Nuclear and chromatin composition of mammalian gametes and early embryos HUGHJ.

CLARKE'

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Department of Obstetrics and Gynecology and Department of Biology, McGiN University, Montrhl, Que., Canada Received March 3, 1992 CLARKE, H. J. 1992. Nuclear and chromatin composition of mammalian gametes and early embryos. Biochem. Cell Biol. 70: 856-866. Changes in nuclear structure and chromatin composition regulate gene activity in many cell types and could play a similar role during early mammalian embryogenesis. Oocytes of the mouse contain the three major lamin species present in somatic cells, although lamin A synthesized by oocytes has a higher molecular mass than the somatic species. Oocyte chromatin contains core histones similar to those of somatic cells, as well as elements that are immunologically related to protamines. In contrast, somatic-type histone H1 is not present. DNA topoisomerase I1 has not yet been identified in mammalian oocytes, but is abundant in frog oocytes. In contrast to oocytes, sperm do not contain a typical nuclear lamina. DNA topoisomerase I1 is detectable until late spermiogenesis. Although the DNA of sperm is associated mainly with protamines, some histone may be retained. There is also evidence that the arrangement of the DNA in the nucleus is nonrandom. These results demonstrate differences in nuclear and chromatin composition between oocytes and sperm. After fertilization, the nuclei of cleavage-stage blastomeres undergo programmed modifications. Lamin B is synthesized, whereas lamin A is not. In addition, a set of nuclear proteins is transiently synthesized in mice at the two-cell stage. Changes in embryonic chromatin composition also occur. The relative abundance of transcripts from different core histone genes differs between mouse oocytes and blastocysts. Furthermore, somatic histone H1 becomes detectable beginning at the mid-four-cell stage. As well, during early cleavage stages, expression of plasmid-borne genes becomes dependent on enhancers. Thus, developmentallyregulated changes in nuclear and chromatin composition occur during early mammalian embryogenesis, and these may be important for the initiation and regulation of embryonic gene activity. Key words: chromatin, nucleus, embryogenesis, gametogenesis, mammals. CLARKE,H. J. 1992. Nuclear and chromatin composition of mammalian gametes and early embryos. Biochem. Cell Biol. 70 : 856-866. Des changements dans la structure nuclkaire et la composition de la chromatine contr6lent l'activitk gtnique dans plusieurs types de cellules et pourraient jouer un r61e similaire durant les premiers moments de I'embryogentse des mammiftres. Les ovocytes de la souris renferment les trois principales esptces de lamines prksentes dans les cellules somatiques, mais la lamine A synthktiskepar les ovocytes posstde une masse molkculaire plus Clevke que I'esptce somatique. La chromatine des ovocytes contient les mCmes histones centrales que celles des cellules somatiques de mCme que les klkments immunologiquement relies aux protamines. En revanche, l'histone H1 de type somatique est absente. La DNA topoisomkrase I1 n'a pas encore kt6 identifiCe dans les ovocytes mammaliens, mais elle est abondante dans les ovocytes de grenouille. Au contraire des ovocytes, les spermatozo'ides n'ont pas de lamina nuclkaire typique. La DNA topoisomkrase I1 est prtsente jusqu'aux derniers instants de la spermiogenbe. Le DNA des spermatozo'ides est surtout associk aux protamines, mais on y trouve une certaine quantitt d'histones. I1 existe kgalement des preuves que l'arrangement du DNA dans le noyau n'est pas au hasard. Ces rCsultats dkmontrent des differences dans la composition nuclkaire et la composition de la chromatine entre les ovocytes et les spermatozoides. Aprts la fkcondation, les noyaw des blastomtres au stade de clivage subissent des modifications programmkes. La lamine B est synthktiske, mais non la lamine A. De plus, un ensemble de protkines nuclkaires sont transitoirement synthktiskes dans les souris au stade de deux cellules. I1 se produit Cgalement des changements dans la composition de la chromatine embryonnaire. L'abondance relative des transcrits des diffkrents gtnes des principales histones diffkre entre les ovocytes et les blastocystes de souris. De plus, I'histone H1 somatique devient visible a partir du milieu du stade de quatre cellules. Aussi, durant les premiers stades de clivage, I'expression des gtnes porteurs de plasmides devient dependante de stimulateurs. Ainsi, au cours du dheloppement, les change-contr6lks dans la composition nuclkaire et la composition de la chromatine arrivent au debut de l'embryogenbe mammalienne et ils peuvent Ctre importants pour l'installation et la rkgulation de l'activitk genique embryonnaire. Mots clPs : chromatine, noyau, embryogentse, gamktogentse, mammiftres. [Traduit par la rkdaction]

Introduction Nuclear and chromatin structure as regulators of gene activity The potential for gene activity to be regulated by nuclear structure and chromatin composition has long been recognized (Comings 1968; Hancock and Boulikas 1982; ABBREVIATIONS: kDa, kilodaltons; EC, embryonal carcinoma; TRC, transcription-requiring complex. ' ~ d d r e s sfor correspondence: Room F3-36, Women's Pavilion, Royal Victoria Hospital, 687, avenue des Pins O., MontrCal, Que., Canada H3A 1Al. Printed in Canada / Imprime au Canada

Manuelidis 1990; Felsenfeld 1992). Chromosomes are not randomly dispersed in the nucleus, but often occupy specific domains. Thus, heterochromatin frequently is found at the nuclear periphery, whereas euchromatin is present in the interior of the nucleus (Manuelidis 1990; Haaf and Schmid 1991). The spatial arrangement of chromatin also can vary during the cell cycle and according to cell type. In postmitotic Purkinje cells, for example, the centromeres move from the nuclear periphery towards the interior (Manuelidis 1990) and when tissue culture cells are exposed to 5-azaqtidine, the centromere of the inactive X-chromosome becomes repositioned in the nucleus (Dyer et al. 1989). Cer-

