CREB-vermittelte Mechanismen der Adaptation und endokrinen

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Gutachter:
Prof. Dr. Dr. Bernd Fischer
Prof. Dr. Gabriele Stangl
Prof. Dr. Heiner Niemann
Verteidigung am: 29.6.2015
Halle
(Saale)
Halle
(Saale)
0
0
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Inhalt
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DIABETES-INSULIN-GLUCAGON-GASTROINTESTINAL
Maternal Diabetes Impairs Gastrulation and Insulin
and IGF-I Receptor Expression in Rabbit Blastocysts
Nicole Ramin,* René Thieme,* Sünje Fischer, Maria Schindler, Thomas Schmidt,
Bernd Fischer, and Anne Navarrete Santos
Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, D-06097 Halle
(Saale), Germany
Women with type 1 diabetes are subfertile. Diabetes negatively affects pregnancy by causing early
miscarriage and poor prenatal outcomes. In this study we examine consequences of maternal type 1
diabetes on early embryo development, metabolic gene expression, and the pattern of insulin receptor
(IR) and IGF-I receptor (IGF-IR) distribution in rabbit blastocysts. In female rabbits, type 1 diabetes was
induced by alloxan treatment. Six-day-old blastocysts were recovered and assessed for receptor distribution and metabolic gene expression. In vitro culture of blastocysts was performed in medium
containing 1 mM, 10 mM, or 25 mM glucose, simulating normo- and hyperglycemic developmental
condition in vitro. The fertility rate of the diabetic rabbits clearly mirrored subfertility with a drop in
blastocyst numbers by 40% (13.3 blastocysts in diabetic vs. 21.9 in control females). In blastocysts onset
and progression of gastrulation was delayed and expression of IR and IGF-IR and their metabolic target
genes (hexokinase, phosphoenolpyruvate carboxykinase), both in vivo and in vitro, was down-regulated. The amount of apoptotic cells in the embryonic disc was increased, correlating closely with the
reduced transcription of the bcl-x(L) gene. Blastocyst development is clearly impaired by type 1 diabetes
during early pregnancy. Insulin-stimulated metabolic genes and IR and IGF-IR are down-regulated,
resulting in reduced insulin and IGF sensitivity and a delay in development. Dysregulation of the IGF
system and embryonic glucose metabolism are potential reasons for diabetogenous subfertility and
embryopathies and start as soon as during the first days of life. (Endocrinology 151: 4158 – 4167, 2010)
omen with type 1 diabetes have a significantly
higher risk of pregnancy loss and embryopathies
compared with healthy women, indicating that a tight
control of glucose and/or insulin is essential for proper
embryo development. One of the most sensitive and vulnerable periods in ontogenesis is the formation and development of the embryoblast and trophoblast during blastocyst formation and differentiation. The mammalian
preimplantation embryo is supplied with nutrients and
growth factors such as glucose, amino acids, insulin, and
IGFs by oviductal and uterine secretions. Glucose is an
essential energy source of the blastocyst (1). Earlier cleavage stages favor pyruvate or lactate as the main energy
substrate. The switch from pyruvate/lactate to glucose is
considered to account for the increased energy require-
ments due to blastocyst expansion and Na⫹/K⫹-ATPases
that facilitate blastocyst cavitation and expansion (2). The
embryo is able to tolerate variations in glucose concentration, but both, a diabetic condition and deprivation
from glucose, are known to be detrimental for the developing embryo. Cell numbers are decreased and can
result in abnormal morphogenesis (3– 6) and developmental arrest (7). Glucose itself is able to modulate the
transcription of many genes, encoding enzymes typical
for glycolytic and lipogenic pathways (8 –10) and gluconeogenesis (11, 12).
Glucose homeostasis in mammals is tightly controlled
by a balanced interaction of peripheral tissues and their
sensitivity to insulin. The glycemic control of insulin is
mediated by the insulin receptor (IR). The IR is highly
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2010 by The Endocrine Society
doi: 10.1210/en.2010-0187 Received February 18, 2010. Accepted June 4, 2010.
First Published Online July 14, 2010
* N.R. and R.T. contributed equally to this study.
Abbreviations: HK, Hexokinase; IGF-IR, IGF-I receptor; IR, insulin receptor; p.c., post coitum;
PEPCK, phosphoenolpyruvate carboxykinase; PVA, polyvinyl alcohol.
W
4158
endo.endojournals.org
Endocrinology, September 2010, 151(9):4158 – 4167
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Endocrinology, September 2010, 151(9):4158 – 4167
endo.endojournals.org
4159
homologue to the IGF-I receptor (IGF-IR). In IR-deficient
cells insulin can mediate its metabolic effects via the
IGF-IR (13). In physiological concentrations insulin acts
only by the IR. The IR is detectable in embryos in all preimplantation stages in most species including the rabbit
(14 –16). Hyperglycemia or type 2 diabetes induce an altered IR expression pattern in adult tissues leading to various degrees of insulin insensitivity (17, 18).
Type 1 diabetes can be experimentally induced by alloxan or streptozotocin, an established type 1 diabetic
model in laboratory animals (19). Alloxan causes a pancreatic ␤ cell-specific necrosis resulting in type 1 diabetes
with insulinopenia and hyperglycemia (20, 21). In mice
both agents were used to investigate the influence of maternal type 1 diabetes on embryo development (22–24).
For rabbits the alloxan-induced diabetes is routinely used
in biomedical and pharmaceutical experimental approaches [see Winiarska et al. (25) for references]. At the
third day after alloxan treatment the blood glucose concentration in the treated rabbits is in a hyperglycemic
range (⬎14 mmol/liter).
The rabbit embryo implants late at d 6 after mating [at
6 d 18 h, (26)]. During d 6 post coitum (p.c.; i.e. during the
preimplantation period), the embryonic disc of the rabbit
blastocysts develops from a nondifferentiated two cell
layer gastrulation stage (epiblast and hypoblast) to three
germ layers, allowing a subtle analysis of diabetogenous
effects on gastrulation in this species. Using the advantages
of the rabbit model such as induced ovulation, high embryo numbers, and large-sized blastocysts (with 2–5 mm
in diameter at d 6 p.c.) and well defined gastrulation stages
before implantation, we investigated the effects of type 1
diabetes on the maternal fertility rate and uterine glucose
and serum insulin concentrations on the one hand and on
the embryonic metabolic and insulin/IGF receptor gene
expression, the early gastrulation, and apoptosis in the
preimplantation blastocyst on the other hand. The in vivo
findings were complemented by in vitro cultures of blastocysts in high glucose media with or without insulin
supplementation.
solution [27.5% (wt/vol)] was injected sc for a subcutaneous
deposit of glucose. Furthermore, the drinking water was supplemented with 5% (wt/vol) glucose. These precautions prevented critical hypoglycemic conditions as the first phase response of the alloxan treatment after 6 – 8 h (see glucose
profile in Fig. 1C). Hyperglycemia of the rabbit with a permanent blood glucose concentration of ⬎14 mmol/liter was
established 36 to 48 h after alloxan injection (Fig. 1C).
Rabbits were hold in diabetic conditions for 9 to 11 d before
they were mated. Ketoacidosis was prevented by regular insulin
supplementation (Insuman Rapid 40 IU/ml, Aventis, Frankfurt
a.M., Germany) after feeding. The blood glucose level was monitored with MediSense Precision Xceed Diabetes Management
System (Abbott, Wiesbaden, Germany) two times a day and kept
in the range of 14 –25 mmol/liter (Fig. 1C). All animal experiments were in accordance with the principles of laboratory animal care and had been approved by the local ethical commission
of the Landesverwaltungsamt Dessau (reference number:
42502-2-812).
Embryo recovery and in vitro culture
Embryos were collected from alloxan and control rabbits
stimulated with 150 IU pregnant mare serum gonadotropin sc
(PMSG, Intervet, Tönisvorst, Germany) 3 d prior to mating.
After mating 75 IU human choriogonadotropin iv (hCG, Scher-
Materials and Methods
Alloxan treatment
Female rabbits were adjusted to a feeding regime with a 3-h
food interval in the morning and in the evening. Ten days before
mating rabbits were fasted overnight, anesthetized with Dormitor
im (0.25 mg/kg body weight, Pfizer, Karlsruhe, Germany) and
analgized with Ketanest im (15 mg/kg body weight, Pfizer,
Karlsruhe, Germany). Alloxan (120 mg/kg body weight, SigmaTaufkirchen, Germany) was injected in the marginal ear
Aldrich,
vein with a Multyfly needle followed by a 10 ml saline injection
(0.9%). Fifteen minutes after alloxan injection 50 ml glucose
FIG. 1. Insulin and glucose concentrations in type 1 diabetes and
normoglycemic rabbits. A, Alloxan-treated type 1 diabetic females had no
or only remnants of insulin-positive cells in pancreatic tissue. The ␤ cells in
the Langerhans Islands (*) were visualized by immunohistochemical
detection (brown color) with an anti-insulin antibody. The plasma insulin
concentrations were significantly decreased (B). A blood glucose profile of
an individual female is shown in C, starting with the day of alloxan
treatment. # indicates the starting point of insulin supplementation. A
constant hyperglycemic level was achieved 3 d after alloxan treatment.
Due to reduced insulin amounts, the glucose concentration was
significantly increased in plasma (D) and uterine fluids (E) of type 1 diabetic
rabbits (mean ⫾ SEM; n ⫽ 3; n ⱖ 5; *, P ⬍ 0.05). Bar, 100 ␮m.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 March 2015. at 22:20 For personal use only. No other uses without permission. . All rights reserved.
4160
Ramin et al.
Maternal Diabetes Restricts Embryo Development
Endocrinology, September 2010, 151(9):4158 – 4167
ing AG, Berlin, Germany) was injected. Mating, embryo recov(27), and embryo culture (28) were performed as described
ery
before. At d 3, 4, and 6 p.c., embryos were flushed from the
oviducts or uteri and washed two times with PBS and processed
for subsequent analyses or in vitro experiments. At d 3 p.c. embryos are in the morula stage and develop to early blastocysts at
d 4 p.c. The d 3 and d 4 embryos were analyzed by RT-PCR
pooled in groups of 10 embryos. Individual d 6 blastocysts were
analyzed directly after recovery or used for in vitro culture. The
gastrulation stage was assessed by morphological examination
of the embryonic disc (embryoblast) according to Viebahn et al.
(29). For in vitro culture, embryos were pooled and randomly
divided among experimental groups. To study the effect of varying glucose concentrations and insulin, d 6 blastocysts were cultured in groups of four to 10 embryos in basal synthetic medium
II at 37 C in a saturated atmosphere of 5% O2, 5% CO2, and
90% N2 in a water-jacketed incubator (BB6060, Heraeus,
Hanau, Germany). The standard glucose concentration in culture media for rabbit embryos is 10 mM (15, 30, 31). In current
in vitro study, we mimicked normal in vivo uterine glucose concentration (1 mM) and hyperglycemic developmental conditions
(25 mM) in vitro. After preculture for 2 h in serum- and insulinfree basal synthetic medium II containing 1 mM, 10 mM, or 25 mM
glucose, 17 nM insulin (Sigma-Aldrich, Taufkirchen, Germany)
was added to the culture medium for further 1 to 4 h. Controls
were cultured in defined glucose concentrations without insulin
but otherwise handled identically. After culture, embryos were
washed twice in ice-cold PBS and transferred into ice-cold 0.05%
polyvinyl alcohol (PVA)/PBS buffer. To investigate gene expression of receptors and key enzymes, blastocyst coverings (neozona, mucin coat) were mechanically removed and the single
blastocysts were stored at ⫺80 C. For Western blot analysis, ten
embryos were randomly pooled in each group. Samples were
homogenized in 100 ␮l RIPA buffer (PBS, 1% Nonidet P-40,
0.5% sodium deoxycholat, 0.1% SDS) with protease and phosphatase inhibitor cocktail (Roche, Mannheim, Germany) and
stored at ⫺80 C.
Insulin-ELISA
The US Insulin ELISA (DRG Instruments GmbH, Marburg,
Germany) was used for measurement of the insulin levels according to the manufacturer’s protocol using EDTA-plasma.
Measurement of glucose concentration
The glucose concentrations in the plasma and uterine fluid of
alloxan-treated and control rabbits were assessed with an enzy-
matic assay based on a turnover of glucose by exogenous added
hexokinase (HK). The product of NADPH was measured spectrophotometrically at a wavelength of 334 nm. 2.8IU HK
(Roche, Mannheim, Germany) was added to semimicro cuvettes
(Brand, Wertheim, Germany) containing 2.5 mM Mg2⫹-ATP
(Sigma-Aldrich, Taufkirchen, Germany) and 0.5 mM NADP
(Applichem, Darmstadt, Germany) in 100 mM triethanol-buffer
with a final volume of 1 ml. Before supplementation of HK,
3.5IU glucose-6-phosphat dehydrogenase (Roche, Mannheim,
Germany) was added to remove endogenous glucose-6-phosphate. The extinction is proportional to the developed NADPH
concentration reflecting the glucose concentration. The uterine
was absorbed directly from the endometrial surface by usfluid
ing glucose-free paper strips without any additional flushing,
getting approximately 5 ␮l of uterine fluid per uterus. Before
measurement the fluid was washed out of the paper with a defined volume of deionized water (dilution 1:3).
RNA extraction, RT reaction, and PCR detection of
insulin
RNA extraction was performed as previously described (15)
by Dynabeads mRNA DIRECT Kit (Dynal, Oslo, Norway) according to the manufacturer’s instructions. The final volume for
cDNA reaction was adjusted to 100 ␮l.
Total RNA extraction from pancreatic tissues and RT-reaction were performed as published before. PCR amplification was
carried out with 3 ␮l cDNA from tissues and blastocysts in a
50-␮l volume containing 200 ␮M each dNTP and 2.5IU Taq
polymerase, using the primer combination insulinfw 5⬘-TTTATACACCCAAGTCCCGCCG-3⬘/ insulinrev 5⬘-GAGCAGATGCTGGTGCAACACT-3⬘ (acc. U03610). Resulting PCR
products of 147 bp were separated by electrophoresis on 2.0%
agarose gel and stained with ethidium bromide.
Cloning and sequencing of partial cDNA for rabbit
HK
A new partial HK (118bp, Acc.No. FJ848380) cDNA was
amplified from rabbit kidney tissue using human primers. The
sequence was identical with human HK-2 to 92% and 98% at the
mRNA and the protein level, respectively, using alignment
BLASTN and BLASTX modus.
Real time RT-PCR for IR, IGF-IR,
phosphoenolpyruvate carboxykinase (PEPCK), HK,
Bcl-x(L), and GAPDH
Samples were analyzed by real-time RT-PCR as described
previously (15). The appropriate primer sets: rabGAPDHfw
5⬘-GCCGCTTCTTCTCGTGCAG-3⬘/ rabGAPDHrev 5⬘-ATGGATCATTGATGGCGACAACAT-3⬘ (acc. L23961), IRfw
5⬘-CCTGAAGGAGGTGGAGGAG-3⬘/IRrev 5⬘-GAGAATCCTGGGACTGTGG-3⬘ (acc. AY339877), IGF-IRfw 5⬘-CCCAAGCTCACGGTCATCACTG-3⬘/IGF-IRrev 5⬘-ATGGGCTTCTCCTCCAAGGTCC-3⬘ (acc. EF616472), rabPEPCKfw
5⬘-TGCGGCCTCCAAAGATGATG-3⬘/rabPEPCKrev 5⬘-CCCTGGAAACCTGGTGACAAGG-3⬘ (acc. EF616471), rabHKfw
5⬘-ACAGCAACCACATCCAGGTCAAAC-3⬘/HKrev5⬘-TTCTCCTCAAGTGGACGAAAGGCT-3⬘ (acc. FJ848380), and rabbcl-x(L)fw 5⬘-GGTATTGGTGAGTCGGATCG-3⬘/rabbcl-x
(L)rev 5⬘-TGTTGCCGTAGAGTTCCACA-3⬘ (acc. AY005131)
for rabbit GAPDH, IR, IGF-IR, PEPCK, HK, and bcl-x(L), respectively. Each assay included duplicates of each cDNA sample and a
no template control for each primer set. Relative mRNA expression for IR, IGF-IR, PEPCK, HK, bcl-x(L), and GAPDH was calculated using the ⌬⌬CT method. The expression of GAPDH RNA
was used to normalize samples for the amount of cDNA used per
reaction. To confirm the amplification, the resulting real-time RTPCR products were analyzed by dissociation curves, visualized in
an agarose gel (GAPDH 144bp, IR 150bp, IGF-IR 347bp, PEPCK
143bp, HK 118bp, bcl-x(L) 113bp) and sequenced. In each realtime RT-PCR, a calibration curve was included that was generated
from serial dilutions (106, 105, 104, 103, 102, and 101 copies) of
primer-specific DNA probes generated from cDNA plasmid
clones. Analysis of the individual data therefore yielded values
relative to these standards. For each group the number of analyzed
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Endocrinology, September 2010, 151(9):4158 – 4167
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4161
washed again in ice-cold PBS and transferred to 0.05% PVA/PBS. The embryonic
disc was mechanically dissected with surgical forceps and scissors (Fine Science Tools
GmbH, Heidelberg, Germany) and either
used immediately for immunohistochemistry or stored in 100% methanol at ⫺20 C
until use. Stored embryonic discs were rehydrated through a series of graded alcohols. The pancreatic tissue was fixed in 4%
paraformaldehyde for 1 h, embedded in paraffin, and fixed on a slide in working sections of 5 ␮m. Nonspecific antibody binding
was blocked with 10% normal goat serum
in PBS at room temperature for 1 h. The
specimens were incubated with the monoFIG. 2. Insulin detection in rabbit embryos. A, Rabbit embryos did not express insulin mRNA
clonal anti-insulin IgG1 mouse-antibody (1:
neither at the morula (d 3), the early blastocyst (d 4), nor at the expanded blastocyst stage (d
200, I2018, Sigma-Aldrich, Taufkirchen,
6) during gastrulation [stage (St.) 0/1, 2, or 3]. RT-PCR analysis was performed at cDNA pools
Germany) in 1% BSA/PBS overnight at 4 C.
of 10 embryos for d 3, d 4, and individual blastocyst for d 6. Representative results of two
Samples were washed with 0.05% PVA/PBS
individual blastocysts per gastrulation stage (A–F) are shown (n ⫽ 2, n ⫽ 3). Pancreas was
used as positive control for RT-PCR amplification. ntc, No template control. B, In in vivo rabbit and incubated with the secondary antiserum goat-antimouse-IgG (Dako EnViblastocysts (control) insulin protein is localized in the cytoplasm of embryoblast (Em) and
sion, Hamburg, Germany). Embryonic discs
trophoblast cells (Tr). The insulin was provided to the embryo by uterine secretions. An in
vitro culture of blastocysts without insulin for 10 h strongly diminished the protein amount,
and tissue sections were examined by light
proving the degradation of insulin in embryonic cells. Blastocysts from type 1 diabetic females
microscopy (Axioplan, Carl Zeiss, Jena,
no detectable insulin protein. No staining was detectable in blastocysts when the
Germany). The specificity of immunostainhad
antiinsulin antibody was preabsorbed with insulin, demonstrating the specificity of the
ing of the primary antibody was proven by
antibody reaction (n ⫽ 3, primary antibody preabsorption control). Bar, 200 ␮m.
blocking the antibody binding with insulin,
using insulin as blocking peptide (bovine 40
blastocysts is given in the diagram bar. Data are expressed as relamg/ml, Sigma-Aldrich, Taufkirchen, Gemany). The antibody
tive values (%) to control blastocysts.
was preincubated with insulin (1:200 in 1% BSA/PBST) for 2 h
at 37 C and further 24 h at 4 C. Subsequent application of the
Protein preparation and immunoblotting
neutralized antibody to the IHC reaction did not show any pos Protein isolation and Western blot analyses were performed itive staining (Fig. 2B, primary antibody preabsorption control),
with 20 ␮g protein from whole blastocysts. The IR and IGF-IR
demonstrating the specificity of the antibody to insulin. The
protein expression was investigated with a monoclonal mouse IR
antibody (1:2000, Calbiochem, Darmstadt, Germany) and rab- specificity of immunostaining of the secondary antibody reaction was proven by the absence of signals in sections processed
bit IGF-IR antibody (1:1000, Santa Cruz, Heidelberg, Gerafter omission of the primary antibody.
many). After blotting, the nylon membrane was stained with
Ponceau. Apparent molecular weights were determined by comparison with PageRuler prestained molecular weight marker
TUNEL
(Fermentas, St. Leon-Rot, Germany). Afterwards the immunoBlastocysts exposed to a diabetic or normoglycemic uterine
reactive signals were visualized by enhanced chemiluminescence
environment were fixed in 4% paraformaldehyde and their emdetection (Millipore, Schwalbach, Germany) and quantified by
ChemiDoc-It Imaging System (LTF Labortechnik, Wasserburg, bryonic discs were obtained as described before. The discs were
permeabilized with 0.1% Tween 20 and incubated in fluoresceGermany). The amounts of receptor protein were evaluated by
in-labeled dUTP and terminal transferase in the dark for 1 h at
the membranes and reblotting with a mouse monoclostripping
37 C to label fragmented 3⬘ DNA (TUNEL, Cell Death In Situ
nal ␤-actin antibody (1:40000, Sigma-Aldrich, Taufkirchen,
Kit, Roche, Mannheim, Germany). A rabbit kidney cell line
Germany). Protein amounts were calculated as ratio of band
(RK13) treated with or without DNase was used to establish and
intensities
(IR
or
IGF-IR
vs.
