Novel imprinted single CpG sites found by global DNA methylation analysis in human parthenogenetic induced pluripotent stem cells

Genomic imprinting is the process of epigenetic modification whereby genes are expressed in a parent-of-origin dependent manner; it plays an important role in normal growth and development. Parthenogenetic embryos contain only the maternal genome. Parthenogenetic embryonic stem cells could be useful for studying imprinted genes. In humans, mature cystic ovarian teratomas originate from parthenogenetic activation of oocytes; they are composed of highly differentiated mature tissues containing all three germ layers. To establish human parthenogenetic induced pluripotent stem cell lines (PgHiPSCs), we generated parthenogenetic fibroblasts from ovarian teratoma tissues. We compared global DNA methylation status of PgHiPSCs with that of biparental human induced pluripotent stem cells by using Illumina Infinium HumanMethylation450 BeadChip array. This analysis identified novel single imprinted CpG sites. We further tested DNA methylation patterns of two of these sites using bisulfite sequencing and described novel candidate imprinted CpG sites. These results confirm that PgHiPSCs are a powerful tool for identifying imprinted genes and investigating their roles in human development and diseases.     

Exposure to diethylhexyl phthalate (DEHP) results in a heritable modification of imprint genes DNA methylation in mouse oocytes

Diethylhexyl phthalate (DEHP) is an estrogen-like compound widely used as a plasticizer in commercial products and is present in medical devices, and common household items. It is considered an endocrine disruptor since studies on experimental animals clearly show that exposure to DEHP can alter epigenetics of germ cells. This study was designed to assess the effects of DEHP on DNA methylation of imprinting genes in germ cells from fetal and adult mouse. Pregnant mice were treated with DEHP at doses of 0 and 40 μg DEHP/kg body weight/day from 0.5 to 18.5 day post coitum. The data revealed DEHP exposure significantly reduced the percentage of methylated CpG sites in Igf2r and Peg3 differentially methylated regions (DMRs) in primordial germ cells from female and male fetal mouse, particularly, in the oocytes of 21 dpp mice (F1), which were produced by the pregnant micetreated with DEHP. More surprisingly, the modification of the DNA methylation of imprinted genes in F1 mouse oocytes was heritable to F2 offspring which exhibit lower percentages of methylated CpG sites in imprinted genes DMRs. In conclusion, DEHP exposure can affect the DNA methylation of imprinting genes not only in fetal mouse germ cells and growing oocytes, but also in offspring’s oocytes.     

Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells

Genomic imprinting is an epigenetic mechanism that causes functional differences between paternal and maternal genomes, and plays an essential role in mammalian development. Stage-specific changes in the DNA methylation patterns of imprinted genes suggest that their imprints are erased some time during the primordial germ cell (PGC) stage, before their gametic patterns are re-established during gametogenesis according to the sex of individuals. To define the exact timing and pattern of the erasure process, we have analyzed parental-origin-specific expression of imprinted genes and DNA methylation patterns of differentially methylated regions (DMRs) in embryos, each derived from a single day 11.5 to day 13.5 PGC by nuclear transfer. Cloned embryos produced from day 12.5 to day 13.5 PGCs showed growth retardation and early embryonic lethality around day 9.5. Imprinted genes lost their parental-origin-specific expression patterns completely and became biallelic or silenced. We confirmed that clones derived from both male and female PGCs gave the same result, demonstrating the existence of a common default state of genomic imprinting to male and female germlines. When we produced clone embryos from day 11.5 PGCs, their development was significantly improved, allowing them to survive until at least the day 11.5 embryonic stage. Interestingly, several intermediate states of genomic imprinting between somatic cell states and the default states were seen in these embryos. Loss of the monoallelic expression of imprinted genes proceeded in a step-wise manner coordinated specifically for each imprinted gene. DNA demethylation of the DMRs of the imprinted genes in exact accordance with the loss of their imprinted monoallelic expression was also observed. Analysis of DNA methylation in day 10.5 to day 12.5 PGCs demonstrated that PGC clones represented the DNA methylation status of donor PGCs well. These findings provide strong evidence that the erasure process of genomic imprinting memory proceeds in the day 10.5 to day 11.5 PGCs, with the timing precisely controlled for each imprinted gene. The nuclear transfer technique enabled us to analyze the imprinting status of each PGC and clearly demonstrated a close relationship between expression and DNA methylation patterns and the ability of imprinted genes to support development.     

