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First published online 19 April 2006
doi: 10.1242/dev.02371
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1 Department of Dermatology, University of Pennsylvania, PA 19104, USA.
2 Department of Biology, University of Pennsylvania, PA 19104, USA.
3 Shanghai Institute of Medical Genetics, Shanghai Jiao Tong University,
Shanghai 200040, People's Republic of China.
4 Center for Research on Reproduction and Women's Health, University of
Pennsylvania, PA 19104, USA.
5 Department of Cell and Developmental Biology, University of Pennsylvania, PA
19104, USA.
* Author for correspondence (e-mail: htsengpe{at}mail.med.upenn.edu)
Accepted 17 March 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Female fertility, Oogenesis, Pre-implantation development, Transcription regulation, Transgenic RNAi
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Schultz, 2002;
Wang et al., 2004). Mammalian
maternal-effect genes have only been identified recently, largely by
gene-knockout approaches [e.g. Mater (Nalp5 - Mouse Genome
Informatics), Hsf1, Zar1, Npm2 and Stella (Dppa3 -
Mouse Genome Informatics)]. Unlike their counterparts in invertebrates and
lower vertebrates, the molecular mechanisms of mammalian maternal-effect genes
have not been well understood. In the strictest sense, mutations in
maternal-effect genes should not affect oocyte development, maturation and
ovulation, but should impair embryo development. Indeed, null mutations of the
mammalian maternal-effect genes identified so far have little effect on
ovulation and fertilization of the affected eggs, and interfere only with
early embryo development (Tong et al.,
2000; Christians et al.,
2000; Wu et al.,
2003; Burns et al.,
2003; Payer et al.,
2003). Hence, genes expressed in oocytes can be classified as
required for oogenesis (i.e. oocyte development, maturation, ovulation and
fertilization) and for embryonic development (i.e. maternal effect)
(Dean, 2002).
A hallmark of oocyte development is the synthesis and accumulation of
macromolecules and organelles that constitute the maternal capital used to
support early development prior to activation of the embryonic genome
(Schultz, 1993). In mammals,
this maternal store includes proteins and RNAs, which are involved in a wide
range of processes, as evidenced by the variety of implicated functions of the
currently identified maternal-effect genes
(Tong et al., 2000;
Christians et al., 2000;
Wu et al., 2003;
Burns et al., 2003;
Payer et al., 2003).
Furthermore, it is still not clear how mammalian oocytes monitor and respond
to a deficiency in maternal-effect genes, even if malfunction of these genes
does not directly affect oocyte maturation itself.
Basonuclin (Bnc1 - Mouse Genome Informatics) is a zinc-finger protein with
a very restricted tissue distribution; it is mainly found in keratinocytes of
the stratified epithelium and germ cells (i.e. male germ cells and oocytes)
(Yang et al., 1997;
Mahoney et al., 1998). There
is highly suggestive evidence that basonuclin regulates rRNA transcription
(Iuchi and Green, 1999;
Tseng et al., 1999). For
example, the zinc fingers of basonuclin interact with three evolutionarily
conserved binding sites in the rDNA promoter
(Tian et al., 2001). In mouse
oocytes, basonuclin co-localizes with RNA polymerase I (Pol I) activity in the
nucleolus, and a dominant-negative mutant of basonuclin interferes with Pol I
transcription in oocytes (Tian et al.,
2001). Basonuclin, however, differs from the dedicated Pol I
transcription factors (e.g. UBF), in that it is also abundant in nucleoplasm
and can interact with Pol II promoters
(Tseng et al., 1999). Such
observations led to the proposal that basonuclin may have target genes other
than rDNA (Tian et al., 2001).
Together, these results led us to propose that basonuclin is necessary for
oocyte rRNA synthesis and possibly other processes, and perturbing basonuclin
function in the oocyte will disrupt oogenesis, leading to infertility. To test
this hypothesis, we employed a recently developed transgenic-RNAi approach
(Svoboda et al., 2000;
Stein et al., 2003) to inhibit
basonuclin function specifically in mouse oocytes.
