(A) Projection images showing meiotic prophase progression in whole gonads from adult hermaphrodites stained with antibodies against SYP-1 (SC; red) and the RING finger protein ZHP-3 (green). The images in the lower panels (pink borders) are from a gonad that was exposed to 7.5% hexanediol just prior to fixation. As previously described (Bhalla et al., 2008), ZHP-3 localizes throughout SCs in early pachytene, and then concentrates at designated CO sites. When the SC is dissolved by hexanediol, the diffusely localized ZHP-3 is also solubilized, but after localization to COs the protein becomes resistant to solubilization by hexanediol. The higher-magnification views on the right show individual nuclei from before and after the relocalization of ZHP-3 upon CO designation (top and bottom, respectively). Scale bars correspond to 10 μm (left) and 2 µm (right). (B) COSA-1 localizes to CO sites and is not dissolved by hexanediol. These projection images are from an adult hermaphrodite expressing GFP-COSA-1 and mRuby-SYP-3. Extruded gonads were exposed to 7.5% 1,6-hexanediol prior to fixation, and counterstained with DAPI (blue). GFP-COSA-1 (green) and mRuby-SYP-3 (red) were visualized by their intrinsic fluorescence. Although SC proteins were dispersed by hexanediol, the 6 CO sites are still marked by GFP-COSA-1. Bottom, magnified view of a single nucleus. Scale bars are 8 μm and 2 μm, for the top and bottom panels, respectively. (C–D) COSA-1 and ZHP-3 are interdependent for their dynamic relocalization during meiotic progression. The outlined regions are shown at higher magnification in Figure 6D–E. (C) Adult htp-3 or htp-3 zhp-3 hermaphrodites expressing GFP-COSA-1 were dissected and stained with antibodies against SYP-2 (SC/polycomplexes; red) and GFP (COSA-1; green) and counterstained with DAPI (blue). GFP-COSA-1 does not form foci associated with polycomplexes in the absence of ZHP-3. Scale bars = 10 μm. (D) Adult htp-3 or htp-3; cosa-1 hermaphrodites were dissected and stained with antibodies against SYP-1 (SC/polycomplexes; red) and ZHP-3 (green), and counterstained with DAPI (blue). In the absence of COSA-1, ZHP-3 remains localized throughout polycomplexes throughout meiotic prophase. Scale bars = 10 μm. (E) Dynamic relocalization of ZHP-3 and COSA-1 is accompanied by different hexanediol sensitivity. Gonads from htp-3 hermaphrodites expressing GFP-COSA-1 were dissected and stained with antibodies against SYP-1 (SC/polycomplexes; magenta), ZHP-3 (orange), and GFP (COSA-1; green), and counterstained with DAPI (blue). Top, higher-magnification view of polycomplexes showing examples of early and late localization patterns. Bottom, upon dissolution of polycomplexes with 1,6-hexanediol, small foci positive for ZHP-3 and GFP-COSA-1 remain visible. The solubilized SC proteins are largely excluded from the volume occupied by chromosomes, resulting in a somewhat filamentous appearance. Scale bars = 2 μm.