REVIEWS Erasure and establishment of parental imprints Imprinting in the male and female germline. Once the An early stage of mammalian Imprint erasure in PGCs. When they arrive at the parental imprints have been erased, new imprints must mbryonic development at genital ridge, which occurs by E11.5, PGCs undergo be re-established according to the gender of the animal. which the first cell lineage extensive epigenetic reprogramming, including the This re-establishment occurs only after sex determina- erasure of parental imprints(FIG. 1). The erasure of tion has been initiated, and male and female germ-cell imprints is reflected by demethylation of the imprinted development diverges to give rise to sperm or oocytes, Spherical structure formed loci, which occurs concomitantly with demethylation respectively. In mice, the gonads of males and females by diferentiating ES cells in of other regions. The erasure occurs at different become morphologically distinguishable by E12.5.In culture, which resembles the imprinted loci at different times between E10.5 and female embryos, germ cells arrest in meiotic prophase E12.5, as shown in a study of cloned embryos that were at around E13. 0, whereas male germ cells enter into produced from PGC nuclei". Since the imprint eras- Gl-phase mitotic arrest at a similar time(FIGS 1,2).A ure at each locus is a rapid process that is completed number of environmental cues, including retinoic acid within one day of development, this might be an active signals from the mesonephroi, determine the timing demethylation process of entry into meiosis by germ cells In somatic cells of female mammals, one of the two G1-arrested male germ cells are called prosper X chromosomes is inactivated so that the dosage of the matogonia or gonocytes(FIG. 1). Paternal methylation genes on this chromosome is equalized between males imprints, which have been identified at just three loci, and females. The inactive X chromosome is reactivated are progressively established in these cells between during female germ-cell development. It had been E14.5 and the newborn stage7-st A germline-specific thought that this reactivation occurs around the time gene-knockout study indicated that the de novo dNA of imprint erasure 39. However, more recent studies methyltransferase, DNMT3A, has a central role in the e novo methylation process of all known paternally at an even earlier stagei. So, X-chromosome reactiva- methylated loci, and another de novo methyltrans tion occurs progressively over a prolonged period and ferase, DNMT3B, is involved only at the Raserfl(ras is completed in post-migratory PGCs. The initiation protein-specific guanine nucleotide-releasing factor 1) of reactivation in migrating PGCs is reminiscent of locus 0. 2. The reason why RasgrfI requires an addi the X reactivation in inner-cell-mass cells of female tional enzyme is unknown, but this could be related to blastocysts, but the mechanisms of these processes are the presence of several retrotransposon sequences at yet to be clarified. this locus(see below ). The establishment of paternal Not all sequences show DNA demethylation in methylation imprints at all loci requires another mem post-migratory PGCs For example, DNA methylation ber of the Dnmt3 family, DNMT3L, which is highly of the intra-cisternal A particle(IAP)retrotransposon expressed in prospermatogonia052-4. This protein has family is only partially reprogrammed. Incomplete no DNA-methyltransferase activity but forms a complex removal of epigenetic marks in the germ line can lead with DNMT3A and/or DNMT3B and stimulates their to epigenetic inheritance from one generation to the activities. next, evidence of which is now accumulating in both The established methylation imprints are then main- factors, it has been suggested that this phenomenon established from neonatal testes, can be maintained could provide a basis for adaptive evolution and/or stably in culture and can give rise to sperm when heritable disease susceptibility without changes in transplanted into testes- possess paternal methyla DNA sequence-[BOX 2). tion imprints, whereas their multipotent derivatives, mGS cells, show partial demethylation at these sites, similar to ES cells. GS cells and mGS cells provide Box 1 Derivation of germ cells from embryonic stem cells invaluable tools for germ-cell study and reproductive Various types of somatic cell, including blood cells and neural cells, have been In the female germline, the initiation of dna obtained from embryonic stem(ES)cells in culture dishes. Recent studies have methylation imprinting occurs after birth, during the revealed that it is also possible to generate gametes from ES cells. Gametes or gamete-like cells were derived when mouse ES cells were cultured under various oocyte growth?. The growing oocytes are at the diplo tene stage of meiotic prophase I, and the de novo methyl differentiation conditions including simple monolayer culture(oocyte), embryoid- ation process is complete by the fully-grown oocyte body formation (sperm)o, embryoid-body formation followed by treatment with retinoic acid (sperm)and retinoic acid induction alone (sperm). In the most ge(FIG. 1). Both DNMT3A and DNMT3L also have successful case, ES-derived sperm cells were able to fertilize oocytes after essential roles in this process 2.9, but DNMT3B seems and survived only up to five months, indicating that reprogramming of the o. K intracytoplasmic injection and support embryonic development to term1.The resultant pups, however, had abnormalities in DNA methylation at imprinted loci Recent studies have started to provide some clues on the mechanism by which the DNMT3A-DNMT3L germ-cell genome was not properly accomplished. When we fully understand the complex recognizes the imprinted loci(and some ret- mechanisms of germ-cell reprogramming, we might be able to derive appropriately rotransposons, see below). A crystallographic analysis reprogrammed, functional gametes from cultured cells, which will allow new of the complexed C-terminal domains of DNMT3A approaches to reproductive engineering, although ethical and safety issues must be and DNMT3L revealed a tetrameric structure with two carefully considered active sites. This structure suggests that DNA regions @2008 Nature Publishing GroupBlastocyst An early stage of mammalian embryonic development at which the first cell lineages become established. Embryoid body Spherical structure formed by differentiating ES cells in culture, which resembles the early embryo. Erasure and