Chromatin remodeling in mammalian zygotes

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Mutation Research, 296 (1992) 43-55 43 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1110/92/$15.00 MUTREV 00395 Chromatin remodeling in mammalian zygotes Sally D. Perreault Reproductive Toxicology Branch (MD-72), Health Effects Research Laboratory, U.S. Environmental t'rotection Agency, Research Triangle Park, NC 27711, USA (Accepted 13 July 1992) ;eywords: Fertilization; Sperm; Oocyte; Protamine; Sperm decondensation; Pronucleus; Zygote Summary With sperm-egg fusion at the time of fertilization the gamete nuclei are remodeled from genetically quiescent structures into pronuclei capable of DNA synthesis. Features of this process that are critical to insure the genetic integrity of the zygote and the success of subsequent embryonic development include: oocyte responses that prevent polyspermy; completion of the 2nd meiotic division by the oocyte; exchange of proteins in the sperm nucleus; and, remodelling of the oocyte chromosomes and sperm nucleus into functional pronuclei. Elucidation of the biological and molecular mechanisms underlying zygote formation and chromatin remodeling should enhance our understanding of the potential vulnera- bility of the zygote to toxicant-induced damage. Introduction Fertilization is an active process whereby highly motile sperm interact with the oocyte in a species-specific manner to bind and traverse the zona pellucida and fuse with the oocyte mem- brane to form the zygote (Yanagimachi, 1988). This active process, however, takes place between Correspondence: Dr. Sally D. Perreault, Reproductive Toxi- cology Branch (MD-72), Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA. Tel.: (919) 541-3826; Fax: (919) 541-5138. E.P.A. Disclaimer: This document has been reviewed in ac- cordance with U.S. Environmental Protection Agency policy and approved for publication. Mention of trade names or commercial products does not constitute endorsement or rec- ommendation for use. gametes that are genetically quiescent. The oocyte is arrested at metaphase II of meiosis, a chromo- somal state that precludes DNA synthesis or RNA transcription (Wassarman, 1988; Crisp, 1992), while the sperm chromatin is uniquely compacted into a highly dense, genetically inert format (Bellve and O'Brien, 1983; Ward and Coffey, 1991). When the sperm fuses with the oocyte, however, it 'activates' the oocyte. The activated oocyte then directs the remodeling of gamete chromatin into male and female pronuclei capa- ble of DNA synthesis (reviewed in Longo, 1985; Poccia, 1986; Zirkin et al., 1989; Perreault, 1990). Thus, the gametes reawaken each other. During fertilization, the oocyte must perform 3 general functions to insure normal zygote forma- tion: (1) fusion with 1 and only 1 sperm; (2) completion of its own meiotic maturation with extrusion of the 2nd polar body and formation of 44 the female pronucleus; and (3) reactivation of the sperm nucleus into a functional male pronucleus. Imperfect zygotes result if any of these 3 func- tions is compromised. The aim of this chapter is to review our knowl- edge of chromatin remodeling in the zygote with emphasis on the potential vulnerability of these processes to disruption by chemical insult. Be- cause the zygote may be uniquely sensitive to genetic insult (see Rutledge, et. al., 1992), an understanding of events surrounding chromatin remodeling may provide insight into potential mechanisms that may underlie mutagenic action. It is also important to consider how disruption of the timing of fertilization or perturbation of oocyte metabolism might alter the ability of the oocyte to process the sperm nucleus and thereby contribute to early embryo failure through epige- netic mechanisms. Synchrony and order in the early zygote Post-fusion events transform the nuclei of both gametes into functional pronuclei within only a few hours (Perreault et al., 1987; Perreault, 1990), and occur in a predictable and coordinated fash- ion as the oocyte proceeds through the cell cycle. Sperm-egg fusion activates calcium transients in the oocyte with consequent oocyte responses. These calcium-mediated responses include corti- cal granule-induced hardening of the zona pellu- cida which establishes a major block to polyspermy, and release of metaphase II arrest in the oocyte which appears to be mediated by cell cycle regulatory factors, particularly maturation- promotion factor (MPF) and the c-mos product cytostatic factor (CSF) (reviewed in Murray and Kirschner, 1989; Cran and Moor, 1990; Mc- Connell, 1991). Morphological consequences of oocyte activation include meiotic spindle rotation, sister chromatid separation as anaphase II and telophase II progress, extrusion of the 2nd polar body and remodeling of the oocyte chromosomes into the female pronucleus. During