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

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    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

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    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