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First published online 21 September 2004
doi: 10.1242/jcs.01368

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Ubiquitin ligase Rad18^Sc localizes to the XY body and to other chromosomal regions that are unpaired and transcriptionally silenced during male meiotic prophase

¹ MGC-Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus MC, University Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands
² Department of Reproduction and Development, Erasmus MC, University Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands
³ Department of Obstetrics and Gynecology, University Medical Center St Radboud, PO Box 9101, 6500 HB Nijmegen, The Netherlands

View larger version (29K): [in a new window]   Fig. 1. Detection of Rad18Sc protein. (A) On an immunoblot of proteins isolated from total testis lysate, the anti-Rad18Sc antibody recognizes a specific protein band around 80 kDa (arrowhead). (B) In vitro transcription-translation using Rad18Sc cDNA results in a protein band with a molecular mass of approximately 80 kDa (arrowhead).

View larger version (32K): [in a new window]   Fig. 2. Relative Rad18Sc protein level in mouse testis at different postnatal developmental time points and in isolated germinal cells. (A) Total testis proteins from mice at different postnatal ages (7-36 days) were analyzed on an immunoblot (WB) using anti-Rad18Sc. The relative Rad18Sc protein level increases after birth to a maximum at 21 days. (B) The Rad18Sc protein level in isolated spermatocytes (scy) is slightly higher than in isolated spermatids (rst). Ponceau S staining of total protein (TP) is shown as a loading control.

View larger version (147K): [in a new window]   Fig. 3. Localization of Rad18Sc protein in mouse and human testis. (A-C) Sections of wild-type mouse testis were stained with anti-Rad18Sc. (A,B) A positive signal was found in spermatogonia (sgo), primary spermatocytes (scy) and round spermatids (rst). (C) Hardly any Rad18Sc was present in elongating spermatids (est). In primary spermatocytes, the highest amount of Rad18Sc was found associated with the XY body (circles). (D) In cross-sections of human testis, anti-Rad18Sc gave marked staining of the XY body (circles) in primary spermatocytes (scy). Bar, 30 µm.

View larger version (50K): [in a new window]   Fig. 4. Rad18Sc and HR6a/b localize to XY body chromatin in mouse primary spermatocytes. Fixed testicular tubule fragments were stained with anti-Rad18Sc (left panel) or anti-HR6a/b (right panel), and nuclei were visualized with DAPI (blue). The arrows indicate the XY body. Bar, 10 µm.

View larger version (92K): [in a new window]   Fig. 5. Rad18Sc protein is associated with XY body chromatin of pachytene and diplotene spermatocytes. Rad18Sc is stained using anti-Rad18Sc (green) and axial elements and lateral elements of SCs are stained with anti-Sycp3 (red) in zygotene (A), pachytene (B), early diplotene (C) and diplotene (D) spermatocytes. Bar, 20 µm.

View larger version (35K): [in a new window]   Fig. 6. Rad18Sc protein localizes to unsynapsed axial loops. (A) The breakpoints (arrows) in chromosomes 1 and 13 of the T(1;13)70H (T, left) and T(1;13)Wa (T', right) mice are illustrated. Meiotic bivalents in T/T' double heterozygote mice are shown in the middle. (B) Immunostaining of Sycp3 (red) and Rad18Sc (green) of T(1;13)70H/T(1;13)Wa double-heterozygous spermatocytes. Rad18Sc protein localizes to the XY body and also to the translocation bivalent that shows incomplete synapsis. The unsynapsed axial loop of the 113 bivalent is often found close to the XY body. The arrow indicates the X and Y bivalent (XY body) and the arrowhead points to the 113 translocation bivalent. Bar, 20 µm.

View larger version (33K): [in a new window]   Fig. 7. Rad18Sc protein localizes to unsynapsed regions of the 113 bivalent. The morphology of the 113 bivalent was classified as partially synapsed A shape (PA, intermediate degree of synapsis), partially synapsed horsheshoe shape (PH, almost complete synapsis), partially synapsed rest (PR, low degree of synapsis), and completely synapsed (CS). The accumulation of Rad18Sc to these regions was analyzed by immunostaining (upper panel). Over groups, the number of nuclei positive for Rad18Sc was found to be increased when synapsis of these bivalents was less complete (lower panel). The arrowhead indicates the 113 bivalent.

View larger version (127K): [in a new window]   Fig. 8. Immunoexpression of RDB proteins downstream of HR6a/b-Rad18Sc is low in XY body chromatin of primary spermatocytes. (A) Left: Immunostaining of UBC13 using -UBC13 (green) shows that UBC13 is absent from a subnuclear region where the XY body is situated (arrow). Right: Co-immunostaining of UBC13 and Sycp3 (red) to visualize the autosomes and the location of the XY body. (B) Left: Immunostaining of the TLS polymerase pol by anti-pol (green). The level of this RDB protein is also very low in the subnuclear region where the sex chromosomes are located (arrow). Right: Co-immunostaining of pol and Sycp3 (red) to visualize the autosomes and the location of the XY body. Bar, 20 µm.

View larger version (100K): [in a new window]   Fig. 9. RNA polymerase II is largely excluded from nuclear regions containing Rad18Sc associated with unsynapsed chromosomes. (A) Left: Immunostaining of the SC using anti-Sycp3 (red) on a wild-type spermatocyte. The arrow points to the XY body. Right: Coimmunostaining of Sycp3 and RNA polymerase II (RNA pol II, green) showing that the RNA polymerase II staining signal is very low in the XY body. (B) Left: Immunostaining of Sycp3 (red) on a T(1;13)70H/T(1;13)Wa double-heterozygous spermatocyte. The arrow points to the XY body and the arrowhead to the 113 translocation bivalent. Right: Co-immunostaining of Sycp3 and RNA polymerase II (RNA pol II, green) shows that the RNA polymerase II staining signal is also very low in the subnuclear region of the 113 translocation bivalent. (C) Left: Immunostaining on a T(1;13)70H/T(1;13)Wa double-heterozygous spermatocyte with anti-RNA polymerase II (RNA pol II, green). The subnuclear regions containing the XY body (arrow) and the 113 translocation bivalent (arrowhead) have a very low level of RNA polymerase II protein. Right: Co-immunostaining of RNA polymerase II and Rad18Sc shows that the regions deficient in RNA polymerase II contain a large amount of Rad18Sc (red). Bar, 20 µm.

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