␤
-actin)
in
the
same
blot
to
correct
test the TUNEL assay in rabbit cells (data not shown). The
for differences in protein loading. The relative expression of IR
TUNEL assays were performed with 9 and 15 blastocysts in
and IGF-IR in embryos of normoglycemic or diabetic animals
was calculated as ratio of expression signals of diabetic to nor- control and diabetic blastocysts, respectively. Counterstaining of
all nuclear DNA was achieved by incubating the embryos with
moglycemic controls.
Hoechst staining (1 mg/ml, Sigma-Aldrich, Taufkirchen, Ger
many) for 20 min. Embryos were visualized using fluorescence
Immunohistochemical localization of insulin
light microscopy (Axioplan, Carl Zeiss, Jena, Germany). The cell
The localization of insulin was investigated in blastocysts and
pancreatic tissue of type 1 diabetic and healthy female rabbits. number of the embryoblast cells was counted with ImageJ software (Fa. Wayne Rasband, National Institutes of Health, BeThe embryonic disc with the surrounding trophoblast was disMD) using the Cell Counter plug-in for digital images. An
sected from whole blastocysts. In vivo derived or in vitro cultured
blastocysts were washed twice in ice-cold PBS and fixed in 4% thesda,
average cell number was counted. The number of apoptotic cells
paraformaldehyde for at least 1 h. The fixed blastocysts were
was related to the total number of counted embryoblast cells.
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4162
Ramin et al.
Maternal Diabetes Restricts Embryo Development
Endocrinology, September 2010, 151(9):4158 – 4167
Statistics
Levels of significance between groups were calculated using
Student’s t test after proving normal distribution (SigmaPlot v.
11.0). Data are expressed as mean ⫾ SEM. The difference in the
mean values of the two groups is greater than would be expected
by chance. There is a statistically significant difference between
input groups. The levels of significance were P ⬍ 0.05, P ⬍
the
0.01, and P ⬍ 0.001.
Results
Insulin and glucose concentration in uterine fluids
and in blastocysts
Alloxan caused a destruction of pancreatic ␤ cells (Fig.
1A) and a massively reduced insulin secretion (Fig. 1B).
In the serum of females with type 1 diabetes (d 6 p.c.)
the plasma insulin concentration was decreased by 85% to
15 pM (Fig. 1B), and blood glucose concentration rose to
ⱖ14 mmol/liter (Fig. 1, C and D). At the third day after
alloxan treatment the blood glucose concentration was
held steady to 18 –25 mmol/liter, defining strong hyperglycemia. All females were profiled twice daily for their
blood glucose levels (example in Fig. 1C). Blood glucose
levels ⱖ25 mmol/liter were prevented by external insulin
supply for each individual female. Hyperglycemia was reflected by 3.5-fold elevated uterine glucose levels (Fig. 1E).
The rabbit blastocyst itself does not express insulin.
The cDNA samples from morulae at d 3, early blastocysts
at d 4, and expanded blastocysts at d 6 from gastrulation
stage 0/1, 2, and 3 were tested negatively for insulin RNA
by RT-PCR (Fig. 2A). The protein, however, was present
in the cytoplasm of in vivo grown blastocysts when analyzed directly after recovery embryoblast and trophoblast
cells (Fig. 2B). In blastocysts of type 1 diabetic rabbits
neither the embryoblast nor the trophoblast showed a positive staining for insulin (Fig. 2B). In vitro culture of blastocysts in serum-free media for up to 10 h diminished the
amount of insulin protein substantially (Fig. 2B).
Developmental competence of blastocysts from
type 1 diabetic rabbits
In type 1 diabetic animals the number of corpora lutea
(Fig. 3A) and flushed blastocysts (Fig. 3B) were significantly lower than in non-alloxan-treated controls. Blastocysts from type 1 diabetic rabbits were developmentally
retarded. At d 6.0 p.c. a lower number had developed to
stage 2, and no embryo had reached gastrulation stage 3
(Fig. 3C).
A
12
100
80
*
60
40
20
0
21
20
control
type 1 diabetes
C
*
10
8
6
4
2
0
9
15
control
type 1 diabetes
embryoblast
control
positive apoptotic cells
per embryoblast [%]
relativve bcl-x(L) transcripts [%]
B
120
type 1 diabetes
FIG. 3. Fertility of type 1 diabetic rabbits. The number of (A) corpora
lutea and (B) blastocysts at d 6 p.c. (n ⫽ 5) of type 1 diabetes (n ⫽ 11)
and
normoglycemic animals (control, n ⫽ 20) were significantly
reduced (mean ⫾ SEM; **, P ⬍ 0.001). C, The developmental
competence was assessed by morphological classification of the
embryonic disc. A significantly higher rate of blastocysts from type 1
diabetes animals was arrested in stage 0/1. Only a low number
developed to stage 2, and no blastocysts in stage 3 were detectable at
d 6.0 p.c. (*, P ⬍ 0.01; **, P ⬍ 0.001). The results of three individual
experiments (mean ⫾ SEM, n ⫽ number in the bar) are shown.
FIG. 4. Apoptosis in blastocysts from type 1 diabetic females. A, RNA
expression of bcl-x(L) in d 6 blastocysts of normoglycemic (control) and
type 1 diabetes rabbits was significantly decreased by type 1 diabetes
developmental conditions (*, P ⬍ 0.001). The result of three individual
experiments are summarized in the diagram (mean ⫾ SEM, n ⫽ number
in the bar). B, Standardized counting of embryoblast resulted in an
increase in TUNEL-positive nuclei in embryos grown under type 1
diabetic conditions. The average number of apoptotic cells per
embryoblast is given in percent of totally counted embryoblast cells
(mean ⫾ SEM, n ⫽ 2, n ⫽ number in the bar; *, P ⬍ 0.001). C,
Embryos were counterstained with the nuclear dye Hoechst (blue). The
panel shows representative embryos grown in a normoglycemic
(control) or type 1 diabetic uterine milieu. Bar, 50 ␮m. In the control
embryoblast the apoptotic cells are marked by arrows.
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Endocrinology, September 2010, 151(9):4158 – 4167
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4163
in two samples of three (with 10 pooled
blastocysts each) (Fig. 5, B and D).
Metabolic target gene expression
in blastocysts from type 1 diabetic
rabbits
The gene expression of the metabolic
key enzymes PEPCK and HK was analyzed by real-time RT-PCR. Compared
with in vivo-derived d 6 blastocysts
from normoglycemic controls, the RNA
levels of both enzymes were significantly
decreased in blastocysts from type 1 diabetic animals. The transcript numbers for
PEPCK (Fig. 6A) and HK (Fig. 6B) were
50% and 60% lower, respectively.
IR, IGF-IR, and metabolic target
gene expression in blastocysts
cultured with 1, 10, or 25 mM
glucose in vitro
To further analyze the hyperglyce
mic and hypoinsulinemic effects observed in vivo, blastocysts from control
rabbits were cultured in vitro with varyFIG. 5. IR and IGF-IR expression in blastocysts of type 1 diabetes rabbits. The mRNA
ing glucose concentrations. Six-day-old
transcripts for both receptors (A and B) were detected by real-time RT-PCR (mean ⫾ SEM; n ⫽ 3).
Differences in IR (C, E) and IGF-IR (D, F) protein expression were determined by Western blot (C,
blastocysts were cultured for 3 to 6 h in
D) and densitometrical analyses (mean ⫾ SEM; n ⫽ 3). The numbers of pooled blastocysts used for
media containing glucose concentraall analysis are inserted in the bars. The type 1 diabetic uterine milieu led to a significant reduction
tions of 1, 10, or 25 mM, respectively,
of receptor quantity (*, P ⬍ 0.05; **, P ⬍ 0.01) on mRNA (IR, IGF-IR) and protein level (only IR)
compared with blastocysts derived from normoglycemic mothers (control).
representing normal glucose concentration in uterine secretions (1 mM), stan
To assess the apoptotic state of the blastocysts grown dard in vitro levels (10 mM), and hyperglycemic developunder type 1 diabetic conditions, the transcription level of ment concentration (25 mM). Analyzed by real-time
the antiapoptotic gene bcl-x(L) and the number of apo- RT-PCR, transcripts of IR, IGF-IR, PEPCK, and HK were
ptotic cells were quantified (Fig. 4). The RNA amount for distinctly changed (Figs. 7 and 8). Increasing glucose conthe bcl-x(L) gene was reduced by 40% (Fig. 4A). Consis- centrations up to 25 mM for 3 h caused a reduction in IR
tent with this decrease in gene expression was a 4.3-fold and IGF-IR expression by 43% and 58%, respectively,
higher percentage of apoptotic cells in the embryoblast compared with blastocysts cultured in medium containing
(Fig. 4, B and C). No differences were found in the averaged embryoblast cells from stage 1 blastocysts from diabetic mothers with 1974 ⫾ 139 cells per embryoblast
compared with controls with 2173 ⫾ 180 cells per embryoblast (P ⫽ 0.43).
IR and IGF-IR expression in blastocysts from type 1
diabetic rabbits
The receptors for insulin (IR) and IGF (IGF-IR) are
present in rabbit blastocysts at d 6 of gestation (15, 16).
When exposed to a hyperglycemic milieu IR RNA and
protein levels decreased dramatically by approximately
80% (Fig. 5, A and C). Also for IGF-IR a significant decrease in transcript numbers was found in all embryos,
whereas the protein amounts where down-regulated only
FIG. 6. PEPCK and HK expression in blastocysts from type 1 diabetes
rabbits. The mRNA transcripts for both enzymes (A and B) were quantified
by real-time RT-PCR (mean ⫾ SEM; n ⫽ 3). Blastocysts were cultured in
pools of three to four embryos, and transcript numbers were quantified in
single blastocysts. The numbers of blastocysts per group are indicated in
the bars. The type 1 diabetic uterine milieu led to a significant reduction in
PEPCK and HK transcript numbers (*, P ⬍ 0.05).
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4164
Ramin et al.
Maternal Diabetes Restricts Embryo Development
Endocrinology, September 2010, 151(9):4158 – 4167
FIG. 7. IR and IGF-IR expression in rabbit blastocysts cultured in vitro.
The blastocysts were cultured in pools of three to four embryos for 3
(3h) or 6 h (6h) in medium containing 1 mM, 10 mM, or 25 mM
glucose. The mRNA transcripts in single blastocysts for IR (A) and IGF-IR
(B) were quantified by real-time RT-PCR (mean ⫾ SEM; n ⫽ 3). The
numbers of blastocysts per group are indicated in the bars. A 3-h
hyperglycemic milieu led to a significant reduction of IR transcripts, an
effect which could be seen also after 6 h (**, P ⬍ 0.01). While IGF-IR
mRNA was decreased by high glucose concentration after 3-h culture,
it was restored after 6 h in 25 mM glucose.
in high glucose concentrations the RNA amount for
PEPCK was decreased with concomitantly increased HK
transcripts. Culture for additional 3 h with 25 mM glucose
did not change the PEPCK level, whereas HK transcript
numbers showed a further 4-fold increase (Fig. 8, A and B).
Insulin treatment of blastocysts cultured with 1 mM or 10
mM glucose significantly reduced the number of PEPCK
transcripts numbers and elevated the HK RNA levels. In
blastocysts from the 25 mM glucose group, neither 3 h nor
6 h insulin stimulation affected PEPCK or HK mRNA (Fig.
8, A and B).
Discussion
Maternal diabetes impairs ovulation and embryo development, a phenomenon which has been confirmed in several mammalian species [rats (32–34); mice (22, 23); rab
bit, current study].
1 mM glucose. A longer incubation for up to 6 h in 25 mM
One hypothesis for the developmental effects of diabeglucose led to an IR expression level down to only 18%
tes is the primary toxic effect of hyperglycemic glucose,
compared with 1 mM glucose concentration (Fig. 7A). In leading as consequence to an accumulation of metabolites
contrast, the same conditions reincreased IGF-IR expres- and programmed metabolic dysfunction. By Moley and
sion to the basic expression level observed in the 1 mM co-authors (35, 36) a metabolic explanation for the degroup (Fig. 7B).
velopmental retardation seen in the preimplantation emA glucose-dependent change was also detectable for bryos from diabetic mice was supposed. In these studies
PEPCK and HK transcripts (Fig. 8). After culture for 3 h only at dramatically high glucose concentrations accumulation of glucose in the embryo and
developmental delay were observed,
clearly disproving the hypothesis of a
primary toxic effect of high glucose.
In the diabetic rabbit we could show
that the uterine glucose concentration
was 3-fold increased. In absolute concentrations, however, it was as low as
1.5 mmol/liter and therefore considerably lower than in hyperglycemic blood
serum or in standard in vitro culture
experiments.
So far, the down-regulation of IR and
IGF-IR in embryos from diabetic mothers
has not been described in other experi
mental models. Our findings strongly
FIG. 8. PEPCK and HK expression in rabbit blastocysts cultured in vitro. The blastocysts were
support the view that the disturbance
cultured in pools of three to four embryos for 3 (3h) or 6 h (6h) in medium containing 1 mM,
10
in insulin-mediated glucose metabolism
mM, or 25 mM glucose with (17 nM insulin, black bars) or without insulin (control, white
causes ramifications of embryo developbars). The mRNA transcripts for PEPCK (A) and HK (B) were quantified by real-time RT-PCR in
single blastocysts (mean ⫾ SEM; n ⫽ 3). The numbers of blastocysts per group are indicated in
ment. In a first step the decrease in the
the bars. A 3-h culture with high glucose concentrations (25 mM) led to a significant
insulin concentration leads to an inhireduction in PEPCK transcription. This down-regulation still persists after 6 h (6h) (**, P ⬍
bition of the insulin-driven glucose
0.01).
Insulin
treatment
in
the
1-m
M and 10-mM glucose groups significantly reduced the
number of transcripts. In 25-mM glucose neither 3-h nor 6-h insulin stimulation affected the
metabolism which then results in an inPEPCK mRNA. The opposite trend was seen for HK mRNA. It was elevated with increasing
tracellular accumulation of nonmetaboglucose concentration, most pronounced after 6-h culture time. An insulin treatment in the
lized glucose as a major pathophysiolog1-mM and 10-mM glucose groups increased the RNA levels, whereas in less than 25 mM
glucose no insulin effect was detectable.
ical mechanism.
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Endocrinology, September 2010, 151(9):4158 – 4167
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4165
We could demonstrate that embryonic insulin sensitivity
is heavily disturbed, as shown by the strongly reduced
receptor expression. The disturbed glucose metabolism is
proven by changes in key enzymes of glycolysis (HK) and
gluconeogenesis (PEPCK) in the blastocyst. PEPCK is a
rate-limiting enzyme of gluconeogenesis in the liver. Its
gene expression is controlled at the transcriptional level
and is induced by glucagon and glucocorticoids and inhibited by insulin (37–39). Earlier studies led to the assumption that gluconeogenesis is an essential metabolic
pathway in the rabbit blastocyst (15). By inhibiting
PEPCK gene transcription, glucose participates in a feedback control loop governing its production by gluconeogenesis. A related feedback regulation was observed for
hexokinase. As a key enzyme in glycolysis, HK initiates the
phosphorylation of glucose to glucose-6-phosphate, an
intermediate for several principal metabolic pathways like
glycogen synthesis, pentose phosphate pathway, and hexosamine biosynthesis. These pathways are of vital importance for the preimplantation embryo (40 – 42). In the adult
liver, glucose is a well-known transcriptional regulator of
lipogenic, glycolytic, and gluconeogenic enzymes (43).
In the rabbit blastocysts we could demonstrate the transcriptional
regulatory capacity of glucose in vitro and in
vivo. The concrete mechanisms involved need to be clarified. Conceivably the transcription factors C/EBP␤ and
FoxO1 could be responsible, which are on the one hand
direct regulators of PEPCK expression (44, 45) and are on
the other hand themselves modulated by glucose (46, 47).
However, short-term exposure of the blastocyst to glucose
mediates embryonic genes involved in glucose homeostasis, both positively and negatively. It is not unexpected that
glucose participates in the regulation of its own production and utilization in the preimplantation embryo.
Elevated glucose levels in the uterine secretions leads to
a decrease in embryonic PEPCK expression. The insulin
stimulus, normally responsible for a decreased PEPCK ex
pression, is missing in blastocysts from mothers with type
1 diabetes. This implicates that glucose is the more potent
regulator for PEPCK in blastocysts than insulin. In case of
HK the opposite effect was observed. In blastocysts from
type 1 diabetic rabbits, HK transcription was down-regulated. The dramatic HK down-regulation can either be
caused by the absence of an adequate insulin stimulus or
the insensitivity of the blastocyst to IGFs.
The down-regulation of both key enzymes, HK and
PEPCK, seems to be a complex reaction of the blastocyst
to the type 1 diabetic situation of the mother. It cannot be
explained by a simple metabolic adaptation. The downregulation of both enzymes has consequences for the utilization of glucose as energy substrate and substrate in
biosynthesis. The deficiency in glucose utilization is the
most likely reason for the impaired preimplantation development of embryos from type 1 diabetic rabbits.
It is known from studies in various cell lines that a
hyperglycemic condition leads to oxidative stress and induction of stress-activated signaling pathways, resulting
in inflammation and apoptosis. Apoptosis in preimplantation embryos exposed to hyperglycemia has been shown
in present study and in a previous study (48). The link
between hyperglycemia, inflammation, and inactivation
of glucose-dependent transcription is currently missing,
but it is supposed that the modulation of intracellular
phosphorylation events leads to the disruption of major
glucose-dependent pathways, thus contributing to metabolic disorders, lipid accumulation, insulin resistance, diabetes, and the metabolic syndrome (49).
In preimplantation embryos insulin also acts as an important mitogenic factor mediating cell proliferation and
initiation of gastrulation, and it prevents apoptosis (14,
30). Under physiological concentrations it exerts these effects mainly via the IR. Due to high homology, it is also
able to bind to the IGF-IR (50, 51). The rabbit blastocyst,
as other mammalian preimplantation embryos too (52–
54), is not able to produce endogenous insulin. The transcription of the insulin gene is switched off as shown by
RT-PCR (Fig. 2B). Preimplantation embryos rely on maternally provided insulin. This insulin was detectable with
a significant staining in the embryoblast and trophoblast
in rabbit blastocysts immediately after recovery from the
uterus (Fig. 2B). The presence of insulin in blastocysts can
be explained by transcytosis of the peptide from maternal
blood through the uterine epithelium into the uterine secretions. Transcytosis of insulin has been shown in the
oviduct and uterus in mouse embryos (52, 54). The maternal insulin was detectable in the cytoplasm of Em and
Tr cells, indicating that the insulin protein was taken up
into the embryonic cells. For insulin it is known that the
aggregated hormone-receptor complex is internalized into
endocytic vesicles (55, 56). In the cytoplasm ligand and
receptor are sorted and insulin is targeted to lysosomes for
degradation. Insulin degradation is inevitably linked to
insulin action. All insulin-sensitive tissues degrade the hormone (57). This is apparently also applicable for the rabbit
blastocyst.
An in vitro culture without insulin reduced the detectable insulin signal in blastocysts within 10 h compared
with in vivo blastocysts (Fig. 2B), demonstrating the clearance of the insulin peptide within a fairly short time period. Blastocysts grown in diabetic mothers do not show
insulin staining (Fig. 2B), indicating that the maternal
plasma insulin level is directly correlated with the insulin
amount in blastocysts provided by uterine secretions.
Next to the loss of insulin, the signaling of the hormone is
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4166
Ramin et al.
Maternal Diabetes Restricts Embryo Development
Endocrinology, September 2010, 151(9):4158 – 4167
impeded due to IR and IGF-IR down-regulation. Nor mally, in adult tissues, the receptor is up-regulated to sensitize cells for insulin when its concentration is low (58). In
the type 1 diabetic situation, however, both receptors are
down-regulated. The in vitro culture results clearly show
that the high glucose concentration is the major factor
responsible for decreased receptor expression. Furthermore, our results show that the time interval of hypergly cemia is not pivotal, as short (6 h in vitro culture; Fig. 7)
and long-time exposure (diabetes; Fig. 5) result in a
strongly reduced receptor expression. We consider this
regulation not to be adaptive but rather a pathological
response, as seen by the missing feedback of insulin-regulated PEPCK and HK transcription under hyperglycemic
in vitro culture condition (Fig. 8).
As shown in the current study, type 1 diabetes during
pregnancy has detrimental effects on glucose metabolism
and differentiation of the blastocyst. The dysregulation of
embryonic development due to maternal lack of insulin
and hyperglycemia appears at a time when the woman is
not yet aware of her pregnancy.
Acknowledgments
We thank Michaela Kirstein and Sabine Schrötter for excellent
technical assistance.
Address all correspondence and requests for reprints to: Dr.
Navarrete Santos, Department of Anatomy and Cell
Biology, Martin Luther University Faculty of Medicine,
Grosse Steinstrasse 52, D-06097 Halle (Saale), Germany.
E-mail: [email protected].
This work was supported by the German Research Council
(DFG; grant number NA 418/4-2) and the Wilhelm Roux Program of the MLU, Faculty of Medicine.
Present address for T.S.: Department of Cell Biology, Ludwig
Maximilians University, 80539 Munich, Germany.
Disclosure Summary: The authors have nothing to declare.
Anne
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Molecular and Cellular Endocrinology 358 (2012) 96–103
Contents lists available at SciVerse ScienceDirect
Molecular and Cellular Endocrinology
journal homepage: www.elsevier.com/locate/mce
Insulin growth factor adjustment in preimplantation rabbit blastocysts and
uterine tissues in response to maternal type 1 diabetes
René Thieme 1, Maria Schindler 1, Nicole Ramin, Sünje Fischer, Britta Mühleck, Bernd Fischer,
Anne Navarrete Santos ⇑
Department of Anatomy and Cell Biology, Martin Luther University Faculty of Medicine, Halle (Saale), Germany
a r t i c l e
i n f o
Article history:
Received 17 October 2011
Received in revised form 17 February 2012
Accepted 12 March 2012
Available online 20 March 2012
Keywords:
IGF1
IGF2
Exp IDD
Rabbit preimplantation embryo
a b s t r a c t
Insulin-like growth factors (IGFs) are well-known regulators of embryonic growth and differentiation. IGF
function is closely related to insulin action. IGFs are available to the preimplantation embryo through
maternal blood (endocrine action), uterine secretions (paracrine action) and by the embryo itself (autocrine action). In rabbit blastocysts, embryonic IGF1 and IGF2 are specifically strong in the embryoblast
(ICM). Signalling of IGFs and insulin in blastocysts follows the classical pathway with Erk1/2 and Akt
kinase activation.
