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First published online 11 December 2002
doi: 10.1242/jcs.00252

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The pairing of X and Y chromosomes during meiotic prophase in the marsupial species Thylamys elegans is maintained by a dense plate developed from their axial elements

¹ Programa de Genética Humana, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
² Unidad de Biología Celular, Departamento de Biología, Universidad Autónoma de Madrid, Madrid, Spain
³ Molecular Genetics Group, Agricultural University of Wageningen, The Netherlands

View larger version (118K): [in a new window]   Fig. 1. Double immunolabelling on spread T. elegans primary spermatocytes with anti-SCP3 (green) and anti-centromere CREST (red) sera. (A) Zygotene. The anti-SCP3 antibody labels axial structures that are polarised in a `bouquet' configuration. Paired regions (arrows) appear thicker than the unsynapsed autosomal AEs (arrowheads). The sex chromosomal AEs (X,Y) are recognisable in the bouquet area, and they are separated. At higher magnification (A') the irregular outline of the X AE can be seen. The AE of the Y chromosome (inset) is regular and the centromere is subterminal. (B-F) Pachytene. The bouquet arrangement is lost and the six autosomal pairs are fully synapsed. (B,B'). Early pachytene. The sex AEs are thickened and separated. The X AE loses its irregular outline. (C,C') Early-mid pachytene. Sex chromosomes start to make contact by means of fine threads labelled in green with anti-SCP3 (arrowheads in C'). (D,D') Mid pachytene. Sex chromosomal AEs (XY) become thinner. An anti-SCP3-labelled structure (arrow) with a horseshoe plate appearance is associated with the ends of the sex AEs. The AE of the Y chromosome is immersed in this plate. (E-E') Mid pachytene. The AE of the X chromosome loses its stiffness and folds. The AE of the Y chromosome is immersed in the anti-SCP3 labelled plate (arrow). (F) Late pachytene. The autosomal SCs are fragmented (arrowheads). (F') Enlargement of the XY body. The AE of the X chromosome is seen as a fine and fragmented thread tracing several loops. X and Y AEs are still associated with the anti-SCP3-labelled plate (arrow). Centromeres are still detectable but are fainter than in previous stages (arrowheads). Bars, 10 µm in (A-F); 2 µm in (A'-F').

View larger version (112K): [in a new window]   Fig. 2. Double immunolabelling on squashed primary spermatocytes with anti-SCP3 (green) and anti-centromere CREST (red) sera. (A-A') Single focal plane through an early pachytene spermatocyte. Sex chromosomes lie on the nuclear periphery. Five autosomal centromeres and the sex chromosomes centromeres are visible. X and Y short (Xp and Yp respectively) arms face each other. (B-B') Single focal plane of an early pachytene spermatocyte in which the sex chromosomes are in contact. Here the association is Xq-Yp and Xp-Yq (arrows). (C-C') A mid pachytene spermatocyte in which both X and Y long arms face each other (arrow). Bars, 5 µm in (A-C); 2 µm (A'-C').

View larger version (65K): [in a new window]   Fig. 3. Double immunolabelling on spread primary spermatocytes with anti-SCP1 (green) (A-C) and anti-SCP3 (red) (D-F) sera. (G-I) Enlargement of the sex chromosomes shown in A-F, where the anti-SCP1 and anti-SCP3 labelling have been superimposed and appear yellow where they colocalise. (A,D) Zygotene spermatocyte where synapsed autosomal regions are labelled with both SCP1 and SCP3 (arrows in A and D), whereas unsynapsed autosomal and sex (XY) AEs are only labelled with anti-SCP3 (arrowheads in D). (G) Enlargement of the sex chromosomes shown in A and D. The end of an autosomal SC appears yellow, whereas the unsynapsed sex chromosomal AEs, associated by one of their ends (arrowhead), only show red labelling corresponding to anti-SCP3. (B,E) Mid pachytene spermatocyte. Autosomes are fully synapsed as indicated by colocalisation of anti-SCP1 and anti-SCP3 labelling. (H) Detail of the sex chromosomes shown in B and E where they are associated by an anti-SCP3-positive structure (arrowhead) that is not labelled with anti-SCP1. (C,F) Late pachytene spermatocyte. Autosomes are fully synapsed. (I) Detail of the X and Y association shown in C and F. The anti-SCP3 positive plate (arrowhead) maintains the association between the sex AEs but no anti-SCP1 labelling is present. Bar, 10 µm in A-F; 2 µm in G-I.