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tain euchromatic regions may be associated with the nuclear matrix, which is the insoluble structure that remains following detergent extraction of nuclei. It has been proposed that specific DNA sequences, which often occur 5' and 3' of genes, may anchor chromatin to the matrix, while the intervening chromatin loops out such that it will be accessible to transcription factors (Gasser et al, 1989; but see Eggert and Jack 1991). These transcription factors may themselves be restricted to specific nuclear domains, as poly(A)containing RNA is localized near the nucleolus (Carter et al. 1991). Furthermore, processing of the newly synthesized premRNA by spliceosomes also probably is spatially restricted, as the latter exist on a reticular network in the nucleus (Spector et al. 1991). Taken together, these results suggest that the position of regions of euchromatin within the nucleus may influence the expression of resident genes. The effect of chromatin composition and structure on gene activity is well illustrated by the relationship between heterochromatin and position-effect variegation (Henikoff 1990). Heterochromatin generally is associated with transcriptional inactivity and it has been observed that if a euchromatic gene is moved, such as by mutagenesis, to a position near heterochromatin, its expression may be inactivated. In some cases, this inactivation occurs only in a fraction of the affected cells. As the active or inactive state is stably transmitted during cell division, clones of nonexpressing and expressing cells are produced and the result is a mosaic or variegated phenotype. Position-effect variegation has been observed in mammals carrying autosomeX-chromosome translocations (Russell 1963) and is particularly well studied in Drosophila. In Drosophila, dominant mutations exist that are defined either as suppressors, when the effect of the mutation is to increase the number of cells expressing the gene, or as enhancers, when the effect of the mutation is to decrease the number of cells expressing the gene. One suppressor gene, suvar3(7), was recently cloned and shown to encode a protein containing five widely spaced zinc-finger domains (Reuter et al. 1990). It was speculated that these domains could contact widely spaced regions of chromatin and essentially pull them together to produce heterochromatin. Mutations in the gene would reduce the amount of heterochromatin present, thus increasing the probability that a gene residing near heterochromatin would be expressed. Several observations implicate histones in the relationship between heterochromatin and position-effect variegation. First, flies containing a reduced number of histone genes, for which hundreds of copies normally exist, show suppression of variegation (Moore et al. 1983). Second, a gene that acts as a suppressor of variegation is associated with an increase in the steady-state level of histone acetylation (Dorn et al. 1986). Third, exposure of flies to butyrate increases the amount of histone acetylation and decreases the extent of variegation (Mottus et al. 1980). These latter results may be interpreted by recalling that acetylated histones are associated with transcriptionally active regions (Turner 1991) and inhibit the ability of histone H1 to condense chromatin in vitro (Ridsdale et al. 1990). These results establish a link between histones, heterochromatinization, and gene expression. It appears that the quantity of histones present in a cell, as well as covalent modifications to the histones, can influence the pattern of gene expression. Nevertheless, the manner in which histones, particularly

histone H1, may regulate gene activity is controversial. On the one hand, histones frequently appear to act as repressors of transcription (Zlatanova 1990; Felsenfeld 1992). Histone H1 is depleted in transcriptionally active chromatin (Kamakaka and Thomas 1990) and in the nonmethylated CpG islands present in promoter sequences (Tazi and Bird 1990). Experiments measuring transcriptional activity of reconstituted chromatin have shown that addition of histone HI dramatically decreases transcription and that addition of specific transcription factors, such as Spl and GAL4-VP16, elevates activity well beyond the level observed even in the absence of histone H1 (Croston et al. 1991; Laybourn and Kadonaga 1991). In a separate series of experiments, it has been shown that amphibian oocyte extracts to which HI-stripped chromatin is added transcribe both oocyte and somatic 5 s RNA genes (Wolffe 1989). Addition of histone H1 to the extracts selectively represses transcription of the oocyte genes. A regulatory role for nucleosomal core histones in gene activity is implied by experiments in yeast, where it has been observed that deletions in the arninoterminal portion of histone H4 cause activation of normally silent mating type loci (Grunstein 1990). On the other hand, deletions in the N-terminal region of histone H4 reduce expression of the GAL1 and pH05 genes, implying a positive regulatory role for histone H4 in these cases (Durrin et al. 1991). As well, histone H1 has been detected on the chromatin axis of a transcriptionally active Balbiani ring gene in Drosophila (Ericsson et al. 1990), suggesting that the presence of histone H1 in chromatin is compatible with transcription. It may be that histones modulate gene activity in several ways in vivo.

Programming of gene activity during early mammalian embryogenesis In view of the established role of nuclear structure and chromatin composition in the regulation of gene expression, it is possible that these may play an important role in patterning gene expression in early embryos. Early embryogenesis is a period of major change in the activity and programming of the genome. Mature spermatozoa and ovulated unfertilized eggs are inactive transcriptionally. Following fertilization, transcription does not resume immediately but only after one or more cleavage divisions have occurred. The precise time of 'embryonic genome activation varies among species (Telford et al. 1990). At the time of transcriptional activation, a specific pattern of gene expression is imposed on the genome; in the mouse, for example, heat-shock proteins (Bensaude et al. 1983) and a group of lamin-like proteins (Conover et al. 1991) are transiently synthesized at the two-cell stage. In addition, the maternally and paternally transmitted genomes are imprinted in some manner, such that certain genetic alleles are expressed differently depending on their parental origin (Surani et al. 1990). Evidence suggests that the effects of imprinting may be manifested as early as during preimplantation development (Surani et al. 1986; Howlett et al. 1987; Renard et al. 1991;Hagemann and First 1992). Developmentally programmed changes in nuclear or chromatin composition could be involved in these events, for example, by ordering the chromatin into transcriptionally competent domains within which transcription of specific genes can be regulated by the activity of specific transcription factors. Evidence supporting such a role for changes in nuclear

