Differential acetylation of histone H4 lysine during development of in ...

[Epigenetics 3:4, 199-209; July/August 2008]; ©2008 Landes Bioscience

Research Paper

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Differential acetylation of histone H4 lysine during development of in vitro fertilized, cloned and parthenogenetically activated bovine embryos

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Walid E. Maalouf,† Ramiro Alberio and Keith H.S. Campbell*

Animal Development and Biotechnology Group; School of Biosciences; Division of Animal Physiology; Sutton Bonington Campus; University of Nottingham; Loughborough, Leicestershire UK address: QMRI; University of Edinburgh; Edinburgh UK

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Introduction

More research is being carried out into understanding the problems that may arise from reprogramming and remodeling of the embryonic genome. Alterations in the epigenetic status can lead to de-regulation of a number of vital genes, and ultimately lead to aberrant genotypic and phenotypic effects.1 Aberrant epigenetic formation, as those observed mostly in embryos reconstructed by nuclear transfer, result in numerous congenital diseases, increased size of the offspring, and increased incidence of neonatal deaths.2,3 The oocyte and the sperm acquire distinct epigenetic marks during their process of maturation and after fertilization, and the donor cell nucleus has to be reprogrammed during nuclear transfer, in order to obtain full developmental competence of putative embryos. Epigenetic markings on the parental gametes are necessary for parent-specific genomic imprinting, re-establishment of totipotency, and for embryonic development to proceed successfully.4 In murine germ cells for instance, the oocyte is de-acetylated between the germinal vesicle (GV) and metaphase of the second meiotic division (MII) stage, while no de-methylation is observed during that period.5 On the other hand, the spermatozoon does not demonstrate staining for histone H4 lysine acetylation,5 and CpG dinucleotides in the acrosomal head of mature sperm are highly methylated.6-8 The ooplasm of a mature oocyte contains maternally inherited mRNA and proteins, which, following normal fertilization, remodel the parental genomes for the support of normal embryo development and the generation of future offspring.9 In addition, nuclear transfer experiments have demonstrated that oocytes also have the ability to reprogram the nuclei of differentiated somatic cells and give rise to a live offspring.10,11 Subsequent to fertilization, the epigenetic status of the embryonic genome is distinctly remodelled such as the de-methylation process of both parental genomes, which is followed by the de novo methylation of genes in later stages of mammalian embryo development (reviewed in ref. 12). The timing of these events varies depending on the species in question.12-15 In a similar illustration, the protamines are rapidly replaced by histones in the paternal pronucleus soon after fertilization, which become transiently hyper-acetylated

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The oocyte is remarkable in its ability to remodel parental genomes following fertilization and to reprogram somatic nuclei after nuclear transfer (NT). To characterize the patterns of histone H4 acetylation and DNA methylation during development of bovine gametogenesis and embryogenesis, specific antibodies for histone H4 acetylated at lysine 5 (K5), K8, K12 and K16 residues and for methylated cytosine of CpG dinucleotides were used. Oocytes and sperm lacked the staining for histone acetylation, when DNA methylation staining was intense. In IVF zygotes, both pronuclei were transiently hyper-acetylated. However, the male pronucleus was faster in acquiring acetylated histones, and concurrently it was rapidly demethylated. Both pronuclei were equally acetylated during the S to G2-phase transition, while methylation staining was only still observed in the female pronucleus. In parthenogenetically activated oocytes, acetylation of the female pronucleus was enriched faster, while DNA remained methylated. A transient de-acetylation was observed in NT embryos reconstructed using a non-activated ooplast of a metaphase second arrested oocyte. Remarkably, the intensity of acetylation staining of most H4 lysine residues peaked at the 8-cell stage in IVF embryos, which coincided with zygotic genome activation and with lowest DNA methylation staining. At the blastocyst stage, trophectodermal cells of IVF and parthenogenetic embryos generally demonstrated more intense staining for most acetylated H4 lysine, whilst ICM cells stained very weakly. In contrast methylation of the DNA stained more intensely in ICM. NT blastocysts showed differential acetylation of blastomeres but not methylation. The inverse association of histone lysine acetylation and DNA methylation at different vital embryo stages suggests a mechanistically significant relationship. The complexities of these epigenetic interactions are discussed.

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Key words: bovine, histone H4, acetylation, DNA methylation, IVF, fertilization, embryo, nuclear transfer, parthenotes, epigenetics, cloning

*Correspondence to: Keith H.S. Campbell; Animal Development and Biotechnology Group; Division of Animal Physiology; Sutton Bonington Campus; University of Nottingham; Loughborough LE12 5RD UK; Tel.: +44.115.951.6298; Fax: +44.115.951.6302; Email: [email protected] Submitted: 04/02/08; Accepted: 06/24/08 Previously published online as an Epigenetics E-publication: http://www.landesbioscience.com/journals/epigenetics/article/6497

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Dynamics of histone acetylation in bovine in vitro produced embryos

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Figure 1. Acetylation pattern of histone H4 K12 (K5, K8 and K16 demonstrated a similar pattern) in (A) germinal vesicle, (B) oocyte at metaphase of meiosis second (MII), (C) oocyte at metaphase II after treatment with 75 μM TSA, and (D) mature spermatozoa. Methylation of DNA in (E) germinal vesicle, (F) oocyte at MII, (G) mature spermatozoa. Red and white arrows indicate the location of metaphase plate and polar body respectively. Scale bar = 10 μm, except for (D and G), scale bar = 4 μm.

