MILI, a PIWI-interacting RNA-binding Protein, Is Required for Germ Line Stem Cell Self-renewal and Appears to Positively Regulate Translation
2008; Elsevier BV; Volume: 284; Issue: 10 Linguagem: Inglês
10.1074/jbc.m809104200
ISSN1083-351X
AutoresYingdee Unhavaithaya, Yi Hao, Ergin Beyret, Hang Yin, Satomi Kuramochi‐Miyagawa, Toru Nakano, Haifan Lin,
Tópico(s)Animal Genetics and Reproduction
ResumoThe Argonaute/PIWI protein family consists of Argonaute and PIWI subfamilies. Argonautes function in RNA interference and micro-RNA pathways; whereas PIWIs bind to PIWI-interacting RNAs and regulate germ line development, stem cell maintenance, epigenetic regulation, and transposition. However, the role of PIWIs in mammalian stem cells has not been demonstrated, and molecular mechanisms mediated by PIWIs remain elusive. Here we show that MILI, a murine PIWI protein, is expressed in the cytoplasm of testicular germ line stem cells, spermatogonia, and early spermatocytes, where it is enriched in chromatoid bodies. MILI is essential for the self-renewing division and differentiation of germ line stem cells but does not affect initial establishment of the germ line stem cell population at 7 days postpartum. Furthermore, MILI forms a stable RNA-independent complex with eIF3a and associates with the eIF4E- and eIF4G-containing m7G cap-binding complex. In isolated 7 days postpartum seminiferous tubules containing mostly germ line stem cells, the mili mutation has no effect on the cellular mRNA level yet significantly reduces the rate of protein synthesis. These observations indicate that MILI may positively regulate translation and that such regulation is required for germ line stem cell self-renewal. The Argonaute/PIWI protein family consists of Argonaute and PIWI subfamilies. Argonautes function in RNA interference and micro-RNA pathways; whereas PIWIs bind to PIWI-interacting RNAs and regulate germ line development, stem cell maintenance, epigenetic regulation, and transposition. However, the role of PIWIs in mammalian stem cells has not been demonstrated, and molecular mechanisms mediated by PIWIs remain elusive. Here we show that MILI, a murine PIWI protein, is expressed in the cytoplasm of testicular germ line stem cells, spermatogonia, and early spermatocytes, where it is enriched in chromatoid bodies. MILI is essential for the self-renewing division and differentiation of germ line stem cells but does not affect initial establishment of the germ line stem cell population at 7 days postpartum. Furthermore, MILI forms a stable RNA-independent complex with eIF3a and associates with the eIF4E- and eIF4G-containing m7G cap-binding complex. In isolated 7 days postpartum seminiferous tubules containing mostly germ line stem cells, the mili mutation has no effect on the cellular mRNA level yet significantly reduces the rate of protein synthesis. These observations indicate that MILI may positively regulate translation and that such regulation is required for germ line stem cell self-renewal. Stem cells are defined by their dual ability to self-renew and to produce numerous differentiated daughter cells. Among adult tissue stem cells, germ line stem cells in mammalian testes serve as the source of spermatogenesis, a robust stem cell-driven process that generates millions of sperm hourly (1Lin H. Sell S. Stem cell Handbook. Humana Press, Inc., Totowa, NJ2004: 57-74Google Scholar). In the testis, germ line stem cells can be identified as a subpopulation of spermatogonia that reside in the basal layer of the seminiferous epithelium. These stem cells divide in a self-renewing fashion to produce differentiating spermatogonia, which then further divide and differentiate into numerous spermatocytes. Spermatocytes undergo meiosis to produce haploid round spermatids, which then undertake drastic morphological changes via a process called spermiogenesis to become motile sperm. Germ line stem cells are not only of paramount importance in reproduction but also are effective models for studying molecular mechanisms that define the fate and behavior of stem cells in general. Germ line stem cell division and differentiation are regulated by both intercellular signaling and cell-autonomous mechanisms (1Lin H. Sell S. Stem cell Handbook. Humana Press, Inc., Totowa, NJ2004: 57-74Google Scholar). The requirement of intercellular signaling is exemplified by mutations in c-Kit receptors or its ligand, stem cell factor, which causes spermatogonial arrest prior to the differentiating (A1) spermatogonia stage (2de Rooij D.G. Okabe M. Nishimune Y. Biol. Reprod. 