Changes in histone methylation during human oocyte maturation and IVF- or ICSI-derived embryo development

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1 Changes in histone methylation during human oocyte maturation and IVF- or ICSI-derived embryo development Jie Qiao, M.D., a Yuan Chen, Ph.D., a Li-Ying Yan, Ph.D., a Jie Yan, Ph.D., a Ping Liu, Ph.D., a and Qing-Yuan Sun, Ph.D. b a Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, People s Republic of China; and b State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, People s Republic of China Objective: To characterize the histone methylation pattern during human oocyte maturation and embryo development after conventional IVF and ICSI. Design: Experimental study. Setting: Reproductive center of hospital. Patient(s): Women underwent IVF or ICSI. Intervention(s): Immature and mature oocytes were collected from patients undergoing ICSI. Tripronuclear and normally fertilized embryos were obtained from patients undergoing IVF or ICSI. Main Outcome Measure(s): The distribution patterns of dimethylated histone H3 lysine 9 (H3K9) and histone H4 arginine 3 (H4R3) in oocytes and embryos were observed by indirect immunofluorescent staining and scanning confocal microscopy. Result(s): H3K9 and H4R3 were dimethylated throughout the meiotic maturation of human oocytes and the tripronuclear embryo development from two-cell to blastocyst stage. However, at the pronuclear stage, approximately half of the tripronuclear IVF zygotes displayed strong staining of MeH3K9 in one pronucleus, whereas MeH4R3 staining was always uniform in all three pronuclei. In the other half of the tripronuclear zygotes, all three pronuclei were strongly stained with MeH3K9 in some cases, and the remaining zygotes were completely unstained. Moreover, with progressively increasing fragmentation of blastomeres in ICSI-derived 2PN embryos with low morphological grade, H3K9 dimethylation decreased when compared with that of IVF embryos. Conclusion(s): Asymmetric distribution of the dimethylation form of H3K9 exists in human zygote pronuclei. The ICSI-derived embryos with low morphological grade are more likely to display H3K9 demethylation than their IVF counterparts. (Fertil Steril Ò 2010;93: Ó2010 by American Society for Reproductive Medicine.) Key Words: Histone methylation, oocyte, tripronuclear zygote, human embryo In mammals, dynamic reprogramming of epigenetic events begins during gametogenesis and continues through embryogenesis. In addition to DNA methylation, covalent modifications of nucleosomal histone are also involved in the processes of epigenetic reprogramming (1). Of the possible modifications, the presence of particular posttranslational modifications of histone, such as methylation, offers a unique platform for the control of chromatin function (2, 3). During specific stages of development, only specific sets of genes are active. However, oocytes cease gene expression before they are fully grown and remain in a transcriptionally silent state during meiotic maturation (4). After Received August 6, 2008; revised January 18, 2009; accepted March 2, 2009; published online April 25, Supported by Major State Basic Research Development Program of China (No. 2007CB948102). J.Q. has nothing to disclose. Y.C. has nothing to disclose. L-Y.Y. has nothing to disclose. J.Y. has nothing to disclose. P.L. has nothing to disclose. Q-Y.S. has nothing to disclose. Reprint requests: Jie Qiao, M.D., Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, 49 North Garden Rd., Beijing , People s Republic of China (FAX: þ ; jie.qiao@263.net). fertilization, the zygote begins to express genes in an embryo-specific pattern that is dynamically altered during preimplantation development (5). Methylation of specific histone residues and the extent of methylation (monomethylation, dimethylation, and/or trimethylation) on the same residues are critical for regulating chromatin structure and gene transcription. Histone H3K9 trimethylation and dimethylation are correlated to gene silencing, whereas histone H4R3 methylation leads to initiation of gene