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and chromatin composition comes from nuclear transplantation studies. In frogs, nuclei from differentiated cells show a limited potential to programme normal development when transplanted into enucleated, fertilized eggs. But, when the donor nuclei are first conditioned by residence in the cytoplasm of unfertilized oocytes and then tested, their developmental potential is substantially enhanced (DiBerardino 1987). This result could be explained by supposing that the chromatin structure or composition of the donor nucleus is modified by factors present in oocyte cytoplasm. In mice, the ability of a transplanted nucleus to direct development is closely linked to the embryonic stage of the donor. Nuclei taken from early two-cell embryos direct development of recipient zygotes at least to the blastocyst stage. But when the nuclei are taken from middle or late two-cell embryos, the recipients rarely progress beyond the four-cell stage. Nuclei taken from cleavage-stage blastomeres or inner-cell-mass cells also are unable to direct development of one-cell embryos (McGrath and Solter 1984; Howlett et al. 1987; Tsunoda et al. 1989). As transcription begins at the two-cell stage in mice, the loss of nuclear ability to direct development in one-cell embryos is correlated with the activation of embryonic transcription. It has been suggested that transcriptional activation modifies nuclei, essentially irreversibly, such that they cannot be reprogrammed after transfer into a transcriptionally inactive one-cell embryo (Howlett et al. 1987). Support for this interpretation comes from the observation that when eight-cell nuclei are transferred into a transcriptionally active two-cell blastomere, the hybrid cell develops well and occasionally to term (Rob1 et al. 1986; Howlett et al. 1987; Tsunoda et al. 1987). These results suggest that activation of transcription is linked to a stable change in nuclear or chromatin structure, which cannot be reversed upon transfer into a one-cell embryo. Several observations suggest, however, that this explanation cannot entirely account for the loss of nuclear potential that occurs during development. Tsunoda et al. (1989) observed that when primordial germ cell nuclei from 14.5to 17.5-day-old embryos are transferred into mouse eggs, some of the recipients develop beyond the stage of implantation. This shows that a transcriptionally active nucleus introduced into a one-cell embryo can direct preimplantation development. As well, live births in sheep (Smith and Wilmut 1989) and cattle (Prather et al. 1987) have been obtained when nuclei from embryos beyond the stage of transcriptional activation, including from the inner cell mass (Smith and Wilmut 1989), are transferred into one-cell embryos. These transcriptionally active nuclei evidently must have been properly reprogrammed within the one-cell embryo. Finally, it is interesting to note that nuclei obtained from late two-cell and older mouse embryos show a progressive reduction in their capacity to support even the first cleavage division of recipient eggs (Howlett et al. 1987; Tsunoda et al. 1989). As this cleavage does not require transcription, it appears that characteristics not directly related to transcriptional activity contribute to the compromised developmental potential of these nuclei. These results strongly suggest that the loss of nuclear potential that occurs during cleavage is due to multiple factors. To begin investigation of the role of changes in nuclear and chromatin composition in the control of gene activity

in preimplantation embryos, it is necessary to identify those changes that occur during this period of development. While these have not been the subject of extensive study to date, the information available at present demonstrates unequivocally that major alterations in nuclear and chromatin structure characterize early mammalian development. The purpose of this article is to review current knowledge, first, of the state of the nucleus and chromatin in the egg and in the sperm, and second, of the changes that have been identified during the preimplantation stages of embryogenesis, spanning the period when the embryonic genome becomes transcriptionally active and the initial pattern of gene expression is established. Most of the available information deals with changes in the nuclear lamina and in the basic chromosomal proteins. I will not discuss DNA methylation, which has been reviewed recently (Reik et al. 1990). Although the focus is on mammals, I will refer to other animals where mammalian information is lacking or where comparative data may assist interpetation of results. Nuclear and chromatin composition of oocytes Nuclear composition Lamins The nucleoplasmic face of the nuclear membrane in most cells is lined by a fibrillar protein network known as the nuclear lamina (Gerace and Burke 1988). The lamina in mammals is primarily composed of three intermediate-type filament proteins: lamins A, B, and C. Lamin B may link the lamina to the nuclear envelope, whereas lamins A and C are thought to interact with chromatin. Direct interactions between lamins A and C and chromatin have recently been documented in vitro (Burke 1990; Glass and Gerace 1990). These interactions are sensitive to treatments that would disrupt the higher order structure of heterochromatin. These results imply that the arrangement of chromatin in the nucleus could be regulated by the composition of the lamina. Several groups have examined the nuclear lamina of germinal vesicle-stage oocytes and fertilized eggs of the mouse, using antibodies recognizing either lamins A and C or lamin B (Schatten et al. 1985; Houliston et al. 1988; Maul et al. 1987; Stewart and Burke 1987). All three lamins have been detected both by immunofluorescenceand by immunoblotting. Similar immunofluorescence data have been obtained from sheep and cows (Prather et al. 1989). Synthesis of lamin A, but not B or C, has been detected in unfertilized mouse eggs (Houliston et al. 1988). It has been noted, however, that lamin A of eggs migrates more slowly during electrophoresis than lamin A from mouse embryos and tissue culture cells, and that this difference is not related to the cell cycle state (Houliston et al. 1988). This suggests that eggs may contain a specific subtype of lamin A. DNA topoisomerase ZI DNA topoisomerase I1 catalyzes the cutting and ligation of double-stranded DNA. In addition to its enzymatic role (Charron and Hancock 1990), it also serves a structural function in the nucleus as a major component of the nuclear matrix and is present in the core of metaphase chromosomes (Earnshaw et al. 1985; Gasser et al. 1986). As association of chromatin with the nuclear matrix can be correlated with increased gene expression (Stief et al. 1989), it is therefore of interest to know whether topoisomerase I1 is present in the nuclei of oocytes and early embryos. In frogs (Luke and Bogenhagen 1989), a 180-kDa pro-

