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First published online March 7, 2007
doi: 10.1242/10.1242/jcs.03394

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Initiation and resolution of interhomolog connections: crossover and non-crossover sites along mouse synaptonemal complexes

Department of Biology, York University, Toronto, Ontario, M3J 1P3, Canada

View larger version (24K): [in this window] [in a new window]   Fig. 1. Development of meiotic chromosome core-associated recombination protein complexes in mouse spermatocytes [adapted from Moens et al. (Moens et al., 2002)]. About 250 to 300 ENs (green), become associated with the chromosome cores (blue) prior to synapsis. They are spherical structures, about 100 nm in diameter, and are identified by antibodies to RAD51 and DMC1 protein (green). They are the sites of programmed, double-strand DNA breaks at meiotic prophase and they are associated with phosphorylated histone H2AX (yellow; this report). The ENs are transformed into 200 synaptonemal complex (SC)-associated, RPA-defined TNs (SCs, parallel blue lines; TN, red) by the acquisition of several proteins – RPA, BLM, MSH4, MSH5 and topoisomerases – while they loose the RAD51-DMC1 and H2AX components. In the mouse spermatocyte nucleus, about 25 of the TNs acquire MLH1 protein (blue) that mark the sites of chiasmata. The remaining TNs are resolved as non-crossovers and they relocate to the outside the SCs (this report).

View larger version (91K): [in this window] [in a new window]   Fig. 2. Development of nodules from leptotene into zygotene. (A) An early leptotene spermatocyte nucleus with the stage defined by the earliest formation of SYCP3 chromosome-core segments (FITC, green). By the internal clock of the progression from zero to 250-plus RAD51-DMC1 foci (Rhodamine, red foci), this nucleus with 100 foci is still developing RAD51-DMC1 sites. The foci are usually connected to a core segment and they are the centres of diffuse H2AX domains (Rhodamine, red). (B) Reorganization of H2AX domains (FITC, green). At a slightly later leptotene stage, the spherical domains rearrange into flare-shaped domains. (C) Alignment of 230 foci and associated H2AX flares (FITC, green). (D,E) Zygotene chromosome cores. Foci of the unpaired cores and recently paired cores (green) are associated with H2AX (Rhodamine, red domains) but the foci of fully synapsed cores have no, or strongly reduced, H2AX domains. Presumably annealing and/or repair has been completed.

View larger version (131K): [in this window] [in a new window]   Fig. 3. An early prophase spermatocyte nucleus where inter-homolog connections are established in the context of RPA-TN foci prior to SC formation [RPA-TN foci, red; SCYP1 cores and centromeres (cen), FITC, green]. Bivalent #2 has broadly aligned cores with one RPA-TN focus at the site of close alignment prior to SC formation. Other TNs are associated with the unpaired cores (caret). Bivalent #1 has two TNs at the distal, closely aligned cores and an additional RPA-TN interhomolog connection at the inflection (i, asterisk). Bivalent #3 has RPA-TN foci along and at both ends of a long closely aligned segment. Close alignment is extensive in bivalent #4 and SC formation (SC) has been initiated in the distal segment.

View larger version (72K): [in this window] [in a new window]   Fig. 4. Progression of SC formation (SC; red). (A) Synaptic forks at the points of synaptic progress are associated with TNs (green foci, arrows). The inflections (i) suggest a structural connection between the two homologs at the site of the TN. At later prophase, the chromosomes are completely synapsed and the SCs can have 10 or more TNs each (SC #1). The centromeres (cen; red) are frequently the last to pair. (B) Immunogold-defined positions of TNs at zygotene. The synaptic fork has two TNs labelled with 5-nm gold particles. The lateral elements (LEs) are labelled with 10-nm gold particles. (C) A mature pachytene SC with lateral elements and medially located TNs (10-nm gold). The width of the SC is approximately 250 nm.

