(A) Projection images of representative late pachytene nuclei stained for GFP-COSA-1 (green) and SYP-1 (red). In the absence of DSB-2 at 24 hr post-L4, an average of 3 bright GFP-COSA-1 foci are detected in each nucleus. However, very few bright GFP-COSA-1 foci are observed when ZHP-2 is co-depleted. By 48 hr post-L4, only ~2 bright GFP-COSA-1 foci are observed per nucleus in the absence of DSB-2, and most nuclei display 0–1 bright GFP-COSA-1 focus when ZHP-2 is also depleted. (B) Quantification of bright GFP-COSA-1 foci at late pachynema, 24 hr post-L4. Worms of the indicated genotypes were maintained in the presence or in the absence of 1 auxin for 24 hr and then dissected for analysis. Data were derived from 4 to 8 gonads for each genotype or condition. n = 165, 348, 172 and 377 nuclei, respectively. The number of GFP-COSA-1 foci differed significantly between dsb-2::AID and zhp-2::AID; dsb-2::AID following auxin treatment (***p<0.0001 by Chi-square test for trend). (C) Quantification of bright GFP-COSA-1 foci in late pachytene nuclei 48 hr post-L4. Data were derived from 4 to 8 gonads for each genotype or condition. n = 252, 558, 263 and 416 nuclei, respectively. Depletion of ZHP-2 significantly reduces the number of GFP-COSA-1 foci in the absence of DSB-2 (***p<0.0001 by Chi-square test for trend). The distribution of GFP-COSA-1 foci is also significantly different between 24 hr and 48 hr post-L4 for auxin treated zhp-2::AID; dsb-2::AID hermaphrodites (***p<0.0001 by Chi-square test for trend). (D) Representative images of mid-pachytene (MP) and late pachytene (LP) nuclei stained for RAD-51 (yellow), GFP-COSA-1 (green), SYP-1 (red), and DNA (blue). GFP-COSA-1 foci in the absence of ZHP-1/2 was visualized following exposure of SPO-11-depleted worms to varying doses of ionizing radiation. L4 worms were maintained in the presence or in the absence of 1 mM auxin for 24 hr, followed by irradiation at the indicated dosage, and then incubated for additional 8 hr to allow irradiated nuclei to progress to late pachynema. Scale bars, 5 µm.