1 Department of Biological Chemistry, University of California, Irvine, CA
92697, USA
2 Developmental Biology Center, University of California, Irvine, CA 92697,
USA
3 Department of Biomedical Science, Cornell University, Ithaca, NY 14852,
USA
* Author for correspondence (e-mail: xdai{at}uci.edu)
Accepted 22 December 2004
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SUMMARY |
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Key words: Ovol1 (movo1), Id2 (Idb2), Spermatogenesis, Germ cell differentiation, Meiosis, Pachytene, Meiotic prophase, Drosophila ovo/svb
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Introduction |
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Meiotic prophase, an extended G2 phase where germ cells transit from the
mitotic to the meiotic cell cycle, is tightly regulated, particularly at
pachytene, the longest stage in prophase. During prophase, chromosomes undergo
dynamic behavioral changes such as movement, pairing, synapsis and
recombination, and checkpoint mechanisms exist to monitor such chromosomal
behavior (Cobb and Handel,
1998; Cohen and Pollard,
2001
). The pachytene stage is also when mRNA synthesis is
particularly active, producing transcripts that encode proteins required for
meiotic and postmeiotic germ cells (Eddy
and O'Brien, 1998
). Although examples of transcriptional control
of meiotic pachytene progression have been documented in yeast
(Chu and Herskowitz, 1998
;
Tung et al., 2000
) and
Drosophila (Lin et al.,
1996
; White-Cooper et al.,
1998
), studies probing such control mechanisms in mammalian
gametogenesis are only beginning to appear
(Eddy, 2002
).
The conserved ovo gene family encodes DNA-binding transcription
factors that lie downstream of the canonical Wg/Wnt signaling pathway
(Li et al., 2002b;
Payre et al., 1999
) and
control the differentiation of a number of tissues in multicellular organisms,
including C. elegans, Drosophila and mice
(Dai et al., 1998
;
Johnson et al., 2001
;
Oliver et al., 1990
;
Payre et al., 1999
).
Therefore, analysis of the regulation and function of Ovo genes
provides an excellent tool with which to investigate the control mechanisms
required for cellular differentiation processes in a complex tissue setting,
and to examine how these mechanisms evolve
(Sucena et al., 2003
;
Wang and Chamberlin,
2002
).
Three distinct mouse ovo family genes, Ovol1, Ovol2
(previously known as movo2 or zinc finger protein 339) and
Ovol3 (previously known as movo3) exist
(Li et al., 2002a). Ablation
of Ovol1 in mice leads to defects in several tissues that express
Ovol1, including testis, skin, kidney and the urogenital tract
(Dai et al., 1998
). Female
mutant mice showed reduced fertility primarily due to structural defects in
the urogenital tract; however, no apparent abnormalities were observed in
oogenesis. Defects are most severe and of highest penetrance in the testis,
where a dramatic decrease in production of spermatozoa, in testis weight and
in male fertility was observed. Although this study suggested a role for
Ovol1 in sperm production, the primary function and cellular
target(s) of Ovol1 in spermatogenesis was not defined due to
complications from secondary events such as massive germ cell degeneration in
the adult mutant testis. In the present study, we examined the first,
relatively synchronous, round of spermatogenesis that spans the first several
postnatal weeks to reveal the primary consequences of the absence of a
functional Ovol1 gene. We report that Ovol1 is expressed
during the pachytene stage of meiotic prophase and is a crucial regulator of
pachytene progression of male germ cells. We also provide molecular evidence
suggesting that Id2 is a Ovol1 target.
