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• W2040463485 abstract "During spermatogenesis, germ cells initially expand exponentially through mitoses. A majority of these cells are then eliminated through p53-mediated apoptosis to maintain germline homeostasis [1Yin Y. Stahl B.C. DeWolf W.C. Morgentaler A. P53 and Fas are sequential mechanisms of testicular germ cell apoptosis.J. Androl. 2002; 23: 64-70Crossref PubMed Scopus (100) Google Scholar, 2Russell L.D. Chiarini-Garcia H. Korsmeyer S.J. Knudson C.M. Bax-dependent spermatogonia apoptosis is required for testicular development and spermatogenesis.Biol. Reprod. 2002; 66: 950-958Crossref PubMed Scopus (190) Google Scholar, 3Knudson C.M. Tung K.S. Tourtellotte W.G. Brown G.A. Korsmeyer S.J. Bax-deficient mice with lymphoid hyperplasia and male germ cell death.Science. 1995; 270: 96-99Crossref PubMed Scopus (1308) Google Scholar, 4Beumer T.L. Roepers-Gajadien H.L. Gademan I.S. van Buul P.P. Gil-Gomez G. Rutgers D.H. de Rooij D.G. The role of the tumor suppressor p53 in spermatogenesis.Cell Death Differ. 1998; 5: 669-677Crossref PubMed Scopus (181) Google Scholar]. However, the activity of p53 must be precisely modulated, especially suppressed in postmitotic spermatogenic cells, to guarantee robustness of spermatogenesis. Currently, how the suppression is achieved is not understood. Here, we show that Pumilio 1, a posttranscriptional regulator, binds to mRNAs representing 1,527 genes, with significant enrichment for mRNAs involved in pathways regulating p53, cell cycle, and MAPK signaling. In particular, eight mRNAs encoding activators of p53 are repressed by Pumilio 1. Deleting Pumilio 1 results in strong activation of p53 and apoptosis mostly in spermatocytes, which disrupts sperm production and fertility. Removing p53 reduces apoptosis and rescues testicular hypotrophy in Pumilio 1 null mice. These results indicate that key components of the p53 pathway are coordinately regulated by Pumilio 1 at the posttranscriptional level, which may exemplify an RNA operon. During spermatogenesis, germ cells initially expand exponentially through mitoses. A majority of these cells are then eliminated through p53-mediated apoptosis to maintain germline homeostasis [1Yin Y. Stahl B.C. DeWolf W.C. Morgentaler A. P53 and Fas are sequential mechanisms of testicular germ cell apoptosis.J. Androl. 2002; 23: 64-70Crossref PubMed Scopus (100) Google Scholar, 2Russell L.D. Chiarini-Garcia H. Korsmeyer S.J. Knudson C.M. Bax-dependent spermatogonia apoptosis is required for testicular development and spermatogenesis.Biol. Reprod. 2002; 66: 950-958Crossref PubMed Scopus (190) Google Scholar, 3Knudson C.M. Tung K.S. Tourtellotte W.G. Brown G.A. Korsmeyer S.J. Bax-deficient mice with lymphoid hyperplasia and male germ cell death.Science. 1995; 270: 96-99Crossref PubMed Scopus (1308) Google Scholar, 4Beumer T.L. Roepers-Gajadien H.L. Gademan I.S. van Buul P.P. Gil-Gomez G. Rutgers D.H. de Rooij D.G. The role of the tumor suppressor p53 in spermatogenesis.Cell Death Differ. 1998; 5: 669-677Crossref PubMed Scopus (181) Google Scholar]. However, the activity of p53 must be precisely modulated, especially suppressed in postmitotic spermatogenic cells, to guarantee robustness of spermatogenesis. Currently, how the suppression is achieved is not understood. Here, we show that Pumilio 1, a posttranscriptional regulator, binds to mRNAs representing 1,527 genes, with significant enrichment for mRNAs involved in pathways regulating p53, cell cycle, and MAPK signaling. In particular, eight mRNAs encoding activators of p53 are repressed by Pumilio 1. Deleting Pumilio 1 results in strong activation of p53 and apoptosis mostly in spermatocytes, which disrupts sperm production and fertility. Removing p53 reduces apoptosis and rescues testicular hypotrophy in