Journal of Cell Science 113, 2897-2907 (2000) 2897 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1314

Somatic linker histone H1 is present throughout mouse embryogenesis and is not replaced by variant H1°

Pierre G. Adenot1,*, Evelyne Campion1, Edith Legouy1, C. David Allis2, Stefan Dimitrov3, Jean-Paul Renard1 and Eric M. Thompson1,4 1Unité de Biologie du Développement, Institut National de la Recherche Agronomique, F-78352 Jouy-en-Josas, France 2Department of Biochemistry and Molecular Genetics, University of Virginia Health Science Center, Charlottesville, Virginia 22908, USA 3Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation, INSERM U 309, Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche, Cedex, France 4Sars International Center, Bergen High Technology Center, Thormøhlensgt. 55, N-5008 Bergen, Norway *Author for correspondence (e-mail: [email protected])

Accepted 9 June; published on WWW 20 July 2000

SUMMARY A striking feature of early embryogenesis in a number of envelopes, somatic H1 was rapidly incorporated onto organisms is the use of embryonic linker histones or high maternal and paternal chromatin, and the amount of mobility group proteins in place of somatic histone H1. The somatic H1 steadily increased on embryonic chromatin transition in chromatin composition towards somatic H1 through to the 8-cell stage. Microinjection of somatic appears to be correlated with a major increase in H1 into oocytes, and nuclear transfer experiments, transcription at the activation of the zygotic genome. demonstrated that factors in the oocyte cytoplasm and the Previous studies have supported the idea that the mouse nuclear envelope, played central roles in regulating the embryo essentially follows this pattern, with the significant loading of H1 onto chromatin. Exchange of H1 from difference that the substitute linker histone might be the transferred nuclei onto maternal chromatin required differentiation variant H1°, rather than an embryonic breakdown of the nuclear envelope and the extent of variant. We show that histone H1° is not a major linker exchange was inversely correlated with the developmental histone during early mouse development. Instead, somatic advancement of the donor nucleus. H1 was present throughout this period. Though present in mature oocytes, somatic H1 was not found on maternal Key words: Histone H1°, Genome activation, Oocyte, Nuclear metaphase II chromatin. Upon formation of pronuclear transfer

INTRODUCTION up and down regulation of the expression of specific genes (Shen et al., 1995; Shen and Gorovsky, 1996; Dou et al., 1999). Linker histones interact with spacer DNA between adjacent A potentially regulatory facet in a number of organisms is the nucleosomal histone octamer cores. The traditional view that use of a repertoire of linker histone variants which differ both they are a stoichiometric structural component of chromatin, in their globular domains, and in modification of the length with an essentially repressive role in regulating transcription, and net charge of the C-terminal domain. In the mouse, for has been undergoing revision. In contrast to the structural example, there are five somatic variants H1a, H1b, H1c, H1d and sequence conservation of the core histones, there is and H1e, a testis specific variant H1t, and the variant H1° considerable divergence in both sequence and structure among which is expressed only in some lineages of differentiated cells linker histones. Metazoan linker histones contain a central (Franke et al., 1998). In the transition from oocyte to somatic globular domain with N- and C-terminal tails, but the 5S rRNA gene expression during Xenopus embryogenesis protozoan, Tetrahymena, has a linker histone which contains (Wolffe, 1989; Bouvet et al., 1994), somatic histone H1 binds only the C-terminal tail (reviewed by Wolffe et al., 1997). The equally to both oocyte and somatic 5S nucleosomal templates C-terminal region is rich in basic amino acids, and it is likely (Howe et al., 1998) but it selectively represses the oocyte that this tail domain interacts with negatively charged linker template through binding to the 3′ end of the nucleosomal DNA to facilitate chromatin condensation (Ramakrishnan, core, resulting in stable positioning of a nucleosome over key 1997). regulatory elements (Sera and Wolffe, 1998). On the somatic The knockout of histone H1 in Tetrahymena revealed two template, H1 binds to the 5′ end of the nucleosomal core, important points; histone H1 is not essential for nuclear leaving key promoter elements accessible. In the chicken, assembly or cell survival, and it appears to be involved in both where the six H1 genes encode different H1 protein sequences 2898 P. G. Adenot and others

