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The ATM signaling cascade promotes recombination-dependent pachytene arrest in mouse spermatocytes.

Pacheco S, Marcet-Ortega M, Lange J, Jasin M, Keeney S, Roig I - PLoS Genet. (2015)

Bottom Line: TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2.Our work supports the conclusion that recombination defects trigger spermatocyte arrest via pathways than are genetically distinct from sex body failure-promoted apoptosis and confirm that the latter can function even when recombination-dependent arrest is inoperative.Implications of these findings for understanding the complex relationships between spermatocyte arrest and apoptosis are discussed.

View Article: PubMed Central - PubMed

Affiliation: Genome Integrity and Instability Group, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain; Cytology and Histology Unit, Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain.

ABSTRACT
Most mutations that compromise meiotic recombination or synapsis in mouse spermatocytes result in arrest and apoptosis at the pachytene stage of the first meiotic prophase. Two main mechanisms are thought to trigger arrest: one independent of the double-strand breaks (DSBs) that initiate meiotic recombination, and another activated by persistent recombination intermediates. Mechanisms underlying the recombination-dependent arrest response are not well understood, so we sought to identify factors involved by examining mutants deficient for TRIP13, a conserved AAA+ ATPase required for the completion of meiotic DSB repair. We find that spermatocytes with a hypomorphic Trip13 mutation (Trip13mod/mod) arrest with features characteristic of early pachynema in wild type, namely, fully synapsed chromosomes without incorporation of the histone variant H1t into chromatin. These cells then undergo apoptosis, possibly in response to the arrest or in response to a defect in sex body formation. However, TRIP13-deficient cells that additionally lack the DSB-responsive kinase ATM progress further, reaching an H1t-positive stage (i.e., similar to mid/late pachynema in wild type) despite the presence of unrepaired DSBs. TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2. These mutant backgrounds nonetheless experience an apoptotic block to further spermatogenic progression, most likely caused by failure to form a sex body. DSB numbers are elevated in Mre11 and Nbs1 hypomorphs but not Chk2 mutants, thus delineating genetic requirements for the ATM-dependent negative feedback loop that regulates DSB numbers. The findings demonstrate for the first time that ATM-dependent signaling enforces the normal pachytene response to persistent recombination intermediates. Our work supports the conclusion that recombination defects trigger spermatocyte arrest via pathways than are genetically distinct from sex body failure-promoted apoptosis and confirm that the latter can function even when recombination-dependent arrest is inoperative. Implications of these findings for understanding the complex relationships between spermatocyte arrest and apoptosis are discussed.

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The MRE11 complex, but not TRIP13 or CHK2, modulates SPO11-oligonucleotide complex levels.SPO11-oligonucleotide complexes were immunoprecipitated from extracts of testes of the indicated genotypes, labelled with terminal transferase and 32P-nucleotide, and resolved by SDS-PAGE. Top image in each panel, autoradiograph; bottom image, SPO11 Western blot where the two major SPO11 isoforms, α and β, are indicated. The vertical lines next to the autoradiographs indicate the signal from SPO11-oligonucleotide complexes. Asterisk indicates non-specific signal from the labelling reaction. Short arrow designates the migration position of the heavy chain of the antibody used to immunoprecipitate SPO11. (A-B) Both Mre11 and Nbs1 mutants have increased levels of SPO11-oligonucleotide complexes compared to littermate controls (1.9 ± 0.4 fold, mean ± SD of the relative signal intensity compared to a wild-type control, n = 3 mice, and 1.8 ± 0.2 fold, n = 3, respectively). (C) SPO11-oligonucleotide complex levels are slightly reduced in Trip13mod/mod samples (0.7 ± 0.2, n = 3), similar to other recombination-deficient mutants, such as Dmc1–/– [27]. The SPO11α isoform is not detected in Trip13mod/mod testes as observed in other mutants that arrest at pachynema [27]. (D) Chk2−/− testes have similar levels of SPO11-oligonucleotide complex as controls (1.0 ± 0.2 fold, n = 3).
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pgen.1005017.g004: The MRE11 complex, but not TRIP13 or CHK2, modulates SPO11-oligonucleotide complex levels.SPO11-oligonucleotide complexes were immunoprecipitated from extracts of testes of the indicated genotypes, labelled with terminal transferase and 32P-nucleotide, and resolved by SDS-PAGE. Top image in each panel, autoradiograph; bottom image, SPO11 Western blot where the two major SPO11 isoforms, α and β, are indicated. The vertical lines next to the autoradiographs indicate the signal from SPO11-oligonucleotide complexes. Asterisk indicates non-specific signal from the labelling reaction. Short arrow designates the migration position of the heavy chain of the antibody used to immunoprecipitate SPO11. (A-B) Both Mre11 and Nbs1 mutants have increased levels of SPO11-oligonucleotide complexes compared to littermate controls (1.9 ± 0.4 fold, mean ± SD of the relative signal intensity compared to a wild-type control, n = 3 mice, and 1.8 ± 0.2 fold, n = 3, respectively). (C) SPO11-oligonucleotide complex levels are slightly reduced in Trip13mod/mod samples (0.7 ± 0.2, n = 3), similar to other recombination-deficient mutants, such as Dmc1–/– [27]. The SPO11α isoform is not detected in Trip13mod/mod testes as observed in other mutants that arrest at pachynema [27]. (D) Chk2−/− testes have similar levels of SPO11-oligonucleotide complex as controls (1.0 ± 0.2 fold, n = 3).