Pumilio 1 null mice. These results indicate that key components of the p53 pathway are coordinately regulated by Pumilio 1 at the posttranscriptional level, which may exemplify an RNA operon. Pum1 null males have significantly reduced sperm count and fertility Pum1 null testes display elevated apoptosis in spermatocytes Among its other target mRNAs, Pum1 binds to and represses multiple activators of p53 p53 deficiency rescues Pum1 phenotype Spermatogenesis in mammals is a complex process in which germline stem cells undergo 9–11 rounds of mitosis, followed by meiosis and a cellular morphogenic process called spermiogenesis that transforms round haploid spermatids into sperm. The multiple rounds of mitoses generate excess spermatogonia that must be eliminated to maintain the homeostasis of spermatogenesis. This is accomplished in part by p53-mediated apoptosis [1Yin Y. Stahl B.C. DeWolf W.C. Morgentaler A. P53 and Fas are sequential mechanisms of testicular germ cell apoptosis.J. Androl. 2002; 23: 64-70Crossref PubMed Scopus (100) Google Scholar, 2Russell L.D. Chiarini-Garcia H. Korsmeyer S.J. Knudson C.M. Bax-dependent spermatogonia apoptosis is required for testicular development and spermatogenesis.Biol. Reprod. 2002; 66: 950-958Crossref PubMed Scopus (190) Google Scholar, 3Knudson C.M. Tung K.S. Tourtellotte W.G. Brown G.A. Korsmeyer S.J. Bax-deficient mice with lymphoid hyperplasia and male germ cell death.Science. 1995; 270: 96-99Crossref PubMed Scopus (1308) Google Scholar, 4Beumer T.L. Roepers-Gajadien H.L. Gademan I.S. van Buul P.P. Gil-Gomez G. Rutgers D.H. de Rooij D.G. The role of the tumor suppressor p53 in spermatogenesis.Cell Death Differ. 1998; 5: 669-677Crossref PubMed Scopus (181) Google Scholar]. The temporally and spatially specific activation of p53 must be precisely controlled so that not only excess spermatogonia are eliminated, but enough germ cells must also survive the elimination to generate a sufficiently large number of sperm. However, how this control is achieved in mammals remains unknown. Here, we show that Pumilio 1 (Pum1), through coordinated posttranscriptional regulation of multiple factors in the p53 pathway, represses p53 activation and apoptosis after spermatogonial division. Pum1 is one of the two members (Pum1 and Pum2) of the PUF (Pumilio/FBF) protein family in the mouse. Its homolog in Drosophila, Pum, is a posttranscriptional regulator essential for a variety of germline processes, such as primordial germ cell proliferation [5Parisi M. Lin H. The Drosophila pumilio gene encodes two functional protein isoforms that play multiple roles in germline development, gonadogenesis, oogenesis and embryogenesis.Genetics. 1999; 153: 235-250Crossref PubMed Google Scholar], germline stem cell self-renewal [6Lin H. Spradling A.C. A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary.Development. 1997; 124: 2463-2476Crossref PubMed Google Scholar, 7Forbes A. Lehmann R. Nanos and Pumilio have critical roles in the development and function of Drosophila germline stem cells.Development. 1998; 125: 679-690Crossref PubMed Google Scholar], ovarian morphogenesis, and oviposition [5Parisi M. Lin H. The Drosophila pumilio gene encodes two functional protein isoforms that play multiple roles in germline development, gonadogenesis, oogenesis and embryogenesis.Genetics. 