(Nakayama et al., 1993), different protein patterns were show that somatic histone H1 was present in chromatin until obtained from a series of mutants cell lines, each lacking one nuclear breakdown during meïotic oocyte maturation, and then of the H1 genes, indicating that H1 variants may play distinct reassembled onto chromatin following pronuclear formation at roles in the transcriptional regulation of specific genes (Takami the onset of embryogenesis. Histone H1° was not detected on et al., 2000). chromatin during this developmental period. The absence of A particular feature of early embryogenesis in some animals, H1 on maternal metaphase II chromosomes, contrasted with its is the absence of somatic linker histones during the initial presence on chromosomes at the first mitosis and a weak cleavage stages. Prior to the mid-blastula transition (MBT) in presence in sperm chromatin. Microinjection of somatic H1 Xenopus embryos, an embryonic variant H1M (or B4) replaces into oocytes did not result in staining of maternal somatic H1 (Smith et al., 1988; Dimitrov et al., 1993). The high chromosomes, but in nuclear transfer experiments, we mobility group protein HMG-1, together with the B4 linker observed that somatic H1 could be loaded onto maternal histone, are major components of chromatin within the nuclei chromosomes only when the transferred nucleus lost its nuclear assembled during the incubation of Xenopus sperm chromatin membrane and formed prematurely condensed chromosomes in Xenopus egg extract (Nightingale et al., 1996). Both proteins (PCC). The extent of transfer of H1 to maternal chromosomes, bind to linker DNA but less tightly than somatic H1 (Ura et al and its removal from PCC, appeared to be mediated by 1996), and thus may facilitate rapid cycles of DNA replication. factors in the oocyte cytoplasm, but also depended on the In Drosophila embryos, an HMG-1 homologue, HMG-D, developmental stage of the transferred nucleus. replaces somatic H1 until the MBT (Ner and Travers, 1994). In the sea urchin, maternal cleavage stage histones are present until the third cell cycle, and the cleavage stage linker histone MATERIALS AND METHODS has a high homology to Xenopus histone B4 (Mandl et al., 1997). However, it remains questionable whether this feature Collection of embryos and oocytes can be extended to early embryogenesis in general, or if it may Female C57/CBA mice, 6-8 weeks old, were superovulated with instead reflect a situation in which rapid DNA replication intraperitoneal injections of 5 i.u. of pregnant mare serum (PMS; occurs in the absence of transcription from the zygotic genome. Folligon, Intervet), followed 46-48 hours later with 5 i.u. human In the mouse, the initial observation by Clarke et al. (1992), chorionic gonadotropin (hCG; Chlorulon, Intervet). To recover mature oocytes in metaphase II (MII), superovulated females were sacrificed that somatic histone H1 is first detected cytochemically in a 15 hours post-hCG (phCG). To obtain fertilized oocytes, females were portion of embryos at the 4-cell stage and by the 8-cell stage caged with C57/CBA males immediately after hCG injection, and in all nuclei in all embryos, suggested that the mouse embryo embryos were collected at the one-cell stage. Oocytes and embryos followed the early cleavage pattern of the sea urchin, Xenopus were incubated after collection in 0.5% hyaluronidase (Sigma) in PB1 and Drosophila, in maintaining the absence of a somatic linker for 1-2 minutes at 37°C to remove cumulus cells, washed extensively histone. The mouse embryo, however, would deviate in two in PB1, and then returned to culture in M16 medium under 5% CO2 important ways; somatic H1 appears one full cell cycle after in air, until fixation. To recover immature oocytes at prophase I, major activation of the zygotic genome (reviewed by Latham, female C57/CBA mice, 11-13 weeks old, were superovulated with 1999), and Clarke et al. (1997) subsequently proposed that the intraperitoneal injections of PMS as described above. Ovaries were differentiation variant H1°, rather than an embryonic variant, removed 45 to 50 hours after injection, and transferred to PB1 prewarmed at 37°C. Fully grown oocytes were freed from peripheral was the substitute linker histone. In the ovary, the fully grown cells by gentle pipetting, and either fixed immediately, or returned to oocyte is a highly specialized cell which results from a culture to be fixed during nuclear maturation. differentiation process during oogenesis. From this point of view, the presence of the differentiation variant H1° in Nuclear transfer procedures chromatin could be considered consistent. The amount of H1° Hybrid cells were reconstructed by electrofusion between MII oocytes increases during terminal cell differentiation (Rousseau et al., and either embryonic, or somatic cells. Following fusion, transferred 1992), and its overproduction results in a decrease of nuclei condense prematurely into chromosomes if the oocyte is not transcriptional activity (Brown et al., 1997). However, the already parthenogenetically activated at the time of cell fusion immediate future of the differentiated oocyte is to become a (reviewed by Campbell, 1999). MII oocytes used as recipient cells totipotent cell following fertilization or parthenogenetic were 16-20 hours phCG at the time of fusion. Blastomeres from mid (43 hours phCG) and late (50 hours phCG) 2-cell embryos, from late activation, and this is difficult to reconcile with the persistence 4-cell embryos (67 hours phCG), and from early 8-cell embryos (72 of H1° in embryonic nuclei beyond activation of the zygotic hours phCG) cleaved during the previous hour, were used as sources genome (Clarke et al., 1997). In studying histones in mouse of embryonic nuclei. Cumulus cells freshly removed from MII oocytes and embryos by radiolabelling coupled to SDS-PAGE, oocytes, as described above, were the source of somatic nuclei. Wiekowski et al. (1997) showed that nascent linker histone in Two nuclear transfer procedures were performed according to the the fully grown oocyte and in the embryo up to the 2-cell stage size of the donor cell. Donor cumulus cells were washed extensively are synthesized from maternal mRNAs and that they migrated in PB1 and then introduced beneath the zona pellucidae of MII as somatic H1 during SDS-gel electrophoresis, suggesting