THE REGULATION OF DNA SYNTHESIS AND MITOSIS IN … · 2005-08-19 · THE REGULATION OF DNA SYNTHESIS AND MITOSIS IN MULTINUCLEATE FROG EGGS C.F.GRAHAM Zoology Department, Parks Road,

J. Cell Sci. i, 363-374 (1966) 363

Printed in Great Britain

THE REGULATION OF DNA SYNTHESIS AND

MITOSIS IN MULTINUCLEATE FROG EGGS

C.F.GRAHAMZoology Department, Parks Road, Oxford

SUMMARY

The timing of DNA synthesis and mitosis in normal eggs, in activated eggs, and in eggs con-taining several accessory nuclei has been studied in Xenopus. DNA synthesis was followed byinjecting PHjthymidine into the egg and detecting its subsequent incorporation into DNA byautoradiography.

It was found that the accessory nuclei which were introduced into the egg during differentphases of the cell cycle incorporated label and entered mitosis in synchrony with the residentegg and sperm nuclei if the introduced nuclei were released from the intact sperm. In eggsactivated by pricking, the egg nucleus had synthesized DNA at a time when it would have doneso during normal fertilization.

INTRODUCTION

The extent to which cytoplasmic factors can regulate DNA synthesis has beeninvestigated in bacteria by studying the synchrony of DNA synthesis in two geneticelements, the episome and the bacterial chromosome, which share a common cyto-plasmic environment (Jacob, Brenner & Cuzin, 1963). The regulation of DNA syn-thesis and mitosis in metazoan nuclei can be studied by an essentially similar procedure.In this work nuclei which were all in the Gx phase were introduced into egg cells indifferent phases of the cell cycle. This makes it possible to determine whether the intro-duced nuclei and the resident egg and sperm nuclei synthesize DNA and entermitosis in synchrony with each other. The results show whether the capacity of theegg cytoplasm to induce DNA synthesis and mitosis in the introduced nuclei changesas the egg passes through the cell cycle.

Accessory sperm were introduced into fertilized eggs of Xenopus laevis at differenttimes after fertilization, when the egg was in different phases of the cell cycle. Thesubsequent induction of DNA synthesis and mitosis in these accessory nuclei wasstudied by following the incorporation of pH]thymidine using autoradiography.

MATERIALS AND METHODS

Ovulation and artificial fertilization

Ovulation of Xenopus laevis (Daudin) was induced by the injection of gonadotrophichormones (Elsdale, Gurdon & Fischberg, i960). Newly laid eggs were artificiallyfertilized in a macerated testis suspension (Gurdon, 1966). The eggs were cultured inmodified Barth X saline solution (Elsdale et al., i960) at 200 ± 1 °C.

364 C. F. GraJiam

Micro-injection and introduction of accessory nuclei

Radioactive thymidine (3H-TdR) was injected into eggs using a micropipetteattached to a glass microsyringe (Elsdale et al, i960). Prior to injection each egg wasirradiated with ultraviolet light at the vegetal pole for 20-30 sec. This treatmentweakens the jelly coat and facilitates the penetration of the injection pipette. Exposure ofthe vegetal pole of the egg to ultraviolet light was found not to interfere with the sub-sequent development of the egg. Each egg was injected with 5 x io"4 ml of 3H-TdR in4/5 modified Barth X. This volume contained 50 m/ic of 3H-TdR (tritiated thymidine14-8 curies/ffiM from the Radiochemical Centre, Amersham, England). Tritiatedthymidine is a specific precursor of DNA in frog embryos (Tencer, 1961).

Sperm were introduced into the egg by covering the vegetal pole of the egg, immedi-ately after it had been exposed to ultraviolet light, with a sperm suspension. Whenlabel was injected into the eggs through this suspension, using a micropipette asdescribed above, several sperm were later found in egg cytoplasm. This operation iscalled 'the introduction of accessory nuclei' to distinguish it from 'the injection ofsperm' in which a sperm suspension is taken up into the micropipette and injecteddirectly into the egg.