The aim of this study was to analyse signalling of IGFs in experimental insulin dependent diabetes (exp
IDD) in pregnancy, employing a diabetic rabbit model with uterine hypoinsulinemia and hyperglycaemia.
Exp IDD was induced in female rabbits by alloxan treatment prior to mating. At 6 days p.c., the maternal
and embryonic IGFs were quantified by RT-PCR and ELISA.
In pregnant females, hepatic IGF1 expression and IGF1 serum levels were decreased while IGF1 and
IGF2 were increased in endometrium. In blastocysts, IGF1 RNA and protein was approx. 7.5-fold and 2fold higher, respectively, than in controls from normoglycemic females. In cultured control blastocysts
supplemented with IGF1 or insulin in vitro for 1 or 12 h, IGF1 and insulin receptors as well as IGF1
and IGF2 were downregulated. In cultured T1D blastocysts activation of Akt and Erk1/2 was impaired
with lower amounts of total Akt and Erk1/2 protein and a reduced phosphorylation capacity after IGF1
supplementation.
Our data show that the IGF axis is severely altered in embryo-maternal interactions in exp IDD pregnancy. Both, the endometrium and the blastocyst produce more IGF1 and IGF2. The increased endogenous IGF1 and IGF2 expression by the blastocyst compensates for the loss of systemic insulin and IGF.
However, this counterbalance does not fill the gap of the reduced insulin/IGF sensitivity, leading to a
developmental delay of blastocysts in exp IDD pregnancy.
Ó 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
IGF1, IGF2 and insulin belong to the insulin-like growth factor
(IGF) family. They play a fundamental role in embryo-maternal
crosstalk and in early embryo development. The specific importance of IGF signalling pathways during early embryogenesis is
pointed out by knockout studies for IGF1, IGF2 and IGF1R which
all result in a reduced birth weight (40–60%) and an increased neonatal lethality (Baker et al., 1993; Liu et al., 1993). A lack of insulin
typically caused by type 1 diabetes mellitus (T1D), leads to maternal subfertility and an increase in miscarriage rates and congenital
⇑ Corresponding author. Address: Department of Anatomy and Cell Biology,
Martin Luther University Faculty of Medicine, Grosse Steinstrasse 52, D-06097 Halle
(Saale), Germany. Tel.: +49 345 5571718x01; fax: +49 345 5571700.
E-mail address: [email protected] (A. Navarrete Santos).
1
Both authors contributed equally to this work.
malformations (Casson et al., 1997; Penney et al., 2003; Verheijen
et al., 2005; Yang et al., 2006).
It is known that IGFs have the ability to compensate for each
other in a distinct manner. Mode and mechanisms of compensation during early pregnancy and diabetic pregnancy, however,
are not known. In the oviductal and uterine lumen, insulin reaches
the preimplantation embryo via transcytosis from the maternal
blood flow (Heyner et al., 1989; Schultz et al., 1992). As the embryo
cannot produce insulin itself (Heyner et al., 1989; Lighten et al.,
1997; Ramin et al., 2010; Rappolee et al., 1992; Schultz et al.,
1992), it is dependent on maternal supply. In contrast, IGF1 and
IGF2 are produced by the embryo itself and by maternal reproductive tissues (Lighten et al., 1997).
IGFs play an essential role not only in early embryo development, growth and differentiation but also in the implantation process. IGF1 has been shown to induce Akt phosphorylation in
human endometrium. Akt phosphorylation was restricted to the
0303-7207/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.mce.2012.03.007
97
R. Thieme et al. / Molecular and Cellular Endocrinology 358 (2012) 96–103
glandular epithelium during implantation (Toyofuku et al., 2006).
Activation of the PI3K-Akt signalling pathway seems to be essential
for successful implantation by maintaining cell survival, trophoblast invasion and induction of extracellular matrix remodelling
(Prast et al., 2008; Toyofuku et al., 2006).
Insulin, IGF1 and IGF2 act via the insulin (IR) and IGF1 receptor
(IGF1R). In women physiological IGF1 concentrations of 8.0 and
10.9 nM were found in the Fallopian tube and in uterine secretions,
respectively (Lighten et al., 1998). Human embryos supplemented
with IGF1 in vitro (1.7 nM) showed an increase in blastocyst development and in the number of ICM cells by 19% and 59%, respectively (Lighten et al., 1998). Compared with healthy patients,
diabetic patients show a decrease by 55% in serum IGF1 due to
an increased GH level (Colao et al., 2008; Shishko et al., 1994).
The predominant source of the circulating IGF1 is the liver, regulated by GH (Schwander et al., 1983), but a local IGF1 synthesis
is seen in many tissues and can potentially compensate for a loss
of systemic IGF1.
Recently, we have shown that the expression of IR and IGF1R is
down-regulated in blastocysts grown in diabetic rabbits
markedly
(Ramin et al., 2010). The aim of current study was to further elucidate maternal and embryonic synthesis of IGFs and potential interactions during early pregnancy in an experimental insulin
dependent diabetes (exp IDD) animal model.
2. Material and methods
2.1. Animals, embryo recovery and in vitro culture
2.1.1. In vivo model of experimental insulin dependent diabetes (exp
IDD) in rabbit preimplantation development
Exp IDD was induced by alloxan treatment as described before
(Ramin et al., 2010). Hyperglycaemia with a permanent blood glucose concentration of >14 mmol/L was established 36–48 h after
alloxan injection. The blood glucose level was monitored two times
a day and kept in the range of 14–25 mmol/L. Ketoacidosis was
prevented by regular insulin supplementation (Insuman Rapid
40 IU/mL, Aventis, Frankfurt a.M., Germany) after feeding. Rabbits
held in diabetic conditions for 9–11 days before they were
were
mated. All animal experiments were in accordance with the principles of laboratory animal care and had been approved by the local
ethics committee (Landesverwaltungsamt Dessau, reference number 42502-2-812).
2.1.2. Embryo sampling
Blastocyst recovery was performed as described before
Santos et al., 2000; Ramin et al., 2010). Briefly, blasto(Navarrete
cysts were collected from sexually mature rabbits which had been
stimulated with 110 I.U. pregnant mare serum gonadotropin
(PMSG, Intervet, Unterschleißheim, Germany) 3 days prior mating
and with 75 I.U. human chorionic gonadotropin (hCG, Intervet,
Unterschleißheim, Germany) after mating. Superovulation allowed
embryos use from the same animals for in vivo and in vitro analyses from control and diabetic animals. The embryos were flushed
from the uteri at day 6.0 post coitum (p.c.), washed two times in
basal synthetic medium II (BSM, serum- and growth-factor-free)
(Maurer, 1978), pooled and randomly divided amongst the
experimental groups.
2.1.3. Embryo in vitro culture
In vitro culture was conducted to verify and strengthen findings
from the in vivo studies. Short cultures of 10 min and 1 h and
longer culture times of 12 h were performed with 6 day old blastocysts. They were cultured in groups of 4 to 8 in 4 mL BSM II medium ± supplementation of IGF1, IGF2 or insulin at 37 °C in a water
saturated atmosphere of 5% O2, 5% CO2 and 90% N2 in a water-jacketed incubator (Heracell 150i, Heraeus, Hanau, Germany). Insulin,
IGF1 or IGF2 were pre-diluted in 100 lL culture medium and then
used in 4 mL culture medium. Either 17 nM insulin (Sigma–Aldrich, Taufkirchen, Germany), 1.3 nM IGF1 (Sigma–Aldrich, Taufkirchen, Germany) or 13 nM IGF2 (Sigma–Aldrich, Taufkirchen,
Germany) were added. These concentrations had been chosen
according to physiological ranges (Chi et al., 2000) and have been
employed in various other studies before (Navarrete Santos et al.,
2000,2004b,2008; Ramin et al., 2010; Thieme et al., 2012). Controls
were handled in the same manner without growth factors supplementation. PD98059 (20 lM) or LY294002 (20 lM) (Sigma–Aldrich, Taufkirchen, Germany) dissolved in DMSO (Sigma–Aldrich,
Taufkirchen, Germany) were used for specific inhibition of the
MAPKK (MEK1) or PI3K and added 30 min prior to IGF2 supplementation. IGF1 treatment was performed at the same day with
Erk and Akt phoshorylation measured in corresponding blastocysts
from control and exp IDD animals.
After culture the blastocysts were washed two times in ice cold
phosphate buffered saline (PBS) and transferred into ice cold 0.05%
polyvinylalcohol (PVA)/PBS buffer. For RNA isolation, the extracellular coverings were removed mechanically. To investigate spatial
expression of IGF2 and to discriminate its effects in Tr and Em, the
embryonic discs were microdissected from the trophoblast under a
stereomicroscope (Navarrete Santos et al., 2004a). Isolated Em and
Tr of single blastocysts were stored separately at 80 °C until processed for gene expression analysis.
2.2. IGF1-ELISA
For the measurement of the IGF1 concentration in EDTA plasma
and in single blastocysts the Active IGF1 ELISA (Diagnostic System
Laboratories Inc., Webster, USA) was used according to the manufacturer’s protocol. Plasma samples were collected from control
and diabetic females at day 6 p.c. and 50 ll plasma was used for
analysis. Determination of embryonic IGF1 concentration was performed on single blastocysts. After measurement of blastocyst size
(by diameter), the extracellular coverings were removed mechanically and the blastocysts were lysed in ice cold 50 ll lysis buffer by
ultrasonication for 10 min.
IGF1 concentrations were quantified by an IGF1 standard curve
in each ELISA. The absolute amount of embryonic IGF1 was related
to the blastocyst surface (in mm2) to correct for differences in blastocysts sizes.
2.3. RNA extraction, RT reaction
RNA extraction for blastocysts was performed as previously described (Navarrete Santos et al., 2004a) using Dynabeads mRNA DIRECT Kit (Dynal, Oslo, Norway) according to the manufacturer’s
instructions. The final volume of the cDNA reaction was adjusted
with water to 100 ll for blastocysts and to 50 ll for separated
embryoblast and trophoblast. Total RNA extraction from liver tissues were performed as published before (Navarrete Santos et al.,
2008). RT-PCR for IGF1 and IGF2 was done as described before
(Navarrete Santos et al., 2004a).
2.4. Cloning and sequencing of partial cDNA for rabbit IGF2
A new partial IGF2 (286 bp) cDNA was amplified from the rabbit
kidney cell line RK13 using human primers. The sequence was
identical with human IGF2 to 92% and 98% at the mRNA and the
protein level, respectively, using the alignment BLASTN and
BLASTX tool.
98
R. Thieme et al. / Molecular and Cellular Endocrinology 358 (2012) 96–103
Table 1
Oligonucleotides.
Gene
Primer sequence
Length
Tm
Acc. No.
Rabbit GAPDH
144 bp
60 °C
L23961
Rabbit IGF1
fw: GCCGCTTCTTCTCGTGCAG
rev: ATGGATCATTGATGGCGACAACAT
fw: TGGTGGATGCTCTCAGTTCGTGT
rev: GCTGATACTTCTGAGTCTTGGGCA
fw: TTCCGATTGCTGGCCATCTCTG
rev: TGTTTCCGCAGCTGTGACCTGG
fw: TGGAAGAACTTGCCCACGGAG
rev: GCTGCATTGCTGCTTACCGC
fw: CGGCATGGCAACCTGTATGACC
rev: TGTCGATGGTCGGGCAGATGTC
fw: CCCAAGCTCACGGTCATCACTG
rev: ATGGGCTTCTCCTCCAAGGTCC
fw: ACCGACTACCTGCTGCTGTT
rev: TGACCAGCGCATAGTTGAAG
237 bp
60 °C
NM_001082026.1
308 bp
60 °C
NM_000612.4
286 bp
60 °C
JN825734.1
127 bp
60 °C
AF339157.1
347 bp
60 °C
EF616472
112 bp
60 °C
AY339877.1
Human IGF2
Rabbit IGF2
Rabbit IGF2R
Rabbit IGF1R
Rabbit IR
***
5
4
3
2
1
0
control
C
B
F1-RNA
IGF
A amoount
3
[per 10 GAPD
DH mo
olecules]
6
1000
1800
1600
1400
1200
1000
800
600
400
200
0
***
800
600
400
200
0
exp
p IDD
reelative IGF1-- and 22-RNA
A amouunts
[% oof controls]
A
F1 conncentraation [n
seru
um IGF
nM]
**
**
IGF1
IGF2
control
exp
p IDD
control
exp IDD
Fig. 1. IGFs in pregnant rabbits. (A) ELISA quantified IGF1 serum concentration and (B) hepatic IGF1 RNA expression are decreased, while (C) endometrial IGF1- and IGF2-RNA
expression are increased in exp IDD animals at day 6 of pregnancy (mean ± SEM; N = 3; n = 6; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001).
B
A
d3
d4
d6
ntc
IGF1
IGF2
GAPDH
reelativee IGF1-- and IGF2-R
I
RNA aamountts
[%
% of tottal IGF
F per blastocy
yst]
120
100
trophoblast
embryoblast
***
***
IGF1
IGF2
80
60
40
20
0
Fig. 2. IGFs expression in the embryoblast and trophoblast of rabbit blastocysts at day 6 (A) Embryonic expression (RT-PCR) of IGF1 and IGF2 was determined in an
ontogenetic series at day 3 (morula), 4 (early blastocyst) and 6 p.c. (expanded blastocyst). (B) Relative amounts of IGF1 and IGF2 mRNA are quantified by real time RT-PCR in
embryoblast and trophoblast. The expression level of both growth factors is clearly higher in the embryoblast. (mean ± SEM, N = 3, n P 4; ⁄⁄⁄p < 0.001).
2.5. Real time RT-PCR
Samples were analysed by real time RT-PCR as reported before
(Navarrete Santos et al., 2008). The primers are given in Table 1.
Each assay included duplicates of each cDNA sample and a no template control for each primer set. The expression of GAPDH RNA
was used as reference for the amount of cDNA used per reaction.
GAPDH levels of day 6 blastocysts from control and exp IDD animals were comparable with ct values for controls 17.67 ± 0.27
and exp IDD 17.41 ± 0.49 (p = 0.6225). In each real time RT-PCR, a
calibration curve was included that was generated from serial dilutions (108–103 copies) of primer-specific DNA probes generated
99
A
1000
**
800
600
control
exp IDD
**
400
200
0
IGF1
IGF2
B
relaative IG
GF1 protein
peer blasttocystss surfacce [ng//mm²]
relatiive IGF
Fs RNA
A amoount
[% off controls]
R. Thieme et al. / Molecular and Cellular Endocrinology 358 (2012) 96–103
#
6
5
4
3
2
1
0
control
exp IDD
3. IGFs mRNA expression and IGF1 protein amounts in blastocysts from exp IDD rabbits. (A) IGF1 and IGF2 transcript amounts were measured in blastocysts from exp
Fig.
IDD and control rabbits by real time RT-PCR. The transcript levels of both, IGF1 (7.5-fold) and IGF2 (5-fold), are increased in blastocysts from diabetic mothers. (B) The IGF1
protein was quantified by ELISA using single blastocysts from control and exp IDD rabbits and is increased under diabetic conditions. The IGF1 protein amount was
normalized by the area of the blastocyst surface (mean ± SEM, N = 3, n P 4; #p = 0.087; ⁄⁄p < 0.01).
A
B
C
D
Fig. 4. IGF2 mediated activation of Erk1/2 (A and B) and Akt (C and D) in the rabbit blastocyst. Day 6 rabbit blastocysts were cultured in groups of 10 with or without 13 nM
IGF2 for 10 min with/without the Erk or Akt specific inhibitors PD98059 (20 lM) or LY294002 (20 lM) 30 min prior to IGF2 supplementation. (A and B) The ratio of
phosphorylated Erk1/2 and (C and D) Akt related to the total Erk1/2 and Akt amount for the same sample and Western blot were calculated with a strong activation by IGF2,
while the use of PD98059 or LY294002 prevented the IGF2 mediated increase in phosphorylated Erk1/2 and Akt, respectively (mean ± SEM; one-way ANOVA, a < 0.001). The
increase in Erk1/2 and Akt phosphorylation is present in both embryonic compartments. Detailed analysis of Erk1/2 (B) and Akt (D) phosphorylation by IGF2 in embryoblast
(Em) (a p < 0.001) and trophoblast (Tr) (b p = 0.019 and c p = 0.004) revealed an increase in both cell lineages, with a strong Erk1/2 phosphorylation in the embryoblast
(d p = 0.003, tow-way ANOVA with p = 0.008 (B) and p = 0.012 (D), mean ± SEM, N = 3, n = 10).
from cDNA plasmid clones. Analysis of the individual data therefore yielded values relative to these standards.
2.6. Protein preparation and immunoblotting
isolation and Western blot analyses were performed
withProtein
20 lg protein. Phosphorylated und non-phosporylated Erk
and Akt expression was investigated with an anti-phospho-Akt1
(S473) (mouse mAb, 1:1000, #4051), anti-Akt1 (mouse mAb,
1:500, #2967), anti-phospho-Erk1/2 (Thr202/Tyr204) (rabbit Ab,
1:1000, #4051) and anti-Erk1/2 (mouse mAb, 1:1000, #9107) antibody (all NEB, Frankfurt, Germany). After blotting, the nylon membranes were stained with Ponceau-S. Molecular weights were
determined by comparison with PageRuler™ prestained molecular
weight marker (Fermentas, St. Leon-Rot, Germany). Afterwards the
immunoreactive signals were visualized by enhanced chemilumi-
nescence detection (Millipore, Schwalbach, Germany) and quantified by ChemiDoc-It Imaging System (LTF Labortechnik,
Wasserburg, Germany). The amounts of phosphorylated protein
were evaluated by re-blotting with an antibody against the nonphosphosphorylated Erk1/2, Akt or the mouse monoclonal beta-actin antibody (1:40,000, Sigma–Aldrich, Taufkirchen, Germany)
after stripping the membranes. Protein amounts were calculated
as ratio of band intensities (pErk1/2 or pAkt vs. Erk1/2 or Akt vs.
beta-actin, respectively) in the same blot in order to correct for differences in protein loading.
2.7. Statistics
If not stated otherwise, levels of significance between groups
were calculated using student’s t-test after proving normal distribution. Multiple comparisons were made by factorial variance
100
R. Thieme et al. / Molecular and Cellular Endocrinology 358 (2012) 96–103
analysis
(SigmaPlot v. 11.0). Data are expressed as mean
value ± standard error (mean ± SEM). Levels of statistical significance are indicated as follows ⁄p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001.
N represents the number of experimental replicates, n the number
of samples (e.g. blastocysts) per experiment.
3. Results
3.1. Maternal IGF1 at day 6 of pregnancy
IGF1 serum levels were clearly reduced in exp IDD rabbits. The
plasma
IGF1 level of 1.9 nM was decreased by 60% compared with
normoglycemic controls (5.3 nM) (Fig. 1A). The hepatic IGF1 transcription, the major source of circulating IGF1, was decreased by
85% in the liver of exp IDD females at day 6 p.c. (Fig. 1B).
3.2. Expression pattern of IGF1 and IGF2 in the endometrium
In exp IDD animals a 11-fold and 13-fold increase in the endometrial IGF1 and IGF2 RNA expression level, respectively, was measured at gestational day 6 (Fig. 1C).
3.5. Kinase content and phosphorylation of Akt and Erk by IGF1 in
blastocysts from exp IDD animals
The total amount of Akt and Erk protein in exp IDD blastocysts
was reduced to 45% and 20%, respectively (Fig. 5A). The relative
phosphorylation levels of both kinases closely resembled those in
control blastocysts (Fig. 5B).
Following supplementation of 1.3 nM IGF1 in vitro, Akt and Erk
phosphorylation was 6-fold and 4.5-fold higher in control blastocysts than in blastocysts from exp IDD animals, demonstrating a
reduced sensitivity to IGF1 in exp IDD blastocysts (Fig. 5C).
3.6. IGF1, IGF2, IR and IGF1R expression in cultured blastocysts
Maternal exp IDD leads to marked decrease in IR and IGF1R
expression in 6 day old rabbit blastocysts (Ramin et al., 2010). In
this study, day 6 blastocysts were cultured with 17 nM insulin or
1.3 nM IGF1 for 1 h or 12 h in vitro. When analysed after 1 h
A
3.3. Embryonic IGF1 and IGF2 at day 6 of pregnancy
IGF1 and IGF2 transcripts were present in all investigated embryo stages from morula (d3), early blastocyst (4d) to expanded
blastocyst at day 6 p.c. (Fig. 2A). IGF1 and IGF2 RNA amounts were
quantified and analysed by real time RT-PCR in separated embryoblast and trophoblast. Transcript numbers of IGF1 and IGF2 in the
embryoblast exceeded those in the trophoblast by approximately
4-fold (Fig. 2B).
In exp IDD blastocysts IGF2 transcript levels and IGF1 RNA and
protein levels were clearly elevated. The rises in IGF1 and IGF2 RNA
levels added up to 7.5 and 5-fold, respectively (Fig. 3A). IGF1 pro(controls)
to
increased
from
2.81 ± 0.25 ng/mm2
tein
4.59 ± 0.82 ng/mm2 (1.6-fold increase) in exp IDD blastocysts. This
increase was consistent and independent from blastocyst size
(Fig. 3B). IGF2 could not be determined due to lack of a specific ELISA detecting rabbit IGF2 protein.