View larger version (55K): [in this window] [in a new window]   Fig. 5. Number and distribution of TNs. (A) A mid-pachytene nucleus with 140 RPA-TN foci (Rhodamine, red, but yellow on a green background) positioned fairly evenly along the SCs (green) (see supplementary material Fig. S4, cell 02). The number of TNs in this nucleus has declined from the original 250+ foci at zygotene. (B) At late pachytene, there remain few TNs – 33 in this nucleus (see supplementary material Fig. S5, cell 09). Uneven loss of TNs leaves an uneven distribution along the SCs and some SCs have no TNs. MLH1 foci (blue) have developed by this stage.

View larger version (141K): [in this window] [in a new window]   Fig. 6. TNs and MLH1 foci in Sycp3–/– oocytes. (A) An early prophase nucleus from a 15.5-day old foetus with 150 TNs (yellow) associated with SC-like structures (bright green) and 250 TNs (red) associated with unpaired cores (weak green). Immunostaining of RPA-TNs: core components SMC3, SYCP1, Rhodamine, red; centromeres, FITC, green. (B-D) Three pachytene oocytes nuclei from one ovary of a 17.5-day old foetus with different levels of synaptic defects. Chromosome cores were immunostained with rabbit anti-SMC3 (Rhodamine, weak red) and SC central elements with rabbit anti-SYCP1 (Rhodamine, bright red). The MLH1 foci are visualized by mouse anti-MLH1 and FITC-conjugated secondary antibody (green). (B) An oocyte nucleus with severe synaptic defects. Arrows mark some of the MLH1 foci that are not associated with brightly stained SYCP1 segments. (C) An oocyte nucleus with intermediate synaptic defects. There are more and longer SYCP1 segments and fewer free MLH1 foci (arrows). (D) A near-normal looking oocyte nucleus with well-developed SYCP1-defined SCs, few unsynapsed cores, and no free MLH1 foci.

View larger version (100K): [in this window] [in a new window]   Fig. 7. RPA-TN and MLH1 dynamics. (A) A full-length pachytene SC with eight RPA-TN foci (TN, carets) and one recombination nodule (RN, elliptical bullet). (B) High resolution of the bracketed segment in A. The 10-nm gold grains mark the positions of RPA protein and the 5-nm particles that of the MLH1 protein. Colocation of 10-nm and 5-nm gold grains at the RN suggests that the RN is derived from a TN. Other TNs have relocated from their positions between the lateral elements of the SC to the periphery of the lateral elements. (C) Same as B but the twist in the SC shows that the RN is on the surface of the SC. (D) Okadaic acid treatment of spermatocytes causes premature separation of the lateral elements (FITC, green). The TNs are no longer between the cores but are associated with the cores. Inset: RNs (green foci), by contrast, remain between the separated lateral elements (red) at the positions of the chiasmata. (E) Summary of RPA-TN and MLH1-RN dynamics for 12 mouse spermatocyte nuclei. MLH1 foci appear when the number of TNs has been reduced to about 100 foci per nucleus. Image in A and data for Table 2 were derived from SCs in the bracketed portion of the graph.

View larger version (114K): [in this window] [in a new window]   Fig. 8. H2AX is associated with DSB of ENs and transcriptional silencing. (A) H2AX domains (red) are associated with the foci (yellow) before synapsis and, when the cores synapse to form an SC (green), the H2AX domains are minimal, which is possibly an indication that DSBs are no longer detectable. (B) At pachytene, histone H2AX of the X-Y sex chromatin is transcriptionally silenced by phosphorylation (FITC, green). As well, there are autosomal H2AX domains (FITC, green) that do not appear to be associated with TNs (red foci) and may represent transcriptional silencing of autosomal loci. (C) H2AX chromatin modification of the sex body and laggards (L). A further indication that pachytene chromatin modification (yellow) may be unrelated to DSBs. Typically, cores (red) that fail to synapse (laggards, L), acquire H2AX domains in association with ATR and TOPBP1 on the unpaired cores. (D) In prematurely separated lateral elements (green) caused by okadaic acid treatment, some of the H2AX domains (yellow) are paired (matched arrows), suggesting silencing of autosomal sites.