Expression and Role of Bcl-2 in Rat Blastocysts Exposed to High D-Glucose
Serge Pampfer,
Sabine Cordi,
Ivo Vanderheyden,
Patrick Van Der Smissen,
Pierre J. Courtoy,
Anne Van Cauwenberge,
Henri Alexandre,
Isabelle Donnay, and
René De Hertogh
From the Physiology of Human Reproduction Research Unit (S.P., S.C.,
I.V., R.D.H.), Université Catholique de
Louvain, Brussels; the Cell Biology Unit (P.V.D.S., P.J.C.), Christian de Duve
Institute of Cellular Pathology, Brussels, Belgium; the Division of Biology
and Embryology (A.V.C., H.A.), Université de
Mons-Hainaut, Mons and the Veterinary Unit (I.D.),
Université Catholique de Louvain,
Louvain-la-Neuve, Belgium.
Address correspondence and reprint requests to Serge Pampfer, PhD, OBST 5330
Research Unit, University of Louvain Medical School, 53 Avenue Mounier, 1200
Brussels, Belgium. E-mail:
pampfer{at}obst.ucl.ac.be
.
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ABSTRACT
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Bcl-2 mRNA expression was detected in rat blastocysts by in situ
hybridization. The distribution of mRNA expression was rather heterogenous,
with
2% of high-expressing cells. In vitro exposure to 28 mmol/l
D-glucose for 24 h resulted in a significant increase in the proportion of
these cells compared with control embryos in either 6 mmol/l D-glucose or 28
mmol/l D+L-glucose. Heterogeneity in the expression of Bcl-2 was also observed
at the protein level by immunocytochemistry. Exposure to 28 mmol/l D-glucose
significantly increased the incidence of chromatin degradation (karyolysis)
and nuclear fragmentation (karyorhexis), two nuclear markers of apoptosis in
rat blastocysts. When two different antisense oligodeoxynucleotides designed
to block Bcl-2 expression were added to 28 mmol/l D-glucose, the incidence of
karyolysis (but not karyorhexis) was increased compared with embryos in 28
mmol/l D-glucose alone. These data suggest that Bcl-2 is involved in the
protective response against the induction of karyolysis in blastocysts on
their exposure to high concentrations of D-glucose in vitro, whereas
karyorhexis appears to result from the activation of an intracellular pathway
that is independent of Bcl-2.
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INTRODUCTION
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In addition to playing a fundamental role in adult homeostasis, apoptosis
is known to be instrumental in the formation and maturation of many
developmental systems (1).
Noticeable developmental cell death occurs at the blastocyst stage
(2,3)
when embryonic cells engage in a subtle process of differentiation that leads
to the emergence of two distinct cell lineages, the fetal precursor inner cell
mass (ICM) and the placental precursor trophectoderm (TE). However, under
normal conditions, the proportion of cells showing signs of self-destruction
remains limited, and this process is predominantly located to the ICM lineage
(4). In contrast to apoptotic
events that are programmed later in the course of development, the restricted
wave of cell death that is detected at the blastocyst stage does not seem to
serve an obvious morphogenetic purpose. The proposed explanation for its
occurrence is that this process may allow for the efficient elimination of
abnormal or redundant ICM cells before gastrulation is initiated.
Previous studies have shown abnormally high incidences of nuclear
fragmentation (karyorhexis)
(5,6)
and chromatin degradation (karyolysis)
(7) in ICM cells of blastocysts
recovered from insulin-dependent diabetic rats, suggesting that excess ICM
cell death may be a factor contributing to the severe embryopathy that has
been associated with preconceptional maternal diabetes
(8,9).
Incubating blastocysts from normal rats in high concentrations of D-glucose
was also found to stimulate karyorhexis
(10) and karyolysis
(7) in the ICM cell lineage of
rat blastocysts, in support of the hypothesis that hyperglycemia plays a
direct role in the induction of the cellular alterations observed in utero.
Observations made on blastocysts from diabetic mice and on mouse embryos after
in vitro culture in high concentrations of D-glucose
(11) largely confirmed the
data reported on rat embryos.
