α1,3-galactosyltransferase deficiency in germ-free miniature pigs increases N-glycolylneuraminic acids as the xenoantigenic determinant in pig-human xenotransplantation.

In this study, we examined whether Hanganutziu-Deicher (H-D) antigens are important as an immunogenic non-α1,3-galactose (Gal) epitope in pigs with a disrupted α1,3-galactosyltransferase gene. The targeting efficiency of the AO blood genotype was achieved (2.2%) in pig fibroblast cells. A total of 1800 somatic cell nuclear transfer (SCNT) embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. The α1,3-galactosyltransferase activity in lung, liver, spleen, and testis of heterozygote α1,3-galactosyltransferase gene knockout (GalT-KO) pigs was significantly decreased, whereas brain and heart showed very low decreasing levels of α1,3-galactosyltransferase activity when compared to those of control. Enzyme-linked lectinosorbent assay showed that the heterozygote GalT-KO pig had more sialylα2,6- and sialylα2,3-linked glycan than the control. Furthermore, the heart, liver, and kidney of the heterozygote GalT-KO pig had a higher N-glycolylneuraminic acid (Neu5Gc) content than the control, whereas the lung of the heterozygote GalT-KO pig had Neu5Gc content similar to the control. Collectively, the data strongly indicated that Neu5Gc is a more critical xenoantigen to overcoming the next acute immune rejection in pig to human xenotransplantation.

Resurrection of an alpha-1,3-galactosyltransferase gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts.

Animals with a targeted disruption of genes can be produced by somatic cell nuclear transfer (SCNT). However, difficulties in clonal selection of somatic cells with a targeted mutation often result in heterogeneous nuclear donor cells, including gene-targeted and non-targeted cells, and impose a risk of producing undesired wildtype cloned animals after SCNT. In addition, the efficiency of cloning by SCNT has remained extremely low. Most cloned embryos die in utero, and the few that develop to term show a high incidence of postnatal death and abnormalities. In the present study, resurrection of an alpha-1,3-galactosyltransferase (αGT) gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts was attempted. Three cloned piglets were produced from the first round of SCNT, including one stillborn and two who died immediately after birth due to respiratory distress syndrome and cardiac dysfunction. Among the three piglets, two were confirmed to be αGT gene-targeted. Fibroblasts derived from postmortem ear skin biopsies were used as nuclear donor cells for the second round of SCNT, and a piglet was produced. As expected, PCR and Southern analyses confirmed that the piglet produced from recloning was αGT gene-targeted. Currently, the piglet is fourteen months of age, and no overt health problems have been observed. Results from the present study demonstrate that loss of an invaluable animal, such as a gene-targeted miniature pig, may be rescued by recloning, with assurance of the desired genetic modification.

Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells.

BACKGROUND: Interspecies somatic cell nuclear transfer (iSCNT) has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF) and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. RESULTS: No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB)-iSCNT, and bovine-bovine (BB)-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA) at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM), we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively. CONCLUSION: The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

Prostacyclin stimulates embryonic development via regulation of the cAMP response element-binding protein-cyclo-oxygenase-2 signalling pathway in cattle.

Prostacyclin (PGI(2)) in oviducal fluid is synthesised from arachidonic acid by cyclo-oxygenase (COX) and prostacyclin synthetase and enhances the implantation and live birth potential of mouse embryos. In the present study, we investigated the developmental competence of bovine embryos by examining the effects of the PGI(2) analogue iloprost on blastocyst development, quality and COX-2 expression during IVF and somatic cell nuclear transfer (SCNT). Bovine IVF and SCNT embryos were cultured in CR1-aa medium supplemented with 0.3% bovine serum albumin in either the presence or absence of 1 mum iloprost at 38.5 degrees C and 5% CO(2). After 3 days of culture, cleaved embryos were cultured for 4 days in the same medium supplemented with 10% fetal bovine serum. For both IVF and SCNT embryos, iloprost improved the blastocyst developmental rate and cell numbers. In the presence of iloprost, the proportion of expanded blastocysts was significantly higher among the IVF embryos and fewer apoptotic cell nuclei were observed. Expression of COX-2 mRNA and protein, evaluated using real-time polymerase chain reaction and immunoblotting, respectively, was increased in the presence of iloprost. These results suggest that PGI(2) improves the developmental competence of embryos via regulation of the cAMP response element-binding protein-COX-2 signalling pathway in cattle.

Aberrant expression of developmentally important signaling molecules in cloned porcine extraembryonic tissues.