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tein that reacts with antitopoisomerase I1 antibodies and cofractionates with topoisomerase I1 activity accumulates in the oocyte during oogenesis and also is synthesized during meiotic maturation. Oocyte topoisomerase I1 is stored primarily in the germinal vesicle. The quantity of topoisomerase I1 present in the germinal vesicle exceeds that present in proliferating cells by several orders of magnitude. This suggests that the oocyte contains a large store to be used in nuclear assembly during the early cleavage cycles. No studies of topoisomerase I1 in mammalian oocytes have yet been reported. It will be interesting to determine whether a similar maternal supply is present.

Chromatin composition Histones Early studies of the nucleoprotein of mammalian oocytes were primarily autoradiographic. Wassarman and Letourneau (1976) have observed an association of lysine-containing proteins with the germinal vesicle of immature oocytes and with the condensing chromosomes of maturing oocytes. When protein synthesis is prevented in these oocytes, the chromosomes condense but no radioactivity becomes associated with them. Similar results have been obtained by Rodman and Barth (1979), using radioactive arginine to label newly synthesized proteins. In pigs and rabbits (Motlik et al. 1978), basic proteins synthesized by immature oocytes accumulate in the germinal vesicle and become associated with the chromosomes at metaphase I and metaphase 11. It is unknown whether this incorporation of basic protein into chromosomes during maturation represents a qualitative change in the oocyte nucleoprotein or a turnover of continuously synthesized species. On the basis of its migration during gel electrophoresis, it has been suggested that the major lysine-containing species synthesized during mouse oocyte maturation is histone H1 (Wassarman and Letourneau 1976). Antibodies raised against the histone H1 subtypes present in somatic cells do not, however, detect any immunoreactive species in oocyte nuclei or chromosomes. By contrast, somatic histone H1 is detectable in the nuclei of ovarian granulosa cells and of oocytes previously injected with histone H1 (Clarke et al. 1992). Furthermore, somatic histone H1 is not detectable in immunoblotted oocyte lysates, although it is present in lysates of preimplantation embryos. The latter observation argues against the possibility that histone H1 is present in oocytes, but masked so that it is immunologically undetectable. These results suggest that oocytes do not contain detectable quantities of somatic histone HI. Rather, as discussed below, the lysine-containing protein synthesized during maturation may represent an oocyte-specific histone H1 subtype. The existence of unusual histone H1 subtypes in oocytes is widespread among animals. Sea urchin eggs contain a maternally encoded histone H1 that disappears after the first few cleavage divisions (Poccia 1986). In the sea worm (Franks and Davis 1983), a subtype designated Hlm is found only in oocytes and embryos up to the blastula stage. Similar results have been observed in the mud snail (Flenniken and Newrock 1987). In amphibians eggs, both somatic and unique histone H1 subtypes may be present. Several groups have detected somatic histone H1 during oogenesis and in unfertilized eggs (Flynn and Woodland 1980; van Dongen et al. 1983). Recently, a novel H1 species, termed HlX, has

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been identified in chromatin assembled in toad and Xenopus egg extracts (Ohsumi and Katagiri 1991). Interestingly, somatic H1 is not detected on this same chromatin (Dilworth et al. 1987; Ohsumi and Katagiri 1991). Histone H1X may correspond to a basic, chromatin-associated protein known as B4, which is a product of maternal mRNA present in Xenopus oocytes and shares sequence homology with histone H1 (Smith et al. 1988). As a rule, the histone H1 subtypes present in eggs are larger and contain less lysine than somatic histone HI. In this context, it may be significant that Wassarman et al. (1979) identified a lysinecontaining protein synthesized by immature oocytes and localized in the nucleus, which migrated slightly more slowly than histone H1 during electrophoresis. The unique histone H1 subtypes present in eggs may fill specific roles in chromatin function during oogenesis or early embryogenesis. Although the evidence suggests that mammalian eggs contain a distinctive histone H1, their core.histone populations are likely similar to those present in somatic cells. mRNAs encoding H2A, H2B, and H3 have been identified in eggs (Giebelhaus et al. 1983; Graves et al. 1985) and synthesis of histone H4 during oogenesis has been directly measured (Wassarman and Mrozak 1981). Furthermore, antisera raised against histones H2B, H3, and H4 all stain nuclei and condensed chromosomes of eggs (Rodman et al. 1981; H.J. Clarke, unpublished observations). In contrast to these results in mammalian eggs, sea urchin eggs contain distinct core histone populations (Poccia 1986). It should, therefore, not be excluded that mammalian eggs may contain core histone subtypes that differ subtly from those present in somatic cells.

Other chromosomal proteins It has also been claimed that oocyte chromosomes contain proteins immunologically related to sperm protamines (Rodman et al. 1981). An antiserum was prepared against a sperm basic protein fraction, including protamines, and shown not to recognize thymus histones by immunoblotting. By immunofluorescence, this antiserum stained the chromosomes of metaphase I1 oocytes, but not those of the first polar body, metaphase I oocytes, or ovarian cumulus granulosa cells. As these cells were fixed using acetic acid, it is possible that the metaphase-11-specific staining reflects a differential extractability of acid-soluble proteins. Some support for the claim of protamine-like proteins in mouse oocyte chromosomes may be derived from the observation that these display a histochemical reaction consistent with the presence of arginine-rich proteins (Da-Yuan and Longo 1983). The availability of cloned mouse protamine genes (Hecht 1986) and antibodies (Nonchev and Tsanev 1990) should make it possible to further investigate these findings. Adducin is a membrane skeletal protein that typically is found associated with complexes containing spectrin and actin. Ovulated eggs reacted with anti-adducin antibodies show a fluorescence localized over the chromosomes and immunoblots of eggs reveal a reactive species that comigrates with adducin (Pinto-Correia et al. 1991). These results suggest that a molecule related to adducin is present on the chromosomes of eggs at metaphase 11. Given the function of adducin in other cell types, it is possible that this protein participates in anchoring the chromosomes near the cortex of the egg, thus ensuring that the second meiotic division will be followed by an asymmetrical cleavage.