There is evidence that several genes of the Bcl-2 and caspase families,
which are known intracellular regulative and executive components of the
apoptotic cascade, are already expressed at the blastocyst stage
(12,13).
Treating mouse blastocysts with a combination of protein synthesis and protein
kinase inhibitor to desensitize them from survival signals was found to induce
massive apoptosis
(14,15),
pointing towards the existence of a fully operational cell death machinery at
that developmental stage. Recent observations also showed that expression of
Bax, a proapoptotic member of the Bcl-2 family, was increased in blastocysts
from diabetic mice (11) and
that expression of clusterin, an apolipoprotein involved in the scavenging of
apoptotic cellular debris, was increased in blastocysts from diabetic rats
(7). These observations were
confirmed in vitro when embryos from normal females were incubated in high
concentrations of glucose
(7,8,9,10,11)
and suggest that exposing preimplantation embryos to hyperglycemia may
directly dysregulate the expression of several genes whose products are
involved in the induction or execution of apoptosis.
In the present work, we examined whether the mRNA and protein expression of
Bcl-2, the antiapoptotic founding member of the eponymous gene family, was
modified in rat blastocysts upon their exposure in vitro to 28 mmol/l
D-glucose (vs. control cultures in 6 mmol/l). In addition, we have analyzed
the consequence of blocking Bcl-2 synthesis in these embryos when exposed to
high D-glucose.
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RESEARCH DESIGN AND METHODS
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Embryo collection and culture. Early blastocysts were recovered from
the uterine horns of Wistar rats and examined immediately after collection or
after culture for 24 h at 37°C in Ham's F-10 medium (Life Technologies)
complemented with 1 mmol/l glutamine, 0.1% bovine serum albumin (BSA), 100
U/ml penicillin, and 100 µg/ml streptomycin. The incubation medium was
supplemented with either 6 mmol/l D-glucose, 28 mmol/l D-glucose, or a
combination of 6 mmol/l D-glucose and 22 mmol/l L-glucose. In some
experiments, blastocysts were maintained for 24 h in 17 mmol/l D-glucose.
In situ hybridization. Blastocysts were fixed in 3% paraformaldehyde
and 0.5% glutaraldehyde in phosphate-buffered saline (PBS), incubated in 10
µg/ml proteinase K in PBS containing 0.1% Triton X-100 (PBS-TR), and washed
in 2 mg/ml of PBS-TR before refixation in 4% paraformaldehyde and 0.2%
glutaraldehyde in PBS. The embryos were treated with 0.1% sodium borohydride,
prehybridized, and then exposed to a combination of two 5'-end
biotinylated oligodeoxynucleotides (custom synthesized by Life Technologies).
These probes were complementary to two distinct regions that were common to
both rat Bcl-2
and Bcl-2ß (Fig.
1). These two antisense oligodeoxynucleotides were code named ISHG
and ISHL and were used at the final concentration of 0.5 µmol/l. Negative
control reactions were performed with a combination of corresponding reverse
complementary oligodeoxynucleotides (sense oligodeoxynucleotides, code named
ISHF and ISHK). After posthybridization and washing in PBS with 0.5% Tween-20
(PBS-T0.5), the embryos were blocked in 0.3% hydrogen peroxide in PBS-T0.5,
permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate, and incubated with
peroxidase-conjugated streptavidin. The embryos were counter-stained with 25
µg/ml Hoechst 33258 in PBS-T0.5 and developed in diaminobenzidine and
nickel chloride. For each blastocyst, the proportion of cells strongly
positive for Bcl-2 mRNA was expressed as a percentage of its total cell
number.

