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The dissection of meiotic chromosome movement in mice using an in vivo electroporation technique.

Shibuya H, Morimoto A, Watanabe Y - PLoS Genet. (2014)

Bottom Line: Further, during bouquet stage, telomeres are constrained near the MTOC, resulting in the transient suppression of telomere mobility and nuclear rotation.In contrast, actin regulates the oscillatory changes in nuclear shape.Our data provide the mechanical scheme for meiotic chromosome movement throughout prophase I in mammals.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chromosome Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan.

ABSTRACT
During meiosis, the rapid movement of telomeres along the nuclear envelope (NE) facilitates pairing/synapsis of homologous chromosomes. In mammals, the mechanical properties of chromosome movement and the cytoskeletal structures responsible for it remain poorly understood. Here, applying an in vivo electroporation (EP) technique in live mouse testis, we achieved the quick visualization of telomere, chromosome axis and microtubule organizing center (MTOC) movements. For the first time, we defined prophase sub-stages of live spermatocytes morphologically according to GFP-TRF1 and GFP-SCP3 signals. We show that rapid telomere movement and subsequent nuclear rotation persist from leptotene/zygotene to pachytene, and then decline in diplotene stage concomitant with the liberation of SUN1 from telomeres. Further, during bouquet stage, telomeres are constrained near the MTOC, resulting in the transient suppression of telomere mobility and nuclear rotation. MTs are responsible for these movements by forming cable-like structures on the NE, and, probably, by facilitating the rail-tacking movements of telomeres on the MT cables. In contrast, actin regulates the oscillatory changes in nuclear shape. Our data provide the mechanical scheme for meiotic chromosome movement throughout prophase I in mammals.

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Related in: MedlinePlus

Visualization of stage-specific chromosome movement in live spermatocytes.A, Time-lapse images (30 sec intervals) of pachytene spermatocytes (from 21 dpp male mouse testis) expressing GFP-TRF1 and GFP-SCP3 with or without nocodazole. Asterisks indicate identical heterochromatin. The trajectories indicate the movements of heterochromatin during the indicated time frame. Whole images are in S1, S2 Movies. B, Time-lapse images (14 sec intervals) of spermatocytes expressing GFP-TRF1 and GFP-SCP3. Leptotene/zygotene spermatocytes were from 14 dpp and pachytene to diplotene spermatocytes were from 21 dpp male mice. Asterisks indicate identical heterochromatin. The trajectories indicate the movements of heterochromatin during the indicated time frame. Whole images are in S5 Figure and S3, S4, S5 Movies. C, Equatorial images of time-lapse observations (7 sec intervals). Arrows indicate identical telomeres. Whole images are in S5 Figure and S3, S4, S5 Movies. D, Quantification of telomere velocities in various meiotic sub-stages. 5 telomeres were traced for 10 continuous time-points (7 sec intervals) for each cell (n = 5 cells), and the 3-dimensional velocities for each time-point interval are plotted. The bars represent average values. Statistical significance (TTEST, two-tailed) was assessed (*P<0.0005). E, Trajectories of a telomere (time point 0:00–3:30) overlaid on a projection of the final time points. F, Spermatocytes stained for SCP3 (blue), γ-Tubulin (red) and SUN1 (green). Bars, 5 µm.
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pgen-1004821-g003: Visualization of stage-specific chromosome movement in live spermatocytes.A, Time-lapse images (30 sec intervals) of pachytene spermatocytes (from 21 dpp male mouse testis) expressing GFP-TRF1 and GFP-SCP3 with or without nocodazole. Asterisks indicate identical heterochromatin. The trajectories indicate the movements of heterochromatin during the indicated time frame. Whole images are in S1, S2 Movies. B, Time-lapse images (14 sec intervals) of spermatocytes expressing GFP-TRF1 and GFP-SCP3. Leptotene/zygotene spermatocytes were from 14 dpp and pachytene to diplotene spermatocytes were from 21 dpp male mice. Asterisks indicate identical heterochromatin. The trajectories indicate the movements of heterochromatin during the indicated time frame. Whole images are in S5 Figure and S3, S4, S5 Movies. C, Equatorial images of time-lapse observations (7 sec intervals). Arrows indicate identical telomeres. Whole images are in S5 Figure and S3, S4, S5 Movies. D, Quantification of telomere velocities in various meiotic sub-stages. 5 telomeres were traced for 10 continuous time-points (7 sec intervals) for each cell (n = 5 cells), and the 3-dimensional velocities for each time-point interval are plotted. The bars represent average values. Statistical significance (TTEST, two-tailed) was assessed (*P<0.0005). E, Trajectories of a telomere (time point 0:00–3:30) overlaid on a projection of the final time points. F, Spermatocytes stained for SCP3 (blue), γ-Tubulin (red) and SUN1 (green). Bars, 5 µm.

