Drs. Park and Jeong contributed equally to this work.
Dynamic DNA methylation reprogramming: Active demethylation and immediate remethylation in the male pronucleus of bovine zygotes
Article first published online: 5 AUG 2007
Copyright © 2007 Wiley-Liss, Inc.
Volume 236, Issue 9, pages 2523–2533, September 2007
How to Cite
Park, J. S., Jeong, Y. S., Shin, S. T., Lee, K.-K. and Kang, Y.-K. (2007), Dynamic DNA methylation reprogramming: Active demethylation and immediate remethylation in the male pronucleus of bovine zygotes. Dev. Dyn., 236: 2523–2533. doi: 10.1002/dvdy.21278
- Issue published online: 17 AUG 2007
- Article first published online: 5 AUG 2007
- Manuscript Accepted: 20 JUN 2007
- KRCF/KRIBB. Grant Number: KGM1310713
- MOST. Grant Number: F104AD010004
- KOSEF. Grant Number: 2006-04082
- bovine embryo;
- male pronucleus;
- active demethylation;
- de novo methylation;
- DNA methylation;
- histone methylation
DNA methylation reprogramming (DMR) is believed to be a key process by which mammalian zygotes gain nuclear totipotency through erasing epigenetic modifications acquired during gametogenesis. Nonetheless, DMR patterns do not seem to be conserved among mammals. To identify uniform rules underlying mammalian DMRs, we explored DMRs of diverse mammalian zygotes. Of the zygotes studied, of particular interest was the bovine zygote; the paternal DNA methylation first decreased and was then rapidly restored almost to the maternal methylation level even before the two-cell stage. The 5-azadeoxycytidine treatment led to complete demethylation of the male pronucleus. The unusually dramatic changes in DNA methylation levels indicate that the bovine male pronucleus undergoes active demethylation, which is followed by de novo methylation. Our results show that, in bovine, the compound processes of active DNA demethylation and de novo DNA methylation, along with de novo H3-K9 trimethylation also, take place altogether within this very narrow window of pronucleus development. Developmental Dynamics 236:2523–2533, 2007. © 2007 Wiley-Liss, Inc.
DNA methylation and the associated assembly of repressive heterochromatin is an important mechanism that stably inactivates genes in a heritable manner (Bird and Wolffe,1999; Wolffe and Matzke,1999). DNA methylation at CpG dinucleotides is involved in regulation of gene expression and is essential for normal mammalian development (Li et al.,1992; Bird,2002). Specialized biochemical processes in which DNA methylation is likely to be involved include allele-specific gene expression (Reik and Walter,2001), X-chromosome inactivation (Heard,2004), and heritable transcriptional silencing of parasitic sequence elements (Yoder et al.,1997).
Despite the stable and heritable features, DNA methylation patterns can be globally reset at certain developmental stages: first in primordial germ cells and then in preimplantation embryos in the mouse. The DNA methylation reprogramming (DMR) process begins with rapid demethylation of single-copy sequences, including imprinted genes, followed by gradual remethylation, which has been shown to be essential for imprinting and erasure of acquired epigenetic modifications, also known as epimutations (Reik et al.,2001; Reik and Walter,2001; Rideout et al.,2001; Surani,2001).
In mice, DMR during preimplantation development leads to genome-wide demethylation of the male pronucleus only hours after fertilization (Mayer et al.,2000; Oswald et al.,2000; Santos et al.,2002), and actively erases differentiation-associated epigenetic traces acquired during gametogenesis from sperm DNA (Morgan et al.,2005). The female DNA, however, seems to be protected from such an active demethylation event at the pronucleus stage, but later, during successive cleavage stages, undergoes a passive demethylation process that is coupled to the DNA replication cycle (Sanford et al.,1984; Monk et al.,1987; Razin and Shemer,1995; Rougier et al.,1998; Mayer et al.,2000; Oswald et al.,2000; Dean et al.,2001). The completion of these re-setting processes may be necessary to “re-write” the epigenetic draft of DNA methylation and to ultimately bring a pluripotent epigenetic feature to the embryonic stem cells (Reik et al.,2001; Rideout et al.,2001; Li,2002; Kang et al.,2003a). Correct DMR in the early stage of development, when new cell lineages are formed, is believed to be essential for regulating gene silencing at specific times and for prevention of damaging or lethal ectopic gene expression (Jaenisch and Bird,2003). Despite the conceptual significance, the DMR patterns do not seem to be conserved among mammalian species (Young and Beaujean,2004). For example, active demethylation has not been detected in sheep (Beaujean et al.,2004a,b), rabbits (Beaujean et al.,2004a; Shi et al.,2004), and pigs (Jeong et al.,2007). These contrasting observations emphasize the risks of extrapolation of methylation reprogramming patterns between mammalian species, and also suggest the existence of additional mammalian types of methylation reprogramming. The developmental outcome of species-specific methylation reprogramming patterns is currently unknown, but the distinct reprogramming strategies used by individual species are likely to yield different gene expression profiles, at least temporarily, and may influence the complicated processes of early embryonic development and differentiation.
