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M31 and macroH2A1.2 colocalise at the pseudoautosomal region during mouse meiosis

James M. A. Turner1, Paul S. Burgoyne1,* and Prim B. Singh2

1 Laboratory of Developmental Genetics, National Institute for Medical Research, Mill Hill London, NW7 1AA, UK
2 Nuclear Reprogramming Laboratory, Division of Gene Expression and Development, Roslin Institute (Edinburgh), Midlothian, UK



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  		Fig. 1. Expression of M31 during XY male mouse meiosis (M31, green; SYCP3, red; DAPI, blue; long arrow, X chromosome; short arrow, Y chromosome; arrowhead, PAR). (A-D) Leptotene nucleus. (A) Axial element formation has just begun. (B) Same nucleus stained for M31, which is concentrated at the centromeric heterochromatin. (C) Superimposition of (A) and (B). (D) Same nucleus stained with DAPI, which highlights areas of centromeric heterochromatin. (E-H) Zygotene nucleus. (E) Synaptonemal complex (SC) formation has just begun. (F) Same nucleus stained for M31, which is concentrated at the centromeric heterochromatin. (G) Superimposition of (E) and (F). (H) Same nucleus stained with DAPI, which highlights areas of centromeric heterochromatin. (I-L) Mid-pachytene nucleus. (I) In contrast to the autosomes, which synapse over their entire length, the X and Y chromosomes show only limited synapsis that includes the PARs. (J) Same nucleus stained with M31. (K) Superimposition of (I) and (J). At this stage, the centromeric heterochromatin of the X chromosome (thin arrow in J) is very compact in comparison to that of the autosomes, and is correspondingly brightly stained with M31. M31 also assembles in the region of X-Y synapsis (open arrowhead in J,K). (L) Same nucleus stained with DAPI. Notice the compact heterochromatin of the X centromere. (M-P) Early diplotene nucleus; the sex chromosomes have partially desynapsed. (N) Same nucleus stained for M31. (O) Superimposition of (M) and (N). At this stage, autosomal centromeric heterochromatin is more compact and brightly stained with M31, whereas that of the X chromosome is barely stained. The focus in the region of X-Y synapsis (open arrowhead in N,O) decorates the very tip of the SC of the sex chromosomes. (P) Same nucleus stained with DAPI. (Q-T) Mid-diplotene nucleus, showing extensive autosomal desynapsis and marked contraction of the XY bivalent. (R) Same nucleus stained with M31. (S) Superimposition of (Q) and (R). At this stage, M31 associates with the sex body (open arrow in R). (T) Same nucleus stained with DAPI. (U-X) Metaphase I nucleus. At this stage, SYCP3 associates exclusively with the centromeric regions of each bivalent. (V) Same nucleus stained with M31. (W) Superimposition of (U) and (V). As well as centromeric heterochromatin, M31 continues to associate with the X and Y chromatin. (X) Same nucleus with X (green) and Y (red) paints. Bars, 10 µm.

 


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  		Fig. 2. Association of M31 with the PAR during male meiosis. (M31, green; SYCP3, red; CREST, which labels the functional centromeres, pseudocoloured white; long arrow, X chromosome; short arrow, Y chromosome; arrowhead, PAR). (A) Schematic representation of the X, Y, XSxra and XY*O chromosomes; circles represent centromeres. Sequences in the PAR are represented by the letters A-C. The XSxra chromosome possesses a complete PAR with some additional Y-chromosome short-arm-derived material attached distally. The XY* chromosome comprises a complete X and Y chromosome, with a compound PAR that is deleted for distal PAR sequence (in this case, ‘C’). The open circle represents the original Y centromere, which, in this context, is functionally inactive (Burgoyne et al., 1998). (B) High power late pachytene XY bivalent. M31 associates with the synapsed sex chromosome PARs; remnants are also present at the centromeric end. (C) Late pachytene XSxra chromosome, with the X centromeric end marked with CREST. M31 associates with the axial element at a less distal position than in the XY bivalent owing to the presence of most of the Y short arm distal to the PAR (compare with Y short arm in B). (D) Late pachytene XY* chromosome, with the X centromeric end again marked with CREST (the inactive Y centromere is negative for CREST). An M31 focus is never seen in the vicinity of the PAR on the XY* chromosome although remnants of M31 remain at the X centromere. Bars, 5 µm.