Mentions: To dissect the meiotic chromosome movements in live spermatocytes, we subjected simultaneous EPs of GFP-Scp3 and GFP-Trf1 transgenes to wild type testis (20 dpp) to visualize chromosome axes and telomeres, respectively, as we demonstrated in the previous study [19]. At 24 hr after EPs, cell suspensions were diluted in Hoechst 33342-containing medium to visualize DNA, the cells were attached to the dishes with Cell-Tak (BD Bioscience) to avoid cell movements, and the cells were then subjected to time-lapse analysis. Consistent with our previous results, we can reproducibly observe the rapid movement of chromosomes within pachytene nuclei (Fig. 3A), that comprise two superimposed types of chromosome movement, random telomere movement and unidirectional rotation of the entire nucleus (rotation is highlighted with trajectories in Fig. 3A). Both of these movements were again significantly suppressed by the addition to the medium of nocodazole, an MT-destabilizing drug, (Fig. 3A, bottom, and S1, S2 Movies), confirming the previously established notion that meiotic chromosome movements in mammals depend totally on the MT polymerization activity, as is the case in S. pombe, C. elegans and perhaps some plant species, but not in S. cerevisiae[12], [30], [31], [32], [33].


The dissection of meiotic chromosome movement in mice using an in vivo electroporation technique.

Shibuya H, Morimoto A, Watanabe Y - PLoS Genet. (2014)

Visualization of stage-specific chromosome movement in live spermatocytes.A, Time-lapse images (30 sec intervals) of pachytene spermatocytes (from 21 dpp male mouse testis) expressing GFP-TRF1 and GFP-SCP3 with or without nocodazole. Asterisks indicate identical heterochromatin. The trajectories indicate the movements of heterochromatin during the indicated time frame. Whole images are in S1, S2 Movies. B, Time-lapse images (14 sec intervals) of spermatocytes expressing GFP-TRF1 and GFP-SCP3. Leptotene/zygotene spermatocytes were from 14 dpp and pachytene to diplotene spermatocytes were from 21 dpp male mice. Asterisks indicate identical heterochromatin. The trajectories indicate the movements of heterochromatin during the indicated time frame. Whole images are in S5 Figure and S3, S4, S5 Movies. C, Equatorial images of time-lapse observations (7 sec intervals). Arrows indicate identical telomeres. Whole images are in S5 Figure and S3, S4, S5 Movies. D, Quantification of telomere velocities in various meiotic sub-stages. 5 telomeres were traced for 10 continuous time-points (7 sec intervals) for each cell (n = 5 cells), and the 3-dimensional velocities for each time-point interval are plotted. The bars represent average values. Statistical significance (TTEST, two-tailed) was assessed (*P<0.0005). E, Trajectories of a telomere (time point 0:00–3:30) overlaid on a projection of the final time points. F, Spermatocytes stained for SCP3 (blue), γ-Tubulin (red) and SUN1 (green). Bars, 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4263375&req=5

