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Telomere-independent homologue pairing and checkpoint escape of accessory ring chromosomes in male mouse meiosis.

Voet T, Liebe B, Labaere C, Marynen P, Scherthan H - J. Cell Biol. (2003)

Bottom Line: Fluorescent in situ hybridization and three-dimensional fluorescence microscopy revealed that ring MCs did not participate in meiotic telomere clustering while MC homologues paired at the XY-body periphery.Unaligned MCs triggered the spindle checkpoint leading to apoptosis of metaphase cells.Our findings indicate a telomere-independent mechanism for pairing of mammalian MCs, illuminate escape routes to meiotic checkpoints, and give clues for genetic engineering of germ line-permissive chromosomal vectors.

View Article: PubMed Central - PubMed

Affiliation: Human Genome Laboratory, Department of Human Genetics, Flanders Interuniversity Institute for Biotechnology, University of Leuven, Belgium.

ABSTRACT
We analyzed transmission of a ring minichromosome (MC) through mouse spermatogenesis as a monosome and in the presence of a homologue. Mice, either monosomic or disomic for the MC, produced MC+ offspring. In the monosomic condition, most univalents underwent self-synapsis as indicated by STAG3, SCP3, and SCP1 deposition. Fluorescent in situ hybridization and three-dimensional fluorescence microscopy revealed that ring MCs did not participate in meiotic telomere clustering while MC homologues paired at the XY-body periphery. Self-synapsis of MC(s) and association with the XY-body likely allowed them to pass putative pachytene checkpoints. At metaphase I and II, MC kinetochores assembled MAD2 and BUBR1 spindle checkpoint proteins. Unaligned MCs triggered the spindle checkpoint leading to apoptosis of metaphase cells. Other MCs frequently associated with mouse pericentric heterochromatin, which may have allowed them to pass the spindle checkpoint. Our findings indicate a telomere-independent mechanism for pairing of mammalian MCs, illuminate escape routes to meiotic checkpoints, and give clues for genetic engineering of germ line-permissive chromosomal vectors.

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MC/XY body associations as revealed by 3D imaging. (A) Disomic pachytene nucleus (2MC+-6) shows the XY body (as defined by XMR IF staining, green) associated with two separated MCs (red signals, α-satellite FISH; arrowheads). The image represents the projection of 10 optical sections encompassing the XY body as obtained by 3D imaging. Bar, 2 μm. (Bi) Deconvoluted 3D volume image from image planes encompassing an XY body from another disomic nucleus. The two paired MCs (one large red signal) are partially embedded (indicated by the shadowed part of the MC signal) in the XY body chromatin (green). (Bii) Shows a projection of the images before volume imaging. (C) Volume reconstruction of a γ-H2ax–stained XY body (red) and associated MCs (green). The paired MCs appear partially embedded in the XY chromatin. (D) Disomic spermatocyte (DNA blue; partial detail) showing the MC bivalent signal distant from the γ-H2ax–stained XY body (red). The paired MCs lack γ-H2ax signals.
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fig5: MC/XY body associations as revealed by 3D imaging. (A) Disomic pachytene nucleus (2MC+-6) shows the XY body (as defined by XMR IF staining, green) associated with two separated MCs (red signals, α-satellite FISH; arrowheads). The image represents the projection of 10 optical sections encompassing the XY body as obtained by 3D imaging. Bar, 2 μm. (Bi) Deconvoluted 3D volume image from image planes encompassing an XY body from another disomic nucleus. The two paired MCs (one large red signal) are partially embedded (indicated by the shadowed part of the MC signal) in the XY body chromatin (green). (Bii) Shows a projection of the images before volume imaging. (C) Volume reconstruction of a γ-H2ax–stained XY body (red) and associated MCs (green). The paired MCs appear partially embedded in the XY chromatin. (D) Disomic spermatocyte (DNA blue; partial detail) showing the MC bivalent signal distant from the γ-H2ax–stained XY body (red). The paired MCs lack γ-H2ax signals.

