RdlF, JSR, and JP conceived and designed the experiments, analyzed the data, and wrote the paper. RdlF, MTP, AV, AC, RG, JAS, JSR, and JP performed the experiments.
The authors have declared that no competing interests exist.
In most eutherian mammals, sex chromosomes synapse and recombine during male meiosis in a small region called pseudoautosomal region. However in some species sex chromosomes do not synapse, and how these chromosomes manage to ensure their proper segregation is under discussion. Here we present a study of the meiotic structure and behavior of sex chromosomes in one of these species, the Mongolian gerbil (
Meiosis is a special kind of cell division that leads to the formation of gametes. During meiosis the number of chromosomes must be halved in the daughter cells, and to do this properly, most organisms use an amazing strategy: during the first of the two meiotic divisions, homologous chromosomes associate in pairs, undergo a reciprocal genetic interchange, and then each member of the pair segregates into a different daughter cell. Genetic exchange, called meiotic recombination, is a key process to ensure that homologous chromosomes remain tightly associated until they segregate. In general, sex chromosomes are subjected to the same processes as the rest of chromosomes. But, of course, exceptions exist. This is the case in the Mongolian gerbil, a mammal whose sex chromosomes pair and segregate during male meiosis without undergoing meiotic recombination. We have found that they are able to do this because some proteins of a meiosis-specific structure, the synaptonemal complex, are reorganized to maintain sex chromosomes associated until they segregate into daughter cells. This kind of behavior resembles the situation found in marsupials and some insect species, indicating a recurrent role of synaptonemal complex components in chromosome segregation when meiotic recombination is missing.
The proper distribution of chromosomes into daughter cells during meiosis depends on the essential phenomena of pairing, synapsis, recombination, and segregation. During early prophase I homologous chromosomes associate in pairs and are held by a proteinaceous structure, the synaptonemal complex (SC) [
It is currently known that these phenomena are intimately related and that they occur in an ordered fashion. Thus, homologous recognition, pairing, and synapsis are promoted by the initiation of recombination events involved in the repair of programmed DNA double strand breaks (DSBs) made by SPO11 protein at the very beginning of first meiotic prophase [
Although this plan is followed by a great majority of species, there are some groups of organisms that show variations in the sequence or even the occurrence of the meiotic hallmarks (for review see [
Sex chromosomes are especially prone to get out of the rules of meiosis [
To shed light on these mechanisms, we have investigated the sequence and the nature of X and Y chromosome association during male meiosis in the Mongolian gerbil (
We first studied the location of SYCP3 protein, the main component of the AE and lateral elements (LEs) of the SC [
Several focal planes have been superimposed and projected in a single plane in each image. Selected sex chromosomes from the whole spermatocyte are detailed in the right column at the same meiotic stage of those of the figures.
(A) Leptotene: AEs are not completely formed, and they are visualized as thin and discrete lines dispersed in the nucleus.
(B) Zygotene: AEs start to form thicker filaments at the regions where synapsis is proceeding. The bouquet configuration is detected by the presence of polarized AE ends in the nucleus (arrowheads).
(C) Pachytene: The autosomes have completed their synapsis. Autosomal LEs present helicoidal twists all along their length (arrowheads in the inset). The AEs of the sex chromosomes appear together and are located in the periphery of the nucleus.
(C′) Shown is detail of a pachytene spermatocyte in which sex chromosomes are arranged in a front view and their AEs are discernible. Note that there is no contact between them.
(D) Early diplotene: SC begins to disorganize, and the LEs appear separated (arrowheads).
(D′) The AEs of the sex chromosomes become tangled.
(E) Late diplotene: SC has almost completely disassembled from the autosomes, only some portions of the LEs are still synapsed (arrowheads).
(E′) SYCP3 on the XY pair redistributes, modifying the AEs morphology, and it begins to accumulate on the Y chromosome.
(F) Diakinesis: Autosomes appear compacted with SYCP3 as discontinuous lines along each chromosome. SYCP3 forms aggregates in the cytoplasm as thick bars (arrow).
(F′) Sex chromosomes can be distinguished from each other. They appear to be distally connected (arrowhead), and SYCP3 is massively accumulated on the Y chromosome, while on the X it appears as an irregular line along the chromosome.
Sex chromosomal AEs are not distinguishable from that of the autosomes during leptotene (
During diplotene sex chromosomes remain associated and located at the nuclear periphery. However, as sex chromosomes increase their condensation their AEs fold (
To test the asynaptic nature of the sex chromosome association in
(A) Leptotene: SYCP3 is detected as short lines dispersed in the whole nucleus. No signal of SYCP1 is detected.