that oocytes. Resulting pairlets were washed in 0.3 M mannitol, placed in somatic H1 may be present during this developmental period. the same solution into a fusion chamber between platinium electrodes, In this study, we have examined whether mouse embryos and then subjected to two DC pulses of 1.5 kV/cm, 100 microseconds each. When the donor cell was a blastomere, the zona pellucida was deviate from the early cleavage pattern of the sea urchin, removed from MII oocytes and embryos by gentle pipetting following Xenopus, and Drosophila, by maintaining somatic linker a treatment with 0.25% Pronase (Sigma) in PB1 for 1 minute at 37°C. histone in chromatin. Using antibodies that recognize somatic Embryos were then incubated 15 minutes at 37°C in PBS without H1 (Dimitrov and Wolffe, 1996), mouse phosphorylated H1 Ca2+ and Mg2+ (Gibco) to dissociate blastomeres. MII oocytes and (Chadee et al., 1995), and mouse H1° (Gorka et al., 1998), we blastomeres were agglutinated in 150 µg/ml phytohaemagglutinin Linker histone H1 in early mouse embryogenesis 2899

(Sigma) in PB1, for 3 minutes at 37°C. Aggregated pairs were washed antibody raised against Xenopus somatic H1 specifically in 0.28 M mannitol and electrofused in this solution. Electrofusion recognized mouse somatic H1 on immunoblot (Fig. 1A, anti- was done as above except that each pulse lasted 60 microseconds. H1 panel), and by immunostaining (Fig. 1B, anti-H1 panel), it Following electrofusion, both oocyte-blastomere and oocyte-cumulus gave an intense nuclear staining in mouse erythroleukemia cell pairlets were rinsed in PB1 and cultured in M16 under 5% CO2 (MEL) cells and in mouse blastula cells which is consistent in air. Fusion between the oocyte and the blastomere occured 5 to 40 with the H1 pattern reported in previous immunocytochemical minutes post pulse, and resulting hybrid cells were fixed 1.5 to 2 hours post fusion. The time of fusion between the oocyte and the cumulus studies (Clarke et al., 1992; Stein and Schultz, 2000). The cell could not be precisely determined because of the small size of a antibody raised against phosphorylated H1 (H1P) in cumulus cell. Therefore, oocyte-cumulus cell pairlets were fixed 1.5 Tetrahymena, which has been demonstrated to recognize only to 2 hours after the electrical pulses. To control for effects of the one of the mouse H1P subtypes (histone H1b; Chadee et al., experimental procedure, hybrid cells were also reconstructed from 1995), reacted with one immunoreactive protein, present only two MII oocytes. in the mouse cell lysate (Fig. 1A, anti-H1P panel), and migrating at the position of mouse H1P (Chadee et al., 1995). Microinjection of oocytes By immunostaining (Fig. 1B, anti-H1P panel), this antibody Microinjection of somatic histone H1 was done essentially as recognized a nuclear protein in MEL cells and in mouse described (Lin and Clarke, 1996). Calf thymus histone H1 blastula cells, with a variable staining level in the nucleus (Boehringer) was dissolved in water at 1 mg/ml. Microinjection into MII oocytes was carried out using a Nikon inverted microscope during interphase, and a much more intense staining on equiped with Narishige micromanipulators and an Eppendorf chromatin during mitosis. At this latter stage, a higher microinjector. About 1-5 pl of histone H1 solution was injected per cytoplasmic staining was also observed. This H1P staining oocyte. Following injection, MII oocytes were returned to culture for pattern is consitent with what is known about H1P levels in 2 hours before fixation. The same injection into the cytoplasm of 1- mammalian proliferative cells (Roth and Allis, 1992; Chadee cell embryos induced an accumulation of linker histones in pronuclei et al., 1995; Bleher and Martin, 1999). The antibody raised within 15 minutes (Lin and Clarke, 1996). against ox liver H1°, which has been demonstrated to specifically recognize an epitope in the region of amino acids Antibodies 20 to 30 in murine H1° on immunoblots (Gorka et al., 1998), Mouse histone H1° was detected with a monoclonal antibody raised reacted strongly with a protein in mouse cell lysates (Fig. 1A, against ox liver H1° (clone 34B10H4, a generous gift from Dr S. Kochbin) and characterized by Dousson et al. (1989). Mouse somatic anti-H1° panel), migrating at the position of H1° (Djondjurov histone H1 was detected with two antisera; one raised against Xenopus et al., 1983), and slightly with somatic H1. By somatic H1 (Dimitrov and Wolffe, 1996), and the other against immunostaining, we observed a clear staining in nuclei of MEL phosphorylated isoforms of Tetrahymena H1 (Lu et al., 1994). cells (Fig. 1B, anti-H1° panel, left column), where histone H1° Secondary antibodies were FITC-conjugated goat anti-rabbit and anti- is present in a substantial amount (Helliger et al., 1992; Gorka mouse IgG (Sigma, 1:400 final dilution). et al., 1998), and an absence of staining in nuclei of mouse STO fibroblasts (Fig. 1B, anti-H1° panel, right column), Confocal immunofluorescence microscopy indicating that the anti-ox liver H1° antibody specifically Oocytes, embryos, hybrid cells and differentiated cells were fixed in recognized H1° but not H1 in fixed cells. 