induction of cleavage in nucleated and enucleated frog … · ovulation was induced by injecting...

J. Cell Sci. 31, 117-135 (i978) 117Printed in Great Britain © Company of Biologists Limited igyS

INDUCTION OF CLEAVAGE IN NUCLEATED

AND ENUCLEATED FROG EGGS BY

INJECTION OF ISOLATED SEA-URCHIN

MITOTIC APPARATUS

Y. MASUIDepartment of Zoology, University of Toronto, Toronto, Ontario

A. FORERDepartment of Biology, York University, Downsview, Ontario

AND A. M. ZIMMERMANDepartment of Zoology, University of Toronto, Toronto, Ontario, Canada

SUMMARYMitotic apparatus (MA) were isolated in glycerol-dimethylsulphoxide solution (MTME)

from zygotes of sea urchins {Strongylocentrotus purpuratus). Freshly isolated MA were storedin. 1/10 strength MTME for varying periods of time and were then injected into unfertilizedfrog (Rana pipiens) eggs. These injections induced 40-60 % of the recipient frog eggs toinitiate cleavage, resulting in the formation of blastula cell clusters. The cleavage-inducingactivity of MA stored in 1/10 MTME at room temperature decreased with time of storagein 1/10 strength MTME, and disappeared by about 6 h. There was no change in the ultra-structure of MA during storage.

MA isolated and stored in MTME at room temperature had a constant level of cleavage-inducing activity during the first 48 h of storage, but this activity slowly declined upon furtherstorage; almost no activity was left after 3 weeks.

MA isolated in hexylene glycol (HG) and immediately transferred into MTME werecompared with MA isolated in MTME; both MA had the same cleavage-inducing activityon the day of isolation, after which the MA isolated in HG quickly lost activity. On the otherhand, MA isolated and stored in HG had little cleavage-inducing activity when tested 3 hfollowing isolation.

Cleavage-inducing agent (CIA) isolated from frog brains induced cleavage and blastulaformation when injected into nucleated frog eggs, but had no such activity when injectedinto enucleated frog eggs. MA isolated in MTME induced cleavage and blastula formationin enucleated frog eggs as well as in nucleated frog eggs. Cytological examination revealedthat blastula cells which developed from MA-injected enucleated eggs contained Feulgen-negative nuclei, whereas cells which developed from CIA-injected nucleated eggs containedFeulgen-positive nuclei. These results suggest that sea-urchin nuclear materials participatein mitosis in frog eggs.

Isolated MA which had been stored in MTME for 3 weeks and which exhibited littlecleavage-inducing activity were injected together with frog brain CIA into either normal orenucleated eggs; normal recipient eggs cleaved with significantly higher frequencies (70%)than those injected with CIA alone (40 %). Furthermore, enucleated eggs injected with CIAalone failed to cleave, while those injected with MA and CIA together cleaved with significantfrequencies (overall 29 %). This result suggests a cooperative interaction between CIA andthe inactivated MA to restore the cleavage-inducing activity of MA.

n 8 Y. Masui, A. Forer and A. M. Zimmerman

INTRODUCTION

The mitotic apparatus (MA) is a transient cell structure which appears onlytemporarily in the cell, during mitosis, and chromosome movement seems to requirea functioning MA. The function of the MA appears to depend upon its structurallability, because during mitosis the MA must change its molecular organization aswell as its morphology, without losing its structural integrity. This unique propertyof the MA has presented difficulties in isolating functional MA. In order to isolatea functional MA two apparently contradictory conditions must be satisfied. First,the structure of the MA must be stabilized in order to protect it from disintegrationduring fractionation of the cell; second, and at the same time, the lability of itsmolecular organization should be preserved.

MA can be isolated to preserve their structure, but not as yet to preserve theirfunction. Since Mazia & Dan (1952) first isolated MA from sea-urchin zygotesusing cold ethanol and digitonin, several techniques to isolate MA from eggs ofmarine animals have been developed using various MA-stabilizing agents such asdithiodiglycol (Mazia, Mitchison, Medina & Harris, 1961), dithiodipropanol (Sakai,1966), hexanediol (Kane, 1962) or hexylene glycol (Kane, 1965). However, MAstabilized with these chemical agents have been found to have properties whichdiffer from those possessed by MA in vivo (summarized in Forer & Zimmerman,

1974)-Recent isolation methods preserve spindle lability better than earlier isolation

methods. These recent methods utilize different MA-stabilizing agents such astubulin (Rebhun, Rosenbaum, Lefebvre & Smith, 1974; Inoue, Borisy & Kiehart,1974), glycerol (Sakai & Kuriyama, 1974), or glycerol-dimethylsulphoxide (Forer &Zimmerman, 1974). MA isolated in these media were found to retain their structurallability to a certain degree. For instance, MA isolated in glycerol-dimethylsulphoxideare highly soluble in 0-5 M KC1 (Forer & Zimmerman, 1974) and retain labilebirefringence, which changes in response to high pressure or to low temperature,even after 2 weeks storage at room temperature (Forer & Zimmerman, 1976 a, b).

Functional MA are labile. If MA in vitro retain a labile molecular organizationthis may indicate that these MA might also function when the MA are placed in theproper environment. The capability of isolated MA to function might be tested byintroducing them into enucleated eggs. Amphibian eggs are very suitable for suchexperiments: not only are they large enough to manipulate, but also they containa cytoplasmic factor which maintains MA at metaphase until activation takes place(Masui & Markert, 1971; Masui, 1974; Meyerhof & Masui, 1977), and they permitnuclei from foreign species to continue to multiply until the eggs reach the blastulastage (Brun, 1973).

