1 Division of Developmental Genetics, National Institute for Medical Research,
Mill Hill, London NW7 1AA, UK
2 Institute of Human Genetics, University of Newcastle Upon Tyne, NE1 7RU,
UK
3 INSERM U25, Hôpital Necker, 75743 Paris Cedex 15, France
4 Department of Animal Biology, School of Veterinary Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania 19104, USA
5 Whitehead Institute for Biomedical Research and Massachusetts Institute of
Technology, Cambridge, Massachusetts 02142, USA
* Author for correspondence (e-mail: pburgoy{at}nimr.mrc.ac.uk)
Accepted 12 August 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Xist/Tsix, Germline expression, Xist mutants, MacroH2A1.2, Meiotic sex chromosome inactivation, Sex body, Spermatogenesis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has been suggested that MSCI may have the same molecular basis as the
somatic X-inactivation process that ensures X-linked dosage compensation
between male (XY) and female (XX) mammals. Somatic X-inactivation requires the
cis-acting Xist RNA, that coats one of the two X-chromosomes in each
female cell (Penny et al.,
1996; Marahrens et al.,
1997
; Brockdorff,
1998
; Jaenisch et al.,
1998
; Lyon, 1999
).
The choice of which X chromosome to silence is mediated in-cis by the
antisense Tsix RNA (Lee et al.,
1999
; Lee and Lu,
1999
; Luikenhuis et al.,
2001
). Recent mutation analysis has demonstrated that the domain
responsible for X-silencing resides at the 5'-end of the Xist
RNA, while localisation of Xist RNA to the X chromosome is mediated
by functionally redundant sequences dispersed throughout the rest of the gene
(Wutz et al., 2002
). In the
male, Xist expression is restricted to the testis, the site at which
MSCI takes place (Richler et al.,
1992
; Salido et al.,
1992
; McCarrey and Dilworth,
1992
). Furthermore, it has been reported that Xist
transcripts coat the X and Y chromosomal axes in the sex body of pachytene
spermatocytes (Ayoub et al.,
1997
). The inactive somatic X chromosome, and the inactive X and Y
chromosomes of spermatocytes, also share other features such as late
replication (Priest et al.,
1967
; Kofman-Alfaro and
Chandley, 1970
; Odartchenko
and Pavillard, 1970
) and enrichment for the H2A variant
macroH2A1.2 (Costanzi and Pehrson,
1998
; Hoyer-Fender et al.,
2000
; Richler et al.,
2000
). Ayoub et al. have proposed that MSCI is brought about by
Xist RNA-mediated spreading of inactivation from the X chromosome to
the Y chromosome, via the region of X-Y pairing
(Ayoub et al., 1997
).
Despite the circumstantial evidence in favour of a common mechanism, a
striking argument against a requirement for Xist function in
spermatogenesis comes from the demonstration that fertility is not impaired in
males with a targeted Xist mutation, although this mutation disrupts
somatic X-inactivation (Marahrens et al.,
1997). This suggests either that MSCI is
Xist-independent, or that MSCI is Xist-dependent but is not
required for efficient spermatogenesis. Here, we differentiate between these
two alternatives by examining sex body formation and MSCI in male mice with
two different Xist disruptions
(Marahrens et al., 1997
;
Csankovszki et al., 1999
). We
also examine the expression profile of Xist and Tsix during
normal spermatogenesis.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
RNA extraction and RTPCR
Total RNA was extracted using Trizol® (Gibco BRL) exactly according to
manufacturer's instructions. Where DNAse treatment was required, 1µg total
RNA was incubated with 1.5 U RQ DNAse 1 (Promega) buffered with 5x First
Strand cDNA Synthesis buffer (Gibco BRL) at 37°C for 60 minutes, with
subsequent heat inactivation at 95°C for 5 minutes. For Xist and
Tsix, strand-specific reverse transcription was carried out according
to Lee et al. (Lee et al.,
1999). Briefly, 3 pmol Xist primer (MIX20
Kay et al., 1993
) or
Tsix primer (JT4-5' CGA CCT ATT CCC TTT GAC GA 3') was
added to 0.2-2µg RNA. The resulting mixture was heated to 70°C for 5
minutes, and equilibrated to 50°C. First strand cDNA was synthesised with
Superscript II reverse transcriptase (200 U, Gibco BRL) for 1 hour at
50°C. The enzyme was then heat inactivated at 90°C for 5 minutes.
Xist/Tsix RTPCR conditions were 1 cycle of 95°C 2 minutes, 35
cycles of 96°C 0.5 minutes, 55°C 0.5 minutes, 72°C 1 minute and 1
cycle 72°C 10 minutes. For Hprt and Dazla, reverse
transcription was primed with oligo-dT primers and RTPCR conditions were as
described by Mahadevaiah et al.
(Mahadevaiah et al., 1998
).
Hprt and Dazla primers were those described by Mahadevaiah
et al. (Mahadevaiah et al.,
1998
) and Cooke et al. (Cooke
et al., 1996
), respectively. In all cases an `H2O +
master mix' control was included in which the cDNA was omitted from the
reaction. All these RTPCRs utilise primers from two exons so any amplification
from contaminating DNA would give rise to a distinctly larger product.
Testis preparations and fluorescence immunostaining
Mouse spermatogenic cells were prepared as squash
(Page et al., 1998) or surface
spread preparations (Peters et al.,
1997
). Following fixation, slides were incubated in PBT (0.15%
BSA, 0.1% Tween-20 in PBS) for 60 minutes prior to incubation overnight at
37°C with primary antibodies diluted in PBT. Mouse anti-COR1 (that
recognises mouse SYCP3, Dobson et al.,
1994
) was used at 1:10. Rabbit anti-SYCP3
(Lammers et al., 1994
) was
used at 1:1000. Rabbit anti-macroH2A1.2
(Costanzi and Pehrson, 1998
)
was used at 1:100. Mouse anti-XY77
(Kralewski et al., 1997
) was
used undiluted. Mouse anti-XLR (Calenda et
al., 1994
) was used at 1:500. Rabbit anti-POLII [C-21, Santa Cruz
(Turner et al., 2000
)] was
used at 1:100. Rat anti-M31 (Turner et
al., 2001
) was used at 1:500. The rabbit anti-RBMY was a
polyclonal antibody raised as follows: amino acids 113-232 of mouse RBMY fused
with GST or thioredoxin were expressed and purified from E. coli BL21 cells as
described previously (Elliott et al.,
2000
). A rabbit was immunised with the GST fusion protein, and the
resulting antiserum affinity purified using the thioredoxin fusion protein
immobilised on a Sulfolink affinity column (Pierce). The antibody recognised a
single testis-specific band of the appropriate molecular mass that was
preabsorbable with the immunising protein (data not shown). Furthermore, as
with a previously reported RBMY antibody
(Mahadevaiah et al., 1998
),
staining of spermatogonia was seen in normal males but was abolished in
XYd1 Sry mice in which most copies of Rbmy are
deleted (J. M. A. Turner, An investigation into the role of sex chromosome
synapsis in meiotic sex chromosome inactivation and fertility, PhD thesis,
University College London, 2000). This RBMY antibody was used at 1:100. Slides
were washed three times for 5 minutes in PBS, followed by application of
secondary antibodies. Secondary antibodies used were goat anti-rabbit Cy3
(Amersham Pharmacia Biotech), goat anti-rabbit Alexa 488 (Molecular Probes),
goat anti-mouse Cy3 (Amersham Pharmacia Biotech), goat anti-mouse Alexa 488
(Molecular Probes) and goat anti-rat Alexa 488 (Molecular Probes). All
secondaries were used at 1:500 in PBS for 60 minutes at room temperature.
Slides were then washed in PBS as described above and were placed in a dark
chamber for 10 minutes. Slides were mounted with Vectashield with DAPI
(Vector). Controls consisted of omission of primary antibodies and replacement
of primary antibodies with preimmune sera. Immunostained cells were examined
and digitally imaged on an Olympus IX70 inverted microscope with a 100 W
mercury arc lamp, using a 100x 1.35 U-PLAN-APO oil immersion objective.
Each fluorochrome image was captured separately as a 12-bit source image using
a computer-assisted (Deltavision) liquid-cooled CCD (Photometrics CH350L;
Sensor: Kodak KAF1400, 1317x1035 pixels). A single multiband dichroic
mirror was used to eliminate shifts between different filters. Captured images
were processed using Adobe Photoshop 5.0.2.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Having verified that Xist was expressed in the testis, we then wished to establish the time course of its expression and whether expression was germ cell-dependent, as would be expected if it were involved in MSCI. Xist and Tsix transcripts were detected at all ages from 11.5 days post coitum (dpc) up to and including adult (Fig. 2b). Notably, Tsix transcript levels were always considerably lower than those of Xist, although we cannot discount the possibility that this reflects differences in the efficiency of JT4 and MIX20 as primers for strand-specific reverse transcription.
To assess whether transcription of Xist and Tsix was germ
cell-dependent, we analysed Xist and Tsix expression in
embryonic We/We testis and testis from adult XO
males transgenic for the testis determinant Sry, both of which are
deficient in germ cells (Mahadevaiah et
al., 1998; Mazeyrat et al.,
2001
). The degree of germ cell deficiency was monitored by
carrying out RTPCR for Dazla, an autosomally encoded germ
cell-specific gene (Cooke et al.,
1996
), and Dazla-positive samples were excluded. Neither
Xist nor Tsix transcripts were detectable in
Dazla-negative We/We and
XOSry testes (Fig.
2c). Collectively, these results indicate that both Xist
and Tsix are expressed in the testis, that expression of both genes
begins from a very early stage of testis development and that expression of
both genes is germ cell-dependent.
Sex body formation in Xisttrun and
Xist1lox spermatocytes
According to Solari (Solari,
1974), sex body formation in the mouse begins during zygotene; at
the light microscope level a morphologically distinct sex body is apparent by
midpachytene (Turner et al.,
2000
). During diplotene, the sex chromosomes migrate towards the
centre of the nucleus and, by metaphase I, they exhibit a similar condensation
state to that of the autosomes. We wished to ascertain if sex bodies were
formed in Xisttrun and Xist1lox
spermatocytes and, if they were, whether the timing of their formation and
disassembly resembled that of controls. To do this, we performed marker
analysis using immunostaining for proteins known to associate preferentially
or specifically with sex chromosomes during different stages of meiosis. In
each case, spermatocytes were substaged by immunostaining for the axial
element protein SYCP3 (Lammers et al.,
1994
).
In order to assess the full time course of sex body formation and loss we
first used immunostaining for the phosphorylated form of histone H2AX, termed
-H2AX (Rogakou et al.,
1999
).
-H2AX marks the chromatin of the X and Y-chromosomes
as they first condense in late zygotene/early pachytene and continues to do so
until the sex body is lost during diakinesis/metaphase I
(Mahadevaiah et al., 2001
). In
Xisttrun (n=142) and Xist1lox
(n=167) spermatocytes just as in normal spermatocytes
(n=107),
-H2AX staining appeared on the sex chromosomes in
late zygotene and persisted throughout pachytene, before being lost at
metaphase I (Fig. 3).
|
As a more specific sex body marker, we used immunostaining for the XY77
protein, which associates with the chromatin of the sex chromosomes of late
pachytene spermatocytes (Kralewski et al.,
1997). This protein does not localise to meiotic sex chromosomes
in circumstances when they are not inactivated, such as in XY female oocytes
(Turner et al., 2000
). This
immunostaining requires squash preparations in which the sex body is less
clearly demarcated, the sex body can nonetheless be identified by DAPI
staining. Sex bodies identified by this criterion in
Xisttrun (n=75) and Xist1lox
(n=80) spermatocytes stained positively for XY77, just as in normal
males (n=81, Fig.
4).
|
Next we analysed the sex body status of diplotene
Xisttrun and Xist1lox spermatocytes
using immunostaining for M31. M31 is a mammalian HP1-like protein that has
been implicated in transcriptional repression, transgene silencing and MSCI
(Jones et al., 2000;
Motzkus et al., 1999
). In
testis sections, M31 has been shown to localise to the sex body during late
prophase (Motzkus et al.,
1999
), specifically during diplotene
(Turner et al., 2001
). Once
again, we detected sex bodies by DAPI staining in wild type (n=42),
Xisttrun (n=45) and Xist1lox
(n=40) spermatocytes and in each case these stained positively for
M31 (Fig. 4).
Finally, we analysed the sex bodies of Xist mutant males by
immunostaining for the core histone macroH2A1.2. MacroH2A1.2 is concentrated
on the somatic inactive X chromosome
(Costanzi and Pehrson, 1998;
Mermoud et al., 1999
;
Rasmussen et al., 2000
) in an
Xist-dependent manner (Csankovszki
et al., 1999
) and is also enriched on the chromatin of the sex
chromosomes of early pachytene spermatocytes
(Hoyer-Fender et al., 2000
;
Richler et al., 2000
).
Consistent with the section data of Hoyer-Fender et al.
(Hoyer-Fender et al., 2000
),
we detected macroH2A1.2 in the sex body of early pachytene spermatocytes from
normal males (n=56) and this localisation was maintained in
Xisttrun (n=62) and Xist1lox
spermatocytes (n=82, Fig.
5).
|
Together, these experiments show that sex body formation proceeds normally in spermatocytes harbouring Xist disruptions.
MSCI in Xisttrun and Xist1lox
spermatocytes
Transcriptional inactivation of the sex chromosomes during meiosis can be
readily demonstrated by visualising the distribution of transcriptional and
pre-mRNA splicing factors in the autosomal versus the sex chromosome domains
(Richler et al., 1994). For
example, RNA polymerase II (RNA POLII) is present at high concentration
throughout the autosomal chromatin but is virtually absent in the sex body
during pachytene (Richler et al.,
1994
; Turner et al.,
2000
). In contrast, RNA POLII uniformly coats all chromosomes in
XX and XY female oocytes, in which no sex bodies are formed
(Turner et al., 2000
). We
therefore examined RNA POLII staining of the sex chromosomes in
Xisttrun and Xist1lox spermatocytes
(Fig. 6). During wild type
spermatogenesis, RNA POLII levels were found to be highest during late
pachytene/early diplotene and it was at this stage that exclusion of RNA POLII
from the sex body could be most easily visualised (data not shown). Analysis
of Xisttrun (n=45) and
Xist1lox (n=49) late pachytene/early diplotene
surface spread spermatocytes (identified by anti-SYCP3 staining) revealed
marked exclusion or absence of RNA POLII from the sex chromosome domain in all
cells analysed (Fig. 6). We
conclude that global transcriptional inactivation of the sex chromosomes is
achieved in Xisttrun and Xist1lox
spermatocytes.
|
In a previous study, Ayoub et al. used an in-situ RTPCR technique to
demonstrate that Xist was present in the sex body during pachytene
where it coated the axes of both the X and Y chromosome
(Ayoub et al., 1997). They
hypothesised that inactivation of the Y chromosome was achieved by
`quasi-cis' spreading of Xist transcripts from the X
chromosome to the Y chromosome via the X-Y pairing (pseudoautosomal) region.
In this model, Y-linked genes would fail to undergo MSCI in
Xist-disrupted spermatocytes. To test this hypothesis, we examined
expression of the multiple-copy Y-linked gene Rbmy during
Xisttrun and Xist1lox spermatogenesis.
In normal males, Rbmy is strongly expressed in spermatogonia, is
transcriptionally repressed during meiosis and is reactivated
post-meiotically, suggesting that it is subject to MSCI
(Mahadevaiah et al., 1998
).
This is supported by our finding that inappropriate expression of RBMY occurs
in pachytene cells of XYY males, in which there is evidence of disrupted sex
body formation (J. M. A. Turner, PhD thesis, University College London, 2000).
RBMY staining was performed on squash preparations in which spermatocytes were
substaged on the basis of immunostaining for the spermatocyte-specific XMR
protein (Calenda et al., 1994
).
The pattern of RBMY staining in Xisttrun and
Xist1lox spermatocytes was indistinguishable from controls
(Fig. 7). Taken together, these
results suggest that MSCI proceeds normally in males with targeted
Xist-disruptions.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Were Xist to mediate MSCI, then we would expect Xist
expression to begin in the germ cells around the time that MSCI commences. Our
data, which show that Xist transcription is germ cell-dependent, are
consistent with those of Salido et al.
(Salido et al., 1992) but not
with those of Kay et al. (Kay et al.,
1993
), who detected Xist transcripts in germ cell
deficient Wv/Wv testes
(Lyon and Searle, 1989
).
However, Wv/Wv testes invariably have some germ
cells remaining, and even with the more penetrant We
allele we have encountered some Dazla-positive (germ cells still
present), Xist-positive testes. There also appears to be a
discrepancy between different reports regarding the timing of Xist
expression. Both the present study and that of Jamieson et al.
(Jamieson et al., 1997
)
detected Xist transcripts from as early as 11.5 dpc. This clearly
does not fit well with the timing of MSCI, which has been estimated by
quantitative RTPCR for selected X-linked genes to commence no earlier than the
spermatogonial A and B stages, i.e. around 6-8 dpp
(Singer-Sam et al., 1990
;
McCarrey et al., 1992
). In
contrast to our results, McCarrey and Dilworth
(McCarrey and Dilworth, 1992
)
did not detect Xist transcripts until after birth, around the time
that primitive type A spermatogonia appear, which more closely corresponds to
the timing of onset of MSCI. However, their RNA samples came from purified
germ cell populations, and it is possible that the low levels of Xist
transcripts are depleted during the purification procedure.
A second prediction of the hypothesis that MSCI is Xist-dependent
is that disruption of Xist function should abolish MSCI and sex body
formation. We have not found this to be the case. We encountered no difference
in the timing of sex body formation, in the protein content of sex bodies as
judged by marker analysis, or in MSCI between wild type,
Xisttrun and Xist1lox spermatocytes.
Interestingly, the Xist1lox mutation disrupts localisation
of macroH2A1.2 to the somatic inactive X-chromosome
(Csankovszki et al., 1999) but
not to the sex body. A possible explanation for this is that the transcript
produced in male spermatogenic cells (see supplementary figure) but not female
somatic cells (Csankovszki et al.,
1999
) carrying this mutation, retains the 3' region of the
transcript necessary for macroH2A1.2 recruitment
(Wutz et al., 2002
). The role
of macroH2A1.2 in somatic X-inactivation remains undetermined, but the timing
of its appearance suggests that macroH2A1.2 functions downstream of the
initiation of X-inactivation (Mermoud et
al., 1999
; Rasmussen et al.,
2000
). In the male macroH2A1.2 similarly appears after the
initiation of MSCI. Perhaps in both cases, macroH2A1.2 plays a role in
initiating heterochromatinisation. However, Perche et al.
(Perche et al., 2000
) have
suggested that macroH2A1.2 localisation to the Barr body is merely a
reflection of increased nucleosomal density resulting from
heterochromatinisation (see also Rasmussen
et al., 2001
; Constanzi and Pehrson, 2001). During spermatogenesis
it is our impression that macroH2A1.2 localisation to X and Y chromatin
immediately precedes condensation and heterochromatinisation but this needs to
be confirmed with a careful time course for these processes.
Since the males with both Xist disruptions studied here produce
truncated Xist transcripts, it could be that the truncated
transcripts are functioning to bring about MSCI. However, this seems unlikely
in the light of the recent mapping of functional domains within the
Xist transcript by Wutz et al.
(Wutz et al., 2002). The
5' repeat domain shown to be required for transcriptional silencing is
deleted in the Xist1lox males and in both mutations the
sequences required for localisation of the transcripts to the X chromosome in
somatic cells, are sufficiently depleted to prevent Xist function in
females. If these truncated transcripts are functioning in MSCI, it must be by
a very different mechanism than that involved in somatic X-inactivation.
If MSCI is indeed completely Xist-independent, the question of why
Xist and Tsix are expressed in the testis deserves
consideration. One explanation is that Xist and Tsix may be
transcribed illegitimately as a result of the programmed demethylation of the
Xist locus that occurs in the male germline
(Norris et al., 1994). This
demethylation event has been proposed to underlie the imprinted paternal
Xist expression that occurs in the trophectoderm and primitive
endoderm during early female embryogenesis
(Kay et al., 1993
;
Norris et al., 1994
;
Rastan, 1994
). Both Kay et al.
(Kay et al., 1993
) and
McCarrey and Dilworth (McCarrey and
Dilworth, 1992
) have highlighted the fact that the expression
level of Xist in the testis is considerably lower than that of female
somatic cells. These observations, together with the inability to detect
Xist transcripts by conventional in-situ analysis
(Salido et al., 1992
;
Ayoub et al., 1997
) throw doubt
on the possible functional significance of such low-level Xist
expression, and we have found the Tsix transcript levels to be even
lower than those of Xist.
Our study highlights an important mechanistic difference between female
somatic and male germline X-inactivation and poses the question of how MSCI is
controlled. Progress in this direction is slow in coming and has to date
relied on identification of proteins that preferentially associate with the
sex body (Smith and Benavente,
1992; Calenda et al.,
1994
; Smith and Benavente,
1995
; Kralewski et al.,
1997
; Bauer et al.,
1998
; Motzkus et al.,
1999
; Parraga and del Mazo,
2000
; Hoyer-Fender et al.,
2000
; Richler et al.,
2000
; O'Carroll et al.,
2000
; Turner et al.,
2000
). Perhaps the most promising candidates are those with proven
roles in transcriptional repression and/or heterochromatinisation, such as M31
(Motzkus et al., 1999
),
macroH2A1.2 (Hoyer-Fender et al.,
2000
; Richler et al.,
2000
) and Suv39h2 (O'Carroll
et al., 2000
). Conditional disruption within the male germ line of
the genes encoding these and other sex body-associating proteins may provide
insights into the mechanism, and even more importantly, the role of MSCI.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Armstrong, S. J., Kirkham, A. J. and Hultén, M. A. (1994). XY chromosome behaviour in the germ-line of the human male: a FISH analysis of spatial orientation, chromatin condensation and pairing. Chromosome Res. 2, 445-452.[Medline]
Ayoub, N., Richler, C. and Wahrman, J. (1997). Xist RNA is associated with the transcriptionally inactive XY body in mammalian male meiosis. Chromosoma 106, 1-10.[CrossRef][Medline]
Bauer, U. M., Schneider-Hirsch, S., Reinhardt, S., Benavente, R. and Maelicke, A. (1998). The murine nuclear orphan receptor GCNF is expressed in the XY body of primary spermatocytes. FEBS Lett. 439,208 -214.[CrossRef][Medline]
Brockdorff, N. (1998). The role of Xist in X-inactivation. Curr. Opin. Genet. Dev. 8, 328-333.[CrossRef][Medline]
Calenda, A., Allenet, B., Escalier, D., Bach, J. F. and Garchon, H. J. (1994). The meiosis-specific Xmr gene product is homologous to the lymphocyte Xlr protein and is a component of the XY body. EMBO J. 13,100 -109.[Abstract]
Cooke, H. J., Lee, M., Kerr, S. and Ruggiu, M.
(1996). A murine homologue of the human DAZ gene is autosomal and
expressed only in male and female gonads. Hum. Mol.
Genet. 5,513
-516.
Costanzi, C. and Pehrson, J. R. (1998). Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals. Nature 393,599 -601.[CrossRef][Medline]
Costanzi, C. and Pehrson, J. R. (2001).
MACROH2A2, a new member of the MACROH2A core histone family. J.
Biol. Chem. 276,21776
-21784.
Csankovszki, G., Panning, B., Bates, B., Pehrson, J. R. and Jaenisch, R. (1999). Conditional deletion of Xist disrupts histone macroH2A localization but not maintenance of X inactivation. Nat. Genet. 22,323 -324.[CrossRef][Medline]
Dobson, M. J., Pearlman, R. E., Karaiskakis, A., Spyropoulos, B.
and Moens, P. B. (1994). Synaptonemal complex proteins:
occurrence, epitope mapping and chromosome disjunction. J. Cell.
Sci. 107,2749
-2760.
Elliott, D. J., Bourgeois, C. F., Klink, A., Stevenin, J. and
Cooke, H. J. (2000). A mammalian germ cell-specific RNA
binding protein interacts with ubiquitously expressed proteins involved in
splice site selection. Proc. Natl. Acad. Sci. USA
97,5717
-5722.
Hendriksen, P. J., Hoogerbrugge, J. W., Themmen, A. P., Koken, M. H., Hoeijmakers, J. H., Oostra, B. A., van der Lende, T. and Grootegoed, J. A. (1995). Postmeiotic transcription of X and Y chromosomal genes during spermatogenesis in the mouse. Dev. Biol. 170,730 -733.[CrossRef][Medline]
Hoyer-Fender, S., Costanzi, C. and Pehrson, J. R. (2000). Histone macroH2A1.2 is concentrated in the XY-body by the early pachytene stage of spermatogenesis. Exp. Cell Res. 258,254 -260.[CrossRef][Medline]
Jablonka, E. and Lamb, M. J. (1988). Meiotic pairing constraints and the activity of sex chromosomes. J. Theor. Biol. 133,23 -36.[Medline]
Jaenisch, R., Beard, C., Lee, J., Marahrens, Y. and Panning, B. (1998). Mammalian X chromosome inactivation. Novartis Found. Symp. 214,200 -209.[Medline]
Jamieson, R. V., Zhou, S. X., Tan, S. S. and Tam, P. P. (1997). X-chromosome inactivation during the development of the male urogenital ridge of the mouse. Int. J. Dev. Biol. 41, 49-55.[Medline]
Jones, D. O., Cowell, I. G. and Singh, P. B. (2000). Mammalian chromodomain proteins: their role in genome organisation and expression. Bioessays 22,124 -137.[CrossRef][Medline]
Kay, G. F., Penny, G. D., Patel, D., Ashworth, A., Brockdorff, N. and Rastan, S. (1993). Expression of Xist during mouse development suggests a role in the initiation of X chromosome inactivation. Cell 72,171 -182.[Medline]
Kofman-Alfaro, S. and Chandley, A. C. (1970). Meiosis in the male mouse. An autoradiographic investigation. Chromosoma 31,404 -420.[Medline]
Kralewski, M., Novello, A. and Benavente, R. (1997). A novel Mr 77,000 protein of the XY body of mammalian spermatocytes: its localization in normal animals and in Searle's translocation carriers. Chromosoma 106,160 -167.[CrossRef][Medline]
Lammers, J. H., Offenberg, H. H., van Aalderen, M., Vink, A. C., Dietrich, A. J. and Heyting, C. (1994). The gene encoding a major component of the lateral elements of synaptonemal complexes of the rat is related to X-linked lymphocyte-regulated genes. Mol. Cell. Biol. 14,1137 -1146.[Abstract]
Lee, J. T. and Lu, N. (1999). Targeted mutagenesis of Tsix leads to nonrandom X inactivation. Cell 99,47 -57.[Medline]
Lee, J. T., Davidow, L. S. and Warshawsky, D. (1999). Tsix, a gene antisense to Xist at the X-inactivation centre. Nat. Genet. 21,400 -404.[CrossRef][Medline]
Lifschytz, E. and Lindsley, D. L. (1972). The role of X chromosome inactivation during spermatogenesis. Proc. Natl. Acad. Sci. USA 69,182 -186.[Abstract]
Luikenhuis, S., Wutz, A. and Jaenisch, R.
(2001). Antisense transcription through the Xist locus
mediates Tsix function in embryonic stem cells. Mol. Cell.
Biol. 21,8512
-8520.
Lyon, M. F. (1999). X-chromosome inactivation. Curr. Biol. 9,R235 -237.[CrossRef][Medline]
Lyon, M. F. and Searle, A. G. (1989). Genetic Variants and Strains of the Laboratory Mouse (2nd edn). Oxford University Press.
Mahadevaiah, S. K., Odorisio, T., Elliott, D. J., Rattigan, A.,
Szot, M., Laval, S. H., Washburn, L. L., McCarrey, J. R., Cattanach, B. M.,
Lovell-Badge, R. and Burgoyne, P. S. (1998). Mouse homologues
of the human AZF candidate gene RBM are expressed in spermatogonia and
spermatids, and map to a Y chromosome deletion interval associated with a high
incidence of sperm abnormalities. Hum. Mol. Genet.
7, 715-727.
Mahadevaiah, S. K., Turner, J. M., Baudat, F., Rogakou, E. P., de Boer, P., Blanco-Rodriguez, J., Jasin, M., Keeney, S., Bonner, W. M. and Burgoyne, P. S. (2001). Recombinational DNA double-strand breaks in mice precede synapsis. Nat. Genet. 27,271 -276.[CrossRef][Medline]
Marahrens, Y., Panning, B., Dausman, J., Strauss, W. and Jaenisch, R. (1997). Xist-deficient mice are defective in dosage compensation but not in spermatogenesis. Genes Dev. 11,156 -166.[Abstract]
Mazeyrat, S., Saut, N., Grigoriev, V., Mahadevaiah, S. K., Ojarikre, O. A., Rattigan, Á., Bishop, C., Eicher, E. M., Mitchell, M. J. and Burgoyne, P. S. (2001). A Y-encoded subunit of the translation initiation factor Eif2 is essential for mouse spermatogenesis. Nat. Genet. 29,49 -53.[CrossRef][Medline]
McCarrey, J. R. and Dilworth, D. D. (1992). Expression of Xist in mouse germ cells correlates with X-chromosome inactivation. Nat. Genet. 2, 200-203.[Medline]
McCarrey, J. R., Dilworth, D. D. and Sharp, R. M. (1992). Semiquantitative analysis of X-linked gene expression during spermatogenesis in the mouse: ethidium-bromide staining of RT-PCR products. Genet. Anal. Tech. Appl. 9, 117-123.[Medline]
McKee, B. D. and Handel, M. A. (1993). Sex chromosomes, recombination and chromatin conformation. Chromosoma 102,71 -80.[Medline]
Mermoud, J. E., Costanzi, C., Pehrson, J. R. and Brockdorff,
N. (1999). Histone macroH2A1.2 relocates to the inactive X
chromosome after initiation and propagation of X-inactivation. J.
Cell Biol. 147,1399
-1408.
Motzkus, D., Singh, P. B. and Hoyer-Fender, S. (1999). M31, a murine homolog of Drosophila HP1, is concentrated in the XY body during spermatogenesis. Cytogenet. Cell Genet. 86,83 -88.[CrossRef][Medline]
Norris, D. P., Patel, D., Kay, G. F., Penny, G. D., Brockdorff, N., Sheardown, S. A. and Rastan, S. (1994). Evidence that random and imprinted Xist expression is controlled by pre-emptive methylation. Cell 77,41 -51.[Medline]
O'Carroll, D., Scherthan, H., Peters, A. H., Opravil, S.,
Haynes, A. R., Laible, G., Rea, S., Schmid, M., Lebersorger, A., Jerratsch, M.
et al. (2000). Isolation and characterization of
Suv39h2, a second histone H3 methyltransferase gene that displays
testis-specific expression. Mol. Cell. Biol.
20,9423
-9433.
Odartchenko, N. and Pavillard, M. (1970). Late DNA replication in male mouse meiotic chromosomes. Science 167,1133 -1134.[Medline]
Odorisio, T., Rodriguez, T. A., Evans, E. P., Clarke, A. R. and Burgoyne, P. S. (1998). The meiotic checkpoint monitoring synapsis eliminates spermatocytes via p53-independent apoptosis. Nat. Genet. 18,257 -261.[CrossRef][Medline]
Page, J., Suja, J. A., Santos, J. L. and Rufas, J. S. (1998). Squash procedure for protein immunolocalization in meiotic cells. Chromosome Res. 6, 639-642.[CrossRef][Medline]
Parraga, M. and del Mazo, J. (2000). XYbp, a novel RING-finger protein, is a component of the XY body of spermatocytes and centrosomes. Mech. Dev. 90, 95-101.[CrossRef][Medline]
Perche, P. Y., Vourc'h, C., Konecny, L., Souchier, C., Robert-Nicoud, M., Dimitrov, S. and Khochbin, S. (2000). Higher concentrations of histone macroH2A in the Barr body are correlated with higher nucleosome density. Curr. Biol. 10,1531 -1534.[CrossRef][Medline]
Penny, G. D., Kay, G. F., Sheardown, S. A., Rastan, S. and Brockdorff, N. (1996). Requirement for Xist in X chromosome inactivation. Nature 379,131 -137.[CrossRef][Medline]
Peters, A. H., Plug, A. W., van Vugt, M. J. and de Boer, P. (1997). A drying-down technique for the spreading of mammalian meiocytes from the male and female germline. Chromosome Res. 5,66 -68.[CrossRef][Medline]
Priest, J. H., Heady, J. E. and Priest, R. E.
(1967). Delayed onset of replication of human X chromosomes.
J. Cell Biol. 35,483
-487.
Rasmussen, T. P., Mastrangelo, M. A., Eden, A., Pehrson, J. R.
and Jaenisch, R. (2000). Dynamic relocalization of histone
MacroH2A1 from centrosomes to inactive X chromosomes during X inactivation.
J. Cell Biol. 150,1189
-1198.
Rasmussen, T. P., Wutz, A. P., Pehrson, J. R. and Jaenisch, R. R. (2001). Expression of Xist RNA is sufficient to initiate macrochromatin body formation. Chromosoma 110,411 -420.[CrossRef][Medline]
Rastan, S. (1994). X chromosome inactivation and the Xist gene. Curr. Opin. Genet. Dev. 4, 292-297.[Medline]
Richler, C., Soreq, H. and Wahrman, J. (1992). X inactivation in mammalian testis is correlated with inactive X-specific transcription. Nat. Genet. 2, 192-195.[Medline]
Richler, C., Ast, G., Goitein, R., Wahrman, J., Sperling, R. and Sperling, J. (1994). Splicing components are excluded from the transcriptionally inactive XY body in male meiotic nuclei. Mol. Biol. Cell 5,1341 -1352.[Abstract]
Richler, C., Dhara, S. K. and Wahrman, J. (2000). Histone macroH2A1.2 is concentrated in the XY compartment of mammalian male meiotic nuclei. Cytogenet. Cell Genet. 89,118 -120.[Medline]
Rogakou, E. P., Boon, C., Redon, C. and Bonner, W. M.
(1999). Megabase chromatin domains involved in DNA double-strand
breaks in vivo. J. Cell Biol.
146,905
-916.
Salido, E. C., Yen, P. H., Mohandas, T. K. and Shapiro, L. J. (1992). Expression of the X-inactivation-associated gene XIST during spermatogenesis. Nat. Genet. 2, 196-199.[Medline]
Singer-Sam, J., Robinson, M. O., Bellve, A. R., Simon, M. I. and Riggs, A. D. (1990). Measurement by quantitative PCR of changes in Hprt, Pgk-1, Pgk-2, Aprt, MTase, and Zfy gene transcripts during mouse spermatogenesis. Nucleic Acids Res. 18,1255 -1259.[Abstract]
Smith, A. and Benavente, R. (1992). Meiosis-specific protein selectively associated with sex chromosomes of rat pachytene spermatocytes. Proc. Natl. Acad. Sci. USA 89,6938 -6942.[Abstract]
Smith, A. and Benavente, R. (1995). An Mr 51,000 protein of mammalian spermatogenic cells that is common to the whole XY body and centromeric heterochromatin of autosomes. Chromosoma 103,591 -596.[CrossRef][Medline]
Solari, A. J. (1974). The behavior of the XY pair in mammals. Rev. Cytol. 38,273 -317.
Turner, J. M. A., Mahadevaiah, S. K., Benavente, R., Offenberg, H. H., Heyting, C. and Burgoyne, P. S. (2000). Analysis of male meiotic sex body proteins during XY female meiosis provides new insights into their functions. Chromosoma 109,426 -432.[Medline]
Turner, J. M., Burgoyne, P. S. and Singh, P. B.
(2001). M31 and macroH2A1.2 colocalise at the pseudoautosomal
region during mouse meiosis. J. Cell Sci.
114,3367
-3375.
Wutz, A., Rasmussen, T. P. and Jaenisch, R. (2002). Chromosomal silencing and localization are mediated by different domains of Xist RNA. Nat. Genet. 30,167 -174.[CrossRef][Medline]