2.5% paraformaldehyde for 20 minutes at room temperature. They were incubated for 30 minutes at 37°C in a blocking solution Somatic histone H1 is present in the nucleus of containing 10% foetal calf serum (FCS) and 0.2% Triton X-100 in mouse oocytes and early cleavage embryos PBS. Subsequent manipulations were performed in PBS/2% FCS/0.1% Triton X-100 solution. Nuclear antigens were detected by indirect The presence of somatic H1 in oocytes and embryos of early immunofluorescence: cells were incubated with the primary antibody cleavage stages was first studied by immunoblot, using the overnight at 4°C, and washed extensively before incubation for 1 hour antibody raised against Xenopus somatic H1. Following at 37°C with the second antibody. Chromatin was stained for 30 autoradiographic exposure of several hours, immunoreactive minutes with 10 µg/ml propidium iodide (Sigma). Cells were mounted proteins, migrating as somatic histone H1 in the blastocyst on well-slides in Vectashield (Vector Laboratories) containing the same embryo (Fig. 2, lane 5), were detected in lysates prepared from concentration of propidium iodide, and were observed under a confocal 1100 MII oocytes or 1-cell embryos (Fig. 2, lanes 1 and 2, laser scanning microscope (Carl Zeiss, CLSM 310). respectively). A very faint signal was obtained with the same Cell lysates, chromatin preparation and western blot number of germinal vesicle (GV) stage oocytes (not shown). analysis At the 2-cell and 4-cell stages when somatic H1 has been Cell lysates were prepared in denaturing buffer L (50 mM Tris-HCl, reported to be present (Clarke et al., 1992; Wiekowski et al., pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate (SDS), 1% 2- 1997; Stein and Schultz, 2000), this protein was detected in mercaptoethanol). Chromatin was prepared as described by lysates prepared from only 300 to 400 embryos (Fig. 2, lanes Wiekowski and DePamphilis (1993). Proteins were electrophoresed 3 and 4, respectively). Thus, somatic H1 was present in mouse in denaturing 15% polyacrylamide-SDS gels and blotted onto oocytes and 1-cell embryos, and it increased substantially by nitrocellulose. Immunodetection by primary antibodies was revealed the 2-cell stage. by a peroxidase-labelled immunoglobulin antiserum and enhanced Histone H1 immunostaining of chromatin was observed in chemiluminescence (Super Signal ULTRA, Pierce). GV oocytes (Fig. 3A,E), and in embryos following pronuclear formation at the 1-cell stage (Fig. 3J-L, and N-R). No H1 RESULTS staining was found on maternal chromatin between GV breakdown (Fig. 3B,F and C,G) and pronuclear formation in The reactivity of anti-histone antibodies was first examined on fertilized or parthenogenetically activated oocytes (not shown). mouse linker histones in cell lysates and in fixed cells. The H1 staining of chromatin was of low intensity in the 1-cell 2900 P. G. Adenot and others

Fig. 1. Recognition of mouse histones by anti-histone antibodies. (A) Immunoblotting of lysates prepared from mouse erythroleukemia (MEL) cells or mouse HC11 cells. Equal amounts of lysates were loaded onto the same gel, together with calf thymus histone H1, subjected to SDS-PAGE, and blotted onto nitrocellulose. The blot was cut and each part was incubated separately with the anti-Xenopus somatic H1 antibody (anti-H1 panel), the anti-Tetrahymena phosphorylated H1 antibody (anti-H1P panel), or the anti-ox liver H1° antibody (anti-H1° panel). Arrows indicate the migrating position of the H1 doublet. (B) Confocal images of immunostained and DNA stained mouse blastula cells (anti-H1 and anti-H1P panels, right columns), mouse STO fibroblasts (anti-H1° panel, right columns), and proliferative MEL cells (left columns) immunolabelled with the anti- Xenopus somatic H1 antibody (anti-H1 panel), the anti-Tetrahymena phosphorylated H1 antibody (anti-H1P panel), or the anti-ox liver H1° antibody (anti-H1° panel). Bar, 30 µm; relative magnification = 1 (MEL cells, anti-H1 and anti-H1P panels), 1.4 (MEL cells, anti-H1° panel), 1.1 (blastula cells), 0.9 (STO cells).

embryo, though several intense spots located at the periphery contrast to meïotic oocyte chromosomes (Fig. 3C,G), somatic of prenucleolar bodies were transiently observed during the histone H1 was present at very low levels in decondensing few hours which followed pronuclear formation (Fig. 3J,N). In sperm chromatin at fertilization (Fig. 3C,G), and on zygotic chromosomes at the first mitosis (Fig. 3K,O). When embryos Fig. 2. Immunodetection of somatic H1 in the mouse oocyte and were fixed at the mid 2-cell stage, nuclear H1 staining intensity embryo with the anti-Xenopus H1 antibody. Lysates were prepared from 1125 MII oocytes (1), 1153 one-cell embryos at 24-26 hours phCG (2), 397 two-cell embryos at 40-45 hours phCG (3), 297 four- cell embryos at 56-66 hours phCG (4), and 209 blastocysts (5). Autoradiographic exposure was for 21 hours (1-2), 23 hours (3-4) or 10 seconds (5). Calf thymus histone H1 served as a marker for position of the H1 doublet (arrows). Linker histone H1 in early mouse embryogenesis 2901