The present paper describes experiments in which MA isolated from sea-urchin(Strongylocentrotuspurpuratus) zygotes were transplanted into frog {Ranapipiens) eggs.The results indicate that isolated MA promote cleavage of the recipient eggs, possibly,as we have argued previously (Forer, Masui & Zimmerman, 1977), by their directparticipation in mitosis.

Sea-urchin MA induce frog egg cleavage 119

MATERIALS AND METHODS

Animals

Sea-urchin (Strongylocentrotus purpuratus) gametes were obtained as described elsewhere(Forer & Zimmerman, 1974, 1976a).

Frogs (Ratta pipiens), purchased from suppliers in Quebec, Vermont and Wisconsin duringthe fall, were kept in cold tapwater until use. Ovulation was induced by injecting one frogpituitary and 1 mg progesterone into each female. The injected frogs were kept at 18 °C for48 h until ovulation was completed.

Solutions

Two isolation media were used for isolation of MA. One, designated as microtubule mediumcontaining EGTA (MTME), consisted of 50% glycerol, 10% dimethylsulphoxide (DMSO),5 HIM MgClj, o-i HIM ethyleneglycol-bis N,N' tetraacetic acid (EGTA) and 10 mM phosphatebuffer (pH 6-8). The other, hexylene glycol (HG) solution (Kane, 1965), consisted of 1 Mhexylene glycol and 10 mM phosphate buffer (pH 6-o).

For isolation of cleavage-inducing agents (CIA) from frog brains, a medium (designatedas CIA medium), containing 02 M sucrose, 5 mM MgSO4, 1 mM EGTA and 01 mM disodiumguanidine triphosphate (GTP) was used following the procedure of Fraser (1971).

Isolation of MA

MA were isolated as previously described (Forer & Zimmerman, 1974). Zygotes fromwhich fertilization membranes had been removed with 1 M urea at pH 78 (Kane, 1962), werelysed at metaphase (75-85 min after insemination), using either MTME or HG. MA were8edimented by centrifugation (3000g, for 15 min), and then washed and stored. IsolatedMA were stored at room temperature either in MTME, MTME containing io"4 M N-N'-p-tosyl-L-lysine chloromethyl ketone HC1 (TLCK, Sigma) or HG. MA isolated in HG werekept at o CC until they were rinsed, after which they were transferred into MTME or HGand stored at room temperature, as described previously (Forer & Zimmerman, 1976a).

Cytoplasm was isolated from zygotes at a stage in which there were no MA: the usualisolation procedure was followed and a pellet of ' cytoplasmic contaminant' was obtained.

To determine the number of MA per ml, known volumes were placed between slide andcoverslip, and the total number of MA found was recorded. This was done on 1 slide each,by 2 separate observers, and the average value was taken as the MA concentration.

Isolation of cleavage-inducing agent (CIA)

Two frog brains were removed immediately following decapitation and were placed ina glass homogenizer containing 5 ml of ice-chilled CIA medium; the brains were then homo-genized by 6 strokes with a teflon pestle. The homogenate was centrifuged at 1020g for 10 minat o °C to remove large particles, after which CIA was sedimented from the supernatant bycentrifugation at i63oog for 15 min at o °C. CIA was rinsed once in cold CIA medium,suspended in 0-5 ml of the same medium and stored at o °C for up to 2 days.

Injection

For injection, MA or cytoplasmic particle suspensions stored in MTME were diluted10-fold with 10 mM phosphate buffer (pH 6-8) containing 1 mM MgCla (P-Mg); we designatethe final solution as 1/10 MTME. The diluted MA (or particles) were sedimented by centri-fugation at iooog for 10 min and the pellets (containing MA or cytoplasmic particles), re-suspended in one-fiftieth of the original volume of 1/10 MTME, were used for injection.Between the time of this final step and the injection into frog eggs the MA were kept at roomtemperature. The CIA suspensions, on the other hand, were kept at o CC.

For injection, frogs were squeezed, and the extruded frog eggs were put into a Petri dishcontaining 0025 M NaHsPO4; eggs were kept like this for 15 min prior to and following

120 Y. Masui, A. Forer and A. M. Zimmerman

injection to prevent them from being activated by the wounding which occurred duringinjection of MA (Ziegler & Masui, 1973). Injection was carried out in the NaHjPO, solutionusing a calibrated micropipette attached to a micromanipulator (Masui & Markert, 1971).Each egg received 20 nl of a suspension. Injected eggs were transferred into tapwaterand were activated by pricking with a glass needle 15 min after being transferred intotapwater.

Enucleation

Enucleation of frog eggs was carried out following the procedure described by Porter(1939). Briefly, a small cut was made across a black spot near the animal pole which appeared15-20 min after pricking eggs and a small amount of cytoplasm was squeezed out: thiscontained the nucleus.

Culture

All the injected eggs, both nucleated and enucleated, were cultured in tapwater at 18 °Cfor 16-24 h, after which time they were fixed for light-microscopic observation.

Microscopy

For electron-microscopic observation of isolated MA, glutaraldehyde was added to thesuspension of MA to give a final concentration of 2 % of glutaraldehyde. MA were processedfurther and examined with an electron microscope as described previously (Forer & Zimmer-man, 1976a).

For light-microscopic observation of frog eggs, the eggs were fixed in Smith's solution(Smith, 1912), embedded in paraffin blocks, and sectioned to give sections 8 fim thick. Sectionswere Feulgen-stained and counterstained with light green (Moore, 1940).

