Figure 1.
Silver Staining of T. elegans Spermatocytes
(A–C) Metaphase I. The autosomal bivalents appear aligned at the cell equator. (A) In an early stage, sex chromosomes (X, Y) appear associated out of the metaphase I plate, near one of the cell poles. There is a wide region of contact between both sex chromosomes (arrow).
(B) In a middle stage, sex chromosomes (X, Y) appear situated at the cell equator, each facing a cell pole. The chromatin that connects both sex chromosomes appears stretched (arrow).
(C) In a late stage, sex chromosomes (X, Y) have initiated their segregation, while the autosomes are still stabilized at the metaphase plate. Note that in this favorable case, silver staining has revealed the kinetochores of chromosomes (arrowheads) and linear elements inside the chromatin (arrows).
(D) Early anaphase I. Autosomes initiate their segregation. Chromatin bridges are seen between the telomeres of some autosomes (arrowheads). Sex chromosomes (X, Y) appear at opposite poles.
(E) Telophase I. Each sex chromosome (X, Y) is incorporated to one pole. Bar: 5 μm.
Figure 2.
Immunolabeling of Spermatocytes with Anti-STAG3 (Green) and Anti-Centromere (Red) Antibodies and Staining of the Chromatin with DAPI (Blue)
Several focal planes have been superimposed and projected in a single plane in each image.
(A–C) Early–middle metaphase I. All bivalents are aligned at the metaphase I plate. Sex chromosomes (X, Y) also appear at the cell equator and their centromeres oriented to opposite poles. Note that the position of the sex pair is not equidistant from the poles. Instead, the X chromosome seems to be closer to the pole than the Y chromosome.
(B) An enlarged autosomal bivalent of the same cell in which STAG3 appears as diffuse lines at the interchromatid domain. (B') Representation of the same bivalent shown in (B). Sex chromosomes are in close proximity, but there is no contact between the STAG-labeled structures (arrow).
(C') Schematic representation of the sex chromosomes in (C).
(D–F) Late metaphase I. Autosomal bivalents are still stabilized at the cell equator. STAG3 labeling is detectable in both autosomes and sex chromosomes (X, Y).
(E) Enlargement of an autosomal bivalent with a subdistal chiasma in the long arm. The signal of STAG3 runs along the interchromatid domain (arrows) but interrupts at the chiasma site (arrowheads).
(E') Schematic representation of the same bivalent shown in (E). Sex chromosomes (detailed in F) have initiated their segregation, even though the STAG3 labeling is still present along their interchromatid domains; arrows in (F).
(F') Representation of the sex chromosomes in (F).
(G and H) Anaphase I. Homologous chromosomes start to segregate to opposite poles. The STAG3 labeling is completely absent from both autosomes and sex chromosomes. The centromere signals in most chromosomes are seen as double dots (arrows), corresponding to sister kinetochores.
Bars: 5 μm in (A), (D), (G), and (H); 1 μm in (B), (C), (E), and (F).
Figure 3.
FISH of Telomeric Repeats (Green) and Chromatin Staining with DAPI (Blue)
(A–A'') Early metaphase I. All chromosomes show distal telomeric signals, and two large bivalents also show a hybridization signal near the centromeric region (arrows). (A') and (A'') show an enlargement of sex chromosomes in the cell shown in (A). Sex chromosomes (X, Y) are associated, each one facing a cell pole, but displaced from the cell equator. The telomeric repeats of the X and Y appear in close apposition, but without contact.
(B–B'') Mid metaphase I. The sex pair is still located out of the cell equator. At this stage, the sex chromosomes show clear signs of being under spindle tension, but their association is not mediated by telomeric repeats, since the distal telomeric signals of both sex chromosomes, although being in close proximity, are not in contact.
(C–C'') Late metaphase I. Sex chromosomes have already initiated their segregation, while the autosomes are still stabilized on the metaphase I plate. No contact between telomeres of the sex chromosomes is detected. Bars: 5 μm in (A–C); 1 μm in (A'–C'').
Figure 4.
Immunolocalization of SCP3 (Green) and Centromeres (Red) and Chromatin Staining with DAPI (Blue)
Several focal planes are superimposed and projected in a single plane in each picture.
Column I: SCP3 and centromere signals. Column II: DAPI staining. Column III: merge of columns I and II. Column IV: detail of the sex chromosomes. Column V: schematic representations of the sex chromosomes shown in the column IV. The DP has been represented in dark green in order to differentiate it from sex chromosomal axes.