In this study, to gain further insight into the aspects of global DNA methylation changes during preimplantation development, we investigated the DMR patterns in bovine zygotes. We report a third type of early methylation reprogramming that is different from those of the mouse and the sheep. Immunocytochemical inspection of bovine zygotes showed that the sperm-derived chromatins were actively and completely demethylated and then re-methylated before the two-cell stage. So far, this is the most dynamic DMR pattern reported to occur in the male pronucleus of a mammalian zygote. We also discuss possible interaction mechanisms among the events of active demethylation, de novo DNA methylation, and H3-K9 trimethylation in the male pronuclei of mammalian zygotes.
Different Patterns of DNA Methylation Reprogramming Among Mammals
We first examined DNA methylation states of various mammalian zygotes at mid-to-late pronucleus stages. The resulting epigenetic “blueprints” of the sperm-derived genomes could be divided into three distinct DMR categories (Fig. 1): type-I species, in which the male pronucleus is actively demethylated (rodents); type-II species, in which the paternal level of DNA methylation is maintained (sheep and pig); and type-III species, in which the male pronucleus seems to be partially demethylated (cow and goat). These results show that mammalian species have adopted various strategies to reprogram incoming sperm DNA (Beaujean et al.,2004a).
Of particular interest was the type-III species (cow and goat), in which the male pronucleus has an intermediate level of DNA methylation compared with type-I and type-II species. As reported previously (Bourc′his et al.,2001; Dean et al.,2001; Beaujean et al.,2004a), we found that bovine zygotes were, for the most part, demethylated to some extent approximately 20 hours postfertilization (hpf). However, we also noticed that paternal DNA methylation states varied among the individual zygotes, ranging from a fully methylated state to an almost demethylated state. This finding prompted us to examine the paternal methylation states at different stages of bovine pronuclear development in detail.
Active DNA Demethylation Is Followed by De Novo Methylation in Bovine Sperm-Derived Pronucleus
We collected bovine zygotes at 10, 20, and 25 hpf. In our in vitro fertilization (IVF) system, most (approximately 70–80%) bovine oocytes that mature in vitro are infiltrated approximately 4–6 hr after co-incubation with capacitated sperm. Figure 2 shows typical staining patterns of 5-methylcytosine (5-MeC) and trimethylated histone H3-lysine 9 (H3-m3K9) at given times. Although the paternal H3-m3K9 level increased steadily during bovine pronucleus development, which was in agreement with previous studies in pigs (Jeong et al.,2007), the paternal 5-MeC level fluctuated markedly. We attempted to measure DNA methylation levels. At 10 hpf, paternal 5-MeC levels were similar or slightly lower than maternal levels (Fig. 3A); the average methylation level of paternal DNA relative to maternal DNA was 84.3% ± 13.1 (mean ± standard deviation; n = 19). This value was reduced significantly to 51.1% ± 19.1 (n = 23, P < 0.001) at 20 hpf, which may be indicative of active demethylation in the male pronucleus, although to a lesser extent than in the mouse (Mayer et al.,2000; Oswald et al.,2000; Santos et al.,2002).
At 25 hpf, bovine zygotes were found at many different developmental stages: group-1 embryos that had already cleaved and progressed to the two-cell stage (15.7%, 8/51); group-2 zygotes with condensed chromosomes in preparation for mitosis (11.8%, Fig. 2e-f′); group-3 zygotes in which the parental pronuclei remained closely associated (41.2%, d); and group-4 zygotes with immature-looking pronuclei with thin and fluffy chromatins (c). Thus, the transition from the one- to two-cell stage might occur at approximately 25 hpf in bovine zygotes. We included group-3 and group-4 zygotes in the quantification. If the two parental pronuclei could not be distinguished by staining, we assessed the H3-m3K9 level: of the two pronuclei, the one with the lower H3-m3K9 level was identified as the male pronucleus (Jeong et al.,2007). Average DNA methylation level of the paternal pronucleus at 25 hpf was 75.0% ± 25.0 (n = 23), which was significantly increased compared with of the level at 20 hpf (P < 0.001). Restoration of the paternal 5-MeC level was also proven by, and thus linked to, the DNA methylation state of the group-2 zygotes, because their sets of condensed parental chromosomes were stained indistinguishably for 5-MeC (Fig. 2e-f′). This quantification result indicates that bovine sperm DNA undergoes active demethylation and then de novo methylation. It is striking that both opposing processes of DNA demethylation and remethylation occur during such a narrow developmental window restricted to the pronucleus stage. Meanwhile, in bovine zygotes, transcripts of the dnmt3a and -3b genes responsible for de novo DNA methylation (Okano et al.,1999) have been shown to be associated with ribosomal fractions, indicating that the active proteins may be present in bovine preimplantation-stage embryos (Golding and Westhusin,2003).