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Materials and methods |
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In situ hybridization
Digoxigenin-labeled sense and antisense cRNA probes were synthesized from
either an 870 bp Ovol1 fragment containing the 3' UTR region, a
1.6 kb Ovol2 cDNA fragment representing a nearly full-length
transcript, or a 754 bp fragment corresponding to nucleotides 11-764 of Id2
mRNA (Accession Number NM_010496). The in situ hybridization procedure was
adapted from Deng and Lin (Deng and Lin,
2002). Specifically, freshly dissected testis samples were fixed
in 4% paraformaldehyde/PBS overnight at 4°C and subsequently passed
through a series of sucrose/PBS solutions of increasing sucrose concentration
(10%, 15%, 20% and 30%). After an overnight incubation in 30% sucrose/PBS:OCT
(1:1), the samples were frozen in 30% sucrose/PBS:OCT (1:3). Sections (8
µm) were cut and dried at 50°C for 2 hours, followed by drying at room
temperature overnight. Dried sections were stored at -80°C until use in
subsequent in situ hybridization experiments. Thawed sections were treated
with proteinase K (30 µg/ml in PBS) for 5 minutes at room temperature,
rinsed briefly in PBS, and re-fixed in 4% paraformaldehyde/PBS for seven
minutes. Following post-fixation washes, the appropriate digoxigenin-labeled
probes were added to the slides at a concentration of 1 µg/ml in
hybridization buffer (1.3xSSC, 50% formamide, 5 mM EDTA, 0.5% CHAPS, 100
µg/ml heparin, 50 µg/ml yeast RNA, 0.5% Tween-20, pH 6.5) and allowed to
hybridize at 60°C overnight. The slides were then washed in several
changes of 0.2xSSC, 50% formamide at 60°C over a period of 1 hour.
Immunological detection of digoxigenin-labeled probes was performed by
incubating the slides with a
-digoxigenin-AP conjugated antibody
[diluted 1:2000 in a buffer containing 2% blocking reagent (Roche), 2% normal
goat serum, 100 mM Tris-Cl, 150 mM NaCl, 0.5 mg/ml levamisole, pH 7.5] for 3
hours at room temperature, followed by a colorimetric reaction using the
NBT/BCIP substrates. Counterstaining with the
-Ldhc4 antibody was
performed using a 1:100 dilution in 10% normal goat serum/PBS at room
temperature for 1 hour followed by incubation in a 1:100 dilution of a
-rabbit-FITC conjugated antibody at room temperature for 1 hour.
BrdU labeling
Juvenile male mice were injected intraperitoneally with BrdU
(5-Bromo-2'-deoxyuridine, Sigma B5002) at a dose of 50 µg/g body
weight, sacrificed 2 hours after injection, and their testes dissected and
fixed in Bouin's fixative as described above. Paraffin sections were incubated
in a 60°C oven overnight, and deparaffinized by a 5-minute wash with
Histoclear and rehydrated by a series of rinses in decreasing concentrations
of ethanol (100%, 95%, 70%, 50%, 0%). The slides were then treated with 50%
formamide in 2xSSC at 65°C for 2 hours, followed by two 5-minute
rinses in 2xSSC, and incubation in 2N HCl at 37°C for 30 minutes.
Samples were neutralized by incubating in 0.1 M boric acid, pH 8.5 for 10
minutes, rinsed briefly in PBS and endogenous peroxidase was quenched by
incubating in freshly prepared 3% H2O2 for 15 minutes.
After three 5-minute washes in PBS, samples were subjected to
immunohistochemical analysis as described above using a mouse monoclonal
-BrdU antibody (Roche). The BrdU-positive cells in P14 mutant and
wild-type testes were counted, and the total number in an area of
2x105 µm2 in size (equivalent to
30
tubular cross-sections) was calculated as an average of three independent
areas.
Histology, immunofluorescence and immunohistochemistry
Testis samples of the desired ages (P14-P21) were fixed in Bouin's fixative
for 12-24 hours, depending on tissue size, processed and embedded in paraffin
wax. Sections (5 µm) were stained with the periodic acid/Schiff sulfite
leucofuchsin (PAS) reaction, Hematoxylin and Eosin, or the appropriate
antibodies. Immunofluorescence using a polyclonal rabbit -Ldhc4
antibody (Hintz and Goldberg,
1977
), a guinea pig
-histone H1t
(Inselman et al., 2003
), or a
monoclonal mouse
-cyclin B1 antibody (Santa Cruz Biotechnology), was as
described (Dai et al., 1998
).