this same time, the nuclear envelope of the sperm breaks down and the sperm chromatin disperses in a process called 'decondensation.' Ultrastructural observations reveal that sperm chromatin decondensation begins in the area of the sperm nucleus that first fuses with the oocyte (Yanagimachi and Noda, 1970; Talbot and Cha- con, 1982) and continues until the entire nucleus is dispersed. The last portion to decondense is the base of the sperm nucleus near the implanta- tion fossa. This area contains the 'sperm nuclear annulus,' recently described by Ward and Coffey b Fig. 1. Phase contrastmicrographs of hamster oocytes after microinjection of a hamster sperm nucleus (aceto-lacmoid stain). (a) One hour after microinjection the sperm nucleus is decondensed fully (arrow) while the activated oocyte has progressed to telophase II of meiosis (x300). (b) Three hours after microinjection the oocyte has completed meiosis and sperm and oocyte chromatin has been remodeled into morphologically mature pronuclei ( x 250). (1989), which is thought to anchor and perhaps organize the sperm DNA. Membrane vesicles, visible near the dispersing chromatin, begin to coalesce to form the pronuclear envelope even before the entire sperm nucleus has dispersed. Thus, under normal conditions, sperm deconden- sation and male pronucleus formation occur as a continuum. Time course studies in rodents and humans have shown that sperm nuclear decondensation, meiotic progression, and formation of male and female pronuclei occur in a coordinated fashion (Perreault et al., 1987; Wright and Longo, 1988; Lassalle and Testart, 1991). In the hamster, for example, the time course of sperm nuclear decon- densation and pronucleus formation was charted over 3 hours after introduction of isolated sperm nuclei into the oocyte (Perreault et al., 1987). Sperm nuclei were introduced by microinjection, rather than fertilization, so that the initial time of sperm contact with the ooplasm was known with precision. In these studies, the sperm nucleus decondensed 45-60 minutes after injection, a time during which the oocyte chromosomes were at anaphase II to telophase II of the meiosis (Fig. la). Extensive decondensation was followed by partial recondensation of the sperm chromatin just before it expanded again during pronucleus formation, a process that has been quantified during hamster and human sperm nuclear pro- gression (Wright and Longo, 1988; Lassalle and Testart, 1991). Extrusion of the 2nd polar body was evident in most zygotes by 75 minutes, when early pronuclei were found in the cytoplasm. Male pronucleus development tended to lag slightly behind female at the early stages, which is the case during in vivo fertilization in this species. By 3 hours after injection, the pronuclei had en- larged and the numerous tiny nucleoli seen at 2 hours, had coalesced into fewer, larger nucleoli (Fig. lb). It was at this time, when the pronuclei appeared morphologically mature, that they be- came capable of DNA synthesis (Naish et al., 1987a). The timing of these events appears to be con- trolled by the oocyte, rather than by the sperm. Indeed, once the oocyte is activated, it proceeds through meiosis whether or not a sperm nucleus is present (Naish et al., 1987b). If sperm nuclear 45 decondensation is experimentally advanced by pretreating the nuclei with disulfide reducing agents, the prematurely decondensed sperm nu- cleus does not form a male pronucleus ahead of its female counterpart. Rather, it awaits condi- tions in the oocyte that permit pronucleus forma- tion around an appropriate template (Perreault et al., 1987). Alternatively, if sperm decondensa- tion is not completed within the normal 'window of opportunity' when the oocyte is proceeding from metaphase through telophase, the oocyte continues its progression into interphase with for- mation of a female pronucleus, but sperm chro- matin remodeling is arrested (Perreault et al., 1988b; Perreault, 1990). Thus, once meiotic events are set into motion, remodeling of the oocyte and sperm chromatin must proceed within a preor- dained time frame. On the other hand, if meiotic progression is arrested, for example by inhibition of microtubule polymerization, then transformation of the de- condensed sperm to a male pronucleus is also arrested (Schatten et al., 1985; Wright and Longo, 1988). Therefore, if the synchrony of events dur- ing fertilization and meiotic maturation is dis- turbed by exposures to toxicants, then the oocyte and/or sperm chromatin may not be remodeled normally and may fail to be replicated completely during the first 'S' phase of embryonic develop- ment. Presumably, this could result in embryonic arrest at whatever stage the incompletely repli- cated DNA becomes needed for development. In addition, the metaphase II oocyte appears capable of remodeling any available