Fig. 3. Confocal images of mouse oocytes and early embryos immunolabelled with the anti-Xenopus somatic H1 antibody (A-C, J-L, Q), or with the pre-immune serum (D,I), and DNA stained (second, fourth and bottom rows). Detector sensitivity was the same for A, B and L, was 3-fold more sensitive for J, K and the insert in B, was 6-fold more sensitive for C, I, and 8-fold more sensitive for D. Images A-P were obtained with a ×63 oil immersion objective and images Q, R, with a ×16 oil immersion objective. (A,E) Immature oocyte; arrow designates the germinal vesicle. (B,F) Oocyte after GV breakdown; insert, the same oocyte at higher detector sensitivity. (C,G and D,H) Fertilized eggs; arrow indicates the position of the very faintly staining sperm head. (I,M and J,N) One-cell pronuclear embryo at 20-22 hours phCG. Dense spots of histone H1 were present at the periphery of prenucleolar bodies (arrowheads), and non-specific staining was found in the sperm tail (arrow). (K,O) One-cell embryos at mitosis. (L,P) Two-cell embryo at 43 hours phCG; a peripheral enrichment of histone H1 in nuclei was observed and was not related to the presence of condensed chromatin in this region. (Q,R) One-cell (a), two-cell (b), four-cell (c) and eight-cell (d) embryos observed in the same field. The quantity of histone H1 in nuclei increased from the one-cell stage to the eight cell stage. Bar, 30 µm. Relative magnification = 1 (A-P), 0.3 (Q,R). had increased strongly, and an enriched perinuclear staining from the 1- to 8-cell stage, and the decreased number of was sometimes observed (Fig. 3L,P). At subsequent cleavage embryos required to obtain a signal by immunoblotting as stages, nuclear H1 staining intensity increased continuously cleavage stage development progressed. (Fig. 3Q,R). Therefore there was a clear correlation between As an additional test to confirm the presence of somatic the increasing immunostaining of nuclei of individual embryos histone H1 in the chromatin of GV oocytes and early embryos, 2902 P. G. Adenot and others

Fig. 4. Confocal images of immunostained (top row) and DNA stained (bottom row) mouse oocytes and early embryos immunolabelled with the anti-Tetrahymena phosphorylated H1 antibody (B-E) or with the pre-immune serum (A). (A,F and B,G) Mouse oocytes at the germinal vesicle stage surrounded by cumulus cells; arrow designates the germinal vesicle and arrowhead a cumulus cell in mitosis. (C,H) 1-cell embryo 23 hours phCG. (D,I) 2-cell embryo 46 hours phCG. (E,J) 4-cell embryo 62 hours phCG. Bar, 30 µm. immunostaining was done with the antibody raised against the experimental procedure, as it was never observed following phosphorylated H1 in Tetrahymena. Nuclei were faintly stained fusion between two MII oocytes (Fig. 5I,L). Interestingly, in oocytes (Fig. 4B,G) and more clearly labelled in 1-cell (Fig. somatic histone H1 was undetectable on oocyte chromatin when 4C,H), in 2-cell (Fig. 4D,I) and in 4-cell (Fig. 4E,J) embryos. the transferred embryonic nucleus did not form PCC (not No chromatin staining was detected in oocytes from GV shown), nor following microinjection of calf thymus H1 into the breakdown to the meïotic arrest at metaphase II (not shown). MII oocyte (not shown). Taken together, these results show that These results confirm the presence of histone H1 in chromatin MII oocytes contain cytoplasmic activities which remove histone of oocytes and early embryos. Thus, we conclude that somatic H1 from chromatin when a nuclear envelope is absent. We histone H1 is located in embryonic nuclei as early as the 1-cell conclude that histone H1 is undetectable on oocyte MII stage, and that substantial nuclear import has taken place by chromosomes because it is removed from chromatin during the 2-cell stage, when major zygotic genome activation occurs. oocyte nuclear maturation. Histone H1 release from chromatin following nuclear Histone H1° is not detected as a replacement linker transfer in MII oocytes histone in mouse oocytes and embryos The lack of detection of somatic histone H1 on meïotic oocyte The low H1 staining of chromatin in the GV oocyte and the 1- chromosomes, and its presence on zygotic chromosomes, cell embryo may reflect the presence of a substitute linker suggest that somatic histone H1 is removed from chromatin histone during this developmental period. Since the during nuclear maturation in oocytes. To test this idea, we differentiation variant H1° has been proposed as a candidate for performed nuclear transfer experiments to simultaneously this replacement, based on cytochemical observations in murine observe both MII chromosomes and embryonic or somatic oocytes and early embryos with an antibody against the related prematurely condensed chromosomes (PCC) in the same oocyte chicken H5 protein, combined with RT-PCR analysis (Clarke et cytoplasmic environment. Resulting hybrid cells were al., 1997), we investigated this proposal using the antibody immunolabelled with the anti-Xenopus H1 antibody (Fig. 5). raised against ox liver H1°, which specifically recognizes murine Following the transfer of a cumulus cell nucleus, the PCC and H1° (Gorka et al., 1998). We did not detect any labelling of GV oocyte chromosomes showed no staining for histone H1 (Fig. stage oocytes (Fig. 6A,E), in chromosomes of MII oocytes (not 5A,D). In contrast, both sets of chromosomes were positively shown), in pronuclei of 1-cell embryos (Fig. 6B,F), nor in nuclei labelled following the transfer of embryonic nuclei. In these of 2-cell (Fig. 6C,G), or 4-cell (Fig. 6D,H) embryos. This was latter hybrid cells, the PCC were less intensely stained than in distinct contrast to the clear detection of H1° in MEL cells corresponding zygotic chromosomes at the same cleavage stage (Fig. 1B). Thus, we conclude that H1° is not the predominant (not shown), indicating that histone H1 was also released from linker histone during early mouse development. chromatin. The PCC formed from mid (Fig. 5B,E and 5C,F) or late (not shown) 2-cell embryonic nuclei remained generally distinguishable by anti-H1 staining, while PCC originating from DISCUSSION late 4-cell (Fig. 5G,J), or early 8-cell (Fig. 5H,K) embryonic nuclei did not. Thus, when using later stage embryonic nuclei, A striking feature of early embryogenesis in the sea urchin, relatively little histone H1 remained on chromatin, and Drosophila, and Xenopus is the use of