1 Programa de Genética Humana, Instituto de Ciencias Biomédicas,
Facultad de Medicina, Universidad de Chile, Santiago, Chile
2 Unidad de Biología Celular, Departamento de Biología,
Universidad Autónoma de Madrid, Madrid, Spain
3 Molecular Genetics Group, Agricultural University of Wageningen, The
Netherlands
* Author for correspondence (e-mail: rfernand{at}machi.med.uchile.cl)
Accepted 31 October 2002
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Summary |
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Key words: Meiosis, Sex chromosomes, Pairing, Marsupials, Thylamys, SCP3, MPM-2
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Introduction |
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Solari, using serial sections and electron microscopy was the first
investigator to show that an SC is formed between the heteromorphic sex
chromosomes in male mice (Solari,
1970). Since then, the occurrence of SC has been demonstrated in
the sex chromosomes of almost all mammalian species studied
(Solari, 1993
). It is
currently known that sex chromosomes in eutherian mammals share regions of
homology, the so-called pseudoautosomal regions (PARs)
(Burgoyne, 1982
), where
crossing-over and chiasma formation occur. It is in the PAR that the SC forms
between sex chromosomes.
Most studies on meiotic sex chromosome pairing have been carried out in
eutherian species. However, little attention has been paid to the meiotic
structure and behaviour of sex chromosomes in the other mammalian taxa, the
monotremes and the marsupials. There is only one report on monotreme meiosis
(Murtagh, 1977), whereas the
meiosis of only some American and Australian marsupial species has been
thoroughly described (Koller,
1936
; Solari and Bianchi,
1975
; Pathak et al.,
1980
; Sharp, 1982
;
Roche et al., 1986
;
Seluja et al., 1987
). Some of
the features found in eutherian males are also observed in marsupial males.
For instance, the sex chromosomes are delayed in pairing relative to the
autosomes, and they form a dense chromatin mass, the sex body, which is
thought to be a result of condensation and transcriptional inactivation of the
sex bivalent during meiosis (Solari,
1974
).
Other features of meiotic sex chromosomes differ between marsupials and
eutherians, including the involvement of SC in mediating their association.
Thus, while eutherian sex chromosomes synapse (i.e. form tripartite SC),
marsupial sex chromosomes develop AEs that do not associate by a SC central
element, but rather by a dense plate (DP) attached to the sex chromosome ends
(Solari and Bianchi, 1975;
Roche et al., 1986
;
Seluja et al., 1987
).
`Balloon' structures between sex chromosome ends have also been described
(Sharp, 1982
;
Roche et al., 1986
). It has
been suggested that the DP and the balloons are related structures
(Roche et al., 1986
), but
their unequivocal correspondence has not been demonstrated so far. It is
widely assumed that marsupial sex chromosomes do not share a region of
homology (Graves and Watson,
1991
). This lack of homology could prevent synapsis and SC
formation between these chromosomes. However, recent studies have revealed
that in some Australian marsupials the X and Y chromosomes share some
sequences (Toder et al., 1997
;
Toder et al., 2000
). Whether
these sequences constitute a PAR is uncertain.
In this study we followed meiotic sex chromosome pairing in males of the marsupial species Thylamys elegans using immunolabelling and electron microscopy. We present immunocytological evidence that sex chromosomes do not form SC during first meiotic prophase, confirming the ultrastructural observations made in this and other marsupial species. We also show that the specific pairing structure of marsupial sex chromosomes, the dense plate, is labelled by antibodies against the SCP3 protein of the AEs. Additionally, we show that component(s) of both sex chromosomal AEs and the DP, but not of the autosomal LEs, are phosphorylated, as revealed by MPM-2 antibody labelling. We propose that DP is formed in mid pachytene as a modification of the sex chromosomal AEs. We discuss the biological significance of the structural and behavioural features displayed by marsupial sex chromosomes during first meiotic prophase.
