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RESEARCH COMMUNICATION
1 Department of Genetics, SOKENDAI, Mishima, Shizuoka 411-8540, Japan; 2 Division of Mammalian Development, National Institute of Genetics, Mishima 411-8540, Japan; 3 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Minato-ku, Tokyo 105-0011, Japan
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Abstract |
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[Keywords: Germ cell; meiosis; nanos; retinoic acid]]
Received September 5, 2007; revised version accepted December 7, 2007.
; Allegrucci et al. 2005).
Recently, it has been reported that retinoic acid (RA) induces Stra8, an RA-responsive gene, in female germ cells, which then leads to meiotic initiation in the embryonic ovary. In contrast, RA signaling has been shown to be inhibited in fetal testes by Cyp26b1, a RA-metabolizing enzyme highly expressed in the somatic cells of the testes (Baltus et al. 2006; Bowles et al. 2006; Koubova et al. 2006). This event is thought to be a key step in the development of the male germ cell. However, it is still unclear how male germ cells continue to escape from entry into meiosis throughout embryogenesis, given that Cyp26b1 expression gradually decreases during the later stages of embryonic development (Bowles et al. 2006; Koubova et al. 2006). In addition, although the mechanisms that trigger the sexual differentiation of the female germ cells are beginning to be revealed, the molecules that promote the male germ cell fate have remained largely unknown up until now.
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Results and Discussion |
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Previously, we identified Nanos2 as a male PGC-specific gene involved in the maintenance of the male germ cells. In the absence of Nanos2, these cells undergo apoptosis from embryonic day 15.5 (E15.5), and most are subsequently lost by birth (Tsuda et al. 2003). This anti-apoptotic function of Nanos also appears to be conserved in several animals (Subramaniam and Seydoux 1999; Sato et al. 2007). However, during embryogenesis, mouse Nanos2 is specifically expressed only in the male germ cells after they have colonized the genital ridges (such cells are often referred to as male gonocytes), where they undergo cell cycle arrest at G1/G0. We speculated that Nanos2 would be involved in this mitotic arrest and, to test this hypothesis, we examined the mitotic activity of male gonocytes by double-immunostaining with anti-phosphorylated histone H3 (pH3, an M-phase marker) and TRA98 (a germ cell marker) (Fig. 1A–D). Although no significant pH3-positive cells could be detected in either Nanos2+/– or Nanos2–/– male gonocytes at E14.5 (Fig. 1A,B), pH3-positive gonocytes became detectable from E15.5 in Nanos2–/– mouse embryos only (Fig. 1D). These data indicate that Nanos2-null male gonocytes appear to undergo cell cycle arrest normally at E14.5 but fail to maintain this arrest state and reinitiate proliferation from E15.5.
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). Our immunohistological analyses revealed that germ cells in the Nanos2–/– testes abnormally express Scp3 at E15.5 (Fig. 1H), whereas very few cells were positive for this protein in the Nanos2+/– male gonad (Fig. 1G). Nanos2-null male gonocytes at this embryonic stage present at both the inside and outside of the tubules due to the active migration of these cells from these structures, as we have reported previously (Tsuda et al. 2003). We found in our current analyses that these cells express Scp3 independently of their location. The Scp3 signals were very low and only marginally detectable in both Nanos2+/– and Nanos2–/– testes at E14.5 (Fig. 1E,F), which suggested that male gonocytes initiate meiosis from E15.5 in the absence of Nanos2.
