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First published online 20 May 2003
doi: 10.1242/jcs.00495

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Temporally and spatially selective loss of Rec8 protein from meiotic chromosomes during mammalian meiosis

Jibak Lee^*,, Toshiharu Iwai, Takehiro Yokota and Masakane Yamashita

Laboratory of Molecular and Cellular Interactions, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan

View larger version (29K): [in a new window]   Fig. 1. Specific expression of Rec8 protein in meiosis-undergoing testis, as revealed by an anti-mouse Rec8 antibody. (A) The anti-mouse Rec8 antibody was raised in a rabbit against the bacterially expressed C-terminal fragment of mouse Rec8 protein. The antigenic C-terminal mouse Rec8 protein (C-terminal Rec8) and nuclear extracts from mouse testis (Testis nuclei) were subjected to western blotting with the anti-Rec8 antibody after pre-absorption of the antibody with (+) or without (-) the antigen. (B) Testis nuclear extracts were immunoprecipitated with anti-Rec8 antibody and treated with (+) or without (-) either alkaline PPase or -PPase. The immunoprecipitates were separated by SDS-PAGE and subjected to western blotting with anti-Rec8 antibody. (C) Nuclear extracts from various mouse tissues were subjected to western blotting with anti-SMC1, anti-SMC3, anti-Rec8 and anti-SCP3 antibodies. K, kidney; L, liver; T, testis. (D) Testis extracts from 0-, 1-, 2-, 3-, and 5-week-old mice were examined in western blotting with anti-SMC1, anti-SMC3, anti-Rec8, anti-SCP3 and anti-tubulin antibodies. Closed arrowheads indicate SMC1, SMC3, Rec8, SCP3 and tubulin protein in the respective blots, while an open arrowhead indicates SMC1ß protein.

View larger version (72K): [in a new window]   Fig. 2. Association of Rec8 with other cohesin proteins in the mouse. Testis nuclear extracts were immunoprecipitated with either of three antibodies, anti-Rec8, anti-SMC1 or anti-SMC3 antibody. The immunoprecipitates were subjected to SDS-PAGE followed by western blotting with the same antibodies plus anti-SCP3 antibody.

View larger version (163K): [in a new window]   Fig. 3. Coexpression of Rec8 protein with SCP3 protein in mouse testis. Frozen sections were incubated with either a mouse polyclonal anti-SCP3 antibody or rabbit polyclonal anti-Rec8 antibody, or both antibodies, and detected with Alexa 488 (green)-conjugated or Alexa 546 (red)-conjugated, anti-mouse or rabbit IgG secondary antibodies. DNA was stained with propidium iodide in single-labeled sections. Broken lines indicate the basement membrane of seminiferous tubules. Spermatogonia (sg) lie next to the basement membrane. Spermatocytes (sc) are larger cells and lie away from the basement membrane. Spermatids (st) are farther from the membrane and have a smaller nuclei. Arrows in lower panels indicate the cells in which Rec8 protein was expressed but in which SCP3 signal was hardly detected. Bar, 50 µm.

View larger version (99K): [in a new window]   Fig. 4. Localization of Rec8 protein on synaptonemal complexes during meiotic prophase. Nuclear spreads from testicular cells were prepared as described in Materials and Methods and subjected to immunofluorescent double labeling with anti-Rec8 and anti-SCP3 antibodies. (A) Leptotene; (B) zygotene; (C) pachytene; (D) diplotene. An arrow indicates an unsynapsed XY bivalent. Bar, 10 µm.

View larger version (147K): [in a new window]   Fig. 5. Localization of Rec8 on chromatin loops in meiotic prophase. Nuclear spreads from mouse testicular cells were prepared by treatment of cells with 85 mM NaCl (A) or 75 mM KCl (B) before fixation in 1% paraformaldehyde with SDS, as described in Materials and Methods. The samples were immunofluorescently stained with anti-Rec8 antibody. DNA was counter-stained with propidium iodide. (C) Magnification of the region indicated in panel B. Bar, 2 µm.

View larger version (147K): [in a new window]   Fig. 6. Localization of Rec8 protein on chromosomes in meiosis I and II. Meiotic chromosome spreads were prepared to prevent chromatin dispersion by SDS during fixation. The chromosome spreads were immunofluorescently stained with anti-Rec8 antibody, and DNA was counter-stained with propidium iodide. (A) Pachytene; (B) diakinesis; (C) metaphase I; (D) early anaphase I; (E) late anaphase I; (F) metaphase II; (G) anaphase II; (H) mitosis. The regions indicated in C and D are magnified in I and J, respectively. Arrows indicate Rec8 signals on the interstices between sister chromatids. White arrowheads indicate Rec8 signals on the overlapped sites of two homologous chromosomes. Blue arrowheads indicate Rec8 signals on the interstitial axes between homologous chromosomes (chromosome arm regions distal to chiasmata). Bar, 5 µm.

View larger version (30K): [in a new window]   Fig. 7. Possible mitotic and meiotic cohesin complexes in yeast and mammals. In budding yeast, one of the mitotic cohesin subunits, Scc1p, is replaced by Rec8p in meiosis. In mammals, by contrast, two types of cohesin complex exist in mitosis; their components are SMC1, SMC3, Rad21 and either one of SA1 and SA2. In meiosis, three proteins, SMC1, Rad21 and SA1/SA2, are likely to be replaced by SMC1ß, Rec8 and SA3/SAX (unidentified meiotic SA homolog) in the meiotic cohesin complexes, respectively.

View larger version (30K): [in a new window]   Fig. 8. Localization of meiotic cohesin complexes during mammalian meiotic divisions. Rec8-containing meiotic cohesin is localized along the chromosomal axes between sister chromatids, including the centromere regions, the arm regions both proximal and distal to chiasmata at metaphase I. At this stage, the arm cohesion distal to chiasmata is responsible for uniting homologous chromosomes together, and the resolution of them triggers separation of the homologs at anaphase I. Rec8 is released from the arm regions concomitantly with the separation of homologs. In metaphase II, Rec8-containing meiotic cohesin is localized at centromere regions at which sister chromatid cohesion is maintained. Release of Rec8 from the centromere regions coincides with the separation of sister chromatids in anaphase II.

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