(B) Zygotene: SYCP3 is detected over the autosomal AEs, which appear partially synapsed (arrowheads). SYCP1 is detected in the regions that have already synapsed. Synapsis proceeds from different points along chromosomes. Sex chromosome AEs also appear labeled with anti-SYCP3 but no SCYP1 labeling is detected (detailed in B′–B′′). X and Y chromosomes appear separated in the nucleus.
(C) Pachytene: SYCP3 and SYCP1 labeling on the autosomes are coincident. Sex chromosomes (enlarged in C′) appear in a close position, but their AEs do not contact and they do not show SYCP1 labeling.
(D) At early diplotene, SYCP1 begins to dissociate from the SC, and the LEs can be seen separated in certain regions along the bivalents (arrowheads). Sex chromosomal AEs appear folded and distally connected. One end of the AE of the Y chromosome (see detail in D′) is in contact with both tips of the X chromosome (arrowhead).
(E) Late diplotene. The bivalents show very little SYCP1 signal. Sex chromosomal AEs are tangled, and their outline is fairly irregular (as seen in E′). The four chromosomal ends seem to be clustered in a single point (arrowhead in E′).
(F) Diakinesis: SYCP3 over the autosomes is detected as a zigzagging and curly signal. Some chiasmata points are clearly identifiable (arrows).
(F′) The sex chromosomes appear to be distally connected (arrow). SYCP3 labeling on the X chromosome is similar to the labeling on the autosomes, while the labeling is massive over the Y chromosome, excepting in the pericentromeric region (arrowhead).
Contrary to what was observed in squashes, sex chromosome AEs can be identified on spreads during zygotene (
As observed in squashed preparations, the morphology of sex chromosomal AEs is modified from diplotene onwards when studied on spreads. Thus, AEs become irregular and folded at diplotene (
In mammals the initiation of SC assembly is dependent on the occurrence of previous recombination events [
(A–A′) At early pachytene RAD51 foci are visible over the autosomes. This protein also appears associated to the AEs of sex chromosomes (enlarged in A′).
(B–B′) At mid pachytene the number of RAD51 foci over the autosomes and sex chromosomes decreases, but RAD51 begins to accumulate over the sex chromatin as a diffuse signal.
(C–C′) At late pachytene RAD51 is absent from autosomes but intensely extended on the sex chromatin.
(D) MLH1 protein is detectable at late pachytene as one single dot over most of the autosomal axes, although some bivalents present two (arrowheads). No signal of this protein is detected over the axes of the sex chromosomes (D′).
RAD51 is detected on the autosomal AEs during zygotene and early pachytene as dots on or very close to the AEs/LEs (
MLH1 is only detected at late pachytene on autosomal SCs. Most bivalents present one MLH1 focus, but some of them may present two foci (
Given the striking modification of SYCP3 location during late stages of prophase I, we analyzed SYCP3 distribution during late stages of first meiotic division to ascertain its potential role in sex chromosome segregation (
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).
At metaphase I, SYCP3 protein remains associated with autosomes at the region of sister chromatid contact (the interchromatid domain) (
The pattern of SYCP3 localization on the X chromosome is visualized as a sinuous and irregular line that runs along its interchromatid domain (
At metaphase I, sex chromosomes are associated and properly bioriented. However, we observed two different configurations. In the first configuration, both arms of the X chromosome are in contact with the Y chromosome (
At the beginning of anaphase I, SYCP3 dissociates from the chromosomes but does not disappear abruptly since it is still detectable during early anaphase I at the interchromatid domains, mainly close to the centromeres (
Taking into account the pattern of SYCP3 distribution on the sex chromosomes up to metaphase I, it is likely that some of the SYCP3 filaments found during anaphase I associate the sex chromosomes. With the aim of identifying the X and Y chromosomes inside these chromatin masses and their relation to SYCP3 filaments, we carried out the double immunolabeling of SYCP3 and γ-H2AX (
Several focal planes have been projected in each image.
(A) Leptotene: γ-H2AX labeling appears distributed throughout the whole nucleus.
(B) During zygotene γ-H2AX distribution is very similar to that observed in the previous stage.
(C) From pachytene onwards, the bulk of anti-γ-H2AX antibody is located onto the sex chromosomes, which appear located at the nuclear periphery and remain so during diplotene (D).
(E–E′) Diakinesis: Sex chromosomes appear distally connected and intensely labeled with γ-H2AX. Note the SYCP3 connection between both arms of the sex chromosomes (arrow in E′). The same situation is found in prometaphase I (F–F′).
