Extended Data Fig. 9: RMMAI accumulation and low frequency of DSB formation at mo-2 regions in oocytes.

a, Examples of zygotene oocytes showing the colocalization between blobs of IHO1 and REC114, MEI4 and MEI1, or ANKRD31 and mo-2 FISH signal (arrowheads). RMMAI blobs colocalized with mo-2 FISH signals in all zygotene oocytes analysed (n > 30) from at least three mice. Scale bars, 2 μm. b, PAR ultrastructure in oocytes, quantified as in Extended Data Fig. 3b. Late-zygotene cells with PAR synapsis are compiled separately from other zygotene cells. Error bars are mean ± s.d. Scale bar, 1 μm. c, Examples of zygotene oocytes showing colocalization of ANKRD31 blobs with enrichment for heterochromatin factors. ANKRD31 colocalized with blobs of heterochromatin factors in all zygotene oocytes analysed (n > 20) from one mouse. Scale bars, 2 μm. d, Representative SIM image of a wild-type late-zygotene oocyte showing neither detectable splitting of the PAR axis nor REC8 enrichment. The absence of spermatocyte-like differentiation of the PAR axis was observed (n > 30 zygotene oocytes) in more than three mice. A modest degree of differentiation was observed in a minority of oocytes (5/45) analysed by SIM, but this did not resemble the typical PAR axis splitting found in spermatocytes. Scale bar, 2 μm. e, Prolonged asynapsis does not allow axis splitting to occur in oocytes. Because synapsis appears to be sufficient to trigger the collapse of the PAR ultrastructure in spermatocytes (Extended Data Fig. 3b), we asked whether preventing synapsis (that is, in a Syce1^−/− mutant) could reveal a tendency towards axis splitting in oocytes. Whereas axis splitting was clearly observed by SIM in Syce1^−/− mutant spermatocytes, PAR axes were not detectably split in oocytes. Axis splitting of chromosome 9 was observed by SIM in multiple (n > 20) Syce1^−/− spermatocytes from three different mice. The centromere-distal axes were also occasionally seen to be split in chromosomes 13 or 4, but we did not quantify this for these chromosomes. In males, the differentiation of the axes of the PAR or chromosome 9 becomes hardly detectable at later stages in some pachytene-like spermatocytes as cells enter apoptosis, similar to Spo11^−/− or Hormad1^−/− mice. However, in Syce1^−/− oocytes, no obvious axis differentiation or splitting was observed by conventional microscopy or by SIM in multiple oocytes (n > 30) from three different mice—similar to what was observed in wild-type oocytes. Scale bars, 2 μm (main image); 1 μm (insets). f, h, Delaying synapsis promotes the formation of DSBs in the PAR in oocytes. Top, representative micrographs of pachytene XY (f) and Syce1^−/− XX (h) oocytes. Middle, RPA2 fluorescence intensity at the border of mo-2 FISH signals from the PAR, chromosome 9 and chromosome 13. Bottom, percentage of oocytes with an RPA2 focus colocalizing with mo-2 regions on the PAR, chromosome 9 and chromosome 13. Graphs show data only for pachytene oocytes in which PARs are synapsed (two mice of each genotype). Red lines indicate mean ± s.d. Scale bars, 2 μm. g, Percentage of pachytene oocytes with one or more RPA2 foci colocalizing with the mo-2 FISH signal from the PAR, chromosome 9 and chromosome 13 in XY pachytene oocytes that had unsynapsed X and Y chromosomes. Scale bars, 2 μm (main image); 1 μm (inset).

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