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(reviewed in ref. 16). Hyper-acetylation of histone lysine residues might occur prior to its association with the chromatin as it happens in nuclei of somatic cells.16-18 In addition, the paternal pronucleus generally replicates more quickly than the maternal one and exhibits increased acetylation of histone lysine19,20 and de-methylated DNA.12 After the first few cell divisions of the embryo, depending on the species and with the right epigenetic configuration, the zygotic genome is activated to take over the maternal control. Histone H4 acetylation and DNA methylation play a key role in the remodelling process of the genome, and are linked with imprinting of genes in the early embryo.21 The objectives of these studies were to characterize the pattern of acetylation of histone H4 lysine -5, -8, -12 and -16 during early development of bovine oocytes matured and fertilized in vitro, and to compare them to the pattern of DNA methylation during the same developmental stages. Even though DNA methylation (5-methyl cytosines within CpG dinucleotides) patterns in early bovine embryos have been well documented in earlier reports,12,22-24 examining this epigenetic mark was considered necessary to be assessed as it may wary with different in vitro culture techniques.1 The functional role of the paternal chromatin on acetylation of lysine residues was assessed by studying parthenogenetically activated bovine oocytes and that of the oocyte factors on the remodelling of donor somatic nucleus was assessed by producing nuclear transfer embryos. 200

Results Acetylation of Histone H4 and methylation of DNA of bovine gametes. Germinal vesicle (GV) stage oocytes stained homogeneously for all lysine residues (Fig. 1A), while strong DNA methylation signal was restricted to the centromeric region of the chromosomes (Fig. 1E). During the breakdown of the GV (GVBD), the acetylation of all histone H4 lysine residues started to decrease whereas DNA methylation increased. By metaphase II (MII), oocytes demonstrated negative staining for H4 K5, K12 and K16 acetylation (Fig. 1B) except for H4K8 which always displayed weak staining (data not shown). In contrast, strong DNA methylation signal was detected in MII oocytes (Fig. 1F). To prove that the reduction in intensity of acetylated H4 was a result of active deaceylation during maturation, we treated oocytes with the deacetylase inhibitor TSA between GV and GVBD. Strong acetylation staining was observed for all lysines at the MII stage (Fig. 1C), indicating that a histone deacetylase is active during the initial steps of the maturation process in bovine oocytes. These experiments demonstrate an inverse correlation between histone lysine acetylation and DNA methylation during bovine oocyte maturation, indicating that matured oocytes have a hypo-acetylated chromatin. We subsequently decided to look at matured sperm to determine whether this was a feature of mature gametes. Viable bull spermatozoa stained strongly positive for

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Figure 2. Acetylation of histone K5, 8, 12 and 16 during the first cycle of monospermic and polyspermic bovine IVF embryos. Blue and green represents DAPI counterstain and acetylation of histone lysine stain respectively. (A) Normospermic zygotes at nine hours post-insemination (hpi); (B) Normospermic zygotes at 18 hpi; (C) Polyspermic zygotes at 18 hpi; and (D) 1-cell until morula (32-cell) stage bovine IVF embryos stained for acetylated H4 K12. DNA methylation in (E) monospermic and (F) polyspermic IVF zygote. Scale bar = 10 μM unless otherwise indicated. Red asterisk indicates the paternal pronucleus and red arrow indicates the location of the inner cell mass.

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DNA methylation (Fig. 1G) and negative for H4 K5-16 acetylation (Fig. 1D). Together these results indicate that matured bovine oocytes and sperm have highly methylated DNA and low histone H4 acetylation at the moment of in vitro fertilization. Acetylation of Histone H4 and methylation of DNA during the first cell cycle of in vitro fertilized, parthenogenetic and cloned embryos. We then determined the pattern of acetylation and DNA methylation after in vitro fertilization. In the early hours after fertilization, the paternal pronucleus expanded quickly and acetylation of H4K5-16 was intense and homogeneous (Fig. 2A). In contrast, the maternal pronucleus was smaller with weak staining for acetylation (Fig. 2A), but only intense and perinuclear for K5 and K12 (Fig. 2A). Later, during the presumptive late S-G2 phase [18 hours post insemination (hpi)], both parental pronuclei were equally hyper-acetylated for all lysines (Fig. 2B), suggesting a strong acetylase activity in zygotes. When polyspermic embryos were produced, we report histone H4 hyper-acetylation in all pronuclei (Fig. 2C), indicating that either a strong acetylase activity is present in oocytes or that the pool of free acetylated histones H4 in oocytes is sufficient to form more than two haploid genomes. In order to determine whether DNA replication was required for hyperacetylation of histone H4, we treated fertilized zygotes for nine and 16 hrs with aphidicolin, a DNA replication inhibitor. Addition of aphidicolin did not prevent the hyper-acetylation of H4K5 between nine hours and 16 hours www.landesbioscience.com

ost-insemination (Table 1), indicating that histone hyperacetylation p is not DNA replication dependent. DNA methylation was also analysed during the first cell cycle. Signal in the paternal pronucleus was significantly reduced 4–6 hpi compared to the maternal pronucleus (data not shown), which maintained a stronger signal even after the S to G2 transition (Fig. 2E). A group of polyspermic embryos stained for DNA methylation after 18 hpi (n = 20) demonstrated the demethylation of only a single pronucleus, which was assumed to be the male pronucleus because of its larger size than the average of other pronuclei. In contrast, the female and the additional male pronuclei remained clearly stained for DNA methylation (Fig. 2F), suggesting that DNA demethylation of the paternal genome is an active process and that the activity mediating it can be titrated out by the presence of an extra male genome. We were interested to determine these chromatin changes in somatic cell nuclear transfer embryos. Somatic cell chromatin fused with an enucleated MII oocyte showed a transient de-acetylation of H4 K5 after two hours (Fig. 5B), whereas after four hrs histone H4K5 staining was visible (Fig. 5C). When using pre-activated oocytes, the nuclear envelope remained intact and no de-acetylation observed (Fig. 5D and E). Eight hours after activation, most cloned embryos formed a pseudo-pronucleus which was hyper-acetylated in all embryos observed (Fig. 5F).