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Materials and Methods |
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Immunofluorescence
The seminiferous tubules were either squashed or spread prior to incubation
with antibodies. For squashing we used a technique described previously
(Page et al., 1998).
Seminiferous tubules were fixed for 10 minutes in 2% formaldehyde in PBS
containing 0.05% Triton X-100. Afterwards, several pieces of the tubules were
placed on a slide and squashed by exerting pressure on the coverslip. The
slides were immersed in liquid nitrogen and the coverslips were removed with a
knife. The slides were washed in PBS for 15 minutes and incubated with primary
antibodies. For spreading of spermatocytes, we followed the drying-down
technique of Peters et al. (Peters et al.,
1997
). Briefly, a testicular cell suspension in 100 mM sucrose was
spread onto a slide dipped in 1% paraformaldehyde in distilled water
containing 0.15% Triton X-100 and left to dry for two hours in a moist
chamber. They were subsequently washed with 0.08% Photoflo (Kodak), air dried
and rehydrated in PBS.
Both squashes and spreads were incubated with the following primary
antibodies diluted in PBS: rabbit serum A1, which recognises SCP3 protein of
the SC lateral elements (Lammers et al.,
1994) at a 1:500 dilution; rabbit serum A2, which recognises SCP1
protein of the transverse filaments and the central element of the SC
(Meuwissen et al., 1992
) at a
1:200 dilution; mAb MPM-2, which recognises mitotic phosphoproteins
(Davis et al., 1983
), kindly
provided by A. Debec (Paris, France) at a 1:1000 dilution; and human CREST
serum 098C7875, which recognises centromeric proteins, kindly provided by
Chantal Andre (Hospital Henry Mondor, Paris, France) at a 1:100 dilution. The
incubations were carried out for 1 hour at 20°C in a moist chamber. Then,
the slides were rinsed in PBS for 3x5 minutes and incubated with the
appropriate secondary antibodies: fluorescein isothiocyanate (FITC)-conjugated
goat anti-rabbit IgG (Jackson) at a 1:100 dilution, Texas Red (TR)-conjugated
goat anti-mouse IgG (Jackson) at a 1:100 dilution, and TR-conjugated goat
anti-human IgG (Jackson) at a 1:150 dilution. 45 minutes of incubation at
20°C in a moist chamber was followed by 3x5 minutes rinse in PBS and
staining with 2 µg/ml DAPI (4',6-diamidino-2-phenylindole). After a
final rinse in PBS, the slides were mounted with Vectashield (Vector
Laboratories). For double immunolocalisation the primary antibodies were
incubated simultaneously except for the double localisation of SCP1 and SCP3.
In this case the slides were first incubated for 1 hour with serum A2 against
SCP1, rinsed in PBS and incubated overnight at 4°C with a FITC-conjugated
Fab' fragment goat anti-rabbit IgG (Jackson) at a 1:100 dilution. After
3x10 minutes rinse in PBS the slides were incubated with serum A1
against SCP3 for 1 hour, rinsed 3x5 minutes in PBS and incubated with
TR-conjugated goat anti-rabbit IgG (Jackson) at a 1:150 dilution.
Observations were made on either a Nikon Optiphot or an Olympus BH2 microscope equipped with epifluorescence optics and the images were photographed on Fujichrome Provia 400F or Kodakchrome 100. Colour slides were scanned in an Agfa DuoScan T-1200 or a Polaroid SprintScan 35 scanner, and images were processed with Adobe Photoshop 6.0 software on a Power Macintosh G3.
Electron microscopy
Seminiferous tubules were processed with conventional techniques for
electron microscopy. They were fixed in 3% glutaraldehyde in 0.067 M
Sörensen phosphate buffer (pH 7.2) for 90 minutes, rinsed and postfixed
in 2% OSO4 for 1 hour and embedded in Embed 812 (EMS).
Serial ultrathin sections were obtained with a DuPont MT2-B ultramicrotome and
contrasted with uranyl acetate and lead citrate.
