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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

Optimization of EP procedures.A, Diagram of in vivo EP procedures for mouse testis highlighting the injection of a DNA solution into the rete testis and the application of an electric pulse. Detailed procedures are shown in S1, S2 Figures. B, Testes from mice of the indicated postnatal ages. The volume of DNA solution and the voltage of the electric pulse are indicated. C, Progression of spermatogenesis analyzed by flow cytometry. Peaks correspond roughly to mitotic cells (2n), spermatocytes (4n) and post-meiotic haploid cells (1n). D, The average GFP-SYCE3 expression efficiencies in testes from mice of the indicated ages. GFP positive cells were counted among the SCP1 positive cell populations (>1000 cells). The right graph indicates the distribution of prophase I sub-stages within testis cell suspensions from mice of the indicated ages. n = 248 (17 dpp), 330 (30 dpp) and 306 cells (60 dpp). The sub-stages are defined by SCP3 and SCP1 stainings. Lep, leptotene; Zyg, zygotene; Pac, pachytene; Dip, diplotene; M I, metaphase I. E, The average GFP-SYCE3 expression efficiencies after EP with various lag times between injection and electroporation examined as in D. Mice aged 17 dpp were used. F, The average GFP-SYCE3 expression efficiencies at various time points after EP. Mice aged 17 dpp were used. G, The localizations of GFP fusion proteins expressed by in vivo EP. Testis cell suspensions were stained with SCP3 (for MIS12, KASH5, SCP3 and RAD21L) or SCP1 (for SYCE3) in red, GFP in green and DAPI in blue. The graph shows the average GFP expression efficiencies as examined by immunofluorescence at the indicated DNA concentrations. GFP positive cells were counted among the SCP3 (for MIS12, KASH5, SCP3 and RAD21L) or SCP1 (for SYCE3) positive cell populations (>1000 cells). Mice aged 17 dpp were used. H, Western-blotting analysis of testis extracts after EP (17 dpp). Asterisks indicate the specific bands corresponding to each fusion protein. α-Tubulin and REC8 were the loading controls. I, Spermatocytes showing diffusive GFP-SCP3 signals stained for SCP3 (red), GFP (green) and DAPI (blue). Each column represents the average of three independent experiments; error bars represent S.E.M. Statistical significance (TTEST, two-tailed) was assessed (*P<0.05, **P<0.005, ***P<0.0005). Bars, 5 µm (unless otherwise indicated).
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pgen-1004821-g001: Optimization of EP procedures.A, Diagram of in vivo EP procedures for mouse testis highlighting the injection of a DNA solution into the rete testis and the application of an electric pulse. Detailed procedures are shown in S1, S2 Figures. B, Testes from mice of the indicated postnatal ages. The volume of DNA solution and the voltage of the electric pulse are indicated. C, Progression of spermatogenesis analyzed by flow cytometry. Peaks correspond roughly to mitotic cells (2n), spermatocytes (4n) and post-meiotic haploid cells (1n). D, The average GFP-SYCE3 expression efficiencies in testes from mice of the indicated ages. GFP positive cells were counted among the SCP1 positive cell populations (>1000 cells). The right graph indicates the distribution of prophase I sub-stages within testis cell suspensions from mice of the indicated ages. n = 248 (17 dpp), 330 (30 dpp) and 306 cells (60 dpp). The sub-stages are defined by SCP3 and SCP1 stainings. Lep, leptotene; Zyg, zygotene; Pac, pachytene; Dip, diplotene; M I, metaphase I. E, The average GFP-SYCE3 expression efficiencies after EP with various lag times between injection and electroporation examined as in D. Mice aged 17 dpp were used. F, The average GFP-SYCE3 expression efficiencies at various time points after EP. Mice aged 17 dpp were used. G, The localizations of GFP fusion proteins expressed by in vivo EP. Testis cell suspensions were stained with SCP3 (for MIS12, KASH5, SCP3 and RAD21L) or SCP1 (for SYCE3) in red, GFP in green and DAPI in blue. The graph shows the average GFP expression efficiencies as examined by immunofluorescence at the indicated DNA concentrations. GFP positive cells were counted among the SCP3 (for MIS12, KASH5, SCP3 and RAD21L) or SCP1 (for SYCE3) positive cell populations (>1000 cells). Mice aged 17 dpp were used. H, Western-blotting analysis of testis extracts after EP (17 dpp). Asterisks indicate the specific bands corresponding to each fusion protein. α-Tubulin and REC8 were the loading controls. I, Spermatocytes showing diffusive GFP-SCP3 signals stained for SCP3 (red), GFP (green) and DAPI (blue). Each column represents the average of three independent experiments; error bars represent S.E.M. Statistical significance (TTEST, two-tailed) was assessed (*P<0.05, **P<0.005, ***P<0.0005). Bars, 5 µm (unless otherwise indicated).

