REVIEWS to phenotypic differences in the next generation(see also adopted zao, because null mutants were lethal. Therefore, BOX 3). Furthermore, it has been reported that sperm the current collection of known epigenetic modifiers that amples from oligospermic patients often contain are essential for germ cells is probably just the tip of an DNA-methylation defects at the imprinted loci 0, 1o. iceberg, which suggests that we will need to study many The significance of these findings and also when and more genes. Although conditional knockout continues how these changes arise are important questions for to be a key technology, recently established GS cells, future studies which can be stably maintained in culture, are genetically In oocytes, except for the rapid deacetylation of modifiable and can differentiate to give rise to functional histones H3 and H4 at the germinal-vesicle break- sperm when transplanted into infertile testes, might offer down mentioned above, histone modifications seem an alternative experimental system 03. to remain unchanged throughout meiosis (FIG. 4]. After Recent advances in epigenomic analysis technolo the first meiotic division, ovulation occurs by releasing gies that allow high-resolution mapping of chromatin the secondary oocyte that is arrested at MIl from the modifications across the genome(for example, REF. 29) ovarian follicle. The ovulated MIl oocyte is now ready also show promise for future progress in this field. to fuse with a sperm cell and the second meiotic divi- These technologies will allow us to know the normal sion is completed after fertilization. Subsequently, the epigenomic landscape of germ cells and epigenomic paternal and maternal genomes of the zygote undergo changes that occur in mutants and disease conditions, The oocyte contributes not only the maternal genome modifiers. However, challenges exist in this respect and its associated epigenetic information, but also some such as the amount of germ-cell samples that are avail factors that are required for post-fertilization reprogram- able to researchers. Further improvement of small-scale ming, which are present in either the cytoplasm or the chromatin immunoprecipitation d unbiased dna nucleus. These factors are important for the develop- amplification methods os will make the technologies ment of both fertilized embryos and nuclear-transferred much more useful. Nuclear-transfer technologies, embryos. Identification of the reprogramming factors, which allow the expansion of cells possessing a specific which might include transcription factors and epigenetic epigenetic profile in cloned embryos " and single-cell modifiers, is an important subject for future study. analysis technologies including live cell imaging are also important. To know the whole picture of the epigenetic regulation of germ-cell development, further studies It has become clear that many epigenetic modifiers, as well as improvements of these key technologies are including DNA methyltransferases, histone-modifica- needed. tion enzymes and their regulatory proteins, have essen- Finally, some of the findings described here will have tial roles in germ-cell development. Mutations that affect an immediate impact on studies of human fertility: mice some of these factors cause early germ-cell loss, whereas harbouring mutations in epigenetic modifier genes can mutations that affect the others cause arrest at specific be models for human infertility conditions. However, meiotic stages and subsequent apoptosis. The functions increasing knowledge of epigenetic regulation of germ of these modifiers are either reprogramming of the germ- cell development will affect a wider biomedical area. cell genome, repression or activation of the downstream For example, one goal of current germ-cell research is target genes, or establishment of a chromatin state that is the efficient and stable derivation of functional gametes appropriate for germ-cell-specific events such as chro- from pluripotent stem cells in culture(BOX 1), success of mosome pairing and genetic crossing over. Furthermore, which depends crucially on knowledge of genetic and the functional links between DNA methylation, histone epigenetic mechanisms of germ cells. Together with modifications and even small-RNA metabolism in germ the use of recently described induced pluripotent stem cells are beginning to be understood, as shown by the cells%.10,, such a technology will allow a new approach recent studies on DNMT3L and MILI (REFS 61, 69). for saving endangered species and overcoming infertil- e Some epigenetic modifiers are specifically expressed ity. In addition, epigenetic changes that are induced by germ cells whereas others are more widely expressed; environmental stress and trans-generational epigenetic this has implications for future studies. The crucial inheritance, which could have relevance to many humai roles of germ-cell-specific genes such as Dnmt3L and diseases, will become an exciting area, for which research iess.5%; the importance of the widely expressed genes implications of these studies are improvements in an. Prdm9 were revealed by conventional knockout stud- on germ cells is particularly important. The far-reachin such as Dnmt3a and Ehmt2 became explicit only when mal cloning, livestock husbandry, assisted reproductive a germline-specific conditional knockout strategy was technologies and human health. 4. Allegrucci, C, Thurston, A, Lucas, E. a Young, L pigenetics: a landscape takes shape. Cell 128. and the germline Reproduction 129 germ cells in the mouse embryo during gastrulation. Surani, M. A, Hayashi, K. Hajkova, P Genetic and 5, Ki ato, M. et al. Identification of PGC7, a new gene s of pluripotency. Cell 128, remodels. a sassone-Corsi,PChromatin Nature434,583-58902005 germ cells. Mech. Dev. 113, 91-94(2002). organ, 6. Reik, w. Stability and flexibility of epigenetic gene 9. Saitou. M. Barton. s C. gulation in mammalian development. Nature 447. mice. Nature418,293-300[2002 38 FEBRU www.nature.com/reviews/genetics @2008 Nature Publishing Groupto phenotypic differences in the next generation (see also BOX 3). Furthermore, it has been reported that sperm samples from oligospermic patients often contain DNA-methylation defects at the imprinted loci100,101. The significance of these findings and also when and how these changes arise are important questions for future