3.4. IGF2 activates Akt and Erk in rabbit blastocysts in vitro
we have reported on Akt and Erk activation by insulin
andRecently
IGF1 in day 6 p.c. rabbit embryos and their differential signalling in embryoblast and trophoblast cells (Navarrete Santos et al.,
2008). Because of endometrial and embryonic increase in IGF2
expression in diabetic animals (Figs. 1 and 2), the IGF2 signalling
was analyzed in preimplantation embryos, too. Therefore 6 day
old blastocysts were stimulated with IGF2 (13 nM) in vitro.
Erk1/2 and Akt phosphorylation increased 8.3-fold and 3.1-fold,
respectively, compared with untreated controls (Fig. 4A and C).
Using the MEK1 specific inhibitor PD98059 and the PI3K specific
inhibitor LY294002 30 min prior to IGF2 supplementation revealed
IGF2 specific signalling as the increase in Erk1/2 and Akt phosthe
phorylation by IGF2 was diminished to control levels (Fig. 4A and
C). To discriminate IGF2 mediated activity between embryoblast
and trophoblast, these cell lineages were separately analysed after
IGF2 treatment. The increase was 7-fold and 3.2-fold for phopsphoErk1/2 (Fig. 4B) and phosphor-Akt (Fig. 4D), respectively, in the
embryoblast and 2.3-fold and 3.3-fold in the trophoblast. Phosphorylation of Erk1/2 was significantly higher in the embryoblast
than in the trophoblast. The activation of Erk and Akt by IGF1
and insulin has recently been shown by Navarrete Santos et al.,
2008.
B
C
Fig. 5. Kinase activation by IGF1 in blastocysts from exp IDD animals. Total Akt and
Erk protein (A) and their phosphorylation levels (B) were analyzed by western blot
in in vivo grown blastocysts from exp IDD and normoglycemic rabbits at day 6 p.c.
There was no change in the ratio of phospho-Akt/Akt and phospho-Erk1/2/Erk1/2
but in the total level of the protein (⁄p < 0.05). (C) In vitro cultured blastocysts from
exp IDD and normoglycemic rabbits supplemented with 1.3 nM IGF1 for 10 min
show a clearly lower increase in the ratio of phospho-Akt/Akt and phospho-Erk2/
Erk2 compared to blastocysts from control animals (mean ± SEM; N = 3; n = 10;
tow-way ANOVA, ⁄⁄p = 0.01; ⁄⁄⁄p < 0.001).
by 55% while IGF1 supplementation decreased the ligands differently by 70% and 50%, respectively (Fig. 6A/B). Like in vivo, both
receptors were downregulated. After 12 h culture, IGF1R RNA
expression was decreased by 70% by IGF1 and insulin, and IR
RNA by 50% by insulin and 65% by IGF1 (Fig. 6C and D), closely
reflecting receptor downregulation in vivo in exp IDD mothers.
4. Discussion
The preimplantation embryo is a sensitive target of IGFs. Except
for insulin, the whole range of the insulin/IGF ligand and receptor
system is expressed during preimplantation development. These
hormones are known key players in embryo growth and differentiation and synchronise and balance the crosstalk between mother
and embryo during early pregnancy. Disturbances of the insulin/
axis by diabetes mellitus or other metabolic diseases result
IGF
in embryonic loss and miscarriages.
In general IGF homoeostases is regulated by the growth hormone and the local synthesis in tissues. During pregnancy the
IGF1 serum level is increased by 3-fold (third trimester) compared
to the serum level of a non-pregnant women. Systemic IGF2 levels
do not change (Gargosky et al. 1990). During gestation in the rabbit
the IGF2 and IGF1 serum levels increase from 4 to 810 nM (IGF2)
and from 23 to 38 nM (IGF1), respectively, from day 0 to 23 (Nason
et al., 1996). The increase in both growth factors is closely correlated with implantation and formation of a functional placenta
(Nason et al., 1996). In this study, in exp IDD rabbits, we show a
remarkable increase in IGF1 and IGF2 production by the embryo
and endometrium to compensate locally, in a paracrine fashion,
for the dramatic loss of circulating insulin (Ramin et al., 2010)
and IGF1 levels (Fig. 1).
Unlike insulin, the IGFs are present in most tissues and cell
types. The liver is the major source for systemic IGFs (Schwander
et al., 1983). This has been shown for IGF1 by a liver specific
knock-down (Yakar et al., 1999) with a 75% drop in systemic
IGF1. If ALS (IGF1-IGFBP3-ALS), a factor essential for IGF1 synthesis, was additionally eliminated in these mice IGF1 serum level decreased to 5% (Yakar et al., 2002). It is important to emphasise that
120
a
80
b
40
b
20
0
no treatment insulin
C
120
a
100
100
60
120
80
40
20
0
no treatment insulin
D
a
100
b
b
60
IGF1
120
rrelativee IR-R
RNA am
mount
% of co
[%
ontrolss]
rrelativee IGF11-RNA
A amou
unt
[% of contro
ols]
B
140
rellative IIGF1R
R-RNA
A amouunt
[%
% of controlss]
A
the relevant IGFs for reproduction are produced locally in the
uterus, creating a micromilieu sufficient for IGF action and to secure pregnancy. These IGFs act paracrine and compensate for the
systemic loss. In diabetic women, this local safeguard regulation
cannot be detected by measurement of serum IGFs.
In the rabbit the IGF expression profiles of the embryoblast and
trophoblast (Fig. 2B) show a distinct pattern with a 4 to 5-fold
higher expression of both growth factors in the embryoblast. IGFs
are known as ‘‘GF of the embryoblast/ICM’’ (Harvey and Kaye,
1992; Kaye, 1997; Navarrete Santos et al., 2008; Pantaleon et al.,
1997). This view is supported by current findings, indicating a
higher local requirement of IGF1 and IGF2 activation in the embryoblast than in the trophoblast. These important growth factors are
not only produced by the blastocyst itself but cross the trophectoderm via transcytosis from the uterine lumen, as shown for IGF1 by
electron microscopy (Smith et al., 1993). In this study we show
that IGF2 is a key player in ICM/embryoblast development, too,
by activating the mitogenic Erk pathway to a greater extend in
embryoblast than in trophoblast cells (Fig. 4B).
The specific importance of IGF1 and IGF2 has been demonstrated in knockout models. Whereas an IGF1 knockout results in
a reduced viability at birth and growth, in developmental defects
of the lung, brain, skeleton, muscle and in infertility of the offspring (Butler and LeRoith, 2001; Powell-Braxton et al., 1993;
Rother and Accili, 2000), an IGF2 knockout leads to a reduced birth
weight and infertility (DeChiara et al., 1990). IGF2 knockout offspring are able to catch up weight postnatally. This suggests a
stronger impact of IGF1 than IGF2 on early embryonic developmental failures (Baker et al., 1993; Rother and Accili, 2000). As seen
in our study expression of IGFs in the preimplantation period can
be altered in specific metabolic conditions with a potential impact
of both studied IGFs on long-term development.
As shown in current study the ‘‘insulin gap’’ in exp IDD can be
bridged during pregnancy. Maternal exp IDD affects both parts of
IGF signalling, receptor and ligand expression, in the embryo and
endometrium. While IR and IGF1R (Ramin et al., 2010); Fig 6) are
down-regulated in exp IDD, expression of both ligands is increased
(Fig. 2). The sensitivity for intracellular downstream activation is
diminished by 55% and 80% of total available Akt and Erk in
rrelativee IGF22-RNA
A amouunt
[% of contro
ols]
in vitro culture, insulin had decreased IGF1 and IGF2 mRNA levels
101
R. Thieme et al. / Molecular and Cellular Endocrinology 358 (2012) 96–103
IGF1
a
100
80
60
b
40
b
20
0
no treatment
insulin
IGF1
80
b
60
b
40
20
0
no treatment insulin
IGF1
Fig. 6. Ligand (IGF1 and IGF2) and receptor (IR and IGF1) expression in cultured rabbit blastocysts. IGF1, IGF2, IR and IGF1R RNA were measured by real time RT-PCR after
h (A and B) and short time 1 h (C and D) in vitro exposure to insulin or IGF1. The transcript numbers of non-treated controls were set to 100%. The expression of IGF1 (A)
12
and IGF2 (B) are decreased by 55% by insulin and by 70% and 50% by IGF1, respectively (mean ± SEM; N = 2; n = 8; one-way ANOVA with a p < 0.001). Insulin and IGF1
decreased (C) IGF1R RNA by 70% and (D) IR RNA by 50% (insulin) and 65% (IGF1), respectively (mean ± SEM; N = 3; n = 10; one-way ANOVA with a p < 0.001).
102
R. Thieme et al. / Molecular and Cellular Endocrinology 358 (2012) 96–103
blastocysts from diabetic mothers (Fig. 5A). Although the proportion of active phosphorylated to non-phosphorylated protein is
the same in blastocysts from control and exp IDD mothers
(Fig. 5B). Exp IDD blastocysts have an impaired metabolic and
developmental competence due to the lower content of Akt and
Erk. Experimental evidence for this view has recently been given
by a delay in development and mesoderm initiation, an increase
in apoptotic cells in the embryonic disc and a decrease in the
expression of enzymes related to glucose homeostasis (HK2 and
PEPCK) reported for exp IDD blastocysts by Ramin et al. (2010)
and Thieme et al. (2012). To further elucidate the responsiveness
of blastocysts from exp IDD mothers we have treated exp IDD blastocysts with physiological concentrations of IGF1 (1.3 nM). Exp
IDD blastocysts show a lower capacity of IGF1 to activate Akt
and Erk (Fig. 5C). The increase in IGF1 and IGF2 ligands can therefore be seen as an adaptation to gap growth factor activity in blastocysts during diabetic pregnancy.
In this context it is important to note that the ability to compensate for kinase activity is different between Erk1 and Erk2. Erk2
contributes to trophoblast formation in the mouse and
activation
proliferation of polar trophectoderm cells (Saba-El-Leil et al.,
2008). However, Erk1 is unable to compensate for a lack in Erk2
activation for trophoblast formation (Saba-El-Leil et al., 2008),
while disturbances in Erk1 do not result in any developmental phenotype (Pagès et al., 1999). A disturbed Erk/MAPK activation has
been shown to result in defective placenta development (Rossant
and Cross, 2001). Erk activation can be seen as one of the most
important proliferative signals to promote cell cycle progression
during whole embryonic development (Li et al., 2011).
5. Conclusion
A direct link may exist between the disarranged IGF/insulin axis
with a lack in kinase activation, disturbed activation of Wnt signalling responsible for mesoderm and heart development in early
embryogenesis, and the reduced insulin and IGF1 receptor expression (Ramin et al., 2010). A disarranged Wnt signalling repertoire
(ligand, receptors and intracellular signalling molecules) in foetal
hearts by maternal diabetes has been shown in mice (Pavlinkova
et al., 2008,2009). In the rabbit insulin has the capacity to initiate
mesoderm formation via Wnt3a and Wnt4, two of the most potent
factors for mesoderm induction, and the Erk signalling pathway
in vitro. Wnt3a expression is down regulated in gastrulating blastocysts from exp IDD (Thieme et al., 2012).
Acknowledgement
We thank Michaela Kirstein, Franziska Knöfel and Sabine
Schrötter for excellent technical assistant. This work was supported by the German Research Council (DFG; grant number NA
418/4-2) and the Wilhelm Roux Programme of the MLU Faculty
of Medicine.
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REPRODUCTION-DEVELOPMENT
cAMP-Responsive Element Binding Protein: A Vital
Link in Embryonic Hormonal Adaptation
Maria Schindler,* Sünje Fischer,* René Thieme, Bernd Fischer, and
Anne Navarrete Santos
Department of Anatomy and Cell Biology, Martin Luther University Faculty of Medicine, D-06097 Halle
(Saale), Germany
The transcription factor cAMP responsive element-binding protein (CREB) and activating transcription factors (ATFs) are downstream components of the insulin/IGF cascade, playing crucial roles
in maintaining cell viability and embryo survival. One of the CREB target genes is adiponectin,
which acts synergistically with insulin. We have studied the CREB-ATF-adiponectin network in
rabbit preimplantation development in vivo and in vitro. From the blastocyst stage onwards, CREB
and ATF1, ATF3, and ATF4 are present with increasing expression for CREB, ATF1, and ATF3 during
gastrulation and with a dominant expression in the embryoblast (EB). In vitro stimulation with
insulin and IGF-I reduced CREB and ATF1 transcripts by approximately 50%, whereas CREB phosphorylation was increased. Activation of CREB was accompanied by subsequent reduction in adiponectin and adiponectin receptor (adipoR)1 expression. Under in vivo conditions of diabetes type
1, maternal adiponectin levels were up-regulated in serum and endometrium. Embryonic CREB
expression was altered in a cell lineage-specific pattern. Although in EB cells CREB localization did
not change, it was translocated from the nucleus into the cytosol in trophoblast (TB) cells. In TB,
adiponectin expression was increased (diabetic 427.8 ⫾ 59.3 pg/mL vs normoinsulinaemic 143.9 ⫾
26.5 pg/mL), whereas it was no longer measureable in the EB. Analysis of embryonic adipoRs
showed an increased expression of adipoR1 and no changes in adipoR2 transcription. We conclude
that the transcription factors CREB and ATFs vitally participate in embryo-maternal cross talk
before implantation in a cell lineage-specific manner. Embryonic CREB/ATFs act as insulin/IGF
sensors. Lack of insulin is compensated by a CREB-mediated adiponectin expression, which may
maintain glucose uptake in blastocysts grown in diabetic mothers. (Endocrinology 154: 2208 –2221,
2013)
distinct activation pattern of transcription factors is
required for normal development and survival of
mammalian embryos. Growth hormones and metabolic
factors like glucose control transcription factor activity.
The family of insulin and IGFs plays a fundamental role in
early embryo development, mediating mitogenic, antiapoptotic, and anabolic effects (1, 2). IGF-I and IGF-II are
produced by the embryo and by reproductive tissues in the
human (3), rabbit (4, 5), and mouse (6, 7). The production
of IGF-I in bovine preimplantation embryos is controver-
sially discussed with publications reporting IGF-I by the
embryo (7) or not (8, 9). Insulin has to be provided by the
mother, because the embryo cannot produce insulin itself
in mammals (3, 4, 6, 7). Insulin and IGFs can partly compensate for each other. So far, few attempts have been
made to investigate mechanisms of insulin compensation
and replacement during early pregnancy (5).
Adiponectin is the most abundantly secreted adipokine
with a 1000 times greater serum concentration than that
of others. Adiponectin exerts its biological function by
ISSN Print 0013-7227
* M.S. and S.F. contributed equally to this work.
A
ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2013 by The Endocrine Society
Received November 1, 2012. Accepted April 1, 2013.
First Published Online April 8, 2013
2208
endo.endojournals.org
Abbreviations: adipoR, adiponectin receptor; ATF, activating transcription factor; BSM,
basal synthetic medium; CRE, cAMP responsive element; CREB, CRE-binding protein;
CREM, CREB modulator protein; EB, embryoblast; exp IDD, experimentally induced insulindependent diabetes; GAPDH, glyceraldehyde-3-phosphate; IGF-IR, IGF-I receptor; IR-A,
insulin receptor isoform A; 3-OMG, 3-O-methyl-d-[1-3H]glucose; p.c., postcoitum; qPCR,
quantitative PCR; TB, trophoblast.
Endocrinology, June 2013, 154(6):2208 –2221
doi: 10.1210/en.2012-2096
The Endocrine Society. Downloaded from press.endocrine.org by [Amanda Price] on 24 February 2015. at 10:44 For personal use only. No other uses without permission. . All rights reserved.
doi: 10.1210/en.2012-2096
endo.endojournals.org
2209
binding to the adiponectin receptor (adipoR)1 or adipoR2. Both receptors are transmembrane proteins and
located in embryoblast (EB) inner cell mass and trophoblast (TB) cells (10). Adiponectin is an important insulin
sensitizing adipokine (11–14). Adiponectin production
and functional signaling has previously been shown in
rabbit and murine preimplantation embryos (10, 15, 16,
for review see Ref. 17). Adiponectin has profound effects
on glucose metabolism by enhancing embryonic glucose
uptake and translocation of the solute carrier family 2
(facilitated glucose transporter), member 4 (also known as
glucose transporter 4) from the cytosol to the cell
membrane.
Overexpression of adiponectin improves insulin sensitivity, and down-regulation of adiponectin goes along
with insulin resistance and obesity (11, 18 –20). The expression of adiponectin and its receptors are regulated by
several extracellular signals and hormones, including
IGF-I, insulin, and TNF␣ (21, 22). A potential mediator
for the transcriptional regulation of adiponectin is the
cAMP responsive element (CRE)-binding protein (CREB)
transcription factor (23–25).
CREB acts as a downstream effector in many signaling
pathways. These pathways are mainly regulated in response to growth factors, stress signals, intracellular
Ca2⫹, and specific peptides. So far, more than 300 different signals are known to activate CREB. The rate-limiting
step of CREB activity is the phosphorylation of CREB at
the residue Ser133 (26). A phosphorylation of CREB is
essential for dimerization and binding to CREB-binding
protein. After binding to the CRE element, the transcription of target genes is activated.
Activating transcription factor (ATF)1 and CREB
modulator protein (CREM) are members of the CREB/
ATF family. Both factors, ATF1 and CREM, can form
heterodimers with CREB and induce transcription (27,
28). Furthermore, CREB and ATF1, sharing an overall
70% sequence homology, are expressed ubiquitously,
whereas CREM is predominantly expressed in neuroendocrine tissue (27, 28).
Previous studies failed to detect CREM in the preimplantation embryo but showed a strong coexpression of
CREB and ATF1 during mouse preimplantation development (29, 30). A deletion of CREB and ATF1 results in
embryonic death before implantation (29). During embryo development, CREB and ATF1 can compensate for
each other. However, ATF1 possesses a lower transcriptional activity than CREB due to a lack of the glutaminerich domain (31). The phosphorylation of CREB and its
nuclear localization has first been observed in the 2-cell
embryo stage of the mouse at the onset of embryonic genome activation (30). In Xenopus laevis, CREB regulates
cell specification in early development (32). An inhibition
of CREB at blastula and early gastrula stages as well as at
the beginning of neurulation has deleterious effects on
embryogenesis with malformations, such as microcephaly
and spina bifida (33)
The functional properties of other family members,
ATF3 and ATF4 (also called CREB-2), are poorly understood, particularly in early embryo development. Both
transcription factors can function as transcriptional activators or repressors. The ATF3 knockout mouse does not
exhibit any distinguishable phenotype (34). However,
mice deficient in ATF4 are 50% smaller than their wildtype counterparts and suffer from a variety of developmental defects (35–37). ATF4 acts as a negative regulator of insulin secretion and insulin sensitivity in liver,
muscle, and fat (38, 39). The ATF4⫺/⫺ mutant mouse
is resistant to diet-induced and age-dependent obesity
and diabetes (37).
We investigated the CREB-ATF network during preimplantation embryo development in the rabbit (40). We
have characterized the expression and localization of
CREB and the ATFs in rabbit blastocyst gastrulation in
normal development and under experimentally induced
insulin-dependent diabetes (exp IDD). Furthermore, we
have stimulated blastocysts with insulin and IGFs in vitro.
Our results show that insulin and IGF-I down-regulate
adiponectin and adipoR1 expression in vitro. A maternal
diabetes mellitus type 1 increases embryonic adiponectin
and adipoR1 expression mediated by CREB and ATF
transcription factors.
Materials and Methods
Embryo recovery
Embryos were collected from sexually mature rabbits stimulated with 110 IU pregnant mare serum gonadotropin sc (Intervet, Unterschleißheim, Germany) 3 days before mating. After
mating, 75 IU human choriongonadotropin was injected iv (Intervent). Mating and embryo recovery were performed as described (41). On days 3, 4, and 6 postcoitum (p.c.), embryos were
flushed from oviducts or uteri, respectively, washed 3 times with
PBS, and randomly divided among the experimental groups. On
day 6 p.c., gastrulation stages can be reliably discriminated in the
rabbit, because implantation starts on day 6 18 hours, ie, half a
day later (30).
In vitro culture
To study the effects of insulin and IGF-I on CREB, adiponectin, and ATF expression, day 6 blastocysts were cultured in
groups of 5-10 at 37°C in a water-saturated atmosphere of 5%
O2, 5% CO2, 90% N2 in a water-jacketed incubator (HERAcell
150i, Heraeus; Thermo Fisher Scientific, Bonn, Germany). Blastocysts were precultured for 2 hours in serum- and insulin-free
basal synthetic medium (BSM) (42). Afterwards, either 17nM
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2210
Schindler et al
Role of CREB in Embryonic Hormonal Adaptation
Endocrinology, June 2013, 154(6):2208 –2221
insulin (Invitrogen, Karlsruhe, Germany), 1.3nM IGF-I, or
13nM IGF-II (Sigma, Taufkirchen, Germany) was added to the
culture medium, and the culture was continued for different time
intervals (10 min to 12 h). The concentrations comply with physiological ranges measured within the Fallopian tube and uterine
cavity in the human (3, 43) and rhesus (43) and have been employed in various other studies before (4, 44 – 46). They are
known to specifically activate IGF-I receptor (IGF-IR) (IGF-I),
insulin receptor isoform A (IR-A) and IGF-IR (IGF-II), and IR-A
and IR-B (insulin) (47).
Controls were cultured without insulin, IGF-I, or IGF-II but
otherwise treated identically. Phosphorylation of CREB was analyzed after culture in the presence or absence of insulin, IGF-I,
or IGF-II for 10 minutes. To analyze transcriptional regulation,
blastocysts were cultured with or without insulin or IGF-I for 6
and 12 hours, respectively.
