All

Experimental

CONTROL

Prmted I” Sweden CopyrIght 0 1979 by Academic Press. Inc. rights of reproduction in any form recerved 0014-48?7/79/1?0?07-13s0?.00/0

Cell Research 123 (1979) 207-219

OF CHROMOSOME CONDENSATION IN THE SEA URCHIN EGG

GEOFFREY W. KRYSTALI

and DOMINIC POCCIA’*

IDepartment of Biology? Stair University oj’Neb ammonia-activated L. picrus culture were removed at intervals and the percent of eggs lacking a maternal pronuclear envelope was determined using Nomarski optics (100 eggs scored for each time point).

Initially, a lower proportion of enucleates is able to induce PCC compared to nucleates. This is probably due to the length of time between when the eggs are actually severed in the centrifuge and when merogons are first fertilized, along with a greater lability of the conditions in the enucleates. Given this greater lability, an explanation for the failure to achieve a 100% PCC response in enucleates produced prior to activation (fig. 4) becomes apparent. In those experiments in which the time between breakage and activation is not a factor, the greater lability described above coupled to a small degree of asynchrony in the culture could make it impossible to achieve a 100% response in the enucleates at any given time. An estimate of the asynchrony with which the PCC-promoting environment appears might be obtained by observing a

recognizable mitotic event, such as the breakdown of the nuclear envelope in a typical culture of NH,-activated eggs (fig. 7). Unlike the assay for PCC, this is essentially an instantaneous measure of the position of the eggs in their cell cycle. By the time the eggs have reached a point where they are capable of promoting PCC, a degree of asynchrony is evident. It takes 30 min between the time when 5% of the envelopes are broken and when 95% are broken. The entire PCC-inducing period in enucleates is shorter than this (fig. 4 and table 2), while in nucleates the period is longer. Furthermore, a greater asynchrony in the population might be introduced by stressing the eggs at the low temperatures and high centrifugal forces used to prepare the merogons. Therefore, the lower maximal response of the enucleates may be a reflection of the instability of the PCC-pro-

Fig. 6. Chromosomes of a fertilized (polyspermic) enu-

Exp Cdl Res 123 (1979)

Fig. 7. Abscissu:

Chromosome condensation/sea urchin egg moting conditions coupled with the asynchrony of the culture. The greater stability seen in the nucleated halves seems to be a result of the presence of the maternal chromosomes or some other components associated with the maternal pronucleus segregating with those chromosomes. RNA synthesis is not required for induction of PCC Although the ability of enucleates to induce PCC rules out a requirement for transcription of the maternal nuclear genome, it does not preclude the possibility that transcription of maternal cytoplasmic DNA or of sperm genomes following fertilization takes place. In order to assess those possibilities, actinomycin D (ActD) (25 pg/ml) and ethidium bromide (EB) (10 pg/ml) were used to inhibit nuclear and cytoplasmic transcription. At these concentrations, nuclear RNA synthesis is inhibited by 94% [19] and cytoplasmic RNA synthesis by 97 % [20]. Eggs were incubated in the presence of the inhibitors for 90 min prior to activation. At intervals after activation, aliquots were removed, fertilized, subcultured for 30 min, and fixed. All steps up to

215

fixation were carried out in the presence of the inhibitors. The results are shown in table 3. PCCpromoting conditions developed in the presence of either or both inhibitors to the same extent as in control cultures. The peak of activity developed more slowly in the presence of ActD, consistent with previous observations that the drug slows down normal development of sea urchin eggs. These experiments rule out a significant dependence of the development of PCC on transcription of cytoplasmic or pronuclear genomes. Induction of PCC is independent oj protein synthesis ufter late S or early G2 Although transcription seems unnecessary to develop conditions promoting PCC, translation of preformed mRNA sequences might occur. Therefore, the effects of continuous exposure to emetine at concentrations which block 98 % of protein synthesis [21] were determined. If emetine was added to the eggs prior to activation, the eggs did not develop PCC-promoting conditions, nor did the maternal chromosomes condense. The same result was obtained when the inhibitor was added at any time up to about

