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{"project":"2_test","denotations":[{"id":"17447844-16580996-85805848","span":{"begin":349,"end":351},"obj":"16580996"},{"id":"17447844-16581510-85805849","span":{"begin":571,"end":573},"obj":"16581510"},{"id":"17447844-9499427-85805850","span":{"begin":1379,"end":1381},"obj":"9499427"},{"id":"17447844-15051956-85805851","span":{"begin":1382,"end":1384},"obj":"15051956"},{"id":"17447844-11591824-85805852","span":{"begin":1722,"end":1724},"obj":"11591824"},{"id":"17447844-16581510-85805853","span":{"begin":2295,"end":2297},"obj":"16581510"},{"id":"17447844-16581510-85805854","span":{"begin":4072,"end":4074},"obj":"16581510"},{"id":"17447844-16809775-85805855","span":{"begin":4075,"end":4077},"obj":"16809775"},{"id":"17447844-15536132-85805856","span":{"begin":4078,"end":4080},"obj":"15536132"},{"id":"17447844-9488723-85805857","span":{"begin":5185,"end":5187},"obj":"9488723"},{"id":"T2977","span":{"begin":349,"end":351},"obj":"16580996"},{"id":"T9467","span":{"begin":571,"end":573},"obj":"16581510"},{"id":"T72574","span":{"begin":1379,"end":1381},"obj":"9499427"},{"id":"T14498","span":{"begin":1382,"end":1384},"obj":"15051956"},{"id":"T43820","span":{"begin":1722,"end":1724},"obj":"11591824"},{"id":"T23359","span":{"begin":2295,"end":2297},"obj":"16581510"},{"id":"T49356","span":{"begin":4072,"end":4074},"obj":"16581510"},{"id":"T34832","span":{"begin":4075,"end":4077},"obj":"16809775"},{"id":"T3513","span":{"begin":4078,"end":4080},"obj":"15536132"},{"id":"T32462","span":{"begin":5185,"end":5187},"obj":"9488723"}],"text":"Abnormal Sex Chromatin in Dmrt7 Mutant Germ Cells\nAlthough the XY body can form during pachynema in Dmrt7 mutants, we considered the possibility that transcriptional silencing might not be properly established. This would be consistent with the Dmrt7 phenotype: pachytene cells that escape from MSCI normally are eliminated prior to late-pachytene [17]. Recently, MSCI has been shown to continue into meiosis II and spermiogenesis, apparently mediated by a distinct chromatin compartment termed postmeiotic sex chromatin (PMSC) that is established starting in diplonema [16]. We therefore asked whether the pachytene germ cell death in Dmrt7 mutants is associated with a failure either to initiate or to maintain sex chromosome inactivation.\nFirst, we examined the mid-pachytene XY body. To examine XY transcriptional status, we carried out Cot-1 RNA fluorescence in situ hybridization (FISH) to detect nascent RNA polymerase II transcription, combined with DAPI staining to locate the XY body on spreads of seminiferous tubules (Figure 8A and 8B). In Dmrt7 mutants, the XY body was formed and excluded Cot-1 hybridization (Figure 8B), indicating that transcriptional silencing is established normally in mutant pachytene cells. We also examined expression of the Y-linked gene Rbmy, which normally is inactivated during pachytene and reactivated after secondary meiosis begins [54,55]. Rbmy was inactivated normally in pachytene cells of Dmrt7 mutants, based on immunofluorescent staining with an anti-RBMY antibody (Figure S5). We also examined heterochromatin protein 1 beta (HP1β), which normally localizes to the X centromere at mid-pachynema and then spreads through the XY body as it internalizes during diplonema [56]. We found that HP1β localization is normal in DMRT7 mutant cells in mid-pachynema (Figure 8C and 8D). These results suggest that XY body formation and initiation of MSCI both occur normally in Dmrt7 mutant germ cells.\nFigure 8 Chromatin Abnormalities in Dmrt7 Mutant Diplotene Germ Cells\n(A and B) MSCI occurs normally in mid-pachytene spermatocyte of Dmrt7 mutant. Arrows indicate XY body in all panels. Cot-1 RNA FISH (red) reveals normal silencing of sex chromosome transcription in XY body. This is consistent with wild-type mid-pachynema as described previously [16].\n(C and D) HP1β localization in mid-pachytene spermatocytes. HP1β localizes to X chromosome centromere (arrowhead) in wild-type (C) and mutant (D).