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Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation.

Mahadevaiah SK, Bourc'his D, de Rooij DG, Bestor TH, Turner JM, Burgoyne PS - J. Cell Biol. (2008)

Bottom Line: In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response.We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs.Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.

View Article: PubMed Central - PubMed

Affiliation: Division of Stem Cell Biology and Developmental Genetics, Medical Research Council National Institute for Medical Research, London NW7 1AA, England, UK.

ABSTRACT
Chromosome synapsis during zygotene is a prerequisite for the timely homologous recombinational repair of meiotic DNA double-strand breaks (DSBs). Unrepaired DSBs are thought to trigger apoptosis during midpachytene of male meiosis if synapsis fails. An early pachytene response to asynapsis is meiotic silencing of unsynapsed chromatin (MSUC), which, in normal males, silences the X and Y chromosomes (meiotic sex chromosome inactivation [MSCI]). In this study, we show that MSUC occurs in Spo11- mouse spermatocytes with extensive asynapsis but lacking meiotic DSBs. In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response. We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs. All four mutants fail to silence the X and Y chromosomes (MSCI failure), which is sufficient to explain the midpachytene apoptosis. Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.

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The majority of Dnmt3l- pachytene spermatocytes are transcribing X or Y genes (MSCI failure). (a and b) RNA/DNA FISH showing strong Zfy2 transcription in a spermatocyte with multiple γH2AX domains (note that some of the γH2AX staining has survived the DNA FISH procedure). (c and d) A rare pachytene spermatocyte with no Zfy2 RNA FISH signal in which the Zfy2 locus (arrow) lies within the sex body–like γH2AX domain (white outline). (e and f) An H1t-positive spermatocyte showing Zfy2 transcription. (g and h) A pachytene spermatocyte with multiple γH2AX domains that is strongly expressing Ddx3x/Usp9x. (i and j) A pachytene spermatocyte with no Ddx3x RNA FISH signal with the locus within the sex body–like γH2AX-positive domain. (k and l) An H1t-positive spermatocyte showing Ddx3x/Usp9x transcription. (a–l) White outlines indicate the extent of the γH2AX domain before the DNA FISH, and arrows point to the FISH signals (either RNA FISH or DNA FISH). Bar, 5 μm.
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fig5: The majority of Dnmt3l- pachytene spermatocytes are transcribing X or Y genes (MSCI failure). (a and b) RNA/DNA FISH showing strong Zfy2 transcription in a spermatocyte with multiple γH2AX domains (note that some of the γH2AX staining has survived the DNA FISH procedure). (c and d) A rare pachytene spermatocyte with no Zfy2 RNA FISH signal in which the Zfy2 locus (arrow) lies within the sex body–like γH2AX domain (white outline). (e and f) An H1t-positive spermatocyte showing Zfy2 transcription. (g and h) A pachytene spermatocyte with multiple γH2AX domains that is strongly expressing Ddx3x/Usp9x. (i and j) A pachytene spermatocyte with no Ddx3x RNA FISH signal with the locus within the sex body–like γH2AX-positive domain. (k and l) An H1t-positive spermatocyte showing Ddx3x/Usp9x transcription. (a–l) White outlines indicate the extent of the γH2AX domain before the DNA FISH, and arrows point to the FISH signals (either RNA FISH or DNA FISH). Bar, 5 μm.

Mentions: In the majority of Dnmt3l−/− pachytene cells, the X and/or Y chromatin was not incorporated within a clear MSUC domain, so we expected that there would be extensive MSCI failure. Once again, we assessed this by gene-specific RNA FISH for Y-located Zfy2 and X-located Ddx3x/Usp9x, the judgement as to whether the cells were in pachytene being made based on the DAPI and γH2AX staining before visualization of the RNA FISH signal. As was the case in Spo11- males, Zfy2 was inappropriately expressed in the majority of these cells (53/55; 96%), including all six cells that we felt confident were pachytene cells based on the DAPI staining despite their having markedly fragmented γH2AX staining (Fig. 5, a and b). In the two cells that failed to express Zfy2, DNA FISH showed that the Zfy2 locus lay within a sex body–like γH2AX-positive domain (Fig. 5, c and d). Similarly, the majority of cells (33/43; 77%) were inappropriately expressing Ddx3x/Usp9x, including all four with fragmented γH2AX staining (Fig. 5, g and h). In the 10 nonexpressing cells, the Ddx3x/Usp9x locus lay within a sex body–like γH2AX domain (Fig. 5, i and j). As a further confirmation that many of the spermatocytes with fragmented γH2AX staining are pachytene cells and that these cells have MSCI failure, we repeated the Ddx3x/Usp9x analysis exclusively on cells with markedly fragmented γH2AX staining using relaxed selection criteria that included cells from late zygotene through to pachytene; 29/52 (56%) of these cells proved to be expressing Ddx3x/Usp9x.


Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation.