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FIG. 1. Position and sequence of the different Bcl-2 probes, primers, and
antisense oligodeoxynucleotides. A: Organization of the Bcl-2 gene
with the position of the first nucleotide (NT#) in the starting ATG codon
(common to Bcl-2 and Bcl-2ß) in the first exon numbered as
position 1. Numbering in the second exon restarts at the first nucleotide of
that exon. The location of the conserved putative intron in exon 1 is
indicated. The alternative mRNA splicing site in exon 1 is indicated by
( ) and defines the two possible Bcl-2 transcripts, which stop in either
exon 2 (Bcl-2 ) or exon 1 (Bcl-2ß). The positions of the in situ
hybridization probes ( labeled ISHG and ISHL) and those of the
amplification primers ( labeled PCR1, PCR2, PCR3, and PCR4) are
indicated relative to the Bcl-2 gene sequence. B: Presentation of the
different domains of the Bcl-2 protein with the first N-terminal amino acid
(AA#) numbered as position 1. The seven functional domains identified in the
Bcl-2 protein are BH4, phosphorylation loop (LOOP), BH3, BH1, pore-formation
(PO), BH2, and transmembrane (TM). The site of the divergence between
Bcl-2 and Bcl-2ß (midway into the BH2 domain), which is secondary
to the alternative splicing decision, is indicated by ( ). The
Bcl-2ß-specific C-terminus has no known function ( ). The positions
of the antisense oligodeoxynucleotides ( labeled ASBH4-Ø,
ASBH2-Ø, and ASTM-Ø) are indicated relative to the different
domains of the Bcl-2 protein. C: Nucleotide sequences of the in situ
hybridization probes, amplification primers, and antisense
oligodeoxynucleotides described in A and B.
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Immunocytochemistry. Blastocysts were treated in acidic Tyrode
solution, transferred onto concanavalin Acoated coverslips, and
centrifuged at 180g for 10 min. The embryos were then fixed in 1.7%
paraformaldehyde in PBS, permeabilized in 1% Triton X-100 in PBS, and
incubated overnight at 4°C in 0.5 µg/ml rabbit anti-human Bcl-2 primary
antibody (Santa Cruz Biotechnology) in PBS with 1% Tween-20 (PBS-T1) and 3%
BSA. Negative control reactions consisted in either replacing the primary
antibody with normal rabbit IgG or omitting the primary antibody from the
procedure. The embryos were then treated with 5.5 µg/ml fluorescein
isothiocyanate (FITC)-conjugated goat anti-rabbit IgG secondary antibody
(Sigma Chemicals) in PBS-T1 for 60 min at 37°C, counter-stained in 1
µmol/l Topro-3 iodide (Molecular Probes) in PBS-T1, and mounted before
examination on a confocal laser scanning microscope. Some embryos were
observed without prior treatment with antibodies to verify the absence of
autofluorescence signal.
Reverse transcription and polymerase chain reaction. Total RNA was
isolated from blastocysts after lysis in 4 mol/l guanidine thiocyanate with
0.5 mol/l 2-mercaptoethanol and then treated with 10 U DNase I per reaction
for 30 min at 37°C in the presence of 40 U RNase inhibitor. After
phenol-chloroformisoamylalcohol extraction, purified RNA was retrotranscribed
with 120 pmoles poly(dT) 15 primers and 50 U reverse transcriptase (Roche
Molecular Biochemicals) per reaction for 45 min at 42°C. Fresh enzyme was
added to the reaction before repeating the incubation. Total cDNA was then
amplified with 25 pmoles rat Bcl-2specific primers and 3.5 U Taq-Pwo
DNA polymerases (Roche Molecular Biochemicals). Upstream primers code named
PCR1 and PCR2 were complementary to sequences in Bcl-2 exon 1 that are common
to Bcl-2
and Bcl-2ß, whereas downstream primer PCR3 was located in
exon 2 and therefore specific for Bcl-2
(Fig. 1). Primer PCR4 was
located in the alternatively transcribed end of exon 1 and hence specific for
Bcl-2ß. Amplicons were visualized by horizontal gel electrophoresis. To
verify their sequence, these amplicons were also directly inserted into pCR
2.1 T/A cloning vectors (In Vitrogen) and analyzed on both strands using M13
reverse or M13 forward primers. Products of thermal cycle sequencing reactions
were analyzed by vertical gel electrophoresis.
Antisense oligodeoxynucleotides. Antisense (AS)
oligodeoxynucleotides were designed against different regions (domains BH4,
BH2, and transmembrane [TM]) of the rat Bcl-2
transcript and code named
accordingly (Fig. 1).
Phosphorothioate linkages were incorporated in the last three 3'-end
internucleotide phosphodiester bridges (custom-synthesized by Biosource).