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9 hours after fertilization

Control Group

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Aphidicolin Treated Group

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Table 1 The staining intensity for acetylated lysine on histone H4 after incubation of bovine zygotes in the presence or absence of aphidicolin in the culture medium for a period of nine or 16 hours post-insemination

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+ indicates the staining intensity for histone lysine residues at 9 hours. ++ indicates a significant increase at 16 hours.

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the methylated cytosines on DNA had been thoroughly tested in previous reports.32,33 To our knowledge, this is the first detailed report characterising the acetylation pattern of lysine 5, 8, 12 and 16 of histone H4 and the methylation of DNA during early bovine embryo development. Germinal vesicle (GV) stage oocytes were strongly positive for all histone H4 lysine residues 5–16. After 6 hours in culture, the oocytes entered the germinal vesicle breakdown (GVBD) stage, acetylation of all histone H4 lysine residues were still positive, but started to decrease significantly when chromosomes started to condense towards metaphase I (MI). By metaphase II (MII), oocytes were negative for H4 K5-16, except for K8 which always displayed weak staining. A recent report in the mouse describes a similar pattern during the maturation process of mouse oocytes.5 The de-acetylation of histones during oocyte maturation is an active process as treatment of oocytes with 75 μM trichostatin A (TSA) which inhibit histone de-acetylase (HDAC) activity during the GVBD period resulted in MII oocytes with strong acetylation staining. Our observation correlates with a recent report in the bovine model.34 Histone acetyl-transferases (HATs) are expected to acetylate histones in MII oocytes that were treated with trichostatin A (TSA). However, oocytes that have reached MII stage and have already de-acetylated histones remained de-acetylated even if TSA was supplemented in the media. This all indicates that the activity of HATs is restricted to the period up to the germinal vesicle breakdown (GVBD) even though bovine oocytes contain a pool of mRNA expression for HAT1 and GCN5—a type A HAT enzyme which increases from GV to MII stages,35 but it appears that these were not translated into proteins or translated but unable to acetylate de-acetylated histone lysine in metaphase chromosomes. A similar observation was reported in metaphase second mouse oocytes which were treated in TSA.36 This de-acetylation activity in the chromatin during meiosis does not take place in somatic cells at the mitotic phase of the cell cycle.37,38 DNA methylation was positive throughout the maturation stages of the oocyte. At the GV stage, DNA methylation was mainly at the centromeric region of the chromosomes due to its spotty pattern on chromosomes, but the number of spots did not add up to 30 (normal karyotype of cattle is 30 pairs of chromosomes). This might be due to the merging of centromeric regions in different pairs of chromosomes. The staining intensity increased significantly between chromosome condensation and progression to MII. Similar observations were reported in mouse oocytes except that they described that

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The extent of paternal influence on the rate and pattern of the epigenetic marks was investigated by producing parthenogenetic embryos. The female pronucleus was hyper-acetylated, and was comparable to that of the male pronucleus of IVF produced embryos fixed at the same respective times (Fig. 3B–E). Parthenotes produced from 30 hours in vitro matured oocytes, activated without CHX and CB, showed a similar intensity and pattern of histone H4 K5 acetylation to the group with CHX and CB, except that they had only one pronucleus (Fig. 3A). There was limited or no global de-methylation activity observed in neither haploid nor diploid parthenogenetic zygotes during the first cell cycle (Fig. 3F and G). Similarly, there was no observed global demethylation up to four hours after fusion of a donor somatic cell into an unactivated or a pre-activated oocyte (data not shown). These observations suggest a strong de-acetylase activity in oocytes before activation or fertilization, the balance of enzyme activity is reversed, and a strong histone acetyltransferase activity takes over after activation or fertilization. The parental pronuclei do not maintain a symmetrical epigenetic modification before syngamy and first cleavage, since the maternal pronucleus is still methylated and the histones are hyper-acetylated, while the paternal pronucleus is de-methylated with hyper-acetylated histones. This maternal epigenetic configuration is not retained in the absence of the paternal pronucleus. Acetylation of histone H4 and methylation of DNA in developing embryos. The pattern of histone H4 acetylation for the different lysines is summarized in Figures 2D and 4. Histone H4 K5 and K12 showed similar patterns, with deacetylation from the one to two-cell stages, and a gradual increase and peak in acetylation at the 8-cell stage. The 16-cell and morula stages showed reduced acetylation, whereas by the blastocyst stage the hypo-acetylated ICM and the hyper-acetylated TE were clearly distinguished (Fig. 3H–K). The differential acetylation of H4 K5, between ICM and TE, was also observed in parthenogenetic and nuclear transfer blastocysts (Figs. 3L and 5G), however, the overall intensity of staining was reduced in parthenotes. The differences in acetylation of H4K-8 and -16 were more subtle during most stages (Fig. 4B and D). Histone H4 hyperacetylation during the 8-cell stage coincided with the transcriptional activation of bovine embryos, as demonstrated by FUrd labelling (Fig. 6), and was inversely correlated with DNA methylation which is detected at its lowest at this developmental stage (Fig. 4E). The differences between ICM and TE cells were also inversely correlated in terms DNA methylation to that of histone acetylation with ICM cells staining more intensely than TE cells in IVF embryos (Fig. 3M). The differential DNA methylation was less apparent in diploid parthenogenetic embryos (Fig. 3N), and to a lesser extent in clone blastocyst (Fig. 5H). The acetylation of histones and the methylation of DNA generally demonstrated an inverse pattern, and this observation was mainly noticeable at ZGA and blastocysts.