Preparation of autoradiographs

The eggs were fixed in Perenyi's fixative and sectioned at 6 fi in paraffin wax. Theywere covered with Kodak AR10 Stripping film and this was exposed to the radio-activity for 1 week at 4 °C. The sections were then stained through the film withMayer's haemalum.

RESULTS

The behaviour of egg and sperm nuclei after fertilization

The following time-table of events that take place after sperm penetration of theegg is based on the development of 3 batches of eggs from different females which werereared at 20 °C. There is slight variation in the timing of these events in batches ofeggs laid by different females.

The egg nucleus does not complete the second meiotic division until about 15 minafter fertilization. At 20 min after fertilization the second polar body is extruded fromthe egg at the animal pole, and the egg nucleus begins to swell as it moves towards thecentre of the egg. At 10 min after fertilization the sperm nucleus is found just belowthe cortex in the animal part of the egg. The condensed chromatin of the intact spermbecomes dispersed at this time and a swollen nucleus is found at 20 min post fertiliza-tion. The two nuclei move towards the centre of the egg and lie adjacent to each other,just above the egg equator, at 40 min post fertilization. Each nucleus is still surroundedby an intact nuclear membrane and the contents of the 2 nuclei are not confluent untilbetween 60 and 70 min post fertilization when the membranes break down and thezygote nucleus enters mitosis. At 70 min post fertilization there is a telophase nucleuson each side of the cleavage furrow which is then clearly seen for the first time. Thesetelophase nuclei consist of groups of vesicles with very dispersed chromatin and

Multinucleate frog eggs 365

exactly resemble the telophase nuclei described in cleaving embryos of Bufo (Bragg,1938). Between 80 and 90 min post fertilization these 2 daughter nuclei of the zygotenucleus each enter the second mitosis at the same time.

Incorporation of3H-TdR after fertilization

The time at which the egg and sperm nuclei begin to incorporate 3H-TdR wasstudied by injecting the label into eggs about 5 min after fertilization. The eggs weresectioned at 20, 30, 40, 50, and 60 min post fertilization, and the numbers of labelledcells were counted at each time (Table 1).

At 20 min post fertilization 50% of the nuclei were labelled; at 30 min post ferti-lization, and at all subsequent times, all the nuclei were labelled (Figs. 2, 3). DNAsynthesis begins in both nuclei between 20 and 30 min post fertilization. The absenceof detectable label in some nuclei at 20 min post fertilization might suggest that theegg nucleus goes through a post-meiotic, pre-DNA-synthesis phase equivalent to theG! phase of the normal cell cycle; it is equally possible that insufficient 3H-TdR waspresent to label a nucleus which was synthesizing DNA very slowly. The sperm nucleusremains in a post-meiotic, pre-DNA-synthesis phase from the completion of gameto-genesis in the male testis until fertilization.

The time at which the egg and sperm nuclei stop incorporating 3H-TdR at the endof the first S phase and the time at which they start to do so again at the end of thefirst mitosis was studied by injecting label into the egg at different times after fertiliza-tion and fixing the eggs 10 min after each injection (Table 2 and Fig. ib). As in theprevious experiment not all nuclei exposed to label between 10 and 20 min afterfertilization incorporate detectable amounts of radioactivity. All nuclei are labelledbetween 20 and 30 min post fertilization (Figs. 4, 5), and at 30 min post fertilizationsome nuclei have stopped incorporating 3H-TdR. After injection at 40 and 50 minpost fertilization no nuclei are labelled. Therefore the 2 pronuclei either have a post-DNA-synthesis, pre-mitosis phase equivalent to the G2 phase or they synthesizeDNA very much more slowly at the end of the S phase and this cannot be detected.

The two daughter nuclei of the zygote nucleus start to incorporate label when theyare in telophase after the first mitotic division, that is at 60—70 min after fertilization.At the end of the second mitosis at 90 min, all nuclei which are not in the metaphaseand the anaphase stages of this division have incorporated label within the preceding10 min. Therefore after the first mitosis there is no Gx phase and DNA synthesis infact begins before this mitosis is complete. After the second mitosis the Gx phase isvery short or absent.