哺乳动物孤雌干细胞与孤雌生殖

未受精的孤雌胚胎衍生的孤雌胚胎干细胞(parthenogenetic embryonic stem cells,pESCs),具有与胚胎干细胞(embryonic stem cells,ESCs)相似的多向分化和自我更新能力,且具备来源广泛、获取高效及低致瘤性等优势,因此成为近年来的研究热点。该文概述了pESCs特殊的组织相容性和基因组印迹特征,综述其在孤雌生殖等方面的应用,并对其在遗传疾病及临床研究中的价值进行了展望。     

Epigenotype switching of imprintable loci in embryonic germ cells

 Expression of imprinted genes is dependent on their parental origin. This is reflected in the heritable differential methylation of parental alleles. The gametic imprints are however reversible as they do not endure for more than one generation. To investigate if the epigenetic changes in male and female germ line are similar or not, we derived embryonic germ (EG) cells from primordial germ cells (PGCs) of day 11.5 and 12.5 male and female embryos. The results demonstrate that they have an equivalent epigenotype. First, chimeras made with EG cells derived from both male and female embryos showed comparable fetal overgrowth and skeletal abnormalities, which are similar to but less severe than those induced by androgenetic embryonic stem (ES) cells. Thus, EG cells derived from female embryos resemble androgenetic ES cells more than parthenogenetic cells. Furthermore, the methylation status of both alleles of a number of loci in EG cells was similar to that of the paternal allele in normal somatic cells. Hence, both alleles of Igf2r region 2, Peg1/Mest, Peg3, Nnat were consistently unmethylated in EG cells as well as in the primary embryonic fibroblasts (PEFs) rescued from chimeras. More strikingly, both alleles of p57kip2 that were also unmethylated in EG cells, underwent de novo methylation in PEFs to resemble a paternal allele in somatic cells. The exceptions were the H19 and Igf2 genes that retained the methylation pattern in PEFs as seen in normal somatic tissues. These studies suggest that the initial epigenetic changes in germ cells of male and female embryos are similar. Received: 1 September 1997 / Accepted: 15 October 1997     

Nuclei of oocytes derived from mouse parthenogenetic embryos are competent to support development to term

Mouse parthenotes result in embryonic death before 10 days of gestation, but parthenogenetic embryos (ng/fg PE) that contain haploid sets of genomes from nongrowing (ng) oocytes derived from newborn fetuses and fully grown (fg) oocytes derived from adults can develop into 13.5-day-old fetuses. This prolonged development is due to a lack of genomic imprinting in ng oocytes. Here, we show maternal genomes of oocytes derived from ng/fg PE are competent to support normal development. After 28 days of culture, the ovaries from ng/fg PE grew as well as the controls, forming vesicular follicles with follicular antrums. The oocytes collected from the developed follicles were the same size as those of the controls. To determine whether maternal primary imprinting had been established in the oocytes derived from ng/fg PE, we examined the DNA methylation status in differentially methylated regions of three imprinted genes, Igf2r, Lit1, and H19. The results showed that maternal-specific modifications were imposed in the oocytes derived from ng/fg PE. Further, to assess nuclear competence to support development, we constructed matured oocytes containing a haploid genome derived from ng/fg PE oocytes by serial nuclear transfer. After in vitro fertilization and culture and embryo transplantation into recipients, two live pups were obtained. One developed normally to a fertile adult. These results revealed that oocytes derived from ng/fg PE can be normally imprinted during oogenesis and acquire competence to participate in development as female genomes.     

A maternal-zygotic effect gene, Zfp57, maintains both maternal and paternal imprints

The mechanisms responsible for maintaining genomic methylation imprints in mouse embryos are not understood. We generated a knockout mouse in the Zfp57 locus encoding a KRAB zinc finger protein. Loss of just the zygotic function of Zfp57 causes partial neonatal lethality, whereas eliminating both the maternal and zygotic functions of Zfp57 results in a highly penetrant embryonic lethality. In oocytes, absence of Zfp57 results in failure to establish maternal methylation imprints at the Snrpn imprinted region. Intriguingly, methylation imprints are reacquired specifically at the maternally derived Snrpn imprinted region when the zygotic Zfp57 is present in embryos. This suggests that there may be DNA methylation-independent memory for genomic imprints. Zfp57 is also required for the postfertilization maintenance of maternal and paternal methylation imprints at multiple imprinted domains. The effects on genomic imprinting are consistent with the maternal-zygotic lethality of Zfp57 mutants.     

