Precise recapitulation of methylation change in early cloned embryos

Change of DNA methylation during preimplantation development is very dynamic, which brings this term to the most attractive experimental target for measuring the capability of cloned embryo to reprogram its somatic genome. However, one weak point is that the preimplantation stage carries little information on genomic sequences showing a site-specific re-methylation after global demethylation; these sequences, if any, may serve as an advanced subject to test how exactly the reprogramming/programming process is recapitulated in early cloned embryos. Here, we report a unique DNA methylation change occurring at bovine neuropeptide galanin gene sequence. The galanin gene sequence in early bovine embryos derived by in vitro fertilization (IVF) maintained a undermethylated status till the morula stage. By the blastocyst, certain CpG sites became methylated specifically, which may be an epigenetic sign for the galanin gene to start a differentiation programme. The same sequence was moderately methylated in somatic donor cell and, after transplanted into an enucleated oocyte by nuclear transfer (NT), came rapidly demethylated to a completion, and then, at the blastocyst stage, re-methylated at exactly the same CpG sites, as observed so in normal blastocysts. The precise recapitulation of normal methylation reprogramming and programming at the galanin gene sequence in bovine cloned embryos gives a cue for the potential of cloned embryo to superintend the epigenetic states of foreign genome, even after global demethylation.     

Aberrant expression of miR-29a/29b and methylation level of mouse embryos after in vitro fertilization and vitrification at two-cell stage

Proper epigenetic modifications during preimplantation embryo development are important for a successful pregnancy. We aim to investigate the putative influence of in vitro fertilization (IVF) and vitrification on DNA methylation in mouse preimplantation embryos. The study groups consisted of blastocyst-derived vitrified two-cell embryos, nonvitrified embryos, and a control group of in vivo derived blastocysts. We assessed developmental competence, global DNA methylation, relative expression levels of miR-29a/29b, and their target genes, Dnmt3a/3b. Vitrified embryos had a lower developmental rate as compared with nonvitrified embryos. There was no significant decrease in blastocyst cell numbers among studied groups, whereas there was a steady decline in DNA methylation after IVF and vitrification. The levels of miR-29a/29b upregulated in the experimental groups as compared with the control group. IVF and vitrification caused Dnmt3a/3b downregulations in blastocysts. The results of this study have suggested that a relationship exists between IVF and embryo vitrification with methylation interruptions in the blastocysts.     

Genome wide abnormal DNA methylome of human blastocyst in assisted reproductive technology

Proper reprogramming of parental DNA methylomes is essential for mammalian embryonic development.However,it is unknown whether abnormal methylome reprogramming occurs and is associated with the failure of embryonic development.Here we analyzed the DNA methylomes of 57 blastocysts and 29 trophectoderm samples with different morphological grades during assisted reproductive technology(ART) practices.Our data reveal that the global methylation levels of high-quality blastocysts are similar(0.30 ± 0.02,mean ± SD).while the methylation levels of low-quality blastocysts are divergent and away from those of high-quality blastocysts.The proportion of blastocysts with a methylation level falling within the range of 0.30 ± 0.02 in different grades correlates with the live birth rate for that grade.Moreover,abnormal methylated regions are associated with the failure of embryonic development.Furthermore,we can use the methylation data of cells biopsied from trophectoderm to predict the blastocyst methylation level as well as to detect the aneuploidy of the blastocysts.Our data indicate that global abnormal methylome reprogramming often occurs in human embryos,and suggest that DNA methylome is a potential biomarker in blastocyst selection in ART.     

Genome-Wide Analysis of DNA Methylation Dynamics during Early Human Development

DNA methylation is globally reprogrammed during mammalian preimplantation development, which is critical for normal development. Recent reduced representation bisulfite sequencing (RRBS) studies suggest that the methylome dynamics are essentially conserved between human and mouse early embryos. RRBS is known to cover 5–10% of all genomic CpGs, favoring those contained within CpG-rich regions. To obtain an unbiased and more complete representation of the methylome during early human development, we performed whole genome bisulfite sequencing of human gametes and blastocysts that covered>70% of all genomic CpGs. We found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts, which could contribute to an increased number of imprinted differentially methylated regions in the human genome. Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation. Furthermore, centromeric satellite repeats were hypermethylated in human oocytes but not in mouse oocytes, which might be explained by differential expression of de novo DNA methyltransferases. These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development. Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.     

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.     

