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Department of Obstetrics and Gynecology (M.M.C., A.L.S., K.H.M.), Department of Cell Biology and Physiology (K.H.M.), Washington University School of Medicine, St. Louis, Missouri 63110
Address all correspondence and requests for reprints to: Dr. Kelle H. Moley, Departments of OB/GYN and Cell Biology and Physiology, Washington University School of Medicine, 4911 Barnes-Jewish Hospital Plaza, St. Louis, Missouri 63110. E-mail: moleyk{at}msnotes.wustl.edu
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Introduction |
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The mechanism for these embryotoxic effects of insulin and insulin-like growth factors has not been explored. High insulin concentrations induce loss of the insulin receptor in several different cell types (10, 11). This phenomenon of hormone-induced receptor loss is dose dependent with a 10–20% reduction at 10 nM and 60–85% at 1 µM (11). The down-regulation is linear over the first 4–6 h and then plateaus, where it reaches a new steady-state. As with the insulin receptor, the IGF-1 receptor (IGF-1R) is regulated by the ambient concentration of the IGF-1 ligand (12). A decrease in receptor number with increasing IGF-1 concentration due to internalization of the complex has been demonstrated in numerous systems, including lymphoid cells, FRTL-5 thyroid cells, endothelial cells, and bovine articular chondrocytes (12, 13). Similarly, it has been suggested that down-regulation occurs in the preimplantation blastocyst (14, 15).
Insulin and IGF-1 stimulate glucose uptake in the preimplantation blastocyst (16), and this uptake has been shown to operate via the IGF-1R, not the insulin receptor. Both receptors are first expressed at the 8-cell stage. It is well established in other cell types that the IGF-1R, activated by its ligands, plays a critical role in regulating programmed cell death vs. maintaining the transformed phenotype. Antibodies to the IGF-1R, antisense expression plasmids to IGF-1R, and dominant negative mutants of the IGF-1R can all reverse the transformed phenotype, inhibit tumorigenesis, and result in an increase in apoptosis (17, 18). De Pablo et al. (19) have also shown that insulin receptor down-regulation by antisense oligonucleotides also leads to increased apoptosis in the neurulating chicken embryo. We propose that similar down-regulation of the IGF-1R is occurring in preimplantation embryos exposed to high IGF-1 concentrations leading to significant apoptosis of the inner cell mass (ICM) or key progenitor cells of the embryo. Such an embryotoxic insult may be responsible for the high incidence of pregnancy loss seen in women with polycystic ovary syndrome.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the studies in Bax null mice, Bax -/- , +/-, and +/+ female mice were superovulated and mated with either Bax +/- males (for the Bax +/- and -/- females) or Bax +/+ males (for the Bax +/+ females). Bax-/- males are infertile, and thus Bax heterozygote males must be mated with Bax null females (21). Embryos were recovered 96 h after mating. For the studies using caspase and ceramide synthase inhibitors, control, wild-type embryos were cultured in high concentrations of IGF-1 with or without either 10 µM z-valine-alanine-aspartate-fluoromethylketone, (zVAD-FMK), (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA), a caspase inhibitor, or 100 µM fumonisin B1 (Sigma, St. Louis, MO), a natural, fungal-derived inhibitor of ceramide synthase.
Terminal dUTP nick end labeling (TUNEL) assays to detect apoptosis
in blastocysts
This technique has been described previously for mouse
blastocysts (22, 23). Fixed blastocysts were
counterstained with propidium iodide to label all nuclear DNA and
fragmented DNA was end labeled with fluoroscein isothiocyanate (FITC)
labeled dUTP using terminal transferase (Cell Death In situ
Kit, Roche Molecular Biochemicals). The embryos were then
observed using confocal immunofluorescent microscopy (Bio-Rad Laboratories, Inc. MRC-600). A complete z-series was performed
for each blastocyst to ensure that each nucleus was sampled and
counted. The degree of apoptosis is expressed as % terminal dUTP nick
end labeling (TUNEL)-positive nuclei (green channel) per total nuclei
(red channel) per embryo. These experiments were performed in
triplicate with 7–10 blastocysts per group for each experiment. This
labeling technique was performed on three different test groups, each
with a nontreatment control. These groups include 1) blastocysts from
Bax -/-, +/-, +/+ mice mated as described
above and cultured in high vs. low IGF-1; 2) blastocysts
cultured in high vs. low IGF-1 with or without added caspase
inhibitor, zVAD-FMK; 3) blastocysts cultured in high vs. low
IGF-1 with or without added ceramide synthase inhibitor, fumonisin
B1.