Heterogeneity in imprinted methylation patterns of pluripotent embryonic germ cells derived from pre-migratory mouse germ cells

Pluripotent stem cells, termed embryonic germ (EG) cells, have been generated from both human and mouse primordial germ cells (PGCs). Like embryonic stem (ES) cells, EG cells have the potential to differentiate into all germ layer derivatives and may also be important for any future clinical applications. The development of PGCs in vivo is accompanied by major epigenetic changes including DNA demethylation and imprint erasure. We have investigated the DNA methylation pattern of several imprinted genes and repetitive elements in mouse EG cell lines before and after differentiation. Analysed cell lines were derived soon after PGC specification, “early”, in comparison with EG cells derived after PGC colonisation of the genital ridge, “late” and embryonic stem (ES) cell lines, derived from the inner cell mass (ICM). Early EG cell lines showed strikingly heterogeneous DNA methylation patterns, in contrast to the uniformity of methylation pattern seen in somatic cells (control), late EG cell and ES cell lines. We also observed that all analysed XX cell lines exhibited less methylation than XY. We suggest that this heterogeneity may reflect the changes in DNA methylation taking place in the germ cell lineage soon after specification.     

Androgenetic mouse embryonic stem cells are pluripotent and cause skeletal defects in chimeras: implications for genetic imprinting

The inviability of diploid androgenetic and parthenogenetic embryos suggests imprinting of paternal and maternal genes during germ cell development, and differential expression of loci depending on parental inheritance appears to be involved. To facilitate identification of imprinted genes, we have derived diploid androgenetic embryonic stem (ES) cell lines. In contrast to normal ES cells, they form tumors composed almost entirely of striated muscle when injected subcutaneously into adult mice. They also form chimeras following blastocyst injection, although many chimeras die at early postnatal stages. Surviving chimeras develop skeletal abnormalities, particularly in the rib cartilage. These results demonstrate that androgenetic ES cells are pluripotent and point to stage- and cell-specific expression of developmentally important imprinted genes.     

Identifying MicroRNA and mRNA Expression Profiles in Embryonic Stem Cells Derived from Parthenogenetic, Androgenetic and Fertilized Blastocysts

MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions. In this study, miRNA and messenger RNA (mRNA) profiles were examined by Illumina microarray in mouse embryonic stem cells (ESCs) derived from parthenogenetic, androgenetic, and fertilized blastocysts. The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression. Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs (aESCs) and fertilized ESCs (fESCs), a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs (pESCs) and fESCs, and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs. In addition, a total of 575, 5 and 376 miRNA-mRNA target pairs were observed in aESCs vs. fESCs, pESCs vs. fESCs, and aESCs vs. pESCs, respectively. Furthermore, 15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR. Finally, transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs. Inhibition of miR-880 increased the expression of Peg3, Dyrk1b, and Prrg2 mRNA, inhibition of miR-363 increased the expression of Nfat5 and Soat1 mRNA, and inhibition of miR-883b-5p increased Nfat5, Tacstd2, and Ppapdc1 mRNA. These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.     

Maternal primary imprinting is established at a specific time for each gene throughout oocyte growth.

Primary imprinting during gametogenesis governs the monoallelic expression/repression of imprinted genes in embryogenesis. Previously, we showed that maternal primary imprinting is disrupted in neonate-derived non-growing oocytes. Here, to investigate precisely when and in what order maternal primary imprinting progresses, we produced parthenogenetic embryos containing one genome from a non-growing or growth-stage oocyte from 1- to 20-day-old mice and one from a fully grown oocyte of adult mice. We used these embryos to analyze the expression of eight imprinted genes: Peg1/Mest, Peg3, Snrpn, Znf127, Ndn, Impact, Igf2r, and p57(KIP2). The results showed that the imprinting signals for each gene were not all imposed together at a specific time during oocyte growth but rather occurred throughout the period from primary to antral follicle stage oocytes. The developmental ability of the constructed parthenogenetic embryos was gradually reduced as the nuclear donor oocytes grew. These studies provide the first insight into the process of primary imprinting during oocyte growth.     

