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First published online January 10, 2007
doi: 10.1242/10.1242/dev.02747

Development 134, 579-590 (2007)
Published by The Company of Biologists 2007
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Mammalian Polycomb Scmh1 mediates exclusion of Polycomb complexes from the XY body in the pachytene spermatocytes

Yuki Takada1, Kyo-ichi Isono1, Jun Shinga1, James M. A. Turner2, Hiroshi Kitamura1, Osamu Ohara1, Gen Watanabe3, Prim B. Singh4, Takehiko Kamijo5, Thomas Jenuwein6, Paul S. Burgoyne2 and Haruhiko Koseki1,*

1 RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro, Tsurumi-ku, Yokohama 230-0045, Japan.
2 Division of Stem Cell Research and Developmental Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
3 Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan.
4 Nuclear Reprogramming Laboratory, Division of Gene Expression and Development, Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, UK.
5 Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Nagano 390-8621, Japan.
6 Research Institute of Molecular Pathology, The Vienna Biocenter, Dr Bohrgasse 7, A-1030 Vienna, Austria.


Figure 1
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  		Fig. 1. Localization of Scmh1 in the adult testes and spermatocytes. (Aa) In situ hybridization using antisense probe. Stages of seminiferous tubules are given. (Ab) Higher magnification view of seminiferous tubule at stage VII shown in a. Arrows and arrowheads indicate pachytene spermatocytes and round spermatids, respectively. (Ac) Control slides using sense probe. (Ba) Immunohistochemical localization of Scmh1 of wild-type testes. (Bb) Higher magnification view of seminiferous tubule shown in a. Arrows and arrowheads indicate pachytene spermatocytes and round spermatids, respectively. (C) Immunocytochemical detection of Scmh1 gene products from zygotene to pachytene stage spermatocytes, which were prepared from day 18 pp wild-type testes. (Ca-Cd) Spermatocyte spreads were substaged into early (a) and late (b) zygotene and early (c) and late (d) pachytene stages based on anti-Scp3 (red) immunostaining and morphology. Scmh1 (green) was localized in the nuclei at each stage, but was mostly excluded from the X and Y chromosome territory at late pachytene stage, as indicated by dotted lines. (Ce) Reciprocal subnuclear localization of Scmh1 and {gamma}H2A.X indicated exclusion of Scmh1 from the XY body. The XY body is indicated by dotted lines. (Cf) Reciprocal subnuclear localization of Scmh1 and dimethylated H3-K9 indicated exclusion of Scmh1 from the XY body. The XY body is indicated by dotted lines.

 

Figure 2
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  		Fig. 2. Expression pattern of other PcG proteins and trimethylated H3-K27 at pachytene stage spermatocytes. (A) Immunocytochemical detection of Prc1 components in pachytene spermatocytes. (Aa,Ab) Phc1 (a) and Phc2 (b) were excluded from the XY body demarcated by extensive accumulation of {gamma}H2A.X. (Ac-Ae) Bmi1 (c), Rnf110 (d) and Cbx2 (e) were excluded from the XY body demarcated by extensive accumulation of uH2A. The XY body is indicated by dotted lines. (Af) Reciprocal subnuclear localization of Phc2 and dimethylated H3-K9 indicated exclusion of Scmh1 from the XY body. (B) Immunocytochemical detection of trimethylated H3-K27 from late zygotene to pachytene stage spermatocytes. Spermatocytes were immunostained by using anti-trimethylated H3-K27 (red) and anti-SCP3 (green). The X and Y chromosome territory is indicated by dotted lines.

 

Figure 3
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  		Fig. 3. Testicular abnormalities in Scmh1-/- mice. (A) Cross-sections of testes from day 35 pp wild-type (Aa) and Scmh1-/- (Ab) mice. Sections were stained with Hematoxylin and Eosin (HE). (B) The frequency of Scmh1-/- mice in which seminiferous tubules were morphologically affected during first-wave spermatogenesis. Days after birth are shown. At each age, more than ten mutants were examined. Mutants over 8 weeks of age were collected and indicated as adults (Ad). (C) Increased apoptotic spermatocytes in Scmh1-/- testes. (Ca-Cc) Incidence of apoptosis in wild-type testes at day 7, 15 and 19 pp. (Cd-Cf) Incidence of apoptosis in Scmh1-/- testes at day 7, 15 and 19 pp. (Cg,Ch) Higher magnification views of individual seminiferous tubules shown in e and f. Outline of seminiferous tubules are indicated by dotted lines. (D) The expression of stage-specific markers during spermatogenesis in wild-type and unaffected and affected Scmh1-/- testes at day 35 pp, as revealed by semi-quantitative RT-PCR. ß-actin was used as a standard to verify the equal amounts of cDNA. Primers used in each reaction are shown in Table 1. Scale bars: 100 µm in A,B,Ca-Cf; 10 µm in Cg,Ch.

