Mol. Hum. Reprod. Advance Access originally published online on February 4, 2005
Molecular Human Reproduction 2005 11(3):167-171; doi:10.1093/molehr/gah145
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DNA methylation patterns in human tripronucleate zygotes
1Program for In Vitro Fertilization, Reproductive Surgery and Infertility, New York University School of Medicine, New York, NY10016, USA and 2Current address: Reproductive Medical Research Center, The 1st Affiliated Hospital of Sun Yat-sen University, Guangzhou, China 510080
3 To whom correspondence should be addressed at: 660 First Avenue 5th Floor, New York, NY 10016, USA. Email: kreyivf{at}yahoo.com
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Abstract |
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In mammals, the dynamic reprogramming of DNA methylation begins during gametogenesis and continues through embryogenesis. Recently, immunofluorescence staining with an antibody against 5-methylcytosine (anti-5-MeC) has revealed active demethylation of the male pronucleus in zygotes beginning at 4–6 h after fertilization. In this study, we characterized the DNA methylation patterns in mouse zygotes and in human tripronucleate (3 PN) zygotes discarded after conventional fertilization or following ICSI. Pronuclei were subjected to fluorescence in-situ hybridization to identify the X and/or Y chromosomes and then stained with anti-5-MeC. In diandric 3 PN zygotes from conventional IVF, we consistently observed one strongly and two weakly stained pronuclei. In contrast, the majority of 3 PN ICSI zygotes, mainly digynic zygotes, displayed two strongly and one weakly stained pronuclei. Two zygotes from ICSI failed to show any staining difference among the three pronuclei. Our results indicate that the active demethylation of male pronuclei occurs in both mouse and human zygotes. It is possible that the abnormal methylation patterns resulting from a dysfunctional cytoplasm may occur in a small number of oocytes and may affect embryonic viability.
Key words: DNA methylation immunostaining/FISH/tripronucleate zygotes
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Introduction |
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DNA methylation of CpG dinucleotides is known to have profound influences on both chromatin structure and gene expression. DNA methylation is essential for regulating and maintaining the active and repressed states of two parental alleles of imprinted genes (Li et al., 1992
; Surani, 1998; Paulsen and Ferguson-Smith, 2001). DNA methylation may also play a role in regulating the expression of tissue-specific genes, as well as inactivating the X chromosome in female mammals (Li et al., 1992; Li, 1999). Recent studies showed that DNA methylation forms a host defence system to control the threats from parasitic sequence elements and maintain genomic stability (Yoder et al., 1997). There is growing evidence relating to DNA methylation with histone methylation, which suggests that DNA methylation is one of the epigenetic codes influencing heterochromatin remodelling (Bannister et al., 2001; Nielsen et al., 2001; Richards and Elgin, 2002).
Dynamic reprogramming of methylation events in mammals begins during gametogenesis and continues through embryogenesis (Kafri et al., 1992). The possible role of post-zygote demethylation is likely to erase the epigenetic modifications acquired during gametogeneisis and prepare for the acquisition of the embryos' own epigenetic modifications. Recent studies using immunofluorescence staining with an antibody against 5-methylcytosine (anti-5-MeC) showed that this process of demethylation includes active demethylation of the male pronucleus in the 1-cell stage and passive demethylation in each subsequent replication after the 2-cell stage. After fertilization, chromosomes in the male pronucleus are actively demethylated within 4–6 h following sperm head decondensation (Mayer et al., 2000; Santos et al., 2002), while those in the maternal pronucleus remain methylated throughout early embryonic development (Rougier et al., 1998). The reason for the selective demethylation of the male pronucleus remains unclear.
Although immunofluorescence staining with the anti-5-MeC has been successfully applied to investigate dynamic methylation changes in early mouse and bovine embryos (Mayer et al., 2000; Bourc'his et al., 2001; Santos et al., 2002), its application in human embryos is limited (Beaujean et al., 2004; Young and Beaujean, 2004). In this study, we used anti-5-MeC to immunostain human tripronucleate (3 PN) zygotes discarded from IVF cycles. In order to provide clues to deduce the parental origin of these pronuclei, fluorescence in-situ hybridization (FISH) was performed before anti-5-MeC staining to identify the sex chromosome status.
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Materials and methods |
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Collection of oocytes and embryos
Female CB6F-1 mice (6–8 weeks old; Charles River Laboratories Wilmington, MA) were ovulation inducted by injection of pregnant mare's serum gonadotrophin (5 IU; Sigma, St Louis, MO) and 5 IU HCG (Sigma), 48 h later (Liu et al., 2000
). After the HCG injection, the mice were mated with male mice overnight. Mice with vaginal plugs were sacrificed the next morning; zygotes were harvested from ampullae of the oviducts. Embryos were cultured in G2.1 culture medium (IVF Science, Vitrolife AB, Gothenberg, Sweden) either briefly or for 24 h prior to fixation. The studies were approved in Protocol A-3517 by the Division of Laboratory Animal Research at NYU School of Medicine.