Parthenogenesis in non-rodent species: developmental competence and differentiation plasticity

An oocyte can activate its developmental process without the intervention of the male counterpart. This form of reproduction, known as parthenogenesis, occurs spontaneously in a variety of lower organisms, but not in mammals. However, it must be noted that mammalian oocytes can be activated in vitro, mimicking the intracellular calcium wave induced by the spermatozoon at fertilization, which triggers cleavage divisions and embryonic development. The resultant parthenotes are not capable of developing to term and arrest their growth at different stages, depending on the species. It is believed that this arrest is due to genomic imprinting, which causes the repression of genes normally expressed by the paternal allele. Human parthenogenetic embryos have recently been proposed as an alternative, less controversial source of embryonic stem cell lines, based on their inherent inability to form a new individual. However many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. Limited information is available in particular on the consequences of the lack of centrioles and on the parthenote's ability to assemble a new embryonic centrosome in the absence of the sperm centriole. Indeed, in lower species, successful parthenogenesis largely depends upon the oocyte's ability to regenerate complete and functional centrosomes in the absence of the material supplied by a male gamete, while the control of this event appears to be less stringent in mammalian cells. In an attempt to better elucidate some of these aspects, parthenogenetic cell lines, recently derived in our laboratory, have been characterized for their pluripotency. In vitro and in vivo differentiation plasticity have been assessed, demonstrating the ability of these cells to differentiate into cell types derived from the three germ layers. These results confirmed common features between uni- and bi-parental embryonic stem cells. However data obtained with parthenogenetic cells indicate the presence of an intrinsic deregulation of the mechanisms controlling proliferation vs. differentiation and suggest their uni-parental origin as a possible cause.     

The testis-specific factor CTCFL cooperates with the protein methyltransferase PRMT7 in H19 imprinting control region methylation

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation—DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation.     

生殖细胞及早期胚胎基因组印记的表观调控

基因组印记是一种区别父母等位基因的表观遗传过程,可导致父源和母源基因特异性表达。印记是在配子发生过程中全基因组表观重编程时获得的,且在早期胚胎发育过程中得以维持。因此,在全基因组重编程过程中,对印记的识别和维持十分重要。本文概述了原始生殖细胞的印记清除、双亲原始生殖细胞的印记获得以及早期胚胎发育过程中印记维持的相关过程,并对在印记区域内保护印记基因免受全基因组DNA去甲基化的表观遗传因子的相关作用机制进行了讨论。     

Diploid parthenogenetic embryos adopt a maternal-type methylation pattern on both sets of maternal chromosomes

Epigenetic modifications are closely associated with embryo developmental potential. One of the epigenetic modifications thought to be involved in genomic imprinting is DNA methylation. Here we show that the maternally imprinted genes Snrpn and Peg1/Mest were nearly unmethylated or heavily methylated, respectively, in their differentially methylated regions (DMRs) at the two-cell stage in parthenogenetic embryos. However, both genes were gradually de novo methylated, with almost complete methylation of all CpG sites by the morula stage in parthenogenetic embryos. Unexpectedly, another maternally imprinted gene, Peg3, showed distinct dynamics of methylation during preimplantation development of diploid parthenogenetic embryos. Peg3 showed seemingly normal methylation patterns at the two-cell and morula stages, but was also strongly de novo methylated in parthenogenetic blastocysts. In contrast, the paternally imprinted genes H19 and Rasgrf1 showed complete unmethylation of their DMRs at the morula stage in parthenogenetic embryos. These results indicate that diploid parthenogenetic embryos adopt a maternal-type methylation pattern on both sets of maternal chromosomes and that the aberrantly homogeneous status of methylation imprints may partially account for developmental failure.     