Paternal 5-MeC and H3-m3K9 Levels Progressively Increase in the Late-Half of Pronucleus Development
The H3-m3K9 levels in the male pronucleus were examined either separately or with the 5-MeC level (Figs. 2, 3B). H3-m3K9 was almost invisible or just faintly stained in the male pronucleus at 10 hpf (21.5 ± 9.4, n = 19). At 20 hpf, when the paternal 5-MeC level was shown to be markedly reduced, the H3-m3K9 level increased to 41.6 ± 15.4 (n = 43; P < 0.001). By 25 hpf, the paternal H3-m3K9 level had further increased by approximately 70% (69.2 ± 24.8; P < 0.001). These results indicate that, in contrast to mouse zygotes, bovine zygotes have an H3-K9 methylation activity that can globally modify the sperm chromatins in the cytoplasm almost to the level of the maternal counterparts. This finding is similar to the pig zygote (Jeong et al.,2007). The amount of H3-m3K9 marks, therefore, is to be a good indicator of both the sexual identity and the maturity of the male pronucleus in mammals, which is because, although the paternal pronucleus of the mouse can be distinguished by a size differential, it has not yet been verified which of the equivalently sized pronuclei are demethylated in the other species (Young and Beaujean,2004).
When the paternal 5-MeC and H3-m3K9 levels of individual zygotes were compared, we found that the levels increase simultaneously (Fig. 3C). At 25 hpf, the paternal levels of the two epigenetic markers in individual zygotes were particularly closely associated; the zygotes with low 5-MeC levels also had low H3-m3K9 levels (ranging from 30–50%, see the lower bracket in Fig. 3C, 25 h), whereas the zygotes with high 5-MeC levels also had high H3-m3K9 levels (80–100%, upper bracket in Fig. 3C, 25 h). The undermethylated group, which amounted to 39% (9/23) of the 25 hpf zygotes, was likely to comprise zygotes that had slower growth because they had similar DNA methylation and H3-m3K9 states as the 20 hpf eggs. If analysis was delayed until 28 hpf, when half of the eggs had either undergone cleavage to the two-cell stage (10/26) or were at mitosis phase (3/26), the paternal DNA methylation level in the rest of the zygotes still having pronucleus increased to a level similar to that of a female (11/26). Therefore, the heavy methylation group in the upper bracket (Fig. 3C, 25 h) could be considered to consist of normal zygotes that develop at the correct time. These observations further demonstrate that bovine zygotes undergo DNA methylation after active demethylation in the male pronucleus before the two-cell stage.
Treatment of Bovine Zygotes With 5-Azadeoxycytidine Brings the Male Pronucleus to a Complete, Not a Partial, Demethylation State
To independently assess the two seemingly consecutive events of active demethylation and de novo methylation, we treated bovine eggs with 5-azadeoxycytidine (5-azadC), a potent inhibitor of DNA methyltransferase I (DNMT1) and, possibly, of DNMT3A and DNMT3B (Christman,2002). The 5-azadC treatment is believed to inhibit maintenance and de novo methylation processes, but not demethylation events. As shown in Figure 4A, 5-MeC signals completely disappeared from the male pronucleus of all zygotes examined (n = 38). If the paternal pronucleus had been “partially” demethylated, a residual amount of 5-MeCs should have been detected after 5-azadC treatment. In the chromosomes of metaphase II-arrested oocyte (Fig. 4 Ba,e) and terminally differentiated cumulus cells (b and f) adhering to the zona pellucida, there were no DNA methylation differences between control and 5-azadC–treated ones. To assess the degree of demethylation, presumably owing to the failure of maintenance DNA methylation, we chemically activated mature oocytes and treated the parthenogenetic eggs with 5-azadC. As shown in Figure 4 Bc,g, DNA methylation signals were reduced in the 5-azadC–treated parthenogenotes (n = 42) compared with the control, while to much lesser extent than in the male pronuclei that were subjected to active demethylation additionally (Fig. 4 Ad–f). Meanwhile, when pig zygotes, which were previously shown to maintain paternal DNA methylation states without active demethylation during the pronucleus stage (Jeong et al.,2007), were treated with 5-azadC, the pig paternal pronuclei did not end in a completely demethylated state (Fig. 4 Bd,h), unlike the bovine male pronuclei. These control experiments demonstrated that the 5-azadC–induced loss of 5-MeCs in the bovine male pronucleus is due to the active demethylation event. Hence, the results of the 5-azadC experiment indicate that the active demethylation occurs throughout the male genome. However, this is not apparent from visual assessment, because the completely demethylated state of the male pronucleus is disguised by the ensuing de novo methylation event. Therefore, the seeming “partial” demethylation observed in the male chromatins is most likely to be due to a combination of complete DNA demethylation and subsequent de novo DNA methylation processes, which overlap so that the latter may start before the former finishes (see also Fig. 7).
