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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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CHK2 is required to maintain early pachytene arrest caused by TRIP13 deficiency.(A-B) Cross-sections of Chk2−/− and Trip13mod/modChk2−/− testes stained with PAS-Haematoxylin. Chk2−/− testes contained all spermatogenic cell types, whereas Trip13mod/modChk2−/− spermatocytes arrested in tubules at epithelial stage IV (red arrowhead). Note the presence of spermatids (green arrowhead) indicating that some Trip13mod/modChk2−/− cells manage to complete meiosis. (C-F) Early and mid/late pachytene Trip13mod/modChk2−/− spermatocytes stained for H1t, γH2AX, and SYCP3. Both early and mid/late pachytene cells exhibit multiple γH2AX patches (arrowheads) corresponding to unrepaired DSBs, as well as accumulation of γH2AX on the chromatin of the sex chromosomes (arrows). (G) Quantification of the number of γH2AX patches in Trip13mod/modChk2−/− spermatocytes. Data for wild type, Trip13mod/mod and Spo11+/−Atm−/− reproduced from Fig. 1D for comparison, N shows the total number of cells counted per each stage and genotype. (H) γH2AX patches in diplotene cells. The plot on the left shows the number of γH2AX patches per cell. Note that overplotting of zero values obscures information about proportions of cells that lack a γH2AX patch, so the plot on the right displays proportions of cells with or without a γH2AX patch. Primary data for panels G and H are provided in S1 Dataset. N shows the total number of cells counted per each stage and genotype. Bar in (A) represents 20 μm and applies to panels (A-B). Bar in (D) represents 10 μm and applies to panels (C-F).
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pgen.1005017.g005: CHK2 is required to maintain early pachytene arrest caused by TRIP13 deficiency.(A-B) Cross-sections of Chk2−/− and Trip13mod/modChk2−/− testes stained with PAS-Haematoxylin. Chk2−/− testes contained all spermatogenic cell types, whereas Trip13mod/modChk2−/− spermatocytes arrested in tubules at epithelial stage IV (red arrowhead). Note the presence of spermatids (green arrowhead) indicating that some Trip13mod/modChk2−/− cells manage to complete meiosis. (C-F) Early and mid/late pachytene Trip13mod/modChk2−/− spermatocytes stained for H1t, γH2AX, and SYCP3. Both early and mid/late pachytene cells exhibit multiple γH2AX patches (arrowheads) corresponding to unrepaired DSBs, as well as accumulation of γH2AX on the chromatin of the sex chromosomes (arrows). (G) Quantification of the number of γH2AX patches in Trip13mod/modChk2−/− spermatocytes. Data for wild type, Trip13mod/mod and Spo11+/−Atm−/− reproduced from Fig. 1D for comparison, N shows the total number of cells counted per each stage and genotype. (H) γH2AX patches in diplotene cells. The plot on the left shows the number of γH2AX patches per cell. Note that overplotting of zero values obscures information about proportions of cells that lack a γH2AX patch, so the plot on the right displays proportions of cells with or without a γH2AX patch. Primary data for panels G and H are provided in S1 Dataset. N shows the total number of cells counted per each stage and genotype. Bar in (A) represents 20 μm and applies to panels (A-B). Bar in (D) represents 10 μm and applies to panels (C-F).

Mentions: The testicular phenotype of Trip13mod/modChk2−/− mice was similar to the Trip13mod/mod single mutant for testis size and histological tubule classification (apoptosis of spermatocytes at epithelial stage IV, but with presence of some spermatids), but also, notably, for the percentage of SYCP3-expressing cells that had progressed beyond pachynema (1.9%, P = 0.2462, Fisher’s exact test; Table 1 and Fig. 5A-B). Thus, CHK2 deficiency does not cause a more penetrant spermatogenic failure in the context of the Trip13mod/mod mutation, unlike MRE11 complex hypomorphs or the absence of ATM itself. Moreover, CHK2-deficient testes displayed similar levels of SPO11-oligonucleotide complexes as wild-type littermates (Fig. 4D). Thus, CHK2 is not required for proper control of meiotic DSB formation, clearly separating CHK2 from both the MRE11 complex and ATM in the regulation of SPO11 activity. These results further support the conclusion above that the synaptic defects and more penetrant block to spermatogenesis observed in TSA triple mutants and in Trip13mod/modMre11ATLD/ATLD and Trip13mod/modNbs1ΔB/ ΔB double mutants are attributable to the increased DSB numbers.


