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Teniposide, a Topoisomerase II Inhibitor, Prevents Chromosome Condensation and Separation but Not Decondensation in Fertilized Surf Clam (Spisula solidissima) Oocytes SHIRLEY J. WRIGHT'ANDGERALDSCHATTEN Integrated
Microscopy
Resource for Biomedical Research, Zoology Research Building, 1117 West Johnson Street, Madison, Wisconsin 53706
University
of Wisconsin,
Accepted July 23, 1990 DNA topoisomerase II has been implicated in regulating chromosome interactions. We investigated the effects of the specific DNA topoisomerase II inhibitor, teniposide on nuclear events during oocyte maturation, fertilization, and early embryonic development of fertilized S;oisula solidissima oocytes using DNA fluorescence. Teniposide treatment before fertilization not only inhibited chromosome separation during meiosis, but also blocked chromosome condensation during mitosis; however, sperm nuclear decondensation was unaffected. Chromosome separation was selectively blocked in oocytes treated with teniposide during either meiotic metaphase I or II indicating that topoisomerase II activity may be required during oocyte maturation. Teniposide treatment during meiosis also disrupted mitotic chromosome condensation. Chromosome separation during anaphase was unaffected in embryos treated with teniposide when the chromosomes were already condensed in metaphase of either first or second mitosis; however, chromosome condensation during the next mitosis was blocked. When interphase two- and four-cell embryos were exposed to topoisomerase II inhibitor, the subsequent mitosis proceeded normally in that the chromosomes condensed, separated, and decondensed; in contrast, chromosome condensation of the next mitosis was blocked. These observations suggest that in Spisula oocytes, topoisomerase II activity is required for chromosome separation during meiosis and condensation during mitosis, but is o 1990 Academic not involved in decondensation of the sperm nucleus, maternal chromosomes, and somatic chromatin. Press, Inc.
Topoisomerase II is a major structural component of the nuclear matrix and is the most abundant nonhistone protein of the metaphase chromosome scaffold (Earnshaw and Heck, 1985; Earnshaw et aL, 1985; Berrios et al., 1985; Gasser et al., 1986; see Heck and Earnshaw, 1988 for a review). Topoisomerase II acts to alter the topological conformation of DNA by making a doublestrand break, passing another double-strand through the break, and then resealing it (Cozarelli, 1980; Gellert, 1981; Liu, 1983; Wang, 1985). Studies with yeast topoisomerase mutants demonstrate that topoisomerase II is required for chromosome condensation and separation during anaphase of mitosis (Uemura and Yanagida, 1984, 1986; Holm et aZ., 1985, 1989; Uemura et aZ., 1987). The role of topoisomerase II in higher eukaryotes is not understood. However, studies using a topoisomerase II inhibitor (Chen et ah, 1984) and cell-free extracts derived from Xenopus oocytes have suggested that topoisomerase II is involved in both chromosome condensation and decondensation (Newport, 1987; Newport and Spann, 1987). If topoisomerase II is involved in chromosome interactions during oocyte maturation and fertilization, blocking topoisomerase II activity should arrest chromosome condensation, separation, and decondensation.
INTRODUCTION
The organization of the nucleus changes dynamically during the cell cycle especially in rapidly dividing cells. Fertilization and early development are particularly well suited for studying these alterations. Spisula solidissima (surf clam) oocytes are spawned at the germinal vesicle stage and are arrested at meiotic prophase until fertilization which triggers both meiotic maturation and zygote development. During fertilization, the maternal chromatin undergoes phases of condensation, separation, and decondensation that occur during germinal vesicle breakdown, polar body formation, and female pronuclear development, while the highly condensed sperm nucleus undergoes phases of decondensation and condensation closely coupled to the meiotic changes of the maternal chromatin (Da-Yuan and Longo, 1983; Luttmer and Longo, 1988). The mechanisms underlying these dramatic changes in chromatin organization during fertilization are not understood, however, the enzyme, DNA topoisomerase II may play an important role. 1 To whom correspondence should be addressed. OOlZ-1606/90 $3.00 Copyright All rights
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
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To test this hypothesis, we have examined the effects of the specific, DNA topoisomerase II inhibitor, teniposide, on nuclear events during meiosis, fertilization, and early embryogenesis of fertilized surf clam (S. solidissima) oocytes. A preliminary account of this investigation has been reported (Wright and Schatten, 1988). MATERIALS
AND
METHODS
Specimen Preparation
Gonads were removed from mature S. solidissima obtained from the Marine Biological Laboratory (Woods Hole, MA). Testes were collected and then filtered through cheesecloth. Sperm were stored undiluted until used. Oocytes, obtained by mincing the ovaries in artificial seawater (ASW) and filtering through cheesecloth, were suspended to 1 liter in ASW, allowed to settle, and the supernatant was decanted. This washing procedure was performed five times. Specimens were exposed to 25 PM teniposide (BristolMyers) either before fertilization (15-60 min) or at specific times during oocyte maturation, fertilization, and early embryogenesis. Teniposide was dissolved in dimethyl sulfoxide (DMSO) as a stock solution of 15.2 mM and was diluted in ASW. The final DMSO concentration which was 0.17% did not affect Spisula development or ultrastructure (Longo, 1972). The teniposide concentration of 25 PM was obtained by exposing oocytes/zygotes to various inhibitor concentrations (0.1-100 PM) and determining the percentage at which polar body formation and cleavage were reversibly inhibited. At 25-100 PM, polar body formation and mitosis were blocked although sperm nuclear decondensation was unaffected. With lower doses (10 FM), only 52% of the embryos exhibited delayed development, and oocytes treated with 1 PM or less developed into trochophore larvae in concert with the controls. To examine nuclear events, synchronous populations of treated and control zygotes incubated at 20°C were either cultured overnight, or fixed at 5- to lo-min intervals and stained with 10 PM Hoechst 33342 (Sigma Chemical Co., St. Louis, MO) as previously described (Luttmer and Longo, 1986,1988). Fluorescence Microscopy
All observations were performed using a Zeiss axiophot microscope equipped with epifluorescence and a Zeiss 100X Plan-Neofluar 1.3 NA oil immersion objective. Specimens were photographed with Kodak Tri-X film (ASA 1600) which was developed in Diaphine (Accuphine, Chicago, IL). The epifluorescence micrographs shown each represent a typical embryo under the control or treatment conditions. Differences between panels reflect actual effects of treatments and do not depend on a particular view of the cell.