template, including heterologous sperm and somatic cell nuclei, into functional pronuclei (Naish et al., 1987b; Czolowska et al., 1984), a feature that forms the basis for embryo cloning (Collas and Robl, 1991). The number of sperm nuclei that can decondense in polyspermic hamster eggs is virtually unlimited, attesting to a large excess of factors required for decondensation (Hirao and Yanagimachi, 1979). However, only a small num- ber (no more than 5 or 6) of decondensed sperm will be transformed into male pronuclei, suggest- ing that oocyte factors required for pronucleus formation (e.g., histones and membrane vesicles for nuclear envelope assembly) are limiting. Ad- ditional studies with such polyspermic eggs have 46 shown that when the sperm nucleus progresses to a mature pronucleus, it can synthesize DNA, but other sperm nuclei at earlier stages (i.e., decon- densed or early pronuclei) in the same egg cannot (Naish et al., 1987a). Thus, only fully remodeled, morphologically mature male pronuclei are capa- ble of DNA synthesis. Fresh eggs make the best zygotes It is well known that mammalian oocytes have a limited functional life span after ovulation (Yanagimachi and Chang, 1961). While reproduc- tive behavior is normally synchronized in rodents, such that fertilization takes place within a few hours of ovulation when the oocytes are 'fresh,' delayed breeding or introduction of sperm by artificial insemination at longer intervals after ovulation may result in abnormal zygote forma- tion (Longo and So, 1982; Juetten and Bavister, 1983; Jedlicki et al., 1986; Smith and Lodge, 1987; Gaulden, 1992). Various abnormalities may have different etiologies. For example, triploidy may occur subsequent to either dispermy (if the cortical reaction fails to induce the zona block to polyspermy), or digyny (if the second polar body is not extruded). Monosomy may result if the oocyte expels all its chromosomes into the 2nd polar body, or if sperm nuclear decondensation is impaired. Finally, chromosomal abnormalities may result from spindle failure. All of these changes have been reported in 'aged' oocytes where the cortical reaction, microtubule function, and sperm decondensing activity may be compro- mised. Therefore, when using rodent models to evalu- ate the potential of environmental contaminants to produce genetically abnormal zygotes or em- bryos, it is important to consider that treatments that influence physiological events such as sperm transport or sperm motility may delay fertilization and thereby indirectly lead to conditions where 'aged' eggs become fertilized abnormally. The genetically imbalanced embryos that result would be expected to fail early in development. In ro- dent toxicologic studies, such indirect effects can be distinguished from more direct, mutagenic ef- fects, by expanding pregnancy outcome studies to include evaluations of fertilized eggs and early embryos recovered during very early pregnancy (Perreault and Mattson, in press). Human reproductive behavior, in contrast to that in rodents, is not synchronized with ovula- tion. This creates an inherently large potential for asynchrony in fertilization which may be a major factor contributing to the high incidence of early pregnancy failure seen in humans as compared with rodents (Hatch, 1988). Obviously, it is criti- cal to understand the etiology of early pregnancy loss in rodent test species when extrapolating from rodents to humans for purposes of estimat- ing genetic risk to the female. Prerequisites for sperm chromatin remodeling In mammals, sperm chromatin is uniquely packaged during spermatogenesis (Ward and Coffey, 1991) when somatic histones are replaced by a series of basic proteins and finally by pro- tamine. In a model proposed by Balhorn (1982), protamines occupy the minor groove of sperm DNA, with DNA-protamine binding most likely at the central polyarginine region (Balhorn, 1989). This conformation permits the chromatin to be packaged in a minimal volume and confers such structural stability that the chromatin is resistant to physical disruption, even by sonication. During epididymal maturation, cysteine residues in the protamine become oxidized to form disulfide cross-links between and among protamine molecules making the chromatin extremely stable (Balhorn, 1989; Hecht, 1989). This unique pack- aging aplSarently serves to protect the sperm chromatin and maintain it in a transcriptionally inert form during epididyrnal maturation and transport through the female tract. As proposed by Bloch many years ago, sperm protamine may also 'erase' the spermatid's developmental his- tory, and afford a mechanism whereby the ga- mete nucleus regains its totipotency (Bloch, 1969). It follows that sperm chromatin must be unpack- aged in the oocyte and its protamine replaced by somatic histones at the time of