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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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Sex body deficiency in Trip13 mutants.(A-D) Wild-type and Trip13mod/modChk2−/− pachytene spermatocytes stained for ATR and SYCP3. Arrowheads indicate the sex chromosomes. Note the relatively continuous ATR staining on the X and Y axes in wild type compared with the focal ATR staining in the mutant. (E) Quantification of the different ATR staining patterns found in the genotypes presented. (F) Left panels, percent of early pachytene-stage spermatocytes expressing Zfx and Scml2 in wild-type, Trip13mod/mod, Trip13mod/modMre11ATLD/ATLD and Trip13mod/modChk2−/− mice. The N value in each bar represents the number of mice analyzed per each gene and genotype. Middle panel, cartoon of a sex body displaying the relative position of Zfx and Scml2 within the X chromosome. Right image, representative Trip13mod/modChk2−/− spermatocyte displaying TOPBP1 (green) and γH2AX (red) immunostaining and Scml2 RNA-FISH signal (white, arrow). Just 10.9% ± 3.8 (mean ± SD) of wild-type spermatocytes (N = 225 cells) express Zfx and 17.1% ± 1.9 express Scml2 (N = 200). Mouse that fail to synapse the X and Y chromosomes, like Trip13mod/modMre11ATLD/ATLD, present more spermatocytes expressing these genes than wild-type mice (for Zfx: 37.1%, N = 35 and for Smcl2: 49.1%, N = 53, P≤0.0001 and P = 0.0002 respectively, Fisher’s exact test). Trip13mod/mod mice present more cells expressing these genes than wild-type mice (for Zfx: 27.2% ± 3.2, N = 158; for Smcl2: 27.5% ± 0.0, N = 160, P<0.005, 1 way Anova). Similarly, Trip13mod/modChk2−/− spermatocytes are more likely to express these X-linked genes than wild-type cells (for Zfx: 28.6%, N = 70; for Smcl2: 30.0%, N = 80, P = 0.0217 and P = 0.0009 respectively, Fisher’s exact test). (G-J) Mid/late pachytene-stage, TUNEL-positive Trip13mod/modChk2−/− spermatocyte immunostained for SYCP3 (green, H), γH2AX (red, I) and H1t (blue, J). Note the presence of an elongated sex body (arrow), multiple γH2AX patches (arrowhead) and H1t in the chromatin of the apoptotic cell. Bars in (C) and (H) represent 10 μm and apply to panels (A,C) and (G-J), respectively. Bar in (F) represents 5 μm.
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pgen.1005017.g006: Sex body deficiency in Trip13 mutants.(A-D) Wild-type and Trip13mod/modChk2−/− pachytene spermatocytes stained for ATR and SYCP3. Arrowheads indicate the sex chromosomes. Note the relatively continuous ATR staining on the X and Y axes in wild type compared with the focal ATR staining in the mutant. (E) Quantification of the different ATR staining patterns found in the genotypes presented. (F) Left panels, percent of early pachytene-stage spermatocytes expressing Zfx and Scml2 in wild-type, Trip13mod/mod, Trip13mod/modMre11ATLD/ATLD and Trip13mod/modChk2−/− mice. The N value in each bar represents the number of mice analyzed per each gene and genotype. Middle panel, cartoon of a sex body displaying the relative position of Zfx and Scml2 within the X chromosome. Right image, representative Trip13mod/modChk2−/− spermatocyte displaying TOPBP1 (green) and γH2AX (red) immunostaining and Scml2 RNA-FISH signal (white, arrow). Just 10.9% ± 3.8 (mean ± SD) of wild-type spermatocytes (N = 225 cells) express Zfx and 17.1% ± 1.9 express Scml2 (N = 200). Mouse that fail to synapse the X and Y chromosomes, like Trip13mod/modMre11ATLD/ATLD, present more spermatocytes expressing these genes than wild-type mice (for Zfx: 37.1%, N = 35 and for Smcl2: 49.1%, N = 53, P≤0.0001 and P = 0.0002 respectively, Fisher’s exact test). Trip13mod/mod mice present more cells expressing these genes than wild-type mice (for Zfx: 27.2% ± 3.2, N = 158; for Smcl2: 27.5% ± 0.0, N = 160, P<0.005, 1 way Anova). Similarly, Trip13mod/modChk2−/− spermatocytes are more likely to express these X-linked genes than wild-type cells (for Zfx: 28.6%, N = 70; for Smcl2: 30.0%, N = 80, P = 0.0217 and P = 0.0009 respectively, Fisher’s exact test). (G-J) Mid/late pachytene-stage, TUNEL-positive Trip13mod/modChk2−/− spermatocyte immunostained for SYCP3 (green, H), γH2AX (red, I) and H1t (blue, J). Note the presence of an elongated sex body (arrow), multiple γH2AX patches (arrowhead) and H1t in the chromatin of the apoptotic cell. Bars in (C) and (H) represent 10 μm and apply to panels (A,C) and (G-J), respectively. Bar in (F) represents 5 μm.