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RESULTS
Normal Development
Fertilization events in untreated specimens were the same as those previously reported (Allen, 1953; Longo and Anderson, 1970a,b; Luttmer and Longo, 1986,1988). A brief description of controls is provided to place the events observed in the treated oocytes and embryos in their proper perspective (Fig. 1). The germinal vesicle (Fig. 1A) broke down by 10 min postinsemination (min PI) and the sperm nucleus initially decondensed. From 20 to 45 min PI, the polar bodies formed with first meiotic metaphase at 20 min PI (Fig. 1B) and second meiotic metaphase at 40 min PI. Second meiotic anaphase occurred by 45 min PI (Fig. 1C). The sperm nucleus partially recondensed during polar body formation (compare Figs. 1B and 1C). Between 45 and 60 min PI, the paternal and maternal chromatin decondensed in concert to form a male and female pronucleus, respectively (Fig. 1D). By 70 min PI, the maternal and paternal chromatin intermixed on the metaphase plate of first mitosis, and by 90 min PI, the 2-cell embryo was in interphase (Fig. 1E). The chromosomes were aligned in metaphase of second mitosis by 105 min PI and were decondensed in the 4-cell embryo by 120 min PI (Fig. 1F). Third and fourth mitosis occurred by 140 and 175 min PI, respectively, and 8- and 16-cell embryos had developed by 160 and 190 min PI, respectively. The topoisomerase II inhibitor, teniposide, was added to either unfertilized oocytes or normally developing oocytes at specific stages of meiotic maturation and development. The effects of teniposide on specific nuclear events were determined, and results of the experiments are categorized below according to the stage of development at the time of teniposide treatment. Teniposide Treatment before Fertilization
When added before fertilization, teniposide did not induce germinal vesicle breakdown in unfertilized oocytes (Fig. 1A’). Moreover, the topoisomerase II inhibitor did not affect sperm motility or fertilization of pretreated oocytes. In oocytes pretreated with teniposide for either 15 or 60 min, nuclear events during the first 20 min PI, such as germinal vesicle breakdown, initial sperm nuclear decondensation, and alignment of the maternal chromatin on the metaphase plate of the first meiotic spindle, were similar to those of the control even at higher (100 PM) teniposide doses (Fig. 1B’). In contrast, later events of meiotic maturation differed markedly from those of the control. Chromosome separation and polar body formation were inhibited with the maternal chromatin remaining in one entangled mass (Fig. 1C’). Although the paternal chromatin
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condensed on schedule, both the paternal and maternal chromatin remained condensed until 65 min PI, well after controls had developed pronuclei and entered first mitotic prophase (Fig. 1D’). When controls were at the two-cell stage, the paternal chromatin and tetraploid mass of maternal chromosomes of teniposide-treated oocytes decondensed and the adjacent male pronucleus became abnormally enlarged (Fig. 1E’). Both the paternal and maternal chromatin partially condensed when controls were at the four-cell stage (Fig. 1F’). Further development was arrested in oocytes pretreated with teniposide in that chromosome condensation during mitotic prophase and first cleavage were blocked (Fig. 1F’). Before fertilization in Spisula, the maternal tetrad chromosomes of the germinal vesicle are still crossed over (Longo, 1983). The failure of oocytes pretreated with teniposide to undergo first polar body formation indicates that topoisomerase II may be functional during first meiosis. One possibility is that topoisomerase II may act to resolve the entangled maternal chromosomes prior to anaphase I of meiosis. Teniposide
Treatment
during
Meiosis
If topoisomerase II plays a role in separating interlocked chromosomes during first meiosis, we would expect teniposide treatment at metaphase I of meiosis to have no affect on chromosome separation during first polar body formation since the chromosomes are already separated at the time of teniposide treatment. However, chromatid separation during second polar body formation may be affected since the chromatids are still entangled when the oocytes are treated with teniposide (at metaphase I of meiosis). As expected, events associated with first polar body formation were not affected by teniposide treatment at 20 min PI, a time when the maternal chromosomes were already condensed in metaphase I of meiosis (Fig. 2A). Chromosome separation during anaphase I and subsequent extrusion of the first polar body were similar to those of the control. In addition, corresponding sperm nuclear
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condensation and orientation of the maternal chromosomes on the second meiotic spindle occurred on schedule. However, teniposide disrupted second polar body formation. Chromatid separation during anaphase II was blocked and a second polar body did not form (Fig. 2B). The maternal chromosomes remained condensed as a diploid mass 5 min longer than those in controls, when they began decondensing into one diploid pronucleus in concert with male pronuclear formation. The zygotes arrested with decondensed pronuclei; consequently, chromosome condensation and cleavage during mitosis were blocked while controls continued development (compare Figs. 1E and 2C). These observations suggest that topoisomerase II may play a role not only in separating chromosome interlocks during first meiosis, but also in chromatid separation during second meiosis. If topoisomerase II has an additional role in resolving interlocked chromatids, teniposide should have no effect on second polar body formation when added after the chromatids have normally separated (e.g., during metaphase II of meiosis). As expected, chromatid separation during second polar body formation was unaffected when fertilized oocytes in metaphase II of meiosis were treated with teniposide at 40 min PI (Fig. 3A). After the second polar body developed, the maternal and paternal chromatin decondensed into pronuclei at the same time as those in controls (Fig. 3B). By 65 min PI, the chromosomes began condensing as those in controls, but normal prophase chromosome condensation into distinct karyomeres was inhibited, resulting in blocked mitosis and cleavage (compare Figs. 1E and 3C). These results suggest that topoisomerase II may not only function in chromosome/chromatid separation during meiosis I and II, but also in chromosome condensation during mitosis.
Teniposide
Treatment
during
Mitosis
Meiosis II is similar to mitosis in that each involves separation of chromatids whereas meiosis I involves sep-
FIG. 1. Teniposide prevents chromosome separation during meiosis and chromosome condensation during mitosis, but not chromosome decondensation in fertilized Spisula oocytes. Controls (A-F) and oocytes treated with 25 PLM teniposide 30 min prior to fertilization (A-F’). A,A’, tetrad maternal chromosomes (arrows) along the periphery of the germinal vesicle of an unfertilized oocyte. After fertilization, the germinal vesicle breaks down (10 min PI) and the maternal chromosomes (f) align on the metaphase plate of meiosis I (20 min PI) while the sperm nucleus (m) decondenses (B). Development of teniposide-treated oocytes appeared normal until after metaphase I of meiosis (B’). Chromosome separation and polar body formation (C) were inhibited while condensation of the paternal chromatin, which normally takes place during polar body development (20-45 min PI), was unaffected (C’). The maternal (f) and paternal (m) chromatin remained condensed (D’) while that of controls decondensed in concert to form pronuclei (D) which were in early prophase. By 90 min PI, two-cell embryos were in interphase (E) while an abnormally large male pronucleus was observed next to the entangled mass of maternal chromosomes in treated oocytes (E’). In controls, second mitosis began by 100 min PI, and by 120 min PI interphase four-cell embryos formed (F); whereas chromosome condensation during mitosis was inhibited in treated oocytes (F’). The time postinsemination (min PI) is at the lower left corner of each panel. “Tenip,” oocytes treated with 25 p&f teniposide 30 min before fertilization; (1) first polar body; (2) second polar body. Bar 10 Frn.
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FIGS. 2 AND 3. Teniposide prevents chromatid separation during meiosis and chromosome condensation during mitosis, but does not affect chromosome decondensation. The time postinsemination (min PI) is at the lower left corner of each panel. Bar, 10 pm. FIG. 2. Fertilized oocytes treated with 25 &fteniposide during metaphase I of meiosis showing in (A) the decondensed sperm nucleus (m) and maternal chromosomes (f) aligned on the metaphase plate. The maternal chromosomes later separated and formed the first polar body (1) but a second polar body did not develop. The haploid paternal and diploid maternal genomes underwent decondensation (B), but did not condense in preparation for mitosis (C). Consequently, mitosis and cleavage were inhibited. FIG. 3. Fertilized oocytes treated with 25 PM teniposide during metaphase II of meiosis showing in (A) the condensed paternal ehromatin (m), first polar body (l), and maternal chromosomes (f) aligned on the metaphase plate. The maternal chromatids later separated and the second polar body (2) formed. Male and female pronuclei developed (8) and the chromosomes partially condensed (C); however, mitosis and cleavage were blocked.
aration of homologous chromosomes. Since chromatid separation and decondensation were not inhibited in oocytes treated with teniposide during metaphase II of meiosis, but chromosome condensation during the next cell cycle was blocked, we expected oocytes treated with teniposide during metaphase of mitosis to complete the present round of mitosis but become arrested in interphase during the next cell cycle. Indeed, zygotes treated with teniposide when the chromosomes were already condensed in metaphase of first mitosis (70 min PI; Fig. 4A) demonstrated normal chromosome separation during anaphase, first cleavage, and nuclear decondensation of two-cell blastomeres (Fig. 4B). However, chromosome condensation during prophase of second mito-
sis failed and nuclei remained decondensed and prominent while controls continued development (compare Figs. 1F and 4B). These results suggest that topoisomerase II may be required for mitotic chromosome condensation, but not decondensation. Similarly, when two-cell embryos were treated with teniposide during metaphase of second mitosis (at 105 min PI), no effects were observed until the following (third) mitosis when the four-cell embryos were arrested in interphase. Chromosome separation, decondensation, and cleavage were similar to those in the control resulting in normal four-cell embryos, but chromosome condensation of the next mitosis was blocked (data not shown). These results confirm that chromosome
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FIGS. 4 AND 5. Teniposide does not prevent chromosome decondensation, but blocks chromosome condensation after one cell cycle. The time postinsemination (min PI) is at the lower left corner of each panel. Bars, 10 pm. FIG. 4. A zygote treated with 25 PM teniposide during metaphase of first mitosis (A) showing condensed chromosomes and the first (1) and second (2) polar bodies. Although chromosome separation, decondensation, and cleavage were unaffected, chromosome condensation during the next mitosis was blocked (B). FIG. 5. A two-cell embryo in interphase at the time of 25 PM teniposide treatment showing two decondensed nuclei (A). Chromosomes condensed and separated during mitosis (B), but chromosome condensation during the next mitosis was blocked and the nuclei remained decondensed (C).