The influence of glucose on adiponectin transcription was
analyzed in blastocysts cultured with 0mM, 10mM, or 25mM
glucose. Day 6 p.c. blastocysts were cultured in groups of 5-6 for
6 hours.
Alloxan treatment
exp IDD was induced in 18- to 20-week-old female nonpregnant rabbits by alloxan (Sigma) treatment as described before
(4). Rabbits were hold in diabetic conditions with permanent
blood glucose concentrations of more than 14 mmol/L by regular
insulin supplementation 4 times per day (Insuman Rapid and
Lantus; Sanofi-Aventis, München, Germany), started at the second day after alloxan treatment. The blood glucose level was
monitored with MediSense Precision Xceed Diabetes Management System (Abbott, Wiesbaden, Germany) 2 times per day.
Therefore, fresh blood was collected at 10 AM and 6 PM by puncturing the vena auricularis lateralis and tested for glucose concentration by a commercial test strip.
All animal experiments were conducted in accordance with the
principles of laboratory animal care, and the experimental protocol
had been approved by the local ethical commission of the Landesverwaltungsamt Dessau (reference number 42502-2-812).
RNA isolation and cDNA synthesis
mRNA of single blastocysts was extracted with Dynabeads
Oligo(dT)
(Invitrogen) and subsequently used for cDNA syn25
thesis. All protocol procedures were carried out according to the
manufacturer’s instructions, with modification described previously by Tonack et al (48).
Reverse transcription-polymerase chain reaction
RT-PCR amplification was carried out with 0.5 ␮L cDNA
from single blastocysts in a 25 ␮L volume containing 200␮M
each deoxyribonucleotide, 2.5 U Taq polymerases, and specific
oligonucleotides for CREB, ATF1, ATF2, ATF4, adiponectin,
and glyceraldehyde-3-phosphate (GAPDH) (primers listed in
Table 1). Nucleotide sequence for rabbit CREB and ATF1 were
determined using human primers for amplification of rabbit liver
cDNA. The amplification was done for 40 cycles (94°C for 45
sec, 60°C for 45 sec, and 72°C for 60 sec). Resulting PCR products were separated by electrophoresis on 2% agarose gel and
stained with ethidium bromide.
RT-qPCR
Real-time analyses (RT-quantitative PCR [qPCR]) were performed as duplicates by using the Applied Biosystems StepOnePlus System (Applied Biosystems, Darmstadt, Germany) with a
no template control for each primer set as described in Thieme et
al (5). The nucleotide sequences of the primers used in this study
are listed in Table 1. GAPDH was simultaneously quantified as
endogenous control, and target gene expression was normalized
to that of GAPDH in each sample. GAPDH was shown to be
unaffected by the treatment, because no variation in the absolute
GAPDH levels compared with GAPDH RNA levels in blastocysts from healthy rabbits were observed (5). Analysis of the
individual data therefore yielded values relative to these standards. Data are expressed as percentage relative to control
blastocysts.
Protein preparation and immunoblotting
Protein preparation, quantification, and Western blotting
were performed with 8-10 blastocysts as described in Fischer et
al (10). For Western blot analysis, 25 ␮g total protein lysates
were subjected to sodium dodecyl sulfate-polyacrylamide electrophoresis and electrotransferred to nitrocellulose membranes.
For detection of phospho-CREB, CREB and ␤-actin membranes
were blocked in Tris-buffered saline containing 0.1% (vol/vol)
Triton X-100 with 3% (wt/vol) nonfat dry milk at room temperature for at least 1 hour. For ATF1 and ATF3 detection,
Table 1. Primers used for RT-PCR and RT-qPCR
Gene
name
GenBank
number
Temperature
(°C)
Fragment
(bp)
Sequence
5ⴕ33ⴕ
Adiponectin
DQ334867
60
158
ATF1
NM_005171
60
197
ATF3
XM_002717521
60
163
ATF4
LOC100339383
60
193
CREB
NM_004379.3
60
169
GAPDH
L23961
60
144
fw: cctggtgagaagggtgaaaa
rev: gctgagcggtagacataggc
fw: caacctggttcagcagttc
rev: tttctgccccgtgtatcttc
fw: cgctggtgtttgaggatttt
rev: ctgactccagtgcagacgac
fw: gcgagaagctggagaagaag
rev: tccagcaggtccttgaggta
fw: gtatgcacagaccacggatg
rev: tgcaggctgtgtaggaagtg
fw: gccgcttcttctcgtgcag
rev: atggatcattgatggcgacaacat
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doi: 10.1210/en.2012-2096
endo.endojournals.org
2211
membranes were blocked in Tris-buffered saline containing
(vol/vol) Triton X-100 with 3% (wt/vol) BSA for 1 hour.
0.1%
The primary antibody was incubated at 4°C overnight. Antibodies were as following: phospho-CREB (no.9196, 1:1000;
Cell Signaling Technology, Beverly, Massachusetts), CREB (no.
9104, 1:1000; Cell Signaling Technology), ATF1 (sc-243,
1:1000; Santa Cruz Biotechnology, Inc, Santa Cruz, California),
ATF3 (sc-81189, 1:500; Santa Cruz Biotechnology, Inc), ␤-actin
(A-5441, 1:40 000; Sigma), and antimouse IgG conjugated to
horseradish peroxidase (115-036-003, 1:45 000; Dianova,
Hamburg Germany). The amounts of phosphorylated proteins
were evaluated by stripping the membranes and reblotting with
the nonphosphorylated protein antibody. The amount of phosphorylated CREB relative to CREB was measured in each sample
by image analysis. Protein phosphorylation was calculated as the
ratio of band intensities (phosphorylated protein vs nonphosphorylated protein) in the same blot to correct for differences in
protein loading.
Immunohistochemical localization of CREB, ATFs,
and adiponectin
Blastocysts were washed twice in ice-cold PBS and fixed in 4%
(wt/vol) paraformaldehyde at 4°C overnight. Preparation and
immunohistochemical protocol were performed as described in
Fischer et al (10). Antibodies for CREB (Cell Signaling Technology) and ATF1 and ATF3 (both Santa Cruz Biotechnology, Inc)
were diluted 1:400 in 3% (wt/vol) BSA/PBS. For adiponectin
detection, the antibody (ab 22554; Abcam, Cambridge, Massachusetts) was diluted 1:100 in (wt/vol) BSA/PBS. The secondary
antibody Dako EnVision ⫹ System-HRP labeled Polymere antimouse (1:1 in PBS, K4001; Dako, Glostrup, Denmark) and
diaminobenzidine (WAK-Chemie Medikal, Steinbac, Germany)
were used for detection.
For immunofluorescence detection, a secondary antibody
conjugated with fluoresceinthiocyanate (1:250 dilution) was
used. The nuclei were counterstained with Hoechst for 5 minutes. All steps were performed within the same experiment, examined microscopically during the same session, using identical
microscope and camera settings.
Adiponectin ELISA
Adiponectin concentrations in embryonic tissue and blastocysts fluid were measured by ELISA (AdipoGen, San Diego, Cal-
ifornia). All protocol procedures were carried out according to
the manufacturer’s instructions. To measure serum adiponectin
concentration, blood samples were collected with S-Monovetten
(Sarstedt, Nümbrecht, Germany), left to coagulate for at least 30
minutes, and centrifuged for 10 minutes by 4°C and 1000g. The
supernatant was stored at ⫺80°C until use. Endometrium samples were collected by opening the uterus on the antimesometrial
side. Endometrium was removed mechanically from the myometrium with a scalpel and stored at ⫺80°C for subsequent protein preparation and analysis by ELISA. Flushed blastocysts were
washed 3 times with ice-cold PBS. Blastocyst stage and size were
determined. Blastocysts were then placed on a dry watch glass.
Extracellular coverings were removed mechanically, blastocysts
were punctured, and the effluent cavity fluid was collected. Blastocyst fluids were stored at ⫺80°C until use. Blastocysts were
then separated into TB and EB for further analyses (45). EBs and
TBs of single blastocyst samples were stored in PBS at ⫺80°C for
subsequent protein preparation and analysis by ELISA. In vitrocultured blastocysts were washed twice in ice-cold PBS and
stored in radioimmunoprecipitation buffer at ⫺80°C until use.
Adiponectin concentrations were quantified in duplicate and
compared with an internal adiponectin standard (0.1-32 ng/mL).
Glucose transport studies
Glucose uptake was measured as previously described (10).
After preculture for 2 hours, embryos were washed 3 times in
glucose-free BSM II media and transferred into 600-␮L pulse
droplets, kept strictly at 37°C for 3 minutes. The glucose-free
pulse medium contained 0.3mM 3-O-methyl-d-[1-3H]glucose
(3-OMG) (37 GBq/L; Amersham, Piscataway, New Jersey) and
25mM 3-OMG (Sigma-Aldrich, Munich, Germany). Uptake was
stopped after 3 minutes by transferring the embryos through 4
washes of ice-cold, glucose-free BSM II media. The diameters of
the embryos were recorded using a calibrated ocular micrometer.
Radioactivity of individual embryos was determined in a Packard 1600TR liquid scintillation analyzer. Uptake of 3-OMG is
expressed as nanomoles per minute per surface area (cm2).
Statistics
All data are expressed as means ⫾ SEM. Levels of significance
between groups were calculated by factorial ANOVA with Bonferroni adjustment. P ⬍ .05 was considered statistically significant. All experiments were conducted at least 3 times.
Results
Expression of CREB in rabbit embryos
In 3-day-old morulae, the transcription of CREB was
not detectable, but it was present in early blastocysts on
day 4 p.c. (Figure 1A). Blastocysts at day 6 showed an
increase in CREB transcript levels during gastrulation
from stage 1 to stage 3 compared with stage 0 (Figure 1B).
A corresponding increase in CREB protein amount from
stage 1 to stage 2 was verified by Western blotting (Figure
1, C and D). Using the same anti-CREB antibody for immunohistochemical detection, CREB was localized in
both EB and TB with the most prominent localization in
the nuclei of EB cells (Figure 1E).
Activation of CREB by insulin, IGF-I, and IGF-II in in
vitro-cultured blastocysts
Transcriptional activation of CREB is commonly associated with phosphorylation at residue Ser133 and nuclear
translocation. To clarify whether CREB can be activated
by insulin and IGFs, we stimulated blastocysts with 17nM
insulin, 1.3nM IGF-I, or 13nM IGF-II for 10 minutes in
vitro. The antibody used in the phosphorylation assay detected specifically CREB phosphorylated at Ser133 (phospho-CREB Ser133). Supplementation led to an increased
amount of phosphorylated CREB (Figure 1G). The rela-
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2212
Schindler et al
Role of CREB in Embryonic Hormonal Adaptation
Endocrinology, June 2013, 154(6):2208 –2221
A ntc d3
d4
d6
st 1
st 2
st 3
CREB
GAPDH
st 1 st 2
C
CREB
B
E
30
D
***
25
**
20
15
10
5
10
relative CREB protein amount
relative CREB mRNA expression [fold change]
β-acn
*
*
8
6
4
2
0
0
st 0
st 1
st 2
st 1
st 3
a)
st 2
b)
c)
EB
TB
F
350
**
H
G
*
300
ctrl
250
ins IGF1 IGF2
p-CREB
*
200
CREB
β-acn
150
100
50
0
120
relative CREB mRNA amount [%]
relative p-CREB amount [% of control]
EB
100
80
*
60
*
40
20
0
control
insulin
IGF1
IGF2
control
insulin
IGF1
Figure 1. Expression of CREB in rabbit preimplantation embryos. (A) Transcripts of CREB were detected in day 3, 4, and 6 p.c. embryos and
blastocysts in gastrulation stage 1 (st 1), stage 2 (st 2), and stage 3 (st 3). A probe without cDNA was used as negative control (ntc). Internal
control was the expression of GAPDH in all probes. (B) Transcript amount of CREB was increased during gastrulation. The amount of CREB mRNA
was measured by RT-qPCR in 9 blastocysts per group in 3 independent experiments (N ⫽ 3, n ⫽ 9). For normalization, RT-qPCR was
performed for the housekeeping gene GAPDH. The results are shown as mean ⫾ SEM (*P ⬍ .05; **P ⬍ .01; ***P ⬍ .001). (D) Relative
CREB protein amount in day 6 p.c. blastocysts was analyzed by Western blotting with an anti-CREB antibody and related to ␤-actin (mean ⫾
SEM; N ⫽ 3, n ⫽ 10; *P ⬍ .05). A representative Western blotting for CREB is shown in C. (E) Immunohistochemical analysis of CREB
revealed a prominent localization of CREB in the nucleus of EB cells (a) (with a higher magnification; b). The CREB protein was visualized by
peroxidase-diaminobenzidine reaction (brown color). A control reaction is shown in c. In vitro stimulation of blastocysts with 17nM insulin
(ins), 1.3nM IGF-I, or 13nM IGF-II led to a significantly higher phospho-CREB amount (*P ⬍ .05; **P ⬍ .01). The phosphorylation of CREB
was analyzed by Western blotting with antiphospho-CREB antibody and set in relation to total CREB protein. A representative Western
blotting for CREB is shown (G). Results of 3 stimulation experiments (N⫽ 3, n ⫽ 10; mean ⫾ SEM) are shown in F. (H) RT-qPCR analysis of
(12 h) in vitro-stimulated blastocysts revealed a reduced amount of CREB RNA. Culture of blastocysts was performed in groups of
long-time
at least 5 blastocysts with or without 17nM insulin and 1.3nM IGF-I and with N ⫽ 3, n ⱖ 5. The results are shown as mean ⫾ SEM
(*P ⬍ .05; **P ⬍ .01). ctrl, control.
tive increase in phosphorylation is shown in Figure 1F.
The in vitro stimulation with insulin or IGF-I for 12 hours
led to a reduction in CREB RNA amounts (Figure 1H),
indicating a negative feedback loop in embryos.
Expression of ATFs in preimplantation embryo
ATF1 is highly related to CREB and can compensate its
absence. Starting on day 4 p.c., ATF1 expression increased
in all embryonic stages investigated (Figure 2, A and B).
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doi: 10.1210/en.2012-2096
endo.endojournals.org
2213
A ntc d3
d4
B
d6
st 1 st 2 st 3
ATF1
ATF3
ATF4
GAPDH
C
D
relative ATF1 mRNA expression [fold change]
12
***
10
8
6
**
4
*
2
0
st 0
st 1
st 2
st 3
ATF1
a)
100
**
40
20
EB
0
control
relative ATF3 mRNA expression [fold change]
**
60
E
TB
b)
80
insulin
IGF1
F a)
ATF3
30
**
25
b)
20
EB
5
0
Figure 2.
st 0
st 1
st 2
st 3
TB
G
relative ATF4 mRNA expression [fold change]
relative ATF1 mRNA amount [%]
120
1,4
1,2
1,0
0,8
0,6
**
0,4
0,2
0,0
st 0
st 1
st 2
st 3
Expression of ATF family in rabbit preimplantation embryos. (A) Analysis by RT-PCR shows an expression from day 4 to 6 p.c. for ATF1
and from day 3 to 6 p.c. for ATF3 and ATF4. Blastocysts were staged in stage 1 (st 1), 2 (st 2), or 3 (st 3). A probe without cDNA was used as
control (ntc). GAPDH was used as internal control. mRNA levels of ATF1 (B), ATF3 (E), and ATF4 (G) were quantified on day 6 p.c. by RTnegative
qPCR. The experiment was performed from 9 blastocyts per stage in (N ⫽ 3, n ⫽ 9). Expression of ATF1, ATF3, and ATF4 was normalized to
GAPDH, and results are shown as mean ⫾ SEM relative to stage 0 (*P ⬍ .05; **P ⬍ .01; ***P ⬍ .001). (C) Long-time in vitro stimulation with or
without insulin or IGF-I was performed in groups of at least 5 blastocysts in each treatment and repeated in 3 independent replicates (N ⫽ 3, n ⱖ
5).
Expression of ATF1 in the nontreated group (control) was set 100%. Culture with insulin or IGF-I led to a decreased ATF1 expression compared
with the corresponding nonstimulated control (*P ⬍ .05; **P ⬍ .01). Immunohistochemical analysis of ATF1 revealed an expression in nuclei of EB
(a) (with a higher magnification, b). ATF3 is shown in F (a) (with a higher magnification, b). Contrary to CREB and ATF3 (EB staining), ATF1
cells
was also localized in the nuclei of TB cells (D).
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2214
Schindler et al
Role of CREB in Embryonic Hormonal Adaptation
Endocrinology, June 2013, 154(6):2208 –2221
C
B
p-CREB
120
normo- exp
insulin IDD
100
80
p-CREB
**
60
CREB
40
20
0
normoinsulinaemic
a1)
E
D
relative protein amount in exp IDD
[% compared to normoinsulinaemic]
A
relative p-CREB amount [%]
500
normoinsulin
**
400
exp
IDD
p-CREB
300
CREB
200
β−acn
*
100
0
exp IDD
CREB/b-actin
p-CREB/b-actin
normoinsulinaemic
b1)
exp IDD
c1)
normoinsulinaemic
d1)
exp IDD
normoinsulinaemic
b2)
exp IDD
c2)
normoinsulinaemic
d2)
exp IDD
a2)
ATF1
G
120
relative ATF1 amount [%]
140
100
b)
exp IDD
ATF1
60
40
**
β−acn
ATF1
0
normoinsulinaemic
I
ATF1
exp IDD
180
relative ATF3 mRNA expression [%]
normoinsulinaemic
a)
80
20
H
normo- exp
insulin IDD
K
ATF3
160
140
120
100
80
60
40
20
0
400
relative ATF4 mRNA expression [%]
F
ATF4
*
300
200
100
0
normoinsulinaemic
exp IDD
normoinsulinaemic
exp IDD
Figure 3. Expression pattern of the CREB family in blastocysts from diabetic rabbits. (A) The amount of phosphorylated CREB was quantified by
Western blotting with an antiphospho-CREB-specific antibody in relation to nonphosphorylated protein. Results are shown as mean ⫾ SEM (N ⫽
3, n ⫽ 10). The amount of phosphorylated CREB was significantly lower in blastocysts from diabetic rabbits (exp IDD) (*P ⬍ .05). (B)
Representative Western blotting. (C) Amounts of CREB protein in blastocysts from normoinsulinaemic and diabetic rabbits (exp IDD) were
quantified by Western blotting and calculated in relation to ␤-actin (mean ⫾ SEM; N ⫽ 3, n ⫽ 10). In blastocysts from diabetic rabbits (exp IDD),
CREB amount was significantly increased (*P ⬍ .05). A representative Western blotting is shown (D). (E) Immunofluorescent detection of CREB
(green color) in blastocysts from normoinsulinaemic (a) and exp IDD (b) rabbits show weaker nuclear localization in TB of diabetic blastocysts. In
the EB, CREB was localized in the nuclei of blastocysts from normoinsulinaemic (c) and diabetic (d) (exp IDD) rabbits. Nuclei were counterstained
with Hoechst (blue) (a2, b2, c2, and d2). Arrows indicate cells with a cytoplasmatic localization (b1 and b2) or a nuclear localization (a1, a2, c1, c2,
d1, and d2) of CREB. (F) Quantification of ATF1 protein revealed a significant decrease in blastocysts from diabetic rabbits (exp IDD) (**P ⬍ .01).
Western blotting was performed by using a specific anti-ATF1 antibody and set in relation to ␤-actin amount in the same probes (mean ⫾ SEM;
N ⫽ 3, n ⫽ 10). (G) Representative Western blotting. (H) Immunohistochemical detection of ATF1 (brown color) showed a weaker nuclear
localization in the EB of blastocysts from diabetic rabbits (exp IDD) (H). The relative amounts of ATF3 (I) and ATF4 (K) mRNA were measured by RTqPCR in at least 8 blastocysts per group. The results are shown as mean ⫾ SEM (*P ⬍ .05).
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doi: 10.1210/en.2012-2096
**
6
5
4
3
2
1
***
250
adiponecn
β−acn
100
50
200
exp IDD
normoinsulinaemic
blastocysts
E
150
100
50
600
EB
TB
**
400
300
200
100
n.d.
0
normoinsulinmaemic
exp IDD
F
adiponectin concentration [ng/ml]
exp IDD
TB
TB
EB
normoinsulinaemic
0,25
0,20
0,15
0,10
0,05
normoinsulinaemic
AdipoR1
I
300
***
250
200
5
4
3
2
150
exp IDD
AdipoR2
adipoR2 copies / 10 GAPDH molecules
relative adiponectin amount
[% of control]
H
100
50
0
**
0,30
0,00
blastocysts fluid
0,35
EB
exp IDD
G
b)
a)
exp IDD
*
500
0
normoinsulinaemic
exp
IDD
150
adiponectin concentration [pg/ml]
relative adiponectin mRNA expression [%]
2215
0
normoinsulinaemic
normoinsulin
200
0
D
C
endometrium
300
relative adiponectin amount
[% of control]
B
serum
7
adiponectin concentration [μg/ml]
A
endo.endojournals.org
normoinsulinaemic
exp IDD
2
1
0
normoinsulinaemic
exp IDD
Figure 4. Adiponectin expression in normoinsulinaemic and diabetic rabbits. (A) In the serum of normoinsulinaemic and diabetic rabbits (exp
IDD), adiponectin concentration was determined by ELISA (mean ⫾ SEM; N ⫽ 3, n ⫽ 11; **P ⬍ .01). (B) Western blot analysis revealed increased
amounts of adiponectin in endometrium of diabetic rabbits (exp IDD) (mean ⫾ SEM; N ⫽ 3, n ⫽ 10; ***P ⬍ .001). (C) Representative Western
blotting. Expression of adiponectin in the endometrium of normoinsulinaemic rabbits was set to 100%. (D) mRNA amount of adiponectin was
quantified by RT-qPCR in blastocysts from normoinsulinaemic and diabetic (exp IDD) rabbits (N ⫽ 3, n ⫽ 9). (E) Adiponectin protein amount was
quantified by ELISA in EB and TB of single blastocysts from normoinsulinaemic and exp IDD rabbits (n.d., not detectable; mean ⫾ SEM; N ⫽ 3, n ⱖ
8; *P ⬍ .05, **P ⬍ .01). (F) Immunohistochemical detection of adiponectin (brown color) showed a diminished nuclear localization in the EB and a
more intensive staining in the TB of blastocysts from diabetic rabbits (exp IDD). (F) Representative examples (a, in blastocyst from a
normoinsulinaemic rabbit; b, from an exp IDD rabbit). (G) Adiponectin concentration was analyzed by ELISA in blastocyst fluid of single blastocysts
from normoinsulinaemic and exp IDD rabbits (mean ⫾ SEM; N ⫽ 3, n ⱖ 8; **P ⬍ .01). adipoR1 and adipoR2 were quantified by RT-qPCR with an
increased adipoR1 RNA (H) and no changes in adipoR2 RNA in blastocysts from diabetic rabbits (exp IDD) (I). The experiments were performed
from 9 blastocysts per stage (mean ⫾ SEM, N ⫽ 3, n ⫽ 9; *** P ⬍ .001).
