Time course and interactions of SC-associated RAD51/DMC1, RPA and BLM protein in mouse spermatocytes. (A,B) RAD51/DMC1 foci initiate prior to detection of RPA. To the left of the white line is a spermatocyte early zygotene nucleus with mostly unpaired centromeres (red), which has about 200 RAD51/DMC1 foci (yellow). A nucleus in a later stage of meiotic prophase to the right of the line has fewer, about 90, foci. The same two nuclei stained for RPA in Fig. 1B show that the early prophase nucleus on the left has few and indistinct RPA foci, whereas the pachytene nucleus to the right of the line has an abundance of RPA foci. (C,D) The shift from RAD51/DMC1 to RPA foci is a developmental progression for the nucleus as a whole, but details seem to be regulated at the level of the individual bivalent. SC#1 and 2 have acquired a full complement of RPA foci (1D) and have lost most RAD51/DMC1 foci (1C), whereas SC#3 still has RAD51/DMC1 foci but few and indistinct RPA foci. (E) The replacement of RAD51/DMC1 by RPA is demonstrated from the relative amounts of the two proteins in individual foci based on electron microscopy of two types of immunogold grains as in Fig. 2. The data from nine of 40 SCs are presented in a bar graph; the top portion is the percentage of foci with only RPA antigen, the middle portion represents the foci with both antigens, and the lower part is the percentage of pure RAD51/DMC1 foci. Each bar represents one SC, and the SCs were sorted according to the prophase stage of the nucleus and by the previously documented decline in RAD51/DMC1 foci from zygotene to pachytene (details in Materials and Methods). The composition of the protein complexes change at successively later stages of meiotic prophase. (F) The line graph shows the number of fluorescent foci at progressively later stages of prophase. RAD51/DMC1 peaks at leptotene. Somewhat later, RPA reaches its maximum and later still, BLM does. These three antigens frequently are present together in individual foci. MLH1 appears at late prophase and is present in low numbers. Each data point is one nucleus, and the staging of the nuclei is described in the Materials and Methods.

Immunoelectron microscopy of RAD51/DMC1 (10 nm gold grains) and RPA (5 nm gold grains) in association with chromosome cores/SCs in an early and later meiotic prophase spermatocyte. (A) At early meiotic prophase, there is an abundance of RAD51/DMC1 foci but relatively few RPA foci in association with partially paired zygotene chromosome cores and SCs. (B; insert) At a later stage, pachytene, there are numerous RPA foci and very few RAD51/DMC1 foci. The RPA foci are concentrated on central element-associated nodules, TN. The width of the SC is about 200 nm. (C) At early pachytene, there are occasionally a few unpaired centromeric ends (15 nm gold grains). One of these `laggards' is shown to have RPA associated with a single, unpaired chromosome core, indicating that RPA is not always associated with RAD51/DMC1 protein. The image further demonstrates the presence of mixed foci containing RAD51/DMC1 and RPA antigen (mix). Incidental is the observation that RPA gold grains occur as a small loop (arrow) in this and other electron micrographs. The width of the SC is about 200 nm.

Colocation of RPA and BLM protein in pachytene spermatocytes. (A,B) The same nucleus immunostained for BLM (A) and for RPA (B). (C) The BLM image of (A) is made into a negative and superimposed on the positive RPA image of (B). Nearly all BLM foci colocate with RPA foci. The relative contributions of the two proteins to the individual foci are semi-quantifiable by the size and the intensity of the immunofluorescent signal, and this is somewhat more efficient than multiple colour combinations. (D-G) Electron micrographs of the mix of RPA 5 nm immunogold grains and 10 nm BLM immunogold grains at phosphotungstic acid-stained SCs and nodules. (H) With our antibodies, there are fewer 10 nm BLM gold grains than there are 5 nm RPA gold grains at the nodules.

RPA at the pseudoautosomal region, PAR, of the X-Y chromosomes. (A) Chromosome cores and centromeres visualized with fluorescent tags. The X and Y chromosomes have a short paired region, PAR. (B) The same chromosome visualized with anti-RPA antibody. The autosomal SCs and the unpaired X-chromosome core have numerous RPA foci. The PA region frequently has the brightest RPA focus or foci. Scale: the SCs are about 15 mm in length. (C; insert) At early pachytene there is extensive non-homologous synapsis between the distal portions of the X and Y chromosomes. RPA is present in the most distal portion, presumably the actual PA region. (D; insert) In the rat, the late RNs are better defined than in the mouse, and the Figure illustrates a group of 5 nm RPA gold grains in association with a nodule.

The association of MSH4p with RPA and RAD51/DMC1p. (A) The images of the red MSH4 foci and the green RPA foci are slightly offset to better demonstrate the colocation of the two types of foci. The short white lines indicate the direction of the offset and examples of colocating foci. The large bright green foci are the centromeres. (B) The colocation is also evident with immune electron microscopy. The 10 nm gold particles mark RPA antigen and the 5 nm grains mark the MSH4 antigen. The grains are associated with a transitional nodule at the central region of the SC. (C) The association of RAD51/DMC1 with MSH4 foci is less pronounced, and evidence from counts of five complete nuclei with some 150 foci each suggest that at most 10% of the foci possibly overlap and that, in general, the MSH4 foci appear after the Rad51/DMC1 foci have declined in numbers. The X chromosome at the base of (C) has, as usual, prolonged presence of RAD51/DMC1 foci. (D,E) At the EM level MSH4 foci (5 nm gold) are mostly separate from RAD51/DMC1 foci (10 nm gold). (E) The MSH4 antigen is associated with the transitional nodule, which has no evidence of RAD51/DMC1 antigen in this double-labeled preparation.