Early embryonic losses are common in cloned embryos in the current cloning system. However, the reasons for embryonic losses in early developmental stages of cloned embryos remain unclear. To elucidate the cause of early defective development in cloned embryos, two porcine clones including extraembryonic tissues were obtained at 26 days of gestation. The expression of various molecules in developmentally important signaling pathways, including Notch, hedgehog (Hh), receptor tyrosine kinase (RTK), Janus kinase/signal transducer and activator of transcription (JAK/STAT), wingless related (Wnt), and transforming growth factor-beta (TGF-beta), was then examined in the extraembryonic tissues. Western blot analysis showed that the expression of key molecules involved in the Notch, Hh, RTK, and JAK/STAT signaling pathways was downregulated, whereas most Wnt and TGF-beta signaling pathway molecules were upregulated in cloned extraembryonic tissues compared to normal extraembryonic tissues. These results indicate that unbalanced coordination of signaling pathways might impair the early development of cloned embryos postimplantation, thereby resulting in embryonic losses during the first trimester.

Dielectrophoretic oocyte selection chip for in vitro fertilization.

This paper reports a new dielectrophoretic separation method of porcine oocytes for in vitro fertilization. Conventional manual selection of oocyte highly depends on the expert's experience and lacks universal standards for identifying the quality of oocyte. In this study, an electrode array chip with castellated shape was developed to evaluate dielectrophoretic velocities of oocytes, under applied bias conditions with an AC 3 V waveform at 1 MHz for 15 s. Based on different dielectrophoresis (DEP) response, the selected group of oocytes that moved showed a better developmental potential than the group of oocytes that stayed, representing a higher rate of blastocyst formation and a lower rate of polyspermic fertilization. In addition, the overall developmental potential of oocytes selected by the DEP device was comparable to that of oocytes selected by conventional manual method. These results demonstrate that the difference in dielectrophoretic velocity can be used to establish an objective criterion for the selection of oocytes. Consequently, this method will open the possibility to develop an automatic tool for oocyte selection, which would be helpful for assisted reproductive technologies such as transgenic and clonal animal production.

Proteomic analysis of pancreas derived from adult cloned pig.

The potential medical applications of animal cloning include xenotransplantation, but the complex molecular cascades that control porcine organ development are not fully understood. Still, it has become apparent that organs derived from cloned pigs may be suitable for transplantation into humans. In this study, we examined the pancreas of an adult cloned pig developed through somatic cell nuclear transfer (SCNT) using two-dimensional electrophoresis (2-DE) and Western blotting. Proteomic analysis revealed 69 differentially regulated proteins, including such apoptosis-related species as annexins, lamins, and heat shock proteins, which were unanimously upregulated in the SCNT sample. Among the downregulated proteins in SCNT pancreas were peroxiredoxins and catalase. Western blot results indicate that several antioxidant enzymes and the anti-apoptotic protein were downregulated in SCNT pancreas, whereas several caspases were upregulated. Together, these data suggest that the accumulation of reactive oxygen species (ROS) in the pancreas of an adult cloned pig leads to apoptosis.

Epigenetic alteration of the donor cells does not recapitulate the reprogramming of DNA methylation in cloned embryos.

Epigenetic reprogramming is a prerequisite process during mammalian development that is aberrant in cloned embryos. However, mechanisms that evolve abnormal epigenetic reprogramming during preimplantation development are unclear. To trace the molecular event of an epigenetic mark such as DNA methylation, bovine fibroblasts were epigeneticallyaltered by treatment with trichostatin A (TSA) and then individually transferred into enucleated bovine oocytes. In the TSA-treated cells, expression levels of histone deacetylases and DNA methyltransferases were reduced, but the expression level of histone acetyltransferases such as Tip60 and histone acetyltransferase 1 (HAT1) did not change compared with normal cells. DNA methylation levels of non-treated (normal) and TSA-treated cells were 64.0 and 48.9% in the satellite I sequence (P < 0.05) respectively, and 71.6 and 61.9% in the alpha-satellite sequence respectively. DNA methylation levels of nuclear transfer (NT) and TSA-NT blastocysts in the satellite I sequence were 67.2 and 42.2% (P < 0.05) respectively, which was approximately similar to those of normal and TSA-treated cells. In the alpha-satellite sequence, NT and TSA-NT embryos were substantially demethylated at the blastocyst stage as IVF-derived embryos were demethylated. The in vitro developmental rate (46.6%) of TSA-NT embryos that were individually transferred with TSA-treated cells was higher than that (31.7%) of NT embryos with non-treated cells (P < 0.05). Our findings suggest that the chromatin of a donor cell is unyielding to the reprogramming of DNA methylation during preimplantation development, and that alteration of the epigenetic state of donor cells may improve in vitro developmental competence of cloned embryos.