Rabbit Zygotes Undergo Similar DNA Demethylation and Remethylation Events During Pronucleus Development
Additional evidence for the consecutive DNA demethylation and remethylation events came from analysis of in vivo-derived rabbit zygotes. Previous studies, which had studied methylation of rabbit zygotes at the late pronucleus stage (24 hpf), did not report demethylation in the paternal pronucleus (Beaujean et al.,2004a; Shi et al.,2004). In the present study, we analyzed methylation status 6 hr earlier, at 18 hpf, in anticipation of a demethylation event. As shown in Figure 5A, the rabbit male pronucleus displayed a sign of demethylation, as the 5-MeC level varied among individual zygotes (61.5% ± 17.5, n = 12; Fig. 5B), as reported for bovine zygotes (Fig. 3A). Combining our 18 hpf result with the previously reported 24 hpf results (Shi et al.,2004), it could be assumed that the rabbit paternal pronucleus undergoes global demethylation first followed by remethylation before the first mitosis, as seen for bovine zygotes. It is likely that such a marked change in the paternal 5-MeC level is not restricted to the bovine zygote but is also used by other mammalian species. In addition, our observations might provide an explanation for some of the conflicting results frequently observed in methylation reprogramming analyses in the same species (Dean et al.,2001; Beaujean et al.,2004a).
Figure 6A summarizes the modes of DMR that occur in the male pronuclei, together with H3-m3K9 installment (HMI), in various mammalian species. The type-I species, including rodents, undergo active DNA demethylation without global H3-m3K9 installment in the male pronucleus. The type-II species, as we recently found with pig zygotes (Jeong et al.,2007), maintain the paternal DNA methylation level but the level of H3-m3K9 progressively increases. In addition to these two opposite DMR patterns, we have identified a third type of DMR. The bovine male genome undergoes global demethylation followed by de novo methylation within a short time period during the pronucleus stage. This marked change in DNA methylation is distinct from the other two types, and can thus be regarded as an additional DMR model. However, the evolutionary significance of the diverse patterns of mammalian methylation reprogramming and the developmental consequence of species-specific reprogramming processes are currently unknown.
A common feature of the three types of species-specific DMR combined with HMI is that the paternal levels of 5-MeC and H3-m3K9 become associated before advancing to the two-cell stage (Fig. 6A, see also Fig. 1). This finding leads to the speculation that, in modulating the sperm-derived chromatins epigenetically, there is an intimate functional communication between H3-K9 and DNA methylating machineries, as seen with other biological systems (Tamaru and Selker,2001; Jackson et al.,2002; Fuks et al.,2003a,b; Tamaru et al.,2003; Geiman et al.,2004). Supporting this finding, our recent comparative study on DMRs in pig and mouse zygotes demonstrated that, of various histone–lysine methylations such as H3-K4, H3-K9, H3-K27, H3-K36, and H4-K20, only H3-K9 methylation is closely associated with DNA methylation (Jeong et al.,2007). If this finding is also the case for other mammalian zygotes, it raises the question of whether H3-K9 methylation or DNA methylation is the dominant process, holding a high rank. The DMR pattern acquired from the 5-azadC result (Fig. 4A) provided some clues for this; we therefore separately considered it as the type “IIIz” (Fig. 6A). As shown in Figure 6B, there are six possible crosstalk combinations for the epigenetic events of active DNA demethylation, de novo DNA methylation, and de novo H3-K9 methylation that occur in the male pronucleus. However, analysis of the DMR+HMI types excluded all but one possible interaction. According to the type I, it is unlikely that active demethylation itself induces de novo DNA methylation (Fig. 6 Ba). Owing to the sequence of events in type III, where de novo DNA methylation follows active demethylation, the former would not be able to trigger the latter (Fig. 6 Bb). In type II, there is a gradual increase in H3-K9 methylation in the absence of active DNA demethylation, so it is unlikely that de novo H3-K9 methylation could trigger active demethylation (Fig. 6 Bc). The type I pattern shows that active demethylation is independent of the de novo H3-K9 methylation event (Fig. 6 Bd). Therefore, active DNA demethylation seems to take place independently of both de novo DNA methylation and de novo H3-K9 methylation. Based on the type III and IIIz results, the possibility that de novo DNA methylation triggers de novo H3-K9 methylation can be excluded, because H3-m3K9 marks are detected even when de novo DNA