Upon the progression of normal meiosis, precursors of the synaptonemal complex show characteristic structures at the zygotene stage known as axial cores (Yuan et al. 2000). However, although the expression of Scp3 is highly up-regulated in Nanos2–/– mice, we observed that almost all of the male gonocytes had morphologically abnormal nuclei and that only a few cells showed axial core formation, a clear indication of meiosis (Fig. 1H, inset). We speculated therefore that Nanos2-null male gonocytes may have entered meiosis but subsequently may have undergone apoptotic cell death prior to axial core formation. To test this possibility, we knocked out the proapoptotic gene, Bax (Knudson et al. 1995; Stallock et al. 2003), together with Nanos2 to suppress apoptosis. We first confirmed that apoptosis was indeed suppressed in these double knockouts by assaying activated caspase3. In Nanos2+/–Bax+/– and Nanos2+/– Bax–/– mice, there were no apoptotic signals detected (Fig. 1I,J). Nanos2–/–Bax+/– mice showed numerous apoptotic male gonocytes (Fig. 1K), similar to Nanos2–/– mice (Tsuda et al. 2003). However, such apoptotic signals were undetectable in the Nanos2–/–Bax–/– background (Fig. 1L). In these double knockouts, many male gonocytes formed axial cores (Fig. 1P), and analysis of chromosome preparations showed that some of these are at the zygotene stage (Fig. 1Q), whereas others were at early pachytene stage (Fig. 1R) and show distinct threads representative of paired chromosomes. Moreover, these cells also expressed Dmc1, a meiosis-specific mammalian RecA homolog and a marker of meiotic cells from the leptotene to zygotene stages (Fig. 1S–U; Pittman et al. 1998; Chuma and Nakatsuji 2001). In contrast to these findings, little or no expression of Scp3 was observed in either Nanos2+/–Bax+/– or Nanos2+/– Bax–/– mice (Fig. 1M,N), and the Nanos2–/–Bax+/– male gonocyte phenotype (Fig. 1O) was similar to that of the Nanos2-single-null mouse (Fig. 1H). Hence, our current data indicate that one of the functions of Nanos2 is to block the male gonocytes from entry into meiosis, which might be the cause of the observed apoptotic response in the knockout animals. However, we cannot at present exclude the possibility that apoptosis is induced independently of abnormal meiosis in these cells.
The relationship between Nanos2 and RA signaling
Our initial data raise two key questions: (1) What is the mechanism by which Nanos2 suppresses meiosis in male gonocytes, and (2) what is the underlying reason for the meiotic entry delay in Nanos2-null male gonocytes compared with their female counterparts? RA signaling is known to be responsible for the induction of germ cell meiosis in the developing ovary, but is inhibited in the fetal testes by Cyp26b1. It has also been reported that abnormal meiosis is induced in Cyp26b1-null male gonocytes. These cells fail to suppress RA signaling, which results in the up-regulation of Stra8, leading to meiotic entry with a similar timing to female meiosis from E13.5 (Baltus et al. 2006; Bowles et al. 2006; Koubova et al. 2006; MacLean et al. 2007).
To evaluate the functions of Nanos2 in the context of RA signaling, we first examined the Stra8 expression levels in Nanos2-null male gonocytes. Although this protein was suppressed in normal male gonocytes, an expected up-regulation was observed for Stra8 in E15.5 Nanos2-null male gonocytes (Fig. 2A–F). We next compared the level and timing of this up-regulation between Nanos2-null testes, wild-type embryonic ovaries, and Cyp26b1-null testes by real-time RT–PCR. In the embryonic ovaries, the Stra8 expression levels were transiently up-regulated from E13.5 to E14.5, and then down-regulated to undetectable amounts after E15.5 (Fig. 2G, green). Similarly, in the Cyp26b1-null testes, the up-regulation of Stra8 was observed from E13.5 to E14.5 at levels comparable with the embryonic ovaries (Fig. 2G, orange). In contrast, Stra8 expression was very low in Nanos2-null testes up to E13.5 but found to be increased from E14.5 and was maintained at higher levels at least until E16.5 (Fig. 2G, red). Very little expression of this gene was evident in normal testes (Fig. 2G, dark blue and blue).