We report here that transgenic-RNAi effectively and specifically reduces basonuclin level in mouse oocytes. The transgenic mice are apparently normal, except females are subfertile. Basonuclin-deficient oocytes display a reduced rate of PoI transcription and perturbation of a large number of Pol II-transcribed genes. Many basonuclin-deficient oocytes appear morphologically and biochemically abnormal, but their maturation and ovulation proceed without apparent problems. Although normal numbers of eggs are ovulated and inseminated, pre-implantation development is severely compromised. Thus, basonuclin joins the short, but growing, list of mammalian maternal-effect genes; among them, it is the first to also perturb oogenesis.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), to yield pB1-rnai-zp3-1 or and pB1rani-zp3-2.
Generating transgenic mice
The transgene construct was used for microinjection at the Transgenic and
Chimeric Mouse Facility at University of Pennsylvania and Transgenic Animal
Model Core at University of Michigan with the approval of the Institutional
Animal Care and Use Committee at the respective universities. The zygotes for
microinjection were produced by mating of B6SJLF1/J mice. Transgenic
out-crossed (TG-OC) mice were obtained by mating transgenic founder males
(B6SJLF1/J) with wild-type females (CF1), and genotyped by PCR using the EGFP
primer pair (5'-CGACGTAAACGGCCACAAGT and 5'-CTCGTTGGGGTCTTTGCTCA)
with 34 cycles at 55°C for 20 seconds, 72°C for 40 seconds and
94°C for 20 seconds.
Fertility
Each founder transgenic female (B6SJLF1/J) was mated with the same three
stud males (B6SJLF1/J) in a random sequence. The same males were also used to
assess the fertility of TG-OC females. Successful mating was verified by
detecting a vaginal plug; the mated females were then housed separately for
observation and recording the number of pups delivered per litter, which was
averaged to assess fertility. Other than the fertility tests, all further
studies were carried out with TG-OC females.
Collection and culture of oocytes and embryos
GV, MII eggs and fertilized eggs were collected as previously described
(Schultz et al., 1983). GV
oocytes were cultured in CZB medium (Chatot
et al., 1989) containing 0.2 mM IBMX (Sigma) to inhibit GV
breakdown; MII eggs were cultured in CZB medium and fertilized eggs cultured
in KSOM medium (Erbach et al.,
1994) and their development was recorded at 36, 60 and 84 hours
post-coitum (hpc).
RNA isolation and real-time PCR
Total RNA was isolated from 20 oocytes and reverse transcribed with
Superscript II reverse transcriptase (Invitrogen) using oligo dT as primer.
The resulting cDNA was quantified by real-time PCR using an ABI Prism 7000
thermocycler (Applied Biosystems). The primers for basonuclin were:
5'-ACTGGACGCTTCAGGATTACATC and 5'-GTCATGATGCTCCAGTGATCCA. PCR
conditions were 40 cycles at 95°C for 15 seconds and 60°C for 60
seconds. UBF (internal standard), was amplified with primers provided by ABI
(assay ID: Mm00456972_m1). Each sample was analyzed in duplicate. The
Ct values were converted to percentage of the wild-type
(non-transgenic) level.
Run-on assay and quantification of the transcription loci
Run-on assays were conducted on meiotically incompetent oocytes obtained
from 13- to 14-day-old mice as previously described
(Tian et al., 2001). Briefly,
oocytes were permeabilized and then incubated at 37°C for 1 hour with 0.2
mM Br-UTP and 100 µg/ml 
-amanitin. Oocytes were then fixed in 2%
paraformaldehyde and the incorporated BrdU was detected with Alexa Fluor
488-conjugated anti-BrdU monoclonal antibody (Molecular Probes) at 1:20
dilution. The amount of incorporated BrdU was quantified by measuring the
overall fluorescence intensity at the largest cross-section of the nucleolus
(recorded on an epi-fluorescent digital micrograph) with the `measurement'
function of the ImageJ, a software package created by W. Rasband
(http://rsb.info.nih.gov/ij/).
This value, in arbitrary units, was subtracted by the background fluorescence
of an area equal in size in the nucleoplasm, which was not labeled in the
presence of -amanitin. The number of labeled transcription foci was
scored using microphotographs, which represented a stack of selected optical
planes of the nucleolus. The microphotograph stacks were produced with a Zeiss
Axiovert 200 using a 100x objective under epifluorescence and by manual
focusing. The informative optical planes containing unique transcription foci
were selected manually and photographed. Scoring transcription foci in a
nucleolus required usually 8 to 12 microphotographs. The foci that had been
scored in the previous plane were marked on photos to avoid repetition. This
method also ensured that no informative optical plane was missed because the
out-of-focus foci image of the previous plane could be seen.
