Chromatin organisation and nuclear architecture in growing mouse oocytes

Molecular and Cellular Endocrinology 234 (2005) 11–17

Review

Chromatin organisation and nuclear architecture in growing mouse oocytes Maurizio Zuccotti a, b, ∗ , Silvia Garagna b , Valeria Merico b , Manuela Monti b , Carlo Alberto Redi b a

Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia, Universita’ degli Studi di Parma, Via Volturno 39, 43100 Parma, Italy b Laboratorio di Biologia dello Sviluppo, Dipartimento di Biologia Animale, Universita’ degli Studi di Pavia, Piazza Botta 9, 27100 Pavia, Italy Received 12 June 2004; accepted 18 August 2004

Abstract Although the female gamete is blocked at the dictyate stage of the first meiotic prophase during the whole folliculogenesis, many important epigenetic changes occur to organise the genome to attend early embryonic development. In this paper, we will describe the results of a number of studies aimed to improve our understanding of the nuclear organization of the mouse oocyte during folliculogenesis. Using silver methods that stain NOR, centromeres and heterochromatin, as well as, the use of specific antibodies for the demonstration of centromeres, we have described the changes to the chromatin organisation and to the spatial localisation of chromocenters and centromeres during oocyte growth; these changes have been correlated to the developmental competence of the resulting antral and metaphase II (MII) oocyte. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Oocyte; Chromatin organisation; Folliculogenesis

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Chromatin organisation during oocyte growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Nuclear architecture during oocyte growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Gene expression in NSN and SN oocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Future studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction At the very early stages of Mammalian preimplantation development, gene expression is regulated by a number of epigenetic mechanisms, which are built up during male and ∗

Corresponding author. Tel.: +39 038 2506 323; fax: +39 038 2506 270. E-mail address: [email protected] (M. Zuccotti).

0303-7207/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2004.08.014

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female gametogenesis (Ferguson-Smith and Surani, 2001; for a review, see Surani, 2001). DNA methylation (Reik et al., 2001), histone acetylation (Cheung et al., 2000), chromatin organisation and nuclear architecture (Vignon et al., 2002; Marshall, 2003) are epigenetic mechanisms involved in modelling and modulating genome functioning in development. Changes in one or more of these epigenetic regulators lead to modifications of the pattern of gene expression (Marshall, 2003) and to alterations in embryonic development, as recent

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somatic cloning experiments have clearly shown. The knowledge of how the epigenetic programme is moulded in the male and female germ cell genomes is crucial to the understanding of the molecular events regulating early development. Although the female gamete is blocked at the dictyate stage of the first meiotic prophase during the whole folliculogenesis, many important epigenetic changes occur to organise the genome to attend early embryonic development (Bestor, 1998). This paper will describe our recent studies, which have been focused on the changes to the chromatin organisation and to the spatial localisation of chromocenters and centromeres during oocyte growth; these changes have been correlated to the developmental competence of the resulting antral and metaphase II oocyte.

2. Chromatin organisation during oocyte growth Within the antral compartment of the mouse ovary, two types of oocytes at the germinal vesicle stage can be distinguished on the basis of their chromatin organization (Fig. 1b and c): one type, referred to as surrounded nucleolus (SN), is characterized by the presence of a rim of Hoechst-positive chromatin that surrounds the nucleolus and a thread-like chromatin organization. The other type, referred to as not surrounded nucleolus (NSN; Mattson and Albertini, 1990; Zuccotti et al., 1995, 1998), has a more diffused Hoechstpositive chromatin, which is not forming a ring around the nucleolus. Oocytes, possessing NSN and SN morphologies, have also been found in a variety of other mammals, including rat (Mandl, 1962), monkey (Lefevre et al., 1989), pig (Crozet et al., 1981) and human (Parfenov et al., 1989). The critical question emerging from these studies is whether both chromatin organisation patterns are indicative of a healthy oocyte competent for development, or whether one is indicative of failure to attain competence or one represents an immature state. The two types of oocytes are found also during oocyte maturation (Zuccotti et al., 1995). Initially, all oocytes in late dictyate are in the NSN state, but while growing, they either continue their development in the NSN configuration or are shifted into the SN configuration. SN oocytes are first found, when the oocytes reach the size of 40–50 ␮m in diameter, constituting about 5% of the whole population of oocytes with this diameter. Their percentage increases up to 50% in the antral compartment, when they reach the size of 70–80 ␮m in diameter. With female ageing, the proportion of SN and NSN antral oocytes changes dramatically. In 1-week-old females, oocytes have not yet reached the antral compartment (<40 ␮m in diameter), and they all show a typical NSN chromatin organisation. In 2-week-old females, only a small number of oocytes have reached the antral compartment and, among these, a small percentage (5.9%) appears with a SN configuration. At about the time when the first ovulation occurs (about 4 weeks of age), a high proportion of follicles have reached the antral stage and NSN oocytes are equal in num-