Mentions: ATR is the kinase responsible for the bulk of H2AX phosphorylation in sex bodies [18,24,25]. In wild type, most pachytene spermatocytes showed ATR staining either as a continuous signal on the sex chromosome axes or spread to the XY chromatin (93.6%, n = 47; Fig. 6A-B), as previously reported [39]. The remainder of the cells displayed stretches of ATR partially covering the X and Y axes. By contrast, only 18.0% of Trip13mod/modChk2−/− pachytene cells had the axial or chromatin-associated ATR staining most commonly found in wild-type cells, 26.2% had short stretches of continuous ATR signal partly covering sex chromosome axes, and most cells had only focal ATR staining (55.7%, n = 61; Fig. 6C-E). Trip13mod/mod spermatocytes had an intermediate phenotype: 38.8% displayed ATR completely covering the XY axes or chromatin, 32.8% showed ATR stretches over the XY axes, and 28.4% had only discrete ATR foci along the X and Y (n = 67, Fig. 6E). These results suggest that CHK2 deficiency may exacerbate a defect in ATR loading on the sex chromosomes in TRIP13-deficient cells.


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)

Sex body deficiency in Trip13 mutants.(A-D) Wild-type and Trip13mod/modChk2−/− pachytene spermatocytes stained for ATR and SYCP3. Arrowheads indicate the sex chromosomes. Note the relatively continuous ATR staining on the X and Y axes in wild type compared with the focal ATR staining in the mutant. (E) Quantification of the different ATR staining patterns found in the genotypes presented. (F) Left panels, percent of early pachytene-stage spermatocytes expressing Zfx and Scml2 in wild-type, Trip13mod/mod, Trip13mod/modMre11ATLD/ATLD and Trip13mod/modChk2−/− mice. The N value in each bar represents the number of mice analyzed per each gene and genotype. Middle panel, cartoon of a sex body displaying the relative position of Zfx and Scml2 within the X chromosome. Right image, representative Trip13mod/modChk2−/− spermatocyte displaying TOPBP1 (green) and γH2AX (red) immunostaining and Scml2 RNA-FISH signal (white, arrow). Just 10.9% ± 3.8 (mean ± SD) of wild-type spermatocytes (N = 225 cells) express Zfx and 17.1% ± 1.9 express Scml2 (N = 200). Mouse that fail to synapse the X and Y chromosomes, like Trip13mod/modMre11ATLD/ATLD, present more spermatocytes expressing these genes than wild-type mice (for Zfx: 37.1%, N = 35 and for Smcl2: 49.1%, N = 53, P≤0.0001 and P = 0.0002 respectively, Fisher’s exact test). Trip13mod/mod mice present more cells expressing these genes than wild-type mice (for Zfx: 27.2% ± 3.2, N = 158; for Smcl2: 27.5% ± 0.0, N = 160, P<0.005, 1 way Anova). Similarly, Trip13mod/modChk2−/− spermatocytes are more likely to express these X-linked genes than wild-type cells (for Zfx: 28.6%, N = 70; for Smcl2: 30.0%, N = 80, P = 0.0217 and P = 0.0009 respectively, Fisher’s exact test). (G-J) Mid/late pachytene-stage, TUNEL-positive Trip13mod/modChk2−/− spermatocyte immunostained for SYCP3 (green, H), γH2AX (red, I) and H1t (blue, J). Note the presence of an elongated sex body (arrow), multiple γH2AX patches (arrowhead) and H1t in the chromatin of the apoptotic cell. Bars in (C) and (H) represent 10 μm and apply to panels (A,C) and (G-J), respectively. Bar in (F) represents 5 μm.