RNase protection assay
RNase protection assays were conducted to quantify rRNA in oocytes.
[32P]-labeled antisense probe for rRNA was prepared using the
Maxiscript T7 kit and pTRI-RNA-28S/pTRI-RNA-18S antisense control templates
(Ambion). Fifty oocytes were transferred to the lysis buffer of the Direct
Protect RPA kit (Ambion). The samples were then incubated with the probe,
treated with nuclease and electrophoresed according to the manufacturer's
protocol. Protected fragments were detected by autoradiography and quantified
by density measurement.
RNA isolation and microarray analysis
Total RNA was extracted from oocyte samples, each originated from one of
the six TG-OC (T#50) and four wild-type eCG-stimulated mice, and subjected to
cRNA preparation (Pan et al.,
2005; Zeng et al.,
2004). The control 1, 3, 4 and transgenic 4, 5, 6 mice were from
one litter, and the rest from another. Each Affymetrix MOE 430 v2 GeneChip was
probed with 15 µg cRNA and processed according to the Affymetrix
instructions. Raw microarray data (*.cel) (available at the Gene
Expression Omnibus Repository, Accession Number GSE4029) were processed using
the GC-RMA or MAS-5 algorithms to obtain the normalized expression
summarization for each probe set. The expression level in our data set ranged
between 1 and 66,582. Genes with an expression level of less than 10 were
excluded before GeneSpring (Silicon Genetics) K-mean hierarchical clustering
and other filter analysis. Genes whose expression was significantly changed in
the transgenic oocytes compared with the control were identified by
Significance Analysis of Microarray (v1.21, two-class, unpaired analysis with
the permutation parameter set at 500, FDR<1%) or by one-way ANOVA analysis
(FDR <5%) and annotated in the GeneSpring. The statistically significant
gene lists obtained by ANOVA were imported into EASE v2.0
(Hosack et al., 2003) to test
for overrepresentation of biological processes in transgenic versus wild-type
samples. An EASE score was calculated for the likelihood of overrepresentation
in the Gene Ontology Consortium annotation categories (i.e. biological
process, cell component, molecular function), as well as KEGG pathways and
SwissProt keywords.
Immunofluorescence staining and immunoblotting
Oocytes/embryos were fixed in 2.5% paraformaldehyde/PBS and
immunocytochemistry was conducted as previously described
(Yu et al., 2004) using an
affinity-purified anti-basonuclin antibody (MBP-1023) diluted at 1:30.
Affinity-purified pre-immune serum was used as a negative control.
For immunoblotting, 75 oocytes/embryos were lysed in 2x SDS-PAGE buffer, run on a 7.5% SDS-PAGE and transferred to PVDF membrane (Hybond-P, Amersham Biosciences). Immunodetection was performed using horseradish peroxidase-conjugated secondary antibodies and ECL Advance reagents (Amersham) according to the manufacturer's instructions.
DNA replication assay
Fertilized eggs were collected at 10 hpc and labeled with 10 µM BrdU for
6 hours. The embryos were then fixed with paraformaldehyde, treated with 2 N
HCl at 37°C for 1 hour, and then neutralized in 0.1 M borate buffer (pH
8.5), for 15 minutes. Incorporated BrdU was visualized with 1:30 diluted Alexa
fluor 488-conjugated anti-BrdU antibody (Invitrogen).
Histology
For histological analysis, adult ovaries were fixed in Bouin's fixative for
16 hours before transfer to 70% ethanol. Samples were then embedded in
paraffin, serially sectioned (5 µm) and stained with Hematoxylin and
Eosin.
TUNEL labeling assay
TUNEL (TdT-mediated dUTP nick end labeling) assays were carried out with
the In Situ Cell Death Detection Kit (Roche Diagnostics) according to the
manufacturer's instructions.
Protein synthesis assay and transcription-requiring complex (TRC) quantification
Oocytes were metabolically radiolabeled with [35S]-methionine
and acid-insoluble radioactivity was then determined following TCA
precipitation as previously described
(Conover et al., 1991).
Quantification of the transcription-requiring complex (TRC) was also conducted
as previously described (Conover et al.,
1991).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). We
first confirmed this basonuclin mRNA expression profile by RT-PCR
(Fig. 1, broken line) and then
established the expression profile for basonuclin protein in oocytes and
pre-implantation embryos by immunoblotting and immunocytochemistry.