Public sequence databases were searched repeatedly to confirm the specificity
of these oligodeoxynucleotides against Bcl-2. Reverse complementary control
sense (SN) oligos were synthesized for each AS oligo. Before studying the
effect of the different anti-Bcl-2 antisense oligodeoxynucleotides on
blastocysts exposed to high D-glucose, the action of these inhibitors was
first tested in a cell-free assay. Synthetic RNA was generated by in vitro
transcription of 0.7 µg of a plasmid containing the entire coding sequence
of mouse Bcl-2
(mBcl-2
/pcDNAI is from C. Borner, University of
Friboug, Switzerland) and translated using an optimized rabbit reticulocyte
lysate (Novagen) supplemented or not with 5 U of RNase H in the presence of 40
µCi of (35S)-L-methionine. Sense and antisense
oligodeoxynucleotides were added at the concentration of 15 µmol/l before
the initiation of the transcription-translation reaction. Protein synthesis
was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
and autoradiography. Preliminary embryotoxicity experiments were then
performed on blastocysts. Embryos were cultured in increasing concentrations
of AS or SN oligos (0-15 µmol/l) for 24 h and then incubated in a hypotonic
solution of 0.9% sodium citrate, fixed in acetic acid and ethanol (3:1), and
stained in 4% Giemsa before cell number counting. Irrespectively of the oligo
tested, no embryotoxicity was detected for concentrations up to 15 µmol/l
(data not shown). In a third set of preliminary experiments, blastocysts were
incubated for 24 h in 15 µmol/l of sense or antisense oligodeoxynucleotides
and then subjected to reverse transcriptase-polymerase chain reaction (RT-PCR)
with different pairs of primers. PCR2 and PCR3 were used to amplify Bcl-2 mRNA
whereas primer sets specifically designed to amplify either rat ß-actin
(code named ACT1 [5'-ATGGGTCAGAAGGACTCCTA-3'], ACT2
[5'-ACACAGAGTACTTGCGCTCA-3']) or rat glyceraldehyde 3-phosphate
dehydrogenase (code named GPD1 [5'-CCATGGAGAAGGCTGGGG-3'], GDP2
[5'-CAAAGTTGTCATGGATGACC-3']) were used in positive control
reactions.
Detection of karyolysis and karyorhexis. Blastocysts were exposed to
0.4% pronase, fixed in 4% paraformaldehyde in PBS, blocked with 0.3% hydrogen
peroxide in methanol, and permeabilized in 0.1% Triton X-100 in 0.1% sodium
citrate. The embryos were then prestained with 25 µg/ml Hoechst 33258 in
PBS with 0.5% Tween-20 (PBS-T0.5) and incubated in 50 U/ml of terminal
transferase and 15 µmol/l fluorescein-conjugated dUTP (Roche Molecular
Biochemicals) for 35 min at 37°C. The addition of terminal transferase was
omitted in control negative reactions. After incubation in
peroxidase-conjugated sheep antifluorescein antibody, terminal
deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining
was developed in diaminobenzamide and nickel chloride. For each blastocyst,
the proportions of cells displaying signs of karyolysis (TUNEL-positive
spherical nucleus) and karyorhexis (Hoechst-stained nuclear fragments) were
expressed as percentages of the total cell number.
Statistical analysis. Differences between control values and
experimental values were compared by one-way analysis of variance coupled with
post hoc Scheffe's F-test. The data were given as means ± SE. For each
experiment blastocysts from at least 5 females were pooled and randomized
before direct observation or assignement to different incubation groups.
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RESULTS
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Bcl-2 mRNA and protein expression in control embryos. Blastocysts
were analyzed by in situ hybridization for the expression of Bcl-2 mRNA using
a mixture of antisense ISHG and ISHL probes. In almost every blastocyst that
was examined at the time of recovery, only 1-2% of the total number of cells
per embryo were strongly positive (Fig.
2). The same proportion of cells with a high ISH signal was found
in blastocysts that had been cultured for 24 h in 6 mmol/l D-glucose (see
below). Cells with a high Bcl-2 mRNA content did not seem to be preferentially
localized in the ICM or TE lineage. Complete absence of staining was found
when blastocysts were hybridized to corresponding control ISHF and ISHK sense
probes (Fig. 2) or when probes
were omitted altogether from the procedure (data not shown).