Fig. 1. Following isolation from the ovaries, antral oocytes (a) were stained with Hoechst 33342 and classified into NSN (b) and SN (c) oocytes on the basis of their chromatin organisation. Following in vitro maturation, insemination and embryo culture, only SN oocytes were capable to reach the four-cell stage (47.4%) and develop to blastocyst (18.4%) (g). On the contrary, NSN oocytes were blocked at the two-cell stage (d).

ber to those with a SN chromatin organisation. Interestingly, when 4 to 6-week-old females were injected with PMSG and the SN/NSN antral oocytes ratio was evaluated 48 h later, the proportion of SN oocytes increased considerably, but the percentage of the two types of oocytes returned back to equal numbers following hCG injection and ovulation. The same number of SN and NSN antral oocytes is maintained until 6 weeks of age then the percentage of SN oocytes increases up to 90% in females of more than 56 weeks of age.

M. Zuccotti et al. / Molecular and Cellular Endocrinology 234 (2005) 11–17

The reduced fertility occurring during aging may be correlated with a decreasing number of NSN oocytes. These experiments suggest that NSN antral oocytes may represent an immature form, which is not ready yet to be ovulated, but requires to reach the SN configuration stage before ovulation. Furthermore, although both types of oocytes are capable of germinal vesicle breakdown in vitro (Wickramasinghe et al., 1991; Debey et al., 1993), observations by Mattson and Albertini (1990) and Zuccotti et al. (1995, 1998) indicate that SN oocytes represent the more advanced stage of preovulatory oocytes. NSN oocytes cultured to metaphase II (MII) and fertilised in vitro are incapable of developing beyond the two-cell stage, whereas SN oocytes are capable of further development to the blastocyst stage (Fig. 1d–g); Zuccotti et al., 1998, 2002). Interestingly, embryonic genome activation (EGA) (also known as zygotic genome activation, ZGA) occurs in the mouse at two-cell stage and represents the first hurdle that the newly formed embryo encounters. During the first 20 h of life, the zygote survives thanks to the maternal RNAs and proteins accumulated during folliculogenesis, but following the first and second mitotic divisions, the embryo transcribes its own genes (Schultz et al., 1999; Hamatani et al., 2004). Lack or wrong EGA leads to embryos death (Schultz et al., 1999). Perhaps, the embryonic genome of zygotes derived from NSN oocytes

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does not attain the epigenetic requirements necessary to correctly express those genes needed to continue beyond the two-cell stage.

3. Nuclear architecture during oocyte growth It is becoming increasingly clear that within the cell nucleus, the chromatin has morphological and functional compartmentalizations (Stewart, 1990; Strouboulis and Wolffe, 1996; Cremer and Cremer, 2001; Marshall, 2003) related to the ribonucleoprotein and deoxyribonucleoprotein activities and to the nuclear organization of gene expression (Misteli and Spector, 1998). This concept derives from Rabl and Boveri (for a review, see Gasser and Laemmli, 1987) whose anticipatory idea was that chromosomes maintain their anaphase–telophase orientation, i.e., individual chromosomes in interphase nuclei tend to occupy exclusive territories rather than to intermingle (Nagele et al., 1998). Clear evidence of the relationships between nuclear compartmentalization and functional states of the cells has been reported (Manuelidis, 1984). More recently, the application of cytochemical techniques combined with three-dimensional (3D) microscopy and computer imaging techniques (reviewed in van der Ploeg, 2000) to specifically

Fig. 2. NOR staining of oocytes of secondary (a), early antral (b) and antral (c and d) follicles. Secondary follicle containing an oocyte that possesses NORsilver-staining deposits throughout its nucleolus. With the increase in size of the nucleolus, silver-staining deposits become separated from one another by more expansive areas of the nucleolar matrix such that its vacuolated appearance is readily apparent in optical sections (arrows). In addition, (1) the NOR staining becomes confined for the most part to the periphery of the nucleolus, and (2) there is the appearance of larger staining deposits at this stage; arrowheads, CHC; bars: 5 ␮m.