RESULTS

Cleavage initiation by MA isolated in MTME

Unfertilized eggs which were in tapwater or Ringer's solution at the time ofinjection always cytolysed shortly after injection with MTME, P-Mg, or CIAmedium. These solutions all contain 1 IIIM MgCl2, and this result corroborates theprevious report that solutions containing Mg are toxic to frog eggs (Fraser, 1971).However, injected eggs did not cytolyse if the quantity of the solution injected wasnot excessive, and if activation of recipient eggs was prevented. To meet theseconditions, each egg was injected with only 20 nl of solution, and MA were injectedinto eggs which were in NaH2PO4 solution, which prevents activation (Ziegler &Masui, 1973). None of the eggs injected under these conditions were activated, andmost could be activated by pricking with a glass needle 15 min after the eggs weretransferred into tapwater. In each activated egg the surface near the animal polebegan to pucker within a few hours after pricking, while in eggs which were notpricked following injection there were no surface changes.

We injected some eggs with 1/10 MTME only. These eggs puckered after theywere activated, but the pucker, once formed, always receded. Hence this is not thesame as a genuine cleavage furrow. Shortly after the pucker receded the cortexbegan to degenerate, showing an irregular pattern of depigmentation and vacuolization.

Other eggs were injected with MA suspensions. These eggs puckered after theywere activated, but in these cases the initial puckering was followed by furrow


formation. Some of these eggs ceased to form additional furrows during culturing,but the furrows did not recede once they were formed. Generally, the eggs werefound to have cleaved into a few or several blastomeres of various sizes. Variablenumbers of eggs continued further furrow formation until near the end of the

Fig. i. A total blastula formed following injection of MA freshly isolated in MTME.Cultured for 18 h at 18 °C. x 22 approx.Fig. 2. A partial blastula formed following injection. Cultured for 12 h at 18 °C.x 22 approx.

Table 1. Cleavage initiation by different samples of MA isolated in MTME

MAsample

3 MS4 MS7 MS

IO MS13 MSio MSOverallCytoplasmicsample

Duration

MTME, h

3-53-53-53-53-52 4

3-5

of storage inA

I / I O MTME,min

10-20

10-20

IO-2O

15-3520-30

12-22

25-55

TMr>IX O.

ofeggs

934 0

32126

2719

337

97

Cleaved eggs,1

TJl .. 1 11

1I

> 1/2 egg

22-6io-o

34'42 6 3

32'52 2 2

2 6 l

A

A

1 . •

< 1/2 egg

28-0

3 2 52 8 1

13-551 942-125-8

0//o

#

Totalf

5°"54 2 56 2 546-07 4 168-451-9*

* % of all eggs cleaved to form blastula cell clusters covering more or less than half theegg surface.

f % of all eggs undergoing cleavage.

culture period (18 h), at which point there were clusters of cells whose sizes werecomparable to those of normal blastula cells. The fraction of the egg which cellclusters occupied varied among individual eggs: in some cases cell clusters coveredthe whole surface of the egg to form a complete blastula (Fig. 1), but in other casescell clusters occupied only part of the egg, forming a partial blastula (Fig. 2). The


percentages of eggs which formed cell clusters of different sizes varied amongbatches of eggs as well as among eggs injected with different samples of isolated MA.Nonetheless, as summarized in Table i, there was no statistically significant variationin the cleavage percentage between different batches of MA-injected eggs when90% of the sibling eggs of the recipients developed normally after insemination.

Table 2. Relation between cleavage initiation and the number of MA injected

Duration of storage inA

f

MTME, h min

333353Overall

20-60

10-20

40-604O-553O-5O25-45

No nfMA/egg

0

4'47 7

14-0

1 4 0

2 1 0

•

nf

eggs

7232634744

126

3 1 2

t As in Table

Cleaved eggs, %A

A

> 1/2 egg < 1/2 egg

0

34'422-2

2 3 44 3 2

3 2 53 0 8

I .

O

2 8 l

4V425-51 3 6

1 3 523 1

Totalf

0

6 2 56674895684 6 0

5 3 9

MA preparations contain some cytoplasmic material as well as MA. In order todetermine which component induced cleavage, the MA or the cytoplasm, we injectedcytoplasmic particles into frog eggs. These injections did not initiate cleavage.Therefore it is the isolated MA, and not the cytoplasmic contamination that isresponsible for cleavage initiation.

In order to determine the optimum number of MA necessary to induce cleavage,the average number of MA injected into a single egg was calculated. As indicatedin Table 2, there was no correlation between the percentage of cleavage inductionand the number of MA injected (MA per egg) in a range from 4 to 20. Therefore, inthe subsequent experiments each egg was injected with 5-10 MA.

Effects of aging of isolated MA on its cleavage-initiating activity

MA freshly isolated in MTME were stored in 1/10 MTME for varying periodsof time prior to injection. The results are summarized in Fig. 3. We conclude fromthese data that the ability of MA to induce cleavage began to decline after the MAhad been stored for 2 h, and almost vanished after 6 h.

MA stored in 1/10 MTME, were studied electron microscopically. There was nosignificant alteration in their ultrastructure, even after 17 h of storage (Fig. 4). Thisimplies that the MA isolated in MTME were structurally stable even after thecleavage-inducing activity had been lost. Therefore we are unable to correlate theloss of the cleavage-inducing activity in MA stored in 1 / io MTME with ultrastructuralchanges observable by electron microscopy.