Mentions: We reasoned that the attenuated ATM signaling in Mre11 and Nbs1 hypomorphic mutants might lead to elevated DSB numbers. To test this, we examined SPO11-oligonucleotide complexes, which provide a measure of whole-testis DSB levels, in Mre11ATLD/ATLD and Nbs1ΔB/ΔB single mutant mice [27]. (Note that this class of hypomorphic MRE11-complex mutation is unlike the Rad50S type of mutation, which in yeast blocks endonucleolytic release of SPO11 from DSB ends [33]). Indeed, we found that Mre11ATLD/ATLD and Nbs1ΔB/ΔB mice displayed an ∼2-fold increase compared to wild-type littermates (Fig. 4A-B). This elevation is less than what is seen in Atm–/–(∼12-fold) or Spo11+/–Atm−/− testes (∼6-fold), presumably because the Mre11 and Nbs1 hypomorphs attenuate but do not eliminate ATM activity [34,35]. (Note that the overall fertility phenotype of the Mre11 and Nbs1 single mutants is also significantly milder than for mice lacking ATM (S1 Table)). These results provide strong evidence that ATM-mediated feedback control of DSBs involves an MRE11 complex-dependent ATM activation pathway similar to the response to ionizing radiation. In contrast, Trip13mod/mod animals had roughly normal levels of SPO11-oligonucleotide complexes (∼70% of wild type, Fig. 4C). Because Trip13mod/mod single mutants have defects in completing DSB repair, we speculate that the autosomal synaptic failure in Trip13mod/modMre11ATLD/ATLD or Trip13mod/modNbs1ΔB/ΔB mice is a synthetic defect caused by an inability of TRIP13-deficient cells to tolerate increased DSB numbers resulting from ATM activation defects. An alternative but not mutually exclusive possibility is that the MRE11 complex and TRIP13 synergistically promote synapsis separately from their effects on recombination.


The ATM signaling cascade promotes recombination-dependent pachytene arrest in mouse spermatocytes.

Pacheco S, Marcet-Ortega M, Lange J, Jasin M, Keeney S, Roig I - PLoS Genet. (2015)

The MRE11 complex, but not TRIP13 or CHK2, modulates SPO11-oligonucleotide complex levels.SPO11-oligonucleotide complexes were immunoprecipitated from extracts of testes of the indicated genotypes, labelled with terminal transferase and 32P-nucleotide, and resolved by SDS-PAGE. Top image in each panel, autoradiograph; bottom image, SPO11 Western blot where the two major SPO11 isoforms, α and β, are indicated. The vertical lines next to the autoradiographs indicate the signal from SPO11-oligonucleotide complexes. Asterisk indicates non-specific signal from the labelling reaction. Short arrow designates the migration position of the heavy chain of the antibody used to immunoprecipitate SPO11. (A-B) Both Mre11 and Nbs1 mutants have increased levels of SPO11-oligonucleotide complexes compared to littermate controls (1.9 ± 0.4 fold, mean ± SD of the relative signal intensity compared to a wild-type control, n = 3 mice, and 1.8 ± 0.2 fold, n = 3, respectively). (C) SPO11-oligonucleotide complex levels are slightly reduced in Trip13mod/mod samples (0.7 ± 0.2, n = 3), similar to other recombination-deficient mutants, such as Dmc1–/– [27]. The SPO11α isoform is not detected in Trip13mod/mod testes as observed in other mutants that arrest at pachynema [27]. (D) Chk2−/− testes have similar levels of SPO11-oligonucleotide complex as controls (1.0 ± 0.2 fold, n = 3).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4358828&req=5