Mentions: We have recently established an efficient DNA EP method for live mouse testes (Fig. 1A; detailed procedures in S1, S2 Figures) [18], [19]. To optimize the EP efficiency, testes from mice of various ages, 17, 30 and 60 dpp (day post-partum), were subjected to EP of a Green Fluorescent Protein (GFP) expression vector harboring the full-length cDNA of SYCE3 (synaptonemal complex central element) (Fig. 1B–D) [21]. The majority of germ cells underwent the first wave of spermatogenesis at 17 dpp, and completed meiosis at 30 dpp and spermatogenesis at 60 dpp (Fig. 1C). We obtained reproducibly high EP efficiencies at 17 dpp (31%) and 30 dpp (22%), but not at 60 dpp (3.5%), although the profiles of the sub-stage distribution of meiotic prophase cells were similar at each point (Fig. 1D). We could detect GFP-SYCE3 expression not only in spermatocytes, but also in mitotic and haploid cells (spermatogonia, round and elongated spermatids) as seen in histological sections of testes after EPs (S3 Figure). Transgene expression was increased when the lag time between DNA injection and EP was lengthened to at least 60 min (Fig. 1E). Further, transgene expression was detected as early as 6 hr after EP, and had already peaked at 12 hr (Fig. 1F). Finally, we noticed that the efficiency of transgene expression estimated by immunofluorescence (IF) largely depends on the DNA concentration used for injection, with 5 µg being close to saturation (Fig. 1G). Thus, we optimized the in vivo EP method applicable for shot-term transgene expression into mouse testes.