The decay of the cleavage-inducing activity of MA also occurred during storagein MTME, though much more slowly than in 1/10 MTME, as summarized in


Fig. 5 A. The MA cleavage-inducing activity decreased upon storage for 48 h ormore at room temperature, and very few eggs were induced to cleave by MA storedfor 3 weeks. This decay was not inhibited by the protease inhibitor TLCK, assummarized in Table 3.

70 r

CO

50

§> 40

30

20

10

154

337

i56

i

97

i69

92n60 120 180 240 300 360

Time in 1/10 MTME, min

Fig. 3. Decay of the cleavage-inducing activity of MA in diluted MTME. MAfreshly isolated in MTME were transferred into 1/10 MTME and injected intofrog eggs at various times after transfer. Ordinate: percentage of eggs undergoingcleavage. Shaded and blank columns represent the formation of blastula cell clusterswhich cover more than the egg hemisphere and those which cover less than thehemisphere, respectively. The number on top of each bar indicates the number ofeggs examined. Abscissa: storage time in min.

The stability of the cleavage-initiating activity was also studied using MA whichhad been first isolated in HG and immediately transferred to and stored in MTME(Fig. 5B). Such MA, when injected on the day of isolation and within 1-5 h afterbeing transferred into 1/10MTME, induced cleavage in a similar percentage ofrecipient eggs as those isolated in MTME, However, as seen in Fig. 5B, the sizes

Legend to Fig. 4, overleaf

Fig. 4. MA fixed at various times after dilution from MTME into 1/10 MTME, asseen electron microscopically. Fig. 4A-C are overviews of MA fixed at various timesafter dilution, while Fig. 4D-F are higher-magnification illustrations of the sameMA:A and D are of an MA fixed immediately upon dilution, B and E are of an MAfixed 1-5 h after dilution, and c and F are of an MA fixed 17 h after dilution. Micro-tubules and associated granular material seem identical in MA fixed at the 3 differenttimes (D-F) ; likewise, the microtubular distributions seem similar in the differentMA (A-C). B is a somewhat oblique section of the MA, whilst A and c are morenearly longitudinal. A-C, x 3150; D-F, X 36500.

Y. Masui, A. Forer and A. M. Zimmerman

B

M*

Fig. 4. For legend see previous page.

Sea-urchin MA induce frog egg cleavage

C E I. 31


50

40

CD

I 30IS

20

10

472 96

I i183

i161

48

2 3 4 5Time in MTME, days

21

50

40

IDCDCO

% 30CD

20

10

-303

I80

54

106

86

48

2 3 4 5Time in MTME, days

21

20

CDto

1 10(J

48 27 51

2 3 4Time in MTME, days

21

Fig. 5A-C. Decay of the cleavage-inducing activity of MA in MTME. MA wereinjected into frog eggs within i h of transfer into I / I O MTME. Ordinates: percentageof eggs undergoing cleavage. Shaded and blank columns represent the formation ofblastula cell clusters which cover more than the egg hemisphere and those whichcover less than the hemisphere, respectively. The number on top of each bar indicatesthe number of eggs examined. Abscissa: storage time in days, A, MA isolated andstored in MTME; B, MA isolated in HG and stored in MTME; c, MA isolatedand stored in HG.


of blastula cell clusters formed in these eggs were generally smaller than thoseinjected with MA isolated in MTME, and their cleavage-inducing activity was rapidlydecreased overnight, and almost disappeared by the third week.

Table 3. Effect of TLCK on decay of CIA activity

Samples

Duration

lvrTlVTPIVL 1 IVlLj,

daysA. No TLCK added

13 MS14 MS13 HMOverall

555

B. TLCK added13 MS14 MS13 HMOverall

555

of storage in

1/10

MTME, min

20-5020-5025-60

20-5020-5025-60

•, f As

±>iU. .Did

eggs >

108

53106

267

8858

163309

in Table 1.

Cleaved eggs,

bxuid ceil

1/2 egg

8-35'76-67 1

8 0I 2 - I

6-88 1

A

Cluster size

< 1/2 egg

1 8 522-61 4 1

1 7 6

1 3 620-71 1 7

1 3 9

0//o

,#

Totalf

2 6 92 8 320-82 4 7

2 1 63 2 81 8 42 2 0

Fig. 6. Abortive cleavage induced in an egg injected with MA freshly isolated andstored in HG for 3 h. The egg was cultured for 18 h at 18 °C. Arrow indicates ablastula cell cluster, x 16.Fig. 7. A total blastula formed by an enucleated egg following injection of MAfreshly isolated in MTME. Cultured for 18 h at 18 °C. The cytoplasmic exovateindicates the site of enucleation. x 20.

In contrast to the MA stored in MTME, MA isolated and stored in HG exhibitedonly weak cleavage-inducing activity, even within 3 h of isolation, and within 1 hfollowing the transfer into 1/10 MTME (Fig. 5 c). In this case, some of the activatedrecipients were found to form cleavage furrows in the first few hours followinginjection, but no more progression of cleavage was observed except for one casewhich formed a small blastula cell cluster (Fig. 6).

9-2


Effect of enucleation on cleavage initiation

There are 2 possible mechanisms whereby isolated MA could promote cleavage:the MA could proceed through its own mitosis in the egg cytoplasm and therebyproduce cleavage, or the MA could merely be a 'cleavage-inducing agent' whichdid not undergo mitosis itself but stimulated the host nucleus to undergo mitosis.To distinguish between these possibilities we removed the nucleus from the eggswhich were to be injected with isolated MA.