 


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  		Fig. 3. Expression of M31 during XYTdym1 and XX female meiosis. (green is M31 except in C and F, where it represents X chromosome paint; red is SYCP3 except in C, where it represents Y chromosome paint; centromeres, white; long arrow, X chromosome; short arrow, YTdym1 chromosome; arrowhead, PAR; open arrowhead, M31 PAR focus). (A) Mid-pachytene XYTdym1 nucleus showing the X and YTdym1 chromosomes, which rarely synapse and do not undergo MSCI or form a sex body. (B) M31 associates with centromeric heterochromatin, with the X and YTdym1 PARs (open arrowheads), and also with the whole chromatin of the YTdym1 chromosome. (C) Same nucleus following X and Y chromosome painting, which unambiguously identifies the sex chromosomes. (D) Mid-pachytene XX nucleus. The XX bivalent cannot be identified by morphology alone. (E) One of the bivalents shows M31 staining of the non-centromeric end of the synaptonemal complex (open arrowhead). (F) With sex chromosome painting, this chromosome is identified as the X chromosome. (G-L) Further examples of YTdym1 chromosomes from XYTdym1 pachytene oocytes, showing that M31 associates with the entirety of the YTdym1 chromatin. Bars, 5 µm.

 


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  		Fig. 4. Association of macroH2A1.2 with the XY bivalent during male meiosis (green is macroH2A1.2 except in I and K, where it represents X chromosome paint; red is COR1 except in I and K, where it represents Y chromosome paint; centromeres, white; DAPI, blue; long arrow, X chromosome; short arrow, Y chromosome; arrowhead, PAR; open arrowhead, macroH2A1.2 PAR focus). (A) Early diplotene XY bivalent. (B) Same nucleus stained for macroH2A1.2. (C) Superimposition of (A) and (B). MacroH2A1.2 forms a sharp focus at the PARs, and, in contrast to M31 at this stage, is abundant at the centromeric heterochromatin of the X and Y chromosomes. (D) Diakinesis XY bivalent. (E) Same nucleus stained for macroH2A1.2. (F) Superimposition of (D) and (E). MacroH2A1.2 continues to associate with the PAR and the heterochromatin of the X and Y chromosome. (G) Metaphase I XY bivalent. (H) Same nucleus stained for macroH2A1.2, which no longer preferentially associates the PAR, but continues to associate with the X and Y centromeric heterochromatin. (I) Same nucleus stained with X and Y chromosome paints, which discriminates these chromosomes from one another. (J) Round spermatids stained for macroH2A1.2, which forms a sharp focus (open arrows) in half of the cells analysed (inset DAPI stained image). (K) Same spermatids stained with X and Y chromosome paints, which demonstrate that the sharp focus is present in Y-bearing, but not X-bearing, spermatids (notice that this focus is preserved after the FISH procedure and appears yellow in this image). (L) The region of heterochromatin from the lowest spermatid stained with CREST and macroH2A1.2. The heterochromatin is due to the clustering of centromeres, and the macroH2A1.2 is closely associated with a single (Y) centromere. Bars, 5 µm.

 


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  		Fig. 5. M31 and macroH2A1.2 colocalise at the PAR (green is M31, red is COR1 in A and macroH2A1.2 in B and C; long arrow, X chromosome; short arrow, Y chromosome; open arrowhead, M31/macroH2A1.2 PAR focus). (A) Late pachytene XY bivalent showing M31 at the PAR and at the X-centromeric heterochromatin. (B) Same nucleus stained for macroH2A1.2, which localises to the PAR and to the X and Y centromeric heterochromatin. (C) Same nucleus stained for M31 and macroH2A1.2, which shows colocalisation at the PAR and at the centromeric heterochromatin of the X but not the Y chromosome. Bars, 5 µm.

 

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© The Company of Biologists Ltd 2001