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Nuclear and chromatin composition of sperm Nuclear composition Lamins Several groups have attempted to determine whether a nuclear lamina composed of lamins exists in sperm nuclei. Maul et al. (1986) identified by immunofluorescence antigens reacting with antibodies directed against lamins A/C and lamin B. Mature sperm contained a small amount of anti-lamin-B immunoreactive material. The anti-laminA/C staining was detectable only in sperm that had been salt extracted and treated with DNase, and no consistent pattern of staining was detected. Schatten et al. (1985) reported a weak reaction of mouse sperm with antibodies recognizing lamins A/C and B. As no immunoblotting results were reported, it is not established that the immunoreactive species were somatic lamins. In rats, a 60-kDa protein detected by immunoblotting using anti-lamin antisera has been reported in testicular and epididyrnal sperm (Sudhakar et al. 1992). This species was also detected in sperm from a variety of other animal species and even from plant meiocytes. In contrast to these results, Longo et al. (1987) were unable to detect a nuclear lamina in mouse sperm by electron microscopy nor could they detect lamins by immunoblotting. Instead, these investigators described a perinuclear theca, apposed to the outer, cytoplasmic face of the nuclear envelope. The theca is chiefly composed of two types of basic proteins, one termed calicin and the other comprising a family of related polypeptides. Protein species that were immunologically related to these thecal components were also present in rat and human sperm. These results may be supported by observations in the chick, where lamins could not be detected, either by immunofluorescence or by immunoblotting, beyond the pachytene stage of spermatogenesis (Lehner et al. 1987). Hamster sperm have also been reported to contain a nuclear matrix of unknown biochemical composition (Ward and Coffey 1989). In sum, the weight of evidence currently available suggests that, while mammalian sperm contain elements that provide structural support to the nucleus, a somatic-type lamina present on the interior face of the nuclear membrane has not been demonstrated. DNA topoisomerase 11 DNA topoisomerase I1 has been detected immunologically on the chromatin cores of pachytene-stage spermatocytes in the rooster (Moens and Earnshaw 1989). As well, topoisomerase I1 activity during rooster spermatogenesis has been examined (Roca and Mezquita 1989) using a DNA unknotting assay based on the activity of topoisomerase I1 to break and rejoin DNA strands. When extracts enriched for sperm at successive stages of spermatogenesis were tested in this assay, topoisomerase I1 activity at a level about four times that present in liver nuclei was detected at all stages up to round spermatids. In mature spermatozoa, the topoisomerase I1 activity dramatically declined. It is not known, however, whether the decline in activity is due to absence of the enzyme in mature sperm. No information is available concerning the fate of topoisomerase I1 during mammalian spermatogenesis.

Chromatin composition Protarnines During spermatogenesis in many organisms, the somatic

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histones are replaced by sperm-specific nucleoproteins (reviewed by Poccia 1986). In some cases, such as the sea urchin, these are also histones. In many other species, including mammals, the somatic histone complement is entirely or extensively (see below) replaced by protamines. Protamines are small proteins that are rich in arginine; mouse protamine 1, for example, contains 50 amino acids, of which 29 are arginine (Hecht 1986). Most mammalian sperm contain only one protamine species, but mice and humans contain two and three species, respectively, encoded by different genes (Hecht 1986). The association of protamines with mammalian sperm chromatin follows the arrest of transcription during spermiogenesis, and it is thought to permit the chromatin to be packed within the sperm nucleus, where it is estimated to be up to sixfold more condensed than in metaphase chromosomes (Ward and Coffey 1991). Balhorn (1982) has proposed a model suggesting how protamines could facilitate such unusually tight packaging of DNA. The central, arginine-rich region of the protamines is.proposed to lie lengthwise in the minor groove of the DNA helix, with the arginine groups neutralizing the negative charge of the DNA and eliminating electrostatic repulsion between neighbouring segments. The tails of the protamines could then fit in the major groove of adjacent DNA molecules, thus linking the strands together in parallel arrays. Disulfide bonds between the cysteine residues of the protamines presumably stabilize the condensed structure. Histones Although protamines are the major nucleoprotein present in mammalian sperm, evidence suggests that some histones are also present. Palmer et al. (1991) have purified an acidsoluble protein from bull sperm nuclei that they identified as CENP-A, a 17-kDa species specifically associated with centromeres. CENP-A was present in similar amounts in acid extracts prepared from sperm and thymus nuclei, whereas the core histone content of the sperm nuclear extract was less than 1% that of the thymus nuclear extract. This suggests that the CENP-A could not have been entirely derived from somatic nuclei present in the sperm nuclear preparation; in fact, the relative enrichment of CENP-A in the sperm nuclear extract permitted its purification. On the basis of its biochemical properties and a partial sequence analysis, CENP-A was classified as a histone sharing some sequence identity with histone H3. These results suggest that specific proteins associated with the centromere may be retained during spermatogenesis. In the case of the human, a more extensive complement of histones (up to 15% of the nucleoprotein; Tanphaichitr et al. 1978) may be present in sperm. Species related to H2B (Tanphaichitr et al. 1978) and H3 and H4 (Gusse et al. 1986) have been reported and variants of each of the four core histones were recently identified in preparations of human sperm (Gatewood et al. 1990). These variants are minor components of the histones present in somatic cells; unfortunately, the quantitative contribution of these histones to sperm nucleoprotein was not reported. The same authors reported that antisera to histone H3 stained human sperm, as detected using immunofluorescence. By contrast, mouse sperm nuclei are not stained by antisera against histone H3 ( ~ o n c h e vand Tsanev 1990) or histone H ~ (Rodman B et al. 1981) and core histones are very rare or absent by biochemical analysis (O'Brien and BellvC 1980). Such species

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differences suggest that a significant histone complement in mature sperm may not commonly exist among mammals.

