Author Summary Many aspects of RNA processing are essential for or prominent in the differentiation of germ cells. Some RNA metabolism in animal germ cells is associated with physical structures surrounding the cell nucleus called nuage. Nuage has a distinct granular appearance prior to the meiotic divisions with unclear functions. We have identi- fied a protein called GASZ, which plays a structural role in this early nuage. In mice lacking GASZ, retrotransposons— endogenous viral-like particles—become released from their typical repressed state in the germline by the loss of small RNAs called piRNAs, resulting in DNA damage and delayed germ cell maturation. Protection of the germline from genetic intruders may require the association of piRNA-synthesizing enzymes and other components of this nuage structure through direct or indirect associations with GASZ. Mutations in GASZ and other nuage compo- nents may contribute to infertility in men who do not produce spermatozoa. mouse VASA homolog (MVH; also called DDX4 or DEAD-box polypeptide 4), tudor-domain containing 1 (TDRD1), tudor- domain containing 6 (TDRD6), and tudor-domain containing 7 (TDRD7) [19–21]. The chromatoid body is not believed to arise merely by coalescence of intermitochondrial cement granules; however, to date there are no examples of proteins localized by electron microscopy specifically to the intermitochondrial cement but absent from the chromatoid body. Nuage are proposed sites for multiple RNA processing events including translational repression, RNA-mediated gene silencing, mRNA degradation, and nonsense-mediated mRNA decay [22]. A number of germ cell-specific mRNAs display translational repression with a lag of up to a week between their transcription and translation [23,24]. The evidence for nuage regulation of mRNA is strongest for the chromatoid body. Translationally regulated mRNAs such as transition protein 2 (Tnp2) have been localized to the chromatoid body [25]. DEAD box helicases, including MVH/DDX4 and DDX25, which can unwind RNA in vitro, are localized to the chromatoid body [26–30]. MicroRNAs (,22 nt non-coding RNAs) and components of the RNA-Induced Silencing Complex (RISC) machinery including Dicer, Argonaute 2 (AGO2), and Argonaute 3 (AGO3) with demonstrated in vitro endonuclease activity, localize to the chromatoid body where they may function in translational control and mRNA stability, but their potential association with the intermitochondrial cement is not described [22,31]. The chromatoid body also contains MILI and MIWI RNA endonucleases that generate piRNAs [32,33]. Most MIWI-associated mRNAs in spermatids are associated with the RNP fraction with a smaller number associated with polysomes suggestive of a function in translational control [34]. Prior to the meiotic divisions, the role of nuage in mRNA metabolism in primordial germ cells, spermatogonia, and spermatocytes is unknown. Whereas MILI is present throughout this period, MIWI2 is restricted to nuage granules in embryonic testes, and MIWI is present in those of pachytene spermatocytes [13,35–37]. The localization of PIWI family proteins to the intermitochondrial cement has not been clearly defined, although MILI interacts with the intermitochondrial cement protein TDRD1, MVH and other nuage proteins [33,38]. Maelstrom (MAEL), the putative 39–59 endonuclease for piRNA 39 end formation, is also associated with nuage in mammals [39–41]. Thus, multiple components necessary for piRNA generation are connected physically to nuage. GASZ Is Essential for Male Meiosis Genetic evidence supports a conserved requirement for proper nuage assembly in retrotransposon control in the germline. In Drosophila, mutants of PIWI, aubergine (AUB), AGO3, the RNA helicases VASA and armitage (ARMI), and TUDOR-domain containing proteins krimper (KRIMP) and spindle-E (SPN-E) have defects in piRNA synthesis and retrotransposon control. In Drosophila, the interaction of two PIWI family members with opposite strand polarity during nuage assembly has also been proposed to facilitate the ‘‘ping-pong’’ mechanism of amplification necessary for retrotransposon inhibition [42]. piRNA defects and derepression of retrotransposons in germ cells also occurs in zebrafish ZILI and ZIWI mutants [43,44]. Consistent with a key role of these nuage-associated proteins in the mammalian male germline, knockouts of Mvh, Mael, Mili, and Miwi2 block at the spermatocyte stage, while knockouts of TDRD1, TDRD6, and MIWI disrupt spermatogenesis at the spermatid stage. With the exception of MVH, which has not been assessed for these defects, all other nuage mutants with a spermatocyte arrest have defects in retrotransposon regulation and piRNA production [7,20,35,38,45–48]. GASZ is a 475 amino acid Germ cell-specific protein with four Ankyrin repeats, a Sterile alpha motif, and a basic leucine Zipper domain [49] that is conserved across vertebrate evolution in amphibians, fish, birds, and mammals [50]. Our previous studies have shown that GASZ localizes to the Balbiani body, a nuage structure in Xenopus laevis oocytes. The high degree of evolutionary conservation of GASZ in vertebrates and the potential localization of GASZ to a conserved germline-specific structure stimulated our interest to determine the expression and essential roles of GASZ in nuage and its germline function in mammals. Results Knockout of GASZ Results in Male Sterility To define the roles of GASZ in mammals, a mutation in Gasz was generated (Figure 1). Gasz+/2 mice were viable and produced pups (1.0060.01 litters/month; 8.2560.29 pups/litter, n = 10) whose genotypes were consistent with Mendelian ratios (25.9% WT, 48.5% Gasz+/2, 25.7% Gasz2/2; n = 495) indicating that GASZ is not essential for embryogenesis. Whereas Gasz2/2 females were fertile (0.9560.02 litters/month; 6.1660.32 pups/ litter), Gasz2/2 males were sterile. Furthermore, although GASZ is a maternal effect protein [49], observed in early preimplantation embryos, the viability of offspring from Gasz females (i.e., oocytes lacking Gasz mRNA) indicates that maternal GASZ is also not required. Gasz2/2 Males Demonstrate a Defect in Meiosis To define the cause of the infertility in Gasz2/2 males, postnatal testes were analyzed grossly and histologically (Figure 2). Testes from Gasz2/2 males were significantly smaller (P,0.0001) than Gasz+/2 or WT littermates (Figure 1A). Six-week-old Gasz2/2 testes (19.261.07 mg; n = 20) were ,20% of the weight of WT (89.965.68 mg; n = 20) and Gasz+/2 (85.865.50 mg; n = 20) testes. Whereas WT and Gasz+/2 testes from adult males demonstrated robust spermatogenesis (Figure 2H), the seminifer- ous tubules of 6-week-old Gasz2/2 testes showed markedly reduced spermatocytes, no post-meiotic spermatids or spermato- zoa, and significant vacuolization (Figure 2I–2K). The most mature meiotic cells in seminiferous tubules from stages VII–XII were early meiotic germ cells while those from stages I–VI showed a mixture of degenerating spermatocytes. Gasz2/2 and Gasz+/2 testes at postnatal day 5 (P5) were similar at the gross and histologic levels (Figure S1). At postnatal day 10 (P10), PLoS Genetics | www.plosgenetics.org 2 September 2009 | Volume 5 | Issue 9 | e1000635