class. Like GASZ, disruption of MILI and MIWI2 result in an absence of repeat-associated piRNAs and a meiotic block prior to the loss of all germ cells from seminiferous tubules by 6 months of age [6,11,38]. The Mael2/2 testis phenotype is unique in showing isolated pachytene piRNA defects but blocking during meiotic prophase with elevated retrotransposons [46]. An important gap in our knowledge is whether Mvh1098/1098 testes display repeat- associated piRNA defects and elevated retrotransposons similar to vasa mutants in Drosophila [55] and the majority of piRNA pathway mutants blocking during meiotic prophase. Germ cell apoptosis in Gasz mice may depend upon retrotransposon expression combined with meiotic defects, representing distinct MILI-dependent functions. The GAG, POL, and PRT proteins are all required for efficient retrotrans- position of IAP [56], but their individual contribution to cellular toxicity has not been described. Line L1 proteins are toxic to cells by several mechanisms including activation of BAX and caspase 3 [57]. Expression of the reverse transcriptase of ORF2 alone causes cellular pathology in cell lines and could inhibit completion of meiotic recombination in nuage protein knockouts through its ability to bind random breaks in DNA [58,59]. Alternatively, the cause of apoptosis in Gasz2/2 spermatocytes and nuage knockouts in the meiotic class may be retrotransposon-independent. Two observations suggest MILI regulates meiosis independent of retrotransposons. TDRD1, by preventing the processing of mRNAs into piRNAs, confers specificity in non-repeat piRNA generation by MILI [48]. Line L1 dysregulation in the Tdrd12/2 mutant must depend upon the decreased mRNAs or increased unannotated RNAs that results from the shift of the substrate RNA used by MILI to generate piRNAs. Since TDRD1 deficient testes do not block during meiotic prophase despite elevated Line L1 expression, spermatocyte death is unlikely to be due to isolated retrotransposon dysfunction. Missense mutants of the zebrafish MILI ortholog ZILI have isolated meiotic defects but no alterations in retrotransposon expression [43], indicating MILI’s contribution to meiosis and retrotransposon control are separable. Knockout models suggest that homologous chromosome synapsis depends upon functional MILI [11,38], MIWI2 [7,11] and MAEL [46]. Mvh testes block at the zygotene-pachytene transition but their ability to complete synapsis has not been assessed [45]. Therefore, the apoptotic loss of Gasz2/2 spermatocytes may not result from retrotransposon-induced DNA damage alone, but could also result from failed MILI-dependent meiotic functions including homologous chromosome synapsis or meiotic recombi- nation. MILI has been proposed to cause general effects on germ cell mRNA translation not limited to control of retrotransposon mRNAs [37]. It is possible that this aspect of MILI function might contribute to the reduction of nuage protein levels in Gasz testes. Translational control of mRNAs necessary for meiotic prophase by MILI may indirectly affect retrotransposon promoter methylation or post-transcriptional processing of retrotransposon mRNAs in addition to MILI’s actions mediated by repeat- associated piRNAs. The piRNA pathway in mammals is specialized to support male germ cell development. Zebrafish and Drosophila share the requirement for PIWI family-dependent piRNA production for fertility of both sexes [42–44]; whereas, it is required only for male fertility in the mouse. GASZ is not correlated with this shift because GASZ orthologs are present in many vertebrates including zebrafish [50]. In the mammalian male germline, GASZ supports piRNA biosynthesis required for initiation of retro- transposon repression during the embryonic period and mainte- nance during meiotic prophase when alterations to chromatin and transcriptional increase would otherwise be favorable toward their GASZ Is Essential for Male Meiosis expression. GASZ is the first mammalian germline- and intermitochondrial cement-specific protein lacking domains for RNA modification which impacts piRNA processing by localizing or stabilizing multiple proteins in the nuage including PIWI family members. Materials and Methods Generation of Gasz Mutant Mice and Fertility Studies We electroporated the linearized Gasz targeting vector (Figure S1A) into the HPRT-negative AB2.2 ES cell line; selected clones in hypoxanthine, aminopterine, thymidine, and 1-(2 -deoxy-2 fluoro-D-arabinofuranosyl)-5-iodouracil; and screened the ES cell DNA by Southern blot as described [60] to identify the mutant Gasz allele, Gasztm1Zuk (herein called Gasz2). Correctly targeted clones were identified by using 59 and 39 probes as shown. Targeted ES cell clones were injected into blastocysts to produce chimeric male mice [61], which were bred to produce C57BL6/ J6129 hybrid F1 Gasz2/2 offspring. Ten homozygous mutant and heterozygous sires were bred with WT females over a 6-month mating period. Similar mating trials were performed with homozygous mutant and heterozygous dams. Except for testis defects, Gasz2/2 mice were grossly indistinguishable from their littermates and lived to become adults. Generation of the Anti-GASZ Antibody and Western Blot Analysis Full-length His-tagged GASZ was injected into guinea pigs to produce polyclonal antibodies (Cocalico Biologicals, Reamstown, PA). Membranes with 20 or 50 mg of total testis lysate per lane were probed with guinea pig anti-GASZ (1:1000), rabbit anti-IAP GAG (1:1000), rabbit anti-TDRD1 (1:1000), rabbit anti-TDRD6 (1:1000), rabbit anti-TDRD7 (1:500), rabbit anti-MVH (1:500), rabbit anti-MAELSTROM (1:250 Abcam ab28661), or rabbit anti-MILI (1:250 Abcam ab36764) polyclonal antibodies. After developing, the membrane was stripped and re-probed with anti- b-actin clone AC-15 (Sigma) at 1:5,000. Secondary anti-guinea pig and anti-mouse horseradish peroxidase-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA) were used at 1:10,000. Immunohistochemistry, Immunofluorescence, and Co- Immunoprecipitation Western Analysis Testes were fixed in Bouin’s fixative (for histology), 4% paraformaldehyde (immunofluorescence) or 4% paraformalde- hyde/6.6% acetic acid (for immunohistochemistry), and embed- ded in paraffin. Antigen retrieval was performed on 5 mm sections by boiling for 20 minutes in citrate buffer pH 6.0. Samples for immunofluorescence were incubated with rabbit anti-cH2AX (1:5,000; 05–636 Upstate, now Millipore, Billerica, MA), guinea pig anti-GASZ (1:300), rabbit anti-MVH (1:500), rabbit anti- TDRD1 (1:100), rabbit anti-MILI (1:500), rabbit anti-IAP GAG (1:500), rabbit anti-ORF1 (1:500), and mouse anti-cytochrome c (1:300, 556433 BD Biosciences San Jose, CA). Alexa594- conjugated anti-guinea pig, Alexa488-conjugated anti-rabbit, and Alexa488-conjugated anti-mouse antibodies (Jackson Immu- noResearch) were used at 1:500. Sections were mounted with Vectashield mounting medium with DAPI or propidium iodide (Vector Laboratories). Representative images for immunofluores- cence were selected and captured on a Zeiss Axiovert s100 2TV. When required, deconvolution was performed with softWoRx v3.3.6 (Applied Precision, Issaquah, WA). One mg of testicular lysates from WT mice were prepared with 0.5% NP-40 lysis buffer as described previously [62]. Pre-cleared PLoS Genetics | www.plosgenetics.org 10 September 2009 | Volume 5 | Issue 9 | e1000635