establishment of parental imprints Imprint erasure in PGCs. When they arrive at the genital ridge, which occurs by E11.5, PGCs undergo extensive epigenetic reprogramming, including the erasure of parental imprints (FIG. 1). The erasure of imprints is reflected by demethylation of the imprinted loci, which occurs concomitantly with demethylation of other regions32. The erasure occurs at different imprinted loci at different times between E10.5 and E12.5, as shown in a study of cloned embryos that were produced from PGC nuclei37. Since the imprint eras￾ure at each locus is a rapid process that is completed within one day of development, this might be an active demethylation process32. In somatic cells of female mammals, one of the two X chromosomes is inactivated so that the dosage of the genes on this chromosome is equalized between males and females. The inactive X chromosome is reactivated during female germ-cell development. It had been thought that this reactivation occurs around the time of imprint erasure38,39. However, more recent studies showed that it is initiated in the migratory stage40 or at an even earlier stage41. So, X-chromosome reactiva￾tion occurs progressively over a prolonged period and is completed in post-migratory PGCs. The initiation of reactivation in migrating PGCs is reminiscent of the X reactivation in inner-cell-mass cells of female blastocysts, but the mechanisms of these processes are yet to be clarified. Not all sequences show DNA demethylation in post-migratory PGCs. For example, DNA methylation of the intra-cisternal A particle (IAP) retrotransposon family is only partially reprogrammed42. Incomplete removal of epigenetic marks in the germ line can lead to epigenetic inheritance from one generation to the next, evidence of which is now accumulating in both mice and humans43–45. Together with the fact that epigenetic marks can be influenced by environmental factors, it has been suggested that this phenomenon could provide a basis for adaptive evolution and/or heritable disease susceptibility without changes in DNA sequence43–45 (BOX 2). Imprinting in the male and female germline. Once the parental imprints have been erased, new imprints must be re-established according to the gender of the animal. This re-establishment occurs only after sex determina￾tion has been initiated, and male and female germ-cell development diverges to give rise to sperm or oocytes, respectively. In mice, the gonads of males and females become morphologically distinguishable by E12.5. In female embryos, germ cells arrest in meiotic prophase at around E13.0, whereas male germ cells enter into G1-phase mitotic arrest at a similar time (FIGS 1,2). A number of environmental cues, including retinoic acid signals from the mesonephroi46, determine the timing of entry into meiosis by germ cells. G1-arrested male germ cells are called prosper￾matogonia or gonocytes (FIG. 1). Paternal methylation imprints, which have been identified at just three loci, are progressively established in these cells between E14.5 and the newborn stage47–51. A germline-specific gene-knockout study indicated that the de novo DNA methyltransferase, DNMT3A, has a central role in the de novo methylation process of all known paternally methylated loci, and another de novo methyltrans￾ferase, DNMT3B, is involved only at the Rasgrf1 (RAS protein-specific guanine nucleotide-releasing factor 1) locus50,52. The reason why Rasgrf1 requires an addi￾tional enzyme is unknown, but this could be related to the presence of several retrotransposon sequences at this locus (see below). The establishment of paternal methylation imprints at all loci requires another mem￾ber of the Dnmt3 family, DNMT3L, which is highly expressed in prospermatogonia50,52–54. This protein has no DNA-methyltransferase activity but forms a complex with DNMT3A and/or DNMT3B and stimulates their activities. The established methylation imprints are then main￾tained throughout the rest of male germ-cell develop￾ment. Notably, germline stem (GS) cells — which are established from neonatal testes, can be maintained stably in culture and can give rise to sperm when transplanted into testes — possess paternal methyla￾tion imprints, whereas their multipotent derivatives, mGS cells, show partial demethylation at these sites, similar to ES cells55. GS cells and mGS cells provide invaluable tools for germ-cell study and reproductive engineering. In the female germline, the initiation of DNA￾methylation imprinting occurs after birth, during the oocyte growth56,57. The growing oocytes are at the diplo￾tene stage of meiotic prophase I, and the de novo methyl￾ation process is complete by the fully-grown oocyte stage (FIG. 1). Both DNMT3A and DNMT3L also have essential roles in this process52,58,59, but DNMT3B seems dispensable52. Recent studies have started to provide some clues on the mechanism by which the DNMT3A–DNMT3L complex recognizes the imprinted loci (and some ret￾rotransposons, see below). A crystallographic analysis of the complexed C-terminal domains of DNMT3A and DNMT3L revealed a tetrameric structure with two active sites60. This structure suggests that DNA regions Box 1 | Derivation of germ cells from embryonic stem cells Various types of somatic cell, including blood cells and neural cells, have been obtained from embryonic stem (ES) cells in culture dishes. Recent studies have revealed that it is also possible to generate gametes from ES cells108–111. Gametes or gamete-like cells were derived when mouse ES cells were cultured under various differentiation conditions including simple monolayer culture (oocyte)108, embryoid￾body formation (sperm)109, embryoid-body formation followed by treatment with retinoic acid (sperm)110 and retinoic acid induction alone (sperm)111. In the most successful case, ES-derived sperm cells were able to fertilize oocytes after intracytoplasmic injection and support embryonic development to term111. The resultant pups, however, had abnormalities in DNA methylation at imprinted loci and survived only up to five months, indicating that reprogramming of the germ-cell genome was not properly accomplished. When we fully understand the mechanisms of germ-cell reprogramming, we might be able to derive appropriately reprogrammed, functional gametes from cultured cells, which will allow new approaches to reproductive engineering, although ethical and safety issues must be carefully considered. R E V I E W S 132 | february 2008 | volume 9 www.nature.com/reviews/genetics © 2008 Nature Publishing Group