pgen-1004821-g003: Visualization of stage-specific chromosome movement in live spermatocytes.A, Time-lapse images (30 sec intervals) of pachytene spermatocytes (from 21 dpp male mouse testis) expressing GFP-TRF1 and GFP-SCP3 with or without nocodazole. Asterisks indicate identical heterochromatin. The trajectories indicate the movements of heterochromatin during the indicated time frame. Whole images are in S1, S2 Movies. B, Time-lapse images (14 sec intervals) of spermatocytes expressing GFP-TRF1 and GFP-SCP3. Leptotene/zygotene spermatocytes were from 14 dpp and pachytene to diplotene spermatocytes were from 21 dpp male mice. Asterisks indicate identical heterochromatin. The trajectories indicate the movements of heterochromatin during the indicated time frame. Whole images are in S5 Figure and S3, S4, S5 Movies. C, Equatorial images of time-lapse observations (7 sec intervals). Arrows indicate identical telomeres. Whole images are in S5 Figure and S3, S4, S5 Movies. D, Quantification of telomere velocities in various meiotic sub-stages. 5 telomeres were traced for 10 continuous time-points (7 sec intervals) for each cell (n = 5 cells), and the 3-dimensional velocities for each time-point interval are plotted. The bars represent average values. Statistical significance (TTEST, two-tailed) was assessed (*P<0.0005). E, Trajectories of a telomere (time point 0:00–3:30) overlaid on a projection of the final time points. F, Spermatocytes stained for SCP3 (blue), γ-Tubulin (red) and SUN1 (green). Bars, 5 µm.
Mentions: To dissect the meiotic chromosome movements in live spermatocytes, we subjected simultaneous EPs of GFP-Scp3 and GFP-Trf1 transgenes to wild type testis (20 dpp) to visualize chromosome axes and telomeres, respectively, as we demonstrated in the previous study [19]. At 24 hr after EPs, cell suspensions were diluted in Hoechst 33342-containing medium to visualize DNA, the cells were attached to the dishes with Cell-Tak (BD Bioscience) to avoid cell movements, and the cells were then subjected to time-lapse analysis. Consistent with our previous results, we can reproducibly observe the rapid movement of chromosomes within pachytene nuclei (Fig. 3A), that comprise two superimposed types of chromosome movement, random telomere movement and unidirectional rotation of the entire nucleus (rotation is highlighted with trajectories in Fig. 3A). Both of these movements were again significantly suppressed by the addition to the medium of nocodazole, an MT-destabilizing drug, (Fig. 3A, bottom, and S1, S2 Movies), confirming the previously established notion that meiotic chromosome movements in mammals depend totally on the MT polymerization activity, as is the case in S. pombe, C. elegans and perhaps some plant species, but not in S. cerevisiae[12], [30], [31], [32], [33].

Bottom Line: Further, during bouquet stage, telomeres are constrained near the MTOC, resulting in the transient suppression of telomere mobility and nuclear rotation.In contrast, actin regulates the oscillatory changes in nuclear shape.Our data provide the mechanical scheme for meiotic chromosome movement throughout prophase I in mammals.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chromosome Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan.

ABSTRACT
During meiosis, the rapid movement of telomeres along the nuclear envelope (NE) facilitates pairing/synapsis of homologous chromosomes. In mammals, the mechanical properties of chromosome movement and the cytoskeletal structures responsible for it remain poorly understood. Here, applying an in vivo electroporation (EP) technique in live mouse testis, we achieved the quick visualization of telomere, chromosome axis and microtubule organizing center (MTOC) movements. For the first time, we defined prophase sub-stages of live spermatocytes morphologically according to GFP-TRF1 and GFP-SCP3 signals. We show that rapid telomere movement and subsequent nuclear rotation persist from leptotene/zygotene to pachytene, and then decline in diplotene stage concomitant with the liberation of SUN1 from telomeres. Further, during bouquet stage, telomeres are constrained near the MTOC, resulting in the transient suppression of telomere mobility and nuclear rotation. MTs are responsible for these movements by forming cable-like structures on the NE, and, probably, by facilitating the rail-tacking movements of telomeres on the MT cables. In contrast, actin regulates the oscillatory changes in nuclear shape. Our data provide the mechanical scheme for meiotic chromosome movement throughout prophase I in mammals.

Show MeSH
Related in: MedlinePlus