For spreading we followed the technique described by Solari
(Solari, 1982). A testicular
cell suspension enriched in pachytene spermatocytes was dropped over a 0.5%
NaCl solution. Spread cells were picked up on plastic-coated slides and fixed
in 4% paraformaldehyde in 0.2 M Na2B4O7
buffer (pH 8) containing 0.03% SDS for 10 minutes. The slides were then washed
in 0.04% Photoflo (Kodak), air-dried and stained with 1% PTA in ethanol.
Selected cells were transferred to parallel bar copper grids. Observations
were made on a Zeiss EM 109 transmission electron microscope operated at 80
KV. Photographs were recorded on Kodalite negative film (Kodak).
For immunocytochemistry, seminiferous tubules were fixed in 4% paraformaldehyde in 0.1 M Sörensen buffer (pH 7.3) for 2 hours. Dehydration was followed by embedding in LR White (London Resins). Silver-gold sections were obtained in a Reichert-Jung Ultracut ultramicrotome and mounted on neckel grids. The grids with the sections were incubated with serum A1 against SCP3 at dilutions of 1:200 and 1:500 for 1 hour, washed in PBS for 4x5 minutes, incubated with 10 nm gold-conjugated goat-anti rabbit IgG (EMS) for 1 hour, washed in PBS, postfixed in 2% glutaraldehyde for 2 minutes and contrasted with uranyl acetate. Observations were made on a Jeol 1010 transmission electron microscope operated at 80 kV.
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Results |
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Differentiation of chromosome AEs
The only labelling detectable with anti-SCP3 serum in leptotene
spermatocytes are small spots homogeneously distributed in the nucleus (data
not shown). Axial structures are first detectable during zygotene, when the
autosomal AEs are seen as faint threads
(Fig. 1A). At this stage, the
chromosomes show a `bouquet' arrangement with the telomeres clustered in one
region of the nucleus. Synapsis of autosomes initiates at telomeres and
extends to the interstitial regions. The synapsed regions appear as short
terminal segments where the anti-SCP3 labelling is thicker than that observed
along the unpaired AEs, which radiate from the bouquet area as arcs that
occupy the whole nuclear space (Fig.
1A). The bouquet arrangement loosens as zygotene progresses and is
completely lost by the beginning of pachytene, when autosomes are completely
synapsed (Fig. 1B-F).
AEs of sex chromosomes are strongly labelled with the anti-SCP3 serum during zygotene (Fig. 1A). They are thicker than expected for a single AE and appear even thicker than the synapsed regions of autosomes. The sex chromosomal AEs are arc-shaped, with their tips broadening at the attachment plates at the nuclear envelope. The outline of the AE of the X chromosome is irregular, showing some narrow regions (Fig. 1A'). The Y chromosome AE is very short and forms a small arc (inset in Fig. 1A'). In this phase the sex chromosomes are located in the bouquet area and usually lie apart from each other. Occasionally they appear end-to-end associated, as shown in Fig. 3G, but they disperse over the nuclear envelope as bouquet polarisation loosens.
|
Pairing of sex chromosomes
Pachytene was divided into substages (early, mid and late) in relation to
the structure and behaviour of sex chromosomes. We did not follow the
classification of meiotic stages of Rattner because in spreads and squashes
the structure of the seminiferous epithelium is disrupted
(Rattner, 1972). However, our
classification is consistent with the pachytene sequence described by Solari
and Bianchi (Solari and Bianchi,
1975
).
