MOESM4 of Silencing markers are retained on pericentric heterochromatin during murine primordial germ cell development

Additional file 4. Summary of the immunosignals at pericentric heterochromatin of PGCs. The table displays whether a certain histone modification or chromatin-binding protein at the pericentric heterochromatin is detected (+), not detected (−) or detected in some but not all (*) nuclei of PGCs at the embryonic stages indicated. Differences in the degree of enrichment between the pericentric heterochromatin of PGCs and somatic cells are only taken into account in the last column, whereby less enrichment or more enrichment at pericentromeric heterochromatin in PGCs compared to the soma is indicated as , respectively. n.d.: not determined

MOESM2 of Silencing markers are retained on pericentric heterochromatin during murine primordial germ cell development

Additional file 2. Immunofluorescent analysis of HP1β in paraffin sections using regular and extended fixation protocols. A Using the regular fixation protocol, HP1β signal is enriched at DAPI (blue)-dense regions of E10.5 and E11.5 PGCs and somatic cells. HP1β is then substantially reduced in E13.5 female and male germ cells. B With the extended fixation protocol, HP1β signal is retained in pericentric heterochromatin of PGCs throughout development. For each stage, two embryos were analysed per fixation protocol and at least 20 nuclei were recorded. E10.5 and E11.5 PGCs were marked with OCT4 (red). E13.5 male and female germ cells were identified by the presence of TRA98 (red). Representative images are shown with germ cells highlighted by dashed yellow circles, and scale bars represent 5 μm

SPO11-dependent and -independent RAD51 foci in mouse meiocytes.

<p>(A–C) The number of RAD51 foci decreases from leptotene to zygotene in <i>Spo11^+/+</i> and <i>Spo11^+/YF</i> spermatocytes, whereas a few foci are detected in <i>Spo11^YF/YF</i> spermatocytes and oocytes at both stages. (A–B) Double immunostaining with anti-SYCP3 (red), anti-RAD51 (green) of spermatocyte (A) and oocyte (B) nuclei from <i>Spo11^+/+</i> (A–B, left panel) and <i>Spo11^YF/YF</i> (A–B, right panel) mice. Arrowheads point to RAD51 foci in <i>Spo11^YF/YF</i> spermatocytes and oocytes, both leptotene and zygotene. Extensive accumulation of RAD51 along axial elements of one or few chromosomes (arrows) can be observed in both <i>Spo11^+/+</i> and <i>Spo11^YF/YF</i> oocyte nuclei (B, lower panel). Size bars represent 10 µm. (C) The number of RAD51 foci was counted in <i>Spo11^+/+</i>, <i>Spo11^+/YF</i>, and <i>Spo11^YF/YF</i> leptotene and zygotene spermatocytes and oocytes. Each dot represents the focus count of one nucleus. Black bars indicate mean number of foci. P values for the indicated comparisons (Mann-Whitney, two-tailed), and genotypes are indicated in the plot. (D) The number of MLH1 foci in pachytene spermatocyte nuclei was counted in <i>Spo11^+/+</i>and <i>Spo11^+/YF</i> mice. Black bars indicate the mean values. (E) Number of RAD51 foci at E17.5 in <i>Spo11^+/+</i> and <i>Spo11^YF/YF</i> oocytes.</p

Number of different subtypes of meiotic nuclei and frequency of nuclei with a pseudo-XY body in E16.5 and E17.5 oocytes from <i>Spo11^YF/YF</i>embryos.

*<p><b>percentage of the total number of counted nuclei.</b></p

Relocalisation of persistent radiation-induced DSBs into a pseudo XY body.

<p>(A) Irradiated <i>Spo11^YF/YF</i> spermatocytes were collected 1 h, 48 h and 120 h upon irradiation and immunostained for RAD51 (green), SYCP3 (red), and γH2AX (blue). Spermatocytes that were irradiated at the leptotene stage, should have reached zygotene and pachytene with respect to the pattern of γH2AX, at 48 and 120 h following irradiation, respectively. (B) Fraction of cells showing a pseudo XY body upon irradiation at the analysed time-points (n = 50). (C) Immunostaining of <i>Spo11^YF/YF</i> spermatocyte 120 hours after irradiation with anti-RNA pol II (green) and anti-γH2AX (red). The intense γH2AX domain (pseudo XY body) corresponds to a nuclear area depleted for RNA pol II.</p

Number of different subtypes of meiotic nuclei and frequency of pachytene nuclei with a pseudo-XY body in E16.5 and E17.5 oocytes from <i>Spo11^+/+</i> and <i>Spo11^+/YF</i>embryos.

*<p><b>percentage of the total number of counted nuclei.</b></p

Model for the roles of SPO11-dependent and -independent meiotic DSBs in synapsis and meiotic silencing.