condensation during later cell cycles, but not decondensation, requires topoisomerase II activity. Teniposide Treatment during Interphase
Since during the first cell cycle, teniposide inhibited chromosome condensation in all cases whether it was added before fertilization or during meiosis, we expected chromosome condensation during the next mitosis to be blocked in embryos treated with teniposide during interphase of later cell cycles. In contrast, when two-cell embryos at interphase were treated with teniposide at 90 min PI (Fig. 5A), chromosome condensation
and separation during second mitosis (Fig. 5B), cleavage, and nuclear decondensation occurred on schedule (Fig. 5C). It was not until the next cell cycle that teniposide prevented the resulting interphase embryos at the four-cell stage from undergoing any further chromosome condensation. Cleavage into eight-cell embryos was blocked with the nuclei remaining decondensed and prominent while controls continued development (Fig. 5C). These results indicate that topoisomerase II functions in chromosome condensation, but not decondensation, during later cell cycles. Similarly, when four-cell embryos at interphase (120 min PI) were treated with teniposide, chromosome con-
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Later Cell Cycles
FIG. 6. Summary of effects of the topoisomerase II inhibitor, teniposide, on chromosome and nuclear cycles during fertilization and the first cell cycle (top), and later cell cycles (bottom). Whether teniposide was added (double arrows) before fertilization (top, first cell) or during metaphase of meiosis I or II (top, second cell), the inhibitor blocked chromosome condensation during mitosis (solid black bar), but not decondensation of the sperm nucleus and maternal chromosomes (top, third cell). During later cell cycles, the effects on chromosome condensation were different. The interphase cell in the bottom display represents one blastomere of an embryo. Chromatin of interphase embryos (bottom, first cell) condensed in the presence of teniposide (double arrows) underwent mitosis and decondensed, but condensation was blocked during the next cell cycle. When mitotic embryos containing condensed chromosomes (bottom, second cell) were treated with teniposide, mitosis continued and chromosomes decondensed but the next mitosis was inhibited. Irrespective of when teniposide was added, the inhibitor blocked chromosome condensation, but not decondensation, suggesting that topoisomerase II may be involved in chromosome condensation but not decondensation.
densation and separation during third mitosis, cleavage, and nuclear decondensation were unaffected (data not shown). However, chromosome condensation during fourth mitosis was inhibited and nuclei of the eight-cell embryos remained decondensed while controls continued development (data not shown). These observations confirm that topoisomerase II activity is also involved in chromosome condensation during later cell cycles, but not in decondensation of somatic chromatin. DISCUSSION
Our results summarized in Fig. 6 show that treatment with teniposide at various stages of oocyte meiotic maturation and early Spisula embryogenesis has two principal effects: during the first cell cycle, it prevents both meiotic chromosome separation and mitotic chromosome condensation; and during later cell cycles, it blocks chromosome condensation only after the embryos have progressed through the next mitotic cycle. Surprisingly, teniposide does not affect decondensation of sperm nuclei, maternal or somatic chromosomes. DNA topoisomerase II has been found in diverse species from bacteria to man (Hsieh and Brutlag, 1980; Baldi et al., 1980; Miller et aZ., 1981; Goto and Wang, 1982;
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Halligan et aL, 1985; Wang, 1985; Rota and Mezquita, 1989). Various studies demonstrate topoisomerase II in gametes and embryos of Drosophila, Xenopus, and chicken, and thus the enzyme is most likely also present in Spisula oocytes and embryos although not directly shown here (Hsieh and Brutlag, 1980; Baldi et aZ., 1980; Rota and Mezquita, 1989). At the molecular level, topoisomerase II separates interlocked loops of kinetoplast DNA (Marini et aZ., 1980; Luke and Bogenhagen, 1989). In somatic cells, it is required to untangle intertwined chromosomal DNA during anaphase of yeast mitosis (DiNardo et aZ., 1984; Uemura and Yanagida, 1984,1986; Holm et ak, 1985,1989; Uemura et aZ., 1987). It has been proposed that the enzyme could play an analogous role in meiosis where it may be responsible for resolution of chromosome interlocks that occur during pairing of homologous chromosomes (Moens and Earnshaw, 1989; Rose et aZ., 1990). Teniposide is known as a specific and potent inhibitor of topoisomerase II. In vitro, teniposide blocks topoisomerase II activity by binding to the DNA-topoisomerase II complex, preventing ligation of the doublestranded DNA cut (Chen et al, 1984; Long et al, 1984). Our investigation shows that teniposide disrupts meiotic maturation by preventing chromosome separation, and suggests an involvement of topoisomerase II in chromosome and chromatid separation during meiosis in Spisula. Consistent with this hypothesis, topoisomerase II has been identified by immunocytochemistry in meiotic chromosomes and its concentration increases during meiotic maturation (Luke and Bogenhagen, 1989; Moens and Earnshaw, 1989). Similarly, topoisomerase II has been identified in mitotic chromosomes and the levels of topoisomerase II fluctuate during the cell cycle of cultured cells, being high during mitosis and rapidly declining at G, (Earnshaw and Heck, 1985; Earnshaw et aZ., 1985; Berrios et al, 1985; Gasser et al, 1986; Heck et al., 1988; Heck and Earnshaw, 1988). During meiosis and the cell cycle, other Spisula proteins such as the cyclins also undergo oscillations in quantity and activity through phosphorylation (Swenson et a& 1986,1989; Westendorf et aL, 1989). Although little is known about the mechanism to modulate topoisomerase II activity, several reports indicate that phosphorylation plays a role in regulating topoisomerase II activity (Ackerman et al., 1985, 1988; Sahyoun et al., 1986; Heck et ah, 1989; Saijo et al, 1990). Perhaps these fluctuations in topoisomerase II may account for the dissimilarity of teniposide reactivity in embryos treated during the first and later cell cycles (Fig. 6). The burst in protein synthesis and phosphorylation that occurs at fertilization could provide a means to achieve this difference (Masui, 1985; Swenson et al, 1989). In older embryos, higher levels of a more active, perhaps phosphor-
WRIGHT AND SCHATTEN
Topoismerase
ylated form of topoisomerase II may explain the dissimilarity of teniposide reactivity in embryos in the first and later cell cycles. Whether embryos were in meiosis, interphase, or mitosis at the time of application, teniposide blocked mitotic chromosome condensation and the nuclei remained decondensed in interphase. This observation is not unexpected since chromosome condensation is teniposidesensitive in Xenopus egg extracts (Newport and Spann, 1987). In addition our results agree with genetic evidence that mutants defective in topoisomerase II are unable to undergo chromosome condensation (Uemura et al, 1987). Thus, we propose that topoisomerase II may be required for chromosome condensation during mitosis in Spisula. In contrast, our results revealed that teniposide did not affect decondensation of sperm nuclei, maternal chromatin, or somatic nuclei, even at higher teniposide doses. This is surprising in light of studies demonstrating that teniposide blocks decondensation of sperm nuclei and metaphase chromosomes in Xenopus egg extracts (Newport, 1987). The reason for this discrepancy is unclear but may be related to the different systems used to study decondensation; namely, intact Spisula oocytes versus cell-free Xenopus egg extracts. That the paternal chromatin of teniposide-treated specimens remained condensed