REVIEWS Box 3 Production of bi-maternal mice genes was upregulated in EHMT2-deficient germ uggesting that silencing of these genes by EhMT2 ocyte-derived genomes and no sperm-derived mediated H3K9 mono-and dimethylation might be genome using tricks(see below)to adjust the expression levels of imprinted genes sential for proper synapsis. ne mice were ini eferred to as parthenogenetic but are now called bi-maternal A functional link between H3K4 methylation and mice. The success relied on the fact that the number of loci that are imprinted in the male germ cells(the paternal meiosis-specific gene expression has also been demon- ed loci) is smaller(only three are known)than hat of the maternally imprinted loci(more than ten). In their previous experiments, strated.PRDM9(PR domain-containing 9, also known eggs were reconstituted so that they have one genome from fully grown(imprinted) as Meisetz) is an H3K4 trimethyltransferase that causes ocytes and the other from newbom or rowing(non-imprinted or default transcriptional activation. It is specifically expressed in ocytes, early meiotic germ cells both in the testis and ovary, and (bottom, middle)developed better than the control parthenogenetic embryos(second analysis of Prdmg-null mice revealed that it is necessary from the right) for three embryonic days. The extended development was probably for synapsis and recombination of homologous chromo due to the fact that, in the reconstituted eggs, all loci except the three paternally somes during meiotic prophase. In PRDM9-deficient imprinted loci from the newborn oocytes had appropriate imprints. To adjust the spermatocytes, expression of a number of autosomal expression levels of the three loci, non-growing oocytes were obtained from mice in genes, including those that are specifically expressed which either one (gf2 (insulin-like growth factor 2 ))or two(af2 and Dik/GtlZ)of the in meiotic germ cells, was repressed. The results sug- ternally imprinted loci were genetically engineered so that the genes in these loci are gest that the genes that are activated by the function of propriately expressed. These engineered non-growing oocytes were then used to reconstitute eggs. This resulted in a low(0.5%, one locus) or surprisingly high (30%, PRDM9 might include those that are involved in the fter correction for the expected frequency of the desired genotype, both loci) synapsis of homologous chromosomes and/or recom- production rate of adult bi-maternal mice(second from the left). The results clearly bination. However, it is also possible that H3K4me3 indicate that imprinting is the main barrier to parthenogenesis in mammals and that that is introduced by PRDM9, well as H3K9me and sperm-derived RNAs or proteins are unnecessary for full development. H3K9me2 that are mediated by EHMT2, is important PGC and non-growing oocyte for specific chromosomal structures that are required for events such as the search for homologous chromo- Imprinting somes and synapsis. Further studies are needed to test Default Maternal imprints 10 loci) Genetic Mechanisms of meiotic sex-chromosome inactivation In male germ cells, X and Y chromosomes undergo syn apsis only within the pseudo-autosomal region during prophase I. Their chromatin is subsequently condensed Male to form a macrochromatin body that is called the XY or sex body and the chromosomes become transcription termal imprints (3 loci) ally silent. This form of X-chromosome inactivation is called meiotic sex-chromosome inactivation(MSCI (FIGS 1.4), and might be necessary for synapsis of the Parthenogenetic Androgenetic pseudo-autosomal regions 3. It is also suggested that the XY body might mask asynapsed axes of non-pseudo- autosomal regions to prevent sensing by the pachytene heckpoint machinery and subsequent meiotic arrest MSCI is mediated by some key molecules including the histone H2A variant, H2AX, and its regulatory pro Developmental potential teins. 8. During the leptotene phase of meiosis, H2AX PGC, primordial germ celL. that is phosphorylated at Ser139(YH2AX), which is known to recruit the DNA repair machinery to damaged chromatin is localized at sites of dna double-strand changes in modification of pericentric chromatin by breaks. yH2AX subsequently disappears from autosomes SUV39H might be necessary for the proper progression by pachytene when synapsis is completed. yH2AX accu- of meiotic prophase. Abnormalities of female germ cells mulates in the XY body at the zygotene-pachytene transi have also been observed in these double mutants, but tion, and analysis of H2AX-deficient mice demonstrated the molecular details of these defects are unknown. that this is essential for XY-body formation, MSCI and A recent study showed that H3K9 mono-and synapsis between the sex chromosomes. The functional dimethylation by EHMT2(euchromatic histone-lysine importance and mechanisms of H2AX phosphorylation N-methyltransferase 2, also known as G9a)is also are suggested by studies of additional molecules such essential for early meiotic progression". A germ-cell- as ATR (ataxia telangiectasia and RAD3-related)and specific homozygous mutation of Ehmt2 caused arrest the tumour suppressor BRCAl(breast cancer 1). ATR, of meiosis at the early pachytene stage, and synapsis a member of the Pl3-like kinase family, co-localizes between homologous chromosomes was not properly with yH2AX only on X and Y chromosomes at the formed in either testis or ovary. In the mutant sper- onset of their inactivation. Studies of BRCAl-deficient matocytes, most H3K9me and H3K9me2 signals were spermatocytes indicated that localization of ATR on lost, but H3K9me3 was unaffected. Expression of some XY chromatin and proper formation of the XY body NATURE REVIEWS GENETICS @2008 Nature Publishing Groupchanges in modification of pericentric chromatin by SUV39H might be necessary for the proper progression of meiotic prophase. Abnormalities of female germ cells have also been observed in these double mutants79, but the molecular details of these defects are unknown. A recent study showed that H3K9 mono- and dimethylation by EHMT2 (euchromatic histone-lysine N-methyltransferase 2, also known as G9a) is also essential for early meiotic progression80. A germ-cell￾specific homozygous mutation of Ehmt2 caused arrest of meiosis at the early pachytene stage, and synapsis between homologous chromosomes was not properly formed in either testis or ovary. In the mutant sper￾matocytes, most H3K9me and H3K9me2 signals were lost, but H3K9me3 was unaffected. Expression of some genes was upregulated in EHMT2-deficient germ cells, suggesting that silencing of these genes by EHMT2- mediated H3K9 mono- and dimethylation might be essential for proper synapsis. A functional link between H3K4 methylation and meiosis-specific gene expression has also been demon￾strated81. PRDM9 (PR domain-containing 9, also known as Meisetz) is an H3K4 trimethyltransferase that causes transcriptional activation. It is specifically expressed in early meiotic germ cells both in the testis and ovary, and analysis of Prdm9-null mice revealed that it is necessary for synapsis and recombination of homologous chromo￾somes during meiotic prophase. In PRDM9-deficient spermatocytes, expression of a number of autosomal genes, including those that are specifically expressed in meiotic germ cells, was repressed. The results sug￾gest that the genes that are activated by the function of PRDM9 might include those that are involved in the synapsis of homologous chromosomes and/or recom￾bination. However, it is also possible that H3K4me3 that is introduced by PRDM9, as well as H3K9me and H3K9me2 that are mediated by EHMT2, is important for specific chromosomal structures that are required for events such as the search for homologous chromo￾somes and synapsis. Further studies are needed to test this possibility. Mechanisms of meiotic sex-chromosome inactivation. In male germ cells, X and Y chromosomes undergo syn￾apsis only within the pseudo-autosomal region during prophase I. Their chromatin is subsequently condensed to form a macrochromatin body that is called the XY or sex body, and the chromosomes become transcription￾ally silent82. This form of X-chromosome inactivation is called meiotic sex-chromosome inactivation (MSCI) (FIGS 1,4), and might be necessary for synapsis of the pseudo-autosomal regions83. It is also suggested that the XY body might mask asynapsed axes of non-pseudo￾autosomal regions to prevent sensing by the pachytene checkpoint machinery and subsequent meiotic arrest83. MSCI is mediated by some key molecules including the histone H2A variant, H2AX, and its regulatory pro￾teins83,84. During the leptotene phase of meiosis, H2AX that is phosphorylated at Ser139 (γH2AX), which is known to recruit the DNA repair machinery to damaged chromatin, is localized at sites of DNA double-strand breaks. γH2AX subsequently disappears from autosomes by pachytene when synapsis is completed. γH2AX accu￾mulates in the XY body at the zygotene–pachytene transi￾tion, and analysis of H2AX-deficient mice demonstrated that this is essential for XY‑body formation, MSCI and synapsis between the sex chromosomes83. The functional importance and mechanisms of H2AX phosphorylation are suggested by studies of additional molecules such as ATR (ataxia telangiectasia and RAD3-related) and the tumour suppressor BRCA1 (breast cancer 1). ATR, a member of the PI3-like kinase family, co-localizes with γH2AX only on X and Y chromosomes at the onset of their inactivation84. Studies of BRCA1-deficient spermatocytes indicated that localization of ATR on XY chromatin and proper formation of the XY body Nature Reviews | Genetics Female Male PGC and non-growing oocyte Imprinting Imprinting Maternal imprints (> 10 loci) Paternal imprints (3 loci) Genetic engineering Fertilized Bi-maternal Parthenogenetic Androgenetic Developmental potential > > > > Default Default PGC Box 3 | Production of bi-maternal mice Kono et al. produced mice with two oocyte-derived genomes and no sperm-derived genome using tricks (see below) to adjust the expression levels of imprinted genes65,66. The mice were initially referred to as parthenogenetic but are now called bi-maternal mice. The success relied on the fact that the number of loci that are imprinted in the male germ cells (the paternally imprinted loci) is smaller (only three are known) than that of the maternally imprinted loci (more than ten). In their previous experiments, eggs were reconstituted so that they have one genome from fully grown (imprinted) oocytes and the other from newborn or non-growing (non-imprinted or default) oocytes, using a serial nuclear-transfer technology that they devised. These eggs (bottom, middle) developed better than the control parthenogenetic embryos (second from the right) for three embryonic days116. The extended development was probably due to the fact that, in the reconstituted eggs, all loci except the three paternally imprinted loci from the newborn oocytes had appropriate imprints. To adjust the expression levels of the three loci, non-growing oocytes were obtained from mice in which either one (Igf2 (insulin-like growth factor 2)) or two (Igf2 and Dlk/Gtl2) of the paternally imprinted loci were genetically engineered so that the genes in these loci are appropriately expressed. These engineered non-growing oocytes were then used to reconstitute eggs. This resulted in a low (0.5%, one locus)65 or surprisingly high (30%, after correction for the expected frequency of the desired genotype, both loci)66 production rate of adult bi-maternal mice (second from the left). The results clearly indicate that imprinting is the main barrier to parthenogenesis in mammals and that sperm-derived RNAs or proteins are unnecessary for full development. PGC, primordial germ cell. R E V I E W S nature reviews | genetics volume 9 | february 2008 | 135 © 2008 Nature Publishing Group