1999; 153: 235-250Crossref PubMed Google Scholar]. Pum binds to a defined motif with a UGUAHAUA core in the 3′ untranslated region (3′ UTR) of its target mRNAs and mediates translational repression and/or mRNA decay [8Wang X. McLachlan J. Zamore P.D. Hall T.M. Modular recognition of RNA by a human pumilio-homology domain.Cell. 2002; 110: 501-512Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 9Zamore P.D. Williamson J.R. Lehmann R. The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins.RNA. 1997; 3: 1421-1433PubMed Google Scholar]. PUF proteins in Caenorhabditis elegans are also known to be involved in multiple steps of germline development [10Parisi M. Lin H. Translational repression: a duet of Nanos and Pumilio.Curr. Biol. 2000; 10: R81-R83Abstract Full Text Full Text PDF PubMed Google Scholar, 11Quenault T. Lithgow T. Traven A. PUF proteins: repression, activation and mRNA localization.Trends Cell Biol. 2011; 21: 104-112Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar]. In contrast to the critical roles of Pum proteins in the Drosophila and C. elegans germline, mouse Pum2 is not essential for spermatogenesis [12Xu E.Y. Chang R. Salmon N.A. Reijo Pera R.A. A gene trap mutation of a murine homolog of the Drosophila stem cell factor Pumilio results in smaller testes but does not affect litter size or fertility.Mol. Reprod. Dev. 2007; 74: 912-921Crossref PubMed Scopus (64) Google Scholar], calling for an understanding of Pum1 in the mouse. We first examined the expression of Pum1 in 11 mouse organs. The mRNA and protein levels were measured by qRT-PCR and immunoblot analysis, respectively. Both analyses showed that Pum1 is highly expressed in the testis (Figures 1A and S1A available online). During the development of the testis, Pum1 is expressed 2 days postpartum (dpp) and then starts to increase at 14 dpp when pachytene spermatocytes first appear (Figure S1B). Immunofluorescence microscopy using an anti-Pum1 antibody further revealed that Pum1 is expressed in the cytoplasm of spermatocytes along with other germ cell types (Figures 1B and S1C). To confirm the subcellular localization of Pum1, we separated adult testicular lysates into cytoplasmic and nuclear fractions. Pum1 was detected almost exclusively in the cytoplasm as indicated by immunoblot analysis (Figure 1C). This is consistent with the reported role of Drosophila and C. elegans PUF proteins in regulating the stability and translation of its target mRNAs [13Wreden C. Verrotti A.C. Schisa J.A. Lieberfarb M.E. Strickland S. Nanos and pumilio establish embryonic polarity in Drosophila by promoting posterior deadenylation of hunchback mRNA.Development. 1997; 124: 3015-3023Crossref PubMed Google Scholar, 14Chagnovich D. Lehmann R. Poly(A)-independent regulation of maternal hunchback translation in the Drosophila embryo.Proc. Natl. Acad. Sci. USA. 2001; 98: 11359-11364Crossref PubMed Scopus (80) Google Scholar, 15Kadyrova L.Y. Habara Y. Lee T.H. Wharton R.P. Translational control of maternal Cyclin B mRNA by Nanos in the Drosophila germline.Development. 2007; 134: 1519-1527Crossref PubMed Scopus (169) Google Scholar, 16Lee M.H. Hook B. Pan G. Kershner A.M. Merritt C. Seydoux G. Thomson J.A. Wickens M. Kimble J. Conserved regulation of MAP kinase expression by PUF RNA-binding proteins.PLoS Genet. 2007; 3: e233Crossref PubMed Scopus (99) Google Scholar, 17Suh N. Crittenden S.L. Goldstrohm A. Hook B. Thompson B. Wickens M. Kimble J. FBF and its dual control of gld-1 expression in the Caenorhabditis elegans germline.Genetics. 2009; 181: 1249-1260Crossref PubMed Scopus (101) Google Scholar]. To understand the role of Pum1 in development, we generated Pum1-null mice for phenotypic analysis (Figure S1D). The loss of Pum1 protein was confirmed by immunoblot analysis of testes from five Pum1−/− mice (Figure S1E). Pum1−/− mice are viable and grow to adulthood without apparent defects except that they are 18% (body weight, ±2.1% SEM; p = 0.0003) smaller than wild-type mice at 8 weeks of age (Figure S1F). However, testicular hypoplasia was observed in all Pum1−/− males examined. The average testicular weight of Pum1−/− mice is 34% (±1.5% SEM; p = 2.28 × 10−16) lower than that of wild-type mice at 8 weeks of age (Figure S1G). In addition, the mature sperm count of Pum1−/− males is reduced by 80% (±2.0% SEM; p = 1.40 × 10−16) at 8 weeks of age and remains at this low level throughout adult life (Figure 1D). Consistently, the fertility of Pum1−/− males is reduced by 41% (±3.2% SEM; p = 1.56 × 10−13) when compared to the wild-type level (Figure 1E). Taken together, these data indicate that spermatogenesis is compromised in Pum1−/− males. To understand which step of spermatogenesis is impaired in the Pum1−/− mice, we examined the testicular histology by hematoxylin and eosin staining (Figures 1F and 1G) and periodic acid-Schiff staining (Figures S2A and S2B). The global morphology of Pum1−/− testes looks normal, containing the complete lineage of germ cells. Acrosome morphogenesis is normal in Pum1−/− testes (Figures S2A and S2B), implicating normal spermiogenesis up to the round spermatid stage. Mature sperm are present (Figure 1D), indicating that spermatogenesis can reach completion for some of the Pum1−/− spermatogenic cells. In addition, Ki-67 immunohistochemistry analysis showed that the division rates of spermatogonia and spermatocytes are not affected in Pum1−/− testes (Figures S2C–S2E). This is confirmed by the normal level of phosphohistone 3, another marker of cell division, in Pum1−/− testes, as revealed by immunoblot analysis (Figures S2F and S2G). Furthermore, serum testosterone level is unchanged in Pum1−/− mice, indicating normal steroidogenesis (Figure S2H). However, Pum1−/− spermatocytes more frequently contain enlarged nuclei and condensed chromatin, suggestive of apoptosis (Figures 1F and 1G). To investigate this defect further, we conducted a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in 16 adult Pum1+/+, Pum1+/−, and Pum1−/− males. This analysis revealed that apoptosis in Pum1+/− and Pum1−/− testes is elevated by 2.7-fold (±0.29 SD; p = 0.0002) and 6.8-fold (±1.02 SD; p = 4.22 × 10−5), respectively (Figures 1H and S2I–S2K). Moreover, Apoptosis is elevated mostly in primary spermatocytes (Figures S2L–S2N) in which Pum1 is normally expressed at high levels. Moreover, in the testes of Pum1−/flox;Vasa-cre+ mice where Pum1 is knocked out only in the germline, apoptosis is also elevated by 7.5-fold (±0.24 SD; p = 1.98 × 10−7) from wild-type level (Figures 1H, S2O, and S2P). This suggests that the apoptosis phenotype in Pum1 mutant testes is germline autonomous. We then tracked the behavior of the apoptotic cells in the reproductive tract and found that the lumen of the epididymis in Pum1−/− mice contains unexpected round cells with aberrant morphology (Figures S3A–S3C). These cells are positive for Miwi (Figures S3D and S3E), a protein specifically expressed in spermatocytes and early spermatids. These cells also showed positive staining for TUNEL (Figures S3F and S3G). This indicates that these cells are apoptotic spermatocytes that are expelled from the testis rather than cells derived from the epididymis epithelium. To understand the mechanism of elevated apoptosis in the Pum1−/− testis, we wanted to know which mRNAs are targeted by Pum1 in the testis. To this end, we performed a genome-wide target identification of Pum1 using ribonucleoprotein-immunoprecipitation followed by microarray (RIP-Chip). The Pum1 ribonucleoprotein (RNP) complexes were precipitated from total testicular lysates with an anti-Pum1 antibody coupled to protein A Sepharose beads. RNAs associated with Pum1 were extracted and analyzed by hybridization to a mouse cDNA microarray (Illumina Mouse Chip M6). To identify nonspecifically precipitated RNAs, we used Pum1−/− testicular lysates in parallel experiments as negative controls. We identified 3,687 transcripts that are consistently associated with Pum1, representing 1,527 Ensembl genes (false discovery rate < 5%; Figure 2A and Table S1). Similar studies performed on human PUM1 protein in HeLa S3 cancer cells identified a similar number of mRNA targets [18Morris A.R. Mukherjee N. Keene J.D. Ribonomic analysis of human Pum1 reveals cis-trans conservation across species despite evolution of diverse mRNA target sets.Mol. Cell. Biol. 2008; 28: 4093-4103Crossref PubMed Scopus (122) Google Scholar, 19Galgano A. Forrer M. Jaskiewicz L. Kanitz A. Zavolan M. Gerber A.P. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system.PLoS ONE. 2008; 3: e3164Crossref PubMed Scopus (205) Google Scholar]. Multiple expectation maximization for motif elicitation (MEME) analysis revealed an eight-nucleotide consensus sequence, UGUAHAUA, that exists among the Pum1 target mRNAs (Figure 2B). This motif predominantly resides in their 3′ UTRs (Figure 2C) and is the same as those found in mRNA targets of yeast Puf3 [20Gerber A.P. Herschlag D. Brown P.O. Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast.PLoS Biol. 2004; 2: E79Crossref PubMed Scopus (513) Google Scholar], Drosophila Pum [9Zamore P.D. Williamson J.R. Lehmann R. The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins.RNA. 1997; 3: 1421-1433PubMed Google Scholar], and human PUM1 and PUM2 [18Morris A.R. Mukherjee N. Keene J.D. Ribonomic analysis of human Pum1 reveals cis-trans conservation across species despite evolution of diverse mRNA target sets.Mol. Cell. Biol. 2008; 28: 4093-4103Crossref PubMed Scopus (122) Google Scholar, 19Galgano A. Forrer M. Jaskiewicz L. Kanitz A. Zavolan M. Gerber A.P. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system.PLoS ONE. 2008; 3: e3164Crossref PubMed Scopus (205) Google Scholar], indicating that the Pum binding motif is highly conserved. Pathway analysis using MetaCore (version 6.7 build 28822) was performed on 656 pathways to which genes on the Illumina mouse chip (M6) can be mapped. Pum1-associtated mRNAs are enriched in 11 pathways (p < 0.001) that regulate p53 activation, cell cycle, and mitogen-activated protein kinase (MAPK) signaling (Table S2 and Figures S4A–S4C), which is also consistent with the pathways targeted by human PUM1 in HeLa S3 cancer cells [18Morris A.R. Mukherjee N. Keene J.D. Ribonomic analysis of human Pum1 reveals cis-trans conservation across species despite evolution of diverse mRNA target sets.Mol. Cell. Biol. 2008; 28: 4093-4103Crossref PubMed Scopus (122) Google Scholar, 19Galgano A. Forrer M. Jaskiewicz L. Kanitz A. Zavolan M. Gerber A.P. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system.PLoS ONE. 2008; 3: e3164Crossref PubMed Scopus (205) Google Scholar]. In the p53-regulating pathways, nine mRNAs encoding factors that regulate p53-mediated apoptosis are Pum1 targets. Among them, Map3k1, Map2k3, and Daxx activate p38 MAPK, which in turn activates p53 [21Bulavin D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation.EMBO J. 1999; 18: 6845-6854Crossref PubMed Scopus (595) Google Scholar, 22Raman M. Chen W. Cobb M.H. Differential regulation and properties of MAPKs.Oncogene. 2007; 26: 3100-3112Crossref PubMed Scopus (1116) Google Scholar, 23She Q.B. Chen N. Dong Z. ERKs and p38 kinase phosphorylate p53 protein at serine 15 in response to UV radiation.J. Biol. Chem. 2000; 275: 20444-20449Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 24Lin T. Mak N.K. Yang M.S. MAPK regulate p53-dependent cell death induced by benzo[a]pyrene: involvement of p53 phosphorylation and acetylation.Toxicology. 2008; 247: 145-153Crossref PubMed Scopus (45) Google Scholar, 25Chang H.Y. Nishitoh H. Yang X. Ichijo H. Baltimore D. Activation of apoptosis signal-regulating kinase 1 (ASK1) by the adapter protein Daxx.Science. 1998; 281: 1860-1863Crossref PubMed Scopus (531) Google Scholar]; Map2k7 together with Map3k1 activates JNK, which in turn activates p53 [22Raman M. Chen W. Cobb M.H. Differential regulation and properties of MAPKs.Oncogene. 