transcription (6, 7). It has been suggested that dimethylation of H3K9 in the maternal genome has a role in preventing histone demethylation in the female pronucleus during the first cell cycle (8). Recently, histone methylation has been found to be crucial for early embryo development and for maining pluripotency of embryonic stem cells (9). To date, dynamic DNA methylation changes during human oocyte meiosis and embryonic development have only been fragmentally reported, and their results showed that the majority of the paternal genome is less methylated than the maternal genome (10, 11). However, detailed changes of histone methylation in meiosis and human embryo development have not been thoroughly examined Fertility and Sterility â Vol. 93, No. 5, March 15, /10/$36.00 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 Because human zygotes differ from mouse zygotes in the aspect of DNA methylation (10, 12, 13), it would be interesting to ask whether the epigenetic differences are also reflected in histone modification. In addition, the most attractive subject would be the analysis of the histone methylation pattern in developing human embryos. Here, we investigated H3K9 and H4R3 dimethylation states in human oocytes and embryos discarded from IVF/ICSI cycles. MATERIALS AND METHODS Human Embryo and Oocyte Collection From July to December 2007, the tripronuclear zygotes were obtained from patients undergoing assisted reproductive technology (ART). These abnormally fertilized zygotes, which were identified to have three pronuclei in the ooplasm and the second polar body, were obtained hours after fertilization and were cultured in G1.2 culture medium (Vitrolife, Kungsbacka, Sweden). Embryos at different developmental phases were collected and then prepared for the following experiments. Sixty-four surplus human embryos originating from normally fertilized (two pronuclei [2PN]) zygotes were collected from patients undergoing IVF or ICSI. Controlled ovarian stimulation was performed using a GnRH agonist and recombinant human FSH (Gonal-F; Serono Laboratories, Inc., Randolph, MA) in a long or short protocol. In the present study, the majority of patients used the long protocol for their ovarian stimulation. Transvaginal oocyte retrieval was performed hours after administration of HCG (Profasi; Serono) and subsequently fertilized by conventional IVF or ICSI and cultured until the day of transfer (day 3) in G1.2 culture medium (Vitrolife). It must be noted that the embryos used for experiment were arrested or in poor morphology, according to the routine standard criteria, and were graded III IV and unsuitable for transfer and freezing. Embryos were graded on a scale of I IV according to morphology as follows: grade (G) I embryos have even and regular blastomeres, with less than 10% fragmentation; GII embryos have uneven blastomeres, with 10% 30% fragmentation; GIII embryos have uneven blastomeres, with 30% 50% fragmentation; G IV embryos have uneven blastomeres, with greater than 50% fragmentation. A total of 98 superfluous morphologically normal oocytes were recruited from patients undergoing routine ICSI. Briefly, oocytes were aspirated hours after HCG injection and the ICSI procedure was performed 4 6 hours later. Mature oocytes were selected for ICSI. Remaining immature fully grown oocytes, arrested either at germinal vesicle (GV) stage or at metaphase I (MI), were used for experiment. Obviously abnormal-shaped or degenerated immature oocytes were excluded. Some of these immature oocytes were fixed immediately, and others completed maturation within a few hours when cultured in IVM-Medium (Cooper Surgical, Sage, CT) supplemented with a final concentration of 75 miu/ml FSH and LH at 37 C in an atmosphere of 5% CO 2 in air with high humidity. A written informed consent was also obtained from each infertile couple before the use of their donated gametes and embryos. The study was approved by the Institutional Review Board of the Peking University Third Hospital. Reagents All chemicals used in this study were purchased from Sigma- Aldrich Chemical Company (St. Louis, MO) except for those specifically mentioned. Rabbit polyclonal antidimethylated lysine 9 of histone H3 (H3K9me2) antibody and antidimethylated Arg 3 of histone 4 (H4R3me2) antibody were purchased from Upstate Biotechnology (Lake Placid, NY). Immunofluorescence and Confocal Microscopy Embryos were fixed in 4% paraformaldehyde in phosphatebuffered saline (PBS; ph 7.4) for 30 minutes at room temperature. After being permeabilized with 0.5% Triton X-100 at room temperature for 1 hour, the embryos were blocked in 1% bovine serum albumin (BSA)-supplemented PBS for 1 hour and incubated overnight at 4 C in the appropriate primary antibodies diluted in 1% BSA-supplemented PBS. The antibodies and the dilutions used in the study were as follows: H3K9me2, (1:300); H4R3me2, (1:300). After three washes in PBS containing 0.1% Tween 20 and 0.01% Triton X-100 for 5 minutes each, the embryos were labeled with 1:100 fluorescein isothiocyanate conjugate (FITC)-conjugated goat-anti-rabbit IgG for 1 hour at room temperature. Nuclear status of embryos was evaluated by staining with 10 mg/ml propidium iodide for 10 minutes. Finally, the embryos were mounted on glass slides and examined with a Confocal Laser-Scanning Microscope (Zeiss LSM 510 META, Jena, Germany). Immunofluorescent staining of H3K9me2 and H4R3me2 in human oocytes was carried out according to the method described above. Each experiment was repeated three times. The instrument settings were kept constant not only for each replicate but also between different groups. RESULTS Distribution Patterns of H3K9me2 and H4R3me2 in Oocytes Of 98 oocytes evaluated, 12 oocytes at GV stage, 10 oocytes at germinal vesicle breakdown (GVBD) stage, 12 oocytes at MI stage, 15 oocytes at MII stage were collected for detecting H3K9me2 distribution. Alternatively, 12 oocytes at GV stage, 10 oocytes at GVBD stage, 12 oocytes at MI stage, and 15 oocytes at MII stage were collected for H4R3me2 staining. The genome of maternal origin was dimethylated at H3K9 in oocytes from the GV-stage to MII-stage (Fig. 1, A1, A2, B1, B2, C1, C2, D1, D2). The H3K9me2 staining was detected in Fertility and Sterility â 1629

3 FIGURE 1 Changes in H3K9 and H4R3 dimethylation during human oocyte meiotic maturation. Oocytes were immunostained with the anti-h3k9me2 antibody or anti-h4r3me2 antibody. Antibodies were localized with an FITC-conjugated secondary antibody (green). The DNA was stained with propidium iodide (red). The staining patterns of H3K9me2 in GV (A1, A2), GVBD (B1, B2), MI (C1, C2) and MII (D1, D2) oocytes are shown. The staining patterns of H4R3me2 in GV (E1, E2), GVBD (F1, F2), MI (G1, G2) and MII (H1, H2) oocytes are shown. GV ¼ oocytes at the germinal vesicle stage; GVBD ¼ oocytes undergoing germinal vesicle breakdown; MI ¼ oocytes at the metaphase of first meiosis; MII ¼ oocytes at the metaphase of second meiosis; scale bar ¼ 20 mm. Qiao. Histone methylation in oocyte and embryo. Fertil Steril a punctuate distribution closely colocalized with the DNA that is surrounding the nucleolus at the GV stage, and was colocalized with chromosome DNA in other maturational stages. The H4R3 dimethylation pattern was similar to that of H3K9 during human oocyte meiotic maturation. H4R3 also maintained a constant dimethylation state during meiotic cell cycle progression through GV to MII stage (Fig. 1, E1, E2, F1, F2, G1, G2, H1, H2). Distribution Patterns of H3K9me2 and H4R3me2 in 3PN Embryos The main origin of tripronuclear zygotes derived from conventional IVF is dispermic fertilization, resulting in one maternal and two paternal pronuclei (14). Although the paternal pronucleus in the mouse can be easily distinguished from the maternal pronucleus by its larger size, both pronuclei in normal human zygotes are the same size when fertilization is assessed (20 22 hours after insemination) (15). Moreover, in the zygote, the maternal pronucleus has been suggested to locate at the nearest distance from the second polar body (16, 17). To test the specificity of this method, we first labeled 54 one-cell-stage embryos collected approximately hours after insemination. Of 54 zygotes evaluated, 28 (52%) showed the H3K9me2 staining limited to the smaller pronucleus, and the staining was intensely distributed at the nucleolar periphery, whereas the larger pronuclei showed a complete lack of staining. We assumed that the pronuclei with lack of labeling pattern were the paternal pronuclei. In 1630 Qiao et al. Histone methylation in oocyte and embryo Vol. 93, No. 5, March 15, 2010