studies. In oocytes, except for the rapid deacetylation of histones H3 and H4 at the germinal-vesicle break￾down mentioned above, histone modifications seem to remain unchanged throughout meiosis (FIG. 4). After the first meiotic division, ovulation occurs by releasing the secondary oocyte that is arrested at MII from the ovarian follicle. The ovulated MII oocyte is now ready to fuse with a sperm cell and the second meiotic divi￾sion is completed after fertilization. Subsequently, the paternal and maternal genomes of the zygote undergo further reprogramming to acquire ultimate totipotency. The oocyte contributes not only the maternal genome and its associated epigenetic information, but also some factors that are required for post-fertilization reprogram￾ming, which are present in either the cytoplasm or the nucleus102. These factors are important for the develop￾ment of both fertilized embryos and nuclear-transferred embryos. Identification of the reprogramming factors, which might include transcription factors and epigenetic modifiers, is an important subject for future study. Conclusion It has become clear that many epigenetic modifiers, including DNA methyltransferases, histone-modifica￾tion enzymes and their regulatory proteins, have essen￾tial roles in germ-cell development. Mutations that affect some of these factors cause early germ-cell loss, whereas mutations that affect the others cause arrest at specific meiotic stages and subsequent apoptosis. The functions of these modifiers are either reprogramming of the germ￾cell genome, repression or activation of the downstream target genes, or establishment of a chromatin state that is appropriate for germ-cell-specific events such as chro￾mosome pairing and genetic crossing over. Furthermore, the functional links between DNA methylation, histone modifications and even small-RNA metabolism in germ cells are beginning to be understood, as shown by the recent studies on DNMT3L and MILI (REFS 61,69). Some epigenetic modifiers are specifically expressed in germ cells whereas others are more widely expressed; this has implications for future studies. The crucial roles of germ-cell-specific genes such as Dnmt3L and Prdm9 were revealed by conventional knockout stud￾ies58,59,81; the importance of the widely expressed genes such as Dnmt3a and Ehmt2 became explicit only when a germline-specific conditional knockout strategy was adopted52,80, because null mutants were lethal. Therefore, the current collection of known epigenetic modifiers that are essential for germ cells is probably just the tip of an iceberg, which suggests that we will need to study many more genes. Although conditional knockout continues to be a key technology, recently established GS cells, which can be stably maintained in culture, are genetically modifiable and can differentiate to give rise to functional sperm when transplanted into infertile testes, might offer an alternative experimental system103. Recent advances in epigenomic analysis technolo￾gies that allow high-resolution mapping of chromatin modifications across the genome (for example, REF. 29) also show promise for future progress in this field. These technologies will allow us to know the normal epigenomic landscape of germ cells and epigenomic changes that occur in mutants and disease conditions, and to identify the downstream targets of epigenetic modifiers. However, challenges exist in this respect, such as the amount of germ-cell samples that are avail￾able to researchers. Further improvement of small-scale chromatin immunoprecipitation104 and unbiased DNA￾amplification methods105 will make the technologies much more useful. Nuclear-transfer technologies, which allow the expansion of cells possessing a specific epigenetic profile in cloned embryos37, and single-cell analysis technologies including live cell imaging are also important. To know the whole picture of the epigenetic regulation of germ-cell development, further studies as well as improvements of these key technologies are needed. Finally, some of the findings described here will have an immediate impact on studies of human fertility: mice harbouring mutations in epigenetic modifier genes can be models for human infertility conditions. However, increasing knowledge of epigenetic regulation of germ￾cell development will affect a wider biomedical area. For example, one goal of current germ-cell research is the efficient and stable derivation of functional gametes from pluripotent stem cells in culture (BOX 1), success of which depends crucially on knowledge of genetic and epigenetic mechanisms of germ cells. Together with the use of recently described induced pluripotent stem cells106,107, such a technology will allow a new approach for saving endangered species and overcoming infertil￾ity. In addition, epigenetic changes that are induced by environmental stress and trans-generational epigenetic inheritance, which could have relevance to many human diseases, will become an exciting area, for which research on germ cells is particularly important. The far-reaching implications of these studies are improvements in ani￾mal cloning, livestock husbandry, assisted reproductive technologies and human health. 1. Goldberg, A. D., Allis, C. D. & Bernstein, E. Epigenetics: a landscape takes shape. Cell 128, 635–638 (2007). 2. Surani, M. A., Hayashi, K. & Hajkova, P. Genetic and epigenetic regulators of pluripotency. Cell 128, 747–762 (2007). 3. Morgan, H. D., Santos, F., Green, K., Dean, W. & Reik, W. Epigenetic reprogramming in mammals. Hum. Mol. Genet. 14, R47–R58 (2005). 4. Allegrucci, C., Thurston, A., Lucas, E. & Young, L. Epigenetics and the germline. Reproduction 129, 137–149 (2005). 5. Kimmins, S. & Sassone-Corsi, P. Chromatin remodelling and epigenetic features of germ cells. Nature 434, 583–589 (2005). 6. Reik, W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447, 425–432 (2007). 7. Ginsburg, M., Snow, M. H. & McLaren A. Primordial germ cells in the mouse embryo during gastrulation. Development 110, 521–528 (1990). 8. Sato, M. et al. Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells. Mech. Dev. 113, 91–94 (2002). 9. Saitou, M., Barton, S. C. & Surani, M. A. A molecular programme for the specification of germ cell fate in mice. Nature 418, 293–300 (2002). R E V I E W S 138 | february 2008 | volume 9 www.nature.com/reviews/genetics © 2008 Nature Publishing Group