methylation is inhibited by 5-azadC treatment (Fig. 6 Be). The final possible mechanism is that de novo H3-K9 methylation directs de novo DNA methylation (Fig. 6 Bf), which is supported by the type III pattern and does not seem to conflict with results for any type of DMR+HMI. In agreement with this, a recent report showed that the male pronucleus derived from micro-insemination using mouse immature, round spermatids undergoes an initial DNA demethylation event followed by remethylation (Kishigami et al.,2006). Interestingly, the authors found that the pronucleus of spermatid origin had a certain level of the H3-m3K9 signal that is normally absent from spermatozoa chromosomes, which suggests H3-m3K9-directed de novo DNA methylation. On the basis of these results, we propose that the presence of paternal H3-m3K9 marks, regardless of whether they are from a de novo process, determine the type of DMR, particularly de novo DNA methylation, in mammalian species. This hypothesis assumes that the DNA methylation asymmetry between the parental pronuclei in mouse zygotes is mainly due to the absence of a de novo H3-K9 methylation event, which leads to a lack of de novo DNA methylation. However, the possibility cannot be excluded that the dependence of de novo DNA methylation on H3-K9 methylation is restricted to either certain species or the pronuclear stage.
Regarding the underlying molecular mechanism, it is unknown how the opposing activities, DNA demethylation and de novo methylation, simultaneously occupy the same space in the bovine male pronucleus, and how newly methylated regions are protected from repeated demethylation. We surmise that structural features of the recently remodeled male chromatins shortly after fertilization are favored by a putative DNA demethylating activity. As the male chromatins become heavily modified, or “mature,” including by H3-K9 trimethylation, it is likely that the male chromatins become resistant to DNA demethylation, probably by imposing restrictions on components of the demethylation machinery. The H3-m3K9-braced repressive chromatins may then form a structure with which de novo DNA methylation machinery can interact. This hypothesis is in agreement with the above proposal that de novo H3-K9 methylation directs de novo DNA methylation. We propose that the sequence of epigenetic events in bovine male pronucleus involves a global demethylation of sperm DNA, gradual H3-K9 methylation, and finally de novo DNA methylation event, as schematically represented in Figure 7. Meanwhile, it was reported that the pronuclei appeared 7–8 hr after the exposure of oocyte to spermatozoa, and the first S phase started around 13–15 hr in bovine zygotes (Comizzoli et al.,2000). Bourc′his et al. observed no methylation difference between parental pronuclei around 8 hpf and a marked reduction of paternal DNA methylation level in the bovine zygotes at 14 hpf, which well concurs with our observation as designated in Figure 7; as for the later-stage zygotes, they primarily examined metaphase chromosome spreads, which is unsuitable to be directly compared with our own results with interphase nuclei (Bourc′his et al.,2001).
Unlike other mammalian zygotes, as we elucidated in this study, the bovine zygote is highlighted by the occurrences of several complex epigenetic processes such as active DNA demethylation, de novo DNA methylation, and de novo H3-K9 trimethylation. Furthermore, these complicated epigenetic processes should be completed at the pronucleus stage before the zygote advances to the two-cell stage. This is likely to explain the difficulty in obtaining bovine clones from somatic cell nuclear transfer (SCNT). The differentiation-associated epigenetic traces should be cleared from a donor cell genome of SCNT oocytes in compliance with the ordered reprogramming processes. However, considering the innate complexity of the epigenetic (re-)programming processes, faults in precise recapitulations of these individual processes in bovine SCNT oocytes may cause particular problems. Although our previous studies showed that DNA demethylation occurs in single-copy sequences in bovine SCNT embryos during cleavage stages (Bourc′his et al.,2001; Kang et al.,2002,2003b), the initial failure to properly reset the donor epigenome during the dynamic pronucleus stage could lead to more developmental defects in bovine clones than in other species.
A monoclonal anti-5-MeC antibody was purchased from Eurogentech (MMS-900S-B). Antibodies that specifically recognize either trimethylation or acetylation at lysine 9 residue of histone H3 were purchased from Upstate biotechnologies. Chicken anti-mouse or anti-rabbit IgG secondary antibodies (Alexa Fluor 488/594–conjugated form, Molecular Probes) were used to detect the individual primary antibodies.
