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Mouse pachytene checkpoint 2 (trip13) is required for completing meiotic recombination but not synapsis.

Li XC, Li X, Schimenti JC - PLoS Genet. (2007)

Bottom Line: The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA.These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes.Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers.

View Article: PubMed Central - PubMed

Affiliation: Cornell University, College of Veterinary Medicine, Department of Biomedical Sciences, Ithaca, New York, United States of America.

ABSTRACT
In mammalian meiosis, homologous chromosome synapsis is coupled with recombination. As in most eukaryotes, mammalian meiocytes have checkpoints that monitor the fidelity of these processes. We report that the mouse ortholog (Trip13) of pachytene checkpoint 2 (PCH2), an essential component of the synapsis checkpoint in Saccharomyces cerevisiae and Caenorhabditis elegans, is required for completion of meiosis in both sexes. TRIP13-deficient mice exhibit spermatocyte death in pachynema and loss of oocytes around birth. The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA. These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes. Double mutant analysis demonstrated that the recombination and synapsis genes Spo11, Mei1, Rec8, and Dmc1 are all epistatic to Trip13, suggesting that TRIP13 does not have meiotic checkpoint function in mice. Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers. To our knowledge, this is the first model to separate recombination defects from asynapsis in mammalian meiosis, and provides the first evidence that unrepaired DNA damage alone can trigger the pachytene checkpoint response in mice.

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Immunohistochemical Analysis of Pachytene Spermatocyte ChromosomesSurface-spread chromosomes were immunolabeled with the indicated antibodies and fluorophores. As indicated in the upper right of each panel, cells were from wild type (WT, either +/+ or Trip13Gt/+) or Trip13Gt/Gt (Mut). There were no differences seen between heterozygotes and +/+ spermatocytes.(A) A mutant pachytene nucleus with full synapsis. Areas of SYCP1/SYCP3 colabeling are yellow.(B–E) Spermatocytes nucleus from 17.5 d postpartum mutant. Asynapsed chromosomes or regions of chromosomes are indicated by white and yellow arrows, respectively. Unlike the normal distribution in wild-type pachytene spermatocytes (C), BLM foci are present on synapsed pachytene chromosomes in the mutant (D). RAD51 foci, which are abundant earlier in prophase, disappear from autosomes in wild-type pachytene nuclei (E) and the bulk of staining is over the XY body (arrow).(F) RAD51 persists on the synapsed mutant chromosomes (arrows).(G) H2AX phosphorylation is restricted to the XY body in WT.(H) In addition to a large area of γH2AX staining (arrow) over the XY body, there is extensive autosomal H2AX phosphorylation (arrows).(I, J) Note that in wild-type pachytene spermatocytes, TOPBP1 is present only over the XY body (yellow arrow). In the mutant (J), an arrow denotes one area of intensive staining that may be over the sex chromosomes, but many other chromosome cores are positively stained.(K, L) RPA persists along synapsed cores in the mutant, not WT.(M, N) Arrows indicate examples of MLH3 foci on SCs.(O) In WT late pachytene spermatocytes, RAD51 is present only at background levels.(P) As in (F), extensive RAD51 staining delineates SCs in mutant pachytene nuclei (indicated by white arcs). MLH1 foci colocalize with these tracts (arrows) at the typical 1–2 foci per chromosome as in (M).
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pgen-0030130-g004: Immunohistochemical Analysis of Pachytene Spermatocyte ChromosomesSurface-spread chromosomes were immunolabeled with the indicated antibodies and fluorophores. As indicated in the upper right of each panel, cells were from wild type (WT, either +/+ or Trip13Gt/+) or Trip13Gt/Gt (Mut). There were no differences seen between heterozygotes and +/+ spermatocytes.(A) A mutant pachytene nucleus with full synapsis. Areas of SYCP1/SYCP3 colabeling are yellow.(B–E) Spermatocytes nucleus from 17.5 d postpartum mutant. Asynapsed chromosomes or regions of chromosomes are indicated by white and yellow arrows, respectively. Unlike the normal distribution in wild-type pachytene spermatocytes (C), BLM foci are present on synapsed pachytene chromosomes in the mutant (D). RAD51 foci, which are abundant earlier in prophase, disappear from autosomes in wild-type pachytene nuclei (E) and the bulk of staining is over the XY body (arrow).(F) RAD51 persists on the synapsed mutant chromosomes (arrows).(G) H2AX phosphorylation is restricted to the XY body in WT.(H) In addition to a large area of γH2AX staining (arrow) over the XY body, there is extensive autosomal H2AX phosphorylation (arrows).(I, J) Note that in wild-type pachytene spermatocytes, TOPBP1 is present only over the XY body (yellow arrow). In the mutant (J), an arrow denotes one area of intensive staining that may be over the sex chromosomes, but many other chromosome cores are positively stained.(K, L) RPA persists along synapsed cores in the mutant, not WT.(M, N) Arrows indicate examples of MLH3 foci on SCs.(O) In WT late pachytene spermatocytes, RAD51 is present only at background levels.(P) As in (F), extensive RAD51 staining delineates SCs in mutant pachytene nuclei (indicated by white arcs). MLH1 foci colocalize with these tracts (arrows) at the typical 1–2 foci per chromosome as in (M).