View larger version (71K): [in a new window]   Fig. 4. Double immunolabelling on squashed primary spermatocytes with anti-SCP3 serum (green) and MPM-2 antibody (red). (A-B) Projection of three focal planes through a middle pachytene spermatocyte to reconstruct the whole XY body. (A) Anti-SCP3 labelling appears on the autosomal SCs (arrow) and the sex AEs (arrowhead). (B) MPM-2 labelling mainly appears on the sex chromosomal AEs (arrowhead) and as diffuse nuclear foci that do not correlate with autosomal SCs. At higher magnification (C,D) the outline of sex chromosomal AEs is clear. The chromosomes are connected at their ends by a fine thread that is recognised by both antibodies (arrowheads in C). The labelling with anti-SCP3 matches perfectly with that of MPM-2. (E,F) Projection of three focal planes through a late pachytene spermatocyte to reconstruct the whole XY body. (E) Autosomal SCs (arrow) are faintly labelled with anti-SCP3. The plate connecting sex chromosomal AEs (arrowhead) has increased in size and includes the four chromosome ends. (F) MPM-2 labelling only appears on the sex body. Higher magnification (G-H) shows details of the XY association. The intensity of labelling is so high on the plate that joins sex chromosomal AEs that it outshines the outline of chromosome AEs. This plate is larger than the region of attachment of AEs to the nuclear envelope. The two labels almost completely overlap. Note that in mid and late pachytene, sex chromosomes are located at the nuclear periphery. Bars, 5 µm in A,B,E,F; 1 µm in C,D,G,H.

View larger version (199K): [in a new window]   Fig. 5. (A-B) Electron micrographs of two consecutive sections of a mid-late pachytene spermatocyte. Sex chromatin has higher electron density than the rest of the chromatin. The sex body is associated with the nuclear envelope (NE). An axis (Ax) emanates from the NE showing an expansion at its base (arrowheads). A layer of electron-dense material corresponding to the dense plate (DP) (arrow) is present on the nucleoplasmic side of the NE. The DP has a granular structure and is continuous with the axis (Ax). The centrioles (Cen) and Golgi (G) are adjacent. (C-E) Electron micrographs of XY pairs from three different spread pachytene spermatocytes. (C) Early pachytene. The AEs are thickened. The AE of the X chromosome shows an irregular outline, with expansions at its ends and in the centromeric region (arrow). The AE of the Y chromosome is less distinct. (D) Mid pachytene. Both AEs are more elongated and thinner than in previous stages. An electron-dense material is present at the ends of AEs (arrowheads). It is composed of fine threads that extend from one extreme to the other. The centromere knob of the X chromosome is visible (arrow). (E) Late pachytene. The sex chromosomal AEs do not touch each other. An electron-dense material, the balloon (BL), mediates the association of AE ends. Bars, 0.5 µm in A,B; 1 µm in C,E.

View larger version (110K): [in a new window]   Fig. 6. Electron microscopic labelling with anti-SCP3 serum in pachytene spermatocyte sections. (A,B) Two serial sections of the same spermatocyte. The sex body (XY) is recognisable by its high electron density. Inside the sex body, an axial structure (Ax) is labelled with gold grains (arrow), so is the dense plate (DP) (arrowheads). (C,D) Higher magnification of two serial sections of another spermatocyte. Gold grains appear on the axial structure (Ax) (arrows) and on the dense plate (DP) (arrowheads). Bars 0.5 µm in A,B; 0.2 µm in C,D.

View larger version (49K): [in a new window]   Fig. 7. Schematic representation of sex chromosome (X, Y) structure and behaviour throughout pachytene. The upper row depicts sex chromosomes as seen in spread spermatocytes, either by electron microscopy or by labelling with anti-SCP3 serum or MPM-2. AEs are initially short and thick and do not touch each other (A). In later stages, AEs become thicker and more loosely organised, and dense material starts to be deposited at their ends (B). Eventually, this material includes the ends of both chromosomes, and the AEs appear as faint threads (C,D). The lower row shows the sex chromosomal AEs as seen in electron microscopy sections and in squashes labelled with the anti-SCP3 serum or MPM-2 antibody. Initially X and Y AEs lie apart from each other, showing a stiff appearance and an irregular outline (A'). Subsequently, sex chromosomes approach and form the sex body, revealed as a dense chromatin mass, and their AEs touch (B'). Thereafter, AEs start to loose their stiffness, and the material composing the AEs is deposited on the nuclear envelope (NE) while the label on the AEs decreases (C'). Finally, AEs get thinner and looped, and the dense plate (DP) forms, extending from the attachment plates of sex chromosomal AEs to the nuclear envelope. The DP extends until it includes the ends of both sex chromosomes (D').