Gene expression profiling of Brwd1^−/− spermatids. (A) Heat map visualization of 286 down-regulated transcripts in mutant testes from 27-d-old animals as detected by gene expression microarray analyses (H1–H4 are heterozygotes, M1–M4 are mutants). (B) Real-time PCR validation of the most down-regulated genes in 27-d-old mice. The hatched boxes represent mutant levels. (C) Flow cytometry count of testicular cells based on DNA content (C) shows that the percentage of haploid spermatids in both WT and mutant testes are the same. The 1C cells are haploid spermatids, the 2C cells correspond to somatic and spermatogonial cells that have not undergone DNA replication, and the 4C cells correspond to spermatocytes (predominantly), spermatogonia, and somatic cells that have completed S phase but not cell division. (D) Real-time PCR shows significant differences in Tnp1, Tnp2, and Prm1 between WT and mutant transcript levels even in 21-d-old mice. Error bars indicate ±SEM. The data are the mean of three independent replicates.