2007; 26: 3100-3112Crossref PubMed Scopus (1116) Google Scholar, 26Liu J. Lin A. Role of JNK activation in apoptosis: a double-edged sword.Cell Res. 2005; 15: 36-42Crossref PubMed Scopus (597) Google Scholar]; Sae1, Uba2, Pias1, and Pias2 are sumoylation ligases that prime p53 for inducing apoptosis [27Stehmeier P. Muller S. Regulation of p53 family members by the ubiquitin-like SUMO system.DNA Repair (Amst.). 2009; 8: 491-498Crossref PubMed Scopus (68) Google Scholar]; and Mdm2 is a ubiquitin ligase for p53 that primes it for degradation [28Haupt Y. Maya R. Kazaz A. Oren M. Mdm2 promotes the rapid degradation of p53.Nature. 1997; 387: 296-299Crossref PubMed Scopus (3707) Google Scholar, 29Kubbutat M.H. Jones S.N. Vousden K.H. Regulation of p53 stability by Mdm2.Nature. 1997; 387: 299-303Crossref PubMed Scopus (2840) Google Scholar]. Because Drosophila Pum promotes the decay of its target mRNAs and/or blocks their translation [13Wreden C. Verrotti A.C. Schisa J.A. Lieberfarb M.E. Strickland S. Nanos and pumilio establish embryonic polarity in Drosophila by promoting posterior deadenylation of hunchback mRNA.Development. 1997; 124: 3015-3023Crossref PubMed Google Scholar, 14Chagnovich D. Lehmann R. Poly(A)-independent regulation of maternal hunchback translation in the Drosophila embryo.Proc. Natl. Acad. Sci. USA. 2001; 98: 11359-11364Crossref PubMed Scopus (80) Google Scholar, 15Kadyrova L.Y. Habara Y. Lee T.H. Wharton R.P. Translational control of maternal Cyclin B mRNA by Nanos in the Drosophila germline.Development. 2007; 134: 1519-1527Crossref PubMed Scopus (169) Google Scholar], we wanted to understand how murine Pum1 regulates its target mRNAs. To this end, we compared the expression levels of the nine regulators of p53 between wild-type, Pum1+/−, and Pum1−/− testes, along with another five Pum1 target mRNAs and seven nontarget mRNAs as controls. qRT-PCR analysis revealed that all seven nontarget mRNAs remain at wild-type levels in Pum1−/− testes, whereas 10 out of 14 Pum1 target mRNAs are slightly elevated in Pum1−/− testes, suggesting that Pum1 plays a minor role in reducing the stability of its target mRNAs (Figure 2D). More significantly, immunoblot analysis revealed the elevation of seven out of the nine Pum1 target genes that are p53 regulators at the protein level in Pum1−/− testes, except for Daxx and Mdm2 (Figure 2E). In addition, Cdk1 and Cdk2, two other Pum1 targets, are elevated at the protein level (Figure 2E). In contrast, the protein levels of Cul3, Map2k2, Map2k4, and Map3k5, which are not Pum1 targets, remain at the same levels in Pum1−/− testes (Figure 2E). The scale of elevation at the protein level (2- to 10-fold) is much larger than that at the mRNA level (mostly within 2-fold; Figure S4D), suggesting that Pum1 regulates its target mRNAs mostly via translational repression. Pum1 represses at least two factors, Map2k3 and Map3k1, that activate p53 by activating the p38 MAPK (Figures 2E, 3A, and 3B). We hypothesized that removing Pum1 would lead to elevated activation of p38. Indeed, immunoblot analysis showed an increase of phosphorylated p38 (Thr180/Tyr182) representing activated p38 in Pum1−/− testes, whereas its total protein level remains unchanged (Figure 3B ). Similarly, the phosphorylated JNK (Thr183/Tyr185), representing activated JNK, is elevated when its upstream kinase, Map2k7, is increased due to Pum1 deficiency (Figures 3A and 3B). Immunofluorescence analysis showed that the strongest activation of p38 under Pum1 deficiency indeed occurs in primary spermatocytes (Figures 3C–3H). This is in agreement with our observations that Pum1 is most abundant in primary spermatocytes and that apoptosis is elevated in these cells under Pum1 deficiency. The activated p38 phosphorylates p53 at Ser15 to prime p53 for proapoptotic transcription [21Bulavin D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation.EMBO J. 1999; 18: 6845-6854Crossref PubMed Scopus (595) Google Scholar, 23She Q.B. Chen N. Dong Z. ERKs and p38 kinase phosphorylate p53 protein at serine 15 in response to UV radiation.J. Biol. Chem. 2000; 275: 20444-20449Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 24Lin T. Mak N.K. Yang M.S. MAPK regulate p53-dependent cell death induced by benzo[a]pyrene: involvement of p53 phosphorylation and acetylation.Toxicology. 