fertilization, in order for it to participate in embryonic develop- ment. Numerous in vitro studies have demonstrated that reduction of protamine disulfide bonds is required before decondensation can be chemi- cally induced by detergent (e.g., SDS), high salt, or polyanions such as polyglutamic acid or hep- arin (Dean, 1983; Wogelmuth, 1983; Zirkin et al., 1985; Huret, 1986; Reyes et al., 1989; Jager et al., 1990). This same requirement exists for sperm decondensation in the oocyte (Perreault et al., 1984), where the reducing power is most likely supplied by glutathione (GSH). For example, ex- posure of oocytes to iodoacetamide, a sulffiydryl blocking agent that limits the reducing power of the ooplasm by depleting GSH and inactivating sulfhydryl-dependent enzymes, arrested both sperm decondensation and meiotic maturation. The latter effect may be due to inhibition of tubulin polymerization (Luduena and Roach, 1991). Similarly, diamide, which temporarily de- pletes GSH by oxidation, blocked decondensation and meiotic progression in a reversible manner. When eggs were transferred to diamide-free medium, the sperm nucleus decondensed and pronucleus formation followed. In mature, metaphase II mammalian oocytes, the timing of decondensation varies directly with the extent of protamine disulfide bonding (Perre- ault et al., 1987). For example, hamster spermatid nuclei, which contain few if any disulfide bonds, decondense within only 5 minutes of injection into hamster oocytes, while cauda epididymal sperm nuclei, which contain maximal numbers of disulfide bonds, require 45-60 minutes to decon- dense, and do so only if the oocytes are main- tained at 37C (Perreault, 1990). Thus, sperm chromatin decondensation in the ooplasm is both time- and temperature-dependent, implying that enzymes such as glutathione reductase may also be required. While reduction of protamine disul- fide bonds is prerequisite, it is not by itself suffi- cient for sperm nuclear decondensation; other oocyte factors are required for chromatin swelling and remodeling. Nevertheless, the requirement for protamine disulfide bond reduction intro- duces the possibility that mutagens such as acryl- amide and ethylene oxide that are capable of binding and depleting GSH (Dearfield et al., 1988; McKelvey and Zemaitis, 1986) could alter the ability of the oocyte to process the sperm chromatin. Thus, such chemicals might act as zygote toxicants through physiological as well as genetic mechanisms (e.g., alkylation of DNA or 47 protein in the zygotic pronuclei, Rutledge, et al., 1992). The ability of the oocyte to initiate deconden- sation also depends on the maturational state of the oocyte (reviewed in Zirkin et al., 1989). For example, Usui and Yanagimachi (1976) showed that immature, germinal vesicle (GV) intact ham- ster oocytes could fuse with hamster sperm but failed to decondense the sperm nucleus. Simi- larly, fertilized eggs (at the pronucleus stage) failed to effect sperm decondensation. Thus, sperm decondensing activity is highest in the metaphase state when MPF activity is also maxi- mal (Cran and Moor, 1990). Oocytes also lose the ability to transform somatic cell. nuclei into pronucleus-like structures shortly after activation, again demonstrating the importance of cell cycle events in chromatin remodeling (Szollosi et al., 1988). Presumably this cell-cycle-dependent mod- ulation of sperm chromatin decondensation is of adaptive value serving to impede fertilization of immature or previously fertilized eggs. The difference in sperm decondensing activity between interphase and metaphase oocytes may be due, at least in part, to differing abilities to reduce protamine disulfide bonds, since 'di- sulfide-poor' sperm nuclei like those isolated from the testis (prior to disulfide bond formation) do decondense in GV or pronucleate eggs (Perreault et al., 1984). Furthermore, ovulated, metaphase II eggs have significantly higher GSH levels than either GV or pronucleate eggs (Perreault et al., 1988a; Boerjan and deBoer, 1990). Also, the ac- quisition of sperm decondensing activity by oocytes maturing in vitro, can be prevented or delayed by inhibiting GSH synthesis with buthio- nine sulfoximine (BSO). Although BSO-treated oocytes matured normally by morphological crite- ria, they were less efficient in decondensing sperm nuclei and contained less GSH than control oocytes (Perreault et al., 1988a). That such a phenomenon could occur in the intact animal was demonstrated by Calvin et al. (1986), who showed that in vivo treatment of mice with BSO depleted both ovarian and oocyte GSH. Furthermore, in vitro fertilization of these oocytes was abnormal, with evidence of arrested sperm decondensation. Thus, toxicants that affect GSH synthesis, metabolism or turnover either during the final 48 maturation of the oocyte in the ovary or during