Expression of IGF-1R protein in response to high ligand
concentrations using confocal microscopy
Blastocysts were exposed to 2% Pronase for 2 min, fixed on
glass slides with 3% paraformaldehyde, and permeabilized with 0.1%
Tween. The embryos were then washed and incubated with a primary
chicken antimouse IGF-1R antibody to the 
-subunit (Upstate Biotechnology, Inc., Lake Placid, NY) for 1 h at
room temperature (10 µg/ml). The embryos were then washed and
incubated with a secondary antibody, donkey anti-chicken FITC-labeled
antibody for 1 h. Nuclear staining was then performed by
incubating the embryos in propidium iodide at a concentration of 0.01
mg/ml. Following extensive washing, confocal immunofluorescent
microscopy (Bio-Rad Laboratories, Inc. MRC-600, Hercules,
CA) was then use to detect fluorescence as previously described
(20). Whole embryo fluorescence was then quantitated using
NIH Image (version 1.6).
Expression of IGF-1R protein by Western analysis
Blastocysts were cultured as above and were collected in groups
of 20–30. The pooled samples of equivalent numbers of embryos were
then solubilized for 30 min at 4 C in a HEPES buffer (50
mM HEPES, 1 mM EDTA, 150 mM NaCl, 1
mM Vanadate, 1%BSA, 1% Triton, pH 7.4) containing
protease inhibitors. The supernatants were then immunoprecipitated
overnight with a rabbit polyclonal antimouse IGF-1R antibody directed
toward the -subunit and not cross-reactive with the insulin receptor
(Upstate Biotechnology, Inc.; 1:1000). Immune complexes
were then bound to Protein-A Sepharose beads (Upstate Biotechnology, Inc.) and washed extensively. The samples were
then added to 2x sample buffer, subjected to 7.5% SDS-PAGE and
transferred to nitrocellulose. IGF-1R was then detected using an
antibody generated against the b-subunit of IGF-1R (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:1000).
125I-Labeled goat antirabbit IgG was used as the
secondary antibody. Radioactive bands were quantitated by a
PhosphorImager SI Analyzer (Molecular Dynamics, Inc. Sunnyvale, CA). All experiments were performed in
triplicate.
Functional analysis of the IGF-1R
Insulin-stimulated glucose transport—an event known to be
regulated by the IGF-1R in the mouse blastocyst. Two-cell embryos
were cultured for 72 h at 37 C in an atmosphere of
5%CO2/5%O2/90%
N2 in HTF containing 1.3 nM or 130
nM IGF-1. Embryos were then washed extensively,
preincubated for 30 min in HTF without IGF-1 containing 0 or 170
nM insulin. Embryos were directly placed in 200
µM 2-deoxyglucose (DG) for 15 min, washed in DG-free,
BSA-free buffer for 1 min, and then quick-frozen on a glass slide.
After freeze-drying overnight, the embryos were extracted in microliter
volumes under oil and single embryos were assayed for DG and
2-deoxyglucose-6-phosphate (DG6P) as described previously
(20). The final measurements are expressed as picomoles
per embryo per 15 min. Experiments were performed in triplicate on
10–15 individual embryos per group for each experiment.
Blocking IGF-1R with blocking antibody IR3
To determine whether blocking IGF-1R signaling would recreate
the apoptotic event seen with high IGF-1 concentrations, two cell
embryos were cultured for 72 h in 1 µg/ml IR3, a monoclonal
antibody (Oncogene Research Products, Manhasset,
NY) known to inhibit IGF-1 binding in the blastocyst and block
activity of the IGF-1R in other cell systems (15). To
confirm that adequate blockade of the receptor was achieved, IGF-1R
autophosphorylation and function were measured in blastocysts using
Western analysis and DG uptake into single blastocysts as described
above. Embryos cultured in IR3 were compared with embryos cultured
in high or low concentrations of IGF-1. After demonstration of
decreased autophosphorylation and function in these embryos, TUNEL was
then used to examine blastocysts exposed to IR3 for evidence of
apoptosis. For these experiments, an additional control group of
embryos were cultured in an equivalent concentration of an
isotype-matched IgG1 negative control antibody, trp E (Oncogene Research Products). The TUNEL methods are described above.