1999; 61: 842-847Crossref PubMed Scopus (118) Google Scholar, 3Ohta H. Tohda A. Nishimune Y. Biol. Reprod. 2003; 69: 1815-1821Crossref PubMed Scopus (82) Google Scholar). Intercellular signaling also involves dose-sensitive regulation by Glial cell line-derived neurotrophic factor, since mice heterozygous for its gene (Gdnf) display aberrant germ line stem cell division (4Meng X. Lindahl M. Hyvonen M.E. Parvinen M. de Rooij D.G. Hess M.W. Raatikainen-Ahokas A. Sainio K. Rauvala H. Lakso M. Pichel J.G. Westphal H. Saarma M. Sariola H. Science. 2000; 287: 1489-1493Crossref PubMed Scopus (1055) Google Scholar). Perhaps the best illustrated cell-autonomous requirements for germ line stem cell maintenance are Plzf and Sox3 genes, both of which encodes transcriptional factors (5Buaas F.W. Kirsh A.L. Sharma M. McLean D.J. Morris J.L. Griswold M.D. de Rooij Braun R.E. Nat. Genet. 2004; 36: 647-652Crossref PubMed Scopus (686) Google Scholar, 6Costoya J.A. Hobbs R.M. Barna M. Cattoretti G. Manova K. Sukhwani M. Orwig K.E. Wolgemuth D.J. Pandolfi P.P. Nat. Genet. 2004; 36: 653-659Crossref PubMed Scopus (741) Google Scholar, 7Raverot G. Weiss J. Park S.Y. Hurley L. Jameson J.L. Dev. Biol. 2005; 283: 215-225Crossref PubMed Scopus (117) Google Scholar). The cell-autonomous mechanism for mammalian germ line stem cell self-renewal probably involves translational regulation, as inferred from studies of germ line stem cells in lower model systems and spermatogenesis in mammals. In Drosophila, translational repression mediated by Pumilio and Nanos is crucial for maintaining the undifferentiated states of germ line stem cells and their precursors, primordial germ cells (8Parisi M. Lin H. Genetics. 1999; 153: 235-250Crossref PubMed Google Scholar, 9Wang Z. Lin H. Science. 2004; 303: 2016-2019Crossref PubMed Scopus (192) Google Scholar, 10Gilboa L. Lehmann R. Curr. Biol. 2004; 14: 981-986Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In Caenorhabditis elegans, ATX-2, a homolog of mammalian ATAXIN 2, binds to cytoplasmic poly(A)-binding protein and is required for translational regulation of germ line stem cell proliferation (11Ciosk R. DePalma M. Priess J.R. Development. 2004; 131: 4831-4841Crossref PubMed Scopus (104) Google Scholar). In mice, translational regulation has been shown to be crucial for spermiogenesis (12Giorgini F. Davies H.G. Braun R.E. Development. 2002; 129: 3669-3679Crossref PubMed Google Scholar), although its role in germ line stem cells is not yet determined. However, studies of a mutation that affects germ line stem cell maintenance, Jsd (juvenile spermatogonial depletion), hint at a role for translation in mammalian germ line stem cell self-renewal. Jsd mutant mice can only undergo one round of spermatogenesis, followed by early (Aal) spermatogonia arrest (13Beamer W.G. Cunliffe-Beamer T.L. Shultz K.L. Langley S.H. Roderick T.H. Biol. Reprod. 1988; 38: 899-908Crossref PubMed Scopus (80) Google Scholar). Molecular analysis indicates that the Jsd mutation affects mUtp14b, a testis-specific retroposed copy of the ubiquitously expressed gene mUtp14a (14Rohozinski J. Bishop C.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11695-11700Crossref PubMed Scopus (60) Google Scholar). This gene is homologous to the yeast UTP14 gene, which is an essential component of a large ribonucleoprotein complex containing the U3 small nucleolar RNA (15Dragon F. Gallagher J.E. Compagnone-Post P.A. Mitchell B.M. Porwancher K.A. Wehner K.A. Wormsley S. Settlage R.E. Shabanowitz J. Osheim Y. Beyer A.L. Hunt D.F. Baserga S.J. Nature. 