 

Figure 4
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  		Fig. 4. Significant reduction of late pachytene spermatocytes in Scmh1-/- testes. (A) Reduction of late pachytene spermatocytes in spermatocyte spread prepared from day 18 pp Scmh1-/- testes in comparison with wild type. Spermatocytes were immunostained using anti-Scp3 (red) and uH2A (green). (Left) Wild-type spermatocytes, in which uH2A is enriched at the XY body, were examined at early or late pachytene stage, based on Scp3 immunostaining and morphology. (Right) Frequency of early and late pachytene spermatocytes was compared between the wild-type and Scmh1-/- testes. (B) Localization of Mlh1 in spermatocytes. (Ba) Spermatocytes of wild type were immunostained by using anti-Scp3 (red) and Mlh1 (green). (Bb) Higher magnification view of chromosomes shown in a. (Bc) Localization of Mlh1 in Scmh1-/- mutant testes. (Bd) Higher magnification view of chromosomes shown in c. Arrows in b and d indicate Mlh1 foci.

 

Figure 5
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  		Fig. 5. Altered chromatin modifications at the XY body in Scmh1-/- spermatocytes. (A) Immunostaining for dimethylated H3-K9, monomethylated H3-K9, phosphorylated RNA pol II, {gamma}H2A.X and uH2A in the spermatocyte spread. (Aa,Ab) Frequency of spermatocytes, in which dimethylated (a) or monomethylated (b) H3-K9 was enriched at the XY body demarcated by uH2A accumulation, was compared (left) and results were summarized (right). (Ac,Ad) Frequency of spermatocytes, in which phosphorylated RNA pol II (c) or acetylated H3-K9 (d) were excluded from the XY body, was compared (left) and results were summarized (right). (Ba,Bb) Frequency of spermatocytes, in which Phc1 (a) or Phc2 (b) were excluded from the XY body, was compared (left) and results were summarized (right). (Bc,Bd) Frequency of spermatocytes, in which trimethylated H3-K27 were excluded from the XY body was compared. Scp3 was used to substage the spermatocytes. (C) Frequency of spermatocytes, in which monomethylated H3-K4 (Ca) and dimethylated H4-K20 (Cb) were accumulated on the XY body, was compared (left) and results were summarized (right).

 

Figure 6
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  		Fig. 6. Restoration of spermatogenic defects in Scmh1-/- testes by Phc2 mutation. (A) Restoration of morphological defects in Scmh1-/- testes by Phc2 mutation. (B) Restoration of fertility in Scmh1-/-;Phc2-/- mice. Results from ten mice with respective genotypes were summarized. (C) Significant reduction of apoptotic outbursts in Scmh1-/-;Phc2-/- testes compared with Scmh1-/- single mutants. (Left) Incidence of apoptosis was examined in wild-type, Scmh1-/- and Scmh1-/-;Phc2-/- testes at day 19 pp by TUNEL staining. (Right) Three hundred seminiferous tubules derived from five mice with respective genotypes were analyzed for the presence of TUNEL-positive cells and the results were summarized. (D) Restoration of spermatocytes, in which dimethylated H3-K9 was enriched at the XY body in Scmh1-/-;Phc2-/- testes (left). Frequency of spermatocytes, in which dimethylated H3-K9 was accumulated on the XY body, was summarized (right). (E,F) Frequency of spermatocytes, in which monomethylated H3-K9 was enriched at (E) and phosphorylated pol II was excluded from (F) the XY body in Scmh1-/-;Phc2-/- testes was compared.

 

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© The Company of Biologists Ltd 2007