Decreasing IGF-1R expression with antisense oligonucleotides
Expression of IGF-1R was blocked with the antisense oligoprobes
to determine whether decreased expression of IGF-1R recreates the
apoptotic event seen with high IGF-1 concentrations. Two-cell embryos
were cultured for 72 h in 0.5 µM IGF-1R antisense
(5'-TCC TCC GGA GCC AGA CTT) or sense (5'-AAG TCT GGC TCC GGA GGA)
oligodeoxynucleotides corresponding to codons 21–26 of the signal
sequence of the ß-subunit IGF-1R preceding the proreceptor sequence.
These oligodeoxynucleotides have been used previously in rodent models
and are known to block expression successfully (17).
Antisense experiments have also been described in a preimplantation
embryo system using the protocol outlined here (24, 25).
Four cell embryos were exposed to 0.01% lysolecithin for 30 min and
then cultured for a further 48 h in control media containing 5
µM IGF-1R sense or antisense. The lysolecithin has been
shown to facilitate transfer of the oligonucleotides into the embryos.
The embryos were cultured in these conditions in 25 µl droplets under
oil and were moved to new equivalent droplets after 24 h. The
oligodeoxynucleotides used were modified to contain phosphorothioate
linkages to decrease degradation. Measuring protein expression and
function using confocal immunofluorescent microscopy and 2-DG uptake
into individual blastocysts respectively assessed the efficiency of the
antisense treatment. After demonstration of decreased IGF-1R expression
and function in these embryos, TUNEL was then used to examine
blastocysts for evidence of apoptosis. These methods are also described
above.
Statistical methods
Differences between the groups with protein expression,
insulin-stimulated glucose uptake and percent TUNEL staining were
compared by one-way ANOVA coupled with Fisher test (Statview 4.5).
Differences between the blastocysts from the different BAX genotypes
exposed to high or low concentrations of IGF-1 also were compared by
ANOVA coupled with Fisher test. Results are expressed as means ±
SE of at least three separate experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGF-1 induces apoptosis in mouse blastocysts
Apoptotic cell assays revealed significantly elevated %
TUNEL-positive or % nuclei containing fragmented DNA/total nuclei in
blastocysts exposed to 130 nM IGF-1 (n = 17)
vs. 1.3 nM (n = 16); 63 ±
9% vs. 6 ± 2%, respectively (P <
0.001) (Fig. 1
). There was no significant
difference between embryos cultured in low IGF-1 vs. control
medium, and thus this lower concentration was used as a control for the
remaining experiments. The DNA fragmentation detected occurred
predominantly in the ICM nuclei. Similarly, high insulin also caused an
increase in DNA fragmentation with 70 ± 9.9% apoptotic nuclei in
embryos cultured in 700 nM insulin (n = 15)
compared with 7 ± 3.1% in 6 nM insulin
(n = 20) (P < 0.001). In contrast, high
concentrations of testosterone (400 nM) failed to
induce increased DNA fragmentation.
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IGF-1-induced apoptosis is BAX-dependent and involves caspases and
ceramide synthesis
To test whether activation of the BAX cell-death effector system
is involved in IGF-induced apoptosis, as is seen in
hyperglycemia-induced apoptosis (23), two cell embryos
from matings of Bax -/- females with Bax
+/- males were cultured for 72 h in HTF containing 1.3
nM (n = 17) or 130 nM
IGF-1 (n = 19). These results were compared with embryos from
Bax +/+ females mated with Bax +/+ males (n
= 15 low IGF-1 and n = 17 high IGF-1). Bax-/- males
are infertile (21) and thus Bax heterozygote
males must be mated with Bax null females. Consistent with
the predicted 50% null embryo mendelian ratio, half the embryos
obtained from the Bax +/- X Bax-/-
matings showed almost complete resistance to IGF-1-induced apoptosis,
5 ± 3% in High IGF-1 (n = 10) vs. 6 ± 4%
in low IGF-1 (n = 14) TUNEL-positive nuclei (Fig. 2). The other half exhibited an apoptotic
phenotype similar to the control wild-type embryos. These results were
in comparison to 64 ± 7% and 7 ± 3% among the embryos
from Bax +/+ matings. These in vitro findings are
consistent with an important role for BAX in high IGF-1-induced embryo
cell death.
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) with only 21 ±
7% TUNEL-positive nuclei per embryo (n = 22) vs.
57 ± 9% in control embryos incubated in high IGF-1 alone (n
= 23). Results were similar in high and low insulin (data not shown).
Despite some findings suggesting a caspase-independent BAXinduced
cell death pathway (30), these results combined with the
Bax-/- embryo findings support the hypothesis of a
hierarchical cell death pathway system with caspases acting downstream
of BAX (31).