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Discussion

Acetylation of histone H4 lysine residues is important in the regulation of higher order chromatin; its modulation regulates access and binding of transcription factors and ultimately transcription.29-31 It facilitates the replacement of histones by protamines in spermatogenesis (reviewed in ref. 32), and protamines by histones in the zygote.19 The specificity of the primary antibodies used in this work against the acetylated lysine residues on histone H4 and against 202

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Figure 3. Cetylation of histone H4 K5 in bovine parthenogenetic zygotes and blastocysts. (A) Haploid parthenogenetic zygote stained at ten hours postactivation (hpa) for acetylated histone H4 K5; (B–E) diploid parthenogenetic zygotes stained at 10 hpa. Methylation of DNA in (F) haploid and (G) diploid parthenogenetic zygotes at 10 hpa. Acetylation of H4 lysines in (H–K) IVF and (L) parthenogenetic blastocysts. Methylation of DNA in (M) IVF and (N) parthenogenetic blastocysts. Blue represents DAPI counterstain and green represents acetylation of histone H4 lysine stain. Scale bar (A–G) = 10 μM and (M–N) = 50 μm. Arrow indicates the location of the inner cell mass.

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DNA methylation was strongly positive throughout the maturation stages.5 Bull spermatozoa were strongly positive for DNA methylation in the acrosome region and strongly negative for H4 K5-16 acetylation which is probably due to the absence of histones in the sperm chromatin. The methylation status of bovine and murine germ cells have been previously reviewed in reference 55. In the early hours after fertilization, the pattern of acetylation of H4 lysine acetylation for the paternal nuclei was intense and homogeneous. However both parental pronuclei by the S to G2 transition were equally acetylated. By comparison, acetylation of H4 K8 and K16 showed a homogeneous distribution of staining throughout the first cell cycle in both pronuclei, but it was stronger in the male pronucleus during the initial hours after fertilization. The enrichment of K5 and K12 acetylation was initially peri-nuclear for the maternal pronucleus. In various species, lysine residues of histone H4 could be di-acetylated on K5 and K12 prior to their association with newly synthesised DNA as happens in somatic cells. This can be accomplished by a cytoplasmic histone acetyl-transferase such as HAT-B39 and are subsequently de-acetylated after nucleosome assembly.16-18,39 This form of acetylated histone deposition is conserved across mammalian species and also in plants, where it extends to the acetylation of H4 K16 prior to the deposition of triacetylated histone H4 in newly replicated chromatin.40 In contrast, H4 K8 and K16 are acetylated after deposition of histones in newly synthesised chromatin.16-18 This could account for the increased www.landesbioscience.com

peri-nuclear staining intensity of H4 K5 and K12, and not K8 and K16, observed at the 1-cell stage. As mentioned earlier, during the initial hours of the first cell cycle the paternal chromatin seems to out-compete the female chromatin for the pool of acetylated histone H4 in the oocyte as it stains more intensely for acetylated histone H4 lysine residues. This pool is not a limiting factor since polyspermic embryos had all their pronuclei transiently hyper-acetylated in the early hours post-fertilization. A similar observation was recently described in the mouse embryo.5 This suggests that the male pronucleus might be preventing the female pronucleus from acquiring acetylated histone lysine residues through some unknown factors. Interestingly, the female pronucleus augmentation in H4 K5 and K12 was peripheral in the initial hours which might imply the use of a pre-acetylated form of H4 lysine from the cytoplasm. During the S to G2 transition of the first cell cycle, the level of acetylation of all histone H4 lysine residues was equivalent in both male and female pronuclei. To elucidate whether the differential acetylation was related to differences in the phases of the cell cycle, we cultured zygotes in the presence or absence of aphidicolin from the time of insemination. Acetylation of histone H4 K5 and K8 patterns of the pronuclei increased between nine and 16 hours following fertilization independent of the presence or absence of aphidicolin, indicating that the temporary enrichment in histone H4 lysine acetylation does not require DNA replication. Similar observations were reported in the

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Figure 4. Acetylation patterns of lysine 5 (A), lysine 8 (B), lysine 12 (C) and lysine 16 (D) residues and methylation of DNA (E) in early pre-implantation bovine embryo produced by standard IVF after correction for area and absorbance. Different letter superscript indicates significant differences (p < 0.05).

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mouse embryos,41 and in Xenopus laevis embryos.42 In addition, the presence of aphidicolin did not prevent the peripheral staining of the female pronucleus for H4 K5 and K12. In bovine parthenotes, the female chromatin became markedly acetylated for K5, K12 and K16, and to a lesser extent for K8, in the early hours after activation. Parthenotes were initially produced by activating MII oocytes with 7% ethanol, followed by six hours incubation in cycloheximide (CHX) and cytochalasin B (CB), and then into mSOFaaBSA until fixation (standard protocol for producing diploid parthenogenetic bovine embryos). The absence of the male pronucleus resulted in a faster enrichment in the acetylation of histone H4 lysine residues of the female pronucleus in parthenotes. Another group of parthenogenetically activated oocytes were produced by activation with 7% ethanol without CHX and CB (due to the fact that CHX is a protein synthesis inhibitor, thus producing haploid embryos), but the same results were obtained. 204