Differential DNA methylation reprogramming of various repetitive sequences in mouse preimplantation embryos

Genome-wide changes of DNA methylation by active and passive demethylation processes are typical features during preimplantation development. Here we provide an insight that epigenetic reprogramming of DNA methylation is regulated in a region-specific manner, not a genome-wide fashion. To address this hypothesis, methylation states of three repetitive genomic regions were monitored at various developmental stages in the mouse embryos. Active demethylation was not observed in the IAP sequences whereas methylation reprogramming of the satellite sequences was regulated only by the active mechanism. Etn elements were actively demethylated after fertilization, passively demethylated by the 8-cell stage, and de novo methylated at the morular and blastocyst stages, showing dynamic epigenetic changes. Thus, our findings suggest that the specific genomic regions or sequences may spatially/temporally have their unique characteristics in the reprogramming of the DNA methylation during preimplantation development.     

MicroRNA‐29b regulates DNA methylation by targeting Dnmt3a/3b and Tet1/2/3 in porcine early embryo development

MicroRNA‐29b (miR‐29b) is a member of the miR‐29 family, which targets DNA methyltransferases (DNMTs) and ten eleven translocation enzymes (TETs), thereby regulating DNA methylation. However, the role of miR‐29b in porcine early embryo development has not been reported. In this study, we examined the effects of miR‐29b in porcine in vitro fertilization (IVF) embryos to investigate the mechanism by which miR‐29b regulated DNA methylation. The interference of miR‐29b by its special miRNA inhibitor significantly up‐regulated Dnmt3a/b and Tet1 but downregulated Tet2/3; meanwhile it increased DNA methylation levels of the global genome and Nanog promoter region but decreased global DNA demethylation levels. The inhibition of miR‐29b also resulted in a decrease in the development rate and quality of blastocysts. In addition, the pluripotency genes Nanog and Sox2 were significantly downregulated, and the apoptosis genes Bax and Casp3 were upregulated, but anti‐apoptosis gene Bcl‐2 was downregulated in blastocysts. Our study indicated that miR‐29b could regulate DNA methylation mediated by miR29b‐ Dnmt3a/b – Tet1/2/3 signaling during porcine early embryo development.     

Sleep deprivation decreases the reproductive capacity by affecting the arrival of morulas in the uterus

A previous animal study by our group found that sleep deprivation during preimplantation was associated with decreased pregnancy maintenance. Given its impact on human society, we aimed in the current study to assess whether sleep deprivation affects blastocyst gene expression and/or the implantation process. For this, pregnant mice (gestational day 0 [GD 0]) were assigned into paradoxical sleep deprivation (SD, 72 hr; multiple platform method) and, a control (CT) group. Animals were euthanized on GD 3.5 and blood, uterus (embryos) and fallopian tube were collected. Then, 89% of CT presented blastocysts in the uterus versus 25% from SD group. Compared to CT, SD presented lighter relative uterus weight, increased plasma concentrations of corticosterone and testosterone, decreased concentrations of progesterone and luteinizing hormone, but no statistical differences in plasma concentrations of 17β‐estradiol and follicle stimulating hormone. There were no differences in uterus and blastocyst gene expression related to embryo implantation and development, and no alteration in blastocysts global DNA methylation. Considering this, the decreased pregnancy maintenance after sleep deprivation seems not to be associated with implantation losses or developmental problems related to the blastocysts. It is likely that complications in morula development and/or its movement through the fallopian tubes affect the pregnancy rate, since only 25% of SD females presented a blastocyst on the GD 3.5. In fact, three out of four females without blastocysts in the uterus presented morula in the fallopian tubes due to a phase delay. Additionally, we suggest that the observed hormonal changes may play a role in this outcome.     

Genome-wide analysis of DNA methylation status of CpG islands in embryoid bodies, teratomas, and fetuses

Differentiation of embryonic stem (ES) cells into embryoid bodies (EBs) provides an in vitro system for the study of early lineage determination during mammalian development. We have previously reported that there are 247 CpG islands that potentially have tissue-dependent and differentially methylated regions (T-DMRs). This provided evidence that the formation of DNA methylation patterns at CpG islands is a crucial epigenetic event underlying mammalian development. Here we present an analysis by the restriction landmark genomic scanning (RLGS) using NotI as a landmark enzyme of the genome-wide methylation status of CpG islands of ES cells and EBs and of teratomas produced from ES cells. These results are considered in relation to the methylation status of CpG islands of genomic DNA from normal fetus (10.5 dpc) and adult tissues. We have prepared a DNA methylation panel that consists of 259 T-DMRs and includes novel T-DMRs that are distinctly methylated or unmethylated in the teratomas. The DNA methylation pattern was complex and differed for the ES cells, EBs, and teratomas, providing evidence that differentiation of cells involves both de novo DNA methylation as well as demethylation. Comparison of the numbers of T-DMRs, that were differentially methylated or unmethylated among the cells and tissue types studied, revealed that the teratomas were the most epigenetically different from ES cells. Thus, analysis of the DNA methylation profiles prepared in this study provides new insights into the differentiation of ES cells and development of fetus, EB, teratoma, and somatic tissues.     