specific cleavage stage numerous foci of histone H1 were observed near the PCC. We linker histones or high mobility group proteins in place of also observed that oocyte chromosomes exhibited more intense somatic histone H1. In the latter two organisms, somatic H1 staining when the PCC also conserved high H1 content, and histone H1 begins to accumulate near the MBT, a time when that the uptake of histone H1 by oocyte chromosomes was not transcription from the zygotic genome begins. In Xenopus at all correlated with the amount of H1 lost from the PCC; embryos, the presence of somatic H1 has been shown to though 4-cell and 8-cell embryonic nuclei had higher H1 content regulate the switch from transcription of oocyte 5S rRNA than 2-cell nuclei, hybrids formed from the former group genes to somatic 5S genes (Sera and Wolffe, 1998) and to exhibited both PCC and oocyte chromosomes with very low H1 restrict the competence of ectodermal cells to differentiate into staining. H1 staining of oocyte chromosomes did not result from mesoderm (Steinbach et al., 1997). This modification of linker Linker histone H1 in early mouse embryogenesis 2903

Fig. 5. Distribution of histone H1 following nuclear transfer and premature chromosome condensation. Hybrid cells were reconstructed by electrofusion between MII oocytes and cumulus cells (A,D), or embryonic blastomeres at the mid 2-cell (B,C,E,F), late 4-cell (G,J), or early 8-cell (H,K) stages. To control for effects of the fusion protocol, MII oocytes were also fused together (I,L). Top and third rows: H1 immunostaining. Second and fourth rows: counterstaining of DNA with propidium iodide. In histone H1 immunostained micrographs, arrows designate chromosomes of the oocyte (long arrow) and the transferred nucleus (short arrow). All images were obtained at the same detector sensitivity. Inserts in G and H show a magnified view where several foci of histone H1 were observed near prematurely condensed chromosomes. Arrow in L indicates two sets of chromosomes corresponding to telophase II in the oocyte hybrid cell. Bar, 25 µm.

Fig. 6. Confocal images of immunostained (top row) and DNA stained (bottom row) mouse oocytes and early embryos immunolabelled with the anti-ox liver H1° antibody. (A,E) Mouse oocytes at the germinal vesicle stage. (B,F) 1-cell embryo 23 hours phCG. (C,G) 2-cell embryo 46 hours phCG. (D,H) 4-cell embryo 62 hours phCG. Bar, 30 µm. histone types during early development is somewhat spermatogenesis. Ausio (1999) has recently reviewed a reminiscent of nature’s experimentation with the replacement proposal of the derivation of protamines (P) from a histone H1 of sperm nuclear basic proteins (SNBPs) during the process of precursor (H1), via protamine-like intermediates (PL), 2904 P. G. Adenot and others concluding that the basic evolutionary H1 → PL → P in earliest cleavage stage mouse embryos. Previous progression connecting these basic proteins has occured immunocytochemical observations, with an antibody that repeatedly on many occasions during metazoan evolution. The recognizes chicken histone H5 and ox liver histone H1°, but apparent random distribution of SNBPs throughout the animal not chicken H1 on immunoblot (Allan et al., 1982), suggested kingdom would be the net result of these events superimposed that the predominant linker histone in post-natal murine on a background of multiple reversions. Whether any oocytes and early embryos was immunologically related to the evolutionary pattern in the use of embryonic variants of linker differentiation variant H1° (Clarke et al., 1997). Using an histones exists, is for the moment, not clear. antibody raised against ox liver H1° (Dousson et al., 1989), which recognizes murine H1° on immunoblot (Gorka et al., Somatic histone H1 is present during the first 1998), and an anti-Xenopus somatic H1 antibody which cleavage stages of mouse development recognizes mouse somatic H1, we detected both H1° and During the last decade, it has been thought that the mouse somatic H1 by immunofluorescence in MEL nuclei, and only embryo followed the early cleavage pattern of the sea urchin, somatic H1 in nuclei of mouse oocytes and early embryos. As Xenopus, and Drosophila, in maintaining the absence of a MEL nuclei contain a substantial amount of H1° (Helliger et somatic linker histone, because somatic H1 was not detected al., 1992; Gorka et al., 1998), we conclude that the on chromatin until the mid 4-cell stage with an anti-rat somatic differentiation variant H1° is not a predominant linker histone H1 antibody (Clarke et al., 1992). Using an antibody raised on chromatin during this developmental period. We have also against Xenopus somatic H1 (Dimitrov and Wolffe, 1996), been unable to detect any proteins recognized by an antibody we have demonstrated by western blotting and directed against the Xenopus embryonic variant B4 immunofluorescence, that somatic H1 was present in the (unpublished observations). The high abundance of xHMG1 unfertilized mouse egg, in 1-cell pronuclei, 2-cell nuclei and and HMG-D in Xenopus and Drosophila cleavage stage in 4-cell nuclei. The amount was low in pronuclei and embryos, followed by a sharp reduction at the MBT, suggests increased through to the 8-cell stage. that they may in part substitute somatic H1 functions (Dimitrov The fact that we detect somatic H1, where Clarke et al. (1992) et al., 1993, 1994; Ner and Travers, 1994), but this is not the did not, may result from the differential sensitivity of the case in mouse embryos, as the profile of HMG1 abundance antibody