Table 4. Cleavage initiation by frog brain CIA in normal and enucleated eggs

Duration of storageCleaved eggs, %

Storage inCIA

Injected material min

No.of

nucleus eggs > 1/2 egg < 1/2 egg Total

Frog brain 20-6D . + 358

CIA Enucleation before activation20-63 . — 91

Enucleation after activation20-62 . — 77

274 134

6-6

408

77

26

Enucleation after activation

Sea-urchinMA

Storage inMTME,

h

3-5

3-5

3-5

2 4

1 2 0

Storage in1/10 MTME,

min

10-50

20-70

140-180

20-40

2O-6D

9853

169

87522 0

115

54269176

1 8 418-9

2 3 71 9 6

0

25-01 3 91 8 56 72 8

35734'O29-6

3 3 326-9io-o1 8 320-41 2 6

9 7

54-i5 2 8

5 3 35 2 92 6 9

3 5 °3 2 2

3 8 91 9 312-5

*, t As in Table 1. + , host nucleus was present; —, host nucleus was removed.

' Enucleation' was carried out using 2 different methods. In the first method, eggswere enucleated in 0-025 M NaH2PO4 before injection by slitting the cortex throughan indentation which indicates the site for expulsion of the first polar body (Ziegler& Masui, 1976). In the second method, eggs were activated, then enucleated througha slit made across a black dot which appears 15-20 min after activation, indicatingthe site for the second polar body (Porter, 1939). The effectiveness of these two'enucleation' methods was tested by injecting eggs with CIA from frog brains. As


seen in Table 4, injection of CIA into normal eggs induced cleavage, in 40 % of therecipients, whereas it induced cleavage in only 7-7% of the eggs enucleated by thefirst method, and in only 2-6% of those enucleated by the second method. Therefore,the cleavage-inducing activity of isolated MA was tested using only eggs enucleatedby the second method of enucleation. The experiments show that whereas CIA failsto initiate cleavage in enucleated eggs, isolated MA induce continued cleavage inenucleated eggs with the same frequency as in nucleated eggs (Table 4 and Fig. 7).Some differences were noted, however. Normal, nucleated eggs which were injectedwith either isolated MA or CIA formed a blastula cell cluster in an area which alwaysincluded the animal pole, whereas enucleated eggs often formed cell clusters in theareas distant from the animal pole (Fig. 8); some clusters were even found solely inthe vegetal hemisphere. This observation may be interpreted in such a way that cleavageis most likely to be initiated in the area where the MA was first localized; wheneggs have their own nucleus this area is near the animal pole, whereas enucleated eggsform cell clusters closest to the area where the sea-urchin zygote MA was injected.

To test whether blastula cell clusters formed in enucleated eggs were derivedfrom the injected MA, injected eggs were studied histologically. In the blastula cellclusters formed in nucleated eggs injected with CIA the nuclei were of uniform sizeand showed uniform, positive staining with Feulgen reagent, with no sign of de-generation (Fig. 9). On the other hand, in blastula cell clusters formed in nucleatedeggs injected with MA the stainability as well as the size of the nuclei were quitevariable: some nuclei were not stained at all, but some were heavily pycnotic. Inblastula cell clusters formed in enucleated eggs injected with MA, nuclei hardly stainedwith the Feulgen reagent (Fig. 10).

We cannot prove the origin of these nuclei, but since only sea-urchin geneticmaterial was present in the enucleated frog eggs, it seems likely that the nuclei arederived from the sea-urchin MA chromosomes. It is relevant in this regard to pointout work by Marshak & Marshak (1955) and Immers (1957) which showed thatsea-urchin nuclei can be Feulgen-negative.

Recovery of the cleavage-inducing activity in the MA stored in MTME

To elucidate the cause of the loss of activity of MA during storage, pellets of theinactive MA were resuspended in freshly prepared CIA and incubated at roomtemperature for varying periods of time. The incubated suspensions were theninjected into nucleated as well as into enucleated eggs to test the cleavage-inducingactivity.

In nucleated eggs, cleavage was induced in at least 60% and on the average, 70%of the recipients of mixtures of MA + CIA. This was significantly higher than the40% induction in eggs injected with CIA alone (P < o-oi), as summarized inTables 4 and 5. Cleavage was also induced in enucleated eggs. Although the averagepercentage of recipients which formed blastula cell clusters (29%) was lower thanthat of the nucleated recipients, it was 10 times higher than that of the enucleatedeggs injected with CIA alone (2-6%), and this difference was highly significant(P < o-ooi) (Table 5).

Sea-urchin MA induce frog egg cleavage

Table 5. Cleavage initiation by MA and CIA in normal and enucleated eggs

Samplesinjected

Duration of MA storagein

A

MTME, 1/10 MTME, Hostdays min nucleus

A. Normal nucleated recipient eggsMA

10 MS17, 18 HM

MA + CIA10 MS17, 18 HM

CIA

B. EnucleatedMA + CIA

10 MS17, 18 HM

CIA

2 1

2 1

Overall

2 1

2 1

Overall

10-20 +10-45 +

20-60 +20-45 +

120-180 +180-240 +

+

recipient eggs

2 1

2 1

Overall

10-60 —10-45 -

120-240 —

N o1 > U.

ofeggs

484 29 0

349 0

555 0

2 2 9

358

362 431

9 1

7 7

Cleaved eggs,

Blastula cell

size

> 1/2 egg <

2 - 1

2-4

2'2

3 5 343-354'54 0 04 4 1

27-4

8-34 2

9 77 71-7

cluster

1/2 egg

4-20

2 ' 2

29-43 2 2

16-422-O

25 8

1 3 4

1 3 92 5 0

1 9 4

1 8 7j . .