while polar body formation was inhibited is consistent with previous studies in Spisula demonstrating that sperm nuclear transformations are coordinate with stages of meiotic maturation (Da-Yuan and Longo, 1983; Luttmer and Longo, 1988). Moreover, that the paternal and maternal chromatin decondensed simultaneously into pronuclei after both processes were delayed, further indicates a coordinate regulation of the maternal and paternal chromatin in Spisula oocytes. An alternative explanation for our results could have been that teniposide exerts other cellular effects besides inhibiting topoisomerase II. Although teniposide is a specific inhibitor of topoisomerase II in vitro, the in viva mechanism of teniposide action has not been elucidated. Teniposide could possibly be toxic, impermeable, or even disrupt microtubules. However, these seem unlikely since: (1) teniposide effects are reversible, (2) the teniposide analog, podophyllotoxin, rapidly penetrates eggs (Schatten and Schatten, 1981), (3) Spisula embryos treated with teniposide develop normal spindles as determined by antitubulin immunofluorescence (data not shown), and (4) teniposide does not affect microtubule assembly (Grieder et al, 1974). The lack of an effect on chromosome decondensation may reflect the need for a higher teniposide concentration, however, the same results were obtained with higher doses indicating that not all nuclear events require topoisomerase II. One could consider that inhibiting polar body formation may
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prevent chromosome separation independent of topoisomerase II activity. However, this seems unlikely since in Spisula oocytes treated with cytochalasin B, chromosome separation and decondensation can occur if cytokinesis is blocked (Longo, 1972; Luttmer and Longo, 1988). Another potential side effect of teniposide may include the numerous cuts in the DNA that persist when teniposide prevents topoisomerase II from resealing the cuts that the enzyme normally makes in the DNA strands. While we can not dismiss the possibility that the numerous cuts in the DNA alone could cause the effects observed on chromosome separation and condensation, we consider this an indirect effect of blocking topoisomerase II activity. Since these potential indirect effects seem unlikely, we think that the effects of teniposide on nuclear events during Spisula fertilization are due to a specific inhibition of topoisomerase II. In summary, we conclude that teniposide disrupts chromosome separation during meiosis and chromosome condensation during mitosis, but does not affect decondensation of sperm nuclei, maternal chromosomes, or somatic chromatin in Spisula. Therefore, we suggest that topoisomerase II may play both a structural and enzymatic role in regulating chromosome interactions during fertilization and development. The authors gratefully acknowledge the generous gift of teniposide from Bristol-Myers. We thank Dr. David J. Wright for his comments on the manuscript. The support of this research by grants from the NIH, including an individual NIH-NRSA Postdoctoral Fellowship to S.J.W. is acknowledged gratefully. The Madison IMR is supported as an NIH Biomedical Research Technology Resource. REFERENCES ACKERMAN, P., GLOVER, C. V. C., and OSHEROFF,N. (1985). Phosphorylation of DNA topoisomerase II by casein kinase II: Modulation of eukaryotic topoisomerase II activity in vitro. Proc. Nat1 Acad. Sci. USA 83,1603-1607.
ACKERMAN, P., GLOVER, C. V. C., and OSHEROFF,N. (1988). Phosphorylation of DNA topoisomerase II in vivo and in total homogenates of Drosophila Kc cells: The role of casein kinase II. J. Biol. Chem. 263, 12,653-12,660. ALLEN, R. D. (1953). Fertilization and artificial activation in the egg of the surf-clam, Stisula solidissima. Biol. Bull. 105,213-239. BALDI, M. I., BENEDETTI, P., MA?TOCCIA, E., and TOCCHINI-VALENTINI, G. P. (1980). 1n vitro catenation and decatenation of DNA and a novel eucaryotic ATP-dependent topoisomerase. Cell 20,461-467. BERRIOS, M., OSHEROFF, N., and FISHER, P. A. (1985). In situ localization of DNA topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix fraction. Proc. Natl. Acad Sci. USA 82,4142-4146. CHEN, G. L., YANG, L., ROWE, T. C., HALLIGAN, B. D., TEWEY, K. M., and LIU, L. F. (1984). Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. J. Biol. Chem. 259, 13,560-13,566. COZARELLI, N. R. (1980). DNA gyrase and the supercoiling of DNA. Science 207,953-960. DA-YUAN, C., and LONGO, F. J. (1983). Sperm nuclear dispersion coordinate with meiotic maturation in fertilized Spisula solidissima eggs. Dev.
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DINARDO, S., BOELKEL, K., and STERNGLANZ, R. (1984). DNA topoisomerase II mutant of Succharomvces ceruisiae: Topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication. Proc. NutL Acad Sci. USA 81,2616-2620. EARNSHAW, W. C., and HECK, M. M. S. (1985). Localization of topoisomerase II in mitotic chromosomes. J. Cell BioL 100,1716-1725. EARNSHAW, W. C., HALLIGAN, B., COOKE, C. A., HECK, M. M. S., and LIU, L. F. (1985). Topoisomerase II is a structural component of mitotic chromosome scaffolds. J. Cell BioL 100,1706-1715. GASSER, S. M., LAROCHE, T., FALQUET, J., BOY DE LA TOUR, E., and LAEMMLI, U. K. (1986). Metaphase chromosome structure involvement of topoisomerase II. J Mol. BioL 188, 613-629. GELLERT, M. (1981). DNA topoisomerases. Annu. Rev. Biochem. 50, 879-910. GOTO, T., and WANG, J. C. (1982). Yeast DNA topoisomerase II, an ATP-dependent type II topoisomerase that catalyzes the catenation, decatenation, unknotting, and relaxation of double-stranded DNA rings. J. BioL Chem 257,5866-5872. GRIEDER, A., MAURER, R., and STAHELIN, H. (1974). Effect of an epipodophyllotoxin derivative (VP 16-213) on macromolecular synthesis and mitosis in mastocytoma cell in vitro. Cancer Res. 34,1788-1793. HALLIGAN, B. D., EDWARDS, K. A., and LIU, L. F. (1985). Purification and characterization of a type II DNA topoisomerase from bovine calf thymus. J. BioL Chem 260.2475-2482. HECK, M. M. S., and EARNSHAW, W. C. (1988). DNA topoisomerase II in chromosome structure and cell proliferation. In “Self-Assembling Architecture” (J. E. Varner, Ed.), 46th Symposium of the Society for Developmental Biology, pp. 243-263. A. R. Liss, New York. HECK, M. M. S., HI’ITELMEN, W. N., and EARNSHAW, W. C. (1988). Differential expression of DNA topoisomerase I and II during the eukaryotic cell cycle. Proc. Natl. Acad Sci. USA 85,1086-1090. HECK, M. M. S., HITPELMEN, W. N., and EARNSHAW, W. C. (1989). In viva phosphorylation of the 170-kDa form of eukaryotic DNA topoisomerase II. J. BioL Chem. 264, 15,161-15,164. HOLM, C., GOTO, T., WANG, J. C., and BOTSTEIN, D. (1985). DNA topoisomerase II is required at the time of mitosis in yeast. Cell 41, 553-563. HOLM, C., STEARNS, T., and BOTSTEIN, D. (1989). DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage. MoL Cell. BioL 9,159-168. HSIEH, T.-S., and BRUTLAG, D. (1980). ATP-dependent DNA topoisomerase from D. melanogaster reversibly catenates duplex DNA rings. Cell 27,115-125. LIU, L. F. (1983). DNA topoisomerases-enzymes that catalyze breaking and rejoining of DNA. CRC Crit. Rev. B&hem. 15,1-23. LONG, B. H., MUSIAL, S. T., and BRA~AIN, M. G. (1984). Comparison of cytotoxicity and DNA breakage activity of congeners of podophyllotoxin including VP16-123 and VM26: A quantitative structureactivity relationship. Biochemistry 23,1183-1188. LONGO, F. J. (1972). The effects of cytochalasin B on the events of fertilization in the surf clam, Spisula solidissima. I. Polar body formation. J. Exp. ZooL 182,321-344. LONGO, F. J. (1983). Meiotic maturation and fertilization. In “The Mollusca” (N. H. Verdonk and J. A. M. van den Biggelaar, Eds.), Vol. 3, pp. 251-298. Academic Press, New York. LONGO, F. J., and ANDERSON, E. (1970a). An ultrastructural analysis of fertilization in the surf clam, Spisula solidissima. I. Polar body formation and development of the female pronucleus. J. Ultrastruct. Res. 33.495-514.