reagents and of detection methods. A mouse oocyte (Spada et al., 1998), follows precisely that described for contains approximatively 60 pg of histone (Wassarman and somatic H1 in this study. Mrozak, 1981). Therefore, in our immunoblots, somatic H1 in the unfertilized egg was detected in a lysate containing about 70 What regulates linker histone association with ng of total histone, whereas Clarke et al. were unable to detect chromatin during early mouse embryogenesis? H1 in a lysate containing twice as much oocyte histone. A more Metabolic radiolabelling studies have shown that histone H1 is recent immunocytochemical study in the early mouse embryo synthesized in the fully grown mouse oocyte (Wassarman and (Stein and Schultz, 2000) showed that the anti-rat H1 antibody Letourneau, 1976; Wiekowski et al., 1997). In vivo, we used in the Clarke et al. study readily detects the presence of observed that somatic histone H1 remained in the germinal somatic H1 on chromatin by the late 2-cell embryonic stage, one vesicle of fully grown oocytes, in contrast to previous reports cleavage stage earlier than previously reported (Clarke et al., (Clarke et al, 1997), and was absent on maternal chromatin 1992). The differential detection of somatic H1 may also be from GV breakdown through to pronuclear formation related to linker histone composition in the early mouse embryo. following fertilization or parthenogenetic activation. Which Somatic H1 subtypes migrate as two prominent bands during activities might be involved in the removal of histone H1 from SDS-gel electrophoresis. Wiekowski et al. (1997) found that the oocyte chromatin? The interaction of histone H1 with two migrating H1 variants are synthesized in the fully grown chromatin is regulated through phosphorylation. It has been oocyte, with the faster migrating H1 variant being predominant. noted that phosphorylation of H1 is increased in highly They also demonstrated that histone H1 synthesis, which is proliferative cells, reduced in quiescent cells, and that arrested when the oocyte undergoes nuclear breakdown and phosphorylation levels are maximal, just prior to, or at nuclear maturation (Wassarman and Letourneau, 1976), resumes metaphase, with a rapid decrease thereafter (reviewed by Roth in the embryo from the late 1-cell/early 2-cell stage, but only the and Allis, 1992). These observations led to the notion that H1 faster migrating H1 variant was detectable in the 2-cell embryo. phosphorylation is important in mitotic chromosome In comparing the two antibodies by western blotting of mouse condensation. Meïotic reinitiation and nuclear maturation in spleen and HC11 cells preparations, we found that the faster the mouse oocyte is a complex process which requires the migrating H1 variant was predominant with the anti-Xenopus gradual activation of the p34cdc2 H1 kinase (Gavin et al., antibody, whereas the slower migrating H1 variant was 1994). This activity first increases 2-fold at GV breakdown, predominant with the anti-rat antibody (data not shown). This and then 8-fold in a protein synthesis-dependent manner as the may explain the increased sensitivity of the anti-Xenopus H1 oocyte progresses to metaphase I. The present study reveals antibody in detecting somatic H1 during early murine that the removal of histone H1 from oocyte chromatin is embryogenesis. coincident with minor p34cdc2 H1 kinase activation. Although phosphorylation should weaken interactions between The differentiation variant H1° is not a predominant nucleosomal DNA and linker histone (Hill et al., 1990; Talasz linker during the first cleavage stages of mouse et al, 1998), the removal of histone H1 from oocyte chromatin development can not be attributed solely to linker histone phosphorylation The low content of somatic H1 in pronuclei means that we can because H1 remains absent from chromatin following p34cdc2 not formally exclude the presence of a substitute linker histone H1 kinase inactivation in fertilized or parthenogenetically Linker histone H1 in early mouse embryogenesis 2905 activated oocytes. It is now known that H1 phosphorylation is oocyte. A clear weakness of the simple dilution argument, not necessary for mitotic chromosome condensation (Guo et however, is that in this case, the microinjection of H1 into al., 1995; Ajiro et al., 1996), and that histone H1 itself is not oocytes should also saturate H1 removal agents in the oocyte required for nuclear assembly (Dasso et al., 1994) or for and result in H1 loading onto MII chromosomes. chromosome condensation in Xenopus (Ohsumi et al., 1993) In a study of remodelling of somatic nuclei in Xenopus egg or Tetrahymena (Shen et al., 1995). Since a similar amount of extracts, Dimitrov and Wolffe (1996) have shown that histone cells was necessary to detect somatic H1 by western blotting H1° is quantitatively removed from chromatin, as is somatic in the MII oocyte and the 1-cell embryo, this suggests that H1 to a lesser extent, and that this process is mediated by egg linker histone H1 is not required to maintain chromatin nucleoplasmin. In the Xenopus oocyte, nucleoplasmin condensation during oocyte nuclear maturation. accumulates in the germinal vesicle until nuclear breakdown Substantial chromatin remodelling occurs in the mouse (Litvin and King, 1988). At fertilization, it removes sperm zygote (Perreault, 1992; Nonchev and Tsanev, 1990; Adenot et specific basic proteins to deposit histones H2A/H2B onto al., 1997). A small, localized distribution of somatic H1 sperm chromatin (reviewed by Laskey et al., 1993). This