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Fig. 3. NOR staining of isolated antral oocytes: (a) NSN oocytes with NOR staining along the periphery of the nucleolus; arrowheads, CHC. (b) SN oocyte demonstrating the absence of NOR-silver-staining. The surface of the nucleolus is associated with CHC that in this case are so plentiful as to form a continuous layer (arrow). CHC (arrowheads) are distributed throughout the germinal vesicle of the NSN oocyte with few associated with the surface of the nucleolus. The insert (a) depicts at higher magnification the characteristic structure of silver-stained CHC when viewed with brightfield; insert, 1 ␮m; bars: 5 ␮m.

reveal DNA, protein components of chromatin and their relationships, has provided a clearer view of the role of chromatin dynamics in nuclear structuring (Abney et al., 1997). Changes in the nuclear architecture during the cell-cycle (Manuelidis, 1984) and cell differentiation (Manuelidis, 1984; Bickmore and Bridger, 1999) are correlated with modifications of gene expression (Misteli and Spector, 1998) and cell function (De

Boni, 1994; Berrios, 1998). The flexible and reversible state of the nuclear architecture has clearly been shown by the successful mammalian cloning experiments (Wilmut et al., 1997; Wakayama et al., 1998). Indeed, the nucleus and its architecture are now seen by molecular morphologists as the structure behind genomic function (Lamond and Earnshaw, 1998). Specifically, the spatial localisation of chromosomes

Fig. 4. Through series of confocal images of the nucleus of an isolated growing oocyte (40–50 ␮m in diameter) reacted with CREST antibodies and counterstained with Hoechst (a). The bright punctate dots indicate centromeres. Positions of centromeres depicted in (b–g) relative to the surface of the nucleolus. Arrowheads indicate two groups of three centromeres each localized on opposite sides of the nucleolus. (h) Stack reconstruction of the whole nucleus made with NIH image. (i) 3D reconstruction of the position of centromeres (green dots) within the oocyte nucleus relative to the nucleolus; bar: 5 ␮m.

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Fig. 5. Confocal images of NSN (a–k) and SN (m–w) oocytes reacted with CREST antibody. The bright punctate dots indicate the centromeres. The 3D reconstruction clearly shows that fewer centromeres are closely associated with the nucleolus of NSN (l) in comparison to the nucleolus of SN (x) oocytes; bar: 5 ␮m.

is central to the global genomic coordination and regulation of basic cellular processes, such as DNA replication and gene expression (Berezney and Wei, 1998; Kozubek et al., 1999). In two recent studies (Longo et al., 2003; Garagna et al., 2004), we have analysed the changes in the pattern of distribution of NORs, centromeres and heterochromatin (chromocenters, CHC) of growing mouse oocytes, using silver-staining, immunocytochemistry and Hoechst staining (that shows A–T preferential binding), respectivelly. Within the nucleolus, the number of NOR-staining deposits was higher in oocytes of primordial/primary to early antral follicles that are heavily engaged in RNA synthesis (Moore et al., 1974; Kaplan et al., 1982). In primordial and primary oocytes, NOR-silver deposits were scattered within the nucleolus (Fig. 2). With further oocyte development and as ribosomal transcripts are accumulated within the nuclear matrix, NORs localise towards the periphery of the nucleolus. NSN oocytes showed a ring of NOR staining along the nucleolar periphery, whereas in SN oocytes, the staining completely disappeared (Fig. 3). The absence of NOR staining in SN oocytes correlates with their transcriptional inactivity. Although the rDNA transcription complex remains spatially associated with the nucleolus of SN oocytes (Zatsepina et al., 2000), the lack of NOR staining may reflect a loss or