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FIG. 2. Bcl-2 mRNA expression in rat blastocysts. A: In situ
hybridization was performed on a freshly collected blastocyst using a
biotinylated antisense probe mixture for Bcl-2. In the small portion of the
blastocyst, featured here at high magnification, two strongly
Bcl-2+ cells are identified ( ). This embryo is representative
of a total of 25 blastocysts that were analyzed for Bcl-2 mRNA expression by
in situ hybridization immediately after recovery from the uterine horns.
B: In situ hybridization using a negative control sense probe mixture
for Bcl-2. No specific ISH signal was detected above weak homogenous
background. The scale bar represents 10 µm in A and
B.
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Blastocysts were examined by immunocytochemistry for the synthesis of
Bcl-2. The cell-to-cell heterogeneity in Bcl-2 expression revealed by ISH
staining was confirmed at the protein level. Moderate levels of Bcl-2 protein
were detected in most of the cells and stronger immunostaining was found in a
small proportion of them. This heterogenous pattern was found in freshly
recovered embryos (data not shown) as well as in cultured blastocysts
(Fig. 3). Cells with a high
immunostaining signal could not be ascribed to a particular cell lineage. In
both moderately and strongly positive cells, Bcl-2 protein expression was
concentrated in the cytoplasmic compartment. No staining was observed in the
blastocoelic cavity. Substitution of the primary antibody with nonimmune
rabbit IgG or complete omission of this step from the procedure resulted in
very low background staining. Omission of both primary and secondary
antibodies indicated that autofluorescence was negligible
(Fig. 3).
Total cDNA from blastocysts was amplified with different pairs of Bcl-2
primers. Use of PCR1 and PCR3 generated an amplicon of
1590 bp in length
(data not shown) in accordance with the possibility that a 220-bp putative
intron, previously described in the Bcl-2
transcript from other
species, is conserved in the rat. Amplification of blastocyst cDNA with PCR2
and PCR3 produced an 887-bp amplicon (Fig.
4) whose sequence was found identical to the corresponding region
of the published rat Bcl-2
cDNA. The combination of PCR2 with PCR4, a
downstream primer specific for Bcl-2ß, generated a 710-bp amplicon whose
sequence was identified as corresponding to the second Bcl-2 isoform.
Transcripts for both Bcl-2
and Bcl-2ß were detected in blastocysts
analyzed at the time of collection as well as after culture for 24 h in either
6 mmol/l or 17 mmol/l D-glucose.
Effect of high D-glucose on Bcl-2 expression. Blastocysts incubated
in either 6 mmol/l or 28 mmol/l D-glucose for 24 h were compared for their
average number of cells per embryo and for the frequencies of cells showing
signs of nuclear fragmentation and chromatin degradation. Exposure to high
D-glucose resulted in a significant 14% decrease in the mean number of cells
per embryo (P
0.01) and in a significant threefold increase in
the incidence of both nuclear apoptotic markers (P
0.01)
(Fig. 5). The proportion of
cells displaying a mitotic figure remained low (<1%) in both culture groups
(data not shown). Incubating the blastocysts in the hyperosmotic control
culture medium had no influence compared with 6 mmol/l D-glucose
(Fig. 5).
Exposure to high D-glucose for 24 h also induced an eight-fold increase in
the proportion of cells that were strongly positive for the transcription of
the Bcl-2 gene when compared with either 6 mmol/l D-glucose or hyperosmotic
control culture medium (P
0.01)
(Fig. 5). Close inspection of
immunostained blastocysts revealed a similar increase in the proportion of
cells that were strongly labeled for the Bcl-2 protein after exposure to high
D-glucose for 24 h against 6 mmol/l D-glucose (data not shown).