2002; 417: 967-970Crossref PubMed Scopus (546) Google Scholar). Deletion of UTP proteins in yeast inhibits 18 S rRNA production, indicating that they are part of the active pre-rRNA-processing complex. Similarly, mice lacking Dazl (deleted in azoospermia-like) contain no differentiated spermatogonia (16Ruggiu M. Speed R. Taggart M. McKay S.J. Kilanowski F. Saunders P. Dorin J. Cooke H.J. Nature. 1997; 389: 73-77Crossref PubMed Scopus (513) Google Scholar), and Dazl has been implicated in translational regulation of meiotic genes but not germ line stem cells (17Collier B. Gorgoni B. Loveridge C. Cooke H.J. Gray N.K. EMBO J. 2005; 24: 2656-2666Crossref PubMed Scopus (174) Google Scholar, 18Reynolds N. Collier B. Maratou K. Bingham V. Speed R.M. Taggart M. Semple C.A. Gray N.K. Cooke H.J. Hum. Mol. Genet. 2005; 14: 3899-3909Crossref PubMed Scopus (145) Google Scholar). Translational regulation of germ line stem cells may involve the PIWI/ARGONAUTE (AGO) protein family, in addition to their known role in transposition repression and epigenetic silencing (19Lin H. Science. 2007; 316: 397Crossref PubMed Scopus (128) Google Scholar, 20Beyret E. Lin H. Appasani MicroRNAs: From Basic Science to Disease Biology. Cambridge University Press, Cambridge, UK2007: 497-511Crossref Scopus (2) Google Scholar). The PIWI/AGO family was first discovered to be required for stem cell maintenance in diverse organisms, such as Drosophila melanogaster, C. elegans, and Arabidopsis (21Cox D.N. Chao A. Baker J. Chang L. Qiao D. Lin H. Genes Dev. 1998; 12: 3715-3727Crossref PubMed Scopus (787) Google Scholar). 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They function in RNA interference and miRNA pathways as essential components of the RNA-induced silencing complex (RISC) and RNA-induced silencing of transcription (RITS) complex to negatively regulate gene expression at the posttranscriptional and transcriptional level, respectively. Recently, mammalian AGO2 has been shown to up-regulate translation via the AU-rich elements (ARE) in mRNA 3′-untranslated regions (25Vasudevan S. Steitz J. Cell. 2007; 128: 1105-1118Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). In contrast, PIWI proteins bind to 24-31-nucleotide PIWI-interacting RNAs (piRNAs) and regulate germ line development, stem cell maintenance, epigenetic regulation, and transposition (26Grivna S.T. Beyret E. Wang Z. Lin H. Genes Dev. 2006; 20: 1709-1714Crossref PubMed Scopus (636) Google Scholar, 27Aravin A. Gaidatzis D. Pfeffer S. Lagos-Quintana M. Landgraf P. Iovino N. Morris P. Brownstein M.J. Kuramochi-Miyagawa S. Nakano T. Chien M. Russo J.J. Ju J. 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Genes Dev. 2007; 21: 2300-2311Crossref PubMed Scopus (266) Google Scholar, 34Yin H. Lin H. Nature. 2007; 450: 304-308Crossref PubMed Scopus (330) Google Scholar). Two PIWI proteins in Drosophila, AUBERGINE and PIWI itself, have been genetically implicated in translational regulation (35Wilson J.E. Connell J.E. Macdonald P.M. Development. 1996; 122: 1631-1639Crossref PubMed Google Scholar, 36Harris A.N. Macdonald P.M. Development. 2001; 128: 2823-2832Crossref PubMed Google Scholar, 37Megosh H.B. Cox D.N. Campbell C. Lin H. Curr. Biol. 2006; 16: 1884-1894Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). In addition, MIWI, a mouse homolog of PIWI, binds to piRNAs as well as to the mRNA cap and polysomes, with the polysome association correlating to the translational activity during spermatogenesis (26Grivna S.T. Beyret E. Wang Z. Lin H. Genes Dev. 2006; 20: 1709-1714Crossref PubMed Scopus (636) Google Scholar, 38Grivna S.T. Pyhtila B. Lin H. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 13415-13420Crossref PubMed Scopus (300) Google Scholar). MILI is a murine PIWI protein that we previously reported to be essential for the early prophase of meiosis (39Kuramochi-Miyagawa S. Kimura T. Ijiri T.W. Isobe T. Asada N. Fujita Y. Ikawa M. Iwai N. Okabe M. Deng W. Lin H. Matsuda Y. Nakano T. Development. 