4 addition, the second polar bodies (maternal chromatin) always showed intensive labeling (Fig. 2, A1, A2, A3).In the remaining 26 zygotes, 19 (35%) with three pronuclei were strongly and homogenously stained (Fig. 2, B1, B2, B3), while the remaining 7 (13%) were completely unstained (Fig. 2, C1, C2, C3). This asymmetric methylation of H3K9 was maintained until syngamy. Surprisingly, when the maternal and paternal pronuclei fused, the methylated H3K9 fluorescence was presented in only half of the chromatin area (Fig. 2, D1, D2, D3). However, the chromatin from both metaphase and interphase stage blastomeres stained positively from 2-cell stage up to hatched blastocyst stage. At the blastocyst stage, the staining of H3K9me2 was homogeneous, without any difference between the inner cell mass and the trophectoderm cells (Fig. 3, A1, A2 F1, F2). As for H4R3me2, its staining was always uniform in both male and female pronuclei in tripronuclear zygotes (Fig. 3, G1, G2). In all of the subsequent developmental stages tested, from two-cell stage to blastocyst stage, chromatin staining was observed in both interphase and metaphase stage blastomeres (Fig. 3, H1, H2 L1, L2). Distribution Patterns of H3K9me2 in 2PN Embryos Derived from IVF and ICSI Obviously, at the blastocyst stage H3K9 methylation patterns were similar between 2PN and 3PN embryos (Fig. 4, H1, H2) derived from IVF. The staining was homogeneous, without any difference between the inner cell mass and the trophectoderm cells, which was in agreement with results in mice (18). Owing to the limited human embryo resource, we only evaluated H3K9 methylation pattern in sixty-four 2PN embryos discarded from clinical practice, in which fragmentation was observed in more than 30% cells. Interestingly, we found that the nuclei in some blastomeres were abnormal, including multinuclei, binuclei, anuclei, and micronuclei, irrespective of the shape of blastomeres. Moreover, of 26 ICSI-derived embryos evaluated, with progressively decreasing regularity of blastomeres and increasing fragmentation of nuclei, 20 (77%) embryos showed decreased H3K9 dimethylation (Fig. 4, A1, A2, A3-D1, D2, D3). However, in 38 IVF-derived embryos, the H3K9me2 staining was constantly colocalized with the DNA (Fig. 4, E1, E2, E3 G1, G2, G3). DISCUSSION Recent studies suggest that IVF and other forms of ART may result in abnormal genomic imprinting, leading to an increased risk of imprinting disorders (19). Histone methylation, which is thought to stabilize chromatin states and regulate gene expressions that are required to maintain cell fate decisions during development, has gained attention as an epigenetic code. This study has investigated the changes in histone methylation patterns during human oocyte maturation and embryo development. Before fertilization, the maternal genome was dimethylated at H3K9 in the germinal vesicles of immature oocytes, the chromosomes of MII-stage oocytes, and in the maternal pronucleus of the zygote, whereas the paternal genome was demethylated. This asymmetric methylation was maintained until syngamy. As for H4R3 methylation, the signals for MeH4R3 persisted in each stage oocytes and were detected continuously at all stages from zygotes to blastocysts. It has been suggested that histone methylation and DNA methylation potentially stabilize epigenetic modifications through critical developmental transitions (20). In general, MeH4R3 modification is thought to facilitate transcription while MeH3K9 and DNA methlyation are hallmarks of the transcriptionally silent state. Sarmento et al. (21) reported that there appeared to be weak or no staining for the MeH4R3 modification in mouse MII oocyte