LONGO, F. J., and ANDERSON, E. (1970b). An ultrastructural analysis of fertilization in the surf clam, Spisula solidissima. II. Development of the male pronucleus and the association of the maternally and paternally derived chromosomes. J. Ultra&w% Res. 33.515-527. LUKE, M., and BOGENHAGEN, D. F. (1989). Quantitation of type II topoisomerase in oocytes and eggs of Xenopus luevis. Dev. BioL 136, 459-468.
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LU~MER, S. J., and LONGO, F. J. (1986). Examination of living and fixed gametes and early embryos stained with supravital fluorochromes (Hoechst 33342 and 3,3’-dihexyloxacarbocyanine iodide). Gamete Res. 15,267-283. LUTI!MER, S. J., and LONGO, F. J. (1988). Sperm nuclear transformations consist of enlargement and condensation coordinate with stages of meiotic maturation in fertilized Spisulu solidissima oocytes. Dev. BioL 128, 86-96. MARINI, J. C., MILLER, K. G., and ENGLUND, P. T. (1980). Decatenation of kinetoplast DNA by topoisomerases. J. BioL Chem. 255, 49764979.
MASUI, Y. (1985). Problems of oocyte maturation and the control of chromosome cycles. Dm. Growth mer. 27,295-309. MILLER, K. G., LIU, L. F., and ENGLUND, P. T. (1981). A homogeneous type II DNA topoisomerase from HeLa cell nuclei. J. BioL Chem. 256,93349339. MOENS, P. B., and EARNSHAW, W. C. (1989). Anti-topoisomerase II recognizes meiotic chromosome cores. Chromosoma g&317-322. NEWPORT, J. (1987). Nuclear reconstitution in vitro: Stages of assembly around protein-free DNA. Cell 48,205-217. NEWPORT, J., and SPANN, T. (1987). Disassembly of the nucleus in mitotic extracts: Membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes. Cell 48, 219-230.
ROCA, J., and MEZQUITA, C. (1989). DNA topoisomerase II activity in nonreplicating, transcriptionally inactive, chicken late spermatids. EMBO
J. 8,1855-1860.
ROSE, D., THOMAS, W., and HOLM, C. (1990). Segregation of recombined chromosomes in meiosis I requires DNA topoisomerase II. Cell 60,1009-1017. SAIJO, M., ENOMOTO, T., HANAOKA, F., and UI, M. (1990). Purification and characterization of type II DNA topoisomerase from mouse FM3A cells: Phosphorylation of topoisomerase II and modification of its activity. Biochemistry 29,583-590. SAHYOUN, N., WOLF, M., BESTERMAN, J., HSIEH, T.-S., SANDER, M., LEVINE, H., III, CHANG, K.-J., and CUATRECASAS, P. (1986). Protein kinase C phosphorylates topoisomerase II: Topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells. Proc. NatL Acad. Sci. USA 83,1603-1607. SCHATTEN, G., and SCHATTEN, H. (1981). Effects of motility inhibitors during sea urchin fertilization. Exp. Cell Res. 135,311-330. SWENSON,K. I., FARRELL, K. M., and RUDERMAN, J. V. (1986). The clam embryo protein cyclin A induces entry into M phase and the resumption of meiosis in Xenopus oocytes. Cell 47,861-870. SWENSON, K., WESTENDORF, J., HUNT, T., and RUDERMAN, J. (1989). Cyclins and regulation of the cell cycle in early embryos. In “Molecular Biology of Fertilization” (H. Schatten and G. Schatten, Eds.), pp. 212-232. Academic Press, New York. UEMURA, T., and YANAGIDA, M. (1984). Isolation of type I and II DNA topoisomerase mutants from fission yeast: Single and double mutants show different phenotypes in cell growth and chromatin organization. EMBO J. 3,1737-1744. UEMURA, T., and YANAGIDA, M. (1986). Mitotic spindle pulls but fails to separate chromosomes in type II DNA topoisomerase mutants: Uncoordinated mitosis. EMBO J. 5,1003-1010. UEMURA, T., OHKURA, H., ADACHI, Y., MORINO, K., SHIOZAKI, K., and YANAGIDA, M. (1987). DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S pornbe. Cell 50, 917-925.
WANG, J. C. (1985). DNA topoisomerases.
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WESTENDORF, J. M., SWENSON, K. I., and RUDERMAN, J. V. (1989). The role of cyclin B in meiosis I. J. Cell BioL 108,1431-1444. WRIGHT, S. J., and SCHATTEN, G. (1988). Chromosome condensation but not pronuclear decondensation is blocked by a topoisomerase II inhibitor during fertilization and mitosis in surf clams, mice and sea urchins. J. Cell Biol. 107,172a.
BIOLOGY
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Teniposide, a Topoisomerase II Inhibitor, Prevents Chromosome Condensation and Separation but Not Decondensation in Fertilized Surf Clam (Spisula solidissima) Oocytes SHIRLEY J. WRIGHT'ANDGERALDSCHATTEN Integrated
Microscopy
Resource for Biomedical Research, Zoology Research Building, 1117 West Johnson Street, Madison, Wisconsin 53706
University
of Wisconsin,
Accepted July 23, 1990 DNA topoisomerase II has been implicated in regulating chromosome interactions. We investigated the effects of the specific DNA topoisomerase II inhibitor, teniposide on nuclear events during oocyte maturation, fertilization, and early embryonic development of fertilized S;oisula solidissima oocytes using DNA fluorescence. Teniposide treatment before fertilization not only inhibited chromosome separation during meiosis, but also blocked chromosome condensation during mitosis; however, sperm nuclear decondensation was unaffected. Chromosome separation was selectively blocked in oocytes treated with teniposide during either meiotic metaphase I or II indicating that topoisomerase II activity may be required during oocyte maturation. Teniposide treatment during meiosis also disrupted mitotic chromosome condensation. Chromosome separation during anaphase was unaffected in embryos treated with teniposide when the chromosomes were already condensed in metaphase of either first or second mitosis; however, chromosome condensation during the next mitosis was blocked. When interphase two- and four-cell embryos were exposed to topoisomerase II inhibitor, the subsequent mitosis proceeded normally in that the chromosomes condensed, separated, and decondensed; in contrast, chromosome condensation of the next mitosis was blocked. These observations suggest that in Spisula oocytes, topoisomerase II activity is required for chromosome separation during meiosis and condensation during mitosis, but is o 1990 Academic not involved in decondensation of the sperm nucleus, maternal chromosomes, and somatic chromatin. Press, Inc.
Topoisomerase II is a major structural component of the nuclear matrix and is the most abundant nonhistone protein of the metaphase chromosome scaffold (Earnshaw and Heck, 1985; Earnshaw et aL, 1985; Berrios et al., 1985; Gasser et al., 1986; see Heck and Earnshaw, 1988 for a review). Topoisomerase II acts to alter the topological conformation of DNA by making a doublestrand break, passing another double-strand through the break, and then resealing it (Cozarelli, 1980; Gellert, 1981; Liu, 1983; Wang, 1985). Studies with yeast topoisomerase mutants demonstrate that topoisomerase II is required for chromosome condensation and separation during anaphase of mitosis (Uemura and Yanagida, 1984, 1986; Holm et aZ., 1985, 1989; Uemura et aZ., 1987). The role of topoisomerase II in higher eukaryotes is not understood. However, studies using a topoisomerase II inhibitor (Chen et ah, 1984) and cell-free extracts derived from Xenopus oocytes have suggested that topoisomerase II is involved in both chromosome condensation and decondensation (Newport, 1987; Newport and Spann, 1987). If topoisomerase II is involved in chromosome interactions during oocyte maturation and fertilization, blocking topoisomerase II activity should arrest chromosome condensation, separation, and decondensation.