From June to August 2002, 30 human 3 PN zygotes were obtained from patients undergoing conventional IVF (n=19) or ICSI (n=11). These abnormally fertilized zygotes were obtained 18–20 h after fertilization with the patients' consent according to protocol H 6902 approved by the Institutional Board of Research Associates at NYU Medical Center. Mean patient age was 35±5 years and their rate for normal (2 PN) fertilization averaged 60%. At retrieval, the oocytes were cultured in microdrops of human tubal fluid (HTF) media (Irvine Scientific, Santa Ana, CA) supplemented with 6% Plasmanate and exposed to sperm overnight; ICSI was performed on the afternoon of oocyte retrieval. The 3 PN zygotes were kept in HTF-Plasmanate (Bayer, Elkhart, IN) media after checking for fertilization at 16–18 h. Cumulus-stripped, and immature GV oocytes at ICSI were also obtained with patient consent and cultured overnight in HTF-Plasmanate media to metaphase II stage before sequential incubation with the Ca++ ionophore A23187
[GenBank]
, followed by cycloheximide and cytocholasin B to initiate pronucleus formation and to prevent extrusion of the second polar body (Liu et al., 2000).
Embryo fixation
After zona pellucida removal by acidified Tyrode's solution, mouse embryos were placed in hypotonic solution (1% sodium citrate) for 2–5 min, then transferred to clean slides and fixed with several drops of a freshly prepared mixture of methanol and acetic acid (3:1). Because of an excess of ooplasm and the fragility of the pronuclear membrane, human 3 PN zygotes were fixed with a Tween-20+HCl method, which protects nuclear integrity better than air-drying techniques (Harper et al., 1994).
FISH
Slides with fixed 3 PN were dehydrated in ethanol (70, 85 and 100%) and 10 µl of an X and Y chromosome probe (X chromosome green, Y chromosome red) was added to each sample according to the manufacturer's protocol (Cytocell Technologies Ltd, Cambridge, UK). After covering with a glass slide and sealing with rubber cement, the pronuclei were denatured with probes on a hotplate at 75 °C for 2 min. Hybridization was completed at 1 h in a humidified chamber at 37 °C. The slides were washed in .4x saline sodium citrate (SSC) at 72 °C for 1.5 min and 2xSSC/0.05% Tween-20 (Sigma) at room temperature for 30 s, respectively, and 10 µl 4',6-diamidino-2-phenylindole (DAPI) (10 µl; Vysis, Downers Grove, IL) was added before observation. After FISH, the slides were washed in phosphate-buffered saline (PBS) (3x5 min) before immunofluorescence staining.
Immunofluorescence staining
Immunofluorescence staining was performed as described previously (Barton et al., 2001). Briefly, mouse or human slides were denatured in 70% formamide (Sigma), 2xSSC at 80 °C for 1 min and then incubated with blocking solution (3% bovine serum albumin, 0.1% Tween-20, 4xSSC) in a Coplin jar for 30 min. Anti-5-MeC antibody (hybridoma supernatant, Eurogentec, Ougree, Belgium), diluted 1:100 with PBT, was added to samples and after incubating in a humidified chamber at 37 °C for 2 h, slides were then washed in PBS for 3x10 min before incubation with fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin G (Molecular Probes, Eugene, OR) for 30 min.
In an additional study, 2-cell mouse embryos were exposed to 100 µM 5.6romo-2'-deoxy uridine (Brdu, Sigma) according to published procedures (Santos et al., 2002). Metaphase chromosome spreads were prepared and doubly stained with anti-5-MeC as above and, after the blocking step, with an Alexafluor-conjugated antibody to BrdU (anti-BrdU; Molecular Probes).
Image observation
The slides were examined under an Olympus BX 70 epifluorescence microscope fitted with a single band pass filter for FITC Texas Red and Aquablue and a triple-band filter for DAPI/Texas Red/FITC. Anti-5-MeC staining was viewed using the FITC filter. Digital images were captured by Sony camera DKC-5000 and saved for subsequent analysis.
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Methylation staining of mouse embryos
Pronuclei of paternal origin were actively demethylated in mouse zygotes. One pronucleus in normally fertilized mouse zygotes and two pronuclei in 3 PN mouse zygotes were weakly stained (Figure 1). Detailed staining of the chromosomes in metaphase spreads revealed a similar pattern (Figure 1C). In additional studies, we exposed mouse embryos to BrdU prior to preparing metaphase spreads, which were then stained with DAPI and with anti-BrdU and anti-5-MeC. Staining intensity was comparable for all the chromosomes when viewed for DAPI and anti-BrdU staining; in contrast, when viewed for anti-5-MeC staining, approximately half the chromosomes were strongly stained and half weakly stained (Figure 1D–F).