Immunohistochemistry using a rat IgM
-mouse Gcna1 monoclonal antibody
(Enders and May, 1994
) was
performed using the SABC kit (Zymed) or the VECTASTAIN elite ABC kit (Vector)
according to the manufacturer's recommendations.
Detection of apoptosis
The terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick
end-labeling (TUNEL) assay was used to detect apoptotic germ cells. Frozen
testis sections from 21-day-old mice were fixed in 4% paraformaldehyde for 10
minutes at room temperature, followed by three 5-minute washes in PBS.
Sections were then processed using the In Situ Cell Death Detection Kit
according to the manufacturer's recommendations (Roche)
Chromosome analysis
Preparation of spermatocyte spreads and the analysis of prophase I
chromosomes by immunostaining of chromosome-associated proteins were as
described (Edelmann et al.,
1999; Kneitz et al.,
2000
).
Transcriptional profiling
Three identical experiments were performed independently. In each
experiment, testes from two to four Ovol1-/- mice together
with two to four wild-type control littermates were taken for RNA preparation,
and total RNA pooled from testes of the same genotype. Total RNA (2 µg)
from each sample group was reverse-transcribed into cDNA, which was then
transcribed into biotin-labeled cRNA (Zhao
et al., 2000). Labeled cRNA (15 µg) was used in each
hybridization to Affymetrix Murine 11K Genechips (SubA and SubB) covering
11,000 genes and ESTs. The Affymetrix GeneChip Analysis Suite software
(MAS 4.0) was used to generate the raw data report. For each entry on the
microarray, the software calculates an average of the difference between
perfect-match and mismatch probes (up to 20 probes per gene). This so-called
average difference is directly related to the level of expression of the
transcript, and is normalized between chips by the use of a global target
value for total chip fluorescence. The program also carries out a
combinatorial evaluation of the performance of each probe set using several
analysis metrics to determine the presence or absence of each transcript. Raw
data were further analyzed using the CyberT program
(Long et al., 2001
), which
runs a T test on the three data sets of the Affymetric output in Excel format
and includes features to allow Bayesian-based approximations of standard
deviations for measurements.
Reporter assays
293T cells were seeded in 24-well plates and transfected at 12-15%
confluence with Ca2+ phosphate as described
(Pear et al., 1993). A typical
transfection mixture contained a total of 0.5 µg of plasmids, including: 50
ng of pGL3-Id2 (where a 1148-bp Id2 gene regulatory fragment was cloned
upstream of the luciferase reporter gene); 0, 5, 10, 15 or 20 ng of the
Ovol1 expression vector (pCB6-Ovol1)
(Dai et al., 1998
); and 0.04
µg of a ß-actin promoter-ß-gal construct. Or a total of 0.5 µg
of plasmids, including: 10 ng of pGL3-Id2; and 0, 10, 20, 40 or 70 ng of the
VP16-Ovol1-expressing vector; and 0.04 µg of a ß-actin
promoter-ß-gal construct. pCB6 (+) (empty vector containing the CMV
promoter) was used as stuffer DNA. Luciferase activity was measured in whole
cell extracts using the Luciferase Assay System (Promega).
ß-Galactosidase activity was measured as previously described
(Eustice et al., 1991
).
