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First published online December 15, 2003
doi: 10.1242/10.1242/jcs.00851

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Localisation of histone macroH2A1.2 to the XY-body is not a response to the presence of asynapsed chromosome axes

¹ III. Department of Zoology-Developmental Biology, University of Göttingen, Humboldtallee 34A, 37073 Göttingen, Germany
² Laboratory of Developmental Genetics, National Institute for Medical Research, Mill Hill, London NM7 1AA, UK
³ Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

View larger version (67K): [in a new window]   Fig. 1. Localisation of histone macroH2A1.2 in surface spread spermatocytes (blue, DAPI staining; green, anti-macroH2A1.2 antibody; red, anti-SCP3 antibody; yellow/orange, co-localisation of anti-macroH2A1.2 and anti-SCP3 antibodies). (A-D) Histone macroH2A1.2 is preferentially concentrated over the forming XY-body of spermatocytes at early pachytene (lower cell) and at mid-pachytene (upper cell with DAPI dense XY-body - arrowhead in A) with a higher concentration at the X centromere (long arrow) and the synapsed PARs (short arrow) evident in the latter. (E-H) In late pachytene spermatocytes histone macroH2A1.2 is no longer concentrated throughout the XY-body, but remains concentrated on the X centromere and the PARs, and now also localises to the centromeric heterochromatin of autosomes. (Labels as for A-D.) Scale bars: 10 µm.

View larger version (90K): [in a new window]   Fig. 2. Co-localisation of macroH2A1.2 and HP1ß in spermatocytes. (blue, DAPI staining; green, anti-macroH2A1.2 antibody; red, anti-HP1ß antibody; yellow/orange, co-localisation of anti-macroH2A1.2 and anti-HP1ß antibodies). (A-D) Histone macroH2A1.2 begins to concentrate over the XY-chromatin in early pachytene spermatocytes (B, arrow) before a DAPI-dense XY-body is detectable (A). HP1ß is concentrated in two foci within the XY-body (C, long and short arrows); these foci are presumably associated with the X centromere and synapsed PARs (Turner et al., 2001). The arrowheads in B mark the filamentous structures detected by macroH2A1.2 antibodies. (E-H) In mid/late pachytene spermatocytes (with DAPI dense XY bodies, arrowhead in E) histone macroH2A1.2 and HP1ß are now both concentrated throughout the XY-body, with one particularly dense focus, presumably associated with the X centromere (long arrow). Both proteins are also concentrated at the centromeric heterochromatin of autosomes. The XY-body-associated macroH2A1.2-positive/HP1ß-negative focus (short arrow) is undoubtedly the Y centromere (Turner et al., 2001). (I-L) In diplotene spermatocytes (with an internalising XY-body, arrowhead in I), macroH2A1.2 and HP1ß remain concentrated at autosomal centromeres. However, the XY-body-located staining pattern is now quite distinct, with the concentration of macroH2A1.2 restricted to the X and Y centromeres and the PAR focus (arrows in J), whereas HP1ß is uniformly concentrated throughout the XY-body. (Note: a DAPI dense sperm head is apparent in I.) Scale bars: 10 µm.

View larger version (109K): [in a new window]   Fig. 3. Histone macroH2A1.2 is not concentrated in X and Y chromatin of early pachytene XY oocytes. (Blue, DAPI; red, anti-SCP3 in A-D and anti-XLR in E-P; green, anti-macroH2A1.2 antibody; yellow/orange, co-localisation of anti-macroH2A1.2 with anti-SCP3 or anti-XLR; white, CREST antibody, which marks the centromeres). (A,B) In spread oocytes from XX mice, macroH2A1.2 is concentrated at the centromeric heterochromatin of all bivalents as well as at the non-centromeric end of one bivalent (arrow); this bivalent must be the X which has a PAR focus (Turner et al., 2001). (C,D) In this spread oocyte from an XYTdym1 mouse in which the X and Y are synapsed, the PAR focus of macroH2A1.2 is clearly visible (arrow). The thin asynapsed non-PAR axes of the X and Y are also distinguishable, but only the centromeric region of the X has a high concentration of histone macroH2A1.2. (E-H) In XX early pachytene oocyte squashes, anti-XLR antibody produces a granular but fairly uniform staining thoughout the nucleus. MacroH2A1.2, however, is concentrated in the regions where the centromeres are clustered. (I-L) In early pachytene XY oocytes, anti-XLR antibody now concentrates on the chromatin of the asynapsed X (arrow). (M-P) In late pachytene XY oocytes, macroH2A1.2 preferentially stains the X centromere (arrow in O) and the PAR focus. Arrow in N indicates the chromatin of asynapsed X. Scale bar: (A-D) 21 µm; (E-P) 7 µm.

View larger version (79K): [in a new window]   Fig. 4. Condensation of gonosomal chromatin starts at zygotene/pachytene transition in spread spermatocytes (blue, DAPI; red, anti-SCP3; green, anti-histone H4). At leptotene (not shown) and zygotene (A-D) histone H4 showed uniform nuclear distribution. At zygotene/pachytene transition (E-H) histone H4 is slightly more concentrated in the forming XY-body located at the nuclear periphery. At mid-pachytene (I-L) and diplotene (not shown) the XY-bodies have a higher histone H4 concentration than the autosomal chromatin. Scale bars: 10 µm.

View larger version (54K): [in a new window]   Fig. 5. Histone macroH2A1.2 begins to concentrate in the XY-body at early pachytene of spermatocytes (blue, DAPI; green, anti-gammaH2AX; red, anti-macroH2A1.2). The gammaH2AX antibody stains the X and Y chromatin before it condenses at late zygotene (AD) where a `tadpole-shaped' structure is visible. At this stage no detectable concentration of macroH2A1.2 could be found. However, once condensation has started at early pachytene (E-H) histone macroH2A1.2 begins to appear over the XY-body. Scale bars: 25 µm.

View larger version (20K): [in a new window]   Fig. 6. Spatial and temporal patterns of histone macroH2A1.2 and HP1ß localisation during prophase progression in male meiosis. The gonosome condensation describes the events as marked by anti-histone H4 and anti-H2AX staining but a DAPI dense XY-body first become apparent at mid-pachytene.

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