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FISH and methylation staining of human 3 PN zygotes
Because human 3 PN zygotes contain three pronuclei, we used FISH to identify the sex chromosome status of each pronucleus in order to provide clues to deduce its parental origin. Prior FISH staining did not influence anti-5-MeC staining outcome since maternal pronuclei displayed comparably bright fluorescence, whether or not they were initially subjected to FISH. When donated human oocytes were matured and chemically activated prior to anti-5-MeC staining, both pronuclei were strongly stained (Figure 2A and B).
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The main origin of 3 PN zygotes derived from conventional IVF is dispermic fertilization resulting in one maternal and two paternal pronuclei (Sathananthan et al., 1999); possible FISH results include XXX, XXY and XYY. After ICSI digyny is the main origin of 3 PN, since only a single sperm is injected into the ooplasm (Grossmann et al., 1997); possible FISH results include XXX and XXY. If the human male pronucleus undergoes active demethylation as in the mouse, then 3 PN zygotes from conventional IVF would show a pattern with one strongly and two weakly anti-5-MeC-stained pronuclei. In contrast, zygotes from ICSI would show a pattern with two strongly and one weakly stained pronuclei.
All 19 zygotes from conventional IVF displayed the pattern of one strongly and two weakly stained pronuclei (Table I). Of these, 34 pronuclei contained an X chromosome, two had XX chromosomes, 19 had a Y chromosome and two had both X and Y chromosomes. All 19 pronuclei with a Y chromosome and the two pronuclei with X and Y chromosomes (disomy) were obviously of paternal origin and showed weak staining (Figure 2E and F). Half of the 34 pronuclei with X chromosome, likely those of paternal origin, also showed weak staining. The remaining 17 pronuclei with an X chromosome and the two pronuclei with XX chromosomes were strongly stained.
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The methylation status in 3 PN zygotes from ICSI was more complex than that in zygotes from conventional IVF. Eight zygotes showed two strongly and one weakly stained pronuclei (Figure 2C and D). One zygote with two Y chromosome pronuclei and one X chromosome pronucleus, which presumably resulted from the injection of two sperm into a single oocyte, displayed one strongly and two weakly stained pattern just like those of diandric zygotes. However, no obvious differences in staining intensity could be identified in the remaining two zygotes with X/X/Y and XX/X/O despite careful observation.
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Discussion |
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Recent reports of genetic anomalies, such as Beckwith–Wiedemann and Prader–Willi/Angelman syndromes in offspring from assisted reproduction technology procedures have raised concerns about the potential interactions between these procedures and gene imprinting (Cox et al., 2002
; De Rycke et al., 2002; Maher et al., 2003). The molecular aspects of gene imprinting are well described and DNA methylation has been shown to be of primary importance in such epigenetic modifications (Li et al., 1992; Surani, 1998; Paulsen and Ferguson-Smith, 2001). Moreover, changes in DNA methylation patterns during fertilization and early embryogenesis have been characterized by anti-5-MeC staining in many species (Rougier et al., 1998; Mayer et al., 2000; Oswald et al., 2000; Santos et al., 2002; Beaujean et al., 2004; Young and Beaujean, 2004). The anti-5-MeC staining results, in the present study, not only confirm the previous studies that the paternal pronucleus is selectively demethylated in mice, but also show that a similar event appears to take place in human zygotes. By identifying paternal pronuclei as the primary site of demethylation, our study extends the preliminary observations of Beaujean et al. (2004) that only one pronucleus is demethylated in normally fertilized human zygotes. Curiously, a zygote's demethylation staining pattern appears highly variable from species to species; differential pronuclear anti-5-MeC staining is striking in mouse and human zygotes but not at all apparent in rabbit and sheep zygotes (Beaujean et al., 2004; Young and Beaujean, 2004).
Additional studies that characterized the physiologic significance of the different anti-5-MeC staining intensities provide support for the foregoing conclusion regarding differential pronuclear demethylation in mouse and human zygotes. First, we confirmed that replication dependent demethylation occurs during the first mitosis in mouse embryos (Rougier et al., 1998). In preliminary studies, we have also noted that anti-5-MeC stained metaphase chromosomes from discarded, normally fertilized human embryos show a similar pattern. Secondly and more importantly for present considerations, we also incorporated BrdU into 2-cell mouse embryos and stained them with an antibody to BrdU to demonstrate that the observed variations in anti-5-MeC stained chromosomes cannot be attributed to problems in antibody accessibility to DNA. Such findings, like those of Santos et al. (2002), provide strong support for the conclusion that chromosomes that weakly stain with this anti-5-MeC protocol are selectively demethylated.