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Meiosis is inhibited in Nanos2-expressing female germ cells
To further clarify the function of Nanos2 in the suppression of meiosis, we attempted to ectopically express this gene in female embryonic germ cells by generating a transgenic mouse line. We initially established a conditional transgenic mouse line, CAG-loxP-CAT-loxP-Flag-tagged-Nanos2, that is capable of expressing Flag-Nanos2 under the control of the CAG promoter after excision of the floxed CAT gene by Cre recombinase. We utilized Nanos3-Cre knock-in mice to induce Flag-Nanos2 expression in the germ cell lineage of the transgenic progeny. By crossing with Nanos3-Cre mice, Cre/loxP-specific CAT excision occurs in Nanos3-expressing cells, and this would have been expected to result in the induction of Flag-tagged Nanos2 expression in the germ cell lineage. Nanos3-cre activity was detected at E7.75 by β-gal reporter activity, and this signal was restricted in the germ cells during both the migration stage and after their entry into the gonad (our unpublished observation). Unfortunately, however, Cre recombinase was found to have activated in only a portion of the PGCs in this Nanos3-cre line (the efficiency is <25%), but we could confirm an appreciable level of Flag-tagged Nanos2 expression in some populations of female germ cells in the double transgenic mice, CAG-CAT-Flag-Nanos2/Nanos3-cre (Fig. 3A,B). Flag-Nanos2 could be detected in the cytoplasm of these female germ cells, and a similar expression pattern was also detectable by anti-Nanos2 antibody staining (Supplemental Fig. S1).
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). The expression of Stra8 is induced in female germ cells from E12.5, transiently increases at E13.5–14.5, and decreases thereafter (Fig. 2G; Menke et al. 2003). However, we could not detect Stra8 in female germ cells expressing Nanos2 at E13.5 (Fig. 3F–H), indicating that Nanos2 can block meiosis by suppressing Stra8 expression. The mechanisms underlying the suppression of Stra8 by Nanos2 are currently under investigation in our laboratory. However, we found in our current experiments that the transcription of Stra8 is up-regulated in Nanos2–/– male gonocytes and down-regulated in Nanos2-expressing female germ cells (Supplemental Fig. S3). Therefore, given that Nanos2 is an RNA-binding protein localized in the cytoplasm, the up- or down-regulation of Stra8 transcription would need to be an indirect effect of Nanos2.
Nanos2 induces male-type differentiation in female germ cells
In addition to the suppression of meiosis, we observed an enlarged nuclear morphology in Nanos2-expressing female germ cells and a reduction in their heterochromatic regions in a similar manner to male gonocytes (Fig. 3C). This prompted us to speculate that these female germ cells might have adopted a male germ cell fate. It has been shown that the chromatin structure in the germ cells changes according to their sexual differentiation, and this can be monitored by the methylation status of the histone proteins. One of the markers associated with this is the dimethylation of histone H3-Lys9 (H3K9me2) (Seki et al. 2005). In wild-type male gonocytes, decreased levels of methylation are evident in comparison with the surrounding somatic cells (Supplemental Fig. S4A–F), but we never observed this in Nanos2-null male gonocytes (Supplemental Fig. S4G–J). In Nanos2-expressing female germ cells, a decrease in H3K9me2 immunoreactivity was also observed (Fig. 3I–K). To further characterize these Nanos2-positive female germ cells, we performed immunostaining analysis of two molecules that are normally expressed in the male gonocytes only. Tudor domain-containing 1 (TDRD1) is a component of chromatoid bodies/nuages in male germ cells. It has been reported that the expression of this protein increases from E13.5 in male gonocytes, but is maintained at much lower levels in female germ cells (Chuma et al. 2003). As reported previously, we occasionally observed weaker signals in the Nanos2-negative female germ cells, but TDRD1 was detectable as discernible granules in the cytoplasm of Nanos2-positive female germ cells (Fig. 3L–N). Dnmt3L is implicated in genomic imprinting and is expressed in male gonocytes from E14.5 to E18.5, but its expression is initiated only after birth in female germ cells (Bourc’his et al. 2001; Bourc’his and Bestor 2004; Sakai et al. 2004). Strikingly, we observed a strong nuclear expression of Dnmt3L only in Nanos2-expressing female germ cells (Fig. 3O–Q).