INTRODUCTION
The organization of the nucleus changes dynamically during the cell cycle especially in rapidly dividing cells. Fertilization and early development are particularly well suited for studying these alterations. Spisula solidissima (surf clam) oocytes are spawned at the germinal vesicle stage and are arrested at meiotic prophase until fertilization which triggers both meiotic maturation and zygote development. During fertilization, the maternal chromatin undergoes phases of condensation, separation, and decondensation that occur during germinal vesicle breakdown, polar body formation, and female pronuclear development, while the highly condensed sperm nucleus undergoes phases of decondensation and condensation closely coupled to the meiotic changes of the maternal chromatin (Da-Yuan and Longo, 1983; Luttmer and Longo, 1988). The mechanisms underlying these dramatic changes in chromatin organization during fertilization are not understood, however, the enzyme, DNA topoisomerase II may play an important role. 1 To whom correspondence should be addressed. OOlZ-1606/90 $3.00 Copyright All rights
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
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To test this hypothesis, we have examined the effects of the specific, DNA topoisomerase II inhibitor, teniposide, on nuclear events during meiosis, fertilization, and early embryogenesis of fertilized surf clam (S. solidissima) oocytes. A preliminary account of this investigation has been reported (Wright and Schatten, 1988). MATERIALS
AND
METHODS
Specimen Preparation
Gonads were removed from mature S. solidissima obtained from the Marine Biological Laboratory (Woods Hole, MA). Testes were collected and then filtered through cheesecloth. Sperm were stored undiluted until used. Oocytes, obtained by mincing the ovaries in artificial seawater (ASW) and filtering through cheesecloth, were suspended to 1 liter in ASW, allowed to settle, and the supernatant was decanted. This washing procedure was performed five times. Specimens were exposed to 25 PM teniposide (BristolMyers) either before fertilization (15-60 min) or at specific times during oocyte maturation, fertilization, and early embryogenesis. Teniposide was dissolved in dimethyl sulfoxide (DMSO) as a stock solution of 15.2 mM and was diluted in ASW. The final DMSO concentration which was 0.17% did not affect Spisula development or ultrastructure (Longo, 1972). The teniposide concentration of 25 PM was obtained by exposing oocytes/zygotes to various inhibitor concentrations (0.1-100 PM) and determining the percentage at which polar body formation and cleavage were reversibly inhibited. At 25-100 PM, polar body formation and mitosis were blocked although sperm nuclear decondensation was unaffected. With lower doses (10 FM), only 52% of the embryos exhibited delayed development, and oocytes treated with 1 PM or less developed into trochophore larvae in concert with the controls. To examine nuclear events, synchronous populations of treated and control zygotes incubated at 20°C were either cultured overnight, or fixed at 5- to lo-min intervals and stained with 10 PM Hoechst 33342 (Sigma Chemical Co., St. Louis, MO) as previously described (Luttmer and Longo, 1986,1988). Fluorescence Microscopy
All observations were performed using a Zeiss axiophot microscope equipped with epifluorescence and a Zeiss 100X Plan-Neofluar 1.3 NA oil immersion objective. Specimens were photographed with Kodak Tri-X film (ASA 1600) which was developed in Diaphine (Accuphine, Chicago, IL). The epifluorescence micrographs shown each represent a typical embryo under the control or treatment conditions. Differences between panels reflect actual effects of treatments and do not depend on a particular view of the cell.
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RESULTS
Normal Development
Fertilization events in untreated specimens were the same as those previously reported (Allen, 1953; Longo and Anderson, 1970a,b; Luttmer and Longo, 1986,1988). A brief description of controls is provided to place the events observed in the treated oocytes and embryos in their proper perspective (Fig. 1). The germinal vesicle (Fig. 1A) broke down by 10 min postinsemination (min PI) and the sperm nucleus initially decondensed. From 20 to 45 min PI, the polar bodies formed with first meiotic metaphase at 20 min PI (Fig. 1B) and second meiotic metaphase at 40 min PI. Second meiotic anaphase occurred by 45 min PI (Fig. 1C). The sperm nucleus partially recondensed during polar body formation (compare Figs. 1B and 1C). Between 45 and 60 min PI, the paternal and maternal chromatin decondensed in concert to form a male and female pronucleus, respectively (Fig. 1D). By 70 min PI, the maternal and paternal chromatin intermixed on the metaphase plate of first mitosis, and by 90 min PI, the 2-cell embryo was in interphase (Fig. 1E). The chromosomes were aligned in metaphase of second mitosis by 105 min PI and were decondensed in the 4-cell embryo by 120 min PI (Fig. 1F). Third and fourth mitosis occurred by 140 and 175 min PI, respectively, and 8- and 16-cell embryos had developed by 160 and 190 min PI, respectively. The topoisomerase II inhibitor, teniposide, was added to either unfertilized oocytes or normally developing oocytes at specific stages of meiotic maturation and development. The effects of teniposide on specific nuclear events were determined, and results of the experiments are categorized below according to the stage of development at the time of teniposide treatment. Teniposide Treatment before Fertilization
When added before fertilization, teniposide did not induce germinal vesicle breakdown in unfertilized oocytes (Fig. 1A’). Moreover, the topoisomerase II inhibitor did not affect sperm motility or fertilization of pretreated oocytes. In oocytes pretreated with teniposide for either 15 or 60 min, nuclear events during the first 20 min PI, such as germinal vesicle breakdown, initial sperm nuclear decondensation, and alignment of the maternal chromatin on the metaphase plate of the first meiotic spindle, were similar to those of the control even at higher (100 PM) teniposide doses (Fig. 1B’). In contrast, later events of meiotic maturation differed markedly from those of the control. Chromosome separation and polar body formation were inhibited with the maternal chromatin remaining in one entangled mass (Fig. 1C’). Although the paternal chromatin
DEVELOPMENTALBIOLOGY VOLUME~~~,199o
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condensed on schedule, both the paternal and maternal chromatin remained condensed until 65 min PI, well after controls had developed pronuclei and entered first mitotic prophase (Fig. 1D’). When controls were at the two-cell stage, the paternal chromatin and tetraploid mass of maternal chromosomes of teniposide-treated oocytes decondensed and the adjacent male pronucleus became abnormally enlarged (Fig. 1E’). Both the paternal and maternal chromatin partially condensed when controls were at the four-cell stage (Fig. 1F’). Further development was arrested in oocytes pretreated with teniposide in that chromosome condensation during mitotic prophase and first cleavage were blocked (Fig. 1F’). Before fertilization in Spisula, the maternal tetrad chromosomes of the germinal vesicle are still crossed over (Longo, 1983). The failure of oocytes pretreated with teniposide to undergo first polar body formation indicates that topoisomerase II may be functional during first meiosis. One possibility is that topoisomerase II may act to resolve the entangled maternal chromosomes prior to anaphase I of meiosis. Teniposide
Treatment
during
Meiosis
If topoisomerase II plays a role in separating interlocked chromosomes during first meiosis, we would expect teniposide treatment at metaphase I of meiosis to have no affect on chromosome separation during first polar body formation since the chromosomes are already separated at the time of teniposide treatment. However, chromatid separation during second polar body formation may be affected since the chromatids are still entangled when the oocytes are treated with teniposide (at metaphase I of meiosis). As expected, events associated with first polar body formation were not affected by teniposide treatment at 20 min PI, a time when the maternal chromosomes were already condensed in metaphase I of meiosis (Fig. 2A). Chromosome separation during anaphase I and subsequent extrusion of the first polar body were similar to those of the control. In addition, corresponding sperm nuclear
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Oocytes
condensation and orientation of the maternal chromosomes on the second meiotic spindle occurred on schedule. However, teniposide disrupted second polar body formation. Chromatid separation during anaphase II was blocked and a second polar body did not form (Fig. 2B). The maternal chromosomes remained condensed as a diploid mass 5 min longer than those in controls, when they began decondensing into one diploid pronucleus in concert with male pronuclear formation. The zygotes arrested with decondensed pronuclei; consequently, chromosome condensation and cleavage during mitosis were blocked while controls continued development (compare Figs. 1E and 2C). These observations suggest that topoisomerase II may play a role not only in separating chromosome interlocks during first meiosis, but also in chromatid separation during second meiosis. If topoisomerase II has an additional role in resolving interlocked chromatids, teniposide should have no effect on second polar body formation when added after the chromatids have normally separated (e.g., during metaphase II of meiosis). As expected, chromatid separation during second polar body formation was unaffected when fertilized oocytes in metaphase II of meiosis were treated with teniposide at 40 min PI (Fig. 3A). After the second polar body developed, the maternal and paternal chromatin decondensed into pronuclei at the same time as those in controls (Fig. 3B). By 65 min PI, the chromosomes began condensing as those in controls, but normal prophase chromosome condensation into distinct karyomeres was inhibited, resulting in blocked mitosis and cleavage (compare Figs. 1E and 3C). These results suggest that topoisomerase II may not only function in chromosome/chromatid separation during meiosis I and II, but also in chromosome condensation during mitosis.