\n(E and F) Cot-1 hybridization in diplotene. Presumptive XY body, based on DAPI and γH2AX localization, is indicated by arrow in (F).\n(G and H) HP1β in diplotene. Wild-type cell (E) has HP1β throughout XY body, whereas mutant cell (H) only has localization to X chromosome centromere (arrowhead).\n(I and J) H3-2meK9 localization in diplotene cells. Mutant cell (J) lacks strong concentration of this mark to the XY body seen in wild-type (I).\n(K and L) H3-3meK9 localization in diplotene cells. Mutant cell (L) has no localization of this mark to the XY body. γH2AX localizes to autosomes in mutant cell, possibly indicating onset of apoptosis.\n(M) Example of mutant diplotene cell with normal HP1β accumulation to the XY body.\nAll images except those in (M) are single Z sections. We next considered the possibility that sex chromatin is established normally but is not properly modified as cells exit pachynema and begin to form PMSC. Although most Dmrt7 mutant cells are eliminated by apoptosis prior to diplonema, we were able to examine epigenetic markers of PMSC in rare Dmrt7 mutant spermatocytes that escaped pachytene arrest and progressed into diplonema. First, we examined nascent transcription by Cot-1 hybridization. Although heterochromatic regions generally showed lower Cot-1 signal than euchromatic regions (Figure 8E and 8F), in some mutant cells the sex chromatin appeared to be incompletely silenced relative to wild-type (Figure 8F). We also examined three epigenetic signatures of PMSC: histone H3 dimethylated or trimethylated at lysine-9 (H3-2meK9, H3-3meK9) and spreading of HP1β through the XY body [16,57,58] (S. H. Namekawa, unpublished data). We observed defects in sex chromatin localization of all three markers in Dmrt7 diplotene cells. Although HP1β localization to the X chromosome centromere initially appeared normal at mid-pachynema, we observed Dmrt7 mutant diplotene cells that failed to show spreading of HP1β to the entire XY body (Figure 8G and 8H). Similarly, we found Dmrt7 mutant diplotene cells lacking accumulation of H3-2meK9 and H3-3meK9 marks onto the sex chromatin (Figure 8I–8L).\nNot all Dmrt7 mutant diplotene cells showed abnormal localization of HP1β to the sex chromatin (Figure 8M). In one experiment, 11/27 mutant cells in diplonema lacked HP1β on the XY body, as compared with 2/22 wild-type cells. We hypothesize that the mutant cells with normal HP1β may be those that can complete meiosis (Figures 4 and 5). Some of the mutant diplotene cells showing abnormal sex chromatin also had abnormal autosomal γH2AX staining (Figure 8L). γH2AX localizes to double-strand DNA breaks, so this staining may indicate that some diplotene mutant cells are approaching or entering apoptosis [59]. We did not observe sex chromatin defects prior to diplonema, but we cannot exclude the possibility that earlier defects exist and the affected cells are rapidly eliminated.\nIn the preceding experiments, we staged cells based on XY body internalization. Because this process could be abnormal in the mutant cells, we also staged mutant cells by chromosome morphology using an antibody to SYCP3 (Figure 9). This independently identified Dmrt7 mutant diplotene cells lacking HP1β accumulation in the XY body, such as the example in Figure 9B. From these results, we conclude that Dmrt7 mutant cells establish a normal XY body in mid-pachynema, but then have multiple epigenetic defects in the sex chromatin transition from pachynema to diplonema.\nFigure 9 Abnormal Sex Chromatin in Cells Staged by Chromosome Pairing Status\n(A) Spread of wild-type germ cell stained with DAPI, anti-SYCP3, and anti-HP1β showing chromosome morphology typical of diplonema and internalized XY body with HP1β accumulation.\n(B) Spread of Dmrt7 mutant germ cell showing normal diplotene chromosome morphology and internalized XY body, but no HP1β accumulation in the XY body.\nXY chromosome pairs are indicated by arrow."}

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PMC:1853120 / 30107-36492 JSON TXT