Table 1. The incorporation of 3H-TdR into the egg and sperm nucleus ofX. laevis after injection of label at about 5 min post fertilization

Time fixed post-fertilization

(min)

Number of eggsPercentage of nuclei labelled

2 0

450

3O

5100

4 0

6100

5°

6100

6 0

SICO

366 C. F. Graliam

100

50

0

% 100

50

0

100

50

(b) Normal eggs

(c) Eggs with extra nuclei

(d) Eggs with extra nuclei

Introductionof sperm

v

ln|ectionof label

10 20 30 40 50 60 70 90

Minutes post fertilization

Fig. i. The labelling of nuclei after (b) normal fertilization; (c) the introduction ofaccessory nuclei io min prior to fixation; and (d) the introduction of accessory nucleiat 40 min post fertilization. In each case the label was injected into the egg 10 minbefore the percentage of labelled interphase nuclei was counted and plotted on thegraph (solid blocks). The percentages of unlabelled interphase nuclei are shown byempty blocks and the percentages of nuclei in mitosis by stippled blocks. The figures arefrom Table 2. The appearance and behaviour of the resident nuclei during normalfertilization are shown in (a).

Introduction and injection of accessory sperm nuclei into fertilized egg cytoplasm

In order to find out how a nucleus behaves when it is exposed to the cytoplasm of acell in different phases of the cell cycle, it is necessary to introduce nuclei which are inexactly the same cell-cycle phase. Sperm nuclei are all in the G2 phase of the cellcycle and they were introduced into the eggs at different times after fertilization. Thetechnique of introducing sperm into the egg consisted of exposing the vegetal poleof the egg to ultraviolet light and then covering this region with a sperm suspension(see Methods). If label was injected into the egg through this suspension then betweenone and 10 accessory sperm nuclei were found in each egg 10 min later. It was notpossible to distinguish these nuclei from the resident nuclei (one egg and one spermnucleus) when the egg was injected at any time up to 50 min post fertilization. Theaccessory nuclei swell and appear exactly the same as the resident nuclei (Fig. 6). If

Multinucleate frog eggs 367

eggs were injected at any time after 60 min post fertilization, the sperm which werefound in the egg cytoplasm were unswollen and retained the typical twisted S-shapeof the intact sperm. The nuclei of these intact sperm appeared not to have been ex-posed to egg cytoplasm and they were therefore unlikely to show the response of thenucleus to the state of the cytoplasm. In order to study this response after 60 minpost fertilization, sperm were introduced into eggs 40 min post fertilization. At thistime nuclei are released from sperm as in normal fertilization and their behaviour couldthen be observed at 70 min post fertilization.

The swollen accessory nuclei found in these experiments are probably derived fromsperm which were previously embedded in the jelly coats and which were probablypushed into the eggs during the injection of label. No accessory sperm nuclei werefound in 10 unfertilized eggs which were activated by pricking with a micropipetteand this shows that these nuclei are unlikely to result from the failure of the egg todiscard the polar body or to a division of the egg nucleus. No accessory sperm nucleiwere found in 10 uninjected eggs and this shows that polyspermy is not common inX. laevis. Sperm which are injected directly into eggs and which are not in pro-longed contact with the jelly coats behave differently. If between 20 and 30 sperm areinjected into the eggs just after fertilization, none are swollen 20 min later, although theresident egg nucleus does swell in this time. The sperm at this time appear intact. Thissuggests that there is some reaction between the sperm and the jelly coats such that thesperm nucleus is later released, soon after the entry of the sperm into egg cytoplasm.This reaction must become ineffective at about 60 min post fertilization since spermintroduced after this time do not swell in egg cytoplasm. However, such contact be-tween the sperm and the jelly coats is not essential for the eventual partial swelling ofthe sperm; if sperm are injected into eggs just after fertilization, then 60 min laterabout 52% have begun to swell (100 sperm scored). These sperm nuclei have onlyswollen to about i/2Oth of the normal volume of the male pronucleus.