alteration of silver reactive proteins in such a manner to render them inaccesible to the silver-staining. The differences in NOR staining between SN and NSN antral oocytes does not entirely explain variations in chromatin patterns as revealed by the fluorochrome Hoechst 33342. Three-dimensional analysis of centromere localisation has shown that during oocyte growth and in NSN antral oocytes, two groups of centromeres (three centromeres each) co-localise with Hoechst-positive heterochromatin and were always associated to the nucleolus surface. These six centromeres, likely correspond to the six NOR-bearing chromosomes present in the CD-1 strain of mice used for these studies. During oocyte growth, centromeres and chromocenters were initially found spread within the nucleus (Fig. 4) and then progressively clustered around the periphery of the nucleolus. In SN antral oocytes, most of the centromeres were juxtaposed and chromatin formed a ring around the nucleolar surface (Fig. 5). Our hypothesis is that the highly homogeneous heterochromatin composition and organisation of mouse centromeres (Redi et al., 1990) may facilitate the nucleolar chromatin association and may be responsible for the clustering of centromeres around the nucleolus. Our results indicate that the oocyte nuclear architecture is developmentally regulated, and they represent the

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necessary knowledge to further investigate the role of nuclear organisation in the regulation of genome functioning during differentiation and development.

Ministero della Salute, Ricerca Finalizzata 2002 “Modelli sperimentali di terapia cellulare e farmacologica per induzione di tolleranza ad alloantigeni”; Istituto Superiore di Sanita’ CS17; the continuous support by Millipore.

4. Gene expression in NSN and SN oocytes The morphological differences between the two types of oocytes has a biological relevance as NSN and SN morphologies in the mouse have been correlated with changes in transcription (Moore et al., 1974; Kaplan et al., 1982; Christians et al., 1999; Liu and Aoki, 2002). NSN antral oocytes remain transcriptionally active and synthesize all classes of RNA, whereas SN oocytes are transcriptionally inactive (Debey et al., 1993; Bouniol-Baly et al., 1999). The number of transcripts of specific genes found in NSN and SN antral oocytes are different. Using a semi-quantitative single-cell retrotranscriptase-polymerase chain reaction (RTPCR) (Gentile et al., 2004), we have analysed the relative profile of abundance of two metabolic genes, Cpt1 (Carnitine palmitoyl transferase 1) and Cpt2 (Carnitine palmitoyl transferase 2) involved in the ␤-oxidation of fatty acids. While Cpt1 transcripts are present only in NSN oocytes, Cpt2 transcripts are present in higher quantity in NSN oocytes. Cpt2 has shown a very interesting pattern of expression (Gentile et al., 2004). Whilst the NSN type of oocytes show a single class of Cpt2 expression, the SN type of oocytes shows two distinct groups of gametes with a significantly different number of Cpt2 transcripts. These two different groups of SN oocytes, as distinguished on the basis of their Cpt2 expression, were found also in MII oocytes, suggesting that both may be ovulated.

5. Future studies The results presented here help to further our understanding of the epigenetic changes occurring in the oocyte nucleus during folliculogenesis. They provide the molecular morphology ground necessary for understanding the regulatory mechanisms that control gene expression at different hierarchical levels during female germ cells differentiation and embryonic development. Our future work will be aimed to improve our knowledge of the nuclear architecture through (a) the analysis of telomeres position and chromosome painting during oocyte maturation and preimplantation embryonic development; (b) the same analysis will be performed on oocytes and preimplantation embryos with altered karyotype structure showing heterozygosity for Robertsonian translocation.

Acknowledgments This studies were made possible thanks to fundings from COFIN 2002, COFIN 2003 to S.G., C.A.R., and M.Z.;