The Endocrine Society. Downloaded from press.endocrine.org by [Amanda Price] on 24 February 2015. at 10:44 For personal use only. No other uses without permission. . All rights reserved.
2216
Schindler et al
Role of CREB in Embryonic Hormonal Adaptation
Endocrinology, June 2013, 154(6):2208 –2221
C
adiponecn - protein
B
120
100
80
*
60
***
40
20
relative adiponectin amount
[% of control]
adiponecn - mRNA
120
100
*
*
80
60
40
20
0
0
control
insulin
D
AdipoR1
120
100
80
**
60
control
IGF1
**
40
20
0
relative ATF3 mRNA expression [%]
A
relative adipoR1 mRNA expression [%]
relative adiponectin mRNA expression [%]
insulin
IGF1
ATF3
120
100
80
*
60
40
20
0
control
insulin
IGF1
control
insulin
IGF1
Figure 5. Regulation of adiponectin, ATF3, and ATF4 by insulin and IGF-I. (A) Adiponectin RNA was quantified by RT-qPCR in single blastocysts
after a 12-hour culture with 17nM insulin or 1.3nM IGF-I. Stimulation with insulin or IGF-I led to a decreased adiponectin amount compared with
the corresponding nonstimulation control (set 100%) (N ⫽ 3, n ⱖ 5; *P ⬍ .05, ***P ⬍ .001). (B) Adiponectin protein concentration was
determined in blastocysts after a 12-hour culture with insulin or IGF-I and compared with the nontreated control (set 100%). Adiponectin
concentration were determined by ELISA (mean ⫾ SEM; N ⫽ 3, n ⱖ 9; *P ⬍ .05). (C) Expression of adipoR1 was quantified after a 12-hour culture
with or without 17nM insulin or 1.3nM IGF-I. Compared with the corresponding nonstimulated controls (set 100%) the expression of adipoR1 was
significantly decreased (N ⫽ 3, n ⱖ 5; **P ⬍ .01). Expression of ATF3 (D) and ATF4 (E) was analyzed by RT-qPCR after in vitro stimulation with or
without insulin or IGF-I in groups of at least 5 blastocysts in each treatment and repeated in 3 independent replicates (N ⫽ 3, n ⱖ 5).
Transcriptions of ATF3 and ATF4 were quantified in blastocysts after culture for 6 and 12 hours, respectively. Amounts in the nontreated group
(control) were set 100%. mRNA level of ATF4 in EB and TB of normoinsulinaemic and diabetic (exp IDD) rabbits was quantified by RT-qPCR (F). The
experiment was performed from 5 blastocysts per stage (mean ⫾ SEM; N ⫽ 3, n ⫽ 5; *P ⬍ .05). Adiponectin transcription level was analyzed after
a 6-hour culture in media containing 0mM, 10mM, or 25mM glucose. The amounts of adiponectin mRNA levels in 0mM group were set 100%.
No effect on adiponectin transcription level was observed (mean ⫾ SEM; N ⫽ 3, n ⱖ 5).
The quantification of ATF1 mRNA in in vitro-stimulated
embryos (17nM insulin or 1.3nM IGF-I) showed a decrease in transcript levels (Figure 2C), as seen for CREB
too. The ATF1 protein was also localized in the nucleus of
embryonic cells, but unlike CREB, the staining had the
same intensity in both cell lineages (Figure 2D).
ATF3 acts as important negative regulator of adiponectin expression in obesity and type 2 diabetes mellitus. As
shown in Figure 2A, ATF3 was detectable in all investigated
embryo stages (d 3; d 4, early blastocyst) with an increased
expression in stage 3 blastocysts compared with stage 0 (Figure 2E). The ATF3 protein was mainly localized in nuclei of
EB cells, with TB cells being hardly stained (Figure 2F).
Unlike CREB, which activates transcription of CRE promoters, the transcription factor ATF4 specifically represses
CRE-dependent transcription. Transcription of ATF4 was
detected in all stages during preimplantation embryo development (Figure 2A). However, in contrast to CREB and
ATF1/3, the transcript level of ATF4 was decreasing during
blastocyst development (Figure 2B). An antibody to determine the ATF4 localization is not commercially available.
Expression of CREB and ATFs in blastocysts from
diabetic rabbits
In blastocysts grown in diabetic mothers (exp IDD),
CREB activation was reduced by 50% (Figure 3, A and B).
The CREB protein amount was increased (Figure 3C, left
column, and D), confirming the regulatory role of insulin
on CREB expression in vivo. However, the total amount
of phosphorylated CREB was significantly decreased (Figure 3C, right column, and D). In the TB of diabetic blastocysts, CREB was mainly localized in the cytosol, indicating its inactivity (Figure 3E, normoinsulinaemic a1⫹a2
and exp IDD b1⫹b2). In the EB, CREB was still located in
the nucleus (Figure 3E, normoinsulinaemic c1⫹c2 and
exp IDD d1⫹d2). Contrary to CREB, ATF1 expression
was dramatically decreased in blastocysts grown under
diabetic conditions (Figure 3, F and G). Furthermore, in
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doi: 10.1210/en.2012-2096
endo.endojournals.org
2217
ATF4
F
250
EB
TB
500
**
200
*
150
100
50
relative ATF4 mRNA expression [%]
ATF4
E
relative ATF4 mRNA expression [%]
*
400
300
200
100
0
0
control
insulin
normoinsulinaemic
IGF1
exp IDD
G
Figure 5. Continued.
the EB of diabetic blastocysts, ATF1 lost its nucleus-specific staining (Figure 3H). No changes on mRNA transcription were observed for ATF3 (Figure 3I), whereas
ATF4 expression was significantly increased in blastocysts
from diabetic rabbits (Figure 3K).
Adiponectin and receptor (adipoRs) expression in
blastocysts from diabetic rabbits
Serum (Figure 4A) and endometrial adiponectin (Fig-
ure 4, B and C) were increased in diabetic rabbits. In blastocysts grown in diabetic females, transcriptional changes
of adiponectin did not reach statistical significance (Figure
4D). Analysis of adiponectin protein, however, revealed a
cell lineage-specific adiponectin distribution pattern. In
EBs from diabetic blastocysts, adiponectin was not detectable (Figure 4E), whereas the adiponectin level in the TB
was profoundly increased (Figure 4E). Immunohistochemical staining was clearly increased in the TB, whereas
the EB almost completely lost its specific staining (Figure
4F). In blastocyst fluid, the adiponectin level was significantly increased (Figure 4G). Compared with corresponding controls, adipoR1 was increased (Figure 4H), whereas
no changes in adipoR2 RNA transcript levels were ob
served (Figure 4I).
Insulin and IGF-I regulate adiponectin, adipoR1,
ATF3, and ATF4 in vitro
To further investigate the compensatory role of adiponectin for insulin in blastocysts (Figure 4), we analyzed
the influence of insulin and IGF-I supplementation on adiponectin and adipoR1 expression in vitro. After culture of
blastocysts for 12 hours with 17nM insulin or 1.3nM
IGF-I, a significant decrease in adiponectin transcript levels and protein amounts (Figure 5, A and B) and adipoR1
mRNA levels (Figure 5C) were observed.
It is known that CREB down-regulates adiponectin
transcription by the intermediate activation of ATF3,
which in turn represses expression of adiponectin (21). An
in vitro stimulation with insulin or IGF-I for 1 hour had no
effect (data not shown), whereas stimulation for 12 hours
led to a decreased expression of ATF3 (Figure 5D). However, the transcript levels of ATF4, acting as a specific
repressor of CRE-dependent transcription, were increased
after a 6-hour in vitro stimulation (Figure 5E). To confirm
the view that ATF4 acts as a potential negative regulator
of adiponectin expression, we analyzed the expression of
ATF4 in a cell compartment-specific manner. As shown in
Figure 5F, the expression of ATF4 was significantly increased in the EB of blastocysts from diabetic mothers,
whereas no difference in ATF4 transcript levels were detectable in the TB, confirming the inhibitory role of ATF4
on adiponectin expression.
Finally, blastocysts were cultured in vitro for 6 hours in
culture medium containing 0mM, 10mM, or 25mM glucose. As shown in Figure 5G, adiponectin transcript levels
were not altered by the different glucose concentrations
studied, supporting our hypothesis that adiponectin re-
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2218
Schindler et al
Role of CREB in Embryonic Hormonal Adaptation
Endocrinology, June 2013, 154(6):2208 –2221
in this phase of development is still unknown. Our data
show that CREB and the ATF family members mediate the
0,15
embryo’s adaptation to insulin by regulating adiponectin
synthesis. Blastocysts react within 10 minutes with an in
sulin- and IGF-dependent CREB phosphorylation after
0,10
stimulation in vitro. Insulin and IGFs were applied in
physiological concentrations, ie, in an optimal range for
0,05
ligand binding and induction of biological effects (43, 47,
49). Other groups have found an IGF-I-dependent CREB
0,00
normoinsulinaemic
exp IDD
phosphorylation after 10 minutes via the MAPK and p38
Figure 6. Uptake of 3-OMG in normoinsulinaemic and diabetic rabbit MAPK pathways (50 –53). We have previously demonblastocysts. Glucose transport was measured by 3-OMG uptake during
strated that insulin activates MAPK also in rabbit blastoa 3-minute pulse period. Data are presented as the mean ⫾ SEM and
cysts (44, 45). A functional signaling cascade in blastoare
expressed
as
nanomoles
of
3-OMG
per
minute
per
surface
area
2
cysts is indicated by a negative feedback loop (mechanism)
(cm ) (see Materials and Methods). Blastocysts grown in a diabetic
environment showed the same uptake of 3-OMG as the corresponding
for CREB, as shown by the reduced expression of CREB
controls (N ⫽ 3, n ⫽ 28).
after a 12-hour in vitro stimulation with insulin and IGF-I.
Patients with type 1 diabetes and patients with a gepression is related to the lack of insulin rather than to
netically
defective IR show increased serum adiponectin
hypo- or hyperglycaemia.
levels (54). Blüher et al (55) found an elevated plasma
adiponectin level in mice lacking IRs in adipocytes. HyGlucose uptake in blastocysts from
perinsulinaemia selectively down-regulates the high monormoinsulinaemic and diabetic rabbits
To test whether the lack of maternal insulin affects glu- lecular weight form of adiponectin (56). In blastocysts, the
cose uptake, we analyzed the rate of 3-OMG uptake in insulin/IGF-dependent CREB activation of adiponectin
has been demonstrated in vitro and in vivo. In our in vivo
normoinsulinaemic and diabetic blastocysts. No difference in 3-OMG uptake was measured (Figure 6), implying model of an experimentally induced diabetes mellitus type
a compensatory role of increased adiponectin levels for 1, an elevated adiponectin level was measured in maternal
serum, endometrium, and in blastocyst cavity fluid. In
glucose uptake under hypoinsulinaemic conditions.
3T3-L1 preadipocytes, insulin led to a reduced adiponec
tin expression in vitro (22). It is noteworthy that EB and
TB cells of diabetic blastocysts showed a distinct different
Discussion
adiponectin regulation. The difference can be explained
Although CREB has been described as an important reg- by different insulin and IGFs signaling in both cell lineages
(Figure 7). Insulin and IGFs act
ulator of early embryo development, its specific function
through their receptors IGF-IR and
IR (IR-A and IR-B). The two IR iso
forms, resulting from alternative
splicing of the primary transcript
(57), are expressed in a distinctly diverse pattern in both cell lineages in
rabbit blastocysts (45). IR-A, the
binding domain for insulin and IGFII, is mainly expressed in the EB,
whereas IR-B is the only detectable
Figure 7. Cell lineage-specific molecular mechanisms of embryonic hormonal adaptation to a
isoform in the TB (44, 45). IGF-IR is
uterine environment Expression of IGF-I and IGF-II is highly increased in blastocysts and
diabetic
predominantly present in the EB and
endometrium from diabetic rabbits. We interpret this as a compensatory mechanism to cover the
lack of insulin normally provided by the mother. However, EB and TB differ in their
to a lower extent in TB cells (45).
responsiveness to IGFs due to distinct differences in IR and IGF-IR expression patterns (45). (A)
Phosphorylation of Erk induced by
Rabbit blastocysts barely express IGF-IR in the TB. Therefore, IGFs cannot compensate for the lack
insulin occurs in the EB and to a
of insulin in these cells in diabetic blastocysts, leading to CREB inactivity as shown by its
lower extent in the TB, and IGF-I excytoplasmic localization. In this case, CREB is not able to inhibit adiponectin production resulting
in higher adiponectin levels in TB cells in diabetic blastocysts. (B) The increased production of IGFclusively activates Erk/MAPKs in the
I and IGF-II and sensitivity of EB cells towards IGFs (expression of IGF-IR and IR-A) sustain CREB
EB (45). Further analysis revealed
activation
and
nuclear
localization,
leading
to
a
down-regulated
adiponectin
synthesis.
Adiponectin was not detectable in EB cells in diabetic blastocysts.
that IGF-I and IGF-II are highly innMol 3-OMG / cm2 * min
0,20
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doi: 10.1210/en.2012-2096
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2219
creased in blastocysts and endometrium of rabbits with an
experimentally induced diabetes mellitus type 1, most
likely as part of a compensatory mechanism to cover the
lack of insulin normally provided by the mother (5). Because IGF-I and IGF-II are considered as growth factors of
the EB in rabbits (45, 46) and mice (58, 59) and insulin is
depleted in diabetic mothers (4), a cell lineage-specific regulation of the CREB-mediated adiponectin expression is
likely. In the EB of blastocysts grown under diabetic conditions, the increased production of IGFs sustains activation and nuclear localization of CREB, correlating closely
with the drop in adiponectin level observed in current
study (Figure 7B). In TB cells, however, both factors, IGF-I
and IGF-II, were not able to compensate for the lack of
insulin due to absence of the IGF-IR and CREB signaling
in these cells (Figure 7A). Although IGFs may maintain
glucose uptake in EB cells, adiponectin may replace insulin
in TB cells, securing embryo development in diabetic
mothers.
CREB regulates the transcription of target genes directly and indirectly. In rabbit blastocysts, adiponectin
transcript levels are decreased by CREB activation, posing
the question regarding potential CREB inhibitors. A potential candidate is ATF3 (23, 60). Noteworthy, in our in
vitro experiments, ATF3 was also down-regulated by insulin and IGF-I. No differences in ATF3 transcript level
and localization were detectable in blastocysts from diabetic rabbits, arguing against ATF3. In contrast, ATF4
represses specifically the CRE-dependent transcription
of target genes. Insulin and IGF-I led to increased
amounts of ATF4 transcripts. Transcript levels of ATF4
were increased in blastocysts from diabetic rabbits, particularly in the EB. Therefore, it is tempting to propose
a role for ATF4 in adiponectin gene expression in rabbit
blastocysts. It is noteworthy, however, that the levels of
adiponectin are not affected in ATF4⫺/⫺ or in mice
overexpressing ATF4 (38). The specific role(s) of
ATF(s) in hormonal adaptation of blastocysts still needs
to be clarified.
Adiponectin exerts its biological function by binding to
adipoR1 or adipoR2. Both receptors are mainly expressed
in the TB but also in the EB of rabbit blastocysts (10, 61).
These results were confirmed by Kim et al (15) in mouse
blastocysts, demonstrating adipoR1 and adipoR2 in EB
and TB cells. A transcriptional regulation of adipoR1 by
insulin has already been shown in rat and mouse tissues
(62– 64). However, the insulin effect on adipoRs depends
on the cell type. In muscle cells, insulin down-regulates
adipoR1 and adipoR2 expression, whereas in fat cells,
adipoR2, but not adipoR1, is up-regulated (64). We could
show insulin- and IGF-I-dependent decreased adipoR1
transcription levels in blastocysts grown in vitro. These
results are supported by increased adipoR1 levels in blastocysts developed in vivo in a hypoinsulinaemic uterine
milieu. No differences in adipoR2 transcription levels
were observed. This suggests that insulin may have different functions in the regulation of adipoR1 and adipoR2.
Therefore the interplay of both receptors has to be kept in
mind when downstream signaling of adipoRs in preimplantation embryo is assessed.
In conclusion, maternal diabetes alters the hormonal
sensitivity of the embryo during preimplantation development. Our results demonstrate that hypoinsulinaemia
elevates adiponectin levels and the expression of adipoR1
in blastocysts. This effect is caused by insulin/IGF, because
no difference in adiponectin transcript levels were observed after high and low glucose administration. Furthermore, we found an insulin- and IGF-I-dependent adipoR1
transcription. The regulation of adiponectin and adipoR
expression may be part of an embryonic adaptation process, compensating for the lack of maternal insulin to
maintain embryonic glucose metabolism (10). This view is
supported by fact that no difference in glucose uptake was
measured in blastocysts from normoinsulinaemic and diabetic rabbits. Thus, CREB-regulated embryonic adiponectin expression may be a functional connecting link
between maternal insulin supply and embryonic metabolic adaptation. Not only adiponectin, but also adipoR1,
is negatively regulated by insulin. In diabetes, this failsafe
system may compensate for the loss of insulin and helps to
maintain embryo development.
Acknowledgments
We thank Michaela Kirstein, Franziska Knöfel, and Sabine
Schrötter for excellent technical assistant.
Address all correspondence and requests for reprints to: Maria Schindler, Diplom-Trophologe, Department of Anatomy and
Cell Biology, Martin Luther University Faculty of Medicine,
Grosse Steinstrasse 52, D-06097 Halle (Saale), Germany.
E-mail: [email protected].
Present address for R.T.: Department of Obstetrics and Fetal
Medicine, University Medical Center Hamburg-Eppendorf,
20246 Hamburg, Germany.
This work was supported by the German Research Council
Grant NA 418/4-2, EU (FP-7 Epihealth 278418), and by the
Wilhelm Roux Programme of the Martin Luther University Faculty of Medicine.
Disclosure Summary: The authors have nothing to disclose.
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REPRODUCTION-DEVELOPMENT
Maternal Diabetes Leads to Unphysiological High Lipid
Accumulation in Rabbit Preimplantation Embryos
Maria Schindler, Mareike Pendzialek, Alexander Navarrete Santos, Torsten Plösch,
Stefanie Seyring, Jacqueline Gürke, Elisa Haucke, Julia Miriam Knelangen,
Bernd Fischer, and Anne Navarrete Santos
Department of Anatomy and Cell Biology (M.S., M.P., S.S., J.G., E.H., J.M.K., B.F., An.N.S.) and
Department of Cardiothoracic Surgery (Al.N.S.), Faculty of Medicine, Martin Luther University, 06097
Halle (Saale), Germany; and Department of Obstetrics and Gynaecology (T.P.), University Medical Center
Groningen, University of Groningen, 9712 CP Groningen, The Netherlands
According to the “developmental origin of health and disease” hypothesis, the metabolic set
points of glucose and lipid metabolism are determined prenatally. In the case of a diabetic pregnancy, the embryo is exposed to higher glucose and lipid concentrations as early as during preimplantation development. We used the rabbit to study the effect of maternal diabetes type 1 on
lipid accumulation and expression of lipogenic markers in preimplantation blastocysts. Accompanied by elevated triglyceride and glucose levels in the maternal blood, embryos from diabetic
rabbits showed a massive intracellular lipid accumulation and increased expression of fatty acid
transporter 4, fatty acid– binding protein 4, perilipin/adipophilin, and maturation of sterol-regulated element binding protein. However, expression of fatty acid synthase, a key enzyme for de
novo synthesis of fatty acids, was not altered in vivo. During a short time in vitro culture of rabbit
blastocysts, the accumulation of lipid droplets and expression of lipogenic markers were directly
correlated with increasing glucose concentration, indicating that hyperglycemia leads to increased
lipogenesis in the preimplantation embryo. Our study shows the decisive effect of glucose as the
determining factor for fatty acid metabolism and intracellular lipid accumulation in preimplantation embryos. (Endocrinology 155: 1498 –1509, 2014)
he global rise in the prevalence of diabetes mellitus is
paralleled by an alarming increase in diabetic pregnant
women. Currently, in the western world 3% to 10% of pregnancies are affected (1). Diabetes mellitus is associated with
abnormalities in lipid metabolism such as hypertriglyceridemia and low levels of high-density lipoprotein (2). Maternal
metabolic disorders are well known risk factors affecting fertility and embryonic and fetal development.