chromatin and pronuclear stage zygotes. Nevertheless, we have detected the signals of MeH4R3 in both human MII oocytes and tripronuclear zygotes. Because there is no transcriptional activity during oocyte maturation (22), the pattern of H4R3 methylation would not be associated with gene expression. Thus, our results may indicate that reprogramming of histone arginine methylation occurs during gametogenesis and this continues through embryogenesis in humans, which may be required for genome-wide alteration of chromatin configuration. In mice, maternal chromosomes undergo H3K9 methylation during oocyte maturation induced by a factor that is still active after fertilization, leaving the paternal pronucleus unmethylated. This epigenetic asymmetry differentiates the parental genomes until the two-cell stage (23). The H3K9 dimethylation patterns in human oocytes and 3PN embryos are similar to those in mice. However, in humans, approximately 50% of pronuclear stage zygotes displayed the same intensity of labeled pronuclei or completely unstained pronuclei in this study. These observations are in agreement with previous results that the process of paternal DNA demethylation was detected in about half of one-cell stage human embryos (10). Moreover, van der Heijden et al. (24) observed pronuclear asymmetry for H3K9me3 and H3K27me3 in tripronuclear human zygotes similar to the patterns of H3K9me2 presented here. In the normal somatic cells, the absence or disruption of H3K9 methylation leads to the chromosome instability and affects chromosomes segregation during mitosis (25). Accordingly, the abnormal H3K9 methylation pattern in maternal or paternal chromosomes may compromise its role in chromosomes segregation. Moreover, the hypomethylation of histone H3K9 in the paternal pronucleus implies that oocyte cytoplasmic histone H3 is demethylated at K9 and/or histone methylase activity in the oocyte is low in fertilized oocytes. The mechanisms responsible for protecting the maternal pronucleus from histone demethylation were not clear until now. Nevertheless, seven embryos displayed undetectable staining in both maternal and paternal PNs simultaneously in this study. A similar demethylation anomaly was observed in a small number of normally fertilized mice embryos (26). In addition, we must realize that Fertility and Sterility â 1631

5 FIGURE 2 Localization of H3K9me2 in tripronuclear zygotes. Fifty-four zygotes were immunostained with the anti- H3K9me2 antibody. Antibody was localized with an FITC-conjugated secondary antibody (green). The DNA was stained with propidium iodide (red). In the merged images, the areas in which H3K9 methylation and DNA were co-localized appear yellow. The typical demethylation pattern in the tripronuclear embryo is shown. Only the smaller (maternal) pronucleus showed intensive labeling with an anti-h3k9me2 antibody, while the larger (paternal) pronuclei were not labeled. The second polar body was also labeled (A1, A2, A3). Arrows indicate the paternal (p) and maternal (m) pronucleus. In 35% of the human zygotes, all pronuclei showed intensive labeling (B1, B2, B3). In 13% of the human zygotes, all pronuclei were not labeled (C1, C2, C3). At one-cell stage, the methylated H3/K9 fluorescence was presented in only half of the chromatin area (D1, D2, D3). Pb ¼ polar body; scale bar ¼ 20 mm. Qiao. Histone methylation in oocyte and embryo. Fertil Steril Qiao et al. Histone methylation in oocyte and embryo Vol. 93, No. 5, March 15, 2010

6 FIGURE 3 Changes in H3K9me2 and H4R3me2 during IVF 3PN embryo development. The 3PN embryos were collected at different developmental phases. In general, day 1 for collecting one-cell stage embryo, day 2 for 2 4 cell stage, day 3 for 8 cell stage, day 4 for morula, day 5 for blastocyst, and days 6 7 for hatched blastocyst. Embryos were immunostained with the antih3k9me2 antibody. Antibody was localized with an FITC-conjugated secondary antibody (green). The DNA was stained with propidium iodide (red). The staining patterns of H3K9me2 in two-cell (A1, A2), four-cell (B1, B2), eight-cell (C1, C2), morula (D1, D2), blastocysts (E1, E2), and hatched embryos (F1, F2) embryos are shown. The staining patterns of H4R3me2 in onecell (G1, G2), two-cell (H1, H2), four-cell (I1, I2), eight-cell (J1, J2), morula (K1, K2), and blastocyst stage (L1, L2) embryos are shown. Scale bar ¼ 20mm. Qiao. Histone methylation in oocyte and embryo. Fertil Steril Fertility and Sterility 1633