0 '

/o

Totalf

6 32-4

4 5

6477567 0 962-0700**

4o-8»»

22'O

2 9 2

2 9 O

264**

2-6*

*, t As in Table 1. MS: MA isolated and stored in MTA1E. HM: MA isolated in HGand stored in MTME. • • x1 values for the difference between MA and MA + CIA injectionsare 259 (P < 001) in the normal recipients, and 474 (P < 0001) in the enucleated recipients.

These observations indicate that some of the MA became capable of initiatingand promoting cleavage in the absence of the recipient nucleus following incubationwith CIA. Thus, it may be concluded that the cleavage-initiating activity of isolatedsea-urchin MA, once lost during storage in MTME, can be expressed again followingan interaction with CIA from frog brains.

Fig. 8. A blastula cell cluster formed in an enucleated egg following injection ofMA freshly isolated in MTME. Cultured for 18 h at 18 °C. The cytoplasmicexovate indicates the site of enuclearion. x 30.

Fig. 9. Blastula cells formed in a CIA-injected egg. Note uniformly stained Feulgen-posirive nuclei, x 850.

Fig. 10. Blastula cells formed in an enucleated egg following injection of MAfreshly isolated in MTME. Note Feulgen-negative nuclei, x 850.


DISCUSSION

Activation of Rana pipiens eggs can be achieved by pricking with a glass needle.Although the egg cortex forms furrows and although the pronucleus initiates DNAsynthesis (Graham, 1966) and eventually metaphase chromosomes form for thefirst mitosis, the egg develops only a single aster and thus fails in mitosis (Battaillon& Tchou-Su, 1934; Aimer & Labrousse, 1975). In order for mitosis to occur, anadditional cytoplasmic factor, called second factor or cleavage-inducing agent (CIA),must be provided by other cells (Battaillon, 1911; Herland, 1913). Recently, several cellcomponents have been identified as having cleavage-inducing activity, including cyto-plasmic particles sedimented at ioooog from body tissues of various animal speciesincluding the frog brain (Shaver, i953;Pfohl, 1970; Fraser, 1971), sea-urchin spermcentriole (Mailer et al. 1976; Iwamatsu, Miki-Noumura & Ohta, 1976; Ohta & Iwam-atsu, 1974), and protozoan basal body (Heideman & Kirschner, 1975). Our study clearlyshows that MA isolated in MTME from sea-urchin zygotes possess CIA activity.

However, as shown in the present experiments, CIA alone cannot initiate cleavagein frog eggs that have no nuclei. On the other hand, MA of sea-urchin zygotesisolated and stored in MTME exhibited cleavage-inducing activity not only innucleated frog eggs, but also in enucleated eggs. Therefore, it is likely that isolatedMA not only possess CIA activity, but can also provide nuclear materials which arerequired for cleavage.

The stability of the MA cleavage-inducing activity depended on the medium inwhich the MA were isolated and stored. MA isolated and stored in MTME maintaintheir cleavage-inducing activity as high as freshly isolated MA for at least 48 h,whereas MA isolated and stored in HG lose their activity within a few hoursfollowing isolation. MA isolated in HG and immediately transferred into MTME,however, retain their cleavage-inducing activity on the same day as they were isolated,but lose most of their activity in the next 24 h.

The rapid decrease in cleavage-inducing activity of MA briefly exposed to HGmay result from the decay of their molecular organization caused by the medium.Various workers have found that the MA isolated and stored in HG rapidly losebirefringence as well as solubility in 0-5 M KC1 (Kane & Forer, 1965; Goldman &Rebhun, 1969; Forer & Goldman, 1969; Forer & Zimmerman, 19766); MA isolatedin HG and immediately transferred into MTME retain a constant level of birefringence,but the lability of the birefringence in 0-5 M KC1 is gradually lost, the first cleardifference being found at 24 h (Forer & Zimmerman, submitted). On the otherhand, MA isolated and stored in MTME were found to maintain not only constantbirefringence but also sensitivity to 0-5 M KC1, pressure and cold treatments for at least2 weeks (Forer & Zimmerman, 1976a). When MA isolated and stored in MTME orMA isolated in HG and stored in MTME are placed in 1/10 MTME both cleavage-inducing activity and birefringence drop with storage time, although some birefringenceis still present at 48 h (Forer & Zimmerman, 19766). Thus, the loss of MA cleavage-inducing activity appears to parallel somewhat the loss of lability of the MA bire-fringent material, which indicates the decay of molecular organization during storage.


It is noted, however, that MA stored in 1/10 MTME for 17 h as well as thosestored in MTME for 2 weeks underwent no changes in their ultrastructure (thisreport for 17-h data, and Forer & Zimmerman, 1976 a) and MA stored for 10 daysin MTME retained as much birefringence which was sensitive to 0-5 M KC1, pressureand cold treatments as freshly isolated MA (Forer & Zimmerman, 1974, 1976a), inspite of the fact that these MA had lost their cleavage-inducing activity. Therefore,if any change in the molecular organization of the isolated MA is responsible forthe loss of their cleavage-inducing activity, it is perhaps a very subtle one, such asthe loss of chemical activities associated with MA rather than the loss of structuralorganization of MA itself. Indeed, it was found that MA once inactivated couldrecover the ability to induce cleavage both in nucleated and enucleated eggs followingincubation with CIA from frog brains. Since injection of CIA alone into enucleatedeggs could not induce cleavage, it is apparent that isolated sea-urchin MA whichhad become incapable of initiating cleavage by itself, still retained the capability offunctioning as a substitute for MA of frog eggs. These results may indicate thatCIA and MA complement each other to induce cleavage. Therefore, MA freshlyisolated in MTME from sea-urchin zygotes can be regarded as a unit which consistsof 2 complementary, functional subunits, i.e. cleavage-inducing activity and thestructural elements. Perhaps ageing of MA isolated in MTME removes the CIAfunction, leaving the structure intact.