It is noteworthy that Nanos2 expression was induced by Nanos3-cre activity in the transgenic mice, resulting in the continuous expression of Nanos2 from as early as E7.75 (our unpublished data). Therefore, the early expression of Nanos2 might account for the fate change of female germ cells. However, when Nanos2 expression was induced under the direct control of the Oct3/4-
PE enhancer, the resulting female mice produced normal oocytes and were fertile (Suzuki et al. 2007). This must be due to the property of the Oct3/4-PE enhancer, which is known to become inactive in the female germ cells after E13.5. Therefore, we conclude that the phenotype we observed in the Nanos2-expressing female germ cells is due to Nanos2 expression after E13.5, and not due to the expression of this protein in the earlier stages.
These findings suggest, therefore, that Nanos2 converts female germ cells to male-type germ cells under our experimental conditions. In agreement with this possibility, the expression of TDRD1 and Dnmt3L was lowered or not induced in Nanos2-null male gonocytes (Supplemental Fig. S5), indicating that these cells cannot initiate normal male pathways in the absence of Nanos2. These findings, together with our results showing the ectopic induction of meiosis, suggested that the male gonocytes had acquired a female germ cell fate. Although we did not observe oocyte-like cells at any stage in the Nanos2/Bax-null testes, we did observe the up-regulation of genes that are required only for female germ cell development, even in the Nanos2–/– testis at E16.5 (Supplemental Fig. S6). This suggests that Nanos2-null male gonocytes have become feminized. The number of germ cells began to reduce gradually after birth, and finally disappeared by 4 wk, even in the Nanos2/Bax-double-null background (Supplemental Fig. S7). We reasoned from this that the germ cells are unable to retain their female characteristics because the surrounding somatic cells in the Nanos2-null testes are not themselves feminized.
Hypothetical model for the sexual differentiation of mouse germ cells
Based on our current results, we devised a working model for the sexual differentiation of mouse germ cells, which is outlined in Figure 4. Both Cyp26b1 and Nanos2-null mice show similar ectopic Stra8 expression phenotypes and the initiation of male gonocyte meiosis. However, the timing of the relevant events is different in these knockouts; in the Cyp26b1-null testes, Stra8 induction occurs at E13.5, which is a similar stage to embryonic ovaries, but does not do so until E14.5 in the Nanos2-null testes (Fig. 4A). The difference in timing between Cyp26b1-null and Nanos2-null male gonocytes is due to different mechanisms operating therein (Fig. 4B). Somatically produced RA strongly induces Stra8 in female germ cells (Fig. 4B, left), which also occurs in Cyp26b1-null male gonocytes. In contrast, Cyp26b1 inhibits RA signaling in normal embryonic testes (Fig. 4B, middle). However, its expression is gradually reduced after E13.5, whereas the high levels of RA are maintained in the male embryonic mesonephros (Bowles et al. 2006). Nanos2 then begins to be expressed at the point Cyp26b1 is down-regulated (Fig. 4B, right). That is why Stra8 is induced in Nanos2-null male gonocytes after E14.5.
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Materials and methods |
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Knockout and transgenic mice
The Nanos2-knockout mouse lines have been established previously. The Baxtm1Sjk mutant mouse was purchased from the Jackson Laboratory. The generation of the Nanos3-Cre knock-in mice will be described elsewhere (our unpublished data). The CAG-loxP-CAT-lox-Flag-tagged-Nanos2 transgenic mouse lines were established using a conventional transgenic technique.
Histological methods
For immunostaining, the gonads of both sexes were directly embedded in O.C.T. compound and frozen in liquid nitrogen. The samples were then sectioned and fixed in 4% PFA. After preincubation with 3% skim milk, the sections were stained with each primary antibody and followed by the appropriate secondary antibody.
Real-time RT–PCR
Total RNA isolates from male gonads were prepared using an RNeasy Mini Kit (Qiagen). One microgram of total RNA was then used for cDNA synthesis by SuperScript III reverse transcriptase (Invitrogen). SYBR Premix Ex-Taq (Takara) was used for the real-time PCR experiments according to the manufacturer’s instructions.
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Acknowledgments |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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Footnotes |
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E-MAIL ysaga{at}lab.nig.ac.jp; FAX 055-981-6828. 
Supplemental material is available at http://www.genesdev.org.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1612708
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References |
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