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Results |
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Inefficient exit from proliferation, incomplete pachytene arrest, and increased apoptosis in Ovol1-/- testis
Meiotic pachytene is a key stage in yeast meiosis after which point a
decision to exit mitosis becomes irreversible
(Shuster and Byers, 1989). The
expression of Ovol1 in pachytene spermatocytes led us to examine
whether exit from proliferation might be affected in prepubertal
Ovol1-/- testis. A small number of mutant tubules
contained multiple layers of bromodeoxyuridine (BrdU)-labeled germ cells
(arrows in Fig. 2B), whereas in
wild-type mice at this age such proliferating cells only occupy the outermost
layer (spermatogonia) of the tubule. This said, there was only a very slight
increase (
7%) in the actual number of BrdU-positive cells in the mutant
testis (not shown). These results suggest that Ovol1 is not essential
for germ cell proliferation per se, but probably plays a modulatory role in
the ability of a germ cell to exit mitosis.
|
|
The pachytene arrest of Ovol1-/- germ cells was associated with aberrant marker expression but not with chromosomal or recombination defects
To further explore the pachytene defect in Ovol1-/-
testis, we examined the biochemistry of prepubertal mutant and wild-type
testis using antibodies to known differentiation markers. These include Gcna1,
which marks the germ cells prior to mid-pachytene
(Enders and May, 1994), and
Ldhc4 and histone H1t (Cobb et al.,
1999
), which mark the mid/late-pachytene spermatocytes. At P14,
nearly all wild-type tubules had multiple rows of Gcna1+ cells
(Fig. 3A), whereas only very
few contained Ldhc4+ cells (Fig.
3E). By P16, Gcna1+ cells were restricted to only one
or two rows at the periphery (Fig.
3C), and the number of Ldhc4+ cells rose significantly
(Fig. 3G). This switch in
marker gene expression was defective in the Ovol1-/-
testis, which at P16 contained more Gcna1+ cells and fewer
Ldhc4+ cells than the controls
(Fig. 3D,H; Table 1). Multiple rows of germ
cells in the P16 Ovol1-/- tubules retained their Gcna1
expression even when they reached the lumen, and the number of
Ldhc4+ cells was more than twofold lower than that in the control
littermates. This was also true at P21 for tubules that had not yet started
massive apoptosis (data not shown). Histone H1t expression overlapped that of
Ldhc4 in both the mutant and control testis
(Fig. 3I-L), confirming a
reduction in the number of mid/late-pachytene spermatocytes in the mutant.
Collectively, these results indicate that Ovol1-deficient germ cells
are unable to efficiently progress beyond mid-pachytene, and support our
morphological observation of a meiotic pachytene defect.
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Genes that were downregulated in Ovol1-/- testis
included those whose products are known to be present in pachytene
spermatocytes, such as Ldhc4 (see above), Adam2 (also called fertilin ß)
(Wolfsberg et al., 1995),
Tctex2 (Rappold et al., 1987
),
S-II-T1 (Ito et al., 1996
),
Odf2 (Turner et al., 1997
),
Tpx1 (Maeda et al., 1999
),
Ldha4 (Thomas et al., 1990
),
Zfp35 (Cunliffe et al., 1990
)
and acrosin (Kremling et al.,
1991
) (see Table S2 in the supplementary material). Northern blot
analysis of selected markers confirmed their reduced levels in
Ovol1-/- mutant testis
(Fig. 6A). By contrast, the
G2-M cell cycle control genes encoding the MPF components Cdc2 and cyclin B1
showed apparently normal levels of mRNA expression in
Ovol1-/- testis, as detected by transcriptional profiling
and northern blot analysis (Fig.
6B). We reasoned that the downregulated genes should include novel
pachytene markers, and chose Ovol2 for additional experiments to test
this possibility. Northern blot analysis confirmed that Ovol2
expression was indeed reduced in juvenile Ovol1-/- testis
(Fig. 6C), and during normal
prepubertal testis development it showed a temporal expression pattern similar
to that of Ovol1 (Li et al.,
2002a
) (Fig. 6D).
