TI - Analysis of Prophase I in Mus81 Mutant Males Reveals Persistence of Unrepaired Double Strand Breaks and Persistence of the Early Meiotic Nodule Component , BLM . AB - To study the progression of synapsis and recombination events during prophase I , chromosome spreads were prepared as described previously [27] . Indirect immunofluorescence on chromosome spreads was performed to localize meiotic proteins SYCP3 , PHOSphorylated H2AX ( gammaH2AX ) and RAD51 on chromosome spread preparations of spermatocyte nuclei ( see materials and methods ) . SYCP3 is a protein that localizes to the lateral elements of the symaptonemal complex during meiosis , and allows the visualization of chromosome cores . Histone H2AX is a ubiquitous member of the histone H2A family that , upon DSB induction , is rapidly PHOSphorylated on serine 139 . This PHOSphorylated form of H2AX , referred to as gamma-H2AX , also localizes to regions of silenced chromatin and is thus used in meiotic cells to mark regions of DSBs , asynapsis and the sex body [28] . Processing of DSBs in early prophase I requires the participation of RecA homologs , RAD51 and DMC1 [29] , components of early meiotic nodules in mice [30] , [31] , [32] . RAD51 , forms a nucleofilamentous structure along single stranded DNA and facilitates strand invasion in the early stages of DSB repair during leptonema and zygonema , and once synapsis occurs , disappears from the chromosome cores , indicating progression of repair events beyond strand invasion [33] . In Mus81-/- males , RAD51 accumulates normally on meiotic chromosomes , but the foci persist to late pachynema in mutant animals , indicating that not all meiotic DSBs are repaired correctly ( Figure 2A , B ) . In addition to these small regions of localized RAD51 staining , some late pachytene spermatocytes ( regions of asynapsis , indicating a role for MUS81 in correct pairing of homologous chromosomes in a subset of spermatocytes , as visualized by the persistence of both RAD51 and gamma-H2AX stains on autosomes in late pachynema ( Figure 2A-D ) . Late pachytene spermatocytes also show increased accumulation of the RecQ helicase Bloom syndrome mutated ( BLM ) , the mammalian ortholog of yeast Sgs1 . Previous studies have demonstrated that BLM accumulates on chromosome cores during Prophase I in WT spermatocytes , appearing early in zygonema and gradually declining through mid-pachynema [34] , [35] . In the current study , a similar increase in BLM foci is observed at zygonema in WT spermatocytes , before decreasing to a few foci in mid-late pachynema ( Figure 3A , B ) . In spermatocytes from Mus81-/- males however , BLM focus numbers rise during zygonema but then remain elevated above WT levels throughout the entire pachytene stage , with increased foci being distributed along all chromosomes in an individual nucleus ( Figure 3C , D ) .