Collection of Mammalian Oocytes and Zygotes
Unless otherwise mentioned, all chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). Mouse fertilized oocytes and embryos were collected from superovulated females on appropriate time post–human chorionic gonadotropin (hCG) injection according to standard procedure (Hogan,1994). The 4- to 6-week-old female BCF1(C57BL/6 X CBA/CA) mice were superovulated by intraperitoneal injections of 5 IU pregnant mares serum gonadotropin (PMSG), followed by injection of 5 IU hCG 48 hr apart. The females were mated with 8-week-old males of the same strain. In 20 hr post-hCG injection, fertilized oocytes were collected from oviducts of the females and the cumulus cells surrounded embryo were removed by treatment with 1 mg/ml hyaluronidase and cultured in M16 medium at 37°C, 5% CO2 in air.
Goat fertilized oocytes were collected from superovulated female 2- to 3-year-old pluriparous Korean native goats (Lee et al.,2000). The estrous cycle of female goats was synchronized by ear implantation of 6 mg of norgestomet implants (Synchromate-B, Rhone Merieux, Inc., Athens, GA) for 14 days. Superovulation was induced by a combined injection of follicle-stimulating hormone (FSH) and hCG. A total of 5.6 mg of FSH (Ovagen, Immuno-Chemical Products, New Zealand) was given to goats over a 4-day period as injection, twice daily, starting 2.5 days before implant removal and continuing to 1 day after removal. A single injection of hCG (100 IU) was given at the time of last FSH injection for the induction of ovulation. Within 24 hr after implant removal, the female goats were mated with male goats. Sixty-eight hours after implant removal, oviducts of female goats were surgically recovered and flushed in retrograde manner with sterile phosphate buffered saline (PBS) as previously reported (Lee et al.,1997).
Rat fertilized oocytes were collected from superovulated females on appropriate time post-hCG (Kato et al.,2004). Briefly, Wistar female rats were superovulated by intraperitoneal injections of 150 IU/kg equine chorionic gonadotrophin (eCG) and 75 IU/kg hCG after an interval of 48 hr, as described previously (Miyoshi et al.,1997). The rats were mated with Wistar male rats. Mating was confirmed by the presence of vaginal plugs. At 24 hours after hCG injection, rat zygotes were recovered from oviductal ampullae of the rats with mR1ECM/bovine serum albumin (BSA; 100 mM NaCl, 3.2 mM KCl, 2.0 mM CaCl2, 0.5 mM sodium pyruvate, 7.5 mM glucose, 2 %(v/v) Minimal Essential Medium (MEM) essential amino acid solution (Gibco), 0.1 mM glutamine, 1% (v/v) MEM nonessential amino acid solution (Gibco) and 0.4% fatty acid free BSA) containing 0.1% hyaluronidase.
Rabbit fertilized oocytes were collected from the oviducts of female New Zealand White rabbits after mating twice and an injection of 100 IU eCG intramuscularly and 100 IU hCG intravenously 72 hr later. Rabbit zygotes were flushed from the oviducts 16 hr post-hCG injection in warm PBS supplemented with 4 mg/ml BSA and incubated in M199 medium containing 5 μg/ml hyaluronidase for 15 min at 38°C. The fertilized oocytes transferred into 50-μl drops of MPM medium supplemented with 10% (v/v) fetal calf serum (Gibco BRL), covered with mineral oil, and cultured at 39°C in a humidified atmosphere of 5% CO2 in air.
Pig ovaries were collected from gilts of Landrace and Large White at a local abattoir. The cumulus-oocyte complex were obtained from follicles and cultured in maturation medium with 10 IU/ml hCG and 10 IU/ml PMSG for 22 hr and then without hCG and PMSG for 22 hr at 38°C in a humidified atmosphere of 5% CO2 in air (Koo et al.,2004). Matured oocytes were in vitro fertilized with pig semen supplied by the Darby Pig Artificial Insermination Center (Anseong, Korea). Fertilized oocytes were cultured in culture medium at 38°C in humidified atmosphere of 5% CO2 in air. In vitro maturation medium was BSA-free North Carolina State University (NCSU) 23 medium supplemented with 10% (v/v) porcine follicular fluid, 0.57 mM cystein, 25 μg/ml gentamycin, 10 ng/ml EGF, 10 IU/ml PMSG, and 10 IU/ml hCG. Medium for in vitro fertilization, designated modified Tris-buffered medium (mTBM), consisted of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2-2H2O, 20 mM Tris (crystallized-free base; Fisher Scientific), 11 mM glucose, 5 mM sodium pyruvate, and no antibiotics. Medium for in vitro culture was NCSU23 supplemented with 4 mg/ml BSA (fatty acid free).
