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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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Trip13mod/modSpo11+/−Atm−/− spermatocytes arrest at epithelial stage IV, but present autosomal asynapsis, multiple unrepaired DSBs and fail to form a sex body at mid/late pachynema.(A-D) Testis cross-sections showing individual seminiferous tubules from the indicated genotypes, stained with PAS-Haematoxylin. (A) In wild type, spermatogonia, spermatocytes and spermatids are present. (B) Stage IV tubule in Trip13mod/mod with apoptotic spermatocytes, corresponding to pachynema (red arrowhead) [22,23]. Only a minor fraction of cells complete meiosis (green arrowhead). (C) Spo11+/−Atm−/− tubule at stage XII, which is characterized by spermatocytes at metaphase I (red arrowhead) [24]. The presence of lagging chromosomes in some metaphase I spermatocytes is thought to be the cause of apoptosis. Green arrowhead denotes a spermatid that has escaped meiotic arrest. (D) Trip13mod/modSpo11+/−Atm−/− tubule at stage IV, presenting multiple apoptotic spermatocytes at pachytene stage (red arrowhead). No spermatids were observed. (E-H) Two Trip13mod/modSpo11+/−Atm−/− spermatocytes, one at preleptonema (left) and the other at mid/late pachynema (right), stained for H1t (blue, F), γH2AX (red, G) and SYCP3 (green, H). A high degree of asynapsis occurs in the H1t-positive cell, where long stretches of axial elements are visible (arrowhead). (I) Enlarged image of a bivalent that has successfully synapsed only a portion of the homologous chromosome pair (arrow). (J) Number of γH2AX patches in mid/late pachytene spermatocytes of the indicated genotypes. Triple and double mutants that generate more DSBs and fail to complete synapsis (highlighted in dark grey) display more γH2AX patches at mid/late pachynema than Trip13mod/modChk2−/− cells (highlighted in light grey). 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 genotype. Asterisk marks statistically significant differences compared to Trip13mod/mod, P≤0.0001, t test. Primary data are provided in S1 Dataset. Bar in (A) represents 20 μm and applies to panels (A-D). Bar in (H) represents 10 μm and applies to panels (E-H).
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pgen.1005017.g002: Trip13mod/modSpo11+/−Atm−/− spermatocytes arrest at epithelial stage IV, but present autosomal asynapsis, multiple unrepaired DSBs and fail to form a sex body at mid/late pachynema.(A-D) Testis cross-sections showing individual seminiferous tubules from the indicated genotypes, stained with PAS-Haematoxylin. (A) In wild type, spermatogonia, spermatocytes and spermatids are present. (B) Stage IV tubule in Trip13mod/mod with apoptotic spermatocytes, corresponding to pachynema (red arrowhead) [22,23]. Only a minor fraction of cells complete meiosis (green arrowhead). (C) Spo11+/−Atm−/− tubule at stage XII, which is characterized by spermatocytes at metaphase I (red arrowhead) [24]. The presence of lagging chromosomes in some metaphase I spermatocytes is thought to be the cause of apoptosis. Green arrowhead denotes a spermatid that has escaped meiotic arrest. (D) Trip13mod/modSpo11+/−Atm−/− tubule at stage IV, presenting multiple apoptotic spermatocytes at pachytene stage (red arrowhead). No spermatids were observed. (E-H) Two Trip13mod/modSpo11+/−Atm−/− spermatocytes, one at preleptonema (left) and the other at mid/late pachynema (right), stained for H1t (blue, F), γH2AX (red, G) and SYCP3 (green, H). A high degree of asynapsis occurs in the H1t-positive cell, where long stretches of axial elements are visible (arrowhead). (I) Enlarged image of a bivalent that has successfully synapsed only a portion of the homologous chromosome pair (arrow). (J) Number of γH2AX patches in mid/late pachytene spermatocytes of the indicated genotypes. Triple and double mutants that generate more DSBs and fail to complete synapsis (highlighted in dark grey) display more γH2AX patches at mid/late pachynema than Trip13mod/modChk2−/− cells (highlighted in light grey). 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 genotype. Asterisk marks statistically significant differences compared to Trip13mod/mod, P≤0.0001, t test. Primary data are provided in S1 Dataset. Bar in (A) represents 20 μm and applies to panels (A-D). Bar in (H) represents 10 μm and applies to panels (E-H).

Mentions: Second, we assessed the timing of apoptosis by histological staging of seminiferous tubules. Many Trip13mod/mod spermatocytes underwent apoptosis in tubules at epithelial stage IV, corresponding to mid pachynema (Fig. 2A,B), as previously shown [23]. In contrast, and consistent with absence of pachytene arrest, Spo11+/−Atm−/− spermatocytes apoptosed in tubules at epithelial stage XII (Fig. 2C), corresponding to metaphase I. Arrest of Spo11+/−Atm−/− spermatocytes at this point is thought to be caused largely by a spindle checkpoint response to achiasmate (unconnected) chromosomes, particularly the X-Y pair [32]. Consistent with the small testis sizes, TSA triple mutant animals showed spermatocyte apoptosis at stage IV (Fig. 2D). Furthermore, whereas few wild-type tubules had >5 TUNEL-positive cells (1.0%, Table 1), both Trip13mod/mod single mutant and TSA triple mutant testes displayed many such apoptotic tubules (21.6% and 26.0% respectively, P≤0.0001 compared to wild type, Fisher’s exact test, Table 1). Surprisingly, no TSA triple mutant cells escaping stage IV apoptosis were observed (Fig. 2B-D), in contrast to Trip13mod/mod or Spo11+/−Atm−/− testes [22–24].


