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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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Metaphase checkpoint response in monosomic and disomic MC+ spermatogenesis. (A and B) IF staining for phosphohistone H3 (pH3, red signal) combined with TUNEL labeling (green) on frozen testes sections of a disomic mouse. XII denotes a part of a seminiferous tubule at stage XII. (Bi) An apoptotic metaphase positive for TUNEL (green) and pH3 labeling (red); (ii) green channel, (iii) red channel, (iv) color inverted, grayscale DAPI image. (C) Table of the mean number of apoptotic nuclei detected in stage XII tubules of two WT, four monosomic (1MC+-64, -66, -73 and -7), and four disomic testes (2MC+-4, -6, -13 and -16). Apoptotic cell death of metaphase cells (≥70% of all apoptotic cells) was increased in disomic as well as monosomic MC+ spermatogenesis. (Di) TUNEL-labeled (green) apoptotic MI that displays a precociously separated MC bivalent (red, arrowheads). One homologue has lost sister cohesion (bottom arrowheads). (Ei) TUNEL-positive apoptotic MI (red) with an MC (arrowhead) off the MI plate. (Aii, Bii, Dii, and Eii) Corresponding, color-inverted, grayscale DAPI images.
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fig7: Metaphase checkpoint response in monosomic and disomic MC+ spermatogenesis. (A and B) IF staining for phosphohistone H3 (pH3, red signal) combined with TUNEL labeling (green) on frozen testes sections of a disomic mouse. XII denotes a part of a seminiferous tubule at stage XII. (Bi) An apoptotic metaphase positive for TUNEL (green) and pH3 labeling (red); (ii) green channel, (iii) red channel, (iv) color inverted, grayscale DAPI image. (C) Table of the mean number of apoptotic nuclei detected in stage XII tubules of two WT, four monosomic (1MC+-64, -66, -73 and -7), and four disomic testes (2MC+-4, -6, -13 and -16). Apoptotic cell death of metaphase cells (≥70% of all apoptotic cells) was increased in disomic as well as monosomic MC+ spermatogenesis. (Di) TUNEL-labeled (green) apoptotic MI that displays a precociously separated MC bivalent (red, arrowheads). One homologue has lost sister cohesion (bottom arrowheads). (Ei) TUNEL-positive apoptotic MI (red) with an MC (arrowhead) off the MI plate. (Aii, Bii, Dii, and Eii) Corresponding, color-inverted, grayscale DAPI images.

Mentions: Homologous centromeres that fail to attach to opposite spindle poles, and hence the lack of tension during MI, may trigger the spindle assembly checkpoint in MI (Rieder et al., 1995; Woods et al., 1999; Shonn et al., 2000; Nicklas et al., 2001). Thus, we determined that in the behavior of MCs during mouse MI and MII in stage XII tubules of monosomic, disomic, and wild-type testes sections by TUNEL labeling and anti-phosphohistone H3 (pH3; a metaphase marker) immunofluorescence, meiotic divisions occur only at this stage of mouse spermatogenesis (Oakberg, 1956). Investigation of >10 stage XII tubule cross sections per testi of each of four monosomic, four disomic, and two wild-type testes revealed a 2–10-fold increase in the mean number of apoptotic cells in monosomic and disomic MC-containing stage XII tubules when compared with wild-type stage XII tubules (Fig. 7 C), with ≥70% of the apoptotic cells being metaphases (Fig. 7, A and B). Furthermore, apoptotic MIs generally revealed one or more MC signals off the MI plate (Fig. 7, D and E), which suggests that precocious separation of MC bivalents and/or noncongressed MCs triggered the spindle checkpoint. In agreement, it was found that MC kinetochores in prometaphase I and II spermatocytes displayed distinct signals of MAD2 (Chen et al., 1996) and BUBR1 (Chan et al., 1998; Fig. 8, A–D), proteins that are central to the spindle checkpoint (Cleveland et al., 2003).