DNA methylation state is preserved in the sperm-derived pronucleus of the pig zygote.

DNA methylation reprogramming (DMR) during preimplantation development erases differentiation-associated, unessential epigenetic information accumulated during gametogenesis, and ultimately brings pluripotency to the resulting embryo. Two patterns of DMR of sperm-derived pronucleus have been reported in mammals. In the first, the male pronucleus is actively demethylated whereas in the second, the methylation state seems to be maintained. The maintenance-type DMR has been seen only through immunocytochemical observations, and waits to be proven by additional molecular-level evidence. We demonstrate that, in pig, paternally derived DNA methylation is preserved during pronucleus development, based on the following observations. First, immunostaining of pig zygotes at different time points showed the DNA methylation state to be balanced between parental pronuclei throughout pronucleus development. Second, bisulfite analysis of PRE-1 repetitive sequences found mono- and polyspermic eggs to have similar methylation states. Third, the methylation state of a human erythropoietin gene delivered by transgenic pig spermatozoa was maintained in the male pronucleus. Finally, 5-aza-2'-deoxycytidine treatment, which blocks re-methylation, did not show the male pronucleus to be stalled in a demethylated state. In pig zygotes, paternally derived cytosine methylation was preserved throughout pronucleus development. These findings from multilateral DMR analyses provide further support to the view that DMR occurs in a non-conserved manner during early mammalian development.

Serial cloning of pigs by somatic cell nuclear transfer: restoration of phenotypic normality during serial cloning.

Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first-generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age- and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development.

Dynamic DNA methylation reprogramming: active demethylation and immediate remethylation in the male pronucleus of bovine zygotes.

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.

Gradual development of a genome-wide H3-K9 trimethylation pattern in paternally derived pig pronucleus.

The cytoplasm of a mature oocyte contains many protein complexes that are programmed to restructure incoming sperm chromatins on fertilization. Of the complicated biochemical events that these functional machineries control, the most impressive and important is epigenetic reprogramming. Despite its importance in epigenetic resetting, or "de-differentiation," of gamete genomes back to an incipient status, the mechanisms of epigenetic reprogramming do not seem to be conserved among mammals. Here, we report that, unlike in the mouse, the pig sperm-derived pronucleus is markedly trimethylated at lysine 9 of histone H3 (H3-m(3)K9), which might be associated with preservation of paternally derived cytosine methylation in pig zygotes. The male H3-m(3)K9 pattern is gradually established during pronucleus development, and this process occurs independently of DNA replication. Considering these unique epigenetic features, the pig zygote is, we believe, suited to serve as another model of epigenetic reprogramming that is antithetical to the well-characterized mouse model.

Cyclopamine treatment of human embryonic stem cells followed by culture in human astrocyte medium promotes differentiation into nestin- and GFAP-expressing astrocytic lineage.

Human embryonic stem cells (hESCs) are able to differentiate into various cell types, including neuronal cells and glial cells. However, little information is available regarding astrocyte differentiation. This report describes the differentiation of hESCs into nestin- and GFAP-expressing astrocytes following treatment with cyclopamine, which is an inhibitor of Hedgehog (Hh) signaling, and culturing in human astrocyte medium (HAM). In hESCs, cyclopamine treatment suppressed the expression of Hh signaling molecules, the Hh signaling target gene, and ESC-specific markers. Clyclopamine also induced the differentiation of the cells at the edges of the hESC colonies, and these cells stained positively for the early neural marker nestin. Subsequent culturing in HAM promoted the expression of the astrocyte-specific marker GFAP, and these cells were also nestin-positive. These findings indicate that treatment with cyclopamine followed by culturing in HAM leads to the differentiation of hESCs into nestin- and GFAP-expressing astrocytic lineage.

Proteomic analysis of the extraembryonic tissue from cloned porcine embryos.

