Gasz2/2 testes with many tubules displaying a Sertoli cell only phenotype (Figure 2L and 2M). To further confirm our histological findings, we analyzed several genes that are expressed in spermatocytes (Figure S2 and Figure S3). cH2AX is expressed from late spermatogonia through pachytene spermatocytes [51]. cH2AX shifts from staining autosomes to the XY body in pachytene spermatocytes. XY body staining by cH2AX was absent from Gasz2/2 testes, in contrast to Gasz+/2 testes (Figure S2). Instead, cH2AX labeled autosomes similar to Gasz+/2 early spermatocytes or dying spermatocytes with increased intensity. Our findings are reminiscent of MILI, MIWI2, and MVH knockout mouse models that demonstrate male sterility due to a zygotene:pachytene block but normal female fertility [7,38,45]. GASZ Is a Component of the Intermitochondrial Cement and Co-Localizes with MILI GASZ is present at low levels in the cytoplasm of type A and B spermatogonia and pre-leptotene spermatocytes, showing peak intensity in middle to late pachytene spermatocytes, and localizing to finer granules in the cytoplasm of round spermatids (Figure S4 and Figure S5). Using antibodies against mitochondrial cyto- chrome c, GASZ was confirmed to localize to the interstices of spermatocyte mitochondrial clusters (i.e., intermitochondrial cement) (Figure 3A and inset), consistent with our previous findings that GASZ localizes to nuage in frog oocytes [50]. GASZ partially co-localizes with TDRD1 and MVH in the intermitochondrial cement (Figure 3B–3I) and displays a substantial overlap with MILI in spermatocytes (Figure 3J–3L). Whereas TDRD1 and MVH relocalize to the chromatoid body in spermatids (Figure 3E and 3F), GASZ fails to do so and also shows no relationship with unclustered mitochondria (Figure 3D). In late pachytene sper- matocytes MIWI and GASZ also overlap (Figure S6). MVH shows intense granular distribution in Gasz+/2 testes but is dramatically reduced in the Gasz2/2 spermatocytes (Figure S7). We also analyzed the relationship of GASZ and MILI at earlier time points to assess their interaction in immature germ cells. MILI was present in perinuclear granules in cell cycle arrested gonocytes of newborn mice (Figure 3N) in a distribution similar to that described in embryonic male germ cells [13]. There is a significant overlap between GASZ and MILI in newborn gonocytes (Figure 3M–3O). We compared the immunostaining of nuage proteins in the newborn Gasz2/2 testis versus controls. TDRD1 was reduced but also diffusely cytoplasmic, failing to localize in a perinuclear granular pattern (Figure 4B). MILI was strikingly absent from the same GASZ gonocytes (Figure 4F). MVH immunostaining also showed reduced granular localization (Figure 4J). Analysis of TDRD1 (Figure 4D) and MVH (Figure 4L) at E16.5 revealed a similar delocalization of these proteins in GASZ gonocytes. However, MILI staining of E16.5 gonocytes (Figure 4H) was variable; in most germ cells, MILI staining was absent, whereas in only 2.25% (5 out of 222 gonocytes) granular MILI staining remained. GASZ Interaction Affects the Level of Intermitochondrial Cement Proteins To study GASZ protein:protein interactions, we screened a P17 testis library by yeast two-hybrid analysis using full-length GASZ as bait. We found GASZ interacts with itself and RANBP9 (Figure 5A), a known MVH interactor that also localizes to nuage and the chromatoid body [52]. MIWI, but not MILI, or MVH, also directly interacts with GASZ (Figure 5B). Antibodies to GASZ could co-immunoprecipitate MIWI, TDRD1, and MVH, but not GASZ Is Essential for Male Meiosis MILI or MAEL from P21 testes (Figure 5C). Conversely, antibodies to MIWI could also co-immunoprecipitate GASZ (Figure 5D). We observed a striking reduction in the amount of multiple intermitochondrial cement proteins in Gasz2/2 testis lysates at P21 as well as at times prior to the observed spermatocyte loss (Figure 5E). Multiple interactions between these nuage proteins suggest that GASZ is a component of this network. Quantitative RT-PCR showed relatively modest reduction (roughly 2-fold) in the intermitochondrial cement mRNAs in contrast to their change in protein abundance (Figure S8 and Table S1). Our findings by Western blot analysis were consistent with reduced MVH immunostaining of spermatocytes (Figure S7) and absence of MILI in gonocytes (Figure 4). We also examined newborn testes by electron microscopy for the presence of intermitochondrial cement. Although we could find nuage material associated with clustered mitochondria in most neonatal gonocytes in controls, we failed to detect this structure in Gasz2/2 germ cells (Figure S9). Our results indicate that GASZ plays a key role in the formation and/or maintenance of this network of RNA processing proteins. Absence of GASZ Causes Hypomethylation of Retrotransposons and Their Increased Expression There is increased retrotransposon transcription in MILI and MIWI2 testes [7,38], and dying Gasz2/2 spermatocytes have a characteristic chromatin pattern similar to these knockouts. Because of these similarities and GASZ association with MIWI, we measured levels of the retrotransposons intracisternal A particle (IAP) and long interspersed nuclear element 1 (Line L1) in the testes of P14 Gasz2/2 mice. Quantitative RT-PCR demonstrated up to a 15-fold increase (p,0.05) in Line L1 and up to a 4-fold increase in IAP mRNA (p,0.05) in the postnatal Gasz2/2 testis as well as similar increases in embryonic testes compared to controls (Figure 6A and 6B). The largest increases were seen in the Line L1 ORF2 (encoding the reverse transcriptase and endonuclease domains) and IAP GAG mRNAs at P14. We saw even more dramatic increases in IAP GAG protein and Line L1 ORF1p. At P14, levels of IAP GAG and Line L1 ORF1p proteins are essentially undetectable in the WT samples but significantly up- regulated in the Gasz2/2 testes (Figure 6C). Likewise, in gonocytes of control newborn (P0) mice, retrotransposon proteins IAP GAG and Line L1 ORF1p were undetectable but were dramatically elevated in the cytoplasm of the Gasz2/2 gonocytes (Figure 6D– 6G). Consistent with compromised transposable element DNA methylation seen in MILI and MIWI2 testes, we found significant hypomethylation of presumed germ cell-derived Line L1 and IAP sequences in Gasz2/2 testes compared to controls at P14 (Figure 6H). Sequences demonstrating appropriate methyla- tion in the Gasz knockout may reflect the presence of admixed somatic cell DNA. Thus, the delayed meiotic initiation and spermatocyte apoptosis in the Gasz2/2 testes are likely secondary to abnormal derepression of retrotransposons in the male germline at the transcriptional and post-transcriptional levels. Suppression of PIWI-Interacting RNAs (piRNAs) in GASZ Null Testes Since increased retrotransposon expression in MILI and MIWI2 germ cells has been ascribed to the loss of repression by repeat-associated RNAs, we evaluated their abundance by small RNA sequencing of P7, P10 and P14 Gasz+/2 and Gasz2/2 testes using the Illumina Next Generation Sequencing platform that yields over 2 million sequence reads per sample. Control testes PLoS Genetics | www.plosgenetics.org 4 September 2009 | Volume 5 | Issue 9 | e1000635