(G–G′) Metaphase I: The autosomal bivalents remain aligned at the metaphase I plate. The connection between the sex chromosomes has broken in the X chromosome short arm (arrowhead), and only the long arm is linked to the Y (arrow).
(H–H′′′) Anaphase I: Sex chromosomes migrate to the poles, but they are delayed compared to autosomes. Note the remnants of SYCP3 over the autosomes and dispersed in the cytoplasm (arrowheads). A thick SYCP3 filament is visible between the X and Y chromosomes (arrow), but no chromatin joining is detected as revealed by DAPI (H′) or γ-H2AX staining (H′′′).
(I–I′′′) Telophase I: Sex chromosomes are clearly lagged in segregation. The SYCP3 filament appears connecting them without chromatin connection (I′–I′′′).
The simultaneous labeling of SYCP3 and γ-H2AX corroborates that SYCP3 occupies almost the entire width of the Y chromosome during diakinesis up to metaphase I (
One of the most striking advances in the understanding of meiosis in the past years has been the realization that the particular processes that take place during this special kind of cell division are tightly interrelated [
Our analysis of the sequence of SC assembly in the Mongolian gerbil revealed that both X and Y chromosome assemble an AE, but they do not synapse. A first explanation for this behavior is that the mechanisms that promote chromosome synapsis in mammals, i.e., occurrence and repair of DNA DSBs [
A second explanation is that sex chromosomes in the Mongolian gerbil do not share a region of homology. Thus, although sex chromosomes can initiate the processes that ultimately culminate in the synapsis with the homologous chromosome, they are unable to complete this process because they have no homologous partner. In this sense, the absence of synapsis between sex chromosomes appears to be a recurrent feature among the species of the family
Therefore, the absence of PAR is to us the most plausible explanation for the absence of synapsis between the X and the Y chromosomes. However, it has been reported that in some species, the marsupial
Our data indicate that pairing of sex chromosomes takes place during zygotene, and they remain associated at pachytene. However, the lack of the PAR between sex chromosomes in
As regards the first topic, one could assume that the polarization of telomeres during the bouquet stage plays an important role in the initial approach of sex chromosomes. Nevertheless, this mechanism would not be sufficient to ensure sex chromosome pairing, since they can appear close together at the very beginning of zygotene, before autosomal AEs are completely formed, and on the contrary, they can remain separated in the nucleus until late zygotene, well after the resolution of the bouquet.
Another possibility, although highly speculative, is that sex chromosome pairing is based on a mechanism of homologous sequence recognition. As mentioned above, the absence of a functional PAR does not imply that there is not homology at all between sex chromosomes. Provided that a certain degree of homology could be conserved, it is possible that the mechanisms of DNA repair mediated by RAD51 and other proteins could promote the approaching and recognition of X and Y chromosomes, although, as stressed before, structural or genetic factors would hamper the formation of a SC. In this sense, this residual homology could not be as efficient as a PAR in promoting the recognition of sex chromosomes, explaining their erratic behavior during zygotene.
Once sex chromosomes recognize each other they remain intimately paired throughout pachytene, even though SC is not formed. In other
An alternative explanation is that the particular chromatin condensation of the sex body may contribute to maintain the association of X and Y chromosomes. It is currently known that sex chromosomes are transcriptionally inactive during most of the first meiotic prophase in mammals and a huge number of proteins, including γ-H2AX, are specifically associated to and/or modified in the sex body [
The correct segregation of chromosomes during first meiotic division depends on their proper alignment and biorientation at the metaphase I plate. In
The AEs of the sex chromosomes appear physically separated at pachytene, but closely related at the periphery of the nucleus. As the chromosomes increase their condensation at early diplotene, the AEs tend to fold and their tips establish an end-to-end contact. At the end of diplotene, SYCP3 reorganizes on the AEs, and acquires a diffuse pattern, mainly on the Y chromosome. At diakinesis sex chromosomes are end-to-end connected, in a conformation that is maintained up to metaphase I, and SYCP3 appears covering the Y chromosome and the distal region of the X chromosome. Our proposal is that the physical connection mediated by SYCP3 at this stage is responsible for maintaining sex chromosome association. Once sex chromosomes have achieved a bipolar orientation on the metaphase I spindle they tend to move to opposite poles. At this point the SYCP3-mediated association may break at the short arm of the X chromosome. At anaphase I chromosomes move to opposite poles. However, an SYCP3 filament remains associating both sex chromosomes, probably causing the delay of sex chromosome migration at anaphase I. This filament ultimately detaches from sex chromosomes during telophase I.