Cloned animals developed from somatic cell nuclear transfer (SCNT) embryos are useful resources for agricultural and medical applications. However, the birth rate in the cloned animals is very low, and the cloned animals that have survived show various developmental defects. In this report, we present the morphology and differentially regulated proteins in the extraembryonic tissue from SCNT embryos to understand the molecular nature of the tissue. We examined 26-day-old SCNT porcine embryos at which the sonogram can first detect pregnancy. The extraembryonic tissue from SCNT embryos was abnormally small compared with the control. In the proteomic analysis with the SCNT extraembryonic tissue, 39 proteins were identified as differentially regulated proteins. Among up-regulated proteins, Annexins and Hsp27 were found. They are closely related to the processes of apoptosis. Among down-regulated proteins, Peroxiredoxins and anaerobic glycolytic enzymes were identified. In the Western blot analysis, antioxidant enzymes and the antiapoptotic Bcl-2 protein were down-regulated, and caspases were up-regulated. In the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay with the placenta from SCNT embryos, apoptotic trophoblasts were observed. These results demonstrate that a major reason for the low birth rate of cloned animals is due to abnormal apoptosis in the extraembryonic tissue during early pregnancy.

Transcriptional profiling of the developmentally important signalling pathways in human embryonic stem cells.

BACKGROUND: Embryonic stem cells (ESC) maintain their 'stemness' by self-renewal. However, the molecular mechanisms underlying self-renewal of human embryonic stem cells (hESC) remain to be elucidated. In this study, expression profiles of the molecules of developmentally important signalling pathways were investigated to better understand the relationships of the signalling pathways for self-renewal in hESC. METHODS: Two human ESC lines were cultured on mouse embryonic fibroblast (MEF) feeder cells. Gene expression was analysed by RT-PCR, real-time RT-PCR and Western blotting. RESULTS: In the bone morphogenetic protein (BMP4), transforming growth factor (TGF-beta) and fibroblast growth factor (FGF4) signalling pathways, ligands and antagonists were highly expressed in hESC compared with human embryoid body (hEB). Human ESC showed abundant transcripts of intracellular molecules in the Wnt, Hh and Notch signalling pathways. No difference was detected in the expression level of the JAK/STAT signalling molecules between hESC and hEB. Western blot analysis showed that the transcriptional levels of the signalling molecules in hESC were consistent with translational levels. From the real-time PCR analysis, expression levels of some genes, such as Oct3/4, Nodal and beta-catenin, were different between two hESC lines. CONCLUSION: The self-renewal of hESC is probably maintained by coordinated regulation of signalling-specific molecules and in a signalling-specific manner.

Inheritable histone H4 acetylation of somatic chromatins in cloned embryos.

A viable cloned animal indicates that epigenetic status of the differentiated cell nucleus is reprogrammed to an embryonic totipotent state. However, molecular events regarding epigenetic reprogramming of the somatic chromatin are poorly understood. Here we provide new insight that somatic chromatins are refractory to reprogramming of histone acetylation during early development. A low level of acetylated histone H4-lysine 5 (AcH4K5) of the somatic chromatin was sustained at the pronuclear stage. Unlike in vitro fertilized (IVF) embryos, the AcH4K5 level remarkably reduced at the 8-cell stage in cloned bovine embryos. The AcH4K5 status of somatic chromatins transmitted to cloned and even recloned embryos. Differences of AcH4K5 signal intensity were more distinguishable in the metaphase chromosomes between IVF and cloned embryos. Two imprinted genes, Ndn and Xist, were aberrantly expressed in cloned embryos as compared with IVF embryos, which is partly associated with the AcH4K5 signal intensity. Our findings suggest that abnormal epigenetic reprogramming in cloned embryos may be because of a memory mechanism, the epigenetic status itself of somatic chromatins.

Effects of in vitro fertilization conditions on preimplantation development and quality of pig embryos.