The enrichment in the presence of CHX further demonstrates that the acetylated histones are from pre-stored proteins in ooplasm or histone acetyl-transferase (HAT) activity is high in the parthenotes. Similar observations have been made in the mouse where a marked increase in staining of H4 K5 in parthenogenetically activated mouse oocytes was reported.19 Another potential for the oocyte is its ability to remodel a donor somatic nuclei into an embryonic state and give rise to a live offspring.10,11 In this work, we show the ability of non-activated MII oocytes to transiently de-acetylate a somatic donor nucleus two hours after fusion in nuclear transfer embryos. This epigenetic modification does not take place using a pre-activated oocyte. A similar observation was reported in mouse nuclear transfer embryos using both cycling cells and M-phase arrested cells by nocodazole. The chromatin form both types of cells were de-acetylated two hours after fusion with non-activated enucleated mouse MII oocyte.36,43

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Figure 5. Acetylation of histone H4 lysine and methylation of DNA in bovine nuclear transfer embryos: (A) Unfused couplet of a donor cell and a oocyte as a positive control; (B and C) two and four hours post-fusion (hpf) using unactivated cytoplasts respectively; (D and E) Two and four using two hours preactivated cytoplasts; (F) Eight hours post-activation; (G and H) bovine NT day 7 blastocysts. Blue represents DAPI counterstain, and green and red represent histone lysine acetylation and DNA methylation stain. Arrow indicates location of ICM. Scale bar = (A–F) 10 μM and (G) 47.62 μM.


Figure 6. FUrd incorporation indicating major transcription in nuclei of bovine IVF (A) 4-cell; (B) 8-cell and (C) 16-cell embryo stages indicating activation of major zygotic transcription at the 8-cell embryo stage. Blue and red represent DAPI counterstain and FUrd incorporation respectively. Scale bar = 10 μM.

The transient de-acetylation observed is most likely associated with the higher activities of MPF and MAPK of non-activated oocytes as activated oocytes lose rapidly the activities of these two key regulators of the cell cycle progression.44 A recent report looking at the acetylation of H4 K12 in mouse nuclear transfer embryos reported again the same observation, except that they argued that this transient de-acetylation is not critical to the development of NT embryos. This is demonstrated by treatment of donor cells and reconstructed embryos with TSA which kept the levels of acetylation high, and which resulted in better development to blastocyst and generation of live offspring.45 Past the first embryonic cycle, there was a gradual increase in acetylated H4 K5 (p < 0.05), K12 (p < 0.05), K16 (not significant) and a decrease in H4 K8 (p < 0.05) towards the 8-cell stage bovine embryo. As mentioned in the introduction, histone H4 K5 acetylation has been correlated with actively transcribing genes.30,31 In addition, histone H4 tails are acetylated in an orderly manner, lysine 16 (K16) occurring first, then followed by K8, K12 and finally K5, which is mainly acetylated in tetra-acetylated H4.32 Epigenetics

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In the last decade, a lot of work has been carried out on the methylation pattern of DNA in animal development. DNA methylation and histone lysine acetylation have been well associated with genomic imprinting and repression of a number of genes in animal development.49,50 Following fertilization, the parental genomes are unequally demethylated for most mammalian species studied so far except for the sheep and the rabbit embryos.51 The paternal pronucleus is demethylated in the first 6–8 hours after fertilization,12,15,51 which probably occurs by active demethylation, the mechanism of which remains unknown. On the other hand, the maternal genome demethylation occurs after several cell divisions22 and most likely results from the exclusion of the DNA methyltransferase—Dnmt1o—from the nucleus.52 A number of hypotheses are proposed for the specific de-methylation of the paternal genome; since methylation is generally associated with repression of transcription, de-methylation of paternal genome may simply reflect an initiation to a transcriptional state of paternal genes, but this suggestion applies more to the mouse embryo development.53 Another hypothesis has to do with the ‘battle of the sexes’ where the paternal interests are to secure nutrients and grow larger, while the maternal one is to secure a durable survival of all of her offspring.54 As epigenetic modifications might vary in different in vitro culture techniques,1 we thought it was necessary to compare our system to other published reports for DNA methylation. Interestingly, de-methylation of the paternal genomes during the first cell cycle is concurrent with our reported enrichment in acetylation of histone H4 K5 and K12 in the bovine. Furthermore, the pool of DNA methyltransferases is not abundant. This is confirmed by staining polyspermic zygotes for DNA methylation. Unlike the staining for acetylation of histone lysine in which all male pronuclei stained positively, all except one pronucleus was de-methylated while all the rest stained positively. This is similar to what observations in the mouse embryo except that they described the threshold for de-methylase activity was for polyspermic embryos with more than five pronuclei.5 Both parental genomes of the bovine IVF embryo are de-methylated by the 8-cell stage, and de novo methylation starts at the 16-cell stage onwards.12 Similarly, we report an increase in acetylation of K5 and ly12 at the 8-cell stage, and a decrease in the stages thereafter. At the 16-cell stage onwards, differential populations of cells in terms of DNA methylation and histone lysine acetylation is observed. By the blastocyst stage, inner cell mass (ICM) cells having a more intense methylation staining than trophectodermal (TE) cells, and which corresponds to previously published work.55 The differential methylation of the two cell populations in the IVF blastocysts was again observed in the bovine parthenogenetic embryos, but the overall intensity was weaker as more exposure time was always required to pick up fluorescence in stained parthenotes. This again is opposite to what we observe for H4 K5 and K12 where ICM cells showed less intense staining than TE cells. Interestingly, this biochemical association does not apply for histone H4 K8, although recent data suggests that different post-translational modifications of the histone tails function simultaneously or in coordination to form the ‘histone code’.18 The highest and lowest staining for histone lysine acetylation and DNA methylation at the 8-cell stage, and the differential staining for these marks that was observed starting from the 8-cell stage onwards can be similar to what was observed in the early