Mahadevaiah SK, Bourc'his D, de Rooij DG, Bestor TH, Turner JM, Burgoyne PS - J. Cell Biol. (2008)

The majority of Dnmt3l- pachytene spermatocytes are transcribing X or Y genes (MSCI failure). (a and b) RNA/DNA FISH showing strong Zfy2 transcription in a spermatocyte with multiple γH2AX domains (note that some of the γH2AX staining has survived the DNA FISH procedure). (c and d) A rare pachytene spermatocyte with no Zfy2 RNA FISH signal in which the Zfy2 locus (arrow) lies within the sex body–like γH2AX domain (white outline). (e and f) An H1t-positive spermatocyte showing Zfy2 transcription. (g and h) A pachytene spermatocyte with multiple γH2AX domains that is strongly expressing Ddx3x/Usp9x. (i and j) A pachytene spermatocyte with no Ddx3x RNA FISH signal with the locus within the sex body–like γH2AX-positive domain. (k and l) An H1t-positive spermatocyte showing Ddx3x/Usp9x transcription. (a–l) White outlines indicate the extent of the γH2AX domain before the DNA FISH, and arrows point to the FISH signals (either RNA FISH or DNA FISH). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2483523&req=5

fig5: The majority of Dnmt3l- pachytene spermatocytes are transcribing X or Y genes (MSCI failure). (a and b) RNA/DNA FISH showing strong Zfy2 transcription in a spermatocyte with multiple γH2AX domains (note that some of the γH2AX staining has survived the DNA FISH procedure). (c and d) A rare pachytene spermatocyte with no Zfy2 RNA FISH signal in which the Zfy2 locus (arrow) lies within the sex body–like γH2AX domain (white outline). (e and f) An H1t-positive spermatocyte showing Zfy2 transcription. (g and h) A pachytene spermatocyte with multiple γH2AX domains that is strongly expressing Ddx3x/Usp9x. (i and j) A pachytene spermatocyte with no Ddx3x RNA FISH signal with the locus within the sex body–like γH2AX-positive domain. (k and l) An H1t-positive spermatocyte showing Ddx3x/Usp9x transcription. (a–l) White outlines indicate the extent of the γH2AX domain before the DNA FISH, and arrows point to the FISH signals (either RNA FISH or DNA FISH). Bar, 5 μm.
Mentions: In the majority of Dnmt3l−/− pachytene cells, the X and/or Y chromatin was not incorporated within a clear MSUC domain, so we expected that there would be extensive MSCI failure. Once again, we assessed this by gene-specific RNA FISH for Y-located Zfy2 and X-located Ddx3x/Usp9x, the judgement as to whether the cells were in pachytene being made based on the DAPI and γH2AX staining before visualization of the RNA FISH signal. As was the case in Spo11- males, Zfy2 was inappropriately expressed in the majority of these cells (53/55; 96%), including all six cells that we felt confident were pachytene cells based on the DAPI staining despite their having markedly fragmented γH2AX staining (Fig. 5, a and b). In the two cells that failed to express Zfy2, DNA FISH showed that the Zfy2 locus lay within a sex body–like γH2AX-positive domain (Fig. 5, c and d). Similarly, the majority of cells (33/43; 77%) were inappropriately expressing Ddx3x/Usp9x, including all four with fragmented γH2AX staining (Fig. 5, g and h). In the 10 nonexpressing cells, the Ddx3x/Usp9x locus lay within a sex body–like γH2AX domain (Fig. 5, i and j). As a further confirmation that many of the spermatocytes with fragmented γH2AX staining are pachytene cells and that these cells have MSCI failure, we repeated the Ddx3x/Usp9x analysis exclusively on cells with markedly fragmented γH2AX staining using relaxed selection criteria that included cells from late zygotene through to pachytene; 29/52 (56%) of these cells proved to be expressing Ddx3x/Usp9x.

Bottom Line: In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response.We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs.Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.

View Article: PubMed Central - PubMed

Affiliation: Division of Stem Cell Biology and Developmental Genetics, Medical Research Council National Institute for Medical Research, London NW7 1AA, England, UK.

ABSTRACT
Chromosome synapsis during zygotene is a prerequisite for the timely homologous recombinational repair of meiotic DNA double-strand breaks (DSBs). Unrepaired DSBs are thought to trigger apoptosis during midpachytene of male meiosis if synapsis fails. An early pachytene response to asynapsis is meiotic silencing of unsynapsed chromatin (MSUC), which, in normal males, silences the X and Y chromosomes (meiotic sex chromosome inactivation [MSCI]). In this study, we show that MSUC occurs in Spo11- mouse spermatocytes with extensive asynapsis but lacking meiotic DSBs. In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response. We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs. All four mutants fail to silence the X and Y chromosomes (MSCI failure), which is sufficient to explain the midpachytene apoptosis. Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.

Show MeSH
Related in: MedlinePlus