It has been speculated that the function of CIA is associated with its ability toorganize microtubule systems in frog eggs which lack this ability, and that the CIAis itself an organized microtubule assembly (Heidemann & Kirschner, 1977; Maileret al. 1976; Fraser, 1971). Evidence to support this contention includes the obser-vations that frog brain CIA can be inactivated by colchicine and stabilized by GTPor D2O (Fraser, 1971), and that basal bodies of protozoa (Heidemann & Kirschner,1975) and sperm centrioles of medaka (Iwamatsu et al. 1974) and of sea urchins(Mailer et al. 1976) actually act as CIA. However, CIA from different origins seemto have different chemical properties. CIA activity of the basal body is destroyedby RNase (Heidemann & Kirschner, 1977), whereas that of frog brain is resistantto RNase (Fraser, 1971). In fact, an electron micrograph of a preparation of frogbrain CIA (Fraser, 1971), shows hardly any microtubules except in a few vesicleswhich enclose tubules, these being found among hundreds of other membranestructures. Therefore, it is possible that frog brain CIA, differing from other CIA,may not be represented by microtubule assemblies.

A variety of cell components other than microtubules are found in isolated MA,including membrane vesicles and ribosome-like particles (Kane, 1962; Goldman &Rebhun, 1969; Forer & Zimmerman, 1974; Harris, 1975; Forer, Kalnins & Zimmer-man, 1976). Although the functions of these components are unknown, these com-ponents may possibly play a role in regulating the dynamic state of MA, as well asin the maintenance of its structural organization. If so, the CIA function of freshlyisolated MA may be due to these non-microtubule components, for which frogbrain CIA can be substituted.

Interaction between CIA and frog MA seems to be required for the eggs to form

134 ^- Masui, A. Forer and A. M. Zimmerman

the amphiaster, since eggs fail to form amphiasters when they do not have eitherMA or CIA. Presumably frog brain CIA interacts in a similar manner with MAisolated from sea-urchin zygotes, whose CIA activity had been lost by ageing inMTME, to restore the original capability to develop asters, which are prerequisitefor triggering cleavage in animal eggs (Rappaport, 1971). As we have argued previously(Forer et al. 1977), we think that the injected MA actually participate in mitosis,though until the events which occur between injection and cleavage have been studiedcytologically other hypotheses cannot be ruled out.

This work was supported by grants from the National Research Council of Canada, andNational Cancer Institute of Canada.

REFERENCES

AIMER, CHR. & LABROUSSE, J. P. (1975). DNA synthesis and evolution of a somatic nucleus,of the female pronucleus after the experimental activation of the egg of Pleurodelts loaltlti.Dev., Growth Diff. 17, 197-207.

BATTAILLON, E. (191 I ) . L'embryogenese provoquee chez l'oeuf vierge d'Amphibiens parinoculation de sang ou de spermie de Mamminifere. Parthenogenese traumatique etempregnation sans amphimixie. C. r. hebd. Sianc. Acad. Sci., Paris 152, 1271-1273.

BATAILLON, E. & TCHOU-SU (1934). L'analyse exp^rimentale de la f^condation et sa definitionpar les processus cine'matiques. Annls Sci. nat., loe Sir. 17, 1-36.

BRUN, R. (1973). Nuclear transplantation-mammalian cells in Xenopus eggs. Nature, NewBiol. 243, 26-27.

FORER, A. & GOLDMAN, R. D. (1969). Comparisons of isolated and in vivo mitotic apparatuses.Nature, Lond. 222, 689-691.

FORER, A., KALNINS, V. I. & ZIMMERMAN, A. M. (1976). Spindle birefringence of isolatedmitotic apparatus: Further evidence for two birefringent spindle components. J. CM Sci.22, 115-131.

FORER, A., MASUI, Y. & ZIMMERMAN, A. M. (1977). A possible bio-assay for chromosomemovement in isolated mitotic apparatus. Expl Cell Res. 106, 430—434.

FORER, A. & ZIMMERMAN, A. M. (1974). Characteristics of sea-urchin mitotic apparatusisolated using a dimethyl sulphoxide/glycerol medium. J. Cell Sci. 16, 481-497.

FORER, A. & ZIMMERMAN, A. M. (1976a). Spindle birefringence of isolated mitotic apparatusanalysed by pressure treatment. J. Cell Sci. 20, 309-327.

FORER, A. & ZIMMERMAN, A. M. (19766). Spindle birefringence of isolated mitotic apparatusanalysed by treatments with cold, pressure and diluted isolation medium. J. Cell Sci. 20,329-339.

FRASER, L. R. (1971). Physico-chemical properties of an agent that induces parthenogenesisin Rana pipiens eggs. J. exp. Zool. 177, 153—172.

GOLDMAN, R. D. & REBHUN, L. E. (1969). The structure and some properties of the isolatedmitotic apparatus. J. Cell Sci. 4, 179-209.

GRAHAM, C. F. (1966). The regulation of DNA synthesis and mitosis in multinucleate frogeggs. J. Cell Sci. 1, 363-374-

HARRIS, P. (1975). The role of membranes in the organization of the mitotic apparatus. ExplCell Res. 94, 409-425.