Since sex chromosomes always appear as laggards at anaphase I it seems that their movement to the poles is somehow obstructed. Physical links between segregating half bivalents at anaphase I have been detected in a wide range of species [
The critical feature in this context is how the AEs components derive in such a structure. Previous studies have shown that the elements of the SC can be transformed into a variety of structures that may remain associated to chromosomes until anaphase I [
Finally, the finding that SYCP3 may form conspicuous aggregates in the cytoplasm, which is a feature common to other species of mammals, is also remarkable [
Different animal groups challenge the rule that synapsis and recombination are required for proper segregation. It is well known that insects represent a wide range of segregation mechanisms of achiasmate chromosomes [
Testes of adult
Slides were incubated overnight at 4 °C with the following primary antibodies diluted in PBS: mouse monoclonal anti-SYCP3 (Abcam, 12452) at a 1:100 dilution; rabbit anti-SYCP3 (Abcam, 15093) at a 1:50 PBS dilution; rabbit anti-SYCP1 (Abcam, 15087) at a 1:100 dilution; mouse monoclonal against histone H2AX phosphorylated at serine 139 (γ-H2AX) (Upstate, 05–636) at a 1:3,000 dilution; rabbit anti-RAD51 (Calbiochem, PC130) at a 1:50 dilution; mouse monoclonal anti-MLH1 (Pharmingen, 551091 ) at a 1:10 dilution; and a human anti-centromere serum that recognizes centromeric proteins (Antibodies Incorporated, 15–235) at a 1:100 dilution. Slides were rinsed 3 × 5 min in PBS and subsequently incubated with secondary antibodies in a moist chamber at room temperature for 1 h: fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG; Texas Red (TR)-conjugated goat anti-mouse IgG; FITC-conjugated goat anti-rabbit IgG; TR-conjugated goat anti-rabbit IgG; and TR-conjugated goat anti-human IgG. All secondary antibodies were from Jackson (Jackson ImmunoResearch Laboratories) and used a 1:100 dilution. Slides were subsequently rinsed in PBS 3 × 5 minutes, stained with DAPI, and mounted with Vectashield (Vector). For double detection of two antibodies raised in mouse, we followed the procedure previously described [
Observations were made on an Olympus BX61 microscope equipped with a motorized Z axis. Images were captured with an Olympus DP70 digital camera using the analySIS software (Soft Imaging System, Olympus) and processed by using public domain ImageJ (National Institutes of Health,
In both cases a late-synapsing autosomal bivalent can be observed (asterisk). We scored sex chromosomes as separated when the distance between their AEs is longer than the length of the X chromosome AE. Frequencies of both situations are detailed below the images.
(2.0 MB TIF)
Only some images from the 3-D reconstruction are projected to observe the sex chromosomes.
(A) Note the signal of SYCP3 as a thin filament in the pericentromeric region of the Y chromosome (arrow). The aggregate of SYCP3 (asterisk) in the long arm of the X chromosome connects it with the Y chromosome (arrowhead).
(B) The chromatin of both chromosomes is not in contact (arrowhead), and it is clear that the aggregate of SYCP3 relies on the distal segment of the X chromosome.
(1.2 MB TIF)
This video, as well as
(2.1 MB MOV)
Double immunolocalization of SYCP3 (green) and ACA (red). Shown is 3-D reconstruction of a pachytene spermatocyte in which the XY pair is labeled on the lower part of the movie.
(2.5 MB MOV)
Double immunolocalization of SYCP3 (green) and ACA (red). SC begins to dissociate from the autosomes and their LEs are detected separated. The XY pair is labeled on the right part of the image.
(2.1 MB MOV)
Double immunolocalization of SYCP3 (green) and ACA (red). By reconstructing the nucleus, X and Y chromosomes can clearly be seen not in contact with each other.
(1.1 MB MOV)
Double immunolocalization of SYCP3 (green) and ACA (red) and staining with DAPI (blue). The sex chromosomes are arranged in the metaphase I plate (labeled), and the short arm of the X chromosome has dissociated from the Y. The 3-D reconstruction allows us to distinguish the chromatin of the short arm separated from the chromatin of the Y chromosome.
(8.9 MB MOV)
This video corresponds to the 3-D reconstruction of the cell in
(1.9 MB MOV)
This video, as well as
(1.1 MB MOV)
Double immunolocalization of SYCP3 (green) and γ-H2AX (red). The X and Y chromosomes are labeled in the image, identified by the γ-H2AX signal. They are clearly linked by a SYCP3 aggregate.
(1.4 MB MOV)
We express our sincere thanks to Juan Luis Santos and Carlos García de la Vega for their critical reading of the manuscript.
axial element
double strand break
lateral element
pseudoautosomal region
synaptonemal complex