Early pachytene
Pachytene is identified by the complete synapsis of autosomes
(Fig. 1B). Labelling with
anti-SCP3 shows a single line along each bivalent representing the two LEs,
one per homologue, of the SC. The position of the centromeres along the
bivalents, revealed by immunolabelling with an anti-centromere CREST serum,
allowed us to identify three long submetacentric bivalents, one mid-sized
submetacentric and two small acrocentrics
(Fig. 1B-F), in agreement with
the somatic karyotype described previously
(Reig et al., 1972). At the
beginning of pachytene, sex chromosomal AEs appear shorter and thicker than in
zygotene, although the outline of the AE of the X chromosome still shows some
irregularities near the telomeres. Sex chromosomes lie separated from each
other and may frequently occupy very distant domains within the nucleus
(Fig. 1B).
However, the behaviour and structure of sex chromosomal AEs change as early pachytene proceeds. They become thinner and the AE of the X chromosome becomes regular. These changes in the morphology of AEs are coincident with a progressive approach of the sex chromosomes, until they eventually touch each other (Fig. 1C'). The contact is always made at the chromosome ends and occurs at the nuclear periphery. This is especially evident in preparations of squashed spermatocytes, where the three-dimensional organisation of chromosomes within the nucleus is maintained (Figs 2 and 4). Contact between sex chromosomal AEs marks the transition to mid pachytene.
|
Double immunolabelling with anti-SCP3 and the anti-centromere serum revealed that the X chromosome is submetacentric, and the Y chromosome is acrocentric (Fig. 1A' and B'). Therefore, we were able to distinguish Xq from Xp and Yq from Yp. We found that all possible configurations of contact occurred: sex chromosomal AEs can make contact by means of one (Fig. 2A',C') or both ends (Fig. 1C', Fig. 2B'), and the contact may be established by both short arms (Fig. 1C', Fig. 2A'), both long arms (Fig. 2C') or by X long arm with Y short arm (Fig. 2B'). Thus, these first contacts do not seem to be arm specific.
Mid pachytene
During mid pachytene sex chromosomal AEs become thinner and more regular
than in early pachytene (Fig.
1D,E). Their ends lose direct touch, becoming associated instead
by a structure labelled by anti-SCP3 (Fig.
1D'). This plate-shaped structure is continuous with the sex
chromosomal AEs and lies in the region where the chromosome ends are attached
to the nuclear envelope (Fig.
2C'). It starts to form around the tips of the sex
chromosomal AEs and increases in size throughout mid pachytene, until it
eventually includes all four ends of sex chromosomal AEs
(Fig. 1E').
Late pachytene
Sex chromosomal AEs elongate and become thinner during late pachytene
(Fig. 1F,F'). The AE of
the X chromosome folds, usually forming several loops
(Fig. 1F'). The AE of the
Y chromosome is difficult to distinguish because it is immersed in the plate
labelled by anti-SCP3, but the centromere signal indicates its presence. The
morphological changes of the sex chromosomal AEs in late pachytene are
accompanied by changes in the autosomal SCs. The labelling with anti-SCP3
starts to fade along the LEs of the bivalents and they appear fragmented
(Fig. 1F). At the end of
pachytene, the spermatocytes enter a diffuse stage characterised by
decondensation of the chromatin and an almost complete disappearance of
labelling with anti-SCP3 and the other antibodies. No later meiotic stages
(diplotene onwards) were found on the spread slides. They were present, but
rare, on squash slides, indicating that they are short-lived phases.
Given that sex chromosomal AEs are not in side-by-side contact during most
of meiotic prophase, it seems that they are not kept together by a tripartite
SC structure. To detect whether a SC central element is formed between sex
chromosomal AEs during any of the stages of first meiotic prophase, we carried
out double immunolabelling with anti-SCP3 and a serum that recognises the SCP1
protein, a component of the CE and the TFs of the SC
(Meuwissen et al., 1992).
During zygotene, anti-SCP1 label covers only those regions where the
homologues are synapsed, whereas the anti-SCP3 serum labels both the AEs and
LEs of autosomes (Fig. 3A,D). Sex chromosomal AEs, revealed by anti-SCP3 labelling, are devoid of signal
with the anti-SCP1 serum, even when sex chromosomal AEs appear occasionally
end-to-end associated (Fig.