The present study was to investigate the effects of in vitro fertilization conditions on in vitro development and structural integrity of pig embryos. Porcine oocytes matured in vitro were co-incubated with four different spermatozoa concentrations (0.6 x 10(5), 1.2 x 10(5), 2.5 x 10(5) and 5 x 10(5) cells/ml) for 6 h, and at a spermatozoa concentration (1.2 x 10(5) cells/ml) for 2, 4 and 6 h, respectively. Spermatozoa penetration and blastocyst formation were observed at 10 and 144 h post insemination, respectively. The allocation of a blastocyst to inner cell mass (ICM) and trophectoderm (TE) cells was determined by using a differential staining method. Polyspermy frequency increased with increasing spermatozoa concentrations. The spermatozoa-oocyte co-incubation period of 2 h provided for decreased in vitro development rate than 4 and 6 h groups (P < 0.05), although no difference was detected in polyspermy frequency between spermatozoa-oocyte co-incubation periods. Interestingly, blastocysts derived from the groups with greater spermatozoa concentrations (2.5 x 10(5) and 5 x 10(5) cells/ml) had significantly fewer ICM cell nuclei as compared with those groups with lesser spermatozoa concentrations (0.6 x 10(5) and 1.2 x 10(5) cells/ml). There was no difference in the structural integrity of blastocysts among the co-incubation periods. Blastocysts derived from respective experiments were individually classified into three groups (I: <20%; II: 20-40% and III: >40%) based on the ratio of ICM to total cells. Proportion of blastocysts in Group II, with a presumptive normal range of structural integrity, was slightly decreased in the groups with greater spermatozoa concentrations (2.5 x 10(5) and 5 x 10(5) cells/ml). The results indicate that the spermatozoa concentration during in vitro fertilization may be important for developmental competence and quality of pig embryos.

Inactivation of MPF and MAP kinase by single electrical stimulus for parthenogenetic development of porcine oocytes.

This study was conducted to examine the activities of maturation-promoting factor (MPF) and mitogen-activated protein (MAP) kinase in the porcine oocytes after artificial activation. To determine optimal electrical activation condition, oocytes were exposed to single DC pulse in a variety of electric field strengths (120, 150, 180, and 210 V/mm) and pulse durations (15, 30, 45, and 60 microsec). After the artificial activation, 40-50 oocytes were cultured in a 50 microl drop of NCSU23 medium supplemented with 0.4% BSA at 39 degrees C, 5% CO2 in air for 6 days. No difference was detected in the preimplantation development of pocine oocytes and the mean nuclei number of blastocysts between electric field strengths. Under the 180 V/mm electric field strength, short pulse durations (15 and 30 microsec) showed a higher preimplantation developmental rate of the oocytes and mean nuclei number of blastocysts than an extended electric pulse (60 microsec) (P < 0.05). Single electrical stimulus (180 V/mm, 15 microsec) resulted in higher preimplantation development of porcine oocytes as compared to other chemical stimulators (P < 0.01). Western blot analyses showed the decrease of MPF and MAP kinase in the electrically-activated oocytes. After single electrical stimulus, the amounts of both cdc2 and ERK in porcine oocytes were remarkably reduced by 4 hr and then further decreased by 8 hr. However, the chemically-stimulated oocytes did not show any significant change at the levels of MPF and MAP kinase. Our results indicate that the optimal single electrical pulse is effective on the inactivation of MPF and MAP kinase, eventually leading to the parthenogenetic development of porcine oocytes.

Varied patterns of DNA methylation change between different satellite regions in bovine preimplantation development.

Global reduction of DNA methylation, a part of genome reprogramming processes, occurs in a gradual manner until before implantation and is recognized as a conserved process in mammals. Here, we reported that in bovine, satellite regions exhibited varied patterns of methylation changes when one-cell egg advanced to the blastocyst; a maintenance methylation was observed in satellite I sequences, a decrease in alpha satellites, and an increase in satellite II regions. Cloned embryos exhibited similar changes for DNA methylation in the satellite I and alpha. We also observed that the satellite I and alpha sequences were methylated more in inner cell mass region of the blastocyst whereas the satellite II showed selective demethylation in this region. Together, these findings point that individual satellite sequences carry their own methylation patterns under the pressure of global demethylation, suggesting that local methylation control system acts on the satellite regions in early bovine embryos.

Methylation changes of lysine 9 of histone H3 during preimplantation mouse development.

Immediately after fertilization, a chromatin remodeling process in the oocyte cytoplasm extracts protamine molecules from the sperm-derived DNA and loads histones onto it. We examined how the histone H3-lysine 9 methylation system is established on the remodeled sperm chromatin in mice. We found that the paternal pronucleus was not stained for dimethylated H3-K9 (H3-m2K9) during pronucleus development, while the maternal genome stained intensively. Such H3-m2K9 asymmetry between the parental pronuclei was independent of HP1b localization and, much like DNA methylation, was preserved to the two-cell stage when the nucleus appeared to be compartmentalized for H3-m2K9. A conspicuous increase in H3-m2K9 level was observed at the four-cell stage, and then the level was maintained without a visible change up to the blastocyst stage. The behavior of H3-m2K9 was very similar, but not identical, to that of 5-methylcytosine during preimplantation development, suggesting that there is some connection between methylation of histone and of DNA in early mouse development.