Mentions: To better characterize the degree of meiotic progression in Trip13Gt/Gt spermatocytes, we immunostained chromosome spreads for SYCP3 and SYCP1, components of the axial/lateral elements and transverse filaments, respectively, of the synaptonemal complex (SC). Pachytene spermatocyte nuclei from postpubertal mutant testes could assemble normal SC cores and exhibited full synapsis of chromosomes as judged by colabeling of SYCP1 and SYCP3 along the full lengths of all autosomes (Figure 4A). Additionally, the X and Y chromosomes were normally synapsed at their pseudoautosomal region. More prepubertal (17.5 d postpartum) mutant spermatocytes contained asynaptic or terminally asynapsed chromosomes than age-matched controls (62.5% versus 25%, respectively; Figure 4B). We attribute this to a delay in the first wave of postnatal spermatogenesis (Figure 2D and 2E), likely related to systemic developmental retardation (Figure 2A and 2B). Nevertheless, since Trip13Gt/Gt spermatocytes progress to pachynema with no gross SC abnormalities, and oocytes were eliminated soon after birth (a characteristic of DNA repair mutants [13]), this suggested that unrepaired DSBs are responsible for eventual meiotic arrest and elimination.


Mouse pachytene checkpoint 2 (trip13) is required for completing meiotic recombination but not synapsis.

Li XC, Li X, Schimenti JC - PLoS Genet. (2007)

Immunohistochemical Analysis of Pachytene Spermatocyte ChromosomesSurface-spread chromosomes were immunolabeled with the indicated antibodies and fluorophores. As indicated in the upper right of each panel, cells were from wild type (WT, either +/+ or Trip13Gt/+) or Trip13Gt/Gt (Mut). There were no differences seen between heterozygotes and +/+ spermatocytes.(A) A mutant pachytene nucleus with full synapsis. Areas of SYCP1/SYCP3 colabeling are yellow.(B–E) Spermatocytes nucleus from 17.5 d postpartum mutant. Asynapsed chromosomes or regions of chromosomes are indicated by white and yellow arrows, respectively. Unlike the normal distribution in wild-type pachytene spermatocytes (C), BLM foci are present on synapsed pachytene chromosomes in the mutant (D). RAD51 foci, which are abundant earlier in prophase, disappear from autosomes in wild-type pachytene nuclei (E) and the bulk of staining is over the XY body (arrow).(F) RAD51 persists on the synapsed mutant chromosomes (arrows).(G) H2AX phosphorylation is restricted to the XY body in WT.(H) In addition to a large area of γH2AX staining (arrow) over the XY body, there is extensive autosomal H2AX phosphorylation (arrows).(I, J) Note that in wild-type pachytene spermatocytes, TOPBP1 is present only over the XY body (yellow arrow). In the mutant (J), an arrow denotes one area of intensive staining that may be over the sex chromosomes, but many other chromosome cores are positively stained.(K, L) RPA persists along synapsed cores in the mutant, not WT.(M, N) Arrows indicate examples of MLH3 foci on SCs.(O) In WT late pachytene spermatocytes, RAD51 is present only at background levels.(P) As in (F), extensive RAD51 staining delineates SCs in mutant pachytene nuclei (indicated by white arcs). MLH1 foci colocalize with these tracts (arrows) at the typical 1–2 foci per chromosome as in (M).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1941754&req=5

