JCS046706 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. INCENP, SYCP2 and RAD21 distribution in Cdk2^+/− pachytene spermatocytes. Double-immunolabelling of SYCP3 (red) with either INCENP (green in A), SYCP2 (green in B) or RAD21 (green in C) in spread spermatocytes. (A) INCENP appears along autosomal SCs, and at the PAR at sex chromosomes. (B,C) SYCP2 and RAD21 colocalise with SYCP3 along autosomal SCs and unsynapsed sex AEs. The sex bivalent (XY) and the PAR (asterisk) are indicated. Scale bar: 10 µm.

• Supplemental Figure S2 -

  Fig. S2. There are internal telomeres in Cdk2^−/− pachytene-like spermatocytes. Anaglyph images of the Cdk2^+/− pachytene (A) and Cdk2^−/− pachytene-like (B) squashed spermatocyte nuclei shown in Fig. 5C-E and 5F-H, respectively. A 3D image can be observed using the appropriate red and cyan/green glasses. SYCP3 is a red-cyan anaglyph and will be visualised in grey, while TRF1 is a red-green anaglyph that will be visualised in yellow. Note the existence of telomeres at the inner portion of the Cdk2^−/− spermatocyte nucleus. Large accumulations of SYCP3 are detected inside the Cdk2^−/− pachytene-like spermatocyte.

• Supplemental Figure S3 -

  Fig. S3. CDK2 distributes at telomeres and recombination nodules. Double-immunolabelling of SYCP3 (red) and CDK2 (green) in wild-type spread spermatocytes. (A) In leptotene spermatocytes CDK2 appears at the ends of some short SYCP3 stretches. (B) At zygotene, CDK2 is located at telomeres of both unsynapsed and synapsed AEs/LEs. (C) During pachytene, CDK2 appears at telomeres and recombination nodules, and at asynapsed sex AEs. (D) In diplotene spermatocytes, CDK2 is restricted to telomeres. The sex bivalent (XY) and the PAR (asterisk) are indicated. Scale bar: 10 µm.

• Movie 1 -

  Movie 1. A sex body (XY) is not detected in Cdk2^−/− pachytene-like spermatocytes. 3D reconstructions of pachytene Cdk2^+/− and pachytene-like Cdk2^−/− squashed spermatocytes immunolabelled for the LE protein SYCP3 (red) and counterstained with DAPI (blue). For the correct visualisation of the movies, please click the loop option in your movie player before running them.

• Movie 2 -

  Movie 2. In Cdk2^−/− pachytene-like spermatocytes some telomeres do not attach to the nuclear envelope. 3D reconstructions of pachytene Cdk2^+/− and pachytene-like Cdk2^−/− squashed spermatocytes immunolabelled for the LE protein SYCP3 (red) and TRF1 (green). In pachytene Cdk2^+/− spermatocytes all telomeres associate to the NE, whereas in Cdk2^−/− spermatocytes some telomeres are found at the nuclear interior.

• Movie 3 -

  Movie 3. Persistence of γ-H2AX-labelled chromatin in Cdk2^−/− pachytene-like spermatocytes. 3D reconstructions of pachytene Cdk2^+/− and pachytene-like Cdk2^−/− squashed spermatocytes immunolabelled for the LE protein SYCP3 (red) and γ-H2AX (green). In pachytene Cdk2^+/− spermatocytes only the sex body (XY) shows an intense γ-H2AX labelling, whereas in Cdk2^−/− spermatocytes there are large γ-H2AX-labelled chromatin masses.

• Movie 4 -

  Movie 4. Persistence of RAD51 early nodules in Cdk2^−/− pachytene-like spermatocytes. 3D reconstructions of pachytene Cdk2^+/− and pachytene-like Cdk2^−/− squashed spermatocytes immunolabelled for the LE protein SYCP3 (red) and RAD51 (green). In pachytene Cdk2^+/− spermatocytes, RAD51 is mostly restricted along the asynapsed AEs of sex chromosomes (XY), whereas in Cdk2^−/− spermatocytes RAD51 persists along autosomal LEs.