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pgen.1005017.g006: Sex body deficiency in Trip13 mutants.(A-D) Wild-type and Trip13mod/modChk2−/− pachytene spermatocytes stained for ATR and SYCP3. Arrowheads indicate the sex chromosomes. Note the relatively continuous ATR staining on the X and Y axes in wild type compared with the focal ATR staining in the mutant. (E) Quantification of the different ATR staining patterns found in the genotypes presented. (F) Left panels, percent of early pachytene-stage spermatocytes expressing Zfx and Scml2 in wild-type, Trip13mod/mod, Trip13mod/modMre11ATLD/ATLD and Trip13mod/modChk2−/− mice. The N value in each bar represents the number of mice analyzed per each gene and genotype. Middle panel, cartoon of a sex body displaying the relative position of Zfx and Scml2 within the X chromosome. Right image, representative Trip13mod/modChk2−/− spermatocyte displaying TOPBP1 (green) and γH2AX (red) immunostaining and Scml2 RNA-FISH signal (white, arrow). Just 10.9% ± 3.8 (mean ± SD) of wild-type spermatocytes (N = 225 cells) express Zfx and 17.1% ± 1.9 express Scml2 (N = 200). Mouse that fail to synapse the X and Y chromosomes, like Trip13mod/modMre11ATLD/ATLD, present more spermatocytes expressing these genes than wild-type mice (for Zfx: 37.1%, N = 35 and for Smcl2: 49.1%, N = 53, P≤0.0001 and P = 0.0002 respectively, Fisher’s exact test). Trip13mod/mod mice present more cells expressing these genes than wild-type mice (for Zfx: 27.2% ± 3.2, N = 158; for Smcl2: 27.5% ± 0.0, N = 160, P<0.005, 1 way Anova). Similarly, Trip13mod/modChk2−/− spermatocytes are more likely to express these X-linked genes than wild-type cells (for Zfx: 28.6%, N = 70; for Smcl2: 30.0%, N = 80, P = 0.0217 and P = 0.0009 respectively, Fisher’s exact test). (G-J) Mid/late pachytene-stage, TUNEL-positive Trip13mod/modChk2−/− spermatocyte immunostained for SYCP3 (green, H), γH2AX (red, I) and H1t (blue, J). Note the presence of an elongated sex body (arrow), multiple γH2AX patches (arrowhead) and H1t in the chromatin of the apoptotic cell. Bars in (C) and (H) represent 10 μm and apply to panels (A,C) and (G-J), respectively. Bar in (F) represents 5 μm.
Mentions: ATR is the kinase responsible for the bulk of H2AX phosphorylation in sex bodies [18,24,25]. In wild type, most pachytene spermatocytes showed ATR staining either as a continuous signal on the sex chromosome axes or spread to the XY chromatin (93.6%, n = 47; Fig. 6A-B), as previously reported [39]. The remainder of the cells displayed stretches of ATR partially covering the X and Y axes. By contrast, only 18.0% of Trip13mod/modChk2−/− pachytene cells had the axial or chromatin-associated ATR staining most commonly found in wild-type cells, 26.2% had short stretches of continuous ATR signal partly covering sex chromosome axes, and most cells had only focal ATR staining (55.7%, n = 61; Fig. 6C-E). Trip13mod/mod spermatocytes had an intermediate phenotype: 38.8% displayed ATR completely covering the XY axes or chromatin, 32.8% showed ATR stretches over the XY axes, and 28.4% had only discrete ATR foci along the X and Y (n = 67, Fig. 6E). These results suggest that CHK2 deficiency may exacerbate a defect in ATR loading on the sex chromosomes in TRIP13-deficient cells.

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