remains in mature sperm chromatin (Pittoggi et al., 1999), and molecular chaperone remains present in nuclei during early a weak somatic H1 staining of sperm chromatin was observed embryogenesis, and then becomes undetectable in adult tissues immediately following fertilization. More substantial (Bürglin et al., 1987; Litvin and King, 1988). It is likely that a accumulation of somatic H1 on maternal and paternal molecular chaperone similar to Xenopus egg nucleoplasmin chromatin began after formation of pronuclear envelopes, exists in the mouse oocyte and during the first cleavage stages though this clearly preceded the detection of nascent linker of early embryogenesis, based on what occurs to mouse sperm histones (Wiekowski et al., 1997), suggesting that the nuclear chromatin following its incubation in Xenopus oocyte extracts envelope may play a role in regulating the loading of H1 onto (Montag et al., 1992), or its microinjection into maturing chromatin in the mouse zygote. It is known that in contrast to oocytes (McLay and Clarke, 1997), immature oocytes, or early core histones, a relatively large pool of H1 is found in the embryos (Maeda et al., 1998). We have recently found a protein cytoplasm of both proliferating and quiescent cells (Zlatanova with molecular and antigenic properties similar to Xenopus egg et al., 1990). Using a digitonin permeabilization assay system, nucleoplasmin that is present in the mouse oocyte and has an Kurz et al. (1997) have shown that nuclear transport of H1 expression pattern restricted to the early cleavage stages of histones meets the criteria of a nuclear localization signal mouse development (unpublished observations). Thus, it is mediated-process, and it has been found recently that import tempting to speculate that removal of H1 from chromatin of H1 into the nucleus requires the cytoplasmic assembly of a during oocyte nuclear maturation is also mediated by a complex including H1, importin β, and importin 7, and the nucleoplasmin-like protein. presence of functional nuclear pore complexes (Jäkel et al., 1999). Conclusions It has been demonstrated that microinjection of somatic H1 In contrast to the absence of somatic H1 during the first into 1-cell embryos results in its rapid uptake onto pronuclear cleavage stages of sea urchin, Xenopus, and Drosophila chromatin (Lin and Clarke, 1996; Stein and Schultz, 2000). In development, we find that somatic H1 is present on chromatin this study, microinjection of somatic H1 into MII oocytes in mouse embryos as soon as pronuclei have formed at the 1- resulted in no uptake of H1 onto MII chromosomes. When cell stage. We have also observed somatic H1 in 1-cell performing nuclear transfer we noted that histone H1 remained pronuclei of cow embryos (unpublished observations) on chromatin in the transferred nucleus and was never found on suggesting that this may be a common feature in mammalian MII chromosomes, provided that the nuclear envelope remained embryos. The evidence in this study also directly contradicts intact (data not shown). If nuclear envelope breakdown the hypothesis (Clarke et al., 1997) that the differentiation occurred, leading to the formation of PCC, the presence of H1 variant H1° serves as a replacement linker histone during early on both PCC and MII chromosomes depended on the mouse embryogenesis. Wiekowski et al. (1997), have shown developmental stage of the donor nucleus. When somatic that new synthesis of histone H1 begins in the late 1-cell cumulus cell nuclei were transferred, H1 was removed from embryo, indicating that the H1 we observe in early pronuclei PCC and was not detected on MII chromosomes. When 8-cell and on immunoblots of MII oocytes is of maternal origin. or 4-cell embryonic nuclei were transferred, foci of H1 Through nuclear transfer experiments, and microinjection of remained in the vicinity of PCC, and very weak H1 staining was somatic H1, we provide evidence that the absence of histone found on MII chromosomes. However, when a 2-cell nucleus H1 on MII chromosomes, despite its presence in the oocyte, is was transferred, some H1 remained on the 2-cell derived PCC due to activities in the oocyte cytoplasm which remove somatic and was also observed on MII chromosomes. These results H1 from chromatin. Both the normal developmental profile of show that the oocyte contains activities which remove somatic H1 on chromatin, and the results of the nuclear transfer H1 from chromatin. There are two potential explanations for the experiments, demonstrate the central role of the nuclear dependance of the extent of this removal on the developmental envelope in regulating the loading of H1 onto chromatin during stage of the donor nucleus. It is possible that the limited release early development. of histone H1 from embryonic nuclei simply results from the In Xenopus and mouse embryos, a basal transcription higher dilution of oocyte cytoplasmic activities upon cell fusion, machinery exists prior to activation of the zygotic genome since a blastomere is much larger than a cumulus cell. The other (Newport and Kirschner, 1982; Bellier et al., 1997). However, possibility is that nuclear factors present in the 2-cell embryo, in Xenopus eggs, a limited translation of stored maternal and to a much lesser extent in 4- and 8-cell embryos, are able mRNAs coding for key components of the transcription to at least partially reverse the removal of somatic H1 by the machinery (Veenstra et al., 1999), and a deficiency in the 2906 P. G. 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