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, A and B).
Only 23 ± 4% TUNEL-positive nuclei per embryo were seen among
the embryos in fumonisin B1 (n = 19) vs. 57 ± 9%
in control embryos incubated in high IGF-1 alone (n = 20).
Although this fungal toxin recently has been shown to have no
preventative effect on mouse oocytes undergoing daunorubicin-induced
ceramide-related apoptosis (33), we have shown that
inhibition of ceramide synthase in the mouse blastocyst partially
prevents high glucose induced apoptosis. Similarly, ceramide synthase
inhibition has been shown in several other cell types to inhibit
stress-activated apoptosis (34). Clearly these findings
provide evidence that generation of ceramide contributes to the
high IGF-1induced apoptotic signaling pathways in the mouse
blastocyst.
High IGF-1 induced decrease in IGF-1R expression
Evidence provided by prior studies of both the IGF-1 and insulin
receptors has suggested that high IGF-1 concentrations trigger
apoptosis by a down-regulation of the IGF-1R (17, 18). To
test the hypothesis, immunofluorescent confocal microscopy was used to
detect protein expression of the IGF-1R. With this technique, 53
± 2% less IGF-1R protein was detected in the embryos exposed to high
IGF-1(n = 7) vs. embryos in low IGF-1 (n = 9)
(Fig. 4A). A similar decrease in IGF-1R
protein expression was seen when immunoprecipitation combined with
Western blot analysis on groups of pooled embryos was used. Blastocysts
were cultured as above except that three different concentrations of
IGF-1, 1.3, 65, 130 nM, were used to determine
whether this down-regulation was dose dependent. This experiment was
performed twice with 40–60 blastocyst per group. By Western analysis,
a 45 ± 9% reduction in IGF-1R protein was seen among embryos
exposed to high IGF-1 levels in vitro vs. low
IGF-1, and this decline in protein expression was dose dependent (Fig. 4B).
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The next hypothesis to test was that this glucose transport event was
down-regulated in response to high IGF-1 as is insulin-stimulated
glucose transport in most other systems in response to high insulin.
Using these techniques, a significant 80-fold reduction in the
insulin-stimulated DG uptake was measured in embryos exposed to high
IGF-1 (basal, n = 21; insulin-stimulated, n = 22) compared
with control embryos stimulated with insulin (1, P <
0.001, basal, n = 25; insulin-stimulated, n = 20, Fig. 5). High insulin exposure for 72 h
followed by insulin-stimulated glucose uptake assay resulted in a
1.6-fold reduction basal, n = 15; insulin-stimulated, n =
12), which likewise was significant (**, P < 0.01). In
contrast, elevated testosterone concentrations had no effect on
insulin-stimulated glucose transport (basal, n = 12,
insulin-stimulated n = 11) (37) .
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IR3 decreases receptor autophosphorylation
and insulin-stimulated glucose uptake and triggers apoptosisIR3 blockade of the receptor
successfully inhibited IGF-1R autophosphorylation upon addition of
insulin (Fig. 6A).
Likewise, IR3 treatment resulted in inhibition of
insulinstimulated glucose uptake at a blastocyst stage (Fig. 6B).
Having confirmed that the blockade worked, apoptosis was examined using
TUNEL. Microscopy revealed increased % TUNEL-positive nuclei in the
embryos exposed to IR3 (46 ± 9; n = 20) compared with
embryos cultured in 1.3 nM IGF-1 (6.0 ± 3; n =
21) and a similar % apoptotic nuclei compared with embryos cultured in
130 nM IGF-1 (63 ± 9; n = 22) (Fig. 6C). This
provides further evidence that a decrease in signaling via the IGF-1R
is responsible for the apoptotic event.
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). This decrease in expression
correlated with a decrease in insulin-stimulated DG uptake (Fig. 7B).