Telomeres The telomeres of eukaryotic chromosomes contain tandem repeats of G-rich sequences (Blackburn 1991). The length of these terminal repeat arrays was examined in a variety of human tissues by Hastie et al. (1990). It was found that the length of the repeats in fetal cells and sperm was approximately twice that found in older tissue. The implication of these results is that during spermatogenesis (and presumably also during oogenesis) a telomerase acts to increase the length of the telomeres of each chromosome. The importance of this change with respect to gene expression in the embryo is unknown. However, a change in telomere length conceivably could affect the spatial arrangement of chromatin in an interphase nucleus. Organization of the DNA in sperm Following fertilization, the sperm chromatin becomes organized in a manner such that the appropriate genes are transcribed in the embryo. Two mechanisms may be proposed that could establish the correct pattern of gene expression. On the one hand, factors present in the egg cytoplasm may programme an unpatterned sperm chromatin. On the other hand, the sperm chromatin may be differentiated in a manner that determines which genes will be expressed in the early embryo. In chicken sperm, for example, differences in the DNA methylation pattern exist between constitutively expressed genes and tissue-specific genes (Groudine and Conklin 1985). Several investigators have attempted to establish whether heterogeneity exists in the packaging of the DNA in the sperm nucleus. Powell et al. (1991) examined the distribution of different classes of repetitive DNA sequences in bovine sperm. In these experiments, sperm were first induced to disperse in vitro by brief proteolysis, thus increasing the volume occupied by the DNA and probably facilitating access of DNA probes. Different DNA probes were then hybridized to the dispersed nuclei. A probe for a highly repeated (70 000 copies) interspersed sequence showed an apparently random pattern of hybridization. By contrast, a probe for centromeric repetitive DNA hybridized along a thin equatorial band, although this pattern was not detectable in extensively decondensed nuclei. The same equatorial localization was seen using a probe for ribosomal DNA, which is located near centromeres. These results suggest that centromeric DNA is positioned nonrandomly in the sperm nucleus. It is interesting to speculate that this could be linked to the presence of specialized histone species associated with centromeres (Palmer et al. 1991). Gatewood et al. (1987) have examined the distribution of low-copy-number sequences in human sperm. Sperm chromatin was subjected to conditions under which histones but not protamines are extracted from DNA. The chromatin containing naked and protamine-bound DNA was then digested using a restriction endonuclease, the restricted DNA was recovered from the supernatant, and the protamineDNA pellet was reextracted to remove protamine and digested using the same restriction endonuclease. This procedure thus produced two populations of DNA, a histoneassociated fraction and a protamine-associated fraction. DNA clones corresponding to low-copy-number sequences were then generated from each fraction. It was found that

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two clones derived from the nucleohistone fraction preferentially hybridized to that fraction, whereas two clones derived from the nucleoprotamine fraction preferentially hybridized to that fraction. This suggests that each fraction was enriched for specific DNA sequences. It was speculated that association of sequences with either histones or protamines in sperm could influence the expression in the embryo of genes within those sequences. The absence of a significant histone complement in sperm of other species, however, suggests that this may not be a general mechanism for marking genes. Nuclear and chromatin composition of early embryos Nuclear composition In 1987, Lehner et al. reported a series of experiments examining the distribution of the major chick lamins during embryogenesis. The results showed that differentiation was generally accompanied by characteristic changes in the species of lamins present and that the timing of these changes varied among tissues. Similarly, in Xenopus, a single lamin species is present in eggs, a second species appears at the midblastula transition, and a third appears at the gastrula stage (Benavente et al. 1985; Stick and Hausen 1985). These results suggest that a link might exist between the biochemical composition of the nuclear lamina and the state of nuclear differentiation. In mice, lamin A is present in extraembryonic tissue shortly after implantation, but appears in embryonic tissue only later during embryogenesis or after birth (Rober et al. 1989). A number of groups have searched for changes in the lamin composition of mammalian preimplantation embryos. These have revealed that lamin B is present at all stages. By contrast, the pattern of distribution of the closely related lamins A and C is controversial. Using immunofluorescent staining, Schatten et al. (1985) detected lamins A/C in oocytes and cleavage-stage blastomeres, but not in morulae or blastocysts. Stewart and Burke (1987) observed a faint nuclear fluorescence in onecell embryos and this became progressively weaker during embryogenesis, such that lamins A/C were undetectable in blastocysts. These workers also were unable to immunoprecipitate radiolabelled lamins A/C from blastocysts, although labelled lamin B was detected. Results similar to these have been obtained in other mammals. Lamins A/C were immunofluorescently detectable in the cow and pig embryos up to the eight-cell stage, but not in morulae or blastocysts (Prather et al. 1989). In the case of the pig, when 16-cell nuclei were transferred into eggs, they acquired lamins A/C. These results all suggest that lamins A/C are present in the egg, but disappear or are diluted out during the early cell divisions so that they become undetectable by the blastocyst stage. This interpretation is supported by the observation that undifferentiated EC cells, which share characteristics with the inner cell mass of the blastocyst, do not contain lamins A/C (Lebel et al. 1987; Stewart and Burke 1987). In contrast to these results is the report of Houliston et al. (1988) who performed an extensive series of experiments using embryos from several mouse strains. Using immunofluorescence and several different fixation protocols, they observed lamins A/C at all examined stages of preimplantation development, including morulae and blastocysts. Lamins A/C also were detected in eggs and in blastocysts