Early embryos are able to store intracellular lipids in the
form
of lipid droplets (3). Lipids play an important role in
energy metabolism during early embryo development by
serving as an energy source and influencing properties and
functions of biological membranes, cell-cell interactions,
cell proliferation, and intercellular and intracellular trans-
port (for a review, see Ref. 4). The size and number of lipid
droplets are considered as markers of embryo vitality (5,
6). Embryos cultured with serum abnormally accumulate
cytoplasmic lipids, probably as a result of incorporation of
lipoproteins from the serum (6 – 8). An increased amount
of intracellular lipids reflects poor embryo quality and
developmental potential. Pig and bovine embryos contain
the highest amounts of lipids. This observation correlates
with the lowest survival rate after cryopreservation (9 –
12). Mouse embryos with low amounts of lipid droplets
are not as sensitive toward adverse effects of vitrification
as embryos from other species (13).
Lipid droplets are coated by lipid droplet–associated proteins (PLIN1– 4). They regulate the assembly, maintenance,
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received August 14, 2013. Accepted December 18, 2013.
First Published Online January 15, 2014
Abbreviations: cSREBP, cytoplasmic sterol-regulated element binding protein; expIDD, experimental insulin-dependent diabetes; FABP, fatty acid– binding protein; FASN, fatty acid
synthase; FATP, fatty acid transporter; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HRP, horseradish peroxidase; nSREBP, nuclear sterol-regulated element binding protein; PLIN, lipid droplet–associated protein; pc, post coitum; PVA, polyvinyl alcohol; qPCR,
quantitative PCR; SREBP, sterol-regulated element binding protein.
T
1498
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Endocrinology, April 2014, 155(4):1498 –1509
doi: 10.1210/en.2013-1760
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doi: 10.1210/en.2013-1760
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1499
and composition of lipid droplets, as well as lipolysis and
lipid efflux (14 –16). The best known are perilipin and adipophilin, also referred as PLIN1 and PLIN2. Adipophilin
expression has been documented previously from day 2 post
coitum (pc) onward in rabbit preimplantation embryos (17).
The first step in long-chain fatty acid utilization is their
uptake across plasma membranes. This process involves 2
components, passive diffusion through the lipid bilayer
and protein-facilitated transfer. In early embryos, the fatty
acid uptake is poorly understood. However, a facilitated
transport is possible, as indicated by the expression of
fatty acid transport protein (FATP) 4 and fatty acid– binding protein (FABP) 4 in mouse blastocysts (18) and in
human trophoblast cells (19, 20).
The expression levels of FATP4 correlate with measures
of obesity and insulin resistance (21). FABP4, also known as
adipocyte fatty acid– binding protein (AFABP) or aP2, affects
intracellular lipid metabolism by transporting fatty acids to
the nucleus for transcriptional regulation, to mitochondria
for ␤-oxidation, and to lipid droplets for storage (22, 23).
New evidence from population studies and experimental animal models indicates that serum FABP4 is a powerful new
risk marker for predicting obesity, insulin resistance, and
dyslipidemia (24, 25). However, the exact secretion mecha
nism of FABP4 is still not clear.
A source of intracellular fatty acids is de novo synthesis.
Fatty acid synthase (FASN), the key enzyme in de novo
lipogenesis, coordinates together with acetyl-CoA carboxylase energy homeostasis by converting excess food intake
into lipids for storage (26, 27). FASN⫺/⫺ null mutant mice
die during preimplantation development, demonstrating
the important role during early embryonic development (28).
The promoter region of FASN contains binding sites for the
transcription factor sterol-regulated element binding protein
(SREBP) 1 (29). SREBPs are synthesized as precursor proteins that remain bound to the endoplasmic reticulum. After
activation, SREBP is processed and translocated to the nucleus (30, 31). The mature nuclear SREBP activates the transcription of genes regulating synthesis and uptake of fatty acids,
triglyceride, and cholesterol (31, 32). Currently, 3 different
isoforms have been described: SREBP1a and SREBP1c, derived from a single gene by alternative transcription start
sites, and SREBP2 (33). SREBP-overexpressing transgenic
animals showed an accumulation of cholesterol and/or fatty
acids in liver cells (32, 34, 35), indicating that the SREBP1
isoforms are more directed forward activation of fatty acid
biosynthesis, whereas SREBP-2 is more specific for controlling cholesterol biosynthesis.
Despite the increasing incidence of metabolic disorders in
women of reproductive age, few attempts have to be made to
analyze the maternal influence on embryonic lipid metabolism (36 –38). Therefore, we have investigated intracellular
lipid accumulation and expression of key lipogenic markers
(such as perilipin, adipophilin, FATP4, FABP4, FASN, and
SREBP1) in response to hyperglycemia in vivo caused by an
induced maternal diabetes mellitus type 1 and hyperglycemia
created in vitro in rabbit preimplantation embryos.
Materials and Methods
Alloxan treatment
Experimental insulin-dependent diabetes (expIDD) was induced in mature 18- to 20-week-old female nonpregnant rabbits
by alloxan treatment (Sigma-Aldrich) as described before (39).
Rabbits were held in the diabetic condition with permanent
blood glucose concentrations of ⬎14 mmol/L by regular insulin
supplementation 4 times/d (Insuman Rapid and Lantus; sanofi
aventis), started on the second day after alloxan treatment. The
blood glucose level was monitored with a MediSense Precision
Xceed Diabetes Management System (Abbott) 2 times/d. For
that, fresh blood was collected at 10:00 AM and 6:00 PM by puncturing the vena auricularis lateralis and tested for glucose concentration by a commercial test strip.
All animal experiments were conducted in accordance with
the principles of laboratory animal care, and the experimental
protocol had been approved by the local ethics commission of the
Landesverwaltungsamt Dessau (reference no. 42502-2-812).
Embryo recovery
Embryos were collected from sexually mature rabbits stimulated with 110 IU of pregnant mare serum gonadotropin sc
(Intervet) 3 days before mating. After mating, 75 IU of human
chorionic gonadotropin was injected iv (Intervet). Mating and
embryo recovery were performed as described previously (40).
On days 3, 4, and 6 pc embryos were flushed from oviducts or uteri,
respectively, washed 3 times with PBS and randomly divided among
the experimental groups. On day 6 pc, gastrulation stages can be
reliably discriminated in the rabbit because implantation starts on
day 6 18 hours, ie, half a day later (41). Only blastocysts from
gastrulation stage 1 and 2 were used for further analyses.
Embryo in vitro culture
To study the effects of different glucose concentrations on
perilipin/adipophilin, FABP4, FATP4, and FASN expression and
intracellular lipid accumulation, day 6 blastocysts were cultured
in groups of 6 to 10 at 37°C in a water-saturated atmosphere of
5% O2, 5% CO2, and 90% N2. Blastocysts were cultured for 6
hours in serum- and insulin-free BSM II medium (42) with 0, 10
(standard culture medium), and 25 mM glucose (hyperglycemic
conditions), respectively.
Quantification of triglycerides in blood samples
Triglyceride concentrations in blood samples were measured
by a commercial kit (DiaSys Triglycerides FS; Diagnostic Systems). All procedures were performed according to the manufacturer’s instructions. To measure the serum triglyceride concentration, blood samples were collected with the S-Monovette
system(Sarstedt), left to coagulate for at least 30 minutes, and
centrifuged for 10 minutes at 4°C and 1000 ⫻ g. The supernatant
was stored at ⫺80°C until use.
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1500
Schindler et al
Lipid Storage in Rabbit Preimplantation Embryos
Endocrinology, April 2014, 155(4):1498 –1509
Oil Red O staining of rabbit blastocysts
Blastocysts were washed twice in ice-cold PBS and fixed in 4%
(wt/vol) paraformaldehyde for 10 min. The fixed blastocysts
were washed again in ice-cold PBS and transferred to 0.05%
(wt/vol) polyvinyl alcohol (PVA)/PBS. Blastocyst coverings were
removed mechanically. The embryonic disk was mechanically diswith surgical forceps and scissors (Fine Science Tools GmbH)
sected
(39) and used immediately for Oil Red O staining. Oil Red O stock
solution (0.5 mg of Red Oil O [Sigma-Aldrich] dissolved in 100 mL
of isopropanol) was diluted 3:2 with distilled water. To remove the
precipitate, the working solution was filtered using a filter with a
pore diameter of 20 ␮m (BD Biosciences).
For Oil Red O staining, the embryonic disc was incubated in
filtered Oil Red O solution for 2 hours. After staining the lipid
droplets, the Oil Red O staining solution was removed by washing
the embryonic disc for 3 hours in 0.05% (wt/vol) PVA/PBS. Subsequently, the embryonic disk was embedded on Superfrost slides
(Menzel Gläser) using 4.8 g of Mowiol reagent (Calbiochem, Germany) dissolved in 12.0 g of glycerol (Merck). Embryonic disks
were examined by light microscopy (BZ 8000; Keyence).
Preparation of a single-cell suspension
For the separation of blastocysts to single-cell suspensions,
the gentleMACS-m-neural-tissue kit (Neural Tissue Dissociation Kit; Miltenyi Biotec) was used. Blastocyst coverings were
removed mechanically, and blastocysts were transferred in a
1.5-mL tube. The preheated papain enzyme mix 1 provided with
the kit was given directly to the blastocysts and incubated according to the manufacturer’s protocol, except that the incubation step was performed at room temperature. Enzyme mix 2,
prepared according to the manufacturer’s protocol, was added
directly to the sample. The specimen was incubated for 15 minutes at room temperature on a rotator.
To remove cell aggregates from the cell suspension, a preseparation filter with a pore diameter of 40 ␮m (BD Biosciences)
was used. After a washing step, the cells were resuspended in
ice-cold 0.05% (wt/vol) PVA/PBS. Nile Red stock solution (100
␮g/mL; Sigma-Aldrich) was prepared in dimethyl sulfoxide and
stored in the dark. For Nile Red staining, the dye was added at
a final concentration of 0.1 ␮g/mL. The cells were then incubated
on ice for 5 minutes, centrifuged, and washed with 0.05% (wt/
vol) PVA/PBS. Afterward they were resuspended in a volume of
0.05% (wt/vol) PVA/PBS and kept on ice in the dark until flow
cytometry analysis.
Flow cytometry
All measurements were performed on a BD FACS Vantage
(BD Biosciences) equipped with 3 lasers (excitation wavelengths:
488, 633, and 351nm) as described previously (43). Cell debris,
doublets, and aggregates were excluded from analysis. Blastocyst
cells without Nile red staining served as a negative control for flow
cytometry analysis. Measurements and data analysis were performed with FACSDiva (version 5.0.3; BD Biosciences). Nile Red
specifically stains intracellular lipid droplets, excites at 485 nm, and
emits at 525 nm. For sorting, a 90-␮m nozzle was used, and the
sorting rate was no more than 1000 events per second.
The visualization by dot plot and histogram shows 2 different
cell populations R1 (P1) and R2 (P2). To prove that region R1
contains embryoblast and R2 contains trophoblast cells, we
sorted both cell populations and used them subsequently for
RT-quantitative PCR (qPCR) analyses for cytokeratin 18 and
cdx2, 2 trophoblast markers (44). Cytokeratin 18 and cdx2 were
highly expressed in the R2 cell population, demonstrating that
these cells were almost exclusively trophoblast cells (Figure 1I).
The analysis of the percentage of cells located in R3 (cell
debris or putative dead cells) revealed no significant differences
between the normohyperglycemic and hyperglycemic groups.
This result suggests that in our experiments increased cell death
in the hyperglycemic group compared with that in the control
group can be excluded.
RNA isolation and cDNA synthesis
mRNA of single blastocysts was extracted with Dynabeads
oligo(dT)25 (Invitrogen) and subsequently used for cDNA synthesis. All protocol procedures were performed according to the
manufacturer’s instructions, with modifications described previously (45).
RT-PCR
RT-PCR amplification was performed with 0.5 ␮L of cDNA
from single blastocysts in a 25-␮L volume containing 200 ␮M
concentrations of each dNTP, 2.5 U of Taq polymerases, and
specific oligonucleotides for adipophilin, FABP4, FATP4, FASN,
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
(primers are listed in Supplemental Table 1 published on The
Endocrine Society’s Journals Online web site at http://end.
endojournals.org). The nucleotide sequence for rabbit FASN
was determined using human primers for amplification of rabbit
liver cDNA. The amplification was done for 40 cycles (94°C for 45
seconds, 60°C for 45 seconds, and 72°C for 60 seconds). The resulting PCR products were separated by electrophoresis on 2%
agarose gels and stained with ethidium bromide. The PCR products
were sequenced and analyzed as described previously (46). Sequence homology was proven by using the alignment BLASTN
tool.
RT-qPCR
Amounts of FAS, FATP4, FABP4, adipophilin, and GAPDH
cDNA were determined by real-time qPCR with SYBR Green
detection by using the StepOnePlus System (Applied Biosystems)
with a no template control for each primer set (described in Ref.
47). The nucleotide sequences of the primers used in this study are
listed in Supplemental Table 1. The amount of cDNA per sample
was normalized by referring to the GAPDH gene. GAPDH was
shown to be unaffected by the treatment because no variation was
observed between the absolute GAPDH levels and GAPDH RNA
levels in blastocysts from healthy rabbits (47). qPCRs for target and
reference genes were always run in duplicate from the same cDNA
dilution taken from the same RT reaction.
The absolute amount of the transcripts was calculated along
with a standard (calibrator) sample. As standards we used defined concentrations of plasmid standards constructed for the
gene of interest and GAPDH. For all genes investigated a partial
sequence was amplified from rabbit tissues. Purified PCR products were ligated into pGEM-T with subsequent transformation
in XL1Blue competent bacteria (described in Ref. 47).
Results are given in molecules of the target gene per GAPDH
molecule. Statistical analysis was performed on the amounts of
molecules. Fold changes were calculated when the results are
expressed as percentages.
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doi: 10.1210/en.2013-1760
endo.endojournals.org
1501
A
exp IDD
normoglycemic
D
E
F
G
EB
C
H
3000
*
2500
2000
1500
1000
500
I
relative transcription [fold change]
B
TB
Nile Red
[Mean of the fluorescence]
25
20
15
10
5
R1
0
0
R2 (P2) normoglycemic
R2 (P2) expIDD
cdx2
cytokeratin 18
Figure 1. Oil Red O staining and Nile Red flow cytometry analyses of blastocyst cells from diabetic rabbits. A, Blastocysts derived from healthy and
diabetic (expIDD) rabbits were stained with Oil Red O, marking intracellular lipid droplets red in trophoblast (TB) and embryoblast (EB) cells (scale
bar corresponds to 50 ␮m). Nuclei were counterstained with hematoxylin in blue. B–G, For flow cytometry analysis blastocyst cells were stained
with Nile Red. A representative analysis is shown. Blastocyst cells were visualized on a dot plot of forward scatter (FSC) vs side scatter (SSC) (B, D,
and F) or in a Nile Red histogram (C, E, and G). The visualization by dot plot, using the parameters granularity and cell size, indicated 2 different
cell populations, region R1 with embryoblast cells and R2 with trophoblast cells, respectively. For further analyses, blastocyst cells were gated in
region R1 and R2. Cell debris and death cells were excluded (region R3). Blastocyst cells were gated in region R1 and R2 from normoglycemic (D)
and diabetic (F) rabbits. Nile Red absorbance of blastocyst cells from regions R1 and R2 is shown for normoglycemic (E) and for diabetic (G) rabbits
in P1 and P2, respectively. H, Nile Red mean fluorescence in R2 (P2) of normoglycemic and diabetic cells determined in 3 independent experiments
is shown. Blastocyst cells were sorted in region R1 and R2 and subsequently used for RT-qPCR analysis of trophoblast markers cdx2 and
cytokeratin18. I, Results are shown as relative amounts and fold change of R1 (means ⫾ SEM; N ⫽ 4, n ⱖ 18). R2 sorted cells were identified as
trophoblast cells with 10- and 5-fold higher cdx2 and cytokeratin 18 RNA amounts, respectively, than those in the R1 cells.
Protein preparation and immunoblotting
Protein preparation, quantification, and Western blotting
performed with 8 to 10 blastocysts as described previously
were
(48). For Western blot analysis, 25 ␮g of total protein lysates was
subjected to SDS-polyacrylamide electrophoresis and electrotransferred to nitrocellulose membranes. For detection of FASN,
SREBP1, and ␤-actin, membranes were blocked in Tris-buffered
saline containing 0.1% (vol/vol) Triton X-100 (0.1% TBST)
with 3% (wt/vol) nonfat dry milk at room temperature for at
least 1 hour. For perilipin detection, membranes were blocked in
0.1% TBST with 3% (wt/vol) BSA for 2 hours. The primary
antibody was incubated at 4°C overnight. The following antibodies were used as follows perilipin (1:1000; Sigma-Aldrich),
FASN (1:1000; Santa Cruz Biotechnology), SREBP1 (1:1000;
Active Motif), ␤-actin (1:40 000; Sigma-Aldrich), anti-rabbit
IgG conjugated to horseradish peroxidase (HRP) (1:15 000;
DAKO Cytomation), and anti-mouse IgG conjugated to HRP
(1:45 000; Dianova). The protein amount was calculated as the
ratio of band intensities (perilipin protein or FASN protein vs
␤-actin protein, respectively) in the same blot to correct for differences in protein loading.
Immunohistochemical localization of FABP4 and FASN
Blastocysts were washed twice in ice-cold PBS and fixed in 4%
(wt/vol) paraformaldehyde at 4°C overnight. The sample was
prepared, and the immunohistochemical protocol was per-
formed as described previously (48). Antibodies for FABP4 and
FASN (both Santa Cruz Technology) were diluted 1:100 and
1:500 in 3% (wt/vol) BSA/PBS, respectively. The secondary antibody Dako EnVision⫹ System HRP-labeled polymer antimouse (1:1 in PBS) and diaminobenzidine (WAK-Chemie
Medikal) were used for detection.
All steps were performed within the same experiment, and
samples were examined microscopically during the same session,
using identical microscope and camera settings (BZ 8000;
Keyence).
FABP4 ELISA
FABP4 concentrations in blastocyst fluids were measured by
ELISA (MyBioSource). All protocol procedures were performed
Table 1. Mean Nile Red Fluorescence Intensity From
Normoglycemic and Diabetic (expIDD) Blastocyst Cells in
Region R1 (P1) and Region R2 (P2)
Nile Red
(Mean ⴞ SEM)
R1 (P1) normoglycemic
R1 (P1) expIDD
R2 (P2) normoglycemic
R2 (P2) expIDD
951 ⫾ 280
1093 ⫾ 202
1566 ⫾ 267
2503 ⫾ 233
P Value
.6
.03
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1502
Schindler et al
Lipid Storage in Rabbit Preimplantation Embryos
Endocrinology, April 2014, 155(4):1498 –1509
A
Results
d6
d3
d4
d5
d6
d8
st.0
st.1
st.2
st.3
+
ntc
Adipophilin
FATP4
FABP4
FASN
GAPDH
B
EB
normoglycemic
exp IDD
C
normoglycemic
exp IDD
EB
TB
D
TB
4
FABP4 concentration [ng/ml]
*
3
2
1
0
normoglycemic
expIDD
2. Expression patterns of the lipogenic markers adipophilin, FATP4, FABP4, and FASN in
Figure
rabbit preimplantation embryos. A, RT-PCR was performed with specific primers for adipophilin,
FATP4, FABP4, and FASN on rabbit morulae at day (d) 3, on blastocysts at days 4, 5, 6, and 8 pc,
and on 6-day-old blastocysts of different gastrulation stages with stage (st) 0, 1, 2, and 3.
Transcripts of adipophilin, FATP4, and FASN were detected in all analyzed developmental stages.
FABP4 transcription was measurable from gastrulation stage 1 (st 1) onward. A probe without
cDNA was used as negative control (ntc). As a positive control, a liver cDNA probe was used (⫹).
The internal control was the expression of GAPDH in all probes. The FABP4 and FASN proteins
were detected by immunohistochemical analysis and visualized by a peroxidase-diaminobenzidine
reaction (brown color) (scale bar corresponds to 50 ␮m). B, In blastocysts from healthy controls
(normoglycemic), FABP4 was localized in the cytosol and nucleus of embryoblast cells (EB). The
trophoblast cells (TB) were not or less stained for FABP4. In blastocysts from diabetic rabbits
(expIDD), the embryoblast and trophoblast cells showed more intense staining for FABP4,
especially in the nuclei of both cell lineages. Nuclei were counterstained in blue with
hematoxylin. C, Immunohistochemical localization revealed FASN in the cytosol of embryoblast
and trophoblast cells. No difference in staining and localization was found between blastocysts
from diabetic and normoglycemic rabbits. D, FABP4 concentrations were analyzed by ELISA in
blastocyst fluids of single blastocysts from normoglycemic and expIDD rabbits (means ⫾ SEM;
N ⫽ 3; n ⱖ 9; *, P ⬍ .05).
according
to the manufacturer’s instructions. Flushed blastocysts were washed 2 times with ice-cold PBS. Blastocyst stage and
size were determined. Blastocysts were then placed on a dry
watch glass. Extracellular coverings were removed mechanically, blastocysts were punctured, and the effluent cavity fluid
was collected. Blastocyst fluids were stored at ⫺80°C until use.
FABP4 concentrations were quantified in duplicate and compared with an internal FABP4 standard (0.625–50 ng/mL).
Statistics
All data are expressed as means ⫾ SEM. Levels of significance
between groups were calculated by factorial ANOVA with Bonferroni adjustment. A value of P ⬍ .05 was considered statistically significant. All experiments were conducted at least 3 times.
In previous studies, we demonstrated
that the increases in maternal blood
glucose concentrations correlate with
increased glucose levels in the uterine
secretions (39). Here, we focused on
the lipid content and lipogenic
markers to test whether a diabetic
disorder during early pregnancy
would affect the embryonic fatty
acid metabolism.