Incorporation of 3H-TdR by accessory sperm nuclei

The time at which accessory sperm nuclei start and stop incorporating 8H-TdR wasstudied by simultaneously introducing sperm and injecting label into the eggs atdifferent times after fertilization. The eggs were fixed at 10 min after each injection.In each egg all the nuclei (with the exception of one partially swollen accessory spermnucleus in one egg) behaved in exactly the same way. The accessory sperm nucleitherefore could not be distinguished from the resident nuclei by their labelling be-haviour and all nuclei are scored together in Table 2. At times when the residentnuclei incorporate label during normal fertilization (between 20 and 40 min postfertilization) a similar percentage of accessory nuclei also do so (Fig. 1 c). All accessorysperm nuclei which had been introduced into the eggs at 20 min post fertilization hadincorporated label 10min later; this shows that sperm nuclei can swell and be inducedto synthesize DNA faster than in normal fertilization when only a few nuclei arelabelled at 20 min after the entry of the sperm into egg cytoplasm. In contrast, spermintroduced into the egg when the resident nuclei had stopped incorporating label, at40 and 50 min post fertilization, were unlabelled 10 min later. The accessory sperm

24 Cell Sci. 1

368 C. F. Graham

nuclei therefore only synthesize DNA at the times when the resident nuclei are in theprocess of doing so.

The accessory sperm nuclei can be induced to enter mitosis if they are introducedinto the egg when the resident nucleus is in mitosis. Accessory sperm nuclei introducedinto the egg at 50 min post fertilization are found 10 min later with their chromosomeson a spindle. These nuclei in mitosis are unlabelled which shows that haploid nucleican be induced to enter mitosis without prior DNA synthesis.

In order to study the behaviour of accessory sperm nuclei after the first mitosis itwas necessary to introduce the nuclei into the egg at 40 min post fertilization (seeprevious section). Label was injected into the eggs containing these nuclei at 50 minpost fertilization and the eggs were fixed 10 min later (Fig. 1 d). At this time theaccessory nuclei were found in pairs in the telophase of mitosis (Fig. 7). These nucleihad presumably divided without prior DNA synthesis since they were only in the eggat times when the resident nucleus does not incorporate label. However, at 70 minpost fertilization they were all labelled and they were therefore incorporating 3H-TdRin synchrony with the 2 daughter nuclei of the zygote nucleus (compare Fig. 1 b and d).

The accessory nuclei always behave in exactly the same way as the resident nucleiwithin 10 min of their introduction into egg cytoplasm. This shows that the egg cyto-plasm changes in state as the egg passes through the cell cycle and that it regulates thebehaviour of accessory nuclei. This regulation only occurs if the sperm nucleus is re-leased from the sperm and injected sperm which remain intact do not synthesize DNA.Sperm injected at fertilization were unswollen and unlabelled 20 min later; 60 minlater about 30 % (100 nuclei scored) of the partially swollen sperm nuclei were labelled.

Incorporation ofzH-TdR by the egg nucleus after activation

In amphibia it is rare for activated eggs to develop parthenogenetically (Tyler,1955) and parthenogenesis has never been reported in X. laevis. This failure of theegg nucleus to support normal development has, in part, been attributed to the absenceof viable centrioles in the unfertilized egg (Fankhauser, 1937) and the egg nucleus isnever found on a mitotic spindle in activated eggs of Xenopus. In order to find out ifthese egg nuclei were unable to synthesize DNA, label was injected into 10 unfertilizedeggs. The penetration of an egg in the course of injection activates the egg and 60 minlater the swollen egg nucleus is found just above the equator of the egg (Fig. 8). Eachof the 10 egg nuclei was labelled at this time (Fig. 9), and this shows that the inabilityof the egg nucleus to support further gynogenetic haploid development is not due to itsfailure to start DNA synthesis. This result also demonstrates that the factor inducingmitosis in the egg nuclei is not the same as that which induces DNA synthesis.