Immunoblotting revealed an apparent decline in basonuclin level from the
immature oocyte (GV-stage) to the mature metaphase II (MII) stages
(Fig. 1). Similar amounts of
basonuclin were present in MII eggs and one-cell embryos, but no basonuclin
was detected in two-cell and morulae/blastocysts, which is consistent with the
disappearance of its mRNA during pre-implantation development.
Immunocytochemical analysis also revealed a similar and progressive decrease
of immunoreactive material from oocytes to pre-implantation embryos
(Fig. 1). Thus, maternal
basonuclin is not replaced by zygotic basonuclin during pre-implantation
development and therefore could have a maternal effect.
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). We
constructed two types of RNAi transgene plasmids, pB1rnai-zp3-1 and
pB1rani-zp3-2 (Fig. 2A); the
details of generating transgenic mice are described in the Materials and
methods. Twenty-four founder mice (13 females and 11 males) were obtained, of
which 14 were generated by pB1rnai-zp3-1 and 10 by pB1rnai-zp3-2.
Transgenic basonuclin-RNAi females are sub-fertile
We analyzed the fertility of both founder transgenic females and female
progeny of the transgenic founder males
(Fig. 2B). To reduce the
influence of male fertility on the outcome, three normal males were mated in a
randomized sequence with each female being tested. Eight of the nine
transgenic founder females showed a wide range of sub-fertility - from
moderate to severe (infertile) - when compared with the average of the seven
non-transgenic littermates in the control group
(Fig. 2B). The sub-fertility
was unlikely to be due to the vector backbone and/or transgene integration
sites because the founder females were derived from multiple injections of the
two transgene-constructs. Southern analysis also failed to establish any
correlation between the integration sites and transgene copy number with the
observed reduced fertility (data not shown).
The fertility of transgenic female progeny of the male founders mirrored
that of the transgenic female founders
(Fig. 2C). Other than female
sub-fertility, the transgenic male and female mice appeared normal. Because
transgenic females were sub-fertile, we used transgenic males to transmit the
transgene and focused subsequent studies on the female progeny of two
transgenic lines, T50 and T8, which had 
10% and 50% normal fertility,
respectively. To date, the female sub-fertility phenotype of these lines has
been stable after two generations of breeding.
Transgenic oocytes contain reduced amounts of basonuclin mRNA and protein
We examined fully grown transgenic oocytes for basonuclin mRNA and protein
to determine the cumulative effect of RNAi during the growth phase. Real-time
PCR demonstrated that the amount of basonuclin mRNA in transgenic oocytes from
both lines was dramatically reduced. For T8 and T50 oocytes, respectively, an
additional 4 and 6 cycles were required to reach threshold, when compared with
non-transgenic counterparts (Fig.
2D). This increase in cycle number translates into a 94% and 98%
reduction in basonuclin mRNA. Indeed, immunocytochemical
(Fig. 2F) and western blot
(Fig. 2G) examinations
confirmed the dramatic decrease in basonuclin protein content in these
transgenic oocytes. These assays, however, failed to distinguish the small
difference in the severely reduced basonuclin protein levels of the two
transgenic lines. Conceivably, the exponential nature of the RT-PCR method is
more sensitive in detecting the small variation between the very low
basonuclin levels, which were below the level of detection of
immunocytochemistry and western blot. Notably, the small difference in mRNA
levels appeared to produce an effect on the fertility in T8 and T50 lines,
which were 50% and 90%, respectively (Fig.
2E) and correlate with the 94% and 98% reduction of basonuclin
mRNA content.
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;
Fedoriw et al., 2004;
Yu et al., 2004). In
transgenic oocytes, no detectable change was observed in the amount of mRNA
for UBF, a ubiquitous Pol I-specific transcription factor
(Fig. 2D). Furthermore, we
observed no difference in the abundance of basonuclin 2 mRNA, which is a
closely related homolog and shares more than 57% of the nucleotide sequence
with basonuclin (Romano et al.,
2004; Vanhoutteghem and Djian,
2004). This high targeting specificity is consistent with a
previous report that transgenic RNAi targeting oocyte Msy2 mRNA has
no effect on the abundance of the closely related Msy4 transcript
(Yu et al., 2004). These
results demonstrate the efficacy and specificity of the transgenic RNAi
approach. Thus, as predicted, perturbing basonuclin function in oocytes
reduces fertility (Tian et al.,
2001).