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)

CHK2 is required to maintain early pachytene arrest caused by TRIP13 deficiency.(A-B) Cross-sections of Chk2−/− and Trip13mod/modChk2−/− testes stained with PAS-Haematoxylin. Chk2−/− testes contained all spermatogenic cell types, whereas Trip13mod/modChk2−/− spermatocytes arrested in tubules at epithelial stage IV (red arrowhead). Note the presence of spermatids (green arrowhead) indicating that some Trip13mod/modChk2−/− cells manage to complete meiosis. (C-F) Early and mid/late pachytene Trip13mod/modChk2−/− spermatocytes stained for H1t, γH2AX, and SYCP3. Both early and mid/late pachytene cells exhibit multiple γH2AX patches (arrowheads) corresponding to unrepaired DSBs, as well as accumulation of γH2AX on the chromatin of the sex chromosomes (arrows). (G) Quantification of the number of γH2AX patches in Trip13mod/modChk2−/− spermatocytes. Data for wild type, Trip13mod/mod and Spo11+/−Atm−/− reproduced from Fig. 1D for comparison, N shows the total number of cells counted per each stage and genotype. (H) γH2AX patches in diplotene cells. The plot on the left shows the number of γH2AX patches per cell. Note that overplotting of zero values obscures information about proportions of cells that lack a γH2AX patch, so the plot on the right displays proportions of cells with or without a γH2AX patch. Primary data for panels G and H are provided in S1 Dataset. N shows the total number of cells counted per each stage and genotype. Bar in (A) represents 20 μm and applies to panels (A-B). Bar in (D) represents 10 μm and applies to panels (C-F).
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pgen.1005017.g005: CHK2 is required to maintain early pachytene arrest caused by TRIP13 deficiency.(A-B) Cross-sections of Chk2−/− and Trip13mod/modChk2−/− testes stained with PAS-Haematoxylin. Chk2−/− testes contained all spermatogenic cell types, whereas Trip13mod/modChk2−/− spermatocytes arrested in tubules at epithelial stage IV (red arrowhead). Note the presence of spermatids (green arrowhead) indicating that some Trip13mod/modChk2−/− cells manage to complete meiosis. (C-F) Early and mid/late pachytene Trip13mod/modChk2−/− spermatocytes stained for H1t, γH2AX, and SYCP3. Both early and mid/late pachytene cells exhibit multiple γH2AX patches (arrowheads) corresponding to unrepaired DSBs, as well as accumulation of γH2AX on the chromatin of the sex chromosomes (arrows). (G) Quantification of the number of γH2AX patches in Trip13mod/modChk2−/− spermatocytes. Data for wild type, Trip13mod/mod and Spo11+/−Atm−/− reproduced from Fig. 1D for comparison, N shows the total number of cells counted per each stage and genotype. (H) γH2AX patches in diplotene cells. The plot on the left shows the number of γH2AX patches per cell. Note that overplotting of zero values obscures information about proportions of cells that lack a γH2AX patch, so the plot on the right displays proportions of cells with or without a γH2AX patch. Primary data for panels G and H are provided in S1 Dataset. N shows the total number of cells counted per each stage and genotype. Bar in (A) represents 20 μm and applies to panels (A-B). Bar in (D) represents 10 μm and applies to panels (C-F).
Mentions: The testicular phenotype of Trip13mod/modChk2−/− mice was similar to the Trip13mod/mod single mutant for testis size and histological tubule classification (apoptosis of spermatocytes at epithelial stage IV, but with presence of some spermatids), but also, notably, for the percentage of SYCP3-expressing cells that had progressed beyond pachynema (1.9%, P = 0.2462, Fisher’s exact test; Table 1 and Fig. 5A-B). Thus, CHK2 deficiency does not cause a more penetrant spermatogenic failure in the context of the Trip13mod/mod mutation, unlike MRE11 complex hypomorphs or the absence of ATM itself. Moreover, CHK2-deficient testes displayed similar levels of SPO11-oligonucleotide complexes as wild-type littermates (Fig. 4D). Thus, CHK2 is not required for proper control of meiotic DSB formation, clearly separating CHK2 from both the MRE11 complex and ATM in the regulation of SPO11 activity. These results further support the conclusion above that the synaptic defects and more penetrant block to spermatogenesis observed in TSA triple mutants and in Trip13mod/modMre11ATLD/ATLD and Trip13mod/modNbs1ΔB/ ΔB double mutants are attributable to the increased DSB numbers.

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