DISCUSSION

The timing of DNA synthesis after normal fertilization

In normal fertilization, the egg and the sperm nucleus of X. laevis begin to syn-thesize DNA at the same time, that is 10-20 min post fertilization. No further in-corporation of label is observed after they come to lie adjacent to each other at the

Multinucleate frog eggs 369

Table 2. Incorporation of 3H- TdR by resident and accessory nuclei ineggs injected with label 10 min prior to fixation

Time of 10 min pulselabel (min after

fertilization).

Number of nucleiper egg

Number of eggsTotal number of nucleifound

Percentage labelledPercentage of inter-phase nuclei labelled

Time of 10 min pulselabel (min after

fertilization)

10-20

<3 >2

2 24 6

25 6725 67

60-70

20-30

r ^

<3 >2

7 313 19

100 100100 100

70-80AI \

30-40A

r \<3 >2

S S9 34

67 6267 62

80-90A

40-50( \

<3 >2

6 49 !9

0 00 0

Pre-injected60-70*

50-60t

< 3

58

0

0

> 2

31 2

0

0

Number of nuclei <3 > 2 <3 >2 <5 >4 <3 >2per egg

Number of eggs 6 — 7 — 8 — 1 9Total number of nuclei 9 — 10 — 19 — 2 68found

Percentage labelled 100 — 50 — 63 — 50 98Percentage of inter- ioo — ioo •— ioo — ioo 100phase nuclei labelled

• The last two columns (Pre-injected 60-70) show the behaviour of accessory nuclei intro-duced into the egg at 40 min and pulsed with label between 60 and 70 min.

As it was impossible to distinguish the accessory nuclei from the resident nuclei by mor-phology or labelling behaviour, the eggs are grouped into those containing the normal numberof nuclei (2 up to 80 min and then 4 at 90 min post fertilization) and those with more than thisnumber and which therefore contained accessory nuclei.

centre of the egg at about 40 min post fertilization. In the mouse there, is a lag of about11 h between penetration of the egg by the sperm and the uptake of label into theDNA of the 2 pronuclei which may occur before the nuclei lie beside each other(Sirlin & Edwards, 1959). In the sand dollar (Simmel & Karnofsky, 1961) and thesea urchin (Hinegardner, Rao & Feldman, 1964) DNA synthesis begins at.aboutthe time of nuclear fusion between 20 and 30 min post fertilization. In mammalsand in echinoderms the egg nucleus, as well as the sperm nucleus, has a sub-stantial post-meiotic, pre-DNA-synthesis phase. A similar phase may occur in thedevelopment of the egg nucleus of Xenopus but the absence of label in these nuclei,which was observed after the completion of the second maturation division, may bedue to the difficulty of detecting synthesis of small amounts of DNA with the auto-radiographic technique. It is certain that if DNA synthesis is occurring immediatelyafter fertilization then it does so at a much slower rate than at 30 min post fertilization.

Before the first mitosis and after the phase of intense DNA synthesis there is aperiod of 20 min during which the pronuclei incorporate no label in Xenopus. Boththe egg and the sperm nucleus, therefore, have a G2 phase, or are synthesizing only

24-2

370 C. F. Graham

traces of DNA at this time. After the first mitosis it was found that nuclei were eitherlabelled, or in mitosis and unlabelled, after they had been exposed to radioactivity for10 min. This situation persists in cleaving embryos up to 5 h after fertilization(Graham & Morgan, 1966). A G2 phase may be present after the first mitosis, but if soit must last considerably less than 10 min and therefore be undetected by the intervalsof observation which were used. In the sea urchin (Hinegardner et al., 1964) and in therat and the mouse (Dalq & Pasteels, 1955), there is a considerable G2 phase after thesynthesis of DNA in the 2 pronuclei and during the subsequent cleavage divisions.The only embryonic cells which have been conclusively shown to lack the G2 phaseare the giant neuroblast cells of the grasshopper embryo (Gaulden, 1956).