The intracellular mechanism(s) responsible for the post-fertilization demethylation of paternal DNA remains elusive. Paternal DNA decondenses in the ooplasm to permit protamine–histone exchange, and this decondensation provides a unique opportunity for demethylating enzyme(s) and/or other protein factors to bind to the ‘exposed’ male chromatin. At present the demethylase(s) is unknown; the only candidate is methyl binding domain protein 2 (MBD 2) (Bhattacharya et al., 1999). However, zygotes of MBD 2 null mice fail to produce abnormal methylation patterns, a result that makes it doubtful that MBD 2 alone is responsible for male pronuclear demethylation (Santos et al., 2002). An alternative explanation is that maternal chromatin may be ‘protected’ from demethylation. A recent study reports rapid and preferential recruitment of heterochromatin protein (HP) 1ß onto the maternal genome at 1–5 h post-fertilization concurrent with paternal genome demethylation (Arney et al., 2002). HP 1ß may not only ‘protect’ the maternal genome from demethylases but also associate with DNA methyltransferases, thereby linking DNA methylation to histone methylation (Arney et al., 2002). However, the only detectable methyltransferase (Dnmet) in oocytes and early embryos is Dnmt1o, which acts on hemimethylated DNA to maintain methylation during replication and this methyltransferase only transiently locates in the nucleus in 8-cell stage embryos to maintain methylation of imprinted genes (Doherty et al., 2000; Howell et al., 2001). That Dnmt1o is preferentially located in the ooplasm of zygotes is not consistent with a hypothesis that it maintains the methylation status of the female pronucleus.
Clearly, whatever the factor(s) and mechanism(s) involved in this selective demethylation event, they are present in more than adequate concentrations in the ooplasm, since multiple pronuclei are actively demethylated in polyspermic zygotes (Fig. 3 and Santos et al., 2002). In fact, using ICSI to inject spterm into zona-free oocytes, Santos and co-workers were able to demonstrate this with up to five supernumerary male pronuclei. It is also important to note that the developmental potential of polyspermic zygotes has been demonstrated in mice and humans; successful microsurgical enucleation of the extra paternal pronucleus in 3 PN zygotes has eventuated in normal live births in both of these species (Feng and Gordon, 1996; Kattera and Chen, 2003). Such observations provide further support that 3 PN zygotes behave normally with respect to demethylation.
Selective paternal DNA demethylation may have both immediate and long-term consequences. Active demethylation of the male's pronuclear DNA is consistent with a significantly higher level of transcription of paternal genes and may be essential for the minor activation of zygotic gene expression that occurs during G2 in the 1-cell embryo (Schultz, 1993; Surani, 2001). In addition, because the epigenetic differences between the parental genomes are enhanced, this demethylation pattern may explain why the majority of methylated imprinted genes are maternal (Reik and Walter, 2001).
Significantly, two embryos did not display obvious male pronucleus demethylation. One embryo was X/X/Y and the other was XX/X/O. These observations suggest that a small number of zygotes may experience demethylation problems at fertilization. However, one can only speculate whether this is a relatively rare or common problem until a larger sample size is examined in future studies. However, similar demethylation anomalies have been reported in mice; 2–4% of normally fertilized embryos exhibited no obvious indications that demethylation occurred during the pronuclear stage (Barton et al., 2001). Failure to demethylate the paternal pronucleus may have an adverse impact on an embryo's later development. Failure to produce distinguishable parental-chromosome methylation patterns after nuclear fusion in cloned bovine embryos results in delayed and incomplete reprogramming of chromosome methylation patterns and these epigenetic anomalies are most likely responsible for the low implantation, high intrauterine fetal demise and abnormality rates reported for these embryos (Bourc'his et al., 2001). In addition, although demethylation takes place in the single-copy sequences of embryos generated following nuclear transfer, the satellite sequences in trophectoderm cells retain the methylation pattern of the transferred donor nucleus and this may result in the placental dysfunctions commonly observed following uterine transfer of these embryos (Kang et al., 2001a,b, 2002).
In summary, the present results are consistent with a conclusion that active demethylation of the paternal pronucleus occurs immediately after post-fertilization in both mouse and man. Additional preliminary observations suggest that replication-dependent demethylation of the maternal chromosomes also occurs during each post-fertilization mitosis in human embryos as it does in mouse embryos (data not shown). It is possible that abnormal methylation patterns may result from dysfunctional cytoplasm in some oocytes and may be an important factor that adversely affects embryonic development and viability. Investigation of methylation patterns of normally fertilized human embryos donated for research may provide further insight into the epigenetic modifications that occur during fertilization and early embryogenesis. In view of the recent observations that preimplantation demethylation dynamics vary among mammals, it will also be important to perform similar analyses for other species as well.
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Acknowledgements |
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The authors would like to thank Anna Blaszczyk and Hui Liu for their comments on this paper.
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References |
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Submitted on May 18, 2004; accepted on December 18, 2004.
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