Immunostaining Early Embryos and Quantitative Analysis of Fluorescent Intensity
Immunostaining procedure appeared elsewhere (Beaujean et al.,2004b). Briefly, embryos were fixed in 4% formaldehyde in PBS (Gibco) for 30 min at 4°C, followed by three 20 min washes in 0.02% Tween 20 (Fisher Scientific) in PBS and permeabilized by 0.25% Triton X-100 (MP Biomedicals) in PBS for 1 hr at room temperature. The embryos were treated with 4 N HCl (Sigma) for 30 min at room temperature, neutralized with 0.1 M Tris-HCl (pH 8.0) and blocked for 1 hr at room temperature in 2% BSA (Sigma), 0.02% Tween 20 in PBS. Primary antibody incubations (diluted by 1:50–500) were carried out in the blocking solution for 1 hr at 37°C, followed by several washes in 0.02% Tween 20 in PBS. Secondary antibodies (diluted to 1:300–500) were incubated for 30 min at room temperature, followed by several washes. Embryos were mounted on Poly-prep slides (Sigma) and, after dry at room temperature, observed with Karl Zeiss Axiovert 200M fluorescence microscope equipped with ApoTome. Most staining experiments were repeated at least three times each with 20 or more embryos. Images were captured digitally using different filter sets and merged using Axiovision (v4.5) or Adobe Photoshop software (v7.0).
For a quantitative analysis, we used the ImageJ program (v1.35s) to measure intensity of optically sectioned pronucleus area, but we also noticed that other programs, including Tina20 (v2.09), yielded similar results. Only the samples with both parental pronuclei placed on the same focal plane in oocyte cytoplasm were considered to be quantified. Percentage methylation described in this study indicates a methylation level of male pronucleus relative to that of female pronucleus.
In Vitro Procedures for Production of Bovine Fertilized Eggs and Parthenogenetically Activated Oocytes
Bovine ovaries were collected from a local slaughter house. Cumulus-oocyte complexes were obtained from follicles and incubated in in vitro maturation medium (Wee et al.,2006) under paraffin oil for 20 hr at 38.5°C in an atmosphere of 5% CO2. The medium for oocytes maturation was TCM-199 (Invitrogen, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen), 10 μg/ml FSH-P (Folltropin-V, Vetrepharm, London, UK), 0.6 mM cysteine, 0.2 mM sodium pyruvate, and 1 μg/ml estradiol-17β. In vitro-matured oocytes were fertilized with frozen–thawed sperm at a concentration of 2 × 106/ml in fertilization medium (Koo et al.,2002). Parthenogenesis was induced with in vitro matured oocytes by chemical activation (Cibelli et al.,1998). Briefly, in vitro-matured oocytes were activated with 5 μM calcium ionophore for 5 min and 2.5 mM 6-dimethylaminopurine (DMAP, Sigma) in CR1aa supplemented with 10% FBS for 4 hr at 38.5°C in an atmosphere of 5% CO2.
5-azadC Treatment of Bovine Zygotes
For treatment of fertilized bovine oocytes with 5-azadC (Sigma Aldrich), the oocytes–spermatozoa complexes were transferred to IVF medium containing 5 μM 5-azadC 5 hr after the commencement of in vitro fertilization, and cultured for another 20 hr. The fertilized embryos were fixed for immunostaining in 4% formaldehyde solution for 30 min at room temperature.
- 2004a. Non-conservation of mammalian preimplantation methylation dynamics. Curr Biol 14: R266–R267. , , , , , , , .
- 2004b. The effect of interspecific oocytes on demethylation of sperm DNA. Proc Natl Acad Sci U S A 101: 7636–7640. , , , , , , , , , , , .
- 2002. DNA methylation patterns and epigenetic memory. Genes Dev 16: 6–21. .
- 1999. Methylation-induced repression—belts, braces, and chromatin. Cell 99: 451–454. , .
- 2001. Delayed and incomplete reprogramming of chromosome methylation patterns in bovine cloned embryos. Curr Biol 11: 1542–1546. , , , , , , .
- 2002. 5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 21: 5483–5495. .
- 1998. Cloned transgenic calves produced from nonquiescent fetal fibroblasts [see comments]. Science 280: 1256–1258. , , , , , , , .
- 2000. Onset of the first S-phase is determined by a paternal effect during the G1-phase in bovine zygotes. Biol Reprod 62: 1677–1684. , , , .
- 2001. Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos. Proc Natl Acad Sci U S A 98: 13734–13738. , , , , , , .
- 2003a. The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase. Nucleic Acids Res 31: 2305–2312. , , , .
- 2003b. The methyl-CpG-binding protein MeCP2 links DNA methylation to histone methylation. J Biol Chem 278: 4035–4040. , , , , , .