Following the first mitosis, the nuclei of Xenopus begin to incorporate label intelophase and the Gx phase is probably absent from all cells at least up to 5 h postfertilization (Graham & Morgan, 1966). The Gx phase is also absent from the cleavingembryos of the sea urchin (Hinegardner et al, 1964) and it is very short or absent in therat from the 2- to the 16-cell stage and in the mouse from the 4- to the 8-cell stage(Dalcq & Pasteels, 1955).

The release of the nucleus from the sperm after penetration of the egg

It was found that of the sperm introduced into the egg, only those which appearedto have been in contact with the jelly coats were able to release their nuclei soon afterthey had entered the cytoplasm of the egg. The jelly coats of the sea urchin or waterwhich has contained the jelly coats can induce an acrosome reaction in the sperm head(Dan, 1952; Afzelius & Murray, 1957). In amphibia it is impossible to fertilize eggsfrom which the jelly coats have been removed (Bataillon, 1919), and antibodies pre-pared against the jelly coats block fertilization even in eggs with intact jelly coats(Shivers & Metz, 1962). The experiments reported here suggest that a similar acti-vation of the sperm by the jelly coats occurs in Xenopus. However, sperm which hadbeen injected into eggs just after fertilization were partially swollen 60 min later and afew of the nuclei of these sperm synthesized DNA. This indicates that a small part ofthe jelly coats may have been pushed into the eggs during injection, or that egg cyto-plasm as well as jelly coats may possess the property of being able to activate sperm.

Cytoplasmic control of the timing of DNA synthesis and mitosis following fertilization

When nuclei which were all in the Gx phase were introduced into eggs in differentphases of the cell cycle they synthesized DNA and underwent mitosis in completesynchrony with the resident nuclei. For this to occur there must be specific signals inthe cytoplasm for the induction of DNA synthesis and the induction of mitosis. Thesesignals affect the whole cytoplasm of the egg since those accessory sperm nuclei whichwere introduced into the heavy yolky regions of the cytoplasm in the vegetal hemi-sphere were in complete synchrony with the resident nuclei in the animal hemisphere.The chemical reactions which either initiate DNA synthesis or block an inhibitor ofDNA synthesis (Jacob et al., 1963) must have the property of an extremely rapidswitch mechanism which acts throughout the egg cytoplasm. In other multinucleatecells the synchrony of DNA synthesis is not complete (Harris, Watkins, Schoefl &

Multinucleate frog eggs 371

Ford, 1966). It is probable that this asynchrony is due to the fact that the hetero-karyons, when they are formed, contain nuclei in different phases of the cell cycle. Ithas been found previously that a percentage of transplanted embryonic frog nucleiequal to the proportion of nuclei in the G2 phase are not induced to synthesize DNAafter 50 min in egg cytoplasm although the resident nucleus does so (Graham, Arms &Gurdon, 1966). The naturally occurring situations in which several nuclei share acommon cytoplasm show that DNA synthesis is usually synchronous; in the slimemould Physarum polycephalum nearly all the nuclei enter DNA synthesis together(Nygaard, Giittes & Rusch, i960). The ciliate heterotrichs, for instance Euplotes, areexceptional in that the macronucleus replicates its DNA at a different time in the cellcycle from the micronucleus (Prescott, Kimball & Carrier, 1962; McDonald, 1962).