References Abney, J.R., Cutler, B., Fillbach, M.L., Axelrod, D., Scalettar, B.A., 1997. Chromatin dynamics in interphase nuclei and its implications for nuclear structure. J. Cell Biol. 137, 1459–1468. Berezney, R., Wei, X., 1998. The new paradigm: integrating genomic function and nuclear architecture. J. Cell Biochem. Suppl. 30–31, 238–242. Berrios, M., 1998. Nuclear structure and function. Methods in Cell Biology, vol. 53. Academic Press, New York. Bestor, T.H., 1998. Cytosine methylation and the unequal developmental potentials of the oocyte and sperm genomes. Am. J. Hum. Genet. 62, 1269–1273. Bickmore, W.A., Bridger, J.M., 1999. A sense of time and place. Chrom. Res. 7, 425–429. Bouniol-Baly, C., Hamraoui, L., Guibert, J., Beaujean, N., Szollosi, M.S., Debey, P., 1999. Differential transcriptional activity associated to chromatin configuration in fully grown germinal vesicle mouse oocytes. Biol. Reprod. 60, 580–587. Cheung, W.L., Briggs, S.D., Allis, C.D., 2000. Acetylation and chromosomal functions. Curr. Opin. Cell Biol. 12, 326–333. Christians, E., Boiani, M., Garagna, S., Dessy, C., Redi, C.A., Renard, J.P., Zuccotti, M., 1999. Gene expression and chromatin organisation during mouse oocyte growth. Dev. Biol. 207, 76–85. Cremer, T., Cremer, C., 2001. Chromosome territories, nuclear architecture and ene regulation in mammalian cells. Nat. Rev. Genet. 2, 292–301. Crozet, N., Motlik, J., Szollosi, D., 1981. Nucleolar fine structure and RNA synthesis in porcine oocytes during early stages of antrum formation. Biol. Cell 41, 35–42. De Boni, U., 1994. The interphase nucleus as a dynamic structure. Int. Rev. Cytol. 150, 149–171. Debey, P., Szollosi, M.S., Szollosi, D., Vautier, D., Girousse, A., Besombes, D., 1993. Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics. Mol. Reprod. Dev. 36, 59–74. Ferguson-Smith, A.C., Surani, M.A., 2001. Imprinting and the epigenetic asymmetry between parental genomes. Science 293, 1086–1089. Garagna, S., Merico, V., Sebastiano, V., Monti, M., Orlandini, G., Gatti, R., Scandroglio, R., Redi, C.A., Zuccotti, M., 2004. Threedimensional localisation and . . . of centromeres in mouse oocytes during folliculogenesis. J. Mol. Histol. 35, 631–638. Gasser, S.M., Laemmli, U.K., 1987. Improved methods for the isolation of individual and clustered mitotic chromosomes. Exp. Cell Res. 173, 85–98. Gentile, L., Monti, M., Sebastiano, V., Merico, V., Garagna, S., Redi, C.A., Zuccotti, M., 2004. Single-cell quantitative RT-PCR analysis of Cpt-1 and Cpt-2 gene expression in mouse antral oocytes and in preimplantation embryos. Cytogen. Genome Res. 105, 215–221. Hamatani, T., Carter, M.G., Sharov, A.A., Ko, M.S., 2004. Dynamics of global gene expression changes during mouse preimplantation development. Dev. Cell 6, 117–131. Kaplan, G., Abreu, S.L., Bachvarova, R., 1982. rRNA accumulation and protein synthetic patterns in growing mouse oocytes. J. Exp. Zool. 220, 361–370. Kozubek, S., Lukasova, E., Mareckova, A., Skalnikova, M., Kozubek, M., Bartova, E., Kroha, V., Krahulcova, E., Slotova, J., 1999. The topological organization of chromosomes 9 and 22 in cell nuclei has a determinative role in the induction of t(9,22) translocations and