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Therefore, acetylation of K5 reflects the total hyper-acetylated state of histone H4 and thus strongly correlates with more active genes. A similar recent report demonstrated an increase in H4K5 acetylation, along with increase in transcription of Ndn and Xist genes, at the 8-cell stage bovine IVF embryo.34 The bovine zygotic genome begins major transcription from the 8-cell stage embryo onwards (reviewed in ref. 44), which further strengthens the association of increased acetylation of H4 K5 in particular, and K12 and K16 in this case, with increased transcription. Our work agrees with similar reports in the mouse,19,46 where increased acetylation of histone H4 K5 was observed at the 2-cell stage embryo which corresponds to the time of the mouse embryo major zygotic genome activation. Another line of evidence comes from the report on mRNA transcript levels of HDACs in pre-implantation bovine embryo stages, where a decline of most HDACs transcripts was observed at the 8-cell embryo.35 We did not observe an increase in acetylation of H4 K8 at the 8-cell stage embryo. Acetylation of K8 has been frequently reported as staining differently than other lysine residues on histone H4. In the mouse, for instance, during germinal vesicle breakdown (GVBD) all lysine residues stained negatively and remained negative in second metaphase (MII), except for K8 which displayed a weak staining in GVBD and MII. Even in cells transplanted into enucleated mouse oocytes, all lysine residues were de-acetylated except for K8 that always showed weak staining.36 In the plant heterochromatin as well, cells treated with trichostatin A (TSA)—a chemical that blocks the activity of HDACs—showed an increase in acetylation of H4 K5, K12 and K16 but not K8.40 The weak intensity staining of K8 acetylation at the 1- and the 8-cell stage bovine embryo could well be involved in the remodelling mechanism of the genome. However, what we observe could as well be due to the location of the acetylated K8 in the three-dimensional structure of the DNA at this embryo stage, which might prevent the access of the HATs or HDACs, or even the primary antibody.47 Histone H4 K5, K12 and K16 were significantly de-acetylated between the 8-cell and 16-cell stage embryos, while it was invariable for K8. At the 16-cell stage onwards and for H4 K5 and K12 mainly, differential acetylation staining of the blastomeres was apparent in IVF and parthenogenetic embryos. At the blastocyst stage, histone H4 K5 and K12 were less acetylated in inner cell mass (ICM) than trophectodermal (TE) cells. A similar observation was recently reported in mouse fertilized embryos at the blastocysts that were stained with hyperacetylated H4 antibody.48 There was no observed difference between ICM and TE cells for acetylation of H4 K8. As an increase in the acetylation of histone lysine residues reflects the active state of genes,30 this may explain the increased K5 and K12 acetylation of TE cells compared to ICM cells, suggesting that TE cells are more transcriptionally active than ICM cells. The difference in acetylation of K16 between ICM and TE cells in blastocysts was not consistent in all embryos as for K5 and K12. This variability may or may not be a measure of embryo quality, but further work hast to be carried out to find out the biological significance of this variability. As for nuclear transfer embryos, there was an observed differential H4 K5 acetylation in the blastomeres of day 7 blastocysts produced but no observed differential DNA methylation. This suggests a limited remodelling ability of the enucleated oocytic factors on the donor somatic nucleus, and this is usually reflected by a very low efficiency of producing cloned offspring using a somatic cell (reviewed in refs. 49 and 50). 206

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All chemicals were purchased from Sigma (Poole, Dorset, UK) and disposable plasticware from Nunc (Nunc, Roskilde, Denmark) unless otherwise stated. In vitro maturation of bovine oocytes. Cumulus oocyte complexes (COCs) were aspirated from follicles of 2 to 8 mm in diameter from ovaries collected at a local slaughterhouse. COCs were matured in vitro in TCM 199 supplemented with 10% FCS, 5 μg/ml each of FSH (Folltropin-V; Vetrepharm, Canada) and LH (Lutropin-V; Vetrepharm, Canada), 1 μg/ml of E2, and Gentamycin at 50 μg/ml, at 39°C in a humidified environment of 5% CO2 for 24 hours. In vitro fertilization and culture of bovine embryos. In vitro matured oocytes were fertilized and cultured as previously reported.25 Briefly, COCs were repeatedly pipetted until 2–3 layers of granulosa cells were left around the oocyte. Groups of 40–50 COCs were then incubated in 0.5 ml of sperm at a concentration of 0.5–1.0 x 106 sperm/ml of fertilization medium and cultured for 20 h at 39°C in a humidified incubator with an atmosphere of 5% CO2 in 95% N2. After 20 hours (day 1), all embryos were washed twice in HEPESbuffered modified synthetic oviductal fluid (H-SOF; reviewed in ref. 26) medium and transferred into mSOFaa media supplemented with 3 mg/ml of BSA for 24 hours (day 2). On day 2, cleaved embryos were transferred into fresh mSOFaa media supplemented with 10% FCS. Embryo culture was carried out at 39°C, in a humidified incubator with a gaseous atmosphere of 5% CO2, 5% O2 and 90% N2. DNA replication was inhibited by culturing presumptive zygotes in the presence of 1 μg/ml aphidicolin from the time of insemination until fixation. Polyspermic embryos were produced by increasing the sperm concentration up to 3 x 106 sperm/ml of fertilization medium. In vitro production of bovine parthenogenetic embryos. Oocytes matured in vitro for 24 hours were denuded by incubating the COCs www.landesbioscience.com

