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Fig. 4. Analysis by immunofluorescence of the localization of BRCA2, H1t, SPO11 and ${gamma}$ -H2AX proteins during male meiosis. Localization of these proteins was compared between control (Brca2 Ko/+; Tg/+, A,C,E,G,I) and rescued (Brca2 Ko/Ko; Tg/+, B,D,F,H,J) in preparations of surface-spread chromatin from spermatocytes. Nuclei were stained with antisera against SYCP3 (in red, except in C and D where SYCP3 is in green). (A) Mouse BRCA2 is present in the nuclei during zygonema, but (B) no signal is detected in the rescued spermatocytes as they lack a functional mouse Brca2 gene. (C,D) The human protein is not detected in control or rescued spermatocytes. (E,F) Nuclei were examined for presence of male germ cell-specific, mid-pachytene marker, histone H1t. Control cells show positive staining but rescued cells lack the staining. (G,H) Both control and rescued spermatocytes exhibit expression of SPO11 protein during leptotene/zygotene stage, with diminished staining by pachynema in control spermatocytes (arrow). (I,J) Localization of phosphorylated histone H2AX ( ${gamma}$ -H2AX), a marker for double-strand break formation, is seen in both genotypes. During pachytene stage, the staining of ${gamma}$ -H2AX disappears except in the sex chromosomes (arrow). Rescued spermatocytes do not show this typical pachytene staining pattern, further evidence that they do not reach this stage.

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