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by immunoblotting. Synthesis of lamins A/C, however, could be detected only in eggs and not in blastocysts. To reconcile these results, it may be proposed that the ratio of lamins A/C : lamin B in nuclei may decrease during mouse preimplantation development. Thus, differences in immunological technique could account for the different irnrnunofluorescence results obtained at stages when relatively little of lamins A/C is present. This proposal also is consistent with the observation (Houliston et al. 1988; Stewart and Burke 1987) that lamins A/C are not synthesized in late cleavage-stage embryos. It seems likely, therefore, that changes in the composition of the nuclear lamina occur during early mouse development. As lamins A/C are thought to link chromatin to the lamina (Gerace and Burke 1988), it might be proposed that when the quantity of lamins A/C is reduced, the chromatin would be less tightly associated with the nuclear periphery. Such a change in the spatial distribution of the chromatin could influence the pattern of gene expression. To investigate a link between the presence of larnins A/C and the state of nuclear activity, Peter and Nigg (1991) transfected a chicken lamin A gene into undifferentiated EC cells. Expression of the chicken lamin A, which is immunologically distinguishable from the mouse homologue, was confirmed by immunofluorescence and by immunoblotting. No effect of the expressed lamin A could be detected, however, either on the growth of the undifferentiated cells or on their pattern of differentiation following treatment with retinoic acid. Although these results evidently do not bear directly on the situation in the embryo, they suggest that the presence of lamin A in the nuclei of embryonic cells does not alter gene expression associated with either the undifferentiated state or the process of differentiation. The biological role of the change in lamina composition during early embryogenesis remains to be identified. An interesting set of nuclear proteins, designated the TRC, that share characteristics with the lamins has recently been characterized by Conover et al. (1991). These proteins have apparent molecular masses of 68,70, and 73 kDa and are related to each other as judged by peptide mapping. Their solubility properties are similar to those of the lamins and they are concentrated in the nucleus. However, they do not comigrate with somatic lamins in two-dimensional gels, nor do they react with anti-lamin antibodies. As well, whereas lamins become soluble following nuclear envelope breakdown, these proteins remain insoluble. Finally, a significant fraction of the TRC is present in the cytoplasm in contrast to the lamins which are essentially exclusively nuclear. The TRC proteins therefore do not appear to be somatic-type lamins. These proteins are synthesized only at the two-cell stage, where they represent up to 5% of total protein synthesis, and are almost completely degraded by the eight-cell stage. These results suggest that embryonic nuclei transiently possess an unusual composition or structure following activation of transcription.

Chromatin composition Histones In the mouse, somatic histone H1 cannot be detected in one-cell or two-cell embryos. A small fraction (less than 10%) of four-cell embryos examined early during the third cell cycle contains somatic histone H1. By contrast, over

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90% of embryos at the late four-cell stage contains somatic histone H1 and it is present in all cell nuclei at the morula and blastocyst stages (Clarke et al. 1992). Thus mouse embryo nuclei acquire somatic histone H1 during the third cell cycle. The appearance of somatic histone H1 shows a dependence on transcription extending into the early fourcell stage. In addition, the nuclear content of somatic histone H1 is vastly reduced when four-cell embryos are prevented from undergoing DNA replication. These results indicate that embryonic chromatin undergoes a major, transcriptiondependent change in its composition at the four-cell stage that is associated with progression through S-phase. The precise timing of the appearance of histone H1 at the four-cell stage may be a product of the combined effects of the timing of transcriptional activation and the control of histone gene expression. In most cell types (though growing oocytes are an exception), the bulk of histone gene expression occurs during S-phase (van Holde 1989) and inhibition of DNA replication leads to a rapid decline in histone gene expression (Graves et al. 1987). In the mouse embryo, it may be speculated that, following widespread activation of embryonic transcription at the late two-cell stage (Flach et al. 1982), histone gene expression is not activated until the next S-phase; thus somatic histone H1 is first synthesized when the four-cell embryos begin DNA replication. It would be interesting to examine when somatic histone becomes detectable in mammals in which transcription begins during embryogenesis (Telford et al. 1990). Switches in the subtype of histone H1 associated with chromatin have been observed in a variety of other species. In the sea urchin, two maternally encoded H1 subtypes appear successively in chromatin of developing embryos and subtypes retained in adult cells are first detected at the late blastula or gastrula stage. In the mud snail, different histone H1 subtypes are synthesized during early cleavage, late cleavage, and organogenesis (Flenniken and Newrock 1987). In the frog, both somatic H1 subtypes and oocyte subtypes are present in cleavage-stage embryos, whereas only the somatic subtypes are found beyond the gastrula stage (Smith et al. 1988; Ohsumi and Katagiri 1991). Thus, developmentally regulated changes in histone H1 subtype are a common feature of the cleavage stages of embryogenesis. It is conceivable that specific histone H1 subtypes are required during oogenesis; thus the subtype switching observed in embryos would serve merely to restore the somatic histone H1 complement. Several lines of evidence suggest, however, that the subtype switches observed in early embryos may be important developmentally. Sea urchin oocytes contain mRNA encoding aH1, but synthesis of the protein cannot be detected until after fertilization (Poccia 1986). Synthesis of histone aH1 terminates at about the blastula stage, when the H1 subtypes present in adults begin to be predominantly synthesized. Histone aH1 thus is transiently present during early embryogenesis. In the mud snail, removing the polar lobe from embryos causes a delay in the timing of the subtype switches, although the normal pattern is eventually established (Flenniken and Newrock 1987). Since this delay occurs even in cells that do not inherit polar lobe cytoplasm, it was proposed by the authors that cell-cell interactions might be a part of the normal switch mechanism. In frogs, both somatic histone H1 and the oocyte subtype H1X (Ohsumi and Katagiri 1991) or B4