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Materials and Methods

with 1 ml of 300 IU of hyaluronidase/ml of normal saline for one minute and then vortexing in the same solution for four minutes in a 15 ml polystyrene conical tube (Bibby Sterilin, Stone, UK). Oocytes with a regular, non-granulated cytoplasm, and exhibiting the first polar body, were selected and washed twice in H-SOF. After 24 hours of maturation, selected oocytes were activated with 7% ethanol in H-SOF for six minutes. This is followed by 5–6 hours incubation in mSOFaaBSA supplemented with 10 μg/ml cycloheximide (CHX) and 7.5 μg/ml cytochalasin B (CB). The oocytes were washed in H-SOF after the CHX-CB step, and transferred into 50 μl microdrops of mSOFaaBSA under oil at 39°C, 5% CO2, 5% O2 and 90% N2. As CHX inhibits protein synthesis, another group of in vitro matured oocytes were activated at 30 hours using the aforementioned method but were transferred straight into mSOFaaBSA without CHX and CB. At day 2, cleaved parthenogenetic embryos were transferred into fresh mSOFaa medium supplemented with 10% FCS until the blastocyst stage. Donor cell culture for nuclear transfer. Primary bovine fetal fibroblasts were isolated from a 30–60 days bovine foetus as previously described27 and cultured for two passages in complete Dulbecco’s Modified Eagle’s Medium (DMEM) medium supplemented with 1.0% (v/v) β-mercaptoethanol, 2.0 mM L-glutamine, 1.0% (v/v) penicillin/streptomycin and 10% FBS. Primary cultures were then stored in liquid N2 until required. For each experiment, cells were thawed, washed in fresh complete DMEM, and cultured until approximately 80–90% confluent, quiescence was then induced by reducing the concentration of FBS to 0.1% for a further 2–3 days. Immediately before use as nuclear donors, a single cell suspension was prepared by trypsinisation (0.25% w/v). The cells were pelleted and resuspended in DMEM plus 0.1% (v/v) FBS and maintained in this medium at 37°C until used as nuclear donors. In vitro production of nuclear transfer embryos. Oocytes were enucleated at metaphase of the 2nd meiotic division (MII). Prior to enucleation the cumulus cells were removed as for parthenotes. For enucleation, cumulus cells were removed at 15–16 hours post onset of maturation (hpm). For enucleation of denuded oocytes, a slit in the zona pellucida was made using the XY clone—a 50X laser objective (Hamilton Thorne, Hamilton, USA) fitted on a Leica DMIRB (Leica, Germany) at 100% power and 100 μsec time. The polar body plus a portion of cytoplasm underneath were aspirated using a 20–25 μm o.d. glass micropipette fitted on Narashige manipulators in a microdrop (50 μl) of manipulation medium (H-SOF containing 4 mg/ml BSA, 7.5 μg/ml cytochalasin B (CB) and 5 μg/ml Hoechst 33342). Enucleation of oocytes was confirmed by visualization of DNA in the aspirated karyoplast using a short exposure to UV light (0.1 sec) and image capture (Simple PCI, Compix, Inc., USA). Quiescent primary fetal fibroblasts used as nuclear donors were fused to enucleated cytoplasts with two DC pulses of 35 V/ cm for 60 μsec in 0.3 M mannitol without calcium ions using an Eppendorf Multiporator (Eppendorf, Germany) and fusion chamber with a 200 μm electrode gap. Fused couplets were cultured in mSOFaaBSA until activated. Activation was carried out in H-SOF medium containing 5 μg/ml A23187 for 5 min, followed by culture in modified mSOFaaBSA medium supplemented with 10 μg/ml of cycloheximide (CHXM) and 7.5 μg/ml CB for 5 h at 39°C in a humidified atmosphere of 5.0% O2, 5% CO2, 90% N2.

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mouse embryo. Exclusion of a DNA methyltransferase (Dnmt1) from the nucleus until the de-methylation of maternal genome is complete. De novo methylation starts between the morula and the blastocyst stage.56 An analogous process might be present in the early bovine embryo. In conclusion, this is the first detailed investigation on the changes of acetylation of lysine residues 5–16 on histone H4 and methylation of CpG dinucleotides on DNA at various stages of the in vitro produced bovine pre-implantation embryo. These two epigenetic marks act in a reverse way at various landmark embryo stages. In the first cell cycle, the paternal pronucleus is enriched with acetylated histone lysine while it gets de-methylated. At the 8-cell IVF embryo stage, the acetylation of H4 lysine is at its peak while the methylation of DNA is at its nadir. At the blastocyst stage, the ICM is methylated and de-acetylated while the TE is de-methylated and acetylated in IVF and parthenogenetic embryo. This difference in the two cell populations in the blastocyst is less obvious in NT embryos for the DNA methylation mark. It is still unclear how histone acetylation and DNA de-methylation are biochemically associated. A number of reports showing that the methyl-binding protein MeCP2, a protein that binds mCpGs and is involved in gene silencing, could recruit a de-acetylase to several gene loci,57,58 which could explain the converse actions of de-acetylation of histone H4 lysine residues with methylation of DNA.

