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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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MCs locate remote from the telomere cluster in bouquet nuclei. (A and B) FISH with a (C3TA2)3 PNA telomere probe (green) and an α-satellite DNA MC probe (red; white arrowhead) on monosomic bouquet stage nuclei (1MC+-30; 12 d post partum). (A) A spermatocyte I with one MC located remote from the telomere cluster at the top of the nucleus (the latter faces the observer). (B) Spermatocyte with the MC located among the tightly clustered telomeres at the bouquet base (yellow arrowhead). Focal plane near the nuclear top. (C and D) Bouquet stage nuclei of a 12-d post partum disomic mouse (2MC+-7). (C) Two separate MCs (white arrowheads) located away from the bouquet base (yellow arrowhead). Focal plane at nuclear equator. (D) Spermatocyte I nucleus with relaxed telomere clustering and two paired MCs (white arrowheads) that create a single large MC signal in the nuclear interior below. Focal plane at the nuclear top. The bar in A represents 5 μm and applies to A–D. (E) Distribution frequencies of MCs with respect to the telomere cluster site. Two MCs were generally absent from the telomere cluster region.
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fig4: MCs locate remote from the telomere cluster in bouquet nuclei. (A and B) FISH with a (C3TA2)3 PNA telomere probe (green) and an α-satellite DNA MC probe (red; white arrowhead) on monosomic bouquet stage nuclei (1MC+-30; 12 d post partum). (A) A spermatocyte I with one MC located remote from the telomere cluster at the top of the nucleus (the latter faces the observer). (B) Spermatocyte with the MC located among the tightly clustered telomeres at the bouquet base (yellow arrowhead). Focal plane near the nuclear top. (C and D) Bouquet stage nuclei of a 12-d post partum disomic mouse (2MC+-7). (C) Two separate MCs (white arrowheads) located away from the bouquet base (yellow arrowhead). Focal plane at nuclear equator. (D) Spermatocyte I nucleus with relaxed telomere clustering and two paired MCs (white arrowheads) that create a single large MC signal in the nuclear interior below. Focal plane at the nuclear top. The bar in A represents 5 μm and applies to A–D. (E) Distribution frequencies of MCs with respect to the telomere cluster site. Two MCs were generally absent from the telomere cluster region.

Mentions: A potential mechanism to instigate meiotic chromosome pairing is meiotic telomere clustering or bouquet formation (Scherthan, 2001; Yamamoto and Hiraoka, 2001). Thus, we prepared three-dimensionally preserved nuclei from testes of monosomic and disomic MC+ mice 12 d post partum, which are moderately enriched in bouquet stage nuclei. Two-color FISH with a (T2AG3)n telomere and an MC-specific alphoid probe was used to identify bouquet nuclei with telomeres clustered in a limited region of the nuclear periphery, which also contain a few peripheral DAPI-bright heterochromatin masses (Scherthan et al., 1996). It was found that 97% of monosomic bouquet stage nuclei (n = 34) displayed the MC away from the bouquet base (Fig. 4, A and E). In the remaining nucleus, the MC was detected at the telomere cluster (Fig. 4, B and E). In all but one disomic bouquet nuclei, both MCs located away from the telomere–cluster site (n = 16; Fig. 4, C–E). Although the MCs failed to locate to the bouquet base, both MC signals coalesced in half of the nuclei analyzed. These data suggest that human ring minichromosomes that lack both functional telomeres and premeiotic homologue association still align and synapse with their homologous partner in mouse prophase I.


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)

MCs locate remote from the telomere cluster in bouquet nuclei. (A and B) FISH with a (C3TA2)3 PNA telomere probe (green) and an α-satellite DNA MC probe (red; white arrowhead) on monosomic bouquet stage nuclei (1MC+-30; 12 d post partum). (A) A spermatocyte I with one MC located remote from the telomere cluster at the top of the nucleus (the latter faces the observer). (B) Spermatocyte with the MC located among the tightly clustered telomeres at the bouquet base (yellow arrowhead). Focal plane near the nuclear top. (C and D) Bouquet stage nuclei of a 12-d post partum disomic mouse (2MC+-7). (C) Two separate MCs (white arrowheads) located away from the bouquet base (yellow arrowhead). Focal plane at nuclear equator. (D) Spermatocyte I nucleus with relaxed telomere clustering and two paired MCs (white arrowheads) that create a single large MC signal in the nuclear interior below. Focal plane at the nuclear top. The bar in A represents 5 μm and applies to A–D. (E) Distribution frequencies of MCs with respect to the telomere cluster site. Two MCs were generally absent from the telomere cluster region.
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Related In: Results  -  Collection

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fig4: MCs locate remote from the telomere cluster in bouquet nuclei. (A and B) FISH with a (C3TA2)3 PNA telomere probe (green) and an α-satellite DNA MC probe (red; white arrowhead) on monosomic bouquet stage nuclei (1MC+-30; 12 d post partum). (A) A spermatocyte I with one MC located remote from the telomere cluster at the top of the nucleus (the latter faces the observer). (B) Spermatocyte with the MC located among the tightly clustered telomeres at the bouquet base (yellow arrowhead). Focal plane near the nuclear top. (C and D) Bouquet stage nuclei of a 12-d post partum disomic mouse (2MC+-7). (C) Two separate MCs (white arrowheads) located away from the bouquet base (yellow arrowhead). Focal plane at nuclear equator. (D) Spermatocyte I nucleus with relaxed telomere clustering and two paired MCs (white arrowheads) that create a single large MC signal in the nuclear interior below. Focal plane at the nuclear top. The bar in A represents 5 μm and applies to A–D. (E) Distribution frequencies of MCs with respect to the telomere cluster site. Two MCs were generally absent from the telomere cluster region.
Mentions: A potential mechanism to instigate meiotic chromosome pairing is meiotic telomere clustering or bouquet formation (Scherthan, 2001; Yamamoto and Hiraoka, 2001). Thus, we prepared three-dimensionally preserved nuclei from testes of monosomic and disomic MC+ mice 12 d post partum, which are moderately enriched in bouquet stage nuclei. Two-color FISH with a (T2AG3)n telomere and an MC-specific alphoid probe was used to identify bouquet nuclei with telomeres clustered in a limited region of the nuclear periphery, which also contain a few peripheral DAPI-bright heterochromatin masses (Scherthan et al., 1996). It was found that 97% of monosomic bouquet stage nuclei (n = 34) displayed the MC away from the bouquet base (Fig. 4, A and E). In the remaining nucleus, the MC was detected at the telomere cluster (Fig. 4, B and E). In all but one disomic bouquet nuclei, both MCs located away from the telomere–cluster site (n = 16; Fig. 4, C–E). Although the MCs failed to locate to the bouquet base, both MC signals coalesced in half of the nuclei analyzed. These data suggest that human ring minichromosomes that lack both functional telomeres and premeiotic homologue association still align and synapse with their homologous partner in mouse prophase I.

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