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First published online 6 February 2003
doi: 10.1242/jcs.00296

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Microtubule distribution during meiosis I in flea-beetle [Alagoasa (Oedionychus)] spermatocytes: evidence for direct connections between unpaired sex chromosomes

¹ Biology Department, York University, Toronto, Ontario M3J 1P3, Canada
² Department of Biological Sciences, Mississippi State University, MS 39762, USA

View larger version (60K): [in a new window]   Fig. 1. Lysis of a living cell in metaphase I. Cells to be processed for immunofluorescence staining were first lysed in a microtubule stabilizing buffer, then fixed in 0.2% glutaraldehyde in phosphate-buffered saline. General cell morphology was preserved by this procedure. (A) Cell 5 minutes prior to lysis. (B) 50 seconds after perfusion of lysis buffer: the lysis buffer is beginning to reach the cell. (C) 1 minute after perfusion: lysis starting. (D) 6 minutes after perfusion: lysis is completed. Bar, 10 µm.

View larger version (41K): [in a new window]   Fig. 2. The cytoplasmic spindle. (A-C) Phase-contrast images of living cells in prophase, revealing spindle-shaped cytoplasmic alignments outside the nucleus. (A) Bar, 10 µm. (B,C) Bar, 5 µm. (D) Labelling of a prophase cell with antibodies to tubulin reveals a spindle-shaped array of microtubules in the cytoplasm. The nucleus is at the bottom of the frame. Bar, 10 µm. (E) Prophase cell triple labelled to reveal acetylated tubulin (green), tyrosinated tubulin (red) and chromosomes (acridine orange labelling, which appears in the green channel). A spindle-shaped structure containing unacetylated microtubules is evident. The nucleus is at the bottom of the frame. Bar, 10 µm.

View larger version (128K): [in a new window]   Fig. 3. Confocal serial section reconstructions of cells in early prometaphase I, showing kinetochore-microtubule associations of autosomes. (A) Prometaphase cell labelled with antibodies against tubulin. Bar, 10 µm. (B) Higher magnification image of cell in (A). The closed arrowhead points to a bivalent with associations to only one pole. Open arrowheads point to a bipolar connection made by a single half-bivalent. The sister half-bivalent has microtubule associations only to the top pole. The arrow points to a half-bivalent with bidirectional microtubule associations, only one of which points towards a pole. Bar, 5 µm. (C-E) Triple-labelled prometaphase cells, stained for acetylated tubulin (green), tyrosinated tubulin (red) and chromosomes (green). Co-localization of acetylated and tyrosinated tubulin would result in a yellow or orange image. (C) Arrowheads point to microtubule associations with a bivalent that has connections to both poles. The arrow points to a kinetochore fibre pointed away from the poles. Bar, 5 µm. (D) Different series of sections from the same cell shown in (C). The closed arrowhead points to a bivalent with attachments only to one pole: notice the two fibres coming from one half-bivalent, one from each sister chromatid. Bar, 5 µm. (E) Arrowheads point to bivalents that have reached the central spindle and established bipolar connections. Bar, 5 µm. (F,G) Stereo images of a prometaphase cell showing the arrangements of microtubules and chromosomes. Bar, 10 µm.

View larger version (125K): [in a new window]   Fig. 4. Confocal Z-series showing microtubule organization at different stages of meiosis I. (A-C) Mid-prometaphase cell labelled with antibodies against tubulin and with acridine orange to reveal the chromosomes. (B) and (C) are stereo images. (A) Bar, 5 µm. (B,C). Bar, 10 µm. (D) Late prometaphase cell stained for acetylated tubulin (left) and tyrosinated tubulin (right). The arrowheads indicate the kinetochore fibres of the two sex chromosomes. Bar, 10 µm. (E) Anaphase cell stained for acetylated tubulin (left) and tyrosinated tubulin (right). Bar, 10 µm.

View larger version (143K): [in a new window]   Fig. 5. Triple-labelled cells revealing microtubule-kinetochore associations of sex chromosomes. Acetylated tubulin and chromosomes are green, tyrosinated tubulin is red: colocalization results in yellow or orange images. (A,B) Prometaphase cells showing bidirectional associations (arrowheads). Bar, 5 µm. (C) Metaphase cell showing syntelic associations (arrowheads). Bar, 10 µm. (D) Metaphase cell showing semi-syntelic associations (arrowheads). Bar, 10 µm.

