GASZ Is Essential for Male Meiosis Figure 4. MILI is lost in embryonic and newborn testes in the absence of GASZ. Immunofluorescent analysis of Gasz+/2 (A,C,E,G,I,K) and Gasz2/2 (B,D,F,H,J,L) testes. Staining is shown for TDRD1 (A–D), MILI (E–H), and MVH (I–L). TDRD1 immunostaining is diffusely cytoplasmic in Gasz2/2 newborn (B) and embryonic (D) gonocytes versus controls (A,C). Staining of the identical germ cells shows MILI is undetectable in newborn gonocytes and present in only a subset of Gasz2/2 E16.5 gonocytes [arrowheads in (F,H)]. MVH staining is less granular in Gasz2/2 (J,L) versus controls (I,K). [Scaling: 5,0006magnification.] doi:10.1371/journal.pgen.1000635.g004 showed an increase in pachytene piRNAs at P14, resulting in a proportional reduction in miRNA reads contributing to the overall small RNA pool. By contrast, Gasz2/2 testes failed to induce piRNAs, and the small RNAome (17–40 nt) was dominated by miRNAs in Gasz2/2 testes compared to controls (71% vs. 44%, Figure 7A). However, the overall miRNA profiles (the subset of miRNAs expressed and relative abundance) were strikingly similar in both genotypes suggesting that miRNA biogenesis and function are likely intact in the absence of GASZ; the relative increase in miRNA read abundance is a consequence of reduced piRNA sequences within a fixed sample of small RNA reads. MicroRNAs have been shown to be present in the chromatoid body, but based on our analysis, they are clearly present prior to the appearance of this structure. This observation suggests that the formation of the intermitochondrial cement and chromatoid body are not required for microRNA biogenesis, but might be important for their action on target mRNAs. After excluding potential contributions to the Gasz phenotype by miRNAs, we analyzed the remaining small RNAs in greater detail. This analysis revealed that most non-repeat- associated piRNAs showed a skewed distribution with 6% contributing 95–99% of the reads. There was a similar low number of known non-repeat-associated piRNAs in the control and samples at P10 (Figure 7A), consistent with previous studies demonstrating a robust increase at P14 pachytene stage in wild-type testes [6]. Non-repeat-associated piRNAs contributed only 14% of the piRNA reads in control testes at this age. However, 99% of the 6500 non-repeat-associated piRNAs detected in control P10 testes were reduced in Gasz2/2 testes, 87% to undetectable levels. The decline was even more dramatic in P14 Gasz2/2 testes when many more piRNAs are produced in the control testes (Figure 7B and Table S2). Although the delayed spermatogenic development and apoptotic loss of spermatocytes could preclude the expression of these piRNAs in Gasz2/2 testes, the concurrent increase in miRNAs and expression of prepachy- tene piRNAs, normally abundant by P8 [6], indicate that these two effects are unlikely to be the cause. Over 1500 of 1700 distinct non-repeat-associated RNAs (92%) with greater abundance at P7 versus P14 were substantially reduced, and 1418 of these (90%) displayed a sustained reduction at subsequent time-points (Table PLoS Genetics | www.plosgenetics.org 6 September 2009 | Volume 5 | Issue 9 | e1000635