Results of in situ hybridization analysis indicated that Ovol2
expression was activated during mid/late pachytene in wild-type testis
(Fig. 6E,F), and that the
number of Ovol2-expressing (also Ldhc4+) tubules was
reduced in Ovol1-/- testis
(Fig. 6G,H). In tubules that
still expressed Ovol2, the signal intensity appeared comparable with
that in the wild-type (Fig. 6G,
compare with 6E). Taken
together, these results showing the downregulation of known and novel
pachytene differentiation markers in juvenile Ovol1-/-
testis provide molecular evidence supporting our morphological and biochemical
observations of a developmental arrest at the pachytene stage in
Ovol1-/- male mice.
|
Both in vivo and in vitro evidence indicates that Id2 expression is repressed by Ovol1
To explore the molecular mechanism by which Ovol1 regulates
pachytene differentiation, we sought to identify its downstream target(s). We
chose Id2 to focus on, because Id2 protein expression was previously detected
in pachytene spermatocytes (Sablitzky et
al., 1998) and due to its demonstrated role in spermatogenesis
(Yokota, 2001
). Northern blot
analysis confirmed that the level of Id2 transcripts was higher in juvenile
Ovol1 mutant testis (Fig.
7A). Id2 is also expressed in Sertoli cells
(Sablitzky et al., 1998
). To
rule out the possibility that the increased Id2 transcript level was due to an
increased contribution from Sertoli cells, we performed in situ hybridization
experiments, which indeed revealed much stronger hybridization signals in
Ovol1-deficient pachytene spermatocytes than that in the wild type
(Fig. 7B, compare signals
indicated by arrows in C' and D'). Furthermore, during normal
prepubertal testis development, the temporal expression pattern of Id2 is just
the opposite of that of Ovol1
(Fig. 7C). Taken together,
these observations are consistent with the possibility that Ovol1
represses Id2 expression in normal pachytene spermatocytes.
|
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Discussion |
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Our work now adds Ovol1 to a growing list of transcriptional
regulators including Egr4 and Mybl1 that are required for meiotic prophase
progression in male germ cells (Toscani et
al., 1997; Tourtellotte et
al., 1999
). Distinct from Egr4-deficient germ cells,
which appeared to arrest at zygotene-early pachytene, Ovol1-deficient
germ cells stalled at a later stage, i.e., during the early- to late-pachytene
transition. Increased germ cell apoptosis, albeit to different extents, was
observed in all three (Egr4, Mybl1 and Ovol1) mouse models.
Assuming that the activity required of these proteins is their transcription
regulatory activity, these observations suggest that at least two crucial
stages of meiotic prophase are subject to the so-called `transcriptional
checkpoint' control (Sassone-Corsi,
1997
): the zygotene-pachytene transition and the pachytene stage.
Interestingly, although the point of arrest in Egr4-/-
testis coincides with the time when the synapsis checkpoint is active, the
point of arrest in Ovol1-/- cells coincides temporally
with the pachytene recombination checkpoint
(Cohen and Pollard, 2001
;
Edelmann et al., 1996
;
Yuan et al., 2001
;
Yuan et al., 2000
).
Recombination proceeds normally in the Ovol1-deficient germ cells,
suggesting that their apoptosis is not due to a recombination failure.
However, it remains possible that some common components of the death pathway
are activated by recombination errors and Ovol1 deficiency. It is
also interesting to note that ectopic expression of a constitutively nuclear
cyclin B1 is sufficient to trigger apoptosis
(Porter et al., 2003
). In this
regard, the precocious nuclear localization of cyclin B1 in
Ovol1-deficient adluminal germ cells provides a possible mechanism by
which Ovol1 ablation leads to apoptosis.
Our microarray experiments have identified a relatively small number of
genes (2%) that are differentially expressed between the juvenile wild-type
and Ovol1-/- testis. The downregulated genes should
provide a useful resource for identifying novel male germ cell or pachytene
markers (see Table S2 in the supplementary material). This list includes
putative regulatory genes such as transcription factors and signaling
molecules, as well as enzymes that are presumably involved in germ cell
metabolism. Our northern and in situ hybridization experiments significantly
extended our previous finding (Li et al.,
2002a) to show that Ovol2 is a new pachytene marker.
