Studies on sea RNA during oogenesis. 95 - 100. Spadafora, C. and ture of sea urchin FEBS Letters, 57,

blastula 25'1, It47

-

G. (1975) chromatin:

The subunit a kinetic

Cooperative Weintraub, E-1. (1976) during chromosome replication in Cell, 9, 415) - 422. exhimide. Yalter, Weintraub, H., K. Ilistones H2A, I-I2R, II3 and in solutions of high salt.

25th

October

451.

1979

of 72,

strucapproach.

82.

Spadafora, C., Bellard, 1!., Compton, J.L., lengths in chromatins (1976) The DNA repeat urchin sperm and gastrula cells are markedly FEBS Letters, 69, 281 - ,285.

Received:

-

Albanese, I. and Giudice, G. urchin oocytes. Il.Synthesis Experimental Cell Research,

Geraci, sperm 79

hatching hcta,

Chambon, I-'. from sea different.

alignment of the presence

and Van Ii4 form Cell,

Accepted:

nu of

bodies cyclo-

Lente, F. (1975) a tetrameric complex 6, 85 - 110.

5th November

1979