<p>In spermatocytes and oocytes, SPO11 generates many meiotic DSBs which are repaired via homologous recombination (HR). This repair process requires the use of the homologous chromosome as a repair template and promotes homologous chromosome synapsis. Once the homologs are in close juxtaposition, synapsis proceeds. Subsequently, repair may occur faster, perhaps now allowing the use of both the homologous chromosome and the sister chromatid as a template for repair. In the absence of a repair template, DSBs persist, inhibiting synapsis between non-homologous partners, although some repair via the sister chromatid on chromosomes that are not synapsed is not excluded. Conversely, asynapsis also contributes to the persistence of DSBs when repair via the sister chromatid remains suppressed. The presence of persistent DSBs on unsynapsed axes, may lead to local accumulation of γH2AX and activate a positive feedback mechanism that involves HORMAD activation, followed by recruitment of ATR, which will lead to rapid spreading of a signal along the unsynapsed axes that will then induce accumulation of γH2AX on the chromatin surrounding these axes. This process always occurs on the XY pair in spermatocytes and leads to MSCI. In the absence of SPO11-induced DSBs, SPO11-independent DNA damage nucleates MSUC via the same mechanism. In spermatocytes, SPO11-independent DNA repair foci may represent remnant DSBs that have formed during the premeiotic S phase. In oocytes (both wild type and SPO11-mutant), SPO11-independent DNA repair foci form late, at a time point corresponding to early pachytene. Such <i>de novo</i> induced DNA repair foci, most likely caused by some form of DNA damage, together with unrepaired SPO11-induced DSBs, and frequently in combination with occasional asynapsis, result in γH2AX accumulation and activation of MSUC. Representative images of the (pseudo) XY body in male and female nuclei from wild type (wt) and <i>Spo11^YF/YF</i> nuclei are shown. The immunostainings show SYCP3 (red), γH2AX (blue) and RAD51 (green).</p

DMC1 preferentially localizes to unsynapsed axes in wild type pachytene oocytes.

<p>(A) Triple immunostaining with anti-TEX12 (red), anti-DMC1 (green), and anti-γH2AX (blue) of pachytene oocyte nuclei from E17.5 wild type embryos. DMC1 foci are detected in the pseudo XY body and localize to synapsed axes (left, close-up), or to unsynapsed axes (middle, close-up). The pseudo XY body is often devoid of DMC1 foci (right, close-up). (B) Quantification of the number of synapsed and unsynapsed axes, present in pseudo XY bodies, that are positive or negative for DMC1 foci (n = 70). Percentages are shown in brackets. (C) Triple immunostaining with anti-TEX12 (red), anti-RAD51 (green), and anti-γH2AX (blue) of oocytes from E17.5 wild type embryos. Axis-wide accumulation of RAD51 in the pseudo XY body was observed on both synapsed (left) and unsynapsed (right) axes. Close-ups separately show TEX12 and RAD51 patterns in the pseudo XY body.</p

Enrichment of DNA repair markers in the pseudo XY body of <i>Spo11^YF/YF</i> spermatocytes.

<p>(A) Nuclei of <i>Spo11^YF/YF</i>zygotene spermatocytes were divided in four subgroups depending on their positivity for the pseudo XY body and for foci of one of the three DNA repair proteins RAD51 (n = 120), DMC1 (n = 227) or RPA (n = 108) as follows: 1) with pseudo XY body and with foci, 2) with pseudo XY body and without foci, 3) without pseudo XY body and with foci, 4) without pseudo XY body and without foci. Spermatocyte nuclei were immunostained with anti-SYCP3 (red), anti-γH2AX (blue), and one of the following antibodies: anti-RAD51 (green, upper panel), anti-DMC1 (green, middle panel) or RPA (green, lower panel). Every panel shows a representative nucleus for each of the four subgroups mentioned above. Numbers in the bottom left corner of every picture represent the percentage of nuclei of this type in the analyzed cell population. (B) The average number of RAD51, DMC1 and RPA foci per nucleus was counted in spermatocytes of the first subgroup (outlined in red). The table also shows the percentage of foci located within a pseudo XY body and the percentage of pseudo XY bodies which contained at least one focus. (C) Scatter plot representing the colocalization percentage in relation to the fraction of the nuclear area occupied by the pseudo XY body. Every dot represents a nucleus. Pearson linear correlation coefficient [Pcorr] = 0.0741.</p

Pseudo XY bodies containing synapsed axes in wild type embryonic oocytes.

<p>(A–B) Triple immunostaining with anti-HORMAD1 (red), anti-TEX12 (green) and anti-γH2AX (blue) of oocyte nuclei from E17.5 wild type embryos. In the lower right corner percentages are reported, representing the frequency of each type of cell in the pachytene oocyte population (n = 244). (A) Representative pictures of early zygotene (EZ), late zygotene (LZ), early pachytene (EP) and pachytene (P) oocytes, from left to right. HORMAD1 levels are decreasing while TEX12 accumulates as synapsis progresses. Parallel to the increase of synapsis and HORMAD1 removal, γH2AX accumulation decreases. (B) Representative pictures of pachytene oocytes with a pseudo XY body. HORMAD1 positive axes totally (left picture) or partially (middle picture) colocalize with the pseudo XY body, or are not present (right picture) in the pseudo XY body. (C) Scatter plot of the total length of synapsed axes in E17.5 wild type pachytene oocytes, belonging to the following categories: HORMAD1 and pseudo XY body absent (blue); HORMAD1 absent and pseudo XY body present (light blue); presence of both HORMAD1 and a pseudo XY body. Every dot represents a nucleus. Black bars indicate the mean values. P values for the indicated comparison (Mann-Whitney, two-tailed) are shown in the plot. (D–E) Triple immunostaining with anti-TEX12 (white), anti-RNA polymerase II (green), and anti-γH2AX (red) of pachytene oocytes from E17.5 wild type embryos, imaged with the Zeiss LSM700 confocal microscope. Depletion of RNA pol II can be observed in the area of the the pseudo XY body marked by γH2AX, both when synapsis is complete (D) and when unsynapsed axes (E) are present in this region (E). Size bars represent 10 µm.</p