Figure 7. Repeat and non-repeat piRNAs regulated by GASZ. (A) Compositional analysis of small RNA populations at postnatal days 7 (P7), 10 (P10), and 14 (P14) in Gasz+/2 (Left) and Gasz2/2 (Right) testes with annotation of small RNA populations as described in experimental procedures. (B–D) Comparison of the relative abundance of several classes of known and putative novel piRNAs between Gasz2/2 and control testes at postnatal days 7, 10, and 14 including known non- repeat-associated piRNAs (B), repeat-associated piRNAs (25–29 nt) (C) and unknown small RNAs (25–29 nt) (D). doi:10.1371/journal.pgen.1000635.g007 GASZ Is Essential for Male Meiosis domains, a sterile alpha motif, and a leucine zipper, may serve to direct the association and/or stabilization of nuage proteins. GASZ self-interaction may suggest the protein forms multimeric complexes as part of this function. A model depicting direct interactions within the nuage and proposed GASZ placement within this protein complex is provided (Figure S11). The Gasz mice phenocopy the loss of MILI. Since the TDRD1:MILI interaction is critical [33,48] and absence of GASZ leads to disruption of the TDRD1 distribution in the cytoplasm (Figure 4), MILI could be physically dislodged from its granule position in Gasz germ cells leading to a subsequent destabilization of MILI. Although formation of intermitochondrial cement is disrupted in both Tdrd12/2 and Gasz2/2 germ cells, MILI is only unstable in Gasz cells indicating a distinct role for GASZ in stabilization of MILI. As we have shown, that all but 2.2% of germ cells at E16.5 contain MILI, and at P0, MILI is absent. Similar to the Mili mice [6,11,38], the absence of MILI in the Gasz knockout, is believed to mechanistically disrupt piRNA synthesis, resulting in an increase in retrotransposons and subsequent catastrophe for the male germ line. The current model for maintenance of transposable element repression in the germline involves the production of piRNAs by PIWI family members MILI and MIWI2 in embryonic and postnatal germ cells [6,7]. Repeat-associated piRNAs are abundant in embryonic germ cells coincident with DNA remethylation of retrotransposons in the male germline at E17.5. GASZ is expressed in embryonic testes and co-localizes with MILI in fetal and newborn gonocytes. Transcriptional regulation of retrotransposons by antisense repeat- associated piRNAs, bound to MIWI2, whose nuclear localization depends upon MILI, has been proposed [13]. Consistent with the proposed mechanism, we observe a similar reduction of promoter methylation of IAP and Line L1 in Gasz2/2 testes as observed in Mili2/2 and Miwi22/2 testes. Mechanistically, absence of Gasz and the consequent loss of MILI and MILI-mediated retro- transposon repression affects fetal gonocytes during the interval that retrotransposon remethylation normally occurs. Although there is a continuity of the cell cycle arrest in male gonocytes between E16.5 and P0, the shift in Gasz testes at E16.5 from slightly detectable MILI to its absence suggests that nuage is changing during this interval despite a lack of cell cycle progression. However, both MVH and TDRD1 displayed similar behavior in Gasz testes at E16.5 and P0. Although we can find a direct interaction between GASZ and MIWI, we could not find evidence for direct interaction between GASZ and MILI. We speculate that a GASZ-MILI interaction occurs indirectly though other mediators. MVH can interact with MILI and MIWI [38], as can TDRD1 [33,48] both of which co-immunoprecipitate with GASZ. As shown in Figure 5A, GASZ interacts with RANBP9 and MIWI, known MVH interactors. Thus a large complex of proteins may be required for co-localization of GASZ with MILI and its support of MILI function in retrotransposon control. The inability to co-immunoprecipitate GASZ and MILI may suggest that MILI is more weakly ‘‘linked’’ to GASZ in this complex. Most of the nuage protein mutants that block in meiotic prophase have defects in repeat-associated piRNAs. Nuage protein knockouts cause two types of spermatogenic defects – those that block during meiotic prophase (including MVH, MILI, MIWI2 and MAEL) and those that block during haploid differentiation (including DDX25, MIWI, TDRD1, and TDRD6). In the latter class, Tdrd12/2 and Miwi2/2 testes do not have altered repeat- associated piRNAs, both of which block at the spermatid stage and do not appear to have defects in stem cell maintenance. Nearly all of the former class possesses some defect in piRNA biosynthesis, but repeat-associated piRNAs are not altered in all mutants of this PLoS Genetics | www.plosgenetics.org 9 September 2009 | Volume 5 | Issue 9 | e1000635