fertilization in the oviduct, could potentially im- pair sperm chromatin remodeling. In regard to the association between sperm decondensing activity and stage of the cell cycle, it has also been hypothesized that oocyte factors responsible for sperm decondensation might be stored in the GV and released into the cytoplasm at GV breakdown to become available or active in the mature (metaphase) oocyte (Usui and Yanagimachi, 1976). Evidence in support of this hypothesis includes an experiment wherein mouse oocytes were bisected at the GV stage, and then allowed to mature in vitro. Only the half that contained the GV was subsequently capable of decondensing a sperm nucleus after GV break- down (Balakier and Tarkowski, 1980). Recently, an acidic nucleoprotein 'nucleo- plasmin,' isolated from immature (GV) amphib- ian (Xenopus laevis) eggs was shown to be re- quired for inducing the early stages of Xenopus sperm decondensation (Philpott et al., 1991). In- deed, nucleoplasmin (or its polyglutamic acid component), has recently proven to be a useful tool for swelling frog sperm nuclei in experiments designed to elucidate mechanisms of nuclear en- velope assembly in cell-free Xenopus egg extracts (Pfaller et al., 1991; Newport and Dunphy, 1992). In these experiments, the number of sperm nuclei that can be decondensed under defined condi- tions is related to the amount of nucleoplasmin added, leading the authors to conclude that the decondensation process itself may result from the stoichiometric displacement of protamines by the acidic tail of nucleoplasmin (Newport and Dun- phy, 1992). Whether or not nucleoplasmin is the critical cytoplasmic factor required for deconden- sation of mammalian sperm nuclei remains to be determined. Since Xenopus sperm nuclei contain a non-mammalian type of protamine that is not stabilized by disulfide bonds (Bloch, 1969), mech- anisms for the early stages of decondensation may differ somewhat in Xenopus and mammals (however, see below regarding protamine re- placement). Protamine replacement by histone The sperm-specific protamines are removed shortly after fertilization and are replaced by histones. It is thought that histone replacement is essential for reactivation of the sperm chromatin, as evidenced by pronuclear DNA replication (Nonchev and Tsanev, 1990). Removal of protamine during fertilization ap- pears to occur as the sperm nucleus decondenses in the ooplasm. For example, radiolabeled (3H- arginine) protamine is lost shortly after sperm entry (Kopecny and Pavlok, 1975; Ecklund and Levine, 1975; Betzalel et al., 1986), and decon- densed sperm no longer react with anti-prot- amine antibody (Rodman et al., 1981). With ei- ther approach, protamine is identifiable in intact or slightly swollen sperm chromatin, but not in markedly decondensed chromatinl In agreement with these studies, we have found that when protamine in hamster sperm nuclei is labeled with the sulfhydryl-specific fluorochrome mono- bromobimane (mBBr), and the nuclei injected into mature hamster oocytes, fluorescence is lost as the sperm decondense, and is absent in the male pronucleus (discussed in Perreault, 1990). Furthermore, fluorescence is lost within the same time frame whether or not metaphase II release occurs, suggesting that factors responsible for protamine replacement are operative in mature, unactivated oocytes (Perreault, unpublished ob- servation). Using another approach based on Feulgen staining as a measure of histone replace- ment, Garagna and Redi (1988) concluded that protamine is replaced by histone just prior to the major increase in sperm nuclear size that accom- panies chromatin dispersion in the mouse and occurs between anaphase II and telophase II. These observations support the contention of Newport and Dunphy (1992) that protamine re- moval and histone deposition are tightly coupled. Further support derives from immunofluores- cence studies in fertilized mouse eggs demon- strating that protamine loss occurs as histones appear in the male pronucleus (Nonchev and Tsanev, 1990). Furthermore, newly synthesized 3H-arginine-labeled proteins (presumably his- tones) have been demonstrated in decondensed boar sperm after fertilization of zona-free ham- ster eggs (Kopecny and Pavlok, 1984) suggesting that there is an immediate shift from protamine to histone during decondensation. However, at least one study provided evidence for the occur- 49 rence of naked sperm DNA after protamine re- moval and before histone deposition (Rodman et al., 1981). All of these studies are consistent with com- plete replacement of protamines by histones prior to the time of pronuclear DNA synthesis, when the sperm-derived chromatin physically resembles that of the oocyte-derived pronucleus (Brandriff and Gordon, 1992). Of relevance to the subject of this volume, sperm chromatin may be more