Table 3. The effect of the inhibition of RNA synthesis on the induction of PCC in whole eggs Time of fertilization after activation (min)

% PCC” Control

ActD

EB

Expt 1 84 114 144

91 83.5 35

43.5 92 69

-

Expt 2 80 110 140

51 2.5 1

-

ActD+EB

44.5 5 1.5

14.5 50 9

Eggs were incubated in the presence of inhibitors actinomycin D (ActD) and ethidium bromide (EB) for 90 min prior to activation. At the specified intervals after activation, ahquots were removed, fertilized, cultured for an additional 30 min, fixed and scored for PCC. a Two hundred eggs counted for each point. Exp Cell Res 123 (1979)

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Krystal and Poccia

Fig. 8. Appearance of emetine-treated eggs fertilized 85 min after activation and fixed 30 min later. Emetine ( 10m4M) added (a) 60 min; (b) 20 min after activation.

Eggs were fixed in Carnoy’s solution and stained with aceto-orcein. X 240.

35 min post-activation. If added at 50 min shown in fig. 10. The results suggest that or later, PCC-promoting conditions and ma- any proteins required for chromosome conternal condensation occurred as in unin- densation are synthesized prior to G2. The hibited controls and chromosomes from as results also eliminate the possibility of a many as 50 male pronuclei/egg could be- need for any contribution from mRNA carried by the sperm cell, since translation may come condensed (fig. 8). These experiments included eggs which be blocked prior to fertilization without had been exposed to [3H]thymidine prior to effect on PCC. activation in order to determine more precisely at what point in the egg cycle the cells DISCUSSION become independent of protein synthesis. As seen in fig. 9, the transition occurs near In this study sperm nuclei were introduced the end of the first round of DNA synthesis into activated egg cytoplasm to assay for at 35-50 min post-activation (late S or early the presence of chromosome-condensing G2). Maternal prophase equivalent began conditions. Two main conclusions emerge. at approx. 70 min; eggs were fertilized at One is that conditions promoting the first 85 min. Since emetine and other protein chromosome condensation in the sea urchin synthesis inhibitors are not readily revers- egg are cytoplasmically programmed to be ible in sea urchin eggs [21], it could not be set in motion by activating agents or, nordetermined whether synthesis might be re- mally, the fertilizing sperm. Once initiated, quired throughout the first 40 min or only the only genetic contribution that may be during some sub-interval. The drug acts required is translation of stored maternal essentially instantaneously, however, as mRNAs prior to G2. The second is that alExp Cd Res 123 (I 979)

Chromosome condensation/sea

urchin egg

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32-

26-

2420- 60 - 50

16-

- 40 - 30 - 20 -

IO

30

40

50

60

70

Fig. IO. Abscissa: time after activation (min); ordinate:

Fig. 9. Abscissa: time after activation (mm); ordinafe: (left) [3H]thymidine incorp. (% of total uptake); (righf) % of control PCC after addition of emetine. 0, [3H]thymidine incorporation; x , time of emetine addition to a particular culture. Effect of the inhibition of protein synthesis on the induction of PCC. Six parallel cultures of L. picks eggs (from the same female, activated at the same time) were established. One served as a control. One was pre-incubated with r3H]thymidine and used to measure DNA synthesis. Emetine (10m4M) was added to the other four, one at a time, at intervals. The emetine-treated cultures and the control were fertilized at 85 min, cultured for an additional 30 min, fixed and scored for PCC (200 eggs scored for each time point).