View larger version (37K): [in a new window]   Fig. 6. Graph showing the distribution of different types of sex-chromosome/microtubule associations at different stages of division. We scored 29 sex chromosomes in early prometaphase (EP), 14 sex chromosomes in mid prometaphase (MP), 25 sex chromosomes in late prometaphase (LP), 20 sex chromosomes in metaphase (M) and 26 sex chromosomes in anaphase (A). (When the number of chromosomes is uneven, one sex chromosome in the group could not be analysed.)

View larger version (137K): [in a new window]   Fig. 7. Confocal Z-series of triple-labelled cells revealing putative microtubule linkages between sex chromosomes (arrowheads). Acetylated tubulin and chromosomes are green, tyrosinated tubulin is red. Areas of colocalization are yellow or orange. (A) Anaphase cell. Bar, 5 µm. (B) Different sections of the same cell shown in (A). Bar, 5 µm. (C) Close up of metaphase cell from Fig. 5C, revealing putative connections between sex chromosomes that were initially classified as having syntelic fibres. Bar, 5 µm. (D) Late anaphase-I cell. Bar, 10 µm. [Fleabeetle spermatocytes are in cysts in the testes and sometimes the cysts survive the spreading procedure; the spindle partially seen in the upper left hand corner is the sister cell (in the cyst) to the one illustrated. Both are primary spermatocytes.] (E) Early anaphase-I cell. Bar, 5 µm. Panels D and E are of cells illustrated in different format (and composed of a different set of Z-series sections) in Fig. 4 of Forer and Wilson (Forer and Wilson, 2000). Only a subset of the original Z-series is shown here.

View larger version (14K): [in a new window]   Fig. 8. Graph of the average angle made between the sex-chromosome arms and their associated fibres ± standard deviation, at different stages of division. We scored 30 sex chromosomes in early prometaphase (EP), 13 sex chromosomes in mid prometaphase (MP), 25 sex chromosomes in late prometaphase (LP), 20 sex chromosomes in metaphase (M) and 26 sex chromosomes in anaphase (A). Using the Student's t test with =0.05, there is no statistical difference between the average angles at different stages from midprometaphase to anaphase. The average angle in early prometaphase is not statistically different from that for midprometaphase, but is different from the other stages. [When the number of chromosomes is uneven, one sex chromosome in the group could not be analysed.]

View larger version (32K): [in a new window]   Fig. 9. Plot of average interkinetochore distances for autosomes and sex chromosomes, kinetochore-to-pole distances and pole-to-pole distances at different stages of division, with standard deviations. To generate the autosomal data, five bivalent pairs per cell were measured and an average distance calculated for each cell. The average distance for data from all cells was then calculated from the individual cell averages. Sex-chromosome data are simply the averages of all data taken. We scored 15 cells in early prometaphase (EP), 7 cells in mid prometaphase (MP), 13 cells in late prometaphase (LP), 10 cells in metaphase (M) and 13 cells in anaphase (A).

View larger version (13K): [in a new window]   Fig. 10. (A) Plot of pole-to-pole distances (open circles) and of distances from the poles of sex-chromosome kinetochores (open and closed triangles, representing partner kinetochores) as a function of autosomal interkinetochore distance (abscissa), for which we used average distances as described in Fig. 9. The lines represent our interpretation: that the poles elongate early in anaphase but not later, and that the sex chromosomes do not move polewards (they remain a constant distance from the poles). (B) The same set of cells, with open circles representing pole-to-pole distances and crosses representing the average distances of the autosomal kinetochores from the spindle poles in these same cells. The lines indicate our interpretation: that autosomal separation during early anaphase is due to spindle elongation and separation later in anaphase is due to movement towards the pole.

View larger version (143K): [in a new window]   Fig. 11. Metaphase primary spermatocyte of Humbertiella, illustrating the resemblance to metaphase flea-beetle primary spermatocytes. Reproduced from Hughes-Schrader (1948) with permission from Springer Verlag (Hughes-Schrader, 1948).