2004; 131: 839-849Crossref PubMed Scopus (600) Google Scholar). Most recently, MILI has also been shown to bind to piRNAs (27Aravin A. Gaidatzis D. Pfeffer S. Lagos-Quintana M. Landgraf P. Iovino N. Morris P. Brownstein M.J. Kuramochi-Miyagawa S. Nakano T. Chien M. Russo J.J. Ju J. Sheridan R. Sander C. Zavolan M. Tuschl T. Nature. 2006; 442: 203-207Crossref PubMed Scopus (1084) Google Scholar) and been implicated in transposon control and DNA methylation (28Aravin A. Sachidanandam R. Girard A. Fejes-Toth K. Hannon G.J. Science. 2007; 316: 744-747Crossref PubMed Scopus (736) Google Scholar, 40Kuramochi-Miyagawa S. Watanabe T. Gotoh K. Totoki Y. Toyoda A. Ikawa M. Asada N. Kojima K. Yamaguchi Y. Ijiri T.W. Hata K. Li E. Matsuda Y. Kimura T. Okabe M. Sakaki Y. Sasaki H. Nakano T. Genes Dev. 2008; 22: 908-917Crossref PubMed Scopus (679) Google Scholar). Here, we show that MILI is a cytoplasmic protein specifically expressed in the germ line during early spermatogenesis. Deleting the mili gene severely affects the self-renewing division and differentiation of germ line stem cells, in addition to its meiotic defects. Furthermore, we provide evidence that MILI positively regulates translation in prepubertal testes that contain only stem cells in the germ line, in contrast to the negative role of AGO proteins and their associated small interfering RNAs and miRNAs. These results suggest a function of MILI in promoting germ line stem cell division and differentiation via translational regulation. Antibody Generation-The MILI antibody for immunoprecipitation was generated by Anaspec Inc. (San Jose, CA). Amino acid residues 108-122 of MILI were synthesized, purified, and conjugated to keyhole limpet hemocyanin for immunization in rabbits. The resulting antisera were affinity-purified using MILI peptide conjugated to activated immunoaffinity support (Affi-Gel 15; Bio-Rad). The purified antibody was bound to protein A-Sepharose beads (Sigma) for immunoprecipitation. A different antibody, DU-PG15, generated in a guinea pig against a 483-amino acid peptide (52.8 kDa) from bp 1634 to 2117 of the Mili open reading frame, was used for immunostaining and Western blotting. Immunoprecipitation and Protein Identification-Testicular lysates were prepared as follows. Mice were asphyxiated with CO2, and testes were dissected. The testes were then decapsulated and homogenized in 1 ml of lysis buffer (100 mm KOAc, 0.1% Triton X-100, 50 mm HEPES, pH 7.4, 2 mm MgOAc, 10% glycerol, 1 mm dithiothreitol, 20 units/ml RNaseOut (Invitrogen), 1× complete mini EDTA-free protease inhibitor mixture (Roche Applied Science)) in a Dounce homogenizer for 20 strokes. The lysate was adjusted with lysis buffer to 10 mg/ml. Antibody-bound protein A-Sepharose (washed with lysis buffer minus RNAseOUT and protease inhibitor) was added to the adjusted lysate. The mixture was tumbled end over end for 2 h at 4 °C. The beads were then washed, and protein samples were eluted by boiling in 2× SDS sample buffer for 5 min. For the blocking experiment, MILI peptide was added to the beads at a final concentration of 100 μg/ml and incubated for 1 h at 4 °C prior to the addition of lysates. RNase A (Sigma) was added to a final concentration of 10 μg/ml prior to a 2-h, 4 °C incubation. The RNase A-treated sample and other samples were tumbled end over end for an additional 10 min at room temperature to ensure RNase activity. Co-immunoprecipitated MILI-interacting proteins were identified by mass spectrometry at the Proteomics Center at Duke University Medical Center. RNA in Situ Hybridization and Immunofluorescence Microscopy-RNA in situ hybridization and immunofluorescence microscopy was performed as described by Deng and Lin (41Deng W. Lin H. Dev. Cell. 2002; 2: 819-830Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar). Affinity-purified polyclonal guinea pig MILI antibody (GP15 against amino acid residues 483-644) was used at 1:15 dilution. Plzf mouse monoclonal antibody from Calbiochem was used at a 1:40 dilution. Human anti-GE-1 serum IC6 from Ref. 42Bloch