Furthermore, the study on the expression of prominin and Mog1 during
prepubertal testis development indicates that these genes are expressed in
testis in a temporally regulated manner. Prominin encodes a protein that is
involved in membrane dynamics and cell shape changes
(Roper et al., 2000
;
Weigmann et al., 1997
). Mog1
encodes a guanine nucleotide release factor for Ran GTPase that is known to
play a role in nucleocytoplasmic transport of macromolecules and spindle
assembly (Clarke and Zhang,
2001
; Nicolas et al.,
2001
). Future studies on the expression and function of these
genes in testis should reveal additional insights into the regulation of
mammalian spermatogenesis.
The developmental pachytene arrest in Ovol1-/- testis
is incomplete, as some Ovol1-deficient germ cells were able to
proceed through the normal differentiation pathway to become functional
spermatozoa (Dai et al.,
1998). That said, our morphological and biochemical findings were
highly reproducible from one mutant mouse to another, and from one genetic
background (129XB6) to another (CD1) (data not shown), indicating a clear-cut
link between Ovol1 ablation and these developmental defects. It has
been shown that Ovol1 and Ovol2 proteins share 77% sequence identity in the
zinc-finger domain and are expressed in overlapping tissues
(Dai et al., 1998
;
Li et al., 2002a
;
Masu et al., 1998
). The
co-expression of Ovol2 with Ovol1 in pachytene spermatocytes
raises the possibility that Ovol2 can partially compensate for the
loss of Ovol1 function. An ultimate understanding of the full
spectrum of Ovol function in mammalian spermatogenesis will require the
analysis of Ovol1/Ovol2 double mutants.
The microarray experiments also identified genes that were upregulated in
the juvenile Ovol1-/- testis. Although previous studies
have detected expression of Id2, Myc and Fgf receptors in primary
spermatocytes (Cancilla and Risbridger,
1998; Sablitzky et al.,
1998
; Wolfes et al.,
1989
), Mgf, activin, Dlk1, Mfge8 and Mif are primarily expressed
in somatic cells of the testis (Jensen et
al., 1999
; Kanai et al.,
2000
; Meinhardt et al.,
2000
; Tanaka et al.,
2002
; Vincent et al.,
1998
). Therefore, the disruption of Ovol1 not only
affected gene expression in germ cells, but also resulted in alterations of
the somatic gene expression program. Undoubtedly, many changes are secondary
consequences of the morphological defect. However, genes that are normally
expressed in pachytene spermatocytes, such as Id2, are possible intracellular
targets of Ovol1. Our in situ experiments revealing an upregulation
of Id2 transcript level in Ovol1-deficient pachytene spermatocytes
support a cell-autonomous effect of Ovol1 on Id2 transcription. This
notion is further supported by the in vitro finding that Ovol1 repressed Id2
promoter activity in reporter assays in a dose-dependent manner. Moreover, the
extent of repression depends on an Ovol1-binding site that is found in the Id2
promoter and is conserved between mouse and human genes, suggesting that this
is a direct repression, and is at least in part mediated by Ovol1 binding to
that site. These results, together with the observation that the chimeric
protein VP16-Ovol1, in which the only DNA-binding motif is the zinc-finger
domain of Ovol1, activated Id2 promoter in a protein dose- and Ovol1 binding
site-dependent manner, clearly establish a direct molecular link between
Ovol1 and Id2. Apparently, the identified Ovol1 binding site could
not account for all the observed repression, leaving open the existence of
additional Ovol1 binding site(s) and/or alternative mechanism(s) of
transcriptional regulation by Ovol1. This mechanistic issue merits further
study that is outside the scope of this work.
Interestingly, Id2 knockout mice are defective in spermatogenesis
(Yokota, 2001). Given the
well-demonstrated role of Id2 as a positive regulator of proliferation and a
negative regulator of differentiation in multiple cell lineages
(Sikder et al., 2003
), it is
tempting to speculate that a negative regulation of Id2 expression might be a
common mechanism by which Ovol1 promotes cellular differentiation in
multiple tissues.
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Supplementary material |
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ACKNOWLEDGMENTS |
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