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(Smith et al. 1988) are present in eggs and cleavage-stage embryos. But when chromatin is assembled in egg (Dilworth et al. 1987) or embryo (Wolffe 1989) extracts, H1X but not H1 can be detected (Ohsumi and Katagiri 1991). This suggests that, at least in vitro, H1X may be selectively employed in chromatin assembly (it is also possible that H1 is lost more easily than H1X during chromatin purification). In sum, these results indicate that the nature of the histone H1 species associated with chromatin is regulated precisely during embryogenesis, which implies an underlying function. It should be possible to specifically address this issue using mammalian embryos by microinjection of mRNA encoding histone H1 or of the protein itself. Changes in the core histone composition also occur during embryogenesis in the mouse. Graves et al. (1985) examined the histone mRNA populations of unfertilized eggs and blastocysts. The same genes were represented in both cell types, but differences existed in the amounts of specific mRNAs that were present. The total amount of histone mRNA declines from the one- to two-cell stage and subsequently increases proportionally to the increase in cell number (Giebelhaus et al. 1983). It therefore appears that the egg and blastocyst mRNA populations represent maternally encoded and embryo-encoded species, respectively. The developmental significance of the change in core histone composition during early embryogenesis is unknown. Enhancer elements and chromatin structure A novel approach to understanding gene regulation in early embryos has been taken by DePamphilis and colleagues (Martinez-Salas et al. 1989; Wiekowski et al. 1991). They have injected plasmids containing a luciferase gene linked to an enhancer-dependent promoter into embryos and examined expression of the reporter gene. The reporter gene could be expressed following injection into two-cell embryos, but not following injection into one-cell embryos, even after these embryos became transcriptionally active. When the injected one-cell embryos were prevented from completing the first cell cycle by incubation in the presence of an inhibitor of DNA replication, however, the reporter gene was expressed beginning at the time of embryonic transcription. This result implies that an event occurring during or after DNA replication of the first cell cycle stably inactivates reporter gene expression. A second series of experiments addressed the role of enhancer elements. In the arrested onecell embryos, luciferase expression occurred independently of enhancers. By contrast, in the two-cell embryos, reporter gene expression was strictly enhancer dependent. These results imply that a mechanism that represses gene expression in the absence of enhancers becomes active at the twocell stage. This mechanism does not appear to be linked to embryonic gene expression, as the arrested one-cell embryos were transcriptionally active. Rather, it was proposed that enhancer-dependent gene expression was linked to first mitosis (Wiekowski et al. 1991). Although these experiments were performed using microinjected plasmid DNA, it may be speculated that the observed phenomena reflect changes occurring in the structure of embryonic chromatin during this time.

Concluding remarks It is clear that nuclei and chromatin undergo developmentally regulated modifications during early mammalian

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embryogenesis, of which the changes in the histone complement of chromatin and the composition of the nuclear lamina provide the clearest present evidence. A task for the future will be to identify other nuclear and chromatin changes occurring during this time. Recent technical advances will facilitate this work. The sensitivity of in situ hybridization techniques means that single copy sequences can be identified in nuclei. This will permit, for example, comparative mapping of the intranuclear location of transgenes passed through the maternal or paternal germ line, which may be correlated with their pattern of expression. The polymerase chain reaction technique allows identification and quantitation of specific mRNAs present in early embryos, despite the small number of cells available for analysis. Peptides synthesized using mRNA sequence information can be used to produce antibodies, which in turn may be used to localize the target protein within cells. Additionally, the relative ease with which eggs can be microinjected permits tests of protein function by injection of antibody or antisense oligonucleotides. Finally, the development of efficient homologous recombination techniques, together with the culture of embryonic stem cells, now means that specific mutations can be generated and introduced into the germ line. Analyses of changes in nuclear and higher order chromatin structure during early development will complement those focussing on other elements regulating gene expression. Oct-3 is a transcription factor containing a homeodomain and a POU domain. It is selectively expressed in the female germ line and can be detected in oocytes and preimplantation embryos, and in the primitive ectoderm cells of early postimplantation embryos (Rosner et al. 1990; Scholer et al. 1989). Oct-3 may thus be related to gene expression characteristic of the pluripotent state. Mouse eggs also contain abundant supplies of the small nuclear RNAs and associated proteins that are required for processing of mRNA precursors (Lobo et al. 1988; Dean et al. 1989; Prather et al. 1990). By integrating the information provided by these two avenues of investigation, we may expect to arrive at a more complete understanding of how a coordinated pattern of gene activity is established during early development.

Acknowledgement Work in the author's laboratory is supported by the Medical Research Council of Canada. Balhorn, R. 1982. A model for the structure of chromatin in mammalian sperm. J. Cell Biol. 93: 298-305. Benavente, R., Krohne, G., and Franke, W.W. 1985. Cell-type specific expression of nuclear lamina proteins during development of Xenopus laevis. Cell, 41: 177-190. Bensaude, O., Babinet, C., Morange, M., and Jacob, F. 1983. Heat shock proteins, first major products of zygotic gene activity. Nature (London), 305: 331-333. Blackburn, E.H. 1991. Structure and function of telomeres. Nature (London), 350: 569-573. Burke, B. 1990. On the cell-free association of lamins A and C with metaphase chromosomes. Exp. Cell Res. 186: 169-176. Carter, K.C., Taneja, K.C., and Lawrence, J.B. 1991. Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus. J. Cell Biol. 115: 1191-1202.

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