(A) Pachytene. SCP3 appears as continuous lines along each autosomal bivalent (arrow) and also depicts the trajectory of sex chromosomal AEs and the DP.
(B) Diffuse stage. SCP3 appears as continuous (arrow) or discontinuous lines (arrowheads) in both autosomal LEs and sex chromosome AES, and also on the DP.
(C) Diplotene. The autosomal LEs appear fragmented. Homologous chromosomes appear separated (arrowheads) except at the chiasma points (arrows). However, the AEs of sex chromosomes (X, Y) appear continuous, and the labeling of the DP is also visible.
(D) Prometaphase I. All centromeres appear polarized to a specific point (asterisk) that presumably corresponds to the position of the centrosomes. SCP3 remains associated with the chromosomes at the interchromatid domain (arrows). This labeling has a dotted-like appearance on the autosomes, but is continuous on the sex chromosomes (X, Y) and the DP. The tips of both sex chromosomes are in contact with the DP.
(E) Early metaphase I. The SCP3 labeling is similar to previous stages, with this protein located at the interchromatid domain of all chromosomes (arrows). The bivalents have adopted a bipolar orientation with the homologous centromeres facing opposite spindle poles. The sex pair (X, Y) appears also bioriented. Both sex chromosomes remain in contact with the DP, but the short arm on the X chromosome appears detached from this structure (arrowhead).
(F) Late metaphase I. Autosomal bivalents appear stabilized at the metaphase I plate. The SCP3 labeling is present at the interchromatid domain in both autosomes and sex chromosomes (X, Y). Sex chromosomes appear separated at this stage. They have lost contact, and each one has initiated its segregation to a different pole. The DP remains associated in this case to the X chromosome long arm.
(G) Anaphase I. The SCP3 labeling disappears at the beginning of anaphase I from both autosomes and sex chromosomes. Only a SCP3 labeling is detected in some centromere regions (arrowhead) and on the DP. The position of the X chromosome is detectable because the DP remains associated in this case to this chromosome. Bars: 5 μm in column I–III; 1 μm in column IV.
Figure 5.
Immunolocalization of SCP3 (Green) and Centromeres (Red) and Chromatin Staining with DAPI (Blue)
Several focal planes are superimposed and projected in a single plane in each picture.
(A–C) Interkinesis. Seven centromeric signals are detected. Each centromere shows two signals corresponding to sister kinetochores (arrows). No SCP3 labeling is detected at all, indicating the absence of this protein during this and subsequent stages.
DAPI staining (B–C) reveals a more condensed chromatin mass inside the nucleus (arrowhead) that presumably corresponds to the X chromosome.
(D–F) Prophase II. The seven centromeric signals are dispersed through the nucleus and the chromatin starts to condense. Chromosomes are individualized, showing their chromatids joined only at the centromeric region (arrows) while the chromatid arms are separated (arrowheads). The putative position of the X chromosome is indicated (X).
(G–I) Metaphase II. All chromosomes are aligned at the metaphase II plate. Note that the distance between sister kinetochores is greater than in prophase II.
(J–L) Anaphase II. All chromosomes separate their chromatids during this stage. Seven single centromeric signals are detected at each pole. Bar: 5 μm.
Figure 6.
Immunolocalization of SCP1 (Green), Centromeres (Red), and Staining of the Chromatin with DAPI (Blue)
Several focal planes are superimposed and projected in a single plane in each picture.
(A–C) Pachytene. SCP1 appears as continuous lines along autosomes and is completely absent on the sex chromosomes. (A' and C'). Detailed view of the sex chromosomes in the same cell.
(D–F) Diplotene. SCP1 mostly appears as fragmented lines that run along chromosomes. These lines are continuous in some regions. SCP1 remains associated at the centromeric regions of autosomes and also appears associated to the X chromosome centromere (arrowheads). A structure labeled with SCP1 appears at the periphery of the sex body (arrow). (D' and F') At a higher magnification this structure is clearly detected near the centromere of the Y chromosome.
(G–I) Prometaphase I. SCP1 signal has completely disappeared from the chromosomes. However, a small structure is clearly labeled (arrow). Bivalents are not oriented at the metaphase plate. Sex chromosomes (X, Y) are clearly discernible, and they remain associated. (G' and I'). Enlargement of the sex chromosomes. The SCP1-labeled structure appears at the region of contact between sex chromosomes (arrow).