Effect of blocking Bcl-2 synthesis on the blastocyst response to high
D-glucose. Antisense oligodeoxynucleotide activity was verified in a
cell-free protein synthesis assay using the complete coding sequence of mouse
Bcl-2
as a template. In the presence of RNase H, the addition of
antisense oligodeoxynucleotides against the BH4 (ASBH4-Ø) or BH2
(ASBH2-Ø) domains of Bcl-2 completely abolished the synthesis of the
expected 236 amino acid-long protein, whereas the addition of the
corresponding sense oligodeoxynucleotides had no such effect
(Fig. 6) (data not shown). The
addition of antisense oligodeoxynucleotides against the TM domain of Bcl-2
(ASTM-Ø), in contrast to ASBH4-Ø and ASBH2-Ø, resulted in
the reproducible formation of a shorter protein that may correspond to a
TM-truncated 209-amino acid version of the protein. The addition of the
corresponding sense oligodeoxynucleotide had no effect on the synthesis of the
Bcl-2 protein (data not shown). Control reactions with RNase H alone showed
that the enzyme had no inhibitory activity on the transcription-translation of
the Bcl-2 cDNA template in the absence of antisense oligonucleotides
(Fig. 6). In a second series of
experiments, rat blastocysts were incubated in the presence or absence of the
different antisense oligodeoxynucleotides and then subjected to RT-PCR with
the Bcl-2specific primer pair PCR2 and PCR3. Amplification for Bcl-2
was found to be specifically inhibited by the addition of antisense
oligodeoxynucleotides ASBH4-Ø, ASBH2-Ø, or ASTM-Ø,
whereas control amplification for either ß-actin or
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) could proceed normally
(Fig. 6). None of the control
sense oligodeoxynucleotides was found to prevent amplification of Bcl-2 mRNA
extracted from treated blastocysts (Fig.
6) (data not shown).

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FIG. 6. Influence of antisense oligodeoxynucleotides on Bcl-2 expression.
A: An acellular transcription-translation assay was performed using a
cDNA encoding the complete sequence of mouse Bcl-2 as template.
Reactions were performed under control conditions (CONTROL), in the presence
of ribonuclease-H alone (RNase-H) or in the presence of ribonuclease-H and
either one of three different antisense oligodeoxynucleotides directed against
the corresponding BH4, BH2, and TM domains of the Bcl-2 protein
(ASBH4-Ø, ASBH2-Ø, and ASTM-Ø, respectively). In this
experiment, a control negative reaction was carried out in parallel using
sense oligodeoxynucleotide SNBH4-Ø combined with ribonuclease-H.
Control negative reactions with the sense oligodeoxynucleotides SNBH2-Ø
and SNTM-Ø were performed in other series of experiments (data not
shown). Reaction products were visualized by Western blot analysis using a
rabbit primary anti-mouse Bcl-2 antibody alongside protein size markers (left
margin, kDa). Complete Bcl-2 protein size is 236 a.a. (right margin, )
and truncated Bcl-2 protein size is 209 a.a. ( ). B: Total cDNA
was prepared from rat thymus (THYMUS) and from rat blastocysts after
incubation for 24 h in either control culture medium (CONTROL) or in the
presence of either one of the three antisense oligodeoxynucleotides
ASBH4-Ø, ASBH2-Ø, and ASTM-Ø. Control negative tests were
carried out in parallel with sense oligodeoxynucleotides SNBH2-Ø and
SNTM-Ø. The absence of influence of sense oligodeoxynucleotide
SNBH4-Ø was verified in other series of experiments (data not shown).
The cDNAs were amplified with primers specific for Bcl-2 , ß-actin,
or GAPDH (right margin) and the reaction products were analyzed by gel
electrophoresis alongside DNA size markers (left margin, in bp). A control
negative amplification was performed in parallel without RNA input during the
reverse-transcription step (RNA-Ø). In the course of the experiments
summarized above, each antisense or sense oligodeoxynucleotide was tested at
least twice with identical results (effect or lack thereof) in the acellular
assay as well as in blastocysts.
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The deleterious impact of 28 mmol/l D-glucose on blastocyst development was
found to be significantly enhanced when the embryos were cotreated with high
D-glucose and antisense oligodeoxynucleotides ASBH4-Ø or
ASBH2-Ø. Cell deficiency was 23% after the combination of high
D-glucose with either one of these two antisense oligodeoxynucleotides
(P
0.05 vs. high D-glucose alone)
(Fig. 7). This combination also
induced a significantly higher increase in the frequency of cells displaying
signs of chromatin degradation (P
0.01 vs. high D-glucose
alone). In contrast, neither ASBH4-Ø nor ASBH2-Ø antisense
oligodeoxynucleotides were found to enhance the occurrence of high
D-glucose-induced nuclear fragmentation. In control experiments, none of the
sense oligodeoxynucleotides had any detectable effect on the different
responses of the blastocysts to high D-glucose
(Fig. 7).