Increased oocyte developmental failure in basonuclin-deficient oocytes
To characterize the cause of the sub-fertility phenotype, we first examined
the histology of transgenic ovaries. Compared with the control
(Fig. 3A,B), histological
sections revealed clear changes in follicular structures in adult transgenic
ovaries (Fig. 3C,D), which
appeared normal in size and weight. Although the number of follicles appeared
unaffected, some follicles exhibited abnormal morphologies, which could be
classified into two discrete categories. In the first category, the follicles
were invariably large and the oocytes contained cytoplasmic vesicles or
cavities (Fig. 3C,D, arrows),
which was not observed in non-transgenic ovaries
(Fig. 3A,B). In the second
category, the follicles were small and irregular in shape, and the oocytes
were apparently being degraded and absorbed
(Fig. 3C,D arrowheads).
Moreover, cumulus cells in contact with the degenerating oocyte had enlarged
nucleoli, which were intensely stained by hematoxylin
(Fig. 3C,D). Transgenic ovaries
contained corpora lutea, suggesting ovulation had occurred. However, when
compared with non-transgenic ovary (Fig.
3A,B), the transgenic corpora lutea contained more cells, which
made them appear darker because of hematoxylin nuclear staining
(Fig. 3C,D). The ovarian
morphology suggested heterogeneity in the transgenic oocyte population, some
oocytes failed, whereas others succeeded in reaching maturity.
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60% of the latter had dark,
granular and even opaque cytoplasm (Fig.
3F, arrowhead shows an example). This cytoplasmic change was
accompanied by a roughing of the oocyte surface, as revealed by differential
interference contrast (DIC) microscopy
(Fig. 3G,H, arrowhead). To
ascertain if oocytes with altered morphology differed in their ability to
mature, GV-intact transgenic oocytes were isolated and divided into two groups
according to their cytoplasm transparency (transparent and light; opaque and
dark) and cultured in vitro. Surprisingly, both the transgenic GV-intact
oocytes with light or dark cytoplasm reached MII, as evidenced by emission of
the first polar body, at incidence similar to the non-transgenic controls
(Table 1). These observations
strongly suggested that the cytoplasmic and cell surface changes did not
interfered with the maturation of this subpopulation. This conclusion was
confirmed when the numbers of MII eggs isolated from PMSG/hCG-primed
transgenic mice were found to be the same when compared with controls
(Table 1).
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-amanitin, incorporation of BrU into
nucleoplasm was abolished, suggesting Pol II transcription was inhibited
(Fig. 4C,D). Under this
condition, fluorescence intensity in the basonuclin-deficient oocytes was
reduced by 38% (Fig. 4E).
Also reduced were the numbers of transcription foci, where fluorescence is
brighter than other regions in the nucleolus, suggesting intensive
incorporation of BrUTP, i.e. a site of Pol I transcription. Because rDNA is a
multicopy gene, these foci probably represent the rDNA copies that are
actively transcribed during the run-on labeling. Scoring the number of
transcription loci by optically sectioning the nucleolus
(Fig. 4F,G) indicated that the
number of foci was reduced by 25% in T50 transgenic oocytes when compared
with controls (P<0.001) (Fig.
4H). Nevertheless, we detected neither a significant change in
mature 28S and 18S rRNA level by RNase-protection assay (data not shown), nor
an apparent change in the rate of protein synthesis in both growing and fully
grown transgenic oocytes (Table
1).
Perturbed Pol II transcription in basonuclin deficient-oocytes
Because of the potential of basonuclin as a Pol II transcription regulator,
we investigated the effect of basonuclin deficiency on Pol II transcription by
analyzing the global gene expression pattern in fully grown, GV-intact oocytes
by microarray analysis. After an active period of transcription during the
first two-thirds of oocyte growth, transcription decreases such that the fully
grown oocyte is essentially transcriptionally quiescent, which stays so during
oocyte maturation until zygotic gene expression initiates in the late one-cell
embryo (Hamatani et al., 2004;
Zeng and Schultz, 2005).
Therefore, the mRNA composition at the GV-intact stage reflects not only the
cumulative effect of Pol II transcription during oocyte growth, but also is
the source of the mRNA at the beginning of oocyte maturation and thus may have
important bearings on early embryonic development.