- 2004. DNMT3B interacts with hSNF2H chromatin remodeling enzyme, HDACs 1 and 2, and components of the histone methylation system. Biochem Biophys Res Commun 318: 544–555. , , , , , .
- 2003. Analysis of DNA (cytosine 5) methyltransferase mRNA sequence and expression in bovine preimplantation embryos, fetal and adult tissues. Gene Expr Patterns 3: 551–558. , .
- 2004. Recent advances in X-chromosome inactivation. Curr Opin Cell Biol 16: 247–255. .
- 1994. Manipulating the mouse embryo: a laboratory manual. Plainview, NY: Cold Spring Harbor Laboratory. 149 p .
- 2002. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416: 556–560. , , , .
- 2003. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33(Suppl): 245–254. , .
- 2007. Gradual development of a genome-wide H3–K9 trimethylation pattern in paternally derived pig pronucleus. Dev Dyn 236: 1509–1516. , , , , .
- 2002. Limited demethylation leaves mosaic-type methylation states in cloned bovine pre-implantation embryos. EMBO J 21: 1092–1100. , , , , , , .
- 2003a. Reprogramming DNA methylation in the preimplantation stage: peeping with Dolly's eyes. Curr Opin Cell Biol 15: 290–295. , , .
- 2003b. Precise recapitulation of methylation change in early cloned embryos. Mol Reprod Dev 66: 32–37. , , , , , , , , , .
- 2004. Donor and recipient rat strains affect full-term development of one-cell zygotes cultured to morulae/blastocysts. J Reprod Dev 50: 191–195. , , , .
- 2006. Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids. Dev Biol 289: 195–205. , , , , , , .
- 2002. Aberrant allocations of inner cell mass and trophectoderm cells in bovine nuclear transfer blastocysts. Biol Reprod 67: 487–492. , , , , , , , , , .
- 2004. A paucity of structural integrity in cloned porcine blastocysts produced in vitro. Theriogenology 62: 779–789. , , , , , , .
- 1997. In vitro development of DNA-injected embryos co-cultured with goat oviduct epithelial cells in Korean native goats (Capra hircus aegagrus). Theriogenology 47: 1115–1123. , , , , , .
- 2000. Embryo recovery and transfer for the production of transgenic goats from Korean native strain, Capra hircus aegagrus. Small Rumin Res 37: 57–63. , , , , , , , , , , , , , , , , , .
- 2002. Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet 3: 662–673. .
- 1992. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69: 915–926. , , .
- 2000. Demethylation of the zygotic paternal genome. Nature 403: 501–502. , , , , .
- 1997. Stage-dependent development of rat 1-cell embryos in a chemically defined medium after fertilization in vivo and in vitro. Biol Reprod 56: 180–185. , , .
- 1987. Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99: 371–382. , , .
- 2005. Epigenetic reprogramming in mammals. Hum Mol Genet 14(Spec No 1): R47–R58. , , , , .
- 1999. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99: 247–257. , , , .
- 2000. Active demethylation of the paternal genome in the mouse zygote. Curr Biol 10: 475–478. , , , , , , , , .
- 1995. DNA methylation in early development. Hum Mol Genet 4: 1751–1755. , .
- 2001. Genomic imprinting: parental influence on the genome. Nat Rev Genet 2: 21–32. , .
- 2001. Epigenetic reprogramming in mammalian development. Science 293: 1089–1093. , , .
- 2001. Nuclear cloning and epigenetic reprogramming of the genome. Science 293: 1093–1098. , , .
- 1998. Chromosome methylation patterns during mammalian preimplantation development. Genes Dev 12: 2108–2113. , , , , , , .
- 1984. Methylation patterns of repetitive DNA sequences in germ cells of Mus musculus. Nucleic Acids Res 12: 2823–2836. , , , , .
- 2002. Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev Biol 241: 172–182. , , , .
- 2004. Methylation reprogramming and chromosomal aneuploidy in in vivo fertilized and cloned rabbit preimplantation embryos. Biol Reprod 71: 340–347. , , , , .
- 2001. Reprogramming of genome function through epigenetic inheritance. Nature 414: 122–128. .
- 2001. A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature 414: 277–283. , .
- 2003. Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa. Nat Genet 34: 75–79. , , , , , , , , .
- 2006. Inheritable histone H4 acetylation of somatic chromatins in cloned embryos. J Biol Chem 281: 6048–6057. , , , , , , , , .
- 1999. Epigenetics: regulation through repression. Science 286: 481–486. , .
- 1997. Cytosine methylation and the ecology of intragenomic parasites. Trends Genet 13: 335–340. , , .
- 2004. DNA methylation in the preimplantation embryo: the differing stories of the mouse and sheep. Anim Reprod Sci 82–83: 61–78. , .