HEIDEMANN, S. R. & KIRSCHNER, M. W. (1975). Aster formation in eggs of Xenopus laevis:Induction by isolated basal bodies. J. Cell Biol. 67, 105-117.

HEIDEMANN, S. R. & KIRSCHNER, M. W. (1977). Evidence for a functional role of RNA incentrioles. Cell 10, 337-350.

HERIANT, M. (1913). Etude sur les bases cytologiques du mechanisme de la parthenogeneseexpe>imentale chez les Amphibiens. Archs Biol., Paris 28, 505-608.

IMMERS, J. (1957). Cytochemical studies of fertilization and first mitosis of the sea urchinegg. Expl Cell Res. 12, 145-153.


INOUE, S., BORISY, G. C. & KIEHART, D. P. (1974). Growth and lability of Chaetopterusoocyte mitotic spindles isolated in the porcine brain tubulin. J. Cell Biol. 62, 175-184.

IWAMATSU, T., MIKI-NOUMURA, I. & OHTA, T. (1976). Cleavage initiating activities of micro-tubules and in vitro reassembled tubulins of sperm flagella. J. exp. Zool. 195, 97-105.

KANE, R. E. (1962). The mitotic apparatus: isolation by controlled pH. J. Cell Biol. 12,

47-55-KANE, R. E. (1965). The mitotic apparatus: physical-chemical factors controlling stability.

J. Cell Biol. 25, 137-144-KANE, R. E. & FORER, A. (1965). The mitotic apparatus: structural changes after isolation.

J. Cell Biol. 25, 31-39.MALLER, J., POCCIA, D., NISHIOKA, D., KIDD, P., GERHART, J. & HARTMAN, H. (1976). Spindle

formation and cleavage in Xenopus eggs injected with centriole-containing fractions fromsperm. Expl Cell Res. 99, 285-294.

MARSHAK, A. & MARSHAK, C. (1955). Quantitative determination of deoxyribonucleic acid inechinoderm germ cells. Expl Cell Res. 8, 126-146.

MASUI, Y. (1974). A cytostatic factor in amphibian oocytes: its extraction and partial charac-terization. J. exp. Zool. 187, 141-147.

MASUI, Y. & MARKERT, C. L. (1971). Cytoplasmic control of nuclear behaviour duringmeiotic maturation of frog oocytes. J. exp. Zool. 177, 129-146.

MAZIA, D. & DAN, K. (1952). The isolation and biochemical characterization of the mitoticapparatus of dividing cells. Prcc. natn. Acad. Sci., U.S.A. 38, 826-838.

MAZIA, D., MITCHISON, J. M., MEDINA, H. & HARRIS, P. (1961). The direct isolation of the

mitotic apparatus. J. biophys. biochem. Cytol. 10, 467-474.MEYERHOF, P. G. & MASUI, Y. (1977). Ca and Mg control of cytostatic factors from Rana

pipiens oocytes which cause metaphase and cleavage arrest. Devi Biol. 61, 214—229.MOORE, B. (1940). Chromosomes of frog eggs and embryos stained by Feulgen method to

avoid excessive staining of yolk granules. Anat. Rec. 78, Suppl. 122.OHTA, T. & IWAMATSU, T. (1974). Initiation of cleavage in fish eggs by injection of flagella

or microrubules of sea urchin spermatozoa. Dev., Growth Diff. 6, 67-74.PORTER, K. R. (1939). Androgenetic development of the egg of Rana pipiens. Biol. Bull. mar.

biol. Lab., Woods Hole 77, 233-257.PFOHL, R. J. (1970). Initiation of cleavage in frog eggs. An analysis of active large granule

fractions from frog liver tissue homogenates. Expl Cell Res. 61, 433-443.RAPPAPORT, R. (1971). Cytokinesis in animal cells. Int. Rev. Cytol. 31, 169-213.REBHUN, L. I., ROSENBAUM, J., LEFEBVRE, P. & SMITH, G. (1974). Reversible restoration of

the birefringence of cold-treated, isolated mitotic apparatus of surf clam eggs with chickbrain tubulin. Nature, Lond. 249, 113-115.

SAKAI, H. (1966). Studies on sulfhydryl groups during cell division of sea-urchin eggs. VIII.Some properties of mitotic apparatus proteins. Biochim. biophys. Acta 112, 132-145.

SAKAI, H. & KURIYAMA, K. (1974). The mitotic apparatus isolated in glycerol-containingmedium. Dev., Growth Diff. 16, 123-134.

SHAVER, J. (1953). Studies on the initiation of cleavage in the frog egg. J. exp. Zool. 122,169-192.

SMITH, B. G. (1912). The embryology of Cryptobranckus allegeheniensis including comparisonswith some other vertebrates. J. Morph. 23, 61-153.

ZIEGLER, D. H. & MASUI, Y. (1973). Control of chromosome behavior in amphibian oocytes.I. The activity of maturing oocytes inducting chromosome condensation in transplantedbrain nuclei. Devi Biol. 35, 283-392.

ZIEGLER, D. H. & MASUI, Y. (1976). Control of chromosome behavior in amphibian oocytes.II. The effect of inhibitors of RNA and protein synthesis on the induction of chromosomecondensation in transplanted brain nuclei by oocyte cytoplasm. J. Cell Biol. 68, 620-628.

{Received n July 1977)

induction of cleavage in nucleated and enucleated frog … · ovulation was induced by injecting...

Documents