This decrease in protein expression and function lead to an increase in
TUNEL-positive nuclei in the embryos cultured in antisense (35 ±
8; n = 27). This increase was significantly greater than embryos
cultured in sense (5 ± 3;n = 22) or embryos in 1.3
nM IGF-1 (6 ± 4; n = 16) and was
similar to embryos cultured in 130 nM IGF-1
(63 ± 10; n = 15) (Fig. 7C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis in the rodent and human preimplantation embryo is a normal process (38, 39, 40). Regulation of this apoptotic process is essential for future development of the embryo. Programmed cell death at this stage of mammalian development is thought to eliminate redundant cells in the inner cell mass, which retain trophectoderm potential after differentiation and thus are deficient in normal developmental capacity (41). In human embryos obtained for in vitro fertilization, apoptosis occurs in normal appearing embryos but is much more prevalent in arrested and fragmented embryos, which are often not used for transfer due to their morphology (38, 42). Studies have also shown increased cell death in mouse preimplantation embryos undergoing retarded, suboptimal development. Suggestions have been made that uncontrolled apoptosis or programmed cell death occurring earlier than a blastocyst stage in preimplantation embryos may lead to embryo demise (38). We have previously shown that high glucose-induced apoptosis causing increased apoptosis at a blastocyst stage results in higher resorption rates in a mouse model. Therefore, up-regulation of the apoptotic pathway may protect the early embryo by eliminating abnormal cells; however, loss of balance in this tissue remodeling may lead to developmental arrest and demise.
Lack of a threshold level of glucose transport triggers apoptosis in the blastocyst (24). This phenomenon occurs in maternal hyperglycemia. In earlier studies, we have demonstrated that hyperglycemia leads to down-regulation of the GLUTS at the blastocyst stage in response to hyperglycemia (20). This occurs concurrently with the onset of increased apoptotic nuclei (23). Blocking GLUT1 expression during the first 72 h in culture with antisense oligonucleotides results in increased apoptosis in the blastocyst stage (24). Similarly, in other cells systems, decreased basal glucose uptake has been shown to initiate the programmed cell death cascade (43, 44, 45) whereas overexpression of GLUT1 can protect against apoptosis (46). The clinical manifestation of this preimplantation event in mice is increased pregnancy loss and congenital malformations, both occurring at a greater frequency in maternal hyperglycemia due to insulin-dependent diabetes mellitus. It should be noted that decrease glucose transport and apoptosis are not the only explanation for the increased pregnancy loss and fetal anomalies among these patients. Several studies using postimplantation models of diabetic embryopathies have implicated other diabetes-associated metabolic alterations. These include increased generation of oxygen free radicals (47), increased sorbitol and ß-hydroxybutyrate (48, 49, 50), and decreased myoinositol levels (51).
At the blastocyst stage, embryonic metabolism switches from using oxidative phosphorylation of pyruvate to glycolysis of glucose (52). This energy switch is believed to occur in preparation for implantation and a temporary anaerobic existence and to afford synthesis of macromolecules from glycolytic intermediates (53, 54). As a result, glucose consumption increases significantly, with the greatest consumption occurring in the inner cell mass (55). Simultaneous to this increase in glucose consumption, the blastocyst exits from the fallopian tube and enters the uterine cavity, at which time in normal physiology insulin and IGF-1 levels both rise (26). It is believed that an insulin/IGF-1 regulated glucose transporter, specifically GLUT8, which we have recently cloned from a blastocyst complementary DNA library (56), responds by translocating to the apical plasma membranes of the blastocyst to maintain glucose homeostasis despite this major shift in energy consumption and glucose utilization. We speculate that with hyperinsulinemia or high IGF-1 levels, the IGF-1/IGFR signaling mechanisms necessary to traffic GLUT8 to the plasma membrane are dysfunctioning, and that without this critical increase in glucose uptake by GLUT8, the lower threshold of glucose is reached and programmed cell death is triggered. This hypothesis agrees with the finding that the TUNEL-positive nuclei are predominantly in the ICM, which at this stage has the greatest demand for glucose consumption. If the degree of programmed cell death is significant enough within the ICM, the pregnancy may result in a fetal resorption.
Women with polycystic ovary syndrome have significantly higher rates of spontaneous miscarriages (1, 2, 3). Hyperinsulinemia in these women is associated with elevated bioactive levels of IGF-1 in serum, follicular fluid and presumably fallopian tube fluid. Elevated concentrations of this growth factor are known to adversely affect embryo development both in vivo and in vitro (8, 9). This study suggests that apoptosis is triggered and accelerated in the mouse blastocyst as a result of IGF-1-induced down-regulation of the receptor and subsequent decreased signaling of IGF-1R associated pathways. Decreased insulin stimulated glucose uptake via GLUT8 may be the inciting metabolic event to trigger apoptosis. This IGF-1-induced embryotoxic insult may be responsible for the high incidence of pregnancy loss seen in women with polycystic ovary syndrome. Given this information, attempts should be made to lower IGF-1 levels by lowering insulin levels in these women to decrease their miscarriage rate.
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Acknowledgments |
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Footnotes |
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Received July 12, 2000.
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