sus- ceptible to alkylating agents during this brief pe- riod of protein exchange, although experimental evidence to this effect is lacking. The necessity of histone replacement as a pre- requisite for pronucleus formation and DNA syn- thesis has also been demonstrated in experiments where nuclear envelopes are reconstituted around protein-free viral DNA, or Xenopus sperm chro- matin in Xenopus egg extracts (Newport, 1987; Pfaller et al., 1991; Newport and Dunphy, 1992). These studies showed that the first step in nu- clear reconstitution is binding of histone to the DNA. Once the nucleosome configuration is re- stored, then nuclear envelope construction fol- lows and requires membrane vesicles and lamin stored in the ooplasm. Finally, DNA replication in the reconstituted nuclei occurs only after nu- clear envelope construction is complete. Several mechanisms by which protamine may be removed from sperm chromatin and replaced by histones have been proposed--although none is definitive at this time. Proteolysis of protamine by trypsin-like enzymes can occur in vitro and is associated with decondensation of the sperm nu- cleus, but a sperm-associated proteinase does not appear to play a required role in vivo (reviewed in Zirkin et al., 1985). Oocyte-associated pro- teinase(s) have been shown to degrade rat prot- amine in vitro (Betzalel et al., 1986). These stud- ies did not determine whether protamine degra- dation is a mechanism for, or occurs subsequent to, protamine removal from the sperm chromatin. Another hypothesis is that protamine removal is dependent upon charge changes induced by its phosphorylation (reviewed in Poccia, 1986). Sperm protamine is phosphorylated in vitro by rabbit oocyte extracts (Wiesel and Schultz, 1981), but direct evidence of protamine phosphorylation during fertilization has not been reported to date. Charge alteration of protamine and/or histone as a mechanism for protein exchange during decon- densation is an attractive hypothesis in light of the importance of phosphorylation and dephos- phorylation reactions associated with the activity of MPF (itself a histone H1 kinase) during oocyte maturation and fertilization (Lohka et al., 1988; Cran and Moor, 1990; McConnell, 1991). Another appealing candidate for modulating protamine-histone exchange is nucleoplasmin, since by binding and transferring histones to chromatin it is known to be involved in nucleo- some assembly (discussed above and in Philpott et al., 1991). Whether nucleoplasmin is required for decondensation and/or protamine exchange remains to be determined. In this regard, it is relevant to note that sperm decondensation in mammalian oocytes can be experimentally uncou- pled from protamine removal. When hamster sperm nuclei are labeled with mBBr and the labeled nuclei injected into GV or PN eggs, the nuclei decondense, but the fluorescence (prot- amine) is not lost, and the nuclei are not trans- formed further into pronuclei (Perreault, unpub- lished observations). These observations are con- sistent with a requirement for nucleoplasmin or other GV factors for protamine replacement by histones, but not necessarily for sperm chromatin swelling. Alkylating agents that bind protamine either during sperm maturation or during fertilization might be expected to alter decondensation and associated events. For example, alkylation of cys- teine residues in testicular spermatid or DTT- treated cauda epididymal sperm nuclei with iodoacetamide blocks formation of disulfide bonds, and these 'disulfide-poor' nuclei decon- dense prematurely in the oocyte (Perreault et al., 1987). As mentioned earlier, such nuclei are transformed into pronuclei but not until the oocyte has completed telophase and is construct- ing the female pronucleus. Whether prematurely decondensed sperm chromatin is at any increased risk of damage by exposure to cytoplasmic factors or to exogenous mutagens prior to formation of the nuclear envelope is not known. However, it is possible, but remains to be demonstrated, that dominant lethal effects of alkylating agents like acrylamide, which bind to spermatid protamine 50 (discussed in Sega, 1990), may occur secondary to altered protamine-DNA interactions that could affect sperm nuclear processing in the oocyte. Oocyte chromatin remodeling and partitioning in the zygote Little is known about spec~hc protein ex- changes in the maternal chromatin, although the autoradiographic studies described above demon- strated the deposition of newly synthesized basic protein in the female, as well as the male, pronu- cleus (Kopecny and Pavlok, 1984). These proteins (presumably histones) were incorporated well ahead of the expected time of DNA synthesis. Immunofluorescent studies also clearly show that histones are present in both pronuclei well before DNA synthesis takes place (Nonchev and Tsanev, 1990). Another