RNAs of oocytes of four non-transgenic and six transgenic mice were
analyzed individually on Affymetrix MOE 430 2.0 gene chips, which contains
more than 34,000 transcripts. The resulting microarray data had a strong
tendency to cluster within control and transgenic groups
(Fig. 5A), when compared with
an unbiased gene list. There were 772 and 253 genes, the mRNA levels of which
were up- or downregulated more than twofold, respectively. As expected,
basonuclin mRNA level was reduced by 210-fold, the largest magnitude of change
among the up- and downregulated genes; the mRNA level of the related
basonuclin 2 was not affected. An EASE analysis
(Hosack et al., 2003)
(Fig. 5B) showed that the genes
whose expression was upregulated more than twofold belonged to four processes:
transcription and DNA binding (39.1%), development (17.5%), intercellular
junction/extracellular space (11.3%) and metal ion binding (6.5%), whereas the
genes downregulated more than twofold were mostly related to cell
motility/adhesion (37.8%), intracellular transport (30.0%) and protein binding
(17.8%). Although it remains to be seen how these perturbations in gene
expression contributed to the phenotype, it is clear that: (1) our RNAi
approach was effective and highly specific; (2) Pol II transcripts were
evidently affected by basonuclin-deficiency; and (3) some of the transcript
upregulation was probably a secondary response to basonuclin deficiency (see
also Discussion). Moreover, these results support the proposal that basonuclin
regulates Pol II transcription, in addition to its function as a Pol I
transcription regulator.
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10
hpc, cultured and scored for developmental stages at 36, 60 and 84 hpc.
Developmental progression of transgenic embryos was clearly delayed or
arrested (Fig. 6A). At 36 hpc,
virtually all non-transgenic embryos reached the two-cell stage, whereas only
75% and 40% of T8 and T50 embryos, respectively, did
(Fig. 6B). At this point, 60%
of the T50 embryos were at the one-cell stage, and during the next 48 hours,
few of them cleaved and progressed beyond the two-cell stage. Overall, very
few T50 embryos could develop beyond the two-cell stage
(Fig. 6A,B). Although
development of T8 embryos was better, 60% of them reached the blastocyst
stage, the rate of development was nonetheless slower than controls. Once the
T8 transgenic embryos reached the eight-cell stage, when an increasing number
of zygotic genes are activated (Hamatani et
al., 2004; Wang et al.,
2004; Zeng et al.,
2004), they readily developed to the blastocyst stage without
further loss (Fig. 6B). We
noted that, overall, at the end of this culture period (84 hpc or 3.5 days),
63±11% and 10±3% of the T8 and T50 embryos, respectively,
developed beyond the four- to eight-cell stages, which could account for the
fertility of these two transgenic lines (53% and 10% respectively). Thus,
early embryonic failure caused the sub-fertility of the transgenic
females.
Because most of the embryo loss occurred during the one- and two-cell
stages, we focused on development during this time. We first assessed if
genome activation, which is required for development beyond the two-cell
stage, occurred in basonuclin-deficient two-cell embryos
(Schultz, 2002). Genome
activation, as assayed by expression of the transcription-requiring complex
(TRC), a set of structurally related proteins that are accepted markers of
genome activation (Conover et al.,
1991), did occur in transgenic embryos to the same extent as in
controls (Fig. 7M). Thus, it is
unlikely that failure to activate the embryonic genome was the basis for
developmental failure and hence we looked at earlier times. We noted a variety
of abnormalities in the one-cell transgenic embryos. First, and reminiscent of
transgenic oocytes (Fig. 4G),
transgenic one-cell embryos appeared `rough' under Normaski optics
(Fig. 7A-D). Nuclear morphology
of the transgenic embryos was also altered; most notably, the male pronuclei
were 18.7% smaller in diameter than that of the control
(Fig. 7E-H,
Table 1), suggesting a
chromatin de-condensation failure. The distribution of chromatin/DNA
surrounding nucleolus in the transgenic pronuclei also differed clearly from
that of controls (Fig. 7F
arrow). The most striking defect, however, was chromatin fragmentation, seen
as additional pieces of DAPI-positive materials, along with the diminutive
pronuclei (Fig. 7G,H,J).
Polyspermy could be a source of additional chromatin, because the frequency of
poly-pronuclei observed in transgenic one-cell embryos (12 hpc) increased by
more than tenfold (Table 1).