pgen-0030130-g004: Immunohistochemical Analysis of Pachytene Spermatocyte ChromosomesSurface-spread chromosomes were immunolabeled with the indicated antibodies and fluorophores. As indicated in the upper right of each panel, cells were from wild type (WT, either +/+ or Trip13Gt/+) or Trip13Gt/Gt (Mut). There were no differences seen between heterozygotes and +/+ spermatocytes.(A) A mutant pachytene nucleus with full synapsis. Areas of SYCP1/SYCP3 colabeling are yellow.(B–E) Spermatocytes nucleus from 17.5 d postpartum mutant. Asynapsed chromosomes or regions of chromosomes are indicated by white and yellow arrows, respectively. Unlike the normal distribution in wild-type pachytene spermatocytes (C), BLM foci are present on synapsed pachytene chromosomes in the mutant (D). RAD51 foci, which are abundant earlier in prophase, disappear from autosomes in wild-type pachytene nuclei (E) and the bulk of staining is over the XY body (arrow).(F) RAD51 persists on the synapsed mutant chromosomes (arrows).(G) H2AX phosphorylation is restricted to the XY body in WT.(H) In addition to a large area of γH2AX staining (arrow) over the XY body, there is extensive autosomal H2AX phosphorylation (arrows).(I, J) Note that in wild-type pachytene spermatocytes, TOPBP1 is present only over the XY body (yellow arrow). In the mutant (J), an arrow denotes one area of intensive staining that may be over the sex chromosomes, but many other chromosome cores are positively stained.(K, L) RPA persists along synapsed cores in the mutant, not WT.(M, N) Arrows indicate examples of MLH3 foci on SCs.(O) In WT late pachytene spermatocytes, RAD51 is present only at background levels.(P) As in (F), extensive RAD51 staining delineates SCs in mutant pachytene nuclei (indicated by white arcs). MLH1 foci colocalize with these tracts (arrows) at the typical 1–2 foci per chromosome as in (M).
Mentions: To better characterize the degree of meiotic progression in Trip13Gt/Gt spermatocytes, we immunostained chromosome spreads for SYCP3 and SYCP1, components of the axial/lateral elements and transverse filaments, respectively, of the synaptonemal complex (SC). Pachytene spermatocyte nuclei from postpubertal mutant testes could assemble normal SC cores and exhibited full synapsis of chromosomes as judged by colabeling of SYCP1 and SYCP3 along the full lengths of all autosomes (Figure 4A). Additionally, the X and Y chromosomes were normally synapsed at their pseudoautosomal region. More prepubertal (17.5 d postpartum) mutant spermatocytes contained asynaptic or terminally asynapsed chromosomes than age-matched controls (62.5% versus 25%, respectively; Figure 4B). We attribute this to a delay in the first wave of postnatal spermatogenesis (Figure 2D and 2E), likely related to systemic developmental retardation (Figure 2A and 2B). Nevertheless, since Trip13Gt/Gt spermatocytes progress to pachynema with no gross SC abnormalities, and oocytes were eliminated soon after birth (a characteristic of DNA repair mutants [13]), this suggested that unrepaired DSBs are responsible for eventual meiotic arrest and elimination.

Bottom Line: The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA.These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes.Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers.

View Article: PubMed Central - PubMed

Affiliation: Cornell University, College of Veterinary Medicine, Department of Biomedical Sciences, Ithaca, New York, United States of America.

ABSTRACT
In mammalian meiosis, homologous chromosome synapsis is coupled with recombination. As in most eukaryotes, mammalian meiocytes have checkpoints that monitor the fidelity of these processes. We report that the mouse ortholog (Trip13) of pachytene checkpoint 2 (PCH2), an essential component of the synapsis checkpoint in Saccharomyces cerevisiae and Caenorhabditis elegans, is required for completion of meiosis in both sexes. TRIP13-deficient mice exhibit spermatocyte death in pachynema and loss of oocytes around birth. The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA. These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes. Double mutant analysis demonstrated that the recombination and synapsis genes Spo11, Mei1, Rec8, and Dmc1 are all epistatic to Trip13, suggesting that TRIP13 does not have meiotic checkpoint function in mice. Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers. To our knowledge, this is the first model to separate recombination defects from asynapsis in mammalian meiosis, and provides the first evidence that unrepaired DNA damage alone can trigger the pachytene checkpoint response in mice.

Show MeSH
Related in: MedlinePlus