2008; 247: 145-153Crossref PubMed Scopus (45) Google Scholar]. Consistently, in wild-type testes, p53 is activated only in a small number of spermatogonia; however, in Pum1−/− testes, the activated form of p53 is extensively present in a large number of spermatocytes (Figures 3I and 3J). To confirm the activation of p53, the transcription of four p53 target genes was examined via qRT-PCR in total testicular lysates. Among them, Bax, Caspase 6, and Noxa showed increased mRNA level in Pum1−/− testes (Figure S4E). The overactivation of p53 can be partially rescued by an inhibitor of p38 (SB239063, intravenously at 15 μg/ml blood), suggesting p53 is overactivated in part via p38 in the Pum1−/− testis. To further confirm the regulation of p53 by Pum1, we crossed the Pum1−/− mice with a Trp53 mutant line [30Jacks T. Remington L. Williams B.O. Schmitt E.M. Halachmi S. Bronson R.T. Weinberg R.A. Tumor spectrum analysis in p53-mutant mice.Curr. Biol. 1994; 4: 1-7Abstract Full Text Full Text PDF PubMed Scopus (1736) Google Scholar]. At 15 dpp, Pum1−/− testes contain significantly smaller seminiferous tubules than wild-type testes (Figures 4D and 4E ), presumably due to an overkill of germ cells. Removing p53 partially restores this morphological defect (Figure 4F). TUNEL analysis of17 mice at 15 dpp revealed that removing p53 effectively reduces apoptosis in Pum1 mutant testes in a dose-dependent manner (Figure 4G). Moreover, removing p53 also rescues the testis hypotrophy in Pum1−/− testes (Figure 4H). These data indicate that p53 is a major mediator of the Pum1 function in maintaining the homeostasis of the mouse testicular germline. Previous studies have shown that p53-mediated apoptosis is used by mammals to eliminate excessive spermatogonia in order to maintain germline homeostasis [1Yin Y. Stahl B.C. DeWolf W.C. Morgentaler A. P53 and Fas are sequential mechanisms of testicular germ cell apoptosis.J. Androl. 2002; 23: 64-70Crossref PubMed Scopus (100) Google Scholar, 2Russell L.D. Chiarini-Garcia H. Korsmeyer S.J. Knudson C.M. Bax-dependent spermatogonia apoptosis is required for testicular development and spermatogenesis.Biol. Reprod. 2002; 66: 950-958Crossref PubMed Scopus (190) Google Scholar, 3Knudson C.M. Tung K.S. Tourtellotte W.G. Brown G.A. Korsmeyer S.J. Bax-deficient mice with lymphoid hyperplasia and male germ cell death.Science. 1995; 270: 96-99Crossref PubMed Scopus (1308) Google Scholar, 4Beumer T.L. Roepers-Gajadien H.L. Gademan I.S. van Buul P.P. Gil-Gomez G. Rutgers D.H. de Rooij D.G. The role of the tumor suppressor p53 in spermatogenesis.Cell Death Differ. 1998; 5: 669-677Crossref PubMed Scopus (181) Google Scholar]. Our data suggest that when a normal number of spermatogonia differentiate into spermatocytes, p53-mediated apoptosis must be suppressed to avoid the overkill of spermatocytes, and that this is achieved at least in part by Pum1 and its coordinated posttranscriptional suppression of multiple activators of p53 (Figure 4I)." @default.
• W2040463485 created "2016-06-24" @default.
• W2040463485 creator A5001330715 @default.
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• W2040463485 date "2012-03-01" @default.
• W2040463485 modified "2023-10-12" @default.
• W2040463485 title "Pumilio 1 Suppresses Multiple Activators of p53 to Safeguard Spermatogenesis" @default.
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