3G). In pachytene, autosomes are fully synapsed and the signals of
anti-SCP1 and anti-SCP3 sera are completely coincident on the autosomal
bivalents (Fig. 3B,C,E,F). No
labelling with anti-SCP1 is found on the sex chromosomes during pachytene,
either when they contact by means of their ends
(Fig. 3H) or by the
anti-SCP3-labelled plate (Fig.
3I).
These results indicate that no canonical SC central element exists between sex chromosomes, even though the sex chromosomes are associated. Instead, the ends of sex chromosomal AEs associate over the nuclear envelope by means a dense plate-like structure, which holds the sex chromosomes in close apposition.
Presence of phosphoproteins on the sex chromosomal AEs
We also followed the behaviour of the sex chromosomes by means of
immunolabelling of squashed spermatocytes with MPM-2, an antibody that
recognises phosphoproteins. This antibody has proved to reveal some
component(s) of the SC in insects (Suja et
al., 1999) and fungi (van
Heemst et al., 1999
). We used MPM-2 in combination with the
anti-SCP3 serum, and we found that whereas anti-SCP3 serum labels the
autosomal LEs of SC and the sex chromosomal AEs (as described above), MPM-2
yields intense labelling only on the sex chromosomal AEs. This labelling
appears in zygotene (data not shown), and it reveals a pattern of
morphological and temporal changes of the sex chromosomal AEs identical to
that described with anti-SCP3 serum. During early-mid pachytene the thickened
sex chromosomal AEs approach each other and make contact
(Fig. 4A-D). The labelling of
both antibodies matches perfectly on the sex chromosomal AEs and the thread
that connects them (Fig. 4C,D). In late pachytene (Fig. 4E-H),
MPM-2 labelling on the X and Y AEs becomes fainter and an intense labelling,
which colocalises with the anti-SCP3 labelled plate, appears in the area of
association of sex chromosomes with the nuclear envelope. The MPM-2 labelling
of sex chromosomes indicates that either sex chromosomal AEs contain exclusive
component(s) or that a component shared with the autosomes is specifically
phosphorylated in sex chromosomal AEs.
Ultrastructure of the sex body
The structure of the sex chromosomes was also studied by means of electron
microscopy. In serial sections of mid-late pachytene spermatocytes, the sex
chromosomes are already paired and can be identified as a condensed chromatin
mass, the sex body, located at the periphery of the nucleus
(Fig. 5A,B). Inside the sex
body, axial structures corresponding to the thickened sex chromosomal AEs can
be discerned, but we never found any SC central element structure associated
with them. The sex chromosomal AEs present conspicuous expansions, the
attachment plates, where they associate with the nuclear envelope (NE)
(Fig. 5B). Additionally, an
electrondense material, the dense plate (DP), is deposited on the inner face
of the NE. The DP is a granular structure that lies on the inner membrane of
the NE, which in this region appears to be stiff and is devoid of nuclear
pores. The DP shows the same electron density as the sex chromosomal AEs.
Indeed, both structures are continuous, as if the DP had been formed by the
expansion of the sex chromosome AEs. An interesting feature of this nuclear
region is the repeated association of the centrioles and the Golgi apparatus
in the adjacent cytoplasmic region (Fig.
5A,B).
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In electron microscopic spreads (Fig.
5C-E) we detected that in early pachytene the sex chromosomal AEs
are thickened and lie apart from each other
(Fig. 5C). The outline of X AE
is irregular, with expansions at the centromeric region and its tips, the
latter representing the attachment plates to the NE. As pachytene proceeds
(Fig. 5D), sex chromosomes
approach, their AEs elongate and become thinner and electron-dense material is
detected at their tips. This material appears at first as fine threads
connecting the AEs ends (arrowheads in Fig.
5D), and as pachytene proceeds, it becomes a round plate in which
the four AEs ends are embedded (Fig.