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)

Trip13mod/modSpo11+/−Atm−/− spermatocytes arrest at epithelial stage IV, but present autosomal asynapsis, multiple unrepaired DSBs and fail to form a sex body at mid/late pachynema.(A-D) Testis cross-sections showing individual seminiferous tubules from the indicated genotypes, stained with PAS-Haematoxylin. (A) In wild type, spermatogonia, spermatocytes and spermatids are present. (B) Stage IV tubule in Trip13mod/mod with apoptotic spermatocytes, corresponding to pachynema (red arrowhead) [22,23]. Only a minor fraction of cells complete meiosis (green arrowhead). (C) Spo11+/−Atm−/− tubule at stage XII, which is characterized by spermatocytes at metaphase I (red arrowhead) [24]. The presence of lagging chromosomes in some metaphase I spermatocytes is thought to be the cause of apoptosis. Green arrowhead denotes a spermatid that has escaped meiotic arrest. (D) Trip13mod/modSpo11+/−Atm−/− tubule at stage IV, presenting multiple apoptotic spermatocytes at pachytene stage (red arrowhead). No spermatids were observed. (E-H) Two Trip13mod/modSpo11+/−Atm−/− spermatocytes, one at preleptonema (left) and the other at mid/late pachynema (right), stained for H1t (blue, F), γH2AX (red, G) and SYCP3 (green, H). A high degree of asynapsis occurs in the H1t-positive cell, where long stretches of axial elements are visible (arrowhead). (I) Enlarged image of a bivalent that has successfully synapsed only a portion of the homologous chromosome pair (arrow). (J) Number of γH2AX patches in mid/late pachytene spermatocytes of the indicated genotypes. Triple and double mutants that generate more DSBs and fail to complete synapsis (highlighted in dark grey) display more γH2AX patches at mid/late pachynema than Trip13mod/modChk2−/− cells (highlighted in light grey). 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 genotype. Asterisk marks statistically significant differences compared to Trip13mod/mod, P≤0.0001, t test. Primary data are provided in S1 Dataset. Bar in (A) represents 20 μm and applies to panels (A-D). Bar in (H) represents 10 μm and applies to panels (E-H).
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pgen.1005017.g002: Trip13mod/modSpo11+/−Atm−/− spermatocytes arrest at epithelial stage IV, but present autosomal asynapsis, multiple unrepaired DSBs and fail to form a sex body at mid/late pachynema.(A-D) Testis cross-sections showing individual seminiferous tubules from the indicated genotypes, stained with PAS-Haematoxylin. (A) In wild type, spermatogonia, spermatocytes and spermatids are present. (B) Stage IV tubule in Trip13mod/mod with apoptotic spermatocytes, corresponding to pachynema (red arrowhead) [22,23]. Only a minor fraction of cells complete meiosis (green arrowhead). (C) Spo11+/−Atm−/− tubule at stage XII, which is characterized by spermatocytes at metaphase I (red arrowhead) [24]. The presence of lagging chromosomes in some metaphase I spermatocytes is thought to be the cause of apoptosis. Green arrowhead denotes a spermatid that has escaped meiotic arrest. (D) Trip13mod/modSpo11+/−Atm−/− tubule at stage IV, presenting multiple apoptotic spermatocytes at pachytene stage (red arrowhead). No spermatids were observed. (E-H) Two Trip13mod/modSpo11+/−Atm−/− spermatocytes, one at preleptonema (left) and the other at mid/late pachynema (right), stained for H1t (blue, F), γH2AX (red, G) and SYCP3 (green, H). A high degree of asynapsis occurs in the H1t-positive cell, where long stretches of axial elements are visible (arrowhead). (I) Enlarged image of a bivalent that has successfully synapsed only a portion of the homologous chromosome pair (arrow). (J) Number of γH2AX patches in mid/late pachytene spermatocytes of the indicated genotypes. Triple and double mutants that generate more DSBs and fail to complete synapsis (highlighted in dark grey) display more γH2AX patches at mid/late pachynema than Trip13mod/modChk2−/− cells (highlighted in light grey). 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 genotype. Asterisk marks statistically significant differences compared to Trip13mod/mod, P≤0.0001, t test. Primary data are provided in S1 Dataset. Bar in (A) represents 20 μm and applies to panels (A-D). Bar in (H) represents 10 μm and applies to panels (E-H).
Mentions: Second, we assessed the timing of apoptosis by histological staging of seminiferous tubules. Many Trip13mod/mod spermatocytes underwent apoptosis in tubules at epithelial stage IV, corresponding to mid pachynema (Fig. 2A,B), as previously shown [23]. In contrast, and consistent with absence of pachytene arrest, Spo11+/−Atm−/− spermatocytes apoptosed in tubules at epithelial stage XII (Fig. 2C), corresponding to metaphase I. Arrest of Spo11+/−Atm−/− spermatocytes at this point is thought to be caused largely by a spindle checkpoint response to achiasmate (unconnected) chromosomes, particularly the X-Y pair [32]. Consistent with the small testis sizes, TSA triple mutant animals showed spermatocyte apoptosis at stage IV (Fig. 2D). Furthermore, whereas few wild-type tubules had >5 TUNEL-positive cells (1.0%, Table 1), both Trip13mod/mod single mutant and TSA triple mutant testes displayed many such apoptotic tubules (21.6% and 26.0% respectively, P≤0.0001 compared to wild type, Fisher’s exact test, Table 1). Surprisingly, no TSA triple mutant cells escaping stage IV apoptosis were observed (Fig. 2B-D), in contrast to Trip13mod/mod or Spo11+/−Atm−/− testes [22–24].

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