In contrast to the sensitizing effect of ASBH4-Ø and ASBH2-Ø,
antisense oligodeoxynucleotides directed against the TM domain of Bcl-2
improved the resistance of blastocysts to the impact of high D-glucose.
Compared with blastocysts incubated in high D-glucose alone, embryos cotreated
with high D-glucose and ASTM-Ø antisense oligodeoxynucleotides had a
significantly lower cell deficit (P
0.01) and incidence of
chromatin degradation (P
0.01)
(Fig. 7). However,
ASTM-Ø anti-sense oligodeoxynucleotides did not influence the induction
of nuclear fragmentation in blastocysts by high D-glucose.
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DISCUSSION
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Although Bcl-2 protein synthesis has been detected in preimplantation
embryos (9,
13) and in derivatives of the
three fetal germ layers during early organogenesis
(16) in the mouse, Bcl-2-null
embryos are able to develop normally throughout the first half-period of
gestation (17). However,
recent reports on mouse blastocysts indicate that Bcl-2 mRNA may be
differentially expressed in the ICM and TE cell lineages
(12) and that Bcl-2 protein
expression is decreased in fragmented embryos
(13), two observations that
indirectly support the possibility that Bcl-2 is important during early
embryogenesis. In the present study, we show that Bcl-2 is expressed at both
mRNA and protein levels in rat blastocysts with no apparent difference between
ICM and TE cells. In both cell lineages, a small proportion of cells were
found to contain high concentrations of Bcl-2 transcripts and proteins.
Heterogeneous distribution of two other Bcl-2-related effectors, Bax and
Bcl-X, has previously been described in human blastocysts
(18) and interpreted as
reflecting the consequence of a cell-specific pattern of gene transcription
activity that may be initiated after fertilization
(4). A broad range of Bcl-2
expression levels has also been described in human cytotrophoblasts
(19), raising the possibility
that their sensitivity to cell-death induction may be predetermined based on a
Bcl-2dependent mechanism.
Rat blastocysts reacted to high D-glucose with an increase in the
frequencies of two nuclear markers of apoptosis, chromatin degradation
(karyolysis) and nuclear fragmentation (karyorhexis), as well as with an
increase in the proportion of cells that were strongly labeled for Bcl-2 mRNA
and protein expression. Transient increases in mRNA and protein Bcl-2
expression levels have been observed in reaction to the induction of apoptosis
in several cell systems, such as in pancreatic ß-cell lines on serum
withdrawal (20), supporting
the concept that elevation in the production of antiapoptotic effectors may be
integral to the survival of certain cells. Interestingly, mouse blastocysts
have previously been found to react to an exposure to high D-glucose with an
increase in pro-apoptotic Bax expression
(11). However, in contrast to
Bcl-2, enhanced Bax expression was evenly distributed across the embryos,
suggesting that antagonizing Bcl-2 and Bax effectors may be differentially
up-regulated by high D-glucose in embryonic cells.
Previous studies have shown that Bcl-2 can occur in two alternatively
spliced isoforms that differ at their C-termini
(21). Compared with shorter
Bcl-2ß, Bcl-2
contains a BH2 domain that is crucial for
dimerization with other Bcl-2like proteins and a transmembrane domain
that anchors Bcl-2
to intracellular membranes. In the present study,
RT-PCR analysis revealed that both Bcl-2
and Bcl-2ß isoforms were
coexpressed in rat blastocysts. Preliminary data were obtained that suggested
a preferential upregulation of Bcl-2
mRNA in blastocysts exposed to
high D-glucose (data not shown), but more experiments will be required to
confirm that observation.
To better delineate the relevance of Bcl-2 in the embryonic response to
high D-glucose, rat blastocysts were pretreated with antisense
oligodeoxynucleotides directed against different sequences of the Bcl-2 mRNA,
which correspond to distinct functional domains in the Bcl-2
protein.