(J–L) Metaphase I. The SCP1-labeled structure is present in the region where sex chromosomes (X, Y) are located. (J'–L') At a higher magnification it can be seen that the SCP1-labeled structure (arrow) is associated to the sex chromosomes.
(M–O). Anaphase I. Homologous chromosomes have segregated and the SCP1-labeled structure is still visible (arrow). However, it does not maintain any association with the chromatin masses near the cell poles. Bar: 5 μm in (A–O); 1 μm in (A'–L').
Figure 7.
Immunolocalization of SCP3 (Red) and SCP1 (Green) and Counterstaining with DAPI (Blue) in a Spermatocyte at Diplotene
Two focal planes are superimposed in each picture.
(A) SCP3 lines representing the autosomal SC appear discontinuous (arrowheads). The sex chromosomes (X, Y) (enlarged in D–F) appear associated and the DP is detectable.
(B) SCP1 follows a similar pattern to that of SCP3 on the autosomes, appearing as fragmented lines.
(C) Merge of SCP3 and SCP1 signals, which co-localize in the autosomes but not in the sex chromosomes.
(D) Enlargement of the sex chromosomes. SCP3 appears along the AEs of the sex chromosomes and also in the DP.
(E) SCP1 does not appear in the AEs of sex chromosomes, but is present in the DP (arrow).
(F) Merge of the signals shown in (D) and (E). Note that the distribution of SCP1 in the DP is more restricted at this stage than that of SCP3.
(G) Schematic representation of the sex chromosomes.
Figure 8.
Triple Immunolocalization of SCP3 (Green), SCP1 (Red), and Centromeres (Red), and Chromatin Staining with DAPI (Blue) in a Spermatocyte at Metaphase I
Two focal planes are superimposed in each picture.
(A) Immunolocalization of SCP1 (red) and DAPI counterstaining (blue) of a spermatocyte in metaphase I. A small structure associated with the chromatin appears labeled (arrow).
(B) Immunolocalization of SCP3 (green) and centromeres (red) on the same spermatocyte shown in (A). SCP3 appears on the three bivalents shown in these focal planes. The labeling runs on the interchromatid domain and interrupts at the chiasma sites (arrowheads). The SCP3 labeling allows the identification of the sex chromosomes (X, Y) and the DP. Sex chromosomes have already initiated their segregation and have lost contact.
(C) Merge of the images (A) and (B) reveals that the structure labeled with anti-SCP1 is the DP, which in this spermatocyte has remained associated with the Y chromosome.
(D) Detail of the sex chromosomes.
(E) Schematic representation of sex chromosomes and the DP.
Bar: 5 μm in (A–C); 1 μm in (D).
Figure 9.
Sequence of Orientation and Segregation of the Sex Chromosomes in T. elegans Meiosis
The DP is formed during pachytene as a structure derived from sex chromosomal AEs that contain SCP3. During diplotene, SCP1 is already present on the DP, where it probably associates with SCP3. During prometaphase I sex chromosomes remain associated by the DP. At this stage the DP reduces in length. We have made an interpretation of the plausible changes on the DP structure during this phase, acquiring a SC-like organization. We propose that this organization is achieved by the bending of the SCP3 outer layer, and this bending is stabilized by the formation of a SC CE-like structure composed of SCP1. The kinetochores of the sex chromosomes, as well as those of the autosomal bivalents, interact with the spindle microtubules. During this stage both centrosomes separate and by metaphase I they form the bipolar spindle. In metaphase I, sex chromosomes remain associated and are already oriented to opposite cell poles. In early metaphase I, both arms of the sex chromosomes are associated with the DP, but as soon as microtubules start to pull sex chromosomes, they begin to lose this contact. The short arms of both sex chromosomes separate from the DP during mid metaphase I. At late metaphase I the long arms of sex chromosomes definitively lose their contact with the DP and they initiate their migration to the cell poles. This segregation occurs before the disappearance of the SCP3 and STAG3 proteins from the interchromatid domain. These proteins disappear at the onset of anaphase I, allowing for the separation of sister chromatids, which remain associated only at the centromeric regions. SCP3 and SCP1 remain detectable during this stage on the DP. Therefore, the SCP3 protein present in the DP is not removed at the same time as it is removed from the interchromatid domain of all chromosomes. Perhaps SCP3 escapes from this process due to its association to SCP1. Telomeric DNA of both sex chromosomes is not associated at any stage of the first meiotic division and therefore does not play any role on their segregation.