Mentions: The data above are suggestive for the presence of a bouquet-independent mode of chromosome alignment and pairing, at least for MCs. Similar numbers of apoptotic spermatocytes in stage I–XI seminiferous tubules of monosomic, disomic, and wild-type mice (Table IV) indicate that MCs manage to bypass checkpoints that putatively monitor synapsis and/or DNA double-strand break (DSB) repair (Roeder and Bailis, 2000; Cohen and Pollard, 2001). A subcompartment of the spermatocyte I nucleus that tolerates the presence of asynapsed chromosome cores is the XY body (Jablonka and Lamb, 1988; Turner et al., 2000). Thus, we tested whether the univalent MC associates with the XY body by X chromosome–specific FISH (Disteche and Adler, 1990) and an MC-specific alphoid probe. In 87–100% of late pachytene/diplotene monosomic nuclei (≥40 nuclei analyzed in each of eight monosomic mice), the MC was associated with the morphologically distinct XY body. Furthermore, ∼90% of the MCs in disomic spermatocytes (≥29 nuclei analyzed in each of two disomic mice) displayed an MC/sex body association. To scrutinize this association in more detail, we stained the MC by FISH and the sex-body chromatin with the XMR antibody (Calenda et al., 1994). 3D wide-field light microscopy disclosed that the MCs were located at the periphery or were partially submerged in the XMR-marked sex body (Fig. 5, A and B) in 97 and 94% of 35 monosomic and 34 disomic pachytene nuclei. Similar results were obtained when we stained the MC by FISH and the XY body with antibodies to phosphorylated histone (γ)H2ax (Fig. 5 C). γ-H2ax IF staining first marks the onset of DSB repair at meiosis, and later is restricted to the XY body, where it is required for meiotic sex chromosome inactivation (Mahadevaiah et al., 2001; Fernandez-Capetillo et al., 2003). At leptotene/zygotene, MCs were covered with the abundant γ-H2ax signals (unpublished data), whereas in pachytene nuclei, the γ-H2ax–sex body signal and MC signal partially colocalized (Fig. 5 C). However, monosomic and disomic MCs that were not associated with the XY body did not display a γ-H2ax signal (Fig. 5 D). This suggests that MCs likely follow the mode of DSB repair of their local chromatin environment.


Telomere-independent homologue pairing and checkpoint escape of accessory ring chromosomes in male mouse meiosis.

Voet T, Liebe B, Labaere C, Marynen P, Scherthan H - J. Cell Biol. (2003)

MC/XY body associations as revealed by 3D imaging. (A) Disomic pachytene nucleus (2MC+-6) shows the XY body (as defined by XMR IF staining, green) associated with two separated MCs (red signals, α-satellite FISH; arrowheads). The image represents the projection of 10 optical sections encompassing the XY body as obtained by 3D imaging. Bar, 2 μm. (Bi) Deconvoluted 3D volume image from image planes encompassing an XY body from another disomic nucleus. The two paired MCs (one large red signal) are partially embedded (indicated by the shadowed part of the MC signal) in the XY body chromatin (green). (Bii) Shows a projection of the images before volume imaging. (C) Volume reconstruction of a γ-H2ax–stained XY body (red) and associated MCs (green). The paired MCs appear partially embedded in the XY chromatin. (D) Disomic spermatocyte (DNA blue; partial detail) showing the MC bivalent signal distant from the γ-H2ax–stained XY body (red). The paired MCs lack γ-H2ax signals.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172825&req=5