though the transition to condensed chromatin is cytoplasmically controlled, a component of the maternal pronucleus may modulate the stability of the chromosome condensing environment. We have demonstrated that preparations for a distinctly nuclear event, chromosome condensation, can occur in activated enucleated cytoplasms. The timing of appearance of chromosome-condensing conditions in these merogons correlates closely with the time of chromosome condensation in normally fertilized eggs. Although to test for these conditions, chromatin in the form of sperm nuclei was introduced, the nuclei

amino acid incorporation (% of total uptake). 0, Control; X , emetine culture. Time course of the inhibition of protein synthesis by emetine. L. pictus eggs were pre-incubated for 20 min in a 10% (v/v) suspension containing 1 &i/ml [“Clvaline. Emetine (10m4M) was added at 39 min after activation.

are considered to be relatively passive indicators of the cytoplasmic conditions, since neither transcription nor translation need occur while the male chromatin is in the egg cytoplasm. The cytoplasmic control of chromosome condensation does not seem to require either transcription of maternal nuclear or cytoplasmic genomes or protein synthesis during the maternal G2 period. A requirement for protein synthesis prior to G2 is suggested by the correlation of inhibition of protein synthesis and inhibition of premature chromosome condensation. The effects of inhibition of RNA and protein synthesis on PCC in polyspermic eggs are consistent with previous observations on mitotic [ 19, 24, 251 and premature chromosome condensation [6, 131. Newly synthesized proteins required for chromosome condensation would have to Exp Cd Res I23 (1979~

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Krystal

and Poccia

be made using stored maternal mRNAs. The role of such proteins is not known. They may interact directly with the chromosomes or be required for some other step which either in a general way allows the cell cycle to proceed or is more specifically related to preparations for chromosome condensation. However such proteins act, requirement for their synthesis ceases well before any visible chromosome condensation takes place. If the proteins were needed in stoichiometric proportions to the chromosomes, they would have to be made in great excess over the normally required amounts, since the eggs can induce PCC in 50 supernumerary pronuclei even if these nuclei are introduced into the egg while protein synthesis is blocked. The proteins, however, might act catalytically. Our second major result is that although PCC-promoting conditions can arise in enucleated merogons, these conditions are much less stable than in nucleated halves. Since more stable conditions are associated with the nucleated merogons prepared manually as well as centrifugally, it would appear unlikely that the differences observed arise from artifactual cytoplasmic segregation. Both types of merogons seem to be activated to the same degree by NH, as judged by protein synthesis. The differences seem to involve the presence or absence of the maternal pronuclei or chromosomes, or some component which segregates with them, but cannot involve transcription of the maternal genome because of the actinomycin D results. It is also unlikely that mRNA sequences which must be translated to confer the increased stability are segregated with the maternal chromosomes, since whole eggs blocked with emetine from G2 on exhibit the same decay of PCC-promoting conditions as uninhibited controls, yet the increased stability is asE.rp Cdl Res 123 (1979)

sociated with the maternal chromosomes even when eggs are broken during the peak of PCC activity, at which time the requirement for protein synthesis has long passed. The increased stability in nucleated merogons suggests that the maternal chromosomes play some non-transcriptional role in stabilizing the cytoplasmic chromosome condensing environment. Mazia [23] has postulated that the chromosome condensation-decondensation cycle and the formation of the mitotic apparatus are coordinately controlled by the same elements. It has been shown that the mitotic apparatus concentrates and organizes potential regulator molecules such as calcium-dependent ATPase [29] and calcium-dependent regulator protein (CDR) [28]. Sluder [30] has suggested that the organizational capabilities of the spindle play an important role in cell cycle timing. He has demonstrated that the length of the period of maximal chromosome condensation is affected by the presence and size of the spindle. In sea urchin eggs treated with colcemid, which completely prevents spindle formation, the prometaphase period is twice as long as in the controls. Similarly, Rao & Johnson [27] have shown a stabilization of PCC-inducing conditions (up to 24 h) in HeLa cells treated with colcemid. Thus, the mitotic apparatus may destabilize the chromosome condensing environment. It is possible that the mitotic apparatus and the chromosomes act antagonistically to produce a coordination of the chromosome condensation-decondensation cycle, chromosome segregation and cytokinesis. A better understanding of the mechanism of chromosome condensation may result from a biochemical analysis of the protein composition of the condensed chromosomes [ 151. Prematurely condensed sperm chromosomes should be of particular value

Chromosome

for several reasons: (1) sperm chromatin has distinctive histones and very little nonhistone protein thus providing a good background against which to measure protein changes associated with condensation; (2) the chromosomes do not go through the previous cell cycle and so alterations in their composition associated with other aspects of the cycle should be minimized; and (3) high polyspermy would allow good yields and reasonable purity of the isolated chromosomes.