pgen.1005017.g004: The MRE11 complex, but not TRIP13 or CHK2, modulates SPO11-oligonucleotide complex levels.SPO11-oligonucleotide complexes were immunoprecipitated from extracts of testes of the indicated genotypes, labelled with terminal transferase and 32P-nucleotide, and resolved by SDS-PAGE. Top image in each panel, autoradiograph; bottom image, SPO11 Western blot where the two major SPO11 isoforms, α and β, are indicated. The vertical lines next to the autoradiographs indicate the signal from SPO11-oligonucleotide complexes. Asterisk indicates non-specific signal from the labelling reaction. Short arrow designates the migration position of the heavy chain of the antibody used to immunoprecipitate SPO11. (A-B) Both Mre11 and Nbs1 mutants have increased levels of SPO11-oligonucleotide complexes compared to littermate controls (1.9 ± 0.4 fold, mean ± SD of the relative signal intensity compared to a wild-type control, n = 3 mice, and 1.8 ± 0.2 fold, n = 3, respectively). (C) SPO11-oligonucleotide complex levels are slightly reduced in Trip13mod/mod samples (0.7 ± 0.2, n = 3), similar to other recombination-deficient mutants, such as Dmc1–/– [27]. The SPO11α isoform is not detected in Trip13mod/mod testes as observed in other mutants that arrest at pachynema [27]. (D) Chk2−/− testes have similar levels of SPO11-oligonucleotide complex as controls (1.0 ± 0.2 fold, n = 3).
Mentions: We reasoned that the attenuated ATM signaling in Mre11 and Nbs1 hypomorphic mutants might lead to elevated DSB numbers. To test this, we examined SPO11-oligonucleotide complexes, which provide a measure of whole-testis DSB levels, in Mre11ATLD/ATLD and Nbs1ΔB/ΔB single mutant mice [27]. (Note that this class of hypomorphic MRE11-complex mutation is unlike the Rad50S type of mutation, which in yeast blocks endonucleolytic release of SPO11 from DSB ends [33]). Indeed, we found that Mre11ATLD/ATLD and Nbs1ΔB/ΔB mice displayed an ∼2-fold increase compared to wild-type littermates (Fig. 4A-B). This elevation is less than what is seen in Atm–/–(∼12-fold) or Spo11+/–Atm−/− testes (∼6-fold), presumably because the Mre11 and Nbs1 hypomorphs attenuate but do not eliminate ATM activity [34,35]. (Note that the overall fertility phenotype of the Mre11 and Nbs1 single mutants is also significantly milder than for mice lacking ATM (S1 Table)). These results provide strong evidence that ATM-mediated feedback control of DSBs involves an MRE11 complex-dependent ATM activation pathway similar to the response to ionizing radiation. In contrast, Trip13mod/mod animals had roughly normal levels of SPO11-oligonucleotide complexes (∼70% of wild type, Fig. 4C). Because Trip13mod/mod single mutants have defects in completing DSB repair, we speculate that the autosomal synaptic failure in Trip13mod/modMre11ATLD/ATLD or Trip13mod/modNbs1ΔB/ΔB mice is a synthetic defect caused by an inability of TRIP13-deficient cells to tolerate increased DSB numbers resulting from ATM activation defects. An alternative but not mutually exclusive possibility is that the MRE11 complex and TRIP13 synergistically promote synapsis separately from their effects on recombination.

Bottom Line: TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2.Our work supports the conclusion that recombination defects trigger spermatocyte arrest via pathways than are genetically distinct from sex body failure-promoted apoptosis and confirm that the latter can function even when recombination-dependent arrest is inoperative.Implications of these findings for understanding the complex relationships between spermatocyte arrest and apoptosis are discussed.

View Article: PubMed Central - PubMed

Affiliation: Genome Integrity and Instability Group, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain; Cytology and Histology Unit, Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain.

ABSTRACT
Most mutations that compromise meiotic recombination or synapsis in mouse spermatocytes result in arrest and apoptosis at the pachytene stage of the first meiotic prophase. Two main mechanisms are thought to trigger arrest: one independent of the double-strand breaks (DSBs) that initiate meiotic recombination, and another activated by persistent recombination intermediates. Mechanisms underlying the recombination-dependent arrest response are not well understood, so we sought to identify factors involved by examining mutants deficient for TRIP13, a conserved AAA+ ATPase required for the completion of meiotic DSB repair. We find that spermatocytes with a hypomorphic Trip13 mutation (Trip13mod/mod) arrest with features characteristic of early pachynema in wild type, namely, fully synapsed chromosomes without incorporation of the histone variant H1t into chromatin. These cells then undergo apoptosis, possibly in response to the arrest or in response to a defect in sex body formation. However, TRIP13-deficient cells that additionally lack the DSB-responsive kinase ATM progress further, reaching an H1t-positive stage (i.e., similar to mid/late pachynema in wild type) despite the presence of unrepaired DSBs. TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2. These mutant backgrounds nonetheless experience an apoptotic block to further spermatogenic progression, most likely caused by failure to form a sex body. DSB numbers are elevated in Mre11 and Nbs1 hypomorphs but not Chk2 mutants, thus delineating genetic requirements for the ATM-dependent negative feedback loop that regulates DSB numbers. The findings demonstrate for the first time that ATM-dependent signaling enforces the normal pachytene response to persistent recombination intermediates. Our work supports the conclusion that recombination defects trigger spermatocyte arrest via pathways than are genetically distinct from sex body failure-promoted apoptosis and confirm that the latter can function even when recombination-dependent arrest is inoperative. Implications of these findings for understanding the complex relationships between spermatocyte arrest and apoptosis are discussed.

Show MeSH
Related in: MedlinePlus