Ma L , Buchold GM , Greenbaum MP , Roy A , Burns KH , Zhu H , Han DY , Harris RA , Coarfa C , Gunaratne PH , Yan W , Matzuk MM .

HD050281 NICHD NIH HHS , K08-CA134746 NCI NIH HHS , T32-HD07165 NICHD NIH HHS , U54-HD07495 NICHD NIH HHS , U01-HD060496 NICHD NIH HHS , T32 HD007165 NICHD NIH HHS , R01 HD050281 NICHD NIH HHS , U54 HD007495 NICHD NIH HHS , K08 CA134746 NCI NIH HHS

	Figure 1. Targeting of the Gasz allele and generation of Gasz mutant mice.(A) Schematic representation of the Gasz gene, structure of the targeting vector, and the resultant mutant allele. Genomic DNA fragments used as 5′ and 3′ homology arms in the targeting vector are indicated by thick lines. Exons 1 and 2 (which encode the Gasz transcriptional start site and the initiation ATG codon) are replaced by a PgkHPRT expression cassette (shaded boxes). The 5′ and 3′ probes (filled boxes) used for Southern blots are indicated. (S) Sph I, (K) Kpn I. The MC1tk expression cassette was used for negative selection. (B) Southern blot analysis of genomic DNA derived from a litter from Gasz+/− (+/−) intercrosses. Similar percentages of male and female mice were genotyped as Gasz homozygous null (−/−). The 5′ probe hybridizes to 7.7 kb (wild-type, WT) and 4.8 kb (mutant, Mut) SphI fragments. (C) RT-PCR analysis of Gasz expression in Gasz+/− (+/−) and Gasz−/− (−/−) testes from E18.5 to post-natal day 14 demonstrating Gasz expression in embryonic testes and the lack of Gasz mRNA in null testes. (D) Western blot analysis of wild type testis samples from different time-points as well as 6-week-old Gasz WT, +/−, and −/− mice using a polyclonal antibody to GASZ (Upper) or an antibody to β-actin as a control for sample loading (Lower). GASZ protein is detected as early as postnatal day 2 and peak abundance is reached after 14 days of age. Absence of the GASZ protein in Gasz−/− testes confirmed that the Gasz−/− mutation was null.
	Figure 2. Gross and histological analysis of postnatal testes.(A) Gross analysis of testes from 7-week-old littermates. (B–M) Histological analysis of testes of Gasz+/− and Gasz−/− mice. (M) is a higher power magnification of (L) to show the lack of germ cells attached to the base of the tubule and sloughing germ cells in the lumen. G, spermatogonia; L, leptotene spermatocytes; M, meiotically dividing spermatocytes; Mit, mitotically dividing spermatogonia; P, pachytene spermatocytes, PL, preleptotene spermatocytes; Ser, Sertoli cells; Z, zygotene spermatocytes; dying spermatocytes with compact chromatin (red arrowhead); and with diffuse chromatin (open arrowhead); sloughing germ cells (black arrowheads). [Scale bars: 100 µm (B–C), 20 µm (D–K).]
	Figure 3. GASZ co-immunolocalization with intermitochondrial cement markers.(A–F) Immunofluorescent analysis of spermatocytes (A–C) and spermatids (D–F). (A,D) GASZ (red) localizes between mitochondrial clusters in spermatocytes using antibodies to cytochrome c (green). (A inset) Higher magnification. (B,E) Staining is shown for GASZ (red), TDRD1 (green). (C,F) Staining for GASZ (red) and MVH (green). Arrows in (E,F) identify the chromatoid body. Note the presence of the GASZ (red) foci and corresponding TDRD1 and MVH (green) foci in spermatocytes (A–C) but not in spermatids (D–F). (G–I) Staining is shown for GASZ (G), TDRD1 (H), and merged (I). (J–L) Staining is shown for GASZ (J), MILI (K), and merged (L). (M–O) Staining of newborn testes is shown for GASZ (M), MILI (N), and merged (O). Note the presence of the GASZ (green) foci and corresponding MILI (red) foci in gonocytes (arrowheads). [Scaling: 10,000×(A–F), 40X(G–L), and 400×(M–O) magnification.]
	Figure 4. MILI is lost in embryonic and newborn testes in the absence of GASZ.Immunofluorescent analysis of Gasz+/− (A,C,E,G,I,K) and Gasz−/− (B,D,F,H,J,L) testes. Staining is shown for TDRD1 (A–D), MILI (E–H), and MVH (I–L). TDRD1 immunostaining is diffusely cytoplasmic in Gasz−/− newborn (B) and embryonic (D) gonocytes versus controls (A,C). Staining of the identical germ cells shows MILI is undetectable in newborn gonocytes and present in only a subset of Gasz−/− E16.5 gonocytes [arrowheads in (F,H)]. MVH staining is less granular in Gasz−/− (J,L) versus controls (I,K). [Scaling: 5,000×magnification.]
	Figure 5. GASZ interactions and reduction of nuage proteins in Gasz null testes.Biosensor quantification of interaction for a full-length GASZ bait by (A) RANBP9 partial clone, and by (B) GASZ and MIWI. (C,D) GASZ co-immunoprecipitation with nuage proteins. Testicular protein lysates from 21-day-old mice were incubated with no primary antibody [lane 1, (C,D)], anti-GASZ antibody [lane 2, (C)] or anti-MIWI antibody [lane 2, (D)] to immunoprecipitate protein complexes. 10 µg of WT lysate [lane 3, (C,D)] and Gasz−/− lysate [(lane 4, (C)] were used as controls. Co-immunoprecipitating proteins were detected by western blot analysis using antibodies against MIWI, TDRD1, MVH, MILI, MAEL, TDRD6, TDRD7, or β-actin. (E) Western blot analysis of testicular protein lysates prepared from 10- and 14-day-old Gasz+/− (+/−, lanes 1 and 3) and Gasz−/− (−/−, lanes 2 and 4) mice. Antibodies against MIWI, TDRD1, MVH, MILI, TDRD6, TDRD7, or an antibody to β-actin show levels of nuage proteins are reduced in Gasz−/− testes prior to spermatocyte apoptosis.
	Figure 6. Dysregulation of transposable elements in Gasz null testes.(A–B) Quantitative RT-PCR analysis of transposable elements in testes from embryonic, newborn, 7- and 14-day-old mice (mean±SEM). (C) Western blot analysis of testis samples from 14-day-old mice by using anti-GAG (Upper), anti-ORF1 (Middle), or anti-β-actin control (Lower) demonstrated increased IAP GAG and LINE L1 ORF1p expression in Gasz−/− testes. (D–G) Immunofluorescent analysis of IAP GAG (D–E) and ORF1p (F–G). Robust staining of IAP GAG and ORF1p is detected in Gasz−/− gonocytes (E,G) but absent from Gasz+/− 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.
	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+/− (Left) and Gasz−/− (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 Gasz−/− 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).

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