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

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

Optimization of EP procedures.A, Diagram of in vivo EP procedures for mouse testis highlighting the injection of a DNA solution into the rete testis and the application of an electric pulse. Detailed procedures are shown in S1, S2 Figures. B, Testes from mice of the indicated postnatal ages. The volume of DNA solution and the voltage of the electric pulse are indicated. C, Progression of spermatogenesis analyzed by flow cytometry. Peaks correspond roughly to mitotic cells (2n), spermatocytes (4n) and post-meiotic haploid cells (1n). D, The average GFP-SYCE3 expression efficiencies in testes from mice of the indicated ages. GFP positive cells were counted among the SCP1 positive cell populations (>1000 cells). The right graph indicates the distribution of prophase I sub-stages within testis cell suspensions from mice of the indicated ages. n = 248 (17 dpp), 330 (30 dpp) and 306 cells (60 dpp). The sub-stages are defined by SCP3 and SCP1 stainings. Lep, leptotene; Zyg, zygotene; Pac, pachytene; Dip, diplotene; M I, metaphase I. E, The average GFP-SYCE3 expression efficiencies after EP with various lag times between injection and electroporation examined as in D. Mice aged 17 dpp were used. F, The average GFP-SYCE3 expression efficiencies at various time points after EP. Mice aged 17 dpp were used. G, The localizations of GFP fusion proteins expressed by in vivo EP. Testis cell suspensions were stained with SCP3 (for MIS12, KASH5, SCP3 and RAD21L) or SCP1 (for SYCE3) in red, GFP in green and DAPI in blue. The graph shows the average GFP expression efficiencies as examined by immunofluorescence at the indicated DNA concentrations. GFP positive cells were counted among the SCP3 (for MIS12, KASH5, SCP3 and RAD21L) or SCP1 (for SYCE3) positive cell populations (>1000 cells). Mice aged 17 dpp were used. H, Western-blotting analysis of testis extracts after EP (17 dpp). Asterisks indicate the specific bands corresponding to each fusion protein. α-Tubulin and REC8 were the loading controls. I, Spermatocytes showing diffusive GFP-SCP3 signals stained for SCP3 (red), GFP (green) and DAPI (blue). Each column represents the average of three independent experiments; error bars represent S.E.M. Statistical significance (TTEST, two-tailed) was assessed (*P<0.05, **P<0.005, ***P<0.0005). Bars, 5 µm (unless otherwise indicated).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004821-g001: Optimization of EP procedures.A, Diagram of in vivo EP procedures for mouse testis highlighting the injection of a DNA solution into the rete testis and the application of an electric pulse. Detailed procedures are shown in S1, S2 Figures. B, Testes from mice of the indicated postnatal ages. The volume of DNA solution and the voltage of the electric pulse are indicated. C, Progression of spermatogenesis analyzed by flow cytometry. Peaks correspond roughly to mitotic cells (2n), spermatocytes (4n) and post-meiotic haploid cells (1n). D, The average GFP-SYCE3 expression efficiencies in testes from mice of the indicated ages. GFP positive cells were counted among the SCP1 positive cell populations (>1000 cells). The right graph indicates the distribution of prophase I sub-stages within testis cell suspensions from mice of the indicated ages. n = 248 (17 dpp), 330 (30 dpp) and 306 cells (60 dpp). The sub-stages are defined by SCP3 and SCP1 stainings. Lep, leptotene; Zyg, zygotene; Pac, pachytene; Dip, diplotene; M I, metaphase I. E, The average GFP-SYCE3 expression efficiencies after EP with various lag times between injection and electroporation examined as in D. Mice aged 17 dpp were used. F, The average GFP-SYCE3 expression efficiencies at various time points after EP. Mice aged 17 dpp were used. G, The localizations of GFP fusion proteins expressed by in vivo EP. Testis cell suspensions were stained with SCP3 (for MIS12, KASH5, SCP3 and RAD21L) or SCP1 (for SYCE3) in red, GFP in green and DAPI in blue. The graph shows the average GFP expression efficiencies as examined by immunofluorescence at the indicated DNA concentrations. GFP positive cells were counted among the SCP3 (for MIS12, KASH5, SCP3 and RAD21L) or SCP1 (for SYCE3) positive cell populations (>1000 cells). Mice aged 17 dpp were used. H, Western-blotting analysis of testis extracts after EP (17 dpp). Asterisks indicate the specific bands corresponding to each fusion protein. α-Tubulin and REC8 were the loading controls. I, Spermatocytes showing diffusive GFP-SCP3 signals stained for SCP3 (red), GFP (green) and DAPI (blue). Each column represents the average of three independent experiments; error bars represent S.E.M. Statistical significance (TTEST, two-tailed) was assessed (*P<0.05, **P<0.005, ***P<0.0005). Bars, 5 µm (unless otherwise indicated).
Mentions: We have recently established an efficient DNA EP method for live mouse testes (Fig. 1A; detailed procedures in S1, S2 Figures) [18], [19]. To optimize the EP efficiency, testes from mice of various ages, 17, 30 and 60 dpp (day post-partum), were subjected to EP of a Green Fluorescent Protein (GFP) expression vector harboring the full-length cDNA of SYCE3 (synaptonemal complex central element) (Fig. 1B–D) [21]. The majority of germ cells underwent the first wave of spermatogenesis at 17 dpp, and completed meiosis at 30 dpp and spermatogenesis at 60 dpp (Fig. 1C). We obtained reproducibly high EP efficiencies at 17 dpp (31%) and 30 dpp (22%), but not at 60 dpp (3.5%), although the profiles of the sub-stage distribution of meiotic prophase cells were similar at each point (Fig. 1D). We could detect GFP-SYCE3 expression not only in spermatocytes, but also in mitotic and haploid cells (spermatogonia, round and elongated spermatids) as seen in histological sections of testes after EPs (S3 Figure). Transgene expression was increased when the lag time between DNA injection and EP was lengthened to at least 60 min (Fig. 1E). Further, transgene expression was detected as early as 6 hr after EP, and had already peaked at 12 hr (Fig. 1F). Finally, we noticed that the efficiency of transgene expression estimated by immunofluorescence (IF) largely depends on the DNA concentration used for injection, with 5 µg being close to saturation (Fig. 1G). Thus, we optimized the in vivo EP method applicable for shot-term transgene expression into mouse testes.

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