7 FIGURE 4 Changes in H3K9me2 during IVF and ICSI-derived 2PN embryo development. Thirty-eight IVF-derived and 26 ICSI-derived embryos were immunostained with the anti-h3k9me2 antibody. Antibody was localized with an FITC-conjugated secondary antibody (green). The DNA was stained with propidium iodide (red). The staining patterns of H3K9me2 in arrested three-cell (A1, A2, A3), four-cell (B1, B2, B3), eight-cell embryos with 30% 50% fragmentation (C1, C2, C3), and embryos with more than 50% fragmentation (D1, D2, D3) derived from ICSI are shown. The staining patterns of H3K9me2 in arrested three-cell (E1, E2, E3), six-cell embryos with 30% 50% fragmentation (F1, F2, F3), and embryos with more than 50% fragmentation (G1, G2, G3) and blastocyst (H1, H2) derived from IVF are shown. Scale bar ¼ 20 mm. Qiao. Histone methylation in oocyte and embryo. Fertil Steril Qiao et al. Histone methylation in oocyte and embryo Vol. 93, No. 5, March 15, 2010

8 embryos used for staining came from patients with different causes of infertility, which may result in this anomaly. Furthermore, our results revealed that methylated H3K9 fluorescence is present in only half of the chromatin area even after the maternal and paternal pronuclei fused. This asymmetric methylation pattern may be due to the different genome origins, suggesting that differential epigenetic reprogramming may occur in the parental genomes, which awaits further experimentation. It has been clearly documented that the aberrant methylation pattern results in further embryonic abnormalities, in the eventual arrest in development, or embryo death (27, 28). Moreover, the inhibition of histone deacetylation during meiosis induces a high frequency of aneuploidy and embryo death (29). In humans, limited information is available on the methylation status of histone during gametogenesis and embryogenesis. As we know, manipulative procedures, such as ICSI or cloning, resulting from damages related to nuclear cytoplasmic interactions, may alter the capability of the oocyte to correctly perform these epigenetic processes, leading to abnormal embryo development (30 32). Therefore, investigation of methylation patterns of normal human oocytes and embryos might provide further insight into the epigenetic modifications that occur during fertilization and early embryogenesis. In our routine practice, selection of embryos for transfer is based mainly on subjective light microscopic morphology analysis, and correlations between morphological structures, such as fragmentation and cleavage stage, with the embryo quality and clinical outcome are well documented (33, 34). Our finding demonstrated that although the shape of blastomeres was regular, the nuclei were fragmented in some embryos. This finding may indicate that a number of these anucleated blastomeres might be cytoplasmic fragments rather than biologically competent blastomeres. Furthermore, H3K9 was demethylated in some low-graded ICSI-derived embryos, even with intact nuclei, when compared with that of IVF-derived embryos. In contrast, recent studies demonstrated no differences in H3K9 methylation pattern among in vivo, in vitro, and ICSI-produced zygotes in mice (18, 26). In addition to the species-specific differences, we cannot exclude the possibility that the 2PN embryos used in this study were arrested or in poor morphology. Accordingly, the methylation patterns in normally developed human embryos warrant further investigation. Taken together, our results show that H3K9 and H4R3 dimethylation was consistent during oocyte maturation and embryo development from two-cell to the blastocyst stage. Nevertheless, their methylation patterns differ at the pronuclear stage. 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