Fig. 1. 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.

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