oocyte factor that would be expected to be important in chromatin remodeling in the zygote is topoisomerase II (topo II). Topo II is a cell-cycle-dependent enzyme found in association with the chromosome scaffold (reviewed in Heck and Earnshaw, 1988). As its name implies, topo II alters DNA topology, relaxing supercoiled DNA, for example. It acts in a stepwise fashion by first inducing double-stranded breaks in DNA, then passing a 2nd, intact double helix of DNA through this break, and finally reannealing the 1st DNA strands (reviewed in Osheroff, 1989). This activity makes topo II important in many aspects of nu- cleic acid metabolism, including DNA replication and transcription and in chromosome segrega- tion. It also participates in chromosome structure and in the conformational changes associated with chromosome condensation (Hirano and Mitchi- son, 1991). Therefore, topo II would be expected to play critical roles in oocyte chromatin remodel- ing, from the initial condensation of chromo- somes that follows GV breakdown (see Albertini, 1992) through both meiotic divisions, pronuclear DNA synthesis and condensation of mitotic chro- mosomes at the G2 to M transition of zygote cleavage. Preliminary results indicate that this is indeed the case (Wright and Schatten, 1988, 1990; Perreault et al., 1991a). Inhibition of topo II with teniposide (a chemotherapeutic agent) during oocyte maturation and fertilization in the surf clam, mouse and hamster results in aberrant chromosome condensation, arrest of first polar body extrusion, inhibition of pronuclear DNA synthesis and failure of pronucleate eggs to cleave (G2 to M blockade). Therefore, acute exposure at the time of oocyte maturation or fertilization to chemicals like teniposide that interfere with topo II activity would be expected to impair zygote formation and could induce DNA damage in the pronuclei. Topo II has also been proposed to play a role in sperm chromatin remodeling during fertiliza- tion. Evidence in support of such a role is derived from experiments in which teniposide arrested nuclear reconstitution from Xenopus sperm cul- tured in Xenopus egg extract (Newport, 1987). However, subsequent fertilization studies cited above (Wright and Schatten, 1988; Perreault et al., 1991a) failed to demonstrate a teniposide-in- duced inhibition of sperm decondensation or male pronucleus formation. Thus, the conformational changes in sperm chromatin during decondensa- tion and pronucleus formation may not be medi- ated by topo II. Furthermore, while topo II is present in Xenopus spermatogenic ceils, it is ab- sent in mature sperm (Morse-Gaudio and Risely, 1991), again suggesting that topo II may not be involved in sperm decondensation. Chromosome partitioning during meiosis and fertilization is dependent upon microtubule and microfilament function. Thus, disruption of these cytoskeletal elements can result in aneuploid or polyploid zygotes (reviewed in Schatten and Schatten, 1987; Albertini, 1992). Immunocyto- chemical and DNA-specific fluorochromes have recently been applied to advance our understand- ing of the roles of various cytoskeletal- and chro- mosome-associated structures including kineto- chores, centromeres and centrosomes during meiosis (reviewed in Wright et al., 1990). In addi- tion, a unique configuration of microtubule asters is assembled in oocytes at the time of sperm incorporation and is responsible for the pronu- clear migration that brings male and female pronuclei together at the egg's center shortly before cleavage (Schatten et al., 1985). Microtubule poisons (like colchicine and col- cemid) cause spindle disintegration and arrest of meiosis, the specific effects of which vary depend- ing upon the time of application (Albertini, 1992; Schatten and Schatten, 1987). For example, expo- sure of mouse eggs to microtubule poisons during in vitro fertilization arrests oocyte chromosomes in the condensed state and prevents female pronucleus formation (Schatten et al., 1985; Wright and Longo, 1988). This effect possibly occurs through a feedback control mechanism such as that proposed recently in yeast, whereby the inactivation of MPF activity and exit from metaphase is prevented until spindle assembly is complete (Li and Murray, 1991). On the other hand, exposures after pronucleus formation pre- vent pronuclear apposition and arrest the zygote at metaphase of the first mitotic division (Schat- ten et al., 1985). Microfilament inhibitors (like cytochalasin B) prevent polar body abscission, so that when sister chromatids separate, each set remains in the oocyte and forms its own pronu- cleus. The resulting egg is then triploid with 2 maternal sets of chromosomes. Early studies (e.g., Edwards, 1954) demon- strated the induction of heteroploidy in preim- plantation embryos of rodents treated with the classic microtubule