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for 15 minutes, permeabilized in 0.2% Triton X-100 for 15 minutes, treated with 2 M HCl for 30 minutes, followed by ten minutes in 10 mM Tris-HCl (pH = 8.5). They were then washed with 0.05% Tween 20 and blocked for one hour in blocking solution (5% BSA, 0.2% Triton X-100, 0.05% Tween 20 and 0.05% Na Azide in PBS). Primary antibody staining was carried out by incubating with a polyclonal rabbit anti-histone H4 lysine 5 (K5; 1:800), lysine 8 (1:600), lysine 12 (1:400), lysine 16 (1:1000; Serotec, UK), or monoclonal mouse anti-5-methyl cytosine mCpG (1:10 Eurogentec, Belgium) diluted in blocking solution overnight at 4°C. Samples were washed well in PBS-PVA before incubation with either 1:200 swine anti-rabbit secondary antibody conjugated with fluorescein isothiocyanate R (FITC; DAKO, Denmark), or goat anti-mouse IgG conjugated to CyTM3 (1:200; Jackson Immunoresearch, USA) in blocking solution for 40 minutes at RT. Embryos were washed and mounted as described earlier. Staining of spermatozoa was carried by adjusting the concentration of frozen thawed bull semen to 50,000– 100,000 sperm per 200–300 μl of PBS. Sperm were centrifuged onto a slide using a cytospin (Centurion 2000 series) at 190 rpm for ten minutes. On the slide, the spermatozoa were fixed in 2.5% PFA, and all subsequent steps were identical to the immunostaining of oocytes and embryos. Microscopy. Samples were examined under epifluorescence. Images were captured and stored with a digital camera (Hammamatsu, ORCA-er, Japan) connected to the microscope (Leica DMR, Germany). Image analysis and quantification were performed using Simple PCI software (Compix Imaging Systems, USA). A laser scanning confocal microscope (LSCM; Leica TCS SP2, Germany) was used to look at sections in the embryos, mainly for the blastocyst stage embryos. Statistical analysis. An average of 20 embryos was examined in each group. Changes in intensities within and between different embryo stages were recorded as a mean ratio of green stain (or red stain for DNA methylation staining) to mean blue counterstain. Overlapping, mitotic, and smashed nuclei were not included in the measurements. In order to verify whether the association of mean green (or red) over mean blue between different embryo stages is not due to the confounding effect of area and absorbance. SPSS 11.0 was used to run a multiple linear regression model with area and absorbance as confounding variables. The graphs generated for acetylation of H4 K5-16 could not be combined and compared together due to the nature of the primary antibodies which are only specific to their respective antigens and have to be used separately and in different concentrations so that each measurement of one lysine does not indicate whether or not adjacent lysine residues are acetylated or not.

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Following activation, reconstructed embryos were transferred into mSOFaaBSA medium and cultured in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 39°C. On 2 day of culture, embryo cleavage was assessed and 5% FBS added to the culture medium. On day 7, development to blastocyst and total cell numbers were assessed. Assessment of nuclear stage. 5-Bromo-2'-deoxy-uridine (BrdU) labelling detection kit (Roche, Germany) was used to assess DNA synthesis. Briefly, BrdU labelling medium was added to the embryo culture medium at 1:1000 dilution (10 μmol). Treated embryos were incubated for one hour, and then fixed with 70% ethanol (diluted in 50 mM glycine buffer, pH 2.0) for 20 minutes at -20°C. BrdU was detected with 1:10 anti-BrdU antibody working solution at 39°C for 40 minutes, followed by incubation with 1:10 anti-mouse Ig-FITC. Embryos were mounted on a glass slide in Vectashield (Vector Laboratories, H-1200), containing 4', 6-diamidino-2-phenylindole (DAPI). Assesment of transcription. Five millimolars 5-Fluoro-5'deoxyuridine (FUrd) was freshly prepared for each experiment in mSOFaa culture medium. Treated embryos were incubated for 45 minutes, before they were fixed with 70% ethanol (diluted in 50 mM glycine buffer, pH 2.0) for 20 minutes at -20°C to measure the ribosomal transcription of RNA polymerase I (POL I).28 Embryos were then washed in PBS with 1% BSA, and incubated in blocking solution (5% BSA, 0.2% Triton X-100, 0.05% Tween 20 and 0.05% Na Azide in PBS). FUrd detection was carried out using Sigma antiBrdU mouse antibody at a 1:1000 dilution in the same blocking solution at 4°C overnight, followed by incubation with 1:200 goat anti-mouse conjugated with cyanine (Jackson Immunoresearch, USA). Embryos were mounted as previously described. Treatment with inhibitors. Trichostatin A (TSA) was used to inhibit histone deacetylase (HDAC) activity. In order to inhibit HDAC activity in meiosis, COCs aspirated form follicles were cultured in oocyte maturation media supplemented with 75 μM of TSA for 4–5 h. COCs were then washed with Hepes buffered TCM-199 supplemented with 10% FCS, and transferred into fresh oocyte maturation media for culture to the MII stage. Another group of oocytes were cultured until MII-stage and then treated for 4-h in maturation medium supplemented with 75 μM TSA. Immunofluorescence. All steps were carried out at room temperature (RT) and all solutions were prepared in PBS-PVA, unless otherwise stated. Oocytes and embryos were collected at different stages of maturation and culture; germinal vesicle (GV) at the time of follicle aspiration, germinal vesicle breakdown (GVBD) after 7 h of maturation, metaphase of the second meiotic division (MII) after 24 h of maturation, 1-cell zygotes at ten and 20 hours post insemination (hpi), 2-cell at 30 hpi, 4- and 8-cell embryos on day 2, 16-cell embryos on day 4, morulaes on day 5 and blastocysts on days 7–8. Parthenogenetic embryos were fixed at ten or 20 hours postactivation (hpa) and nuclear transfer embryos were fixed either at different time points of the first cell cycle after fusion or activation, or at the blastocyst stage on day 7–8. COCs were denuded by incubation in hyaluronidase at 300 IU/ ml for one minute, followed by four minutes of vortexing. The zona pellucida was removed by incubating in 2 mg/ml pronase for 2–3 minutes at 37°C on a heated stage and gentle pipetting. All subsequent steps were similar for both oocytes and embryos. Oocytes and embryos were fixed in 2.5% paraformaldehyde (PFA) 208

Acknowledgements

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