Quantitative proteomics analysis of parthenogenetically induced pluripotent stem cells

Parthenogenetic embryonic stem (pES) cells isolated from parthenogenetic activation of oocytes and embryos, also called parthenogenetically induced pluripotent stem cells, exhibit pluripotency evidenced by both in vitro and in vivo differentiation potential. Differential proteomic analysis was performed using differential in-gel electrophoresis and isotope-coded affinity tag-based quantitative proteomics to investigate the molecular mechanisms underlying the developmental pluripotency of pES cells and to compare the protein expression of pES cells generated from either the in vivo-matured ovulated (IVO) oocytes or from the in vitro-matured (IVM) oocytes with that of fertilized embryonic stem (fES) cells derived from fertilized embryos. A total of 76 proteins were upregulated and 16 proteins were downregulated in the IVM pES cells, whereas 91 proteins were upregulated and 9 were downregulated in the IVO pES cells based on a minimal 1.5-fold change as the cutoff value. No distinct pathways were found in the differentially expressed proteins except for those involved in metabolism and physiological processes. Notably, no differences were found in the protein expression of imprinted genes between the pES and fES cells, suggesting that genomic imprinting can be corrected in the pES cells at least at the early passages. The germline competent IVM pES cells may be applicable for germ cell renewal in aging ovaries if oocytes are retrieved at a younger age.     

Temporal and spatial selection against parthenogenetic cells during development of fetal chimeras

The fate of parthenogenetic cells was investigated during development of fetal and early postnatal chimeras. On day 13 of embryonic development, considerable contribution of parthenogenetic cells was observed in all tissues of chimeric embryos, although selection against parthenogenetic cells seemed to start before day 13. Between days 13 and 15 of development, parthenogenetic cells came under severe selective pressure, which was most striking in tongue. The disappearance of parthenogenetic cells from tongue coincided with the beginning of myoblast fusion in this tissue. Severe selection against parthenogenetic cells was also observed in pancreas and liver, although in the latter, parthenogenetic cells were eliminated later than in skeletal muscle or pancreas. In other tissues, parthenogenetic cells may persist and participate to a considerable extent throughout the gestation period and beyond, although a significant decrease was observed in all tissues. Parthenogenetic in equilibrium fertilized chimeras were significantly smaller than their non-chimeric littermates at all developmental stages. These results suggest that the absence of paternal chromosomes is largely incompatible with the maintenance of specific differentiated cell types. Furthermore, paternally derived genes seem to be involved in the regulation of proliferation of all cell types, as indicated by the drastic growth decceleration of parthenogenetic in equilibrium fertilized chimeras and the overall decrease of parthenogenetic cells during fetal development. Chromosomal imprinting may have a role in maintaining a balance between cell growth and differentiation during embryonic development. The major exception to the selective elimination of parthenogenetic cells appear to be the germ cells; viable offspring derived from parthenogenetic oocytes were detected, sometimes at a high frequency in litters of female parthenogenetic in equilibrium fertilized chimeras.     

Oocyte-like Cells Induced from Mouse Spermatogonial Stem Cells

ABSTRACT: BACKGROUND: During normal development primordial germ cells (PGCs) derived from the epiblast are the precursors of spermatogonia and oogonia. In culture, PGCs can be induced to dedifferentiate to pluripotent embryonic germ (EG) cells in the presence of various growth factors. Several recent studies have now demonstrated that spermatogonial stem cells (SSCs) can also revert back to pluripotency as embryonic stem (ES)-like cells under certain culture conditions. However, the potential dedifferentiation of SSCs into PGCs or the potential generation of oocytes from SSCs has not been demonstrated before. RESULTS: We report that mouse male SSCs can be converted into oocyte-like cells in culture. These SSCs-derived oocytes (SSC-Oocs) were similar in size to normal mouse mature oocytes. They expressed oocyte-specific markers and give rise to embryos through parthenogenesis. Interestingly, the Y- and X-linked testis-specific genes in these SSC-Oocs were significantly down-regulated or turned off, while oocyte-specific X-linked genes were activated. The gene expression profile appeared to switch to that of the oocyte across the X chromosome. Furthermore, these oocyte-like cells lost paternal imprinting but acquired maternal imprinting. CONCLUSIONS: Our data demonstrate that SSCs might maintain the potential to be reprogrammed into oocytes with corresponding epigenetic reversals. This study provides not only further evidence for the remarkable plasticity of SSCs but also a potential system for dissecting molecular and epigenetic regulations in germ cell fate determination and imprinting establishment during gametogenesis.