5E). It is particularly noteworthy that this structure observed on
spread spermatocytes by electron microscopy, which some authors refer to as
`balloons' (Sharp, 1982), and
the plates revealed by immunolabelling with anti-SCP3 serum, are very similar
(compare Fig. 1 to
Fig. 5), indicating that they
may in fact represent the same structure.
Finally, we analysed labelling with anti-SCP3 serum at the ultrastructural level. Fig. 6A,B shows two consecutive sections of a pachytene spermatocyte, in which the sex body is clearly discernible as a dense mass at the nuclear periphery. At the region of association of the sex body to the NE, the DP can be discerned as a structure with higher electron density than the rest of the NE. Inside the sex body the AE of the X chromosome can be seen. The anti-SCP3 labelling mainly appears over the AE and the DP (Fig. 6C,D).
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Discussion |
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The pairing of sex chromosomes
The differentiation of sex chromosomal AEs in T. elegans, as
revealed by the labelling with the anti-SCP3 serum, starts during zygotene and
is coincident with the beginning of autosome synapsis. In zygotene, sex
chromosomes lie in the bouquet area where they are clearly distinguishable
from the autosomal AEs or SCs, but they are usually separated from each other,
although they are occasionally found joined by their ends
(Fig. 3G), or even joined to
autosomes (data not shown). These associations seem to be unstable since by
the beginning of pachytene sex chromosomes are always separated and without
contact and remain so throughout early pachytene. However, sex chromosomes
approach and pair during the transition between early and mid pachytene. This
is accompanied by changes in the organisation of sex chromosomal AEs: they
become thinner at the time of pairing, the outline of X chromosome AE becomes
regular and the trajectory of Y chromosome AE is clearly discernible. In
contrast to the occasional zygotene associations, pairing of sex chromosomes
during early-mid pachytene occurs in every nucleus, is stable and lasts (at
least) until the entry into the diffuse stage.
Since during early pachytene sex chromosomes lie apart from each other,
sometimes occupying very different nuclear domains, the confluence of the sex
chromosomes during the transition from early to mid pachytene is the result of
a secondary polarisation. In this sense, the close location of the centrioles
and Golgi complex in the adjacent cytoplasm is noteworthy
(Solari and Bianchi, 1975;
Roche et al., 1986
) (this
work). Association of the centrioles with the zygotene bouquet polarisation
has been found in man (Berrios and
Fernández-Donoso, 1990
) and other species (for a review,
see Zickler and Kleckner,
1998
). It is likely that the centrioles play some role in the late
polarisation of sex chromosomes, leading to the formation of a pachytene `late
bouquet' where the sex chromosomes ultimately find each other. It is
remarkable that this late polarisation affects sex chromosomes but not
autosomes. The remaining question is how do sex chromosomes recognise each
other. We found that during pachytene in T. elegans the first contact
of sex chromosomal AEs is established at their ends. One possibility is that
sex chromosomes bear terminal homologous regions involved in their
recognition. However, the fact that the association of sex chromosome AEs
could involve any of their ends indicates that it does not depend upon
specific chromosome arm recognition. Therefore, sex chromosomes seem to
recognise each other, but this recognition is not based on the pairing of a
specific chromosome region. This strongly supports the absence of a
pseudoautosomal region.
The asynaptic nature of the XY association
In T. elegans, we did not find sex chromosomal SC either by light
or electron microscopy. In spreads and serial sections for electron microscopy
the sex chromosomal AEs were always found too far apart to be connected by SC
central region, and no structural components of the CE were detected.
Moreover, sex chromosomes are devoid of labelling with the anti-SCP1
serum.
Several hypotheses have been postulated to explain the absence of SC in
marsupial sex chromosomes. Solari and Bianchi proposed that the small size of
the Y chromosome could be an obstacle for a proper alignment of the sex
chromosomes (Solari and Bianchi,
1975). However, SC is not formed even in species with a long Y
chromosome (Roche et al.,
1986
), suggesting that chromosome size is not a determinant
factor. On the other hand, some authors have postulated that the lack of SC is
due to the absence of shared homologous sequences between both sex
chromosomes. It is currently accepted that sex chromosomes in marsupials would
have undergone a sequential process of differentiation from each other,
leading eventually to a complete loss of homology
(Graves and Watson, 1991
). In
this context, the absence of homology may prevent the formation of SC and
therefore recombination and chiasma formation
(Hayman, 1990
). However, it has
been reported that shared sequences between sex chromosomes are present in
some marsupials as a result of translocation of the nucleolar organiser
regions (NORs) to both sex chromosomes
(Hayman, 1990
;
Toder et al., 1997
;
Toder et al., 2000
). NORs
usually form a normal SC when they are present in autosomal chromosomes in
most mammalian species, including marsupials (S.B., unpublished), but in
marsupials no SC is found between NORs when they are present in sex
chromosomes (Sharp, 1982
;
Hayman, 1990
).
Thus, it seems that marsupial sex chromosomes inhibit the formation of an
SC, even in the presence of putative homologous regions
(Hayman, 1990). This contrasts
with the behaviour of eutherian sex chromosomes, in which SC frequently
extends from the PAR to the non-homologous segments during early pachytene
(Solari, 1970
). We suggest
that the lack of SC in marsupial sex chromosomes may result from structural
modifications of their AEs. One of the features present in all marsupial sex
chromosomes studied up to now is the conspicuous thickening of their AEs. This
feature has been reported in a variety of eutherian mammals
(Solari, 1974
;
Solari, 1993
). In these cases,
the thickening mainly involves the unpaired regions of chromosomes, whereas
the LEs that participate in the formation of SC in the PAR are about as thick
as autosomal LEs. One possible explanation is that structural modifications of
sex chromosomal AEs, starting in zygotene, prevent the SC formation.
Our findings that sex AEs are the only axial structures labelled with MPM-2
antibody in T. elegans spermatocytes supports such a model. Because
SCP3 and MPM-2 colocalise on the sex chromosomes throughout meiotic prophase,
it could be possible that MPM-2 recognises a hyperphosphorylated form of SCP3.
Alternatively, MPM-2 could recognise a different component that is exclusive
to sex chromosomes. In either case, the specific presence and/or
phosphorylation of some proteins on the sex chromosomal AEs could contribute
to an inhibition of SC formation. All these structural and biochemical
modifications of the sex chromosomal AEs could be related to the programme of
meiotic sex chromosome inactivation, preventing the establishment of
heterologous interactions between sex chromosomes and other chromosomes, and
also contributing to by-passing the meiotic arrest that should be induced by
the unsynapsed sex chromosomal AEs (Handel
and Hunt, 1992; McKee and
Handel, 1993
).
Maintaining marsupial sex chromosomes together: the dense plate
In the absence of SC, it is clear that the DP maintains the association of
sex chromosomes. The DP was first described in spermatocyte sections of
Monodelphis dimidiata (Solari and
Bianchi, 1975). Afterwards, Sharp described the presence of
`balloons' in spermatocyte spreads of about twenty marsupial species
(Sharp, 1982
). Solari proposed
that the balloons could represent remnants of the nuclear envelope that would
remain attached to the sex chromosomal AEs in spreads
(Solari, 1993
). From our
results it seems clear that both the DP and the balloon are different
manifestations of the same structure, as suggested previously
(Roche et al., 1986
). The
detailed analysis throughout first meiotic prophase of sex chromosomes
structure and behaviour by means of immunofluorescence, electron microscopy
spreads and sections and immunocytochemistry techniques enabled us to
establish a morphological, temporal and compositional correlation between the
SCP3-labelled structures (Figs
1,
4 and
6), the balloons
(Fig. 5C-E) and the DP
(Fig. 5A-B, Fig. 6). Therefore, we can
conclude that all these structures are in fact the same, and we propose to
refer to them as the dense plate, as they were first named
(Solari and Bianchi,
1975
).
Our data also establish an unequivocal correlation between the sex chromosomal AEs and the DP. We show that both structures share some components, since they are labelled with anti-SCP3 and also with MPM-2, and that the development of the DP is coincident in time with the progressive thinness of the sex chromosomal AEs. Furthermore, the DP seems to be originated by the expansion of the sex chromosomal AEs ends attached to the nuclear envelope. This agrees with observations made in spermatocyte sections (Fig. 5) that show both structures are continuous. It is likely that the DP arises as a specific modification of the sex chromosomal AEs. Such a model is represented in Fig. 7. It is interesting to note that there is an inverse correlation between the anti-SCP3 labelling on sex chromosomal AEs and that found on the DP. Our results with MPM-2 also support this inverse correlation. The DP seems to be formed by the material detached from the AEs, but we cannot reject the hypothesis that the DP is also formed by the accumulation of newly synthesised components.
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The presence of SCP3 in the DP is of special interest. Despite the
polyclonal origin of the rabbit anti-SCP3 serum, the labelling on meiotic
chromosomes of T. elegans follows the same pattern described for
eutherian mammals (Lammers et al.,
1994; Dobson et al.,
1994
). Thus, anti-SCP3 labels the unpaired AEs, during zygotene
and the paired LEs in pachytene. Moreover, the presence of SCP3 in the DP has
been corroborated by the use of a guinea pig polyclonal serum against the SCP3
protein (Alsheimer and Benavente,
1996
) (data not shown). SCP3 has been related to the organisation
of the AEs and LEs of the SC, and also, as remnants of these structures, in
the maintenance of sister chromatid cohesion during meiosis
(Moens and Spyropoulos, 1995
).
We demonstrate that SCP3 participates in the formation of the DP, which is a
non-axial structure. Therefore, we report a novel unsuspected role for this
protein in marsupial meiosis.
A remaining question to be answered is whether the DP maintains the
association of sex chromosomes until they segregate during anaphase-I. Solari
and Bianchi described a folded sheet associated to the sex chromosomes in
metaphase-I in Monodelphis dimidiata
(Solari and Bianchi, 1975).
This structure most probably represents the remains of the DP. However, these
authors did not attribute any role to the folded sheet in the maintenance of
the integrity of the sex bivalent, since they assumed that a chiasma must
exist between sex chromosomes. Further studies on the orientation and
segregation of marsupial sex chromosomes during first meiotic division are
under way in order to test these hypotheses (J.P., unpublished).
From the results presented here it is clear that marsupials and eutherians
have striking differences in the meiotic process concerning the structure and
behaviour of sex chromosomes. Perhaps the most important difference is the
development of the DP as a differentiation of the sex chromosomal AEs, which
ensures their association at least during first meiotic prophase. This
indicates that some components of the SC, in this case their AEs, are capable
of a surprising plasticity, generating new structures that have been useful to
respond to new situations such as maintaining the association of two
chromosomes that have become non-homologous. Examples of asynaptic sex
chromosomes have also been reported in eutherian mammals
(Solari and Ashley, 1977).
However the DP seems to be an exclusive marsupial feature that has no
counterpart in any other vertebrate. In the current context of knowledge,
these observations open new ways of interpretation for the evolutionary
history of mammalian sex chromosomes, their meiotic behaviour and how meiotic
cells deal with non-exchange chromosomes
(Wolf, 1994
). The remarkable
fact that marsupial sex chromosomes do not form SC, together with observations
of the conspicuous modifications of the sex chromosomal AEs, indicate that the
formation of the DP may be part of an asynaptic programme that cannot be
overcome easily, even in the presence of homologous sequences. This could have
placed marsupial sex chromosomes in an evolutionary pathway completely
different from that followed by the eutherian sex chromosomes.
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