Teniposide
Treatment
during
Mitosis
Meiosis II is similar to mitosis in that each involves separation of chromatids whereas meiosis I involves sep-
FIG. 1. Teniposide prevents chromosome separation during meiosis and chromosome condensation during mitosis, but not chromosome decondensation in fertilized Spisula oocytes. Controls (A-F) and oocytes treated with 25 PLM teniposide 30 min prior to fertilization (A-F’). A,A’, tetrad maternal chromosomes (arrows) along the periphery of the germinal vesicle of an unfertilized oocyte. After fertilization, the germinal vesicle breaks down (10 min PI) and the maternal chromosomes (f) align on the metaphase plate of meiosis I (20 min PI) while the sperm nucleus (m) decondenses (B). Development of teniposide-treated oocytes appeared normal until after metaphase I of meiosis (B’). Chromosome separation and polar body formation (C) were inhibited while condensation of the paternal chromatin, which normally takes place during polar body development (20-45 min PI), was unaffected (C’). The maternal (f) and paternal (m) chromatin remained condensed (D’) while that of controls decondensed in concert to form pronuclei (D) which were in early prophase. By 90 min PI, two-cell embryos were in interphase (E) while an abnormally large male pronucleus was observed next to the entangled mass of maternal chromosomes in treated oocytes (E’). In controls, second mitosis began by 100 min PI, and by 120 min PI interphase four-cell embryos formed (F); whereas chromosome condensation during mitosis was inhibited in treated oocytes (F’). The time postinsemination (min PI) is at the lower left corner of each panel. “Tenip,” oocytes treated with 25 p&f teniposide 30 min before fertilization; (1) first polar body; (2) second polar body. Bar 10 Frn.
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FIGS. 2 AND 3. Teniposide prevents chromatid separation during meiosis and chromosome condensation during mitosis, but does not affect chromosome decondensation. The time postinsemination (min PI) is at the lower left corner of each panel. Bar, 10 pm. FIG. 2. Fertilized oocytes treated with 25 &fteniposide during metaphase I of meiosis showing in (A) the decondensed sperm nucleus (m) and maternal chromosomes (f) aligned on the metaphase plate. The maternal chromosomes later separated and formed the first polar body (1) but a second polar body did not develop. The haploid paternal and diploid maternal genomes underwent decondensation (B), but did not condense in preparation for mitosis (C). Consequently, mitosis and cleavage were inhibited. FIG. 3. Fertilized oocytes treated with 25 PM teniposide during metaphase II of meiosis showing in (A) the condensed paternal ehromatin (m), first polar body (l), and maternal chromosomes (f) aligned on the metaphase plate. The maternal chromatids later separated and the second polar body (2) formed. Male and female pronuclei developed (8) and the chromosomes partially condensed (C); however, mitosis and cleavage were blocked.
aration of homologous chromosomes. Since chromatid separation and decondensation were not inhibited in oocytes treated with teniposide during metaphase II of meiosis, but chromosome condensation during the next cell cycle was blocked, we expected oocytes treated with teniposide during metaphase of mitosis to complete the present round of mitosis but become arrested in interphase during the next cell cycle. Indeed, zygotes treated with teniposide when the chromosomes were already condensed in metaphase of first mitosis (70 min PI; Fig. 4A) demonstrated normal chromosome separation during anaphase, first cleavage, and nuclear decondensation of two-cell blastomeres (Fig. 4B). However, chromosome condensation during prophase of second mito-
sis failed and nuclei remained decondensed and prominent while controls continued development (compare Figs. 1F and 4B). These results suggest that topoisomerase II may be required for mitotic chromosome condensation, but not decondensation. Similarly, when two-cell embryos were treated with teniposide during metaphase of second mitosis (at 105 min PI), no effects were observed until the following (third) mitosis when the four-cell embryos were arrested in interphase. Chromosome separation, decondensation, and cleavage were similar to those in the control resulting in normal four-cell embryos, but chromosome condensation of the next mitosis was blocked (data not shown). These results confirm that chromosome
WRIGHT
ANDSCHA~TEN
Topoimmerase
II Inhibition
on Spisula
Oocytes
FIGS. 4 AND 5. Teniposide does not prevent chromosome decondensation, but blocks chromosome condensation after one cell cycle. The time postinsemination (min PI) is at the lower left corner of each panel. Bars, 10 pm. FIG. 4. A zygote treated with 25 PM teniposide during metaphase of first mitosis (A) showing condensed chromosomes and the first (1) and second (2) polar bodies. Although chromosome separation, decondensation, and cleavage were unaffected, chromosome condensation during the next mitosis was blocked (B). FIG. 5. A two-cell embryo in interphase at the time of 25 PM teniposide treatment showing two decondensed nuclei (A). Chromosomes condensed and separated during mitosis (B), but chromosome condensation during the next mitosis was blocked and the nuclei remained decondensed (C).
condensation during later cell cycles, but not decondensation, requires topoisomerase II activity. Teniposide Treatment during Interphase
Since during the first cell cycle, teniposide inhibited chromosome condensation in all cases whether it was added before fertilization or during meiosis, we expected chromosome condensation during the next mitosis to be blocked in embryos treated with teniposide during interphase of later cell cycles. In contrast, when two-cell embryos at interphase were treated with teniposide at 90 min PI (Fig. 5A), chromosome condensation
and separation during second mitosis (Fig. 5B), cleavage, and nuclear decondensation occurred on schedule (Fig. 5C). It was not until the next cell cycle that teniposide prevented the resulting interphase embryos at the four-cell stage from undergoing any further chromosome condensation. Cleavage into eight-cell embryos was blocked with the nuclei remaining decondensed and prominent while controls continued development (Fig. 5C). These results indicate that topoisomerase II functions in chromosome condensation, but not decondensation, during later cell cycles. Similarly, when four-cell embryos at interphase (120 min PI) were treated with teniposide, chromosome con-
230
DEVELOPMENTALBIOLOGY Fertilization
and First Cell Cycle
Later Cell Cycles
FIG. 6. Summary of effects of the topoisomerase II inhibitor, teniposide, on chromosome and nuclear cycles during fertilization and the first cell cycle (top), and later cell cycles (bottom). Whether teniposide was added (double arrows) before fertilization (top, first cell) or during metaphase of meiosis I or II (top, second cell), the inhibitor blocked chromosome condensation during mitosis (solid black bar), but not decondensation of the sperm nucleus and maternal chromosomes (top, third cell). During later cell cycles, the effects on chromosome condensation were different. The interphase cell in the bottom display represents one blastomere of an embryo. Chromatin of interphase embryos (bottom, first cell) condensed in the presence of teniposide (double arrows) underwent mitosis and decondensed, but condensation was blocked during the next cell cycle. When mitotic embryos containing condensed chromosomes (bottom, second cell) were treated with teniposide, mitosis continued and chromosomes decondensed but the next mitosis was inhibited. Irrespective of when teniposide was added, the inhibitor blocked chromosome condensation, but not decondensation, suggesting that topoisomerase II may be involved in chromosome condensation but not decondensation.