Elevated triglyceride levels in
female rabbits with expIDD at
day 6 pc
In blood samples of diabetic (expIDD) pregnant rabbits, triglyceride
levels were increased approximately
6-fold (3.55 ⫾ 0.43 mM; mean ⫾
SEM) compared with that in the
healthy controls (0.57 ⫾ 0.05 mM)
[number of independent experiments
used for blastocysts (N) ⫽ 3; total
number of blastocysts used for analysis (n) ⫽ 9; P ⬍ .001], respectively.
Accumulation of intracellular
lipid droplets in normoglycemic
and diabetic (expIDD)
blastocysts
The effect of alloxan-induced maternal diabetes mellitus type 1 on intracellular lipid accumulation was
examined by Oil Red O staining.
Blastocysts from diabetic rabbits
showed a higher accumulation of intracellular lipids in the embryoblast
and trophoblast, indicated by the
higher amount of red-stained lipid
droplets (Figure 1A). To further support these data, we used a quantitative method to determine the cellular lipid content based on Nile Red staining
and flow cytometry analyses. In Figure 1, B to G, the results of a representative flow cytometry analysis are illustrated. Blastocyst cells were visualized by dot plot and
histogram. A higher Nile Red fluorescence intensity was
found in blastocyst cells from diabetic rabbits in both cell
populations R1 (P1) and R2 (P2). However, in contrast to the
R2 (Figure 1I and Table 1), the difference in the Nile Red
fluorescence mean in the R1 between normoglycemic and
diabetic blastocysts was not significant (data not shown).
The mean Nile Red absorbance is given in Table 1.
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doi: 10.1210/en.2013-1760
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1503
Table 2. Relative Protein Amount of Lipogenic Markers
in Blastocysts from Normoglycemic and Diabetic
(expIDD) Rabbits
Protein
Perilipin
FASN
nSREBP1
cSREBP1
Ratio: nSREB1/
cSREBP1
Normoglycemic
expIDD
P Value
100.0 ⫾ 3.5
100.0 ⫾ 6.8
100.0 ⫾ 7.2
100.0 ⫾ 12.7
0.51 ⫾ 0.03
136.8 ⫾ 10.5
105.3 ⫾ 1.2
107.2 ⫾ 5.2
76.9 ⫾ 5.8
0.70 ⫾ 0.03
.048
.473
.437
.138
.005
are given relative to protein amounts in blastocysts from
Results
healthy rabbits (normoglycemic) (means ⫾ SEM; N ⫽ 3; n ⫽ ⱖ10).
Adipophilin and perilipin expression in
preimplantation embryos from diabetic rabbits
Intracellular lipid droplets are coated by the lipid droplet–
associated proteins perilipin and adipophilin. The transcription of adipophilin was detectable in the rabbit preimplantation embryos from day 3 old morulae onward in all
analyzed days and stages (Figure 2A). Transcription levels in
blastocysts (day 6 pc stage 1) from diabetic rabbits (expIDD)
were significantly increased compared with those from normoglycemic rabbits (Table 2). Quantification of adipophilin
in the embryoblast and trophoblast revealed that the increase
in the adipophilin amount resulted from transcriptional upregulation, mainly in embryoblast cells (Table 4). In addition,
the perilipin protein amount was significantly increased in
blastocysts from diabetic rabbits (Table 3).
FATP4 and FABP4 expression in preimplantation
embryos from diabetic rabbits
FATP4 and FABP4 are expressed in rabbit blastocysts.
Whereas FATP4 transcription was detectable in all investigated embryonic stages, starting on day 3 pc (Figure 2A),
FABP4 expression was only measurable in expanded and
gastrulating blastocysts on day 6 pc, from stage 1 onward
and on day 8 pc (Figure 2A). Analysis of FABP4 transcription, however, revealed a cell lineage–specific FABP4 distribution pattern with the main expression in the embryoblast (Figure 2B). Only a few cells stained positive for
FABP4 in the trophoblast.
In diabetic conditions, the transcription levels for both
FATP4 and FABP4 were increased in blastocysts (Table 2).
FATP4 mRNA was increased in the embryoblast and trophoblast (Table 4). For FABP4, the RNA amount was
increased only in the embryoblast (Table 4). Compared
with embryos from healthy rabbits, blastocysts from diabetic mothers showed more intense FABP4 staining in
both cell lineages, with the most prominent localization in
the nuclei (Figure 2B).
Because FABP4 can be secreted from cells and is present
in the circulation, we analyzed the FABP4 concentration in
blastocyst fluid. Analysis by ELISA showed that FABP4 is
present in blastocyst fluid and that the amount is increased
in the cavity fluid from diabetic blastocysts (Figure 2D).
FASN expression in preimplantation embryos from
diabetic rabbits
The key enzyme of de novo lipogenesis, FASN, was
detectable form day 3 old morulae (d3) onward in all analyzed stages (Figure 2A). Neither FASN transcription
(Table 2) nor protein (Table 3) amounts were significantly
different in blastocysts from diabetic rabbits. In addition,
no alterations of FASN transcription levels in embryoblasts and trophoblasts from normoglycemic and diabetic
rabbits were detectable. However, a cell lineage–specific
transcription was observed (Table 4), with higher transcripts in the embryoblast. FASN protein was localized in
both embryoblast and trophoblast cells with a cytoplasmic
localization (Figure 2C). In blastocysts from diabetic rabbits, no differences in staining intensity or in cellular localization were observed.
SREBP1 expression in preimplantation embryos
from diabetic rabbits
Under hyperglycemic conditions, no changes in total
SREBP1 protein amounts were observed (Table 2). However, the ratio of cytoplasmic (cSREBP1) and mature, nuclear SREBP1 (nSREBP1) was influenced. Blastocysts
from diabetic rabbits had relatively more SREBP1 located
in the nucleus (based on the total SREBP1 protein amount
and the ratio of nSREBP1 to cSREBP1) compared with
that of blastocysts from normoglycemic rabbits (Table 2),
indicating a higher transcriptional regulation by SREBP1
in blastocysts from diabetic rabbits.
Table 3. Relative mRNA Amount of Lipogenic Target Genes in Blastocysts from Normoglycemic and Diabetic
(expIDD) Rabbits
mRNA
Normoglycemic
expIDD
%
P Value
Adipophilin
FABP4
FATP4
FASN
51.76 ⫾ 15.28
4.45 ⫾ 2.03
855.4 ⫾ 124.1
0.66 ⫾ 0.18
167.78 ⫾ 3.99
9.91 ⫾ 2.27
1382.4 ⫾ 179.6
0.38 ⫾ 0.06
287.4 ⫾ 29.9
222.6 ⫾ 51.1
188.9 ⫾ 23.2
58.7 ⫾ 10.1
.020
.041
.038
.195
Results are given as molecules and as a percentage of transcription levels in normoglycemic blastocysts (means ⫾ SEM; N ⫽ 3; n ⫽ ⱖ10).
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1504
Schindler et al
Lipid Storage in Rabbit Preimplantation Embryos
Endocrinology, April 2014, 155(4):1498 –1509
Table 4. Relative mRNA Amount of Lipogenic Target Genes in Embryoblast and Trophoblast From Normoglycemic
and Diabetic (expIDD) Rabbits
Embryoblast
Trophoblast
mRNA
Normoglycemic
expIDD
Normoglycemic
expIDD
Adipophilin
FABP4
FATP4
FASN
100.0 ⫾ 8.3
100.0 ⫾ 17.1
100.0 ⫾ 6.4
100.0 ⫾ 23.5
131.3 ⫾ 11.8a
293.3 ⫾ 71.2a
147.2 ⫾ 15.3a
91.4 ⫾ 23.4
118.1 ⫾ 6.6
14.0 ⫾ 3.5b
92.8 ⫾ 10.4
25.9 ⫾ 6.7b
125.1 ⫾ 17.7
9.9 ⫾ 1.8b
148.6 ⫾ 15.1a
15.8 ⫾ 2.3b
Results are given as a percentage of the amount in the embryoblast from healthy rabbits (normoglycemic) (means ⫾ SEM; N ⫽ 3; n ⫽ ⱖ10).
a
b Amount significantly different between control and expIDD (P ⬍ .05).
Amount significantly different between embryoblast and trophoblast (P ⬍ .05).
Glucose-dependent expression of lipogenic genes
To clarify whether glucose can regulate the expression
of lipogenic genes, we cultured blastocysts without glucose (0 mM) and with 10 mM (standard culture media) or
25 mM (hyperglycemia in vitro) for 6 hours in vitro. The
quantification of adipophilin and FABP4 transcript levels
and perilipin protein showed increased expression of all
markers in blastocysts cultured under hyperglycemic conditions (Figure 3, A, B, C, and E). No significant differences were measured for the FATP4 transcript levels in
response to the various glucose concentrations (Figure
3D). In contrast to diabetic conditions in vivo, the FASN
protein amount was increased in blastocysts cultured with
10 and 25 mM compared with that in blastocysts cultured
with 0 mM, respectively (Figure 3, F and G). Total protein
amounts of SREBP1 were increased in a glucose-dependent manner with a higher accumulation of mature
nSREBP1 in the nucleus (Figure 3, H–K).
Glucose-dependent accumulation of lipid droplets
Excess energy intake leads to an accumulation of intra
cellular lipids. To analyze whether a high concentration or
the lack of glucose influences the amount of lipid droplets in
embryos, we cultured blastocysts (day 6 pc) from normoglycemic and diabetic rabbits for 6 hours with either 0, 10, or 25
mM glucose and afterward stained intracellular lipid droplets with Oil Red O. Blastocyst cells from both normoglycemic and diabetic (Figure 4) rabbits showed a glucose-dependent accumulation of intracellular lipids. High glucose
concentrations led to a higher amount of lipid droplets compared with 0 and 10 mM in blastocysts from normoglycemic
rabbits. The lowest amount of lipid droplets was found in
blastocysts cultured with 0 mM. A comparable glucose-dependent increase in lipid droplets was observed in blastocysts
from diabetic rabbits. However, the amount of lipid droplets
in each group (0 –25 mM) was higher in diabetic blastocysts
than in the corresponding controls.
Discussion
Since the initial observation by Mills in 1982 (49), a great
number of studies have shown the determining effect of
maternal hyperglycemia on pregnancy outcome, offspring
malformation, and the risk of later life diseases (39, 50 –
55). Adverse effects of high glucose concentrations have
been shown by in vitro and in vivo culture experiments,
such as retarded embryo development and increased apoptosis (39, 55– 63). The current study shows for the first
time the direct influence of maternal diabetes in vivo and
hyperglycemia in vitro on embryonic lipid storage. One
central finding of this study is the highly increased amount
of intracellular lipid droplets in blastocysts from diabetic
rabbits. Embryos with higher amounts of cytoplasmic lipids are more sensitive to chilling and cryopreservation (64)
and have a lower developmental efficiency (10, 11). Therefore, the increased amount of lipid droplets in blastocysts
may contribute to the adverse effects of maternal diabetes
on preimplantation embryo development.
The massive lipid accumulation in blastocysts from diabetic rabbits raises the question of whether the embryo
produces lipids from excess maternal glucose supply or by
an increased transport of fatty acids into the cell. Type 1
diabetes mellitus is not only characterized by high serum
glucose concentrations but also by increased serum triglyceride concentrations. These phenomena are closely reflected by hyperglycemia and hypertriglyceridemia in the
diabetic rabbit model. Diabetes mellitus is associated with
maternal and fetal dyslipidemia, which manifests as high
plasma triglyceride concentrations, elevated concentrations of nonesterified fatty acids, increased concentrations
of low-density lipoprotein cholesterol, and decreased levels of high-density lipoprotein cholesterol (20, 65– 68). An
increase in fatty acids enhances the formation of lipid
droplets and expression of adipophilin and FABP4 (20,
69 –71). Therefore, it is likely that the diabetes-associated
hyperlipidemia is a potent regulator of embryonic lipid
metabolism. This view is supported by the observation
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doi: 10.1210/en.2013-1760
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1505
G
relative perilipin amount [%]
1,6
1,4
1,2
1,0
0,8
0,6
0,4
C
*
180
160
0 mM 10 mM 25 mM
140
Perilipin
120
100
80
β-acn
60
40
20
0,2
0
0,0
0 mM
10 mM
0 mM
25 mM
E
1,2
1,0
0,8
0,6
0,4
0,2
0,0
10 mM
F
5
200
*
4
3
2
1
10 mM
25 mM
0 mM
10 mM
β-acn
J
0 mM 10 mM 25 mM
cSREBP1
(125kDa)
0 mM
10 mM
25 mM
*
1,6
250
200
150
100
50
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
0
nSREBP1
50
25 mM
*
300
0 mM 10 mM 25 mM
FASN
100
I
H
*
150
0
0
0 mM
25 mM
relative FASN amount [%]
D
200
ratio nSREBP1 / cSREBP1
1,8
FABP4 transcription [fold change]
B
*
2,0
relative SREBP1 amount [%]
FATP4 transcription [fold change]
A
adipophilin transcription [fold change]
0 mM
10 mM
25 mM
0 mM
10 mM
25 mM
(68kDa)
β-acn
Figure 3.
Relative expression of lipogenic genes in rabbit blastocysts cultured in vitro with 0, 10, or 25 mM glucose. A, In vitro culture of
blastocysts for 6 hours with 0, 10, or 25 mM glucose led to a significantly higher adipophilin transcription level in blastocysts of the 25 mM group
(*, P ⬍ .05). Blastocysts were cultured in groups of at least 5 blastocysts (N ⫽ 3; n ⱖ 5). mRNA levels were analyzed by RT-qPCR, and results are
shown as means ⫾ SEM (*, P ⬍ .05). B and C, Relative perilipin amounts after 6 hours of cultivation without or with 10 or 25 mM glucose were
analyzed by Western blotting with anti-perilipin antibody and set in relation to total ␤-actin. A representative Western blot for perilipin is shown in
panel C. Results of 3 independent stimulation experiments (N ⫽ 3, n ⱖ 9; means ⫾ SEM) are shown in panel B. The group without glucose (0
mM) was set at 100%. D and E, RT-qPCR analysis of FATP4 (D) or FABP4 (E) after in vitro culture (for 6 hours) of blastocysts with 0, 10, or 25 mM
shows a distinctly different expression pattern. In vitro culture with 10 or 25 mM or without glucose was performed in groups of at least 5
blastocysts in each treatment and repeated in 3 independent replicates (N ⫽ 3; n ⱖ 5). Results are shown as means ⫾ SEM (*, P ⬍ .05). F and G,
FASN protein amount was analyzed by Western blot and the 0 mM group (control) was set at 100% (*, P ⬍ .05). Results (F) and a representative
Western blot (G) are shown. H–K, Relative SREBP1 amounts after 6 hours of cultivation without or with 10 or 25 mM glucose were analyzed by
Western blotting with anti-SREBP1 antibody, which specifically detects cSREBP1 (125 kDa) and nSREBP1 (68 kDa). Culture was performed in
groups of at least 5 blastocysts in each treatment and repeated in 3 independent replicates (N ⫽ 3; n ⱖ 5). H, Relative SREBP1 protein amounts
(nSREBP1 and cSREBP1) in relation to those in the 0 mM group, which was set at 100%. I, Ratio of nSREBP1 vs cSREBP1. J, Representative Western
blot for SREBP1.
that both normoglycemic and diabetic blastocysts cultured
for 6 hours in vitro showed a lower amount of lipid droplets
than that of their in vivo counterparts, implying that meta-
bolic factors other than glucose might play a role, too. Furthermore, FASN expression was not altered in blastocysts
from diabetic rabbits. Because lipogenesis encompasses fatty
acid and subsequent triglyceride synthesis, it is tempting to
speculate that the unchanged FASN expression in vivo is the
result of an increased lipid supply by the diabetic mother and
the increased expression of FABP4 and FATP4 in their blastocysts (Figure 5). On the other hand, it is also possible that
the 3-fold increase in glucose in uterine secretions (20) is not
high enough to regulate FASN expression. Finally, the increased FABP4 amount in the cavity fluid from diabetic blastocysts may play a pivotal role by trafficking free fatty acids
to blastocyst cells.
In our in vitro model, we can create a hyperglycemic situation independent of any triglyceride influence. In the case
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1506
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Lipid Storage in Rabbit Preimplantation Embryos
Endocrinology, April 2014, 155(4):1498 –1509
nSREBP1/cSREBP1 ratio in vivo.
Therefore, SREBP1 could belong to
the connecting link between glucose
normoand lipid storage in preimplantation
glycemic
embryos.
Several studies clearly show that
glucose is a main source and key signal for SREBP1 induction and cleav
age of SREBP1 precursor protein in a
dose-dependent manner (77–79), as
exp IDD
shown by our in vitro experiments,
too. This effect is not due to osmotic
properties, because glucose could
not be replaced by the glucose anaFigure 4. Intracellular lipid accumulation in blastocysts cultured in vitro with 0, 10, and 25 mM
glucose. Six-day-old blastocysts were removed from healthy (normoglycemic) and diabetic
logs 2-deoxyglucose or 3-O-methyl
(expIDD) rabbits and cultured in vitro with 0, 10, or 25 mM glucose for 6 hours. Then lipid
glucopyranose (77, 79). SREBP1 acdroplets were stained red with Oil Red O (red dots). Nuclei were counterstained blue with
tivity is necessary not only for full
hematoxylin. Representative pictures from trophoblasts are shown (scale bar corresponds to 50
␮m). Culture was performed in groups of at least 4 blastocysts in each treatment and repeated in induction of de novo fatty acid syn3 independent replicates (N ⫽ 3; n ⱖ 4).
thesis but also for intracellular lipid
droplet accumulation by increasing
of an oversupply of glucose in vitro, the embryo shows a cholesterol biogenesis and activation of the low-density
glucose-dependent regulation of lipogenic marker genes, including FASN (Figure 5). We interpret these findings as in- lipoprotein receptor (80 – 82), leading to increased
lipogenesis.
creased de novo lipogenesis, the first step of lipogenesis.
The present results are particularly important with reHowever, FATP4 expression was not affected in vitro bespect to possible short- and long-term consequences of in
cause the blastocysts were not supplied with lipids from the
vitro biotechnologies such as assisted reproductive techoutside. Glucose can stimulate de novo lipogenesis via sevnologies, in which embryos are cultured for various times
eral mechanisms. First, glucose can be converted into lipids
with high glucose concentrations before transfer. Experfor storage (72). Furthermore, embryos cultured with high imental evidence suggests that culture conditions influglucose concentrations show a slightly increased uptake of
ence normal development and may contribute to prenatal
palmitic acid and an enhanced uptake of arachidonic acid
(36). That high glucose and enhanced fatty acid uptake in- and/or postnatal disorders (83– 86). We show for the first
crease the accumulation of lipid droplets has also been shown time that high glucose concentrations in vitro lead to a
in renal tubal epithelium and pancreatic ␤-cells (73–76). The higher lipid accumulation and an elevated expression of
up-regulation of FASN was SREBP1 dependent (74 –76). We key lipogenic target genes, indicating that the glucose confound a glucose-dependent accumulation of lipid droplets, a centration is critical for metabolic set points of embryonic
higher nuclear SREBP1 amount in vitro and an increased cells. Embryonic cells adapt to changes in glucose concentration in the surrounding milieu and may retain the in
formation during later differentiation, as has been shown
in embryonic stem cells (87).
However, we cannot rule out the possibility that factors
other than hyperglycemia or hyperlipidemia may also influ
ence intracellular lipid accumulation and lipogenic gene expression. Insulin and IGFs, which are known to be regulated
by diabetic developmental conditions (39, 47), are potent
regulators of FASN expression and enzyme activity (88 –90),
Figure
5.
Schematic
mechanism
of
intracellular
lipid
accumulation
in
FABP4 expression (91, 92), and SREBP1 transcription and
rabbit blastocysts in diabetic conditions in vivo and hyperglycemia in
maturation (78, 93, 94). Therefore, the altered insulin/IGF
vitro. In the preimplantation embryo, hyperglycemia leads to a
system and/or a potential interplay between insulin/IGFs, hynonphysiological lipid accumulation with increased expression of the
adipogenic markers adipophilin, perilipin, FABP4, and SREBP1. In in
perlipidemia, and hyperglycemia has to be kept in mind,
vivo and in vitro conditions, the embryonic gene regulation differed
when the underlying mechanism of aberrant embryonic lipid
only
in
FATP4
or
FASN
with
increases
in
FATP4
in
vivo
and
FASN
in
vitro.
storage in diabetic blastocysts is discussed.
0 mM
10 mM
25 mM
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doi: 10.1210/en.2013-1760
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1507
In conclusion, maternal diabetes alters the concentration
of a variety of maternal nutrients, which in turn modifies the metabolic uterine environment for the developing
embryo. Exposure to such an altered nutrient profile can
disrupt normal development or, less dramatically, change
embryonic metabolism. We demonstrated that embryonic
lipid storage is altered under induced maternal diabetes
mellitus in the rabbit. Intracellular lipid accumulation and
expression of key genes for lipid storage (perilipin and adipophilin), fatty acid transport, and metabolism (FATP4 and
FABP4) and lipogenesis (SREBP1) are increased. This effect can be explained in part by the fact that hyperglycemia
in vitro increases the expression of lipogenic target genes
and the amount of intracellular lipid droplets. Because
preimplantation embryo development is one of the most
critical periods in an individual’s life, future health trajectories may be (mis)programmed with severe consequences
later in life.
7.
8.
9.
10.
11.
12.
13.
14.
Acknowledgments
15.
We thank Sabine Schrötter and Michaela Kirstein for excellent
technical assistance.
Address all correspondence and requests for reprints to:
Maria
Schindler, Dipl. troph., Department of Anatomy and Cell
Biology, Martin Luther University Faculty of Medicine, Grosse
Steinstrasse 52, D-06097 Halle (Saale), Germany. E-mail:
[email protected].
This work was supported by the German Research Council
(DFG) (Grant NA 418/4-2), the European Union (FