Department of Cell Biology, Duke University, Durham NC 27710, USA
Author for correspondence (e-mail:
b.capel{at}cellbio.duke.edu)
Accepted 14 August 2003
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SUMMARY |
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Key words: Primordial Germ cells, Meiosis, Sex Determination, Gonad, Testis, Sry
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Introduction |
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In the absence of the Sry gene, ovarian fate proceeds
(Gubbay et al., 1992;
Hawkins et al., 1992
). In
contrast to the case in the XY gonad, germ cells are crucial for the formation
and maintenance of ovarian structure. In the absence of germ cells, ovarian
follicles do not assemble, and when germ cells are lost, ovarian follicles
rapidly degenerate (McLaren,
1988
). By 13.5 dpc, germ cells in the XX gonad enter meiosis and
arrest in prophase I by birth (McLaren,
1988
). The timing of germ cell entry into meiosis appears to be
based on an intrinsic clock. Germ cells enter meiosis around 13.5 dpc even
when they develop in regions outside the gonad such as adrenal glands and the
mesonephros (Zamboni and Upadhyay,
1983
), or when they are assembled in lung aggregates in culture
(McLaren and Southee,
1997
).
Several pieces of evidence indicate that the male pathway must be initiated
within a narrow window in development. During normal gonad development, male
and female fates are mutually exclusive; testis and ovarian structures
normally do not co-exist. One exception is the formation of ovotestes in
hermaphrodites where the YPOS chromosome from Mus domesticus
poschiavinus is crossed onto Mus musculus musculus strains,
notably C57BL/6. These ovotestes typically consist of testis cords in the
mid-region of the gonad and ovarian structure in the polar regions
(Bradbury, 1987). Based on
these data, it was hypothesized that there is a requirement for the
testis-determining gene to act during a narrow window of time, and above a
crucial threshold, to initiate the testis pathway and avert the competing
ovarian pathway (Burgoyne and Palmer,
1991
; Eicher and Washburn,
1986
). Consistent with this idea, recent molecular evidence has
provided a strong correlation between delayed and/or lowered expression of
Sry, and ovotestis development in C57BL/6 XYPOS mice. If
Sry expression is delayed by 24 hours, complete or partial sex
reversal occurs in XY gonads (Eicher et
al., 1995
; Nagamine et al.,
1998
; Washburn et al.,
2001
).
Organ culture experiments provide further evidence for a narrow
developmental window for the initiation of testis development. Cellular events
downstream of Sry, including mesonephric cell migration, can be
induced in XX gonads in organ culture during the bi-potential stage. However
induction of these elements of the testis pathway can occur in XX gonads only
prior to 13.5 dpc (Tilmann and Capel,
1999). These results are consistent with the window for the
initiation of testis development predicted on the basis of the C57BL/6
XYPOS ovotestis (Burgoyne and
Palmer, 1991
; Eicher and
Washburn, 1986
). These experiments strongly suggested that the
timing of the initiation of testis development is crucial because of changes
that occur in the gonad that result in loss of the bipotentiality of the organ
primordia. What was not clear from these experiments was whether these changes
were dependent on somatic or germ cells in the XX gonad.
To test whether somatic or germ cells are responsible for the resistance of
XX gonads to induction of the testis pathway after 12.5 dpc, we compared
mesonephric cell migration in organ culture assays using XX gonads with or
without meiotic germ cells. We also investigated whether XX germ cells or
somatic cells interfere with testis cord formation by generating XXXY
recombinant aggregates in culture. Results of these experiments indicate that
the physical presence of germ cells inhibits initiation of the testis pathway.
We show that the stage when germ cells from XX gonads inhibit the male pathway
is temporally correlated with the time that germ cells spontaneously enter
meiosis. We propose that once germ cells commit to meiosis, they are
antagonistic to the testis pathway as the result of signaling changes that
occur at this stage.
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Materials and methods |
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Germ cell depletion by busulfan treatment
Pregnant females were injected IP with 100 µl busulfan solution (16
mg/ml of 50% DMSO) or 50% DMSO (control) at 10.5 dpc. Busulfan at this
concentration was effective at depleting more than 98% of germ cells in rat
(Merchant, 1975) and in mouse
gonads based on alkaline phosphatase staining
(De Felici et al., 1989
).
Embryos from the treated females were obtained at 11.5, 13.5 or 14.5 dpc for
isolation of the gonad for mesonephric cell migration assays.
Mesonephric cell migration assay
XY gonads from CD1 embryos (12.5 dpc), XX gonads from +/+,
KitW/W-v embryos or busulfan-treated embryos (11.5, 13.5
or 14.5 dpc), and mesonephroi from 11.5 dpc GFP embryos were obtained and
assembled as illustrated in Fig.
3. The recombinant explants were assembled on an 1.5% agar block
and cultured for 48 hours in Dulbecco's Minimal Eagle Medium (DMEM)
supplemented with 10% fetal calf serum (Hyclone) and 50 µg/ml ampicillin at
37°C with 5% CO2/95% air
(Martineau et al., 1997).
Migration images were obtained using a Leica MZFLIII dissecting microscope
with a GFP filter. After 48 hours of culture, the explants were fixed and
processed for either immunocytochemistry or in situ hybridization.
|
Generation of recombinant aggregates
After the germ cell and somatic cell fractions were obtained as described
above, the cell suspension was centrifuged at 2000 g for 5
minutes and cell pellets were resuspended in 100 µl pre-warmed culture
medium. The number of germ cells in the cell suspensions were estimated by
counting alkaline phosphatase-positive cells present in aliquots according to
De Felici and colleagues (De Felici et al.,
1989). Volumes were adjusted such that 50 µl of the somatic
cell fraction from 12.5 dpc XY gonads, combined with 50 µl of the germ cell
or somatic cell test fractions at different stages, resulted in a cell ratio
of
1.5:1.0. One µl phytohemagglutinin-P (Sigma #L9132, 5 mg/ml in
water) was added to the cell mixture. The cell mixture (about 100 µl) was
back-loaded into an ordinary sequencing gel loading tip sealed with a flame.
The loaded tip was placed in a 15 ml Corning tube and centrifuged at 2000
g for 5 minutes. The sealed end of the tip was cut off and the
aggregate was expelled to a groove in an agar block using a filtered mouth
pipette. The aggregates were cultured in Dulbecco's Minimal Eagle Medium
(DMEM) supplemented with 10% fetal calf serum (Hyclone) and 50 ug/ml
ampicillin for 48 hours at 37°C with 5% CO2/95% air.
Immunocytochemistry
For immunostaining against laminin, samples were fixed overnight in 4%
paraformaldehyde in PBS at 4°C. For immunostaining against PECAM-1,
phosphorylated H2AX (H2AX) and SYN/COR, samples were fixed for 1 hour
in 1% paraformaldehyde at 4°C. Samples were then processed and cut into 8
µm frozen sections as described (Karl
and Capel, 1998
). Sections were blocked for 1 hour at room
temperature in blocking solution (PBS/10% heat inactivated goat serum/0.1%
Triton-X 100). Primary antibody incubations were carried out overnight at
4°C in blocking solution (1:200 dilution of rabbit anti-laminin1 antibody,
provided by Harold Erickson; 1:500 dilution of rat anti-PECAM-1 antibody,
PharMingen; 1:800 dilution of a rabbit polyclonal antibody against SYN1/COR1,
provided by Peter Moens; 1:800 dilution of a rabbit polyclonal antibody
against
H2AX, provided by William Bonner). Sections were washed three
times in washing solution (PBS/1% heat inactivated goat serum/0.1% Triton-X
100) for 5 minutes each. Secondary antibody incubations were performed
overnight at 4°C with 1:500 dilution of fluorescently conjugated secondary
antibodies (FITC- or Cy5-conjugated goat anti-rabbit antibody and
Cy3-conjugated goat anti-rat antibody, Jackson Immunochemicals). Sections were
washed three times for 5 minutes each in washing solution and mounted on glass
slides in DABCO. Images were collected on a Zeiss LSM confocal microscope and
processed using Adobe Photoshop.
LysoTracker assay for identification of apoptotic cells
Gonads were isolated and incubated in 1 ml DMEM with 5 µl LysoTracker
Red (DND-99, Molecular Probes) for 30 minutes at 37°C, washed three times
in PBS, then three times in PBT (0.1% Triton X100 in PBS) for 30 minutes per
wash. Gonads were fixed overnight in 4% paraformaldehyde and processed for
immunocytochemistry.
Whole-mount in situ hybridization
Samples were fixed overnight in 4% paraformaldehyde in PBS at 4°C and
processed according to Henrique et al.
(Henrique et al., 1995). A
digoxigenin labeled RNA probe for Sox9 was detected using an alkaline
phosphatase-conjugated anti-digoxigenin antibody.
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Results |
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Temporal and spatial patterns of SYN/COR and H2AX in germ
cells of XX mouse gonads
There is still no explanation for why ovarian sectors in ovotestes form in
the polar regions of the gonad. We speculated that the temporal and spatial
pattern of germ cell entry into meiosis might provide a clue as to why distal
regions of the gonad are more vulnerable to sex reversal. To investigate the
possibility that germ cells commit to meiosis earlier in polar regions of the
gonad, we obtained antibodies that recognize several different components of
meiotic chromosomes. Antibodies against SYN/COR are a complex mixture that
recognizes elements of the synaptonemal complex of meiotic chromosomes,
reportedly during the zygotene-pachytene transition
(Dobson et al., 1994).
Phosphorylated gamma histone 2AX (
H2AX) is known to associate with the
DNA double-strand break points in meiotic chromosomes during leptotene in
mouse spermatogenic cells (Mahadevaiah et
al., 2001
).
In female gonads, antibodies against SYN/COR detected a punctate pattern in
the nuclei of some germ cells at 13.5 dpc
(Fig. 4A, green staining),
indicating that these germ cells in XX gonads have committed to meiosis by
13.5 dpc. By 14.5 dpc, SYN/COR staining in germ cells showed a filamentous
pattern associated with the assembly of homologous chromatid pairs in
zygotene/pachytene (Fig. 4A,
arrowheads). Germ cells positive for H2AX were first detected at 14.5
dpc (Fig. 4B). There was no
evidence that germ cells in the most distal regions of the gonad entered
meiosis earlier than in the central domain. Instead, germ cells in XX gonads
became positive for both of these meiotic markers first at the anterior end of
the gonad (Fig. 4, arrows). The
leading edge of SYN/COR-positive germ cells appeared to follow the leading
edge of
H2AX-positive germ cells
(Fig. 4; consecutive 8 µm
sections are shown). At 15.5 dpc, germ cells throughout the gonad, in the
cortex and medulla, were positive for both of these markers. SYN/COR staining
was clearly associated with condensed chromosome pairs in all positive cells,
whereas
H2AX staining showed a diffuse pattern concentrated at the
periphery of some nuclei (Fig.
4B).
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Discussion |
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A molecular profile of germ cell meiosis in embryonic gonads
The light microscope has previously been used to monitor the meiotic status
of germ cells in embryonic gonads. Using antibodies that detect stages of
meiotic progression, we established a molecular profile for meiotic events
during early gonad development. These observations show that the commitment of
XX germ cells to meiosis occurs by 13.5 dpc. We observed punctate staining for
SYN/COR at 13.5 dpc, 24 hours earlier than the first appearance of
H2AX. This pattern probably represents staining of the COR1 proteins,
precursors of the lateral elements of the synaptonemal complex that first form
as short discrete stretches in leptotene
(Mahadevaiah et al., 2001
).
SYN staining marks the assembly of the synaptonemal complex from zygotene
onward (Mahadevaiah et al.,
2001
). In spermatogenic cells, COR1 and
H2AX appear at the
same time (Mahadevaiah et al.,
2001
). Our results in the XX gonad are slightly different:
H2AX staining was not visible in early leptotene at 13.5 dpc, when COR1
first appeared. However, at 14.5 dpc, when the anterior to posterior pattern
is visible,
H2AX staining appears slightly ahead of SYN/COR. This
discrepancy may be due to a mechanistic or timing difference in meiotic
chromatin assembly between XX and XY germ cells.
Germ cells are believed to enter meiosis based on an intrinsic clock. It is
therefore somewhat surprising that they do not enter meiosis simultaneously.
Progression of meiosis from anterior to posterior might be expected if there
is a regional signaling source that affects the process. Thus far, the
evidence has not supported the idea of a local inductive signal, as germ cells
enter meiosis with similar timing in all environments (other than the testis)
in which they have been studied (McLaren
and Southee, 1997). Alternatively, this pattern could result from
differences in the time that germ cells populate the anterior versus the
posterior of the gonad.
Both SYN/COR and H2AX markers also detected a small, transient group
of meiotic germ cells in XY gonads, at the anterior junction between the gonad
and mesonephros. This group of cells has been mentioned in a previous report
(McLaren, 1984
). Our markers
revealed that at 14.5 dpc, these meiotic germ cells were located inside testis
cords, outside testis cords and in the mesonephros. By 15.5 dpc, somatic and
germ cells in this region of the XY gonad are undergoing apoptosis, and
meiotic cells were no longer found. Although the significance of this region
of meiotic germ cells in the XY gonad remains obscure, it is nonetheless
spatially coincident with the region where disruptions in cord formation
contribute to the structural reorganization of the junctions between testis
cords, rete testis and efferent ductules. It is possible that meiotic germ
cells in this region destabilize the tubules and contribute to their
reorganization or, alternatively, that focal disruptions of cord structures
trigger germ cell entry into meiosis.
Timing is crucial
In this study, we have directly tested whether XX germ cells or somatic
cells inhibit the testis pathway after 14.5 dpc. We provide evidence that 14.5
dpc germ cells can block cell migration into XX gonads and can interfere with
the organization of testis cords. Mesonephric cell migration is a
male-specific event required for testis cord formation
(Buehr et al., 1993;
Tilmann and Capel, 1999
). In
XYPOS mice that form ovotestes, mesonephric cells always migrate
into the central testicular region but not into the polar ovarian regions of
the ovotestes (Albrecht et al.,
2000
). The stereotypic compartmentalization of the ovotestes in
C57BL/6 XYPOS gonads is not explained by earlier meiosis in distal
regions. Instead, this compartmentalization may simply reflect the fact that
delayed migration reaches only the regions most proximal to the mesonephros
prior to the stage when germ cells become inhibitory.
It would be expected that if the ovarian pathway is initiated by meiotic
germ cells, removal of germ cells would eliminate the ovarian pathway and
therefore, rescue the sex reversal phenotype in C57BL/6 XYPOS mice.
However, the results using mouse mutants devoid of germ cells did not support
this notion. In certain alleles of the sterile mutants Kit and Kit
ligand (Kitl), the absence of germ cells exacerbated the male to
female sex reversal in C57BL/6 XYPOS mice
(Burgoyne and Palmer, 1991;
Cattanach et al., 1988
;
Nagamine and Carlisle, 1996). One possible explanation for these results is
that germ cells at pre-meiotic stages play a positive role in supporting
testis cord formation. Although the presence of germ cells is not required for
formation of testis cords, cord formation is delayed in the absence of germ
cells in KitW/W-v mutants and in other cases where germ
cells are lost (H.H.-C.Y. and B.C., unpublished). Recent studies by Adams and
McLaren demonstrated that germ cells generate a masculinizing feedback loop by
producing prostaglandin D2 which promoted Sertoli cell differentiation
(Adams and McLaren, 2002
). Loss
of germ cells might critically impair the cord forming process in C57BL/6
XYPOS gonads and contribute to failure of Kit mutants to
rescue sex reversal in XYPOS mice
(Burgoyne and Palmer,
1991
).
How do meiotic germ cells interfere with the testis pathway?
We found that when an XX gonad with meiotic germ cells was cultured on top
of an XY gonad, there was no inhibitory effect on the adjacent XY tissue: cord
formation and the expression of male markers occurred normally in nearby
cells. These experiments cannot rule out the possibility that long-range
signals from the XX gonad are blocked by signals from somatic or germ cells in
the XY gonad. However, these sandwich cultures suggest that the inhibitory
effect of meiotic germ cells is mediated through cell-cell interactions or
through short-range diffusible factors. We suggest that the adhesion molecules
on the surface of germ cells or short-range diffusible factors may be
permissive to testis cord formation only until germ cells commit to meiosis
(Fig. 6). There are several
surface molecules known to exhibit sexually dimorphic patterns as germ cells
enter meiosis. Erb2 and Erb3 are present on the germ cell surface between
11.5-13.5 in both sexes, but disappear after 13.5 dpc in XX gonads
(Toyoda-Ohno et al., 1999).
E-cadherin expression follows a similar pattern with downregulation in XX
gonads when germ cells enter meiosis (Di
Carlo and de Felici, 2000
). However the involvement of these
surface molecules in testis development has not been investigated.
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ACKNOWLEDGMENTS |
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Footnotes |
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