CO-Associated Markers Appear Normally in the Absence of TRIP13
The persistence of BLM on Trip13Gt/Gt spermatocyte chromosomes suggests that at least a subset of the unrepaired DSBs correspond to sites of defective NCO recombinational repair. To assess whether CO recombination occurs in the mutant, we examined the distribution of the mismatch repair proteins MLH1 and MLH3, which are normally detectable as foci in mid-late pachynema and mark the locations of chiasmata [30,31]. Remarkably, MLH1/3 foci were formed; we observed 1–2 foci/chromosome as in wild type and at typical overall levels (MLH3 = 23 ± 2, N = 10; [30,32]) on mid-late pachytene chromosomes (Figure 4M and 4N; MLH1 not shown). Since <1% of Trip13Gt/Gt pachytene nuclei had normal repair (as judged by absence of persistent DSB repair markers; see above), but most of the pachytene nuclei had MLH1/3 foci, it was unlikely that the MLH1/3 foci formed only on chromosomes with fully repaired DSBs. To test this directly, we conducted double staining for MLH1 and RAD51/DMC1. MLH1 foci were present on chromosomes that also contained numerous RAD51/DMC1 foci (Figure 4O and 4P).
To assess whether these MLH1/3 foci in Trip13Gt/Gt pachytene spermatocytes represent CO events completed to a point where they could maintain interhomolog attachments though the end of prophase I, we treated testicular cells from 17.5–20.5-d-old control (+/+), Trip13Gt/Gt, and Dmc1 −/− mice with the protein phosphatase inhibitor okadaic acid (OA), a chemical that induces degradation of the SC, chromosome condensation, and premature progression to metaphase I [33]. Fifteen metaphase spreads were identified for each genotype. Whereas all of the Dmc1 −/− spreads had ∼35 or more condensed chromosomes, all of the +/+ and Trip13Gt/Gt spreads had 20–25, suggesting that the MLH1/3 foci in Trip13Gt/Gt pachytene spermatocytes represent sites of completed, or near-completed, COs. Because the preparations were made from whole testes, it is possible that the univalent-containing metaphases from Dmc1 −/− mice were from spermatogonia, not spermatocytes.