(a and b) RNA/DNA FISH showing strong 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 RNA FISH signal in which the locus (arrow) lies within the sex body–like γH2AX domain (white outline). (e and f) An H1t-positive spermatocyte showing transcription. (g and h) A pachytene spermatocyte with multiple γH2AX domains that is strongly expressing /. (i and j) A pachytene spermatocyte with no RNA FISH signal with the locus within the sex body–like γH2AX-positive domain. (k and l) An H1t-positive spermatocyte showing / 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.<p><b>Copyright information:</b></p><p>Taken from "Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation"</p><p></p><p>The Journal of Cell Biology 2008;182(2):263-276.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483523.</p><p></p

(a and b) A rare pachytene spermatocyte with asynapsis restricted to what are probably the X and Y axes

ATR has been recruited to the asynapsed axes and has spread to the associated chromatin, and there is phosphorylation of H2AX throughout the associated chromatin. (c–f) With increasing asynapsis, the ATR becomes more focal and axially restricted, and the γH2AX staining becomes weaker and progressively more fragmented. Arrows indicate axes with partner exchange indicative of nonhomologous synapsis. (g and h) Staining of a spermatogenic cell squash preparation for the midpachytene marker H1t and for γH2AX shows that the majority of H1t-positive cells have fragmented γH2AX staining; only rare cells have a single sex body–like γH2AX-positive domain (bottom insets). In the control (top insets), cells with fragmented γH2AX staining (presumed to be zygotene) are H1t negative, whereas all H1t-positive spermatocytes have a single γH2AX-positive sex body (the small H1t-positive cells are round spermatids). (i) Quantitation of the whole nuclear γH2AX signal for rare pachytene spermatocytes with only the X and Y axes asynapsed and for pachytene spermatocytes with increasing levels of asynapsis shows that the amount of γH2AX does not increase in response to asynapsis (fitted blue regression line). The projected increase in γH2AX signal if it was in proportion to the amount of asynapsed axis is denoted by the red line. Bars: (a–f) 10 μm; (g and h) 15 μm.<p><b>Copyright information:</b></p><p>Taken from "Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation"</p><p></p><p>The Journal of Cell Biology 2008;182(2):263-276.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483523.</p><p></p

(a) Pachytene cell showing unsynapsed h21 (arrow), which is positive for BRCA1

(b) Pachytene cell with self-synapsed h21, which is negative for BRCA1. (c and d) The unsynapsed h21 is also positive for ATR and γH2AX (c; inset), whereas the self-synapsed h21 (d) is negative. (e and f) Human Cot1 RNA FISH/chromosome painting (arrows) showing that when h21 lies outside the γH2AX domain, it is transcriptionally active. (g and h) When h21 (arrow in h) lies within the γH2AX domain, it is transcriptionally silenced. (i) Pachytene cell showing RAD51 signals on the unsynapsed h21 and X chromosome; in the inset, the SCYP3 signal has been reduced so that the RAD51 foci are more visible. (j) In late pachytene, RAD51 signals disappear from both chromosomes. (i and j) In the insets, the SCYP3 signal is reduced to emphasize the lack of Rad51 staining. (k and l) In h21 carriers, apoptosis is not increased in stage IV tubules relative to controls (wt; arrows point to TUNEL-positive cells). (a–l) Arrows point to the h21. Bars: (a–d, i, and j) 10 μm; (e–h) 5 μm; (k and l) 40 μm.<p><b>Copyright information:</b></p><p>Taken from "Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation"</p><p></p><p>The Journal of Cell Biology 2008;182(2):263-276.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483523.</p><p></p

(a and b) Cot1 RNA FISH staining detects nascent transcripts within the nucleus; the pseudo–sex body marked by γH2AX is a transcriptionally repressed domain

(c and d) In -null pachytene spermatocytes, as a result of asynapsis/nonhomologous synapsis, the two autosomal loci are nearly always well separated. In the cell shown, there is a single RNA FISH signal (arrows); DNA FISH shows that the second non-transcribing locus lies within the pseudo–sex body domain (white outline). (e and f) A pachytene spermatocyte showing transcription of the Y chromosomal gene when it is not located in the pseudo–sex body. (g and h) A nontranscribing locus lying within the pseudo–sex body. (i–n) RNA/DNA FISH for the / X chromosomal BAC showing two transcribing pachytene spermatocytes with the locus outside the pseudo–sex body and one nontranscribing pachytene spermatocyte in which the locus lies within the pseudo–sex body. (a–n) White outlines indicate the extent of the γH2AX domain before DNA FISH, and arrows point to the FISH signals (either RNA FISH or DNA FISH). Bar, 5 μm.<p><b>Copyright information:</b></p><p>Taken from "Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation"</p><p></p><p>The Journal of Cell Biology 2008;182(2):263-276.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483523.</p><p></p

(a–c) SYCP3 and BRCA1 staining of control spermatocytes showing the appearance of BRCA1 foci on the forming axial elements in leptotene, their loss after synapsis during zygotene, and BRCA1 accumulation on the asynapsed X and Y axes in pachytene

(d) The kinase ATR appears throughout the chromatin of the asynapsed X and Y axes in pachytene of control males, and there is an associated phosphorylation of H2AX (γH2AX), which defines the sex body domain (inset). (e–g) In -null spermatocytes, which lack meiotic DSBs, BRCA1 foci still appear on the axial elements in leptotene, are retained throughout the asynaptic zygotene stage (magnified in insets), and are lost in conjunction with the nonhomologous synapsis apparent in pachytene (highlighted in insets, which show synapsed and asynapsed axes with BRCA1 staining offset). There is concurrent BRCA1 accumulation on a subset of asynapsed axes (arrow) that mark the location of the pseudo–sex body. (h) Staining with ATR or γH2AX (inset) marks the entire chromatin domain of the pseudo–sex body. (i and j) In , leptotene and zygotene spermatocytes, no foci are detected, confirming the specificity of the BRCA1 antibody. (k and l) A -null pachytene spermatocyte stained with SYCP3 and SYCP1. The synapsed regions appear light blue because of colocalization; the pseudo–sex body domain marked with ATR is restricted to the chromatin of asynapsed axes but only encompasses a small proportion of such chromatin. Bars,10 μm.<p><b>Copyright information:</b></p><p>Taken from "Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation"</p><p></p><p>The Journal of Cell Biology 2008;182(2):263-276.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483523.</p><p></p

(a and b) In pachytene spermatocytes, increasing asynapsis is associated with increasingly focal BRCA1 staining

In the two spermatocytes shown, the Y chromosome has failed to synapse with the X chromosome. (c and d) Enlargements of the X (presumed) and Y chromosomes show that BRCA1 covers the axes of both chromosomes from the spermatocyte with only the X and Y asynapsed, but, in the spermatocyte with more extensive asynapsis, the BRCA1 has become more focal on the presumed X and is restricted on the Y chromosome to a single focus. (e) Quantitation of the axially located BRCA1 in pachytene spermatocytes shows that it does not increase with increasing asynapsis (fitted blue regression line). The projected increase in BRCA1 signal if it was in proportion to the amount of asynapsed axis is denoted by the red line. (f) The focal BRCA1 staining seen when asynapsis is extensive is largely DSB associated, as indicated by costaining for RAD51. Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation"</p><p></p><p>The Journal of Cell Biology 2008;182(2):263-276.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483523.</p><p></p