fig5: MC/XY body associations as revealed by 3D imaging. (A) Disomic pachytene nucleus (2MC+-6) shows the XY body (as defined by XMR IF staining, green) associated with two separated MCs (red signals, α-satellite FISH; arrowheads). The image represents the projection of 10 optical sections encompassing the XY body as obtained by 3D imaging. Bar, 2 μm. (Bi) Deconvoluted 3D volume image from image planes encompassing an XY body from another disomic nucleus. The two paired MCs (one large red signal) are partially embedded (indicated by the shadowed part of the MC signal) in the XY body chromatin (green). (Bii) Shows a projection of the images before volume imaging. (C) Volume reconstruction of a γ-H2ax–stained XY body (red) and associated MCs (green). The paired MCs appear partially embedded in the XY chromatin. (D) Disomic spermatocyte (DNA blue; partial detail) showing the MC bivalent signal distant from the γ-H2ax–stained XY body (red). The paired MCs lack γ-H2ax signals.
Mentions: The data above are suggestive for the presence of a bouquet-independent mode of chromosome alignment and pairing, at least for MCs. Similar numbers of apoptotic spermatocytes in stage I–XI seminiferous tubules of monosomic, disomic, and wild-type mice (Table IV) indicate that MCs manage to bypass checkpoints that putatively monitor synapsis and/or DNA double-strand break (DSB) repair (Roeder and Bailis, 2000; Cohen and Pollard, 2001). A subcompartment of the spermatocyte I nucleus that tolerates the presence of asynapsed chromosome cores is the XY body (Jablonka and Lamb, 1988; Turner et al., 2000). Thus, we tested whether the univalent MC associates with the XY body by X chromosome–specific FISH (Disteche and Adler, 1990) and an MC-specific alphoid probe. In 87–100% of late pachytene/diplotene monosomic nuclei (≥40 nuclei analyzed in each of eight monosomic mice), the MC was associated with the morphologically distinct XY body. Furthermore, ∼90% of the MCs in disomic spermatocytes (≥29 nuclei analyzed in each of two disomic mice) displayed an MC/sex body association. To scrutinize this association in more detail, we stained the MC by FISH and the sex-body chromatin with the XMR antibody (Calenda et al., 1994). 3D wide-field light microscopy disclosed that the MCs were located at the periphery or were partially submerged in the XMR-marked sex body (Fig. 5, A and B) in 97 and 94% of 35 monosomic and 34 disomic pachytene nuclei. Similar results were obtained when we stained the MC by FISH and the XY body with antibodies to phosphorylated histone (γ)H2ax (Fig. 5 C). γ-H2ax IF staining first marks the onset of DSB repair at meiosis, and later is restricted to the XY body, where it is required for meiotic sex chromosome inactivation (Mahadevaiah et al., 2001; Fernandez-Capetillo et al., 2003). At leptotene/zygotene, MCs were covered with the abundant γ-H2ax signals (unpublished data), whereas in pachytene nuclei, the γ-H2ax–sex body signal and MC signal partially colocalized (Fig. 5 C). However, monosomic and disomic MCs that were not associated with the XY body did not display a γ-H2ax signal (Fig. 5 D). This suggests that MCs likely follow the mode of DSB repair of their local chromatin environment.

Bottom Line: Fluorescent in situ hybridization and three-dimensional fluorescence microscopy revealed that ring MCs did not participate in meiotic telomere clustering while MC homologues paired at the XY-body periphery.Unaligned MCs triggered the spindle checkpoint leading to apoptosis of metaphase cells.Our findings indicate a telomere-independent mechanism for pairing of mammalian MCs, illuminate escape routes to meiotic checkpoints, and give clues for genetic engineering of germ line-permissive chromosomal vectors.

View Article: PubMed Central - PubMed

Affiliation: Human Genome Laboratory, Department of Human Genetics, Flanders Interuniversity Institute for Biotechnology, University of Leuven, Belgium.

ABSTRACT
We analyzed transmission of a ring minichromosome (MC) through mouse spermatogenesis as a monosome and in the presence of a homologue. Mice, either monosomic or disomic for the MC, produced MC+ offspring. In the monosomic condition, most univalents underwent self-synapsis as indicated by STAG3, SCP3, and SCP1 deposition. Fluorescent in situ hybridization and three-dimensional fluorescence microscopy revealed that ring MCs did not participate in meiotic telomere clustering while MC homologues paired at the XY-body periphery. Self-synapsis of MC(s) and association with the XY-body likely allowed them to pass putative pachytene checkpoints. At metaphase I and II, MC kinetochores assembled MAD2 and BUBR1 spindle checkpoint proteins. Unaligned MCs triggered the spindle checkpoint leading to apoptosis of metaphase cells. Other MCs frequently associated with mouse pericentric heterochromatin, which may have allowed them to pass the spindle checkpoint. Our findings indicate a telomere-independent mechanism for pairing of mammalian MCs, illuminate escape routes to meiotic checkpoints, and give clues for genetic engineering of germ line-permissive chromosomal vectors.

Show MeSH
Related in: MedlinePlus