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GASZ is essential for male meiosis and suppression of retrotransposon expression in the male germline.

Ma L, Buchold GM, Greenbaum MP, Roy A, Burns KH, Zhu H, Han DY, Harris RA, Coarfa C, Gunaratne PH, Yan W, Matzuk MM - PLoS Genet. (2009)

Bottom Line: MILI is prominent in embryonic and early post-natal germ cells in nuage also called germinal granules that are often associated with mitochondria and called intermitochondrial cement.We also find global shifts in the small RNAome, including down-regulation of repeat-associated, known, and novel piRNAs.These studies provide the first evidence for an essential structural role for GASZ in male fertility and epigenetic and post-transcriptional silencing of retrotransposons by stabilizing MILI in nuage.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Nuage are amorphous ultrastructural granules in the cytoplasm of male germ cells as divergent as Drosophila, Xenopus, and Homo sapiens. Most nuage are cytoplasmic ribonucleoprotein structures implicated in diverse RNA metabolism including the regulation of PIWI-interacting RNA (piRNA) synthesis by the PIWI family (i.e., MILI, MIWI2, and MIWI). MILI is prominent in embryonic and early post-natal germ cells in nuage also called germinal granules that are often associated with mitochondria and called intermitochondrial cement. We find that GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) co-localizes with MILI in intermitochondrial cement. Knockout of Gasz in mice results in a dramatic downregulation of MILI, and phenocopies the zygotene-pachytene spermatocyte block and male sterility defect observed in MILI mice. In Gasz testes, we observe increased hypomethylation and expression of retrotransposons similar to MILI testes. We also find global shifts in the small RNAome, including down-regulation of repeat-associated, known, and novel piRNAs. These studies provide the first evidence for an essential structural role for GASZ in male fertility and epigenetic and post-transcriptional silencing of retrotransposons by stabilizing MILI in nuage.

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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 (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.
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pgen-1000635-g006: 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 (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.

Mentions: There is increased retrotransposon transcription in MILI and MIWI2 testes [7],[38], and dying Gasz−/− 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 Gasz−/− 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 Gasz−/− 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 Gasz−/− 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 Gasz−/− gonocytes (Figure 6D–6G).


GASZ is essential for male meiosis and suppression of retrotransposon expression in the male germline.

Ma L, Buchold GM, Greenbaum MP, Roy A, Burns KH, Zhu H, Han DY, Harris RA, Coarfa C, Gunaratne PH, Yan W, Matzuk MM - PLoS Genet. (2009)

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 (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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2727916&req=5

pgen-1000635-g006: 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 (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.
Mentions: There is increased retrotransposon transcription in MILI and MIWI2 testes [7],[38], and dying Gasz−/− 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 Gasz−/− 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 Gasz−/− 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 Gasz−/− 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 Gasz−/− gonocytes (Figure 6D–6G).

Bottom Line: MILI is prominent in embryonic and early post-natal germ cells in nuage also called germinal granules that are often associated with mitochondria and called intermitochondrial cement.We also find global shifts in the small RNAome, including down-regulation of repeat-associated, known, and novel piRNAs.These studies provide the first evidence for an essential structural role for GASZ in male fertility and epigenetic and post-transcriptional silencing of retrotransposons by stabilizing MILI in nuage.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Nuage are amorphous ultrastructural granules in the cytoplasm of male germ cells as divergent as Drosophila, Xenopus, and Homo sapiens. Most nuage are cytoplasmic ribonucleoprotein structures implicated in diverse RNA metabolism including the regulation of PIWI-interacting RNA (piRNA) synthesis by the PIWI family (i.e., MILI, MIWI2, and MIWI). MILI is prominent in embryonic and early post-natal germ cells in nuage also called germinal granules that are often associated with mitochondria and called intermitochondrial cement. We find that GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) co-localizes with MILI in intermitochondrial cement. Knockout of Gasz in mice results in a dramatic downregulation of MILI, and phenocopies the zygotene-pachytene spermatocyte block and male sterility defect observed in MILI mice. In Gasz testes, we observe increased hypomethylation and expression of retrotransposons similar to MILI testes. We also find global shifts in the small RNAome, including down-regulation of repeat-associated, known, and novel piRNAs. These studies provide the first evidence for an essential structural role for GASZ in male fertility and epigenetic and post-transcriptional silencing of retrotransposons by stabilizing MILI in nuage.

Show MeSH
Related in: MedlinePlus