The control mechanism for mitosis must also possess the property of a rapid switch.Most multinucleate cells have synchronous mitoses (reviewed by Mazia, 1961; Agrell,1963) and the many accessory nuclei in the polyspermic eggs of the newt show syn-chrony of an abortive mitosis (Fankhauser, 1955). When nuclei which are about toenter mitosis are transplanted into eggs in which the resident nucleus is in interphase,they enter mitosis only when the resident nucleus is ready to do so. This has beendemonstrated by the transplantation of nuclei from embryonic frog cells into eggcytoplasm (Graham et al, 1966) and by the transplantation of nuclei into the hetero-trich ciliate Stentor (de Terra, 1963). In this work it has been shown that accessorysperm nuclei can be induced to enter mitosis without prior DNA synthesis and itfollows that the stimulus for the initiation of mitosis is not dependent on the previousreplication of the genetic material. Taken together these results show that the nucleushas little autonomy in the control of DNA synthesis and mitosis; the state of the cyto-plasm controls these functions precisely. The dominant role of the cytoplasm duringearly cleavage is also shown by the study of sea urchin androgenetic haploid hybrids.In these the time of the first cleavage division is the same as that of the egg species, andnot the same as the sperm-producing species (Moore, 1933)-

The author would like to thank Dr J. B. Gurdon, Mr P. J. Ford and Miss K. Arms for theiruseful comments on this work. The financial support of the Medical Research Council isgratefully acknowledged.

REFERENCESAFZELIUS, B. A. & MURRAY, A. (1957). The acrosomal reaction of spermatozoa during fertiliza-

tion. Expl Cell Res. 12, 325-327.AGRELL, I. (1963). Mitotic gradients in the early insect embryo. Archiv. Zool. 15, 143-149.BATAILLON, E. (1919). Analyse de l'activation par la technique des oeufs nus et la polyspermie

exp^rimentale chez les batrachiens. Attnh Sci. nat. Zool. (10), 2, 1-38.BRAGG, A. N. (1938). The organization of the early embryo of Bufo cognatus as revealed es-

pecially by the mitotic index. Z. Zellforsch. mikrosk. Anat. 28, 154-178.DALCQ, A. & PASTEELS, J. (1955). Determination photome'trique de la teneur relative en DNA

des noyaux dans les ceufs en segmentation du rat et de la souris. Expl Cell Res. (Suppl. 3),72-97-

DAN, J. C. (1952). Studies on the acrosome. 1. Reaction to egg water and other stimuli. Biol.Bull. mar. biol. Lab., Woods Hole, 103, 54-66.

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ELSDALE, T. R., GURDON, J. B. & FISCHBERG, M. (i960). A description of the technique fornuclear transplant in Xenopus lands. J. Embryol. exp. Morph. 8, 437-444.

FANKHAUSER, G. (1937). The development of fragments of the fertilized Triton egg with theegg nucleus alone ('gynomerogony')- J- exp- Zool. 15, 413-469.

FANKHAUSER, G. (1955). The role of nucleus and cytoplasm. In Analysis of Development (ed.B. H. Willier, P. A. Weiss & V. Hamburger), pp. 126-150. New York: Saunders.

GAULDEN, M. E. (1956). DNA synthesis and X-ray effects at different mitotic stages in thegrasshopper neuroblast. Genetics, Princeton 41, 645.

GRAHAM, C. F., ARMS, K. & GURDON, J. B. (1966). The induction of DNA synthesis by frogegg cytoplasm. Devi Biol. (in the press).

GRAHAM, C. F. & MORGAN, R. M. (1966). Changes in the cell cycle during the early embryonicdevelopment of Xenopus laevis. Devi Biol. (in the press).

GURDON, J. B. (1966). Xenopus laevis, the South African Clawed Frog. In Techniques in De-velopmental Biology (ed. F. Wilt & N. K. Wessells). New York: Crowell.

HARRIS, H., WATKINS, J. F., SCHOEFL, G. I. & FORD, C. E. (1966). Artificial heterokaryons ofanimal cells from different species. J. Cell Set 1, 1-30.

HINEGARDNER, R. T., RAO, B. & FELDMAN, B. E. (1964). The DNA synthesis period duringearly development of the sea urchin. Expl Cell Res. 36, 53-61.

JACOB, F., BRENNER, S. & CUZIN, F. (1963). On the regulation of DNA replication in bacteria.Cold Spring Harb. Symp. quant. Biol. 28, 329-348.

MCDONALD, B. B. (1962). Synthesis of deoxyribonucleic acid by micro- and macro-nuclei ofTetrahymena pyriformis. J. Cell Biol. 13, 193-203.

MAZIA, D. (1961). Mitosis and the physiology of cell division. In The Cell, vol. 3 (ed. J. Brachet& A. E. Mirsky), pp. 77-412. New York and London: Academic Press.

MOORE, A. R. (1933). Is cleavage rate a function of the cytoplasm or of the nucleus? J. exp.Biol. 10, 230-236.

NYGAARD, O. F., GOTTES, S. & RUSCH, H. P. (i960). Nucleic acid metabolism in a slime mouldwith synchronous mitosis. Biochim. biophys. Acta 38, 298-306.

PRESCOTT, D. M., KIMBALL, R. F. & CARRIER, R. F. (1962). Comparison between the timing ofmicronuclear and macronuclear DNA synthesis in Euplotes eurystromes. J. Cell Biol. 13,175-176.

SHIVERS, C. A. & METZ, C. B. (1962). Inhibition of fertilization in frog eggs by univalent frag-ments of rabbit antibody. Proc. Soc. exp. Biol. Med. n o , 385-387.

SIMMEL, E. K. & KARNOFSKY, D. A. (1961). Observations on the uptake of tritiated thymidine inthe pronuclei of fertilized sand dollar embryos. J. biophys. biochem. Cytol. 10, 59—65.

SIRLIN, J. L. & EDWARDS, R. G. (1959). The timing of DNA synthesis in ovarian oocyte nucleiand pronuclei of the mouse. Expl Cell Res. 18, 190—194.

TENCER, R. (1961). Incorporation of tritium labelled thymidine in Bufo^xRana temporana <Jhybrid embryos. Nature, Lond. 190, 100-101.

TERRA, N. DE (i960). A study of nucleo- cytoplasmic interactions during cell division in Stentorcoeruleus. Expl Cell Res. ai, 41-48.

TYLER, A. (1955). Gametogenesis, fertilization and parthenogenesis. In Analysis of Develop-ment (ed. B. H. Willier, P. A. Weiss & V. Hamburger), pp. 175-212. New York: Saunders.

(Received 16 March 1966)

Multinucleate frog eggs 373

For figures see following page.

374 C. F. Graham

Figs. 2, 3. An autoradiograph showing the egg and the sperm nuclei beside each otherat the centre of the egg at 60 min post fertilization. 3H-TdR was injected into the eggimmediately after fertilization and both nuclei are labelled (Fig. 3).Figs. 4, 5. The egg and sperm nuclei in an egg fixed at 30 min post fertilization(marked with arrows). 3H-TdR was injected into the egg at 20 min post fertilizationand the intense labelling of both nuclei shows that exposure to the radioactivity for10 min is sufficient to reveal DNA synthesis.Fig. 6. This egg was fixed at 40 min post fertilization and accessory sperm nuclei wereintroduced into it 10 min earlier. At the top of the photograph there are two nucleiwith vesicles which are presumably the resident egg and sperm nuclei. One othernucleus (shown in the bottom of the photograph) was found in another section and isprobably an introduced accessory sperm nucleus, which has swollen to the same extentas the resident nuclei.Fig. 7. Sperm were introduced into this egg at 40 min post fertilization and the eggwas fixed at 70 min, 10 min after the injection of label. It contained 6 different pairsof accessory sperm nuclei and this autoradiograph shows one pair which had com-pleted an abnormal mitosis. All the accessory nuclei were labelled at the same time as theresident nuclei.Figs. 8, 9. The incorporation of 3H-TdR by the egg nucleus of an activated egg. Thenucleus is found at the centre of the egg at 60 min post activation and injection oflabel (Fig. 8) and it is heavily labelled (Fig. 9).

Journal of Cell Science, Vol. i, No. 3

50//

• • " • • . %

50 /<

5

'A

.*/*.

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C. F. GRAHAM (Facing p. 374)