D.B. Gulick T. Bloch K.D. Yang W.H. RNA. 2006; 12: 707-709Crossref PubMed Scopus (18) Google Scholar was used at a 1:200 dilution to recognize GE-1 in cells. The m7GTP Cap Binding Assay-Testicular extract was prepared as described above and incubated for 2 h at 4 °C with either 60 μl of protein A-Sepharose beads (Sigma) or m7GTP Sepharose beads (Amersham Biosciences) in lysis buffer. The beads were then washed, and protein samples were eluted by boiling in 2× SDS sample buffer for 5 min. For the competition experiment, m7GTP was added to the testicular extract to a final concentration of 200 μg/ml and rotated end over end at 4 °C for 1 h prior to incubation with m7GTP-Sepharose. RNA Isolation and Labeling-RNAs were isolated from the bound fraction of immunoprecipitation using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. For small RNA labeling, one-half of the RNA co-immunoprecipitated with MILI from 6 mg of testicular extract was dephosphorylated using calf intestinal phosphatase (New England Biolabs) at 10 units/30-μl reaction in 1× calf intestinal phosphatase reaction buffer at 37 °C for 2 h. The dephosphorylated RNA was then precipitated overnight at -20 °C by ethanol and NaAc, supplemented by Glyco Blue glycogen (Invitrogen). End-labeling of small RNAs was performed using polynucleotide kinase (New England Biolabs), at 10 units/30-μl reaction, using 2 μl of γ-ATP (6000 Ci/mmol). The reaction was performed in 1× T4PNK buffer at 37 °C for 2 h and terminated by adding 1 μl of 0.5 m EDTA. The sample was then put through a Sephadex G-50 RNA purification column (Roche Applied Science) to remove the unincorporated radioactive nucleotides. Formamide sample buffer was added to one-half of the labeled RNA sample, which was resolved on 15% urea polyacrylamide gel. The signal from labeled small RNAs was visualized via autoradiography. For mRNA labeling, one-half of the extracted RNA from immunoprecipitation was used for first-strand cDNA synthesis using Superscript reverse transcriptase H- (Invitrogen) according to the instructions. Oligo(dT)20 was used as primer, and [α-32P]ATP (3000 Ci/mmol) was used as the labeling source. Samples were resolved on 1% agarose gel, transferred to nylon membrane, and visualized by autoradiography. Polysome Fractionation-Testicular extract and sucrose gradient fractionation were carried out according to Cataldo et al. (43Cataldo L. Mastrangelo M.A. Kleene K.C. Exp. Cell Res. 1997; 231: 206-213Crossref PubMed Scopus (28) Google Scholar) with the following modifications. One pair of mouse testes were dissected, decapsulated, and then homogenized in 2 ml of lysis buffer (100 mm NaCl, 3 mm MgCl, 20 mm HEPES, pH 7.5, 0.1% Triton, 1 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, 20 units/ml RNase Out (Invitrogen)), using 20 strokes in the Dounce homogenizer. The lysates were treated with cycloheximide, a translational elongation inhibitor (44Godchaux III, W. Adamson S.D. Herbert E. J. Mol. Biol. 1967; 27: 57-72Crossref PubMed Scopus (69) Google Scholar), at a final concentration of 200 μm to stabilize polysomes. The lysate was then centrifuged at 13,000 × g at 4 °C for 2 min. The supernatant was then layered on to the top of a 15-50% (w/w) linear sucrose gradient in the lysis buffer. The gradient was centrifuged in Beckman SW40 rotor for 3 h at 150,000 × g in an SW41 rotor (Beckman). The centrifugation was stopped without applying a break. The fractions were then precipitated using 7.2% trichloroacetic acid (final concentration) and dissolved in SDS sample buffer to 2× final concentration. For micrococcal nuclease experiment, micrococcal nuclease (60 units/ml final concentration; Sigma) was added to the lysate, which is supplemented with 2 mm CaCl2 and incubated at room temperature for 20 min. The reaction was stopped by adding EGTA to a final concentration of 25 mm prior to layering on the sucrose gradient, which was supplemented with EGTA as well. [35S]Methionine Pulse-Chase Labeling of Protein Synthesis in Seminiferous Tubules-The [35S]methionine metabolic labeling