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12. - Adv cell mol biol 3 (1974) 135. 13. Matsui, S, Yoshida, H, Weinfeld, H & Sandberg, A, J natl cancer inst 47 (1971) 401. 14. Rao, P N & Johnson, R T, Control of proliferation in animal cells (ed B Clarkson & R Baserga) vol. 1, p. 785. Cold Spring Harbor Press, N.Y. (1974). 15. Poccia, D, Krystal, G, Nishioka, D & Salik, J, ICN-UCLA symposium on molecular and cellular biology. Cell reproduction (ed E R Dirksen & D Prescott) p. 197. Academic Press, New York (1978). 16. Mazia, D, Proc natl acad sci US 71 (1974) 690. 17. Nishioka, D & Mazia, D, Cell biol int rep 1 (1977) 23. 18. Harvey, E B, The American Arbacia and other sea urchins. Princeton University Press, Princeton, N.J. (1956). 19. Gross, P R & Cousineau, G H, Exp cell res 33 (1964) 368. We wish to thank Dr B. A. Palevitz for many helpful 20. Craig; S P& Piatigorsky, J, Dev biol24 (1971) 214. discussions and his assistance in the microscopy por- 21. Hogan. B & Gross. P R. J cell biol49 (1971) 692. 22. Humason, G L, Animal tissue techniques, 2nd tion of this work. This work was supported by NIH edn, p. 309. W H Freeman, San Francisco (1967). Grant HD 09654 to D. L. P. 23. Mazia, D, Cell cycle controls (ed G M Padilla, I L Cameron & A Zimmerman) D. 265. Academic Press, New York (1974). 24. Young, C W, Hendler, F J & Karnofsky, DA, Exp REFERENCES cell res 58 (1969) 15. 1. Johnson, R T & Rao, P N, Biol rev Cambridge phi1 25. Wagenaar, E B & Mazia, D, ICN-UCLA symsot 46 (1971) 97. posium on molecular and cellular biology. Cell 2. Brachet, A, Arch bio132 (1922) 205. reproduction (ed E R Dirksen & D Prescott) p. 3. Graham, C F, J cell sci 1 (1966) 363. 539. Academic Press, New York (1978). 4. Gurdon, J B, J embryo1 exp morph01 20 (1968)401. 26. Black, R E & Baptist, E & Piland, J, Exp cell res 5. Ziegler, D & Masui, Y, Dev biol 35 (1973) 283. 48 (1967) 431. 27. Rao, P N & Johnson, R T, J cell sci 10 (1972) 495. 6. - J cell biol68 (1976) 620. 7. Johnson, R T & Rao, P N, Nature 226 (1970) 717. 28. Welsh, M J, Dedman, J R, Brinkley, B R & Means, 8. Batakier, H & Czolowska, R, Exp cell res 110 A R. Proc natl acad sci US 75 (1978) 1867. 29. Mazia, D, Petzelt, C, Williams, R 0 & Meza, I, (1977) 466. 9. Sunkara, P S, Al-Bader, A A & Rao, P N, Exp cell Exp cell res 70 (1972) 325. 30. Sluder, G, J cell biol 80 (1979) 674. res 107(1977) 444. 10. Johnson, R T, Rao, P N & Hughes, H D, J cell physio176 (1970) 151. Received October 6, 1978 11. Rao, P N & Johnson, R T, J cell physiol78 (1971) Revised version received May 28, 1979 217. Accepted June 1, 1979