densation and separation during third mitosis, cleavage, and nuclear decondensation were unaffected (data not shown). However, chromosome condensation during fourth mitosis was inhibited and nuclei of the eight-cell embryos remained decondensed while controls continued development (data not shown). These observations confirm that topoisomerase II activity is also involved in chromosome condensation during later cell cycles, but not in decondensation of somatic chromatin. DISCUSSION
Our results summarized in Fig. 6 show that treatment with teniposide at various stages of oocyte meiotic maturation and early Spisula embryogenesis has two principal effects: during the first cell cycle, it prevents both meiotic chromosome separation and mitotic chromosome condensation; and during later cell cycles, it blocks chromosome condensation only after the embryos have progressed through the next mitotic cycle. Surprisingly, teniposide does not affect decondensation of sperm nuclei, maternal or somatic chromosomes. DNA topoisomerase II has been found in diverse species from bacteria to man (Hsieh and Brutlag, 1980; Baldi et al., 1980; Miller et aZ., 1981; Goto and Wang, 1982;
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1'&1990
Halligan et aL, 1985; Wang, 1985; Rota and Mezquita, 1989). Various studies demonstrate topoisomerase II in gametes and embryos of Drosophila, Xenopus, and chicken, and thus the enzyme is most likely also present in Spisula oocytes and embryos although not directly shown here (Hsieh and Brutlag, 1980; Baldi et aZ., 1980; Rota and Mezquita, 1989). At the molecular level, topoisomerase II separates interlocked loops of kinetoplast DNA (Marini et aZ., 1980; Luke and Bogenhagen, 1989). In somatic cells, it is required to untangle intertwined chromosomal DNA during anaphase of yeast mitosis (DiNardo et aZ., 1984; Uemura and Yanagida, 1984,1986; Holm et ak, 1985,1989; Uemura et aZ., 1987). It has been proposed that the enzyme could play an analogous role in meiosis where it may be responsible for resolution of chromosome interlocks that occur during pairing of homologous chromosomes (Moens and Earnshaw, 1989; Rose et aZ., 1990). Teniposide is known as a specific and potent inhibitor of topoisomerase II. In vitro, teniposide blocks topoisomerase II activity by binding to the DNA-topoisomerase II complex, preventing ligation of the doublestranded DNA cut (Chen et al, 1984; Long et al, 1984). Our investigation shows that teniposide disrupts meiotic maturation by preventing chromosome separation, and suggests an involvement of topoisomerase II in chromosome and chromatid separation during meiosis in Spisula. Consistent with this hypothesis, topoisomerase II has been identified by immunocytochemistry in meiotic chromosomes and its concentration increases during meiotic maturation (Luke and Bogenhagen, 1989; Moens and Earnshaw, 1989). Similarly, topoisomerase II has been identified in mitotic chromosomes and the levels of topoisomerase II fluctuate during the cell cycle of cultured cells, being high during mitosis and rapidly declining at G, (Earnshaw and Heck, 1985; Earnshaw et aZ., 1985; Berrios et al, 1985; Gasser et al, 1986; Heck et al., 1988; Heck and Earnshaw, 1988). During meiosis and the cell cycle, other Spisula proteins such as the cyclins also undergo oscillations in quantity and activity through phosphorylation (Swenson et a& 1986,1989; Westendorf et aL, 1989). Although little is known about the mechanism to modulate topoisomerase II activity, several reports indicate that phosphorylation plays a role in regulating topoisomerase II activity (Ackerman et al., 1985, 1988; Sahyoun et al., 1986; Heck et ah, 1989; Saijo et al, 1990). Perhaps these fluctuations in topoisomerase II may account for the dissimilarity of teniposide reactivity in embryos treated during the first and later cell cycles (Fig. 6). The burst in protein synthesis and phosphorylation that occurs at fertilization could provide a means to achieve this difference (Masui, 1985; Swenson et al, 1989). In older embryos, higher levels of a more active, perhaps phosphor-
WRIGHT AND SCHATTEN
Topoismerase
ylated form of topoisomerase II may explain the dissimilarity of teniposide reactivity in embryos in the first and later cell cycles. Whether embryos were in meiosis, interphase, or mitosis at the time of application, teniposide blocked mitotic chromosome condensation and the nuclei remained decondensed in interphase. This observation is not unexpected since chromosome condensation is teniposidesensitive in Xenopus egg extracts (Newport and Spann, 1987). In addition our results agree with genetic evidence that mutants defective in topoisomerase II are unable to undergo chromosome condensation (Uemura et al, 1987). Thus, we propose that topoisomerase II may be required for chromosome condensation during mitosis in Spisula. In contrast, our results revealed that teniposide did not affect decondensation of sperm nuclei, maternal chromatin, or somatic nuclei, even at higher teniposide doses. This is surprising in light of studies demonstrating that teniposide blocks decondensation of sperm nuclei and metaphase chromosomes in Xenopus egg extracts (Newport, 1987). The reason for this discrepancy is unclear but may be related to the different systems used to study decondensation; namely, intact Spisula oocytes versus cell-free Xenopus egg extracts. That the paternal chromatin of teniposide-treated specimens remained condensed while polar body formation was inhibited is consistent with previous studies in Spisula demonstrating that sperm nuclear transformations are coordinate with stages of meiotic maturation (Da-Yuan and Longo, 1983; Luttmer and Longo, 1988). Moreover, that the paternal and maternal chromatin decondensed simultaneously into pronuclei after both processes were delayed, further indicates a coordinate regulation of the maternal and paternal chromatin in Spisula oocytes. An alternative explanation for our results could have been that teniposide exerts other cellular effects besides inhibiting topoisomerase II. Although teniposide is a specific inhibitor of topoisomerase II in vitro, the in viva mechanism of teniposide action has not been elucidated. Teniposide could possibly be toxic, impermeable, or even disrupt microtubules. However, these seem unlikely since: (1) teniposide effects are reversible, (2) the teniposide analog, podophyllotoxin, rapidly penetrates eggs (Schatten and Schatten, 1981), (3) Spisula embryos treated with teniposide develop normal spindles as determined by antitubulin immunofluorescence (data not shown), and (4) teniposide does not affect microtubule assembly (Grieder et al, 1974). The lack of an effect on chromosome decondensation may reflect the need for a higher teniposide concentration, however, the same results were obtained with higher doses indicating that not all nuclear events require topoisomerase II. One could consider that inhibiting polar body formation may
II Inhibition
an Spisula
Oocytes
231
prevent chromosome separation independent of topoisomerase II activity. However, this seems unlikely since in Spisula oocytes treated with cytochalasin B, chromosome separation and decondensation can occur if cytokinesis is blocked (Longo, 1972; Luttmer and Longo, 1988). Another potential side effect of teniposide may include the numerous cuts in the DNA that persist when teniposide prevents topoisomerase II from resealing the cuts that the enzyme normally makes in the DNA strands. While we can not dismiss the possibility that the numerous cuts in the DNA alone could cause the effects observed on chromosome separation and condensation, we consider this an indirect effect of blocking topoisomerase II activity. Since these potential indirect effects seem unlikely, we think that the effects of teniposide on nuclear events during Spisula fertilization are due to a specific inhibition of topoisomerase II. In summary, we conclude that teniposide disrupts chromosome separation during meiosis and chromosome condensation during mitosis, but does not affect decondensation of sperm nuclei, maternal chromosomes, or somatic chromatin in Spisula. Therefore, we suggest that topoisomerase II may play both a structural and enzymatic role in regulating chromosome interactions during fertilization and development. The authors gratefully acknowledge the generous gift of teniposide from Bristol-Myers. We thank Dr. David J. Wright for his comments on the manuscript. The support of this research by grants from the NIH, including an individual NIH-NRSA Postdoctoral Fellowship to S.J.W. is acknowledged gratefully. The Madison IMR is supported as an NIH Biomedical Research Technology Resource. REFERENCES ACKERMAN, P., GLOVER, C. V. C., and OSHEROFF,N. (1985). Phosphorylation of DNA topoisomerase II by casein kinase II: Modulation of eukaryotic topoisomerase II activity in vitro. Proc. Nat1 Acad. Sci. USA 83,1603-1607.
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