to measure total protein synthesis in wild type or mutant testes was based on the method of Kleene (43Cataldo L. Mastrangelo M.A. Kleene K.C. Exp. Cell Res. 1997; 231: 206-213Crossref PubMed Scopus (28) Google Scholar, 45Kleene K.C. Dev. Biol. 1993; 159: 720-731Crossref PubMed Scopus (63) Google Scholar). Briefly, seminiferous tubules from 7-day postpartum (dpp) mili+/- or mili-/- testes were dissected in RPMI 1640 medium supplemented with 1 mm pyruvic acid and 6 mm lactic acid and then transferred to RPMI medium minus methionine. [35S]methionine (20 μCi/ml) was then added, and the tubules were incubated at 32 °C for 30 min. Total proteins were then extracted and precipitated by 20% trichloroacetic acid. The precipitated proteins were then examined on the SDS-polyacrylamide gel to evaluate the quality of labeling and to examine whether the synthesis of specific proteins is significantly changed in the mutant. The total protein synthesis activity was quantified by scintillation counting of the radioactivity of 20% trichloroacetic acid pellet. Microarray Analysis of the Levels of Total and MILI-associated mRNAs in Wild Type and Mili-/- Mutant-Total RNA from groups of 9-dpp mili+/- or mili-/- testes was isolated using TRIzol reagent (Invitrogen) without any nucleic acid carrier. The high molecular weight RNAs were then further enriched against the low molecular weight RNAs (<200 nucleotides) by selective precipitation in 12.5% polyethylene glycol 8000 and 1.25 m NaCl. The samples were treated with RQ1 RNase-free DNase (Promega) to remove any contaminating DNA and cleaned with RNeasy columns (Qiagen) according to the manufacturer’s instructions. The quantity and quality of the RNA samples were evaluated by both spectrophotometry and gel electrophoresis. For the microarray analysis, 10 μg of each sample was reverse transcribed with oligo(dT) primers and SuperscriptII reverse transcriptase (Invitrogen). cDNA samples were labeled with Cy3-conjugated nucleotides during the reverse transcription and directly hybridized to a mouse AROS version 3.0 oligonucleotide array (Operon) representing gene transcripts in the mouse. Cy5-labeled oligonulceotides complementary to the probes on the array were externally included in the sample during hybridization as a reference set for initial normalization of the raw data. Likewise, MILI-associated mRNAs were isolated using the TRIzol reagent from the anti-MILI immunoprecipitates from adult (2-4 months old) wild type testicular extract. After one round of amplification with the MessageAmp II RNA amplification kit (Ambion Inc.), the cDNAs were prepared, labeled, and subjected to the microarray chips as above. For the negative control, anti-MILI was blocked with the peptide antigen before applying for immunoprecipitation. The negative control immunoprecipitates were processed and analyzed side by side with the experimental sample in the same way. The same amount and batch of antibodies and extract were used for the negative control as for the experimental sample. The enriched mRNAs in the experimental sample relative to the negative control were dubbed MILI-associated mRNAs, and their expression levels were evaluated in the mili+/- and mili-/- microarray samples. The represented results were derived from five independent replicas for total RNAs from mili+/- and mili-/- and four replicas for MILI-associated mRNAs (reverse transcription, amplification, and hybridization were conducted by the Duke Institute of Genome Sciences and Policy). Bioinformatic Analysis of Microarray Data-Data on relative abundances of mRNAs within total testicular lysates, MILI immunoprecipitation samples, and mock immunoprecipitation samples were generated as follows. Microarray signals from replicates of total lysates were mean-normalized and averaged, respectively, to generate standard gene expression profiles of mouse testicular tissue. The ratio of the signal of each gene from each MILI immunoprecipitation sample to the standard gene expression profile was then calculated. Based on this ratio, a