M. Zuccotti et al. / Molecular and Cellular Endocrinology 234 (2005) 11–17 in the pathogenesis of t(9, 22) leukemias. Chromosoma 108, 426– 435. Lamond, A.I., Earnshaw, W.C., 1998. Structure and function in the nucleus. Science 280, 547–553. Lefevre, B., Gougeon, A., Nome, F., Testart, J., 1989. In vivo changes in oocyte germinal vesicle related to follicular quality and size at midfollicular phase during stimulated cycles in the cynomolgus monkey. Reprod. Nutr. Dev. 29, 523–532. Liu, H., Aoki, F., 2002. Transcription activity associated with meiotic competence in fully grown mouse GV oocytes. Zygote 10, 327–332. Longo, F., Garagna, S., Merico, V., Orlandini, G., Gatti, R., Scandroglio, R., Redi, C.A., Zuccotti, M., 2003. Nuclear localization of NORs and centromeres in mouse oocytes during folliculogenesis. Mol. Reprod. Dev. 66, 279–290. Mandl, A.M., 1962. Preovulatory changes in the oocyte of the adult rat. Proc. R. Soc. Lond. (Biol.) 158, 105–118. Manuelidis, L., 1984. Different CNS cell types display distinct and nonrandom arrangements of satellite DNA sequences. Proc. Natl. Acad. Sci. U.S.A. 181, 3123–3127. Marshall, W.F., 2003. Gene expression and nuclear architecture during development and differentiation. Mech. Dev. 120, 1217–1230. Mattson, B.A., Albertini, D.F., 1990. Oogenesis: chromatin and microtubule dynamics during meiotic prophase. Mol. Reprod. Dev. 25, 374–383. Misteli, T., Spector, D.L., 1998. The cellular organization of gene expression. Curr. Opin. Cell Biol. 10, 323–331. Moore, G.P.M., Lintern-Moore, S., Peters, H., Faber, M., 1974. RNA synthesis in the mouse oocyte. J. Cell Biol. 60, 416–422. Nagele, R.G., Freeman, T., Fazekas, J., Lee, K.M., Thomson, Z., Lee, H.Y., 1998. Chromosome spatial order in human cells: evidence for early origin and faithful propagation. Chromosoma 107, 330–338. Parfenov, V., Potchukalina, G., Dudina, L., Kostyuchek, D., Gruzova, M., 1989. Human antral follicles: oocyte nucleus and the karyosphere formation (electron microscopic and autoradiographic data). Gamete Res. 22, 219–231. Redi, C.A., Garagna, S., della Valle, G., Bottiroli, G., Dell’Orto, P., Viale, P., Peverali, F., Raimondi, E., Forejt, J., 1990. Differences in the organization and chromosomal allocation of satellite DNA between the european long tailed house mice of the domesticus and musculus groups. Chromosoma 99, 11–17.

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Reik, W., Dean, W., Walter, J., 2001. Epigenetic reprogramming in mammalian development. Science 293, 1089–1093. Schultz, R.M., Davis Jr., W., Stein, P., Svoboda, P., 1999. Reprogramming of gene expression during preimplantation development. J. Exp. Zool. 285, 276–282. Stewart, A., 1990. The functional organization of chromosomes and the nucleus. Trends Genet. 6, 377–379. Strouboulis, J., Wolffe, A.P., 1996. Functional compartmentalization of the nucleus. J. Cell Sci. 109, 1991–2000. Surani, M.A., 2001. Reprogramming of genome function through epigenetic inheritance. Nature 414, 122–128. van der Ploeg, M., 2000. Cytochemical nucleic acid research during the twentieth century. Eur. J. Histochem. 44, 7–42. Vignon, X., Zhou, Q., Renard, J.P., 2002. Chromatin as a regulative architecture of the early developmental functions of mammalian embryos after fertilization or nuclear transfer. Cloning Stem Cells 4, 363– 377. Wakayama, T., Perry, A.C.F., Zuccotti, M., Johnson, K.R., Yanagimachi, R., 1998. Full term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–374. Wickramasinghe, D., Ebert, K.M., Albertini, D.F., 1991. Meiotic competence acquisition is associated with the appearance of M-phase characteristics in growing mouse oocytes. Dev. Biol. 143, 162– 172. Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J., Campbell, K.H., 1997. Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–813. Zatsepina, O.V., Bouniol-Baly, C., Amirand, C.P., Debey, P., 2000. Functional and molecular reorganization of the nucleolar apparatus in maturing mouse oocytes. Dev. Biol. 223, 354–370. Zuccotti, M., Giorgi Rossi, P., Martinez, A., Garagna, S., Forabosco, A., Redi, C.A., 1998. Meiotic and developmental competence of mouse antral oocytes. Biol. Reprod. 58, 700–704. Zuccotti, M., Piccinelli, A., Giorgi Rossi, P., Garagna, S., Redi, C.A., 1995. Chromatin organization during mouse oocyte growth. Mol. Reprod. Dev. 41, 479–485. Zuccotti, M., Ponce, R.H., Boiani, M., Guizzardi, S., Govoni, P., Scandroglio, R., Garagna, S., Redi, C.A., 2002. The analysis of chromatin organisation allows selection of mouse antral oocytes competant for development to blastocysts. Zygote 10, 73–78.