poison colchicine. More re- cent studies have characterized the dose and tem- poral sensitivity of such effects. For example, in vivo exposure of female mice to nocodazole (a benzimidazole compound that inhibits tubulin polymerization) at the time of fertilization (mei- osis II) caused early pregnancy loss character- ized by pre- and early post-implantation loss (Generoso et al., 1989). Similar effects were found in hamsters after carbendazim (a fungicide struc- turally related to nocodazole) exposure during oocyte maturation (meiosis I) or fertilization (meiosis II) (Perreault et al., 1992). Cytogenetic analyses of zygotes obtained from mice (Generoso et al., 1989) and metaphase II oocytes obtained from hamsters (Perreault et al., 1991b) have pro- vided direct evidence of the induction of aneu- ploidy and/or polyploidy, demonstrating the sen- sitivity of the spindle to disruption during these critical periods. Construction of the pronuclear envelope As described earlier, the pronuclear envelope forms around the decondensing sperm nucleus, 51 and the male pronucleus emerges more or less in synchrony with its female counterpart (Yanagi- machi, 1988). Little is known about the specific requirements for pronuclear envelope assembly in mammalian eggs. However, recent evidence derived from nuclear reconstitution experiments using Xenopus egg extracts and viral or Xenopus sperm DNA indicates that the first step in nu- clear envelope assembly is histone addition to DNA (Newport, 1987) which would restore the nucleosome configuration typical of somatic cells. Association of this chromatin with membrane vesicles in the ooplasm follows (Newport, 1987; Pfaller et al., 1991; Newport and Dunphy, 1992). This is mediated indirectly by MPF and occurs via a membrane-bound receptor and a chromatin- bound ligand in a manner that requires dephos- phorylation of the receptor. Cell cycle depen- dence was demonstrated by showing that vesicles are released from the chromatin if a mitotic ex- tract (with MPF activity) is added. Finally, the membrane vesicles fuse to form the nuclear enve- lope, nuclear lamins are added, and the nucleus becomes capable of DNA synthesis (Newport, 1987). The Xenopus work supports a model for sperm chromatin remodeling in mammalian oocytes that would have disulfide bond reduction as a prereq- uisite for protamine replacement by histones as the nucleus decondenses. Then once the histone is on board, the chromatin is in a form suitable for complexing with membrane vesicles which then coalesce to form the nuclear envelope. The metaphase II arrested ooplasm may be in an ideal state for effecting sperm decondensation and protamine replacement. However, with acti- vation, conditions soon change to permit mem- brane-chromatin association, pronuclear enve- lope formation and subsequent DNA replication. Conclusions The nuclear events that proceed during fertil- ization can thus be seen as a continuum, inti- mately linked to the cell cycle. When allowed to progress unimpeded, they remain tightly coordi- nated and support an impressive remodeling of the sperm chromatin within only a few minutes, with subsequent transformation of male and fe- 52 male chromatin into the pronuclei within only a few hours. An understanding of the cellular and molecular mechanisms of these transformations may provide testable hypotheses regarding the vulnerabil ity of the zygote to toxicant insult. Based on our current understanding of fertil ization, we suggest that zygote toxicity may occur through a number of routes including metabolic inhibition, cell cycle disruption, or direct alkylation of DNA or protamine. Studies that combine approaches taken in re- productive toxicology (e.g., cellular evaluation of fertil ization and zygote function) with those taken in genetic toxicology (e.g., chromosome analysis, dominant lethal studies) are needed to character- ize zygote toxicity fully (Darney, 1991). Goals include the development of better, more specific markers of oocyte 'health, ' so that we can identify adverse effects of toxicants during oocyte matura- tion, and the application of more informative measures of chromatin remodel ing during sperm decondensat ion and pronucleus formation to dis- tinguish between direct DNA damage and indi- rect perturbat ions in the synchrony of fertiliza- tion events. It is not unreasonable to hypothesize that the complex protein exchanges in the sperm nucleus during pronucleus formation (discussed above) and the unusual ly loose packaging of pronuclear chromatin (discussed in Brandriff, 1992) may confer a unique vulnerabil ity to the zygote DNA. Acknowledgements The author thanks Drs. Jerome Goldman and David Albert in i for critically reviewing the manuscript and contr ibuting valuable insights. References Albertini, D.F. 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