GASZ Is Essential for Male Meiosis Figure 6. Dysregulation of transposable elements in Gasz testes. (A–B) Quantitative RT-PCR analysis of transposable elements in testes from embryonic, newborn, 7- and 14-day-old mice (mean6SEM). (C) Western blot analysis of testis samples from 14-day-old mice by using anti-GAG (Upper), anti-ORF1 (Middle), or anti-b-actin control (Lower) demonstrated increased IAP GAG and LINE L1 ORF1p expression in Gasz2/2 testes. (D–G) Immunofluorescent analysis of IAP GAG (D–E) and ORF1p (F–G). Robust staining of IAP GAG and ORF1p is detected in Gasz2/2 gonocytes (E,G) but absent from Gasz+/2 controls (D,F). (H) CpG methylation analysis of IAP and LINE L1 using bisulfite-converted testicular genomic DNA. Methylated CpG dinucleotides remain unconverted as cytosine (filled circles) and unmethylated cytosines are converted to uracils and amplified as thymidines (open circles). Percentages of CG dinucleotide methylation are given. doi:10.1371/journal.pgen.1000635.g006 versus 0.91%) (Figure 7D). These findings indicate that GASZ plays a major role prior to the pachytene stage in facilitating the production of multiple types of piRNAs, including those associated with repeats involved in regulation of retrotransposons. Discussion GASZ was initially identified by our group as a male and female germ cell and maternal effect gene product [49]. Herein, we show that GASZ is not essential for fertility in the female germline. In contrast, absence of GASZ leads to male sterility due to a block at the zygotene-pachytene transition, reminiscent of the defect that is observed in knockouts of two PIWI family members, MILI and MIWI2 [7,11,38]. In frog oocytes, GASZ is expressed in the Balbiani body, a nuage structure [50]. Similarly, we show here that GASZ localizes to nuage in testicular primordial germ cells, gonocytes, spermatogonia, and spermatocytes. Using various markers, GASZ appears to be a component of the nuage termed intermitochondrial cement. We show that many nuage proteins depend upon GASZ for their normal levels. Aside from MIWI, which is expressed in germ cell types absent in the Gasz knockout, the reduction of MILI, MAEL, MVH, TDRD1, TDRD6, and TDRD7 in Gasz2/2 testicular lysates likely reflects destabilization of this entire ultrastructural feature. TDRD1 and MVH become mislocalized in gonocytes and they become subsequently lost during postnatal spermatogenesis, suggesting that the loss of nuage proteins may result secondary to degradation following persistent failure to localize to germinal granules. The lesser change in MVH protein in GASZ testes at P10 may indicate that MVH is less dependent upon GASZ than are the remaining nuage proteins. Perhaps this effect is due to additional proteins functionally redundant with GASZ or that MVH has a greater intrinsic stability in the absence of GASZ than other nuage proteins. Protein-protein interaction motifs in GASZ, including ankyrin PLoS Genetics | www.plosgenetics.org 8 September 2009 | Volume 5 | Issue 9 | e1000635