Another cause of DNA fragmentation was probably due to stress on segregating
chromatin during mitotic division, which led to unequal distribution of DNA
between daughter cells (Fig.
7K,L). Cytoplasm fragmentation was just as frequent as nuclear
fragmentation (Fig. 7L). There
was, however, no single common defect observed in the transgenic embryos.
Despite these failures, the apoptotic pathway was not activated when assayed
by TUNEL (not shown). It is very likely that this range of abnormalities
underlies the reduced developmental competence of embryos derived from
basonuclin-deficient oocytes and, in particular, cell cycle regulation.
Consistent with this, in transgenic one-cell embryos, our result indicated a
reduction or block in DNA replication (Fig.
7M), which, in non-transgenic embryos, was observed between 8 and
16 hours post-fertilization as previously reported
(Moore et al., 1996).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Basonuclin appears to be another member of a growing family of mammalian
maternal-effect genes. The source of embryonic basonuclin is maternal and
targeted depletion of basonuclin in oocytes leads to sub-fertility. The
observed embryonic failure at the one- to two-cell stages, which accounts for
the reduced fertility of basonuclin-RNAi transgenic females, is similar to the
phenotypes of several recently described mouse maternal-effect genes [e.g.
Mater, Hsf1, Zar1, Npm2 and Stella
(Tong et al., 2000;
Christians et al., 2000;
Wu et al., 2003;
Burns et al., 2003;
Payer et al., 2003)]. Like
basonuclin-deficient embryos, these embryos show perturbed fertilization
(Zar1-/-), fragmentation or abnormal cleavage at the first
mitosis (Mater-/-, Stella-/- and
Npm2-/-), and delayed cell cycle progression leading to
cleavage arrest during one- to two-cell transition
(Zar1-/-). Although we suspect that genome activation in
the early embryo was compromised by the failure of chromatin reorganization,
the apparent normal expression of the TRC suggests that at least some aspect
of genome activation occurs in basonuclin-deficient transgenic embryos.
Our observations suggest an interesting difference between basonuclin and
other maternal-effect genes reported so far. Most of the known homozygous-null
maternal-effect mutants have little effect on the histology of the ovary,
follicular structure, oocyte morphology and ovulation
(Tong et al., 2000;
Christians et al., 2000;
Howell et al., 2001;
Wu et al., 2003;
Burns et al., 2003;
Payer et al., 2003), i.e.
there appears to be little, if any, overt problem with oocyte development. By
contrast, basonuclin-deficient oocytes appear abnormal and are often present
in degenerating follicular structures, which may reflect improper
communication between the oocyte and follicle cells that is required for
proper and coordinated follicle development
(Matzuk et al., 2002). The
transgenic oocyte population is apparently heterogeneous, suggesting either a
variable expression of the transgene or a variable resilience to
basonuclin-deficiency. The observation that normal numbers of eggs are
ovulated implies that mouse ovary can compensate for the loss of oocytes
during their development and maintain the overall output. However, very little
is known about how ovary determines and regulates its output. Another level of
compensation might take place within basonuclin-deficient oocytes. Consistent
with this proposal is that although we detect reduced rates of Pol I
transcription using the run-on assay, the level of mature rRNA and protein
synthesis remain unchanged in fully grown basonuclin-deficient oocytes.
Another sign that a compensatory mechanism exists is that microarray analysis
of basonuclin-deficient oocytes reveals a large number of upregulated genes,
and the most notable among them is a 16-fold increase in abundance of
Gli2 mRNA, which encodes a transcriptional effector (GLI2) that can
upregulate basonuclin transcription (Cui et
al., 2004). Upregulation of Gli2 in basonuclin-deficient
oocytes suggests an apparently futile attempt of the transgenic oocytes to
compensating for the reduced level of basonuclin mRNA by increasing its
transcription. Another likely source of compensation comes from basonuclin 2,
the transcript of which is also abundantly present in mouse oocytes and not
affected by RNAi targeting basonuclin. It is clear, however, that the putative
compensation of basonuclin 2 can be only partial, because of the observed
embryonic developmental failure of embryos derived from basonuclin-deficient
oocytes. Thus, the detrimental effect of basonuclin deficiency, be it
reduction of Pol I and Pol II-mediated transcription or other impairments, may
be alleviated by the ability of the oocyte to compensate for these
perturbations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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