percentile rank of each gene relative to all genes in each immunoprecipitated replicate was calculated. The percentile ranks in the three replicates of MILI-immunoprecipitated samples were averaged. Student's t test was utilized to determine if the average percentile ranks of enrichment of individual genes were significantly higher (p value) than the mean enrichment of all genes in the immunoprecipitation samples. To determine the MILI-associated genes, the following criteria were used: 1) average percentile ranks of enrichment were greater than the mean enrichment of all genes in MILI immunoprecipitation with p < 0.01; 2) average signal in MILI immunoprecipitation replicates was significantly greater than the background signal (including 2× S.D.), with background signal and S.D. calculated based on signals from empty spots on each microarray; 3) criterion 1 was not satisfied for the same gene in the corresponding control immunoprecipitation. Using the above criteria, 802 mRNAs were designated as MILI-associated mRNAs. We further compared the expression profiles of MILI-associated mRNAs as well as nonassociated mRNAs in wild type versus mili-/- testis. All cDNA microarray signals were median-normalized. The relative abundances of individual mRNAs from replicates were averaged. MILI Is Required for Germ Line Stem Cell Self-renewal-We previously reported that mili is required for meiosis (39Kuramochi-Miyagawa S. Kimura T. Ijiri T.W. Isobe T. Asada N. Fujita Y. Ikawa M. Iwai N. Okabe M. Deng W. Lin H. Matsuda Y. Nakano T. Development. 2004; 131: 839-849Crossref PubMed Scopus (600) Google Scholar), which echoes post-germ line stem cell function of piwi and pumilio in the Drosophila ovary (8Parisi M. Lin H. Genetics. 1999; 153: 235-250Crossref PubMed Google Scholar, 46Lin H. Spradling A.C. Development. 1997; 124: 2463-2476Crossref PubMed Google Scholar). Although the mili mutant displays a terminal arrest phenotype at the pachytene stage of meiosis, we notice that only 2 ± 2.5% of the mutant spermatogonia actually reach this stage of differentiation (analysis detailed below), indicating an earlier spermatogenic defect prior to meiosis. To characterize the early spermatogenic defect, we examined the mutant testes at critical stages of early spermatogenesis. In mouse testes, the precursors of germ line stem cells, called gonocytes, start proliferation at 3 dpp to establish the population of germ line stem cells (also known as primitive Type A spermatogonia) at 6 dpp. These stem cells then undergo self-renewing divisions to produce differentiated Type A spermatogonia (47Bellve A.R. Millette C.F. Bhatnagar Y.M. O'Brien D.A. J. Histochem. Cytochem. 1977; 25: 480-494Crossref PubMed Scopus (294) Google Scholar). To determine whether this defect occurs prior to stem cell formation at 6 dpp, we examined the number and proliferation rate of gonocytes at 5 dpp by immunofluorescence microscopy, using the gonocyte/spermatogonium-specific EE2 antibody (48Koshimizu U. Nishioka H. Watanabe D. Dohmae K. Nishimune Y. Mol. Reprod. Dev. 1995; 40: 221-227Crossref PubMed Scopus (31) Google Scholar) to label gonocytes and anti-phosphohistone 3 antibody to label mitotic cells. The mili-/- and mili+/- tubules were similar in size (51.0 ± 4.0 versus 48.1 ± 5.1 μm in diameter) and contained an equivalent number of gonocytes that proliferated at a similar rate (Fig. 1, A and M). This indicates that the mili mutation does not affect gonocyte proliferation. At 8 dpp, the number of spermatogonia in mili-/- and mili+/- tubules remained similar (Fig. 1, C and D), indicating that the germ line stem cell population had been established normally. However, at 8 dpp, the mili-/- germ line stem cells already showed a reduced mitotic rate (2.1 ± 0.40% versus 3.6 ± 0.4%; p < 0.006; Fig. 1M), indicating that spermatogonia division was compromised in the mili mutant. We then investigated the effects of the mili mutation on spermatogonial division and differentiation at representative stages of p
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