The first antisense oligodeoxynucleotide, ASBH4-Ø, was designed to
hybridize to the BH4 domain that is common to the two Bcl-2 isoforms.
Pretreatment with ASBH4-Ø was found to decrease the expression of Bcl-2
and to sensitize the blastocysts to the impact of high D-glucose on cell
proliferation and chromatin degradation. The addition of ASBH2-Ø, an
oligodeoxynucleotide against the BH2 dimerization domain that is absent in
Bcl-2ß, had the same effects as ASBH4-Ø. Thus, in support of the
general hypothesis that Bcl-2 is an antiapoptotic protein, inhibiting its
synthesis resulted in increased D-glucose embryotoxicity.
In contrast, the addition of ASTM-Ø, an oligodeoxynucleotide
hybridizing just upstream of the Bcl-2
transmembrane domain, protected
the blastocysts against high D-glucose. In vitro protein synthesis assays
showed that production of a smaller Bcl-2 protein occurred in the presence of
ASTM-Ø, suggesting that hybridization of ASTM-Ø to Bcl-2
transcripts would not lead to mRNA degradation but rather induce modifications
in Bcl-2 mRNA splicing (22) or
block the completion of later translational events
(23) and thereby produce a
truncated Bcl-2 protein. The former possibility may also explain the absence
of Bcl-2 amplicon when mRNA from ASTM-Øtreated blastocysts was
examined by RT-PCR. Previous studies have shown that Bcl-2
proteins
lacking a transmembrane domain can increase cell resistance to cytotoxic
agents under certain conditions
(24,
25). Whether this explanation
applies in ASTM-Øtreated blastocysts remains to be
investigated.
None of the antiBcl-2 antisense oligodeoxynucleotides influenced the
susceptibility of rat blastocysts to the induction of nuclear fragmentation by
high D-glucose. This suggests that, in contrast to chromatin degradation,
Bcl-2 is not involved in this second nuclear apoptotic event. Experiments in
progress show that specific inhibition of either caspase-3 or
caspase-activated deoxyribonuclease in rat blastocysts also failed to block
the induction of nuclear fragmentation by high D-glucose (data not shown), an
indication that the intracellular cascades leading to nuclear fragmentation
and chromatin degradation may be either completely independent or diverge
downstream of a common trigger mechanism.
There is now convincing evidence that severe developmental anomalies
leading to fetal resorption or malformation can occur as consequences of
subtle damages inflicted to the embryos before or at the time of implantation
(26). One of these primary
damages may be the disruption of the highly regulated gene program that
controls the expression pattern of crucial developmental determinants during
early embryogenesis, including apoptosis
(9). The present study shows
that rat blastocysts express the antiapoptotic effector Bcl-2 and that their
exposure to high D-glucose in vitro induces an increase in Bcl-2 expression in
a limited number of cells. Blocking Bcl-2 synthesis with antisense
oligodeoxynucleotides sensitized the blastocysts to the apoptotic impact of
D-glucose. If the implication of Bax
(11) and Bcl-2 in the control
of the apoptotic cascade that is triggered by high D-glucose in blastocysts
thus appears established, then the precise nature of the signals that are
acting upstream and downstream of these effectors remains to be
investigated.
 |
ACKNOWLEDGMENTS
|
---|
This work was supported by an Action de Recherche
Concertée de la Direction
Générale de la
Recherche de la Communauté
Française de Belgique (grant 96/01-96), the
Juvenile Diabetes Foundation International, and the Fonds de la Recherche
Scientifique Médicale de Belgique (grant
3.4527.99). S.P. is Maître de Recherche of the
Fonds National de la Recherche Scientifique de Belgique.
 |
FOOTNOTES
|
---|
AS, antisense; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate;
ICM, inner cell mass; PBS, phosphate-buffered saline; PBS-T, PBS with Tween;
PBS-TR, PBS containing Triton; RT-PCR, reverse transcriptase-polymerase chain
reaction; SN, control sense; TE, trophectoderm; TM, transmembrane; TUNEL,
terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
Received for publication June 7, 2000
and accepted in revised form September 11, 2000
 |
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