Global remodeling of the mouse DNA methylome during aging and in response to calorie restriction

Aging is characterized by numerous molecular changes, such as accumulation of molecular damage and altered gene expression, many of which are linked to DNA methylation. Here, we characterize the blood DNA methylome across 16 age groups of mice and report numerous global, region‐ and site‐specific features, as well as the associated dynamics of methylation changes. Transition of the methylome throughout lifespan was not uniform, with many sites showing accelerated changes in late life. The associated genes and promoters were enriched for aging‐related pathways, pointing to a fundamental link between DNA methylation and control of the aging process. Calorie restriction both shifted the overall methylation pattern and was accompanied by its gradual age‐related remodeling, the latter contributing to the lifespan‐extending effect. With age, both highly and poorly methylated sites trended toward intermediate levels, and aging was accompanied by an accelerated increase in entropy, consistent with damage accumulation. However, the entropy effects differed for the sites that increased, decreased and did not change methylation with age. Many sites trailed behind, whereas some followed or even exceeded the entropy trajectory and altered the developmental DNA methylation pattern. The patterns we observed in certain genomic regions were conserved between humans and mice, suggesting common principles of functional DNA methylome remodeling and its critical role in aging. The highly resolved DNA methylome remodeling provides an excellent model for understanding systemic changes that characterize the aging process.     

DNA methylation in mouse embryonic stem cells and development

Mammalian development is associated with considerable changes in global DNA methylation levels at times of genomic reprogramming. Normal DNA methylation is essential for development but, despite considerable advances in our understanding of the DNA methyltransferases, the reason that development fails when DNA methylation is deficient remains unclear. Furthermore, although much is known about the enzymes that cause DNA methylation, comparatively little is known about the mechanisms or significance of active demethylation in early development. In this review, we discuss the roles of the various DNA methyltransferases and their likely functions in development.     

FGF10 enhances yak oocyte fertilization competence and subsequent blastocyst quality and regulates the levels of CD9, CD81, DNMT1, and DNMT3B

The fusion of sperm and oocytes determines the fertilization competence and subsequent development of embryos, which, in turn, can be affected by various proteins and DNA methylation. However, several factors in this whole regulation process remain unknown, especially in yaks. Here, we report that fibroblast growth factor 10 (FGF10) is an important growth factor that can enhance the maturation rate of yak oocytes and the motility of frozen spermatozoa. Subsequent blastocyst quality was also improved by increasing the total cell number and level of pregnancy-associated protein in blastocysts. These effects were significantly high in the group that received the 5 ng/ml FGF10 treatment, during both in vitro maturation (IVM) and capacitation. Our data show that the effects of FGF10 were dose-dependent at vital steps of embryogenesis in vitro. Furthermore, quantitative polymerase chain reaction, western blot analysis, and immunofluorescence demonstrated that the levels of CD9, CD81, DNMT1, and DNMT3B in both mature cumulus-oocyte complexes and capacitated sperms were regulated by FGF10, which was also highly expressed in the group treated with 5 ng/ml FGF10 during both IVM and capacitation. From our present study, we concluded that FGF10 promotes yak oocyte fertilization competence and subsequent blastocyst quality, and could also regulate CD9, CD81, DNMT1, and DNMT3B to optimize sperm–oocyte interactions and DNA methylation during fertilization.     

Vitrification of murine mature metaphase II oocytes perturbs DNA methylation reprogramming during preimplantation embryo development

Accurate reprogramming of DNA methylation occurring in preimplantation embryos is critical for normal development of both fetus and placenta. Environmental stresses imposed on oocytes usually cause the abnormal DNA methylation reprogramming of early embryos. However, whether oocyte vitrification alters the reprogramming of DNA methylation (5 mC) and its derivatives in mouse preimplantation embryo development remains largely unknown. Here, we found that the rate of cleavage and blastocyst formation of embryos produced by IVF of vitrified matured oocytes was significantly lower than that in control counterparts, but the quality of blastocysts was not impaired by oocyte vitrification. Additionally, although vitrification neither altered the dynamic changes of 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5 fC) before 4-cell stage nor affected the levels of 5 mC and 5-carboxylcytosine (5caC) throughout the preimplantation development, vitrification significantly reduced the levels of 5hmC and 5 fC from 8-cell stage onwards. Correspondingly, vitrification did not alter the expression patterns of Tet3 in preimplantation embryos but apparently reduced the expression levels of Tet1 in 4-cell and 8-cell embryos and increased the expression levels of Tet2 at morula stage. Taken together, these results demonstrate that oocyte vitrification perturbs DNA methylation reprogramming in mouse preimplantation embryo development.