Figure 4  Immunohistochemical Analysis of Pachytene Spermatocyte Chromosomes
Surface-spread chromosomes were immunolabeled with the indicated antibodies and fluorophores. As indicated in the upper right of each panel, cells were from wild type (WT, either +/+ or Trip13Gt/+) or Trip13Gt/Gt (Mut). There were no differences seen between heterozygotes and +/+ spermatocytes.
(A) A mutant pachytene nucleus with full synapsis. Areas of SYCP1/SYCP3 colabeling are yellow.
(B–E) Spermatocytes nucleus from 17.5 d postpartum mutant. Asynapsed chromosomes or regions of chromosomes are indicated by white and yellow arrows, respectively. Unlike the normal distribution in wild-type pachytene spermatocytes (C), BLM foci are present on synapsed pachytene chromosomes in the mutant (D). RAD51 foci, which are abundant earlier in prophase, disappear from autosomes in wild-type pachytene nuclei (E) and the bulk of staining is over the XY body (arrow).
(F) RAD51 persists on the synapsed mutant chromosomes (arrows).
(G) H2AX phosphorylation is restricted to the XY body in WT.
(H) In addition to a large area of γH2AX staining (arrow) over the XY body, there is extensive autosomal H2AX phosphorylation (arrows).
(I, J) Note that in wild-type pachytene spermatocytes, TOPBP1 is present only over the XY body (yellow arrow). In the mutant (J), an arrow denotes one area of intensive staining that may be over the sex chromosomes, but many other chromosome cores are positively stained.
(K, L) RPA persists along synapsed cores in the mutant, not WT.
(M, N) Arrows indicate examples of MLH3 foci on SCs.
(O) In WT late pachytene spermatocytes, RAD51 is present only at background levels.
(P) As in (F), extensive RAD51 staining delineates SCs in mutant pachytene nuclei (indicated by white arcs). MLH1 foci colocalize with these tracts (arrows) at the typical 1–2 foci per chromosome as in (M).