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)

Metaphase checkpoint response in monosomic and disomic MC+ spermatogenesis. (A and B) IF staining for phosphohistone H3 (pH3, red signal) combined with TUNEL labeling (green) on frozen testes sections of a disomic mouse. XII denotes a part of a seminiferous tubule at stage XII. (Bi) An apoptotic metaphase positive for TUNEL (green) and pH3 labeling (red); (ii) green channel, (iii) red channel, (iv) color inverted, grayscale DAPI image. (C) Table of the mean number of apoptotic nuclei detected in stage XII tubules of two WT, four monosomic (1MC+-64, -66, -73 and -7), and four disomic testes (2MC+-4, -6, -13 and -16). Apoptotic cell death of metaphase cells (≥70% of all apoptotic cells) was increased in disomic as well as monosomic MC+ spermatogenesis. (Di) TUNEL-labeled (green) apoptotic MI that displays a precociously separated MC bivalent (red, arrowheads). One homologue has lost sister cohesion (bottom arrowheads). (Ei) TUNEL-positive apoptotic MI (red) with an MC (arrowhead) off the MI plate. (Aii, Bii, Dii, and Eii) Corresponding, color-inverted, grayscale DAPI images.
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fig7: Metaphase checkpoint response in monosomic and disomic MC+ spermatogenesis. (A and B) IF staining for phosphohistone H3 (pH3, red signal) combined with TUNEL labeling (green) on frozen testes sections of a disomic mouse. XII denotes a part of a seminiferous tubule at stage XII. (Bi) An apoptotic metaphase positive for TUNEL (green) and pH3 labeling (red); (ii) green channel, (iii) red channel, (iv) color inverted, grayscale DAPI image. (C) Table of the mean number of apoptotic nuclei detected in stage XII tubules of two WT, four monosomic (1MC+-64, -66, -73 and -7), and four disomic testes (2MC+-4, -6, -13 and -16). Apoptotic cell death of metaphase cells (≥70% of all apoptotic cells) was increased in disomic as well as monosomic MC+ spermatogenesis. (Di) TUNEL-labeled (green) apoptotic MI that displays a precociously separated MC bivalent (red, arrowheads). One homologue has lost sister cohesion (bottom arrowheads). (Ei) TUNEL-positive apoptotic MI (red) with an MC (arrowhead) off the MI plate. (Aii, Bii, Dii, and Eii) Corresponding, color-inverted, grayscale DAPI images.
Mentions: Homologous centromeres that fail to attach to opposite spindle poles, and hence the lack of tension during MI, may trigger the spindle assembly checkpoint in MI (Rieder et al., 1995; Woods et al., 1999; Shonn et al., 2000; Nicklas et al., 2001). Thus, we determined that in the behavior of MCs during mouse MI and MII in stage XII tubules of monosomic, disomic, and wild-type testes sections by TUNEL labeling and anti-phosphohistone H3 (pH3; a metaphase marker) immunofluorescence, meiotic divisions occur only at this stage of mouse spermatogenesis (Oakberg, 1956). Investigation of >10 stage XII tubule cross sections per testi of each of four monosomic, four disomic, and two wild-type testes revealed a 2–10-fold increase in the mean number of apoptotic cells in monosomic and disomic MC-containing stage XII tubules when compared with wild-type stage XII tubules (Fig. 7 C), with ≥70% of the apoptotic cells being metaphases (Fig. 7, A and B). Furthermore, apoptotic MIs generally revealed one or more MC signals off the MI plate (Fig. 7, D and E), which suggests that precocious separation of MC bivalents and/or noncongressed MCs triggered the spindle checkpoint. In agreement, it was found that MC kinetochores in prometaphase I and II spermatocytes displayed distinct signals of MAD2 (Chen et al., 1996) and BUBR1 (Chan et al., 1998; Fig. 8, A–D), proteins that are central to the spindle checkpoint (Cleveland et al., 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.

Show MeSH
Related in: MedlinePlus