Figure 4. Immunolabeling of Squashed Spermatocytes with Anti-SYCP3 (Green) and Anti-Centromere (Red) Antibodies.

Several focal planes have been superimposed and projected in each image.

(A) Metaphase I. Bivalents are correctly bioriented in the metaphase plate, including the XY pair.

(A′–A′′) SYCP3 is detected on the X chromosome as an irregular line (with small splittings and excrescences) covering the interchromatid domain. The Y chromosome is completely labeled with the anti-SYCP3 antibody. Comparison of SYCP3 and DAPI images shows that this massive labeling also involves the distal region of the X chromosome long arm (asterisk in A′–A′′). The distal region of the long arm of the X chromosome contacts with the Y chromosome (arrowhead) while an SYCP3 filament overpasses the X short arm and links to the distal region of the X long arm and the Y chromosome (arrow).

(A′′′) Schematic illustration of the XY pair in this stage. The limit between both sex chromosomes is marked.

(B) Shown is an autosomal bivalent in metaphase I, and (B′) its schematic representation. SYCP3 signal runs along the interchromatid domain and interrupts at the chiasma point.

(C–C′′) Metaphase I: In this spermatocyte bivalents are correctly bioriented in the metaphase plate, including the XY pair. Some aggregates of SYCP3 are detected in the cytoplasm (arrowheads). In this case, the SYCP3 bridge from the X chromosome short arm is broken (arrow in C′ and C′′), while the long arm is still in contact with the Y chromosome (asterisk).

(D–D′′) Early anaphase I: SYCP3 begins to dissociate from the autosomes as they migrate to the poles, but some SYCP3 is still present near some centromeres (arrowheads) and along some regions of the chromosome arms. Bar-shaped SYCP3 aggregates appear in the spindle area of the cytoplasm (b). Although sex chromosomes initiate their migration they remain linked by an SYCP3 bridge. Note that in this case the filament protruding from the X short arm is still visible (arrow in D′). SYCP3 signal exceeds the end of the X chromosome short arm (arrow in D′′). SYCP3 is detected as a filament in the pericentromeric region of the Y chromosome (arrowhead in D′), in contrast with the massive signal observed on it. The SYCP3 massive labeling has partially disorganized but it does not disappear (asterisk). During mid (E–E′′) and late anaphase I (F–F′′), this massive SYCP3 joining between X and Y chromosomes disorganizes, making it difficult to unequivocally identify sex chromosomes as they move apart from each other (their putative location has been indicated as X and Y). A series of thick filaments are present between chromatin masses migrating to opposite poles (asterisk).

(G–G′′) At telophase I thick SYCP3 filaments are visible in the cytoplasm between the cell poles (arrow) and also around some centromere regions (arrowheads).

(H) Interkinesis. Minute bars of SYCP3 are still detected in the cytoplasm (arrowheads).