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Functional dynamics of H3K9 methylation during meiotic prophase progression

2007; Springer Nature; Volume: 26; Issue: 14 Linguagem: Inglês

10.1038/sj.emboj.7601767

1460-2075

Makoto Tachibana, Masami Nozaki, Naoki Takeda, Yoichi Shinkai,

DNA Repair Mechanisms

Article28 June 2007free access Functional dynamics of H3K9 methylation during meiotic prophase progression Makoto Tachibana Corresponding Author Makoto Tachibana Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Masami Nozaki Masami Nozaki Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan Search for more papers by this author Naoki Takeda Naoki Takeda Center for Animal Resources and Development, Kumamoto University, Kumamoto, Japan Search for more papers by this author Yoichi Shinkai Corresponding Author Yoichi Shinkai Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Makoto Tachibana Corresponding Author Makoto Tachibana Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Masami Nozaki Masami Nozaki Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan Search for more papers by this author Naoki Takeda Naoki Takeda Center for Animal Resources and Development, Kumamoto University, Kumamoto, Japan Search for more papers by this author Yoichi Shinkai Corresponding Author Yoichi Shinkai Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Author Information Makoto Tachibana 1, Masami Nozaki2, Naoki Takeda3 and Yoichi Shinkai 1 1Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan 2Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan 3Center for Animal Resources and Development, Kumamoto University, Kumamoto, Japan *Corresponding authors: Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. Tel.: +81 75 751 3991; Fax: +81 75 751-3991; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2007)26:3346-3359https://doi.org/10.1038/sj.emboj.7601767 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Histone H3 lysine 9 (H3K9) methylation is a crucial epigenetic mark of heterochromatin formation and transcriptional silencing. G9a is a major mammalian H3K9 methyltransferase at euchromatin and is essential for mouse embryogenesis. Here we describe the roles of G9a in germ cell development. Mutant mice in which G9a is specifically inactivated in the germ-lineage displayed sterility due to a drastic loss of mature gametes. G9a-deficient germ cells exhibited perturbation of synchronous synapsis in meiotic prophase. Importantly, mono- and di-methylation of H3K9 (H3K9me1 and 2) in G9a-deficient germ cells were significantly reduced and G9a-regulated genes were overexpressed during meiosis, suggesting that G9a-mediated epigenetic gene silencing is crucial for proper meiotic prophase progression. Finally, we show that H3K9me1 and 2 are dynamically and sex-differentially regulated during the meiotic prophase. This genetic and biochemical evidence strongly suggests that a specific set of H3K9 methyltransferase(s) and demethylase(s) coordinately regulate gametogenesis. Introduction In all eukaryotes, genetic information is stored as chromatin, which consists of genomic DNA, histones, and a wide array of chromosomal proteins. The most fundamental unit of chromatin is the nucleosome, which is comprised of an octamer of core histones, H2A, H2B, H3, and H4 wrapped around 147 bp of DNA (Luger et al, 1997). The N-terminal tails of core histones are subject to various chemical modifications such as phosphorylation, acetylation, methylation, ubiquitination, and ADP ribosylation. The ‘histone code’ hypothesis (Strahl and Allis, 2000; Turner, 2000; Jenuwein and Allis, 2001) predicts that different modifications at specific amino-acid residues in histones or combinations of these modifications are translated into functionally distinct nuclear processes. For example, lysine methylation on histone H3 or H4 is involved in a variety of distinct biological processes including transcriptional regulation, DNA recombination and repair, heterochromatin formation, and X-chromosome inactivation (Lachner and Jenuwein, 2002; Lachner et al, 2003; Cao and Zhang, 2004). The mechanisms by which epigenetic chromatin modification regulate development, differentiation, and many cellular functions remain unclear. In this regard, germ cell development represents an excellent system for addressing these issues. Germ-lineage cells are the only population from which parental genetic and epigenetic information can be transferred to progeny. It was shown that epigenetic cellular memories were dynamically reorganized during germ cell development (Reik et al, 2001; Seki et al, 2005; Payne and Braun, 2006). In addition, germ cells must pass through meiosis to develop into mature haploid gametes, which requires the transcriptional activation of meiosis-specific genes and the concomitant suppression of other genes that inhibit the meiotic process. Chromosomes in the meiotic prophase also must form a specific structure termed the synaptonemal complex. Genetic analyses have revealed that several chromatin modification enzymes play essential roles in germ cell development and meiotic progression. First, the loss of Dnmt3L, which is a catalytically inactive form of DNA methyltransferase but a key regulator for de novo CpG methylation, causes a meiotic prophase defect specifically in males (Bourc'his and Bestor, 2004; Webster et al, 2005; Hata et al, 2006). Similarly, the catalytically active de novo DNA methyltransferase, Dnmt3a, is essential for male gametogenesis (Kaneda et al, 2004). In the former case at least, meiotic progression defects accompany the reactivation of retrotransposable elements, suggesting that silencing of such widely dispersed DNA elements is required for proper synapsis. Second, meiotic progression defects are also induced in two lines of knockout (KO) mice lacking histone lysine methyltransferases (HKMTases). Male germ cells in mice lacking Suv39h, which catalyzes H3K9 tri-methylation at pericentric heterochromatin, exhibit abnormal synapsis and mis-segregation of chromosomes at the male meiotic prophase (Peters et al, 2001). Meiosis-specific H3K4 tri-methyltransferase, Meisetz, which is involved in transcriptional activation of meiotic genes, is also essential for meiotic progression (Hayashi et al, 2005). Germ cells lacking Meisetz are unable to complete double-stranded break repairs at the pachytene stage in both sexes, possibly due to a defect in the appropriate activation of meiosis essential gene(s). G9a is a major mammalian H3K9 mono- and di-methyltransferase which mainly localizes at euchromatin (Tachibana et al, 2001, 2002; Peters et al, 2003; Rice et al, 2003) and contributes to transcriptional silencing (Ogawa et al, 2002; Tachibana et al, 2002; Shi et al, 2003; Gyory et al, 2004; Nishio and Walsh, 2004; Roopra et al, 2004). Our previous biochemical studies revealed that G9a forms a multiprotein complex, which consists of at least other two polypeptides. G9a-related H3K9 methyltransferase GLP and the zinc-finger protein Wiz were integrated stoichiometrically into active G9a-complexes and formation of this tripartite complex was crucial for H3K9 methyltransferase function in vivo (Tachibana et al, 2005; Ueda et al, 2006). G9a and GLP were both crucial for embryogenesis since deletion of either G9a or GLP leads to embryonic lethality at mid-gestation. However, the ubiquitous expression profile of G9a/GLP/Wiz genes predicts important biological functions in other organs or tissues beyond embryogenesis (Tachibana et al, 2005). To overcome the embryonic lethality of germline G9a deletion, we generated conditional mutant mice in which G9a can be inactivated by the Cre/loxP recombination system in a cell type-specific manner. Here, we focus on the role(s) of G9a in germ cell development, and report that G9a function is essential for meiotic prophase progression. We also present evidence for the genome-wide dynamics of H3K9 methylation in the meiotic prophase, and the potential involvement of specific HKMTase(s) and demethylase(s) in this reorganization of the germ cell epigenome. Results G9a protein expression is restricted to spermatogonia and early leptotene spermatocytes To elucidate the functional role of G9a during germ cell development, we first analyzed G9a protein expression in testis using immunoblot analysis. As shown in Figure 1A, both G9a and GLP were abundant from postnatal day (P) 2 to P11, but signals for both gradually decreased with developmental progression. We used antibodies against PLZF as a marker for undifferentiated A-type spermatogonia (Buaas et al, 2004; Costoya et al, 2004) and the mouse VASA homologue (MVH) as a pan-germ cell marker (most strongly expressed in pachytene spermatocytes and round spermatids) (Toyooka et al, 2000). The expression profile of G9a/GLP was similar to that of PLZF. Taken collectively, these data suggest that G9a/GLP protein expression is not constant, but temporally regulated during spermatogenesis. As it was shown that formation of the G9a/GLP complex is crucial for exerting H3K9 methyltransferase activity in vivo, we examined their interactions in testicular cells and confirmed a stoichiometric interaction of G9a/GLP in testicular cells (Figure 1B). Figure 1.Temporal G9a/GLP expression in germ cell development. (A) Postnatal testicular cells prepared at various stages were subjected to immunoblot analysis. Total amounts of proteins loaded were normalized by determining tubulin content. ES cell lysate (TT2 strain) served as a positive control for G9a/GLP proteins. PLZF and mouse VASA homologue (MVH) were marker proteins for undifferentiated spermatogonia and pan-germ cells, respectively. (B) G9a/GLP complex formation was conserved in testicular cells. (C) Double-immunofluorescent staining profiles with anti-G9a/MVH on P10 testes sections. G9a protein is expressed in a subpopulation of male germ cells (arrowhead). The arrow represents a G9a-negative germ cell. (D) Double-immunofluorescent staining profiles with anti-G9a/PLZF on P10 testes sections. PLZF-positive spermatogonia are G9a positive (arrowhead). A subpopulation of G9a-positive cells is PLZF negative (arrow). (E) Double-immunofluorescent staining profiles with anti-G9a/c-Kit on P10 testes sections. G9a-positive germ cells are c-Kit positive (arrowhead). Scale bars in (C–E), 20 μm. (F) G9a expression profile during male meiosis. G9a protein is detected in only early leptotene (designated as E-leptotene) nuclei. Arrowheads indicate XY bodies. Download figure Download PowerPoint To further confirm developmental dynamics of G9a expression, we carried out immunohistochemical staining using testes sections at P10, which contained germ cells at several developmental stages ranging from undifferentiated spermatogonia to leptotene spermatocytes (Figure 1C–E). Combinational staining with anti-G9a/MVH showed that G9a protein is expressed in germ cells located near the basal lamina, suggesting its expression in spermatogonia (Figure 1C). Double staining with anti-G9a/PLZF indicated that PLZF-positive spermatogonia were all G9a positive, suggesting that G9a protein is expressed in As to Apr spermatogonia (Figure 1D). Notably, there are G9a-positive germ cells that lack PLZF signal. We performed immunostaining analysis with anti-G9a and anti-c-Kit, which is expressed from A1 spermatogonia until the leptotene spermatocyte stage (Schrans-Stassen et al, 1999; Payne and Braun, 2006). As shown in Figure 1E, some G9a-positive germ cells were c-Kit positive, suggesting that the expression of G9a protein is maintained after differentiation into A1 spermatogonia. To examine G9a-expression during meiosis, we carried out immunostaining analysis using meiotic chromosome spreads using antibodies against G9a and synaptonemal complex protein 3 (SCP3), which is a lateral component of the synaptonemal complex. As shown in Figure 1F, expression of G9a protein is highly restricted to the early leptotene stage and the expression was hardly detectable from the leptotene through the diplotene stages of spermatocyte development. We further examined G9a expression in round or elongating spermatids from adult testes, but were unable to detect G9a-positive cells (data not shown). The expression profile of GLP was identical to that of G9a (data not shown). To summarize, G9a/GLP protein is expressed from the undifferentiated spermatogona until the early leptotene spermatocyte stage during spermatogenesis. We also examined G9a/GLP expression during the female meiotic prophase and the kinetics were similar to those observed for male germ cells (Supplementary Figure S1). Germ-lineage specific deletion of G9a produces infertility accompanied with a drastic loss of germ cells in adult gonads To generate germ-lineage-specific conditional G9a-KO mice, we generated a conditionally defective G9a allele containing target sites for the Cre/loxP recombination system (Supplementary Figure S2). Mice carrying the G9aflox mutation were crossed with tissue-nonspecific alkaline phosphatase (TNAP)-Cre knock-in mice, which express the Cre recombinase in primordial germ cells from E9.5 to late gestation (Lomeli et al, 2000). The germ-lineage-specific G9a-KO mice (genotyped as TNAP-Cre, G9aflox/Δ) were obtained by crossing G9aflox/flox females with TNAP-Cre, G9a+/flox males. TNAP-Cre, G9aflox/Δ mice were born in a slightly lower Mendelian ratio, that is, we obtained 30 TNAP-Cre, G9aflox/Δ mice out of 200 progeny, while the expected number was 50. As leaky Cre expression in somatic tissues during embryogenesis is observed for this TNAP-Cre line (Kaneda et al, 2004), we speculate that some of the TNAP-Cre, G9aflox/Δ embryos died due to the inactivation of G9a in non-germ cells. However, the delivered germ-lineage G9a-KO mice were indistinguishable from wild-type (WT) mice in appearance, reached normal adult body size, and lived more than 2 years. To examine fertility in the KO mice, we crossed them with WT animals. There were no apparent defects for the mice genotyped as G9aflox/flox (both alleles active, referred to as WT hereafter), G9aflox/Δ (single allele active), and TNAP-Cre, G9a+/flox mice (single allele active, referred to as heterozygous hereafter) in both sexes, and they were all fertile. In contrast, when the germ-lineage G9a-KO male mice (n=8) were crossed with WT females, no pregnancy features were observed in the partner females. In the case of females, only three out of 22 germ-lineage G9a-KO females were pregnant and delivered pups, whereas the other 19 females did not. The fertility of the three G9a-KO females is listed in Table I. The average number of the pups per a delivery was smaller (3.0) than in WT females (7.1). Moreover, we did not observe any deliveries from these G9a-KO females after 7 months old age, suggesting that germ-lineage-specific G9a-KO females had a paucity of oocytes, even when they were fertile. Importantly, some of the progeny from the G9a-KO females carried the G9aflox allele, indicating that TNAP-Cre-mediated depletion of the floxed G9a locus was not complete during female germ cell development (see legend of Table I). Table 1. Fertility of germ cell G9a-KO female mice Pup numbers (mother age at deliverya) #1 7 (2) 3 (3) 4 (4) 3 (5) #2 1 (3) 3 (4) 1 (5) 1 (6) #3 4 (2) Wild-type ♀ 8 (3) 10 (4) 7 (5) 9 (6) 4 (8) 6 (9) 6 (10) a Natural mating was started using germ KO female mice at 1 month old age and wild-type fertile males at 4 months old age, and then the mating was kept until the female reached to 1 year old age. Out of 22 females, only three delivered pups described above. Average numbers per a delivery are 3.0 for KO (27/9) and 7.1 for wild-type (50/7) mice. Out of 27 offspring from KO females, five carried G9aflox allele. To examine G9a protein expression in the germ-lineage of these animals, we performed immunocytochemical analyses on embryonic or postnatal gonads (Figure 2A and B). Although G9a protein was ablated in the vast majority of germ cells in E12.5 females and males (Figure 2A and not shown) and P7 males (Figure 2B), we observed a subpopulation of germ cells that remained G9a positive. The ratio of G9a-positive versus -negative germ cells is summarized in Table II. The efficiencies of G9a depletion were 80–90% in both sexes at E12.5. In contrast, the depletion efficiencies of P7-spermatogonia reached nearly 100%. The high efficiency of G9a depletion at P7 compared with E12.5 may derive from prolonged exposure of the conditional allele to Cre enzyme. Figure 2.Loss of germ cells in G9a-KO gonads. (A–B) Depletion of G9a protein in G9a-KO gonads. Enzymatically dissociated cells from E12.5 female gonads (A) and P7 testes (B) were squashed onto slide glass and stained with the indicated antibodies. Calculated efficiencies of G9a depletion are shown in Table II. Arrows and arrowheads indicate germ cells and control somatic cells, respectively. Bars, 10 μm. (C) Gross morphology of wild-type (WT) and germ cell G9a-KO (KO) testes at 3-month old age. (D) Testes weight of WT and KO mice at 3 months of age. The average weights were 98 and 15 mg, respectively. Error bars represent standard deviation. (E) Histological examination of WT and KO testes from 3-month-old mice. Paraffin-embedded sections were stained with hematoxylin and eosine. Seminiferous tubules devoid of germ cells were indicated by an asterisk. Leptotene-like spermatocytes and spermatogonia were indicated by arrowheads and arrows, respectively. Scale bars, 100 μm. (F) Histological examination of WT and KO ovaries at 2 months of age. Paraffin-embedded sections were stained with hematoxylin and eosine. The KO ovary was significantly smaller than that of WT mice. Only one oocyte was recognized in a section of KO ovary (arrowhead). Scale bar, 200 μm. (G) Gross numbers of oocytes per ovary at 2 months of age. Download figure Download PowerPoint Table 2. Efficiency of G9a depletion by TNAP-Cre at several developmental stages Genotype Stage sex Cells examined Ratio of depletion (%) MVH+/G9a+ MVH+/G9a− Het #1 E12.5 ♀ 50 0 — Het #2 E12.5 ♀ 50 0 — KO #1 E12.5 ♀ 9 41 82 KO #2 E12.5 ♀ 7 43 86 MVH+/G9a+ MVH+/G9a− Het #3 E12.5 ♂ 50 0 — Het #4 E12.5 ♂ 50 0 — KO #3 E12.5 ♂ 5 45 90 KO #4 E12.5 ♂ 8 42 84 PLZF+/G9a+ PLZF+/G9a− Het #5 P7 ♂ 300 0 — Het #6 P7 ♂ 300 0 — KO #5 P7 ♂ 2 298 99.3 KO #6 P7 ♂ 7 293 97.7 Enzymatically dissociated cells from embryonic or postnatal gonads were squashed onto slide glass, and then were stained with antibodies against germ cell marker protein and G9a as described in Figure 2A and B. To assess the cause of infertility, we dissected gonads from the G9a-KO adult mice. As shown in Figure 2C and D, adult testes of KO males were markedly smaller than those of WT. Histological examinations showed that the majority (∼70%) of seminiferous tubules in the KO testes lacked any type of germ cells, and the remainder (∼30%) contained only a few spermatogonia and leptotene-like cells (Figure 2E). A typical result from histological analysis of a KO female ovary is shown in Figure 2F. The numbers of maturating and primordial oocytes were greatly reduced (only one oocyte per section) in comparison with those of WT (more than 10 per section). To evaluate these defects quantitatively, we counted the gross numbers of oocytes by scanning serial sections of adult ovaries (Figure 2G). The WT female contained 520 and 777 ooctyes per ovary, whereas those from four independent KO females were one- or two orders fewer in magnitude (2 and 2, 13 and 4, 55 and 59, 2 and 5). Thus, we conclude that infertility in the germ-lineage-specific G9a-KO mice stems from a complete loss or a drastic reduction of sperm or oocytes, respectively. Developmental defects at the pachytene stage in G9a-KO male germ cells To determine the development stage of germ cells perturbed by the G9a deficiency, we examined the first round of spermatogenesis using juvenile testes (Bellve et al, 1977) (Figure 3A). In P7 testes, difference between heterozygous and KO tubules was not apparent, suggesting that differentiation into spermatogonia was normal. Importantly, we observed nearly complete depletion of G9a in the PLZF-positive spermatogonia at this stage (Figure 2B and Table II). In contrast, seminiferous tubules in the KO testes were apparently abnormal at P14 and P21, exhibiting a consistent increase in apoptotic cell death by TUNEL analysis (data not shown). A subpopulation of heterozygous spermatocytes at P14 had differentiated into the pachytene stage of meiotic prophase, while apparent pachytene spermatocytes were hardly detectable in KO tubules. In P21 heterozygous testes, subpopulations of germ cells had completed meiosis and differentiated into haploid spermatids; however, KO tubules at P21 completely lacked spermatids. These data strongly suggest that the G9a mutation affects meiotic progression. To determine more precisely when meiosis was blocked in KO testes, we examined several time points for the appearance of apoptotic cells. As shown in Figure 3B, apoptotic nuclei were frequently detected in KO tubules in which spermatocytes developed into the early pachytene stage based on their light microscopic characteristics. These data indicate that meiosis was aborted during the early pachytene stage in G9a-KO male germ cells. Figure 3.Developmental defects at the pachytene stage in G9a-KO male germ cells. (A) Paraffin-embedded sections (3 μm) of testes at various developmental time points (postnatal day (P) 7, and 14, and 21) were prepared and stained with hematoxylin. Pachytene spermatocytes at P14 and haploid spermatids at P21 in heterozygous testes were indicated by arrow and arrowhead, respectively. Scale bar, 50 μm. (B) Magnification of KO tubules. Apoptotic nuclei were detected both at P14 (top) and P21 (bottom) in KO tubules (arrows). Black and white arrowheads indicate Intermediate spermatogonia and early pachytene spermatocytes. Scale bar, 50 μm. (C–D) Histogram of male meiotic prophase stages at P14 (C) and P21 (D) between heterozygous and KO spread preparations. Developmental stages were classified by staining profiles with anti-SCP3/γH2AX. Bars represent the average of two samples per genotype (>150 cells were analyzed per sample). <Pachytene represents incomplete pachytene spermatocyte. (E–F) Perturbed synapsis formation in KO spermatocyte. P14 spermatocytes were stained with a combination of anti-SCP3/γH2AX (E) and anti-SCP1/SCP3 antibodies (F). Typical pachytene spermatocytes from heterozygous testes and incomplete pachytene spermatocytes (<Pachytene) from KO testes were represented. Arrowheads indicate XY bodies. (G) Immunofluorescence staining profile with anti-Rad51 antibodies in KO spermatocytes. At the pachytene stage, the signals of Rad51 protein were retained only at the XY body in heterozygous nuclei (arrowhead). In contrast, the signals were still retained on asynapsed chromosomes in KO spermatocyte (right). Download figure Download PowerPoint To further confirm the block in meiotic prophase, the frequency of the meiotic stage from leptotene until diplotene spermatocytes in juvenile testes were calculated (Figure 3C and D). Spermatocytes were classified by their immnunofluorescent staining profiles with antibodies against SCP3 and histone H2AX phosphorylated on serine 139 (γH2AX), which marks sites of double-strand breaks (Mahadevaiah et al, 2001). As shown in Figure 3C, we could not detect full pachytene spermatocytes in P14 KO testes, whereas more than 30% of spermatocytes reached the full pachytene stage in heterozygous testes. Despite the lack of full pachytene spermatocytes, incomplete pachytene spermatocytes were frequently observed in KO testes. Diplotene spermatocytes emerged at P21 in heterozygous testes but were not observed in KO testes (Figure 3D). Next, we examined details of the meiotic defects in G9a-KO testis (P14) using testicular cell spreads. At the leptotene and zygotene stages, there was no apparent difference in staining profiles between the G9a heterozygous and KO spermatocytes using anti-SCP3/γH2AX (data not shown). However, perturbed synapsis formation was easily detected in G9a-KO spermatocytes at more advanced pachytene stage (Figure 3E). The autosomes in heterozygous pachytene spermatocytes were fully synapsed and characterized as entire thick SCP3 distribution and negative for γH2AX (left panel). In contrast, G9a-KO spermatocytes contained a mixture of fully synapsed, partially synapsed, and asynapsed chromosomes (the asynapsed portion is characterized as thin SCP3 signal with massive γH2AX accumulation) (right panel). We further confirmed these incomplete synaptonemal complex (SC) formation by double staining with antibodies against SCP3/SCP1, which is a central component of the SC localized at synapsed regions of chromosomes. As shown in Figure 3F, G9a-KO spermatocytes again contained both synapsed (SCP3/1 double positive) and asynapsed chromosomes (SCP3 single positive). We rarely identified spermatocytes with such incomplete SC formation in heterozygous mice (summarized in Figure 3C and D). To examine whether the double-stranded break (DSB) repair pathway progresses normally in KO spermatocytes, we performed double staining analysis with anti-SCP3/Rad51 (Figure 3G). Signals corresponding to Rad51 protein were detected as numerous foci along all meiotic chromosomes in KO spermatocytes at the zygotene stage (left panel), which was indistinguishable from that observed in heterozygotes (not shown). These data suggest that the loading of Rad51 protein onto early meiotic chromosomes occurs normally in the KO spermatocytes. However, Rad51 protein persisted on asynapsed chromosomes in incomplete pachytene spermatocytes from KO testes (right panel). These data as well as γH2AX staining profiles suggest that the processing of DSB repair is incomplete in asynapsed meiotic chromosomes of KO spermatocytes. Collectively, our data indicate that the loss of G9a arrests meiosis in male germ cells at the early pachytene stage and is characterized by disordered progression of SC formation. G9a deficiency leads to a global loss of H3K9me2 and 1 but not H3K9me3 at male meiotic prophase To elucidate the contribution of G9a to H3K9 methylation during the meiotic prophase, we examined the levels and kinetics of H3K9 mono-, di-, and tri-methylation in male meiotic nuclei. As shown in Figure 4A and C, G9a-heterozygous spermatocytes from the early leptotene until the zygotene stage displayed disperse nuclear staining profile for H3K9me2 and me1. Unexpectedly, these signals disappeared in pachytene spermatocytes even in the heterozygous testes (right panels in Figure 4A and C). As the meiotic prophase proceeds without an S phase, the rapid loss of H3K9me marks at this stage might be induced by an active mechanism rather than a passive one such as histone dilution accompanied by DNA synthesis. Importantly, H3K9me2/1 signals were hardly detectable in the G9a-KO nucleus at any stages of the meiotic prophase (Figure 4B and D). Figure 4.The kinetics of H3K9 methylation during male meiotic prophase. (A–B) Kinetics of H3K9me2 in male meiotic prophase. Meiotic nuclear spreads were prepared from P14 testes of heterozygous (A) and KO (B) male mice, and the status of H3K9me2 was monitored in combination with anti-SCP3 antibody. (C–D) Kinetics of H3K9me1 in male meiotic prophase in nuclei of heterozygous (C) and KO spermatocytes (D). (E–F) Kinetics of H3K9me3 in male meiotic prophase in nuclei of heterozygous (E) and KO spermatocytes (F). Download figure Download PowerPoint Considering the restricted expression of G9a/GLP proteins in germ cells from spermatogonia to early leptotene spermatocytes (Figure 1), it seems likely that the H3K9me2/1 marks are added before initiation of synapsis and are maintained up to the completion of synapsis. Subsequently, these marks are rapidly and actively removed at the pachytene stage. In contrast, H3K9me3 kinetics was distinct from those of me2/1. Large blocks of H3K9me3 signals were not erased until the pachytene stage (Figure 4E), as shown previously (Peters et al, 2001). The H3K9me3 signals in G9a-KO nuclei were indistinguishable from heterozygous nuclei (Figure 4F), indicating that most H3K9me3 is G9a independent in spermatocytes, similar to what was observed previously in ES cells (Peters et al, 2003; Rice et al, 2003). Loss of germ cells during meiotic prophase in G9a-KO female gonads To investigate whether the loss of G9a also affects meiosis in females, we assessed the reduction of germ cells at several developmental stages in G9a-KO embryonic ovaries. Figure 5A shows ovarian sections stained with anti-MVH antibodies at different developmental periods. Primordial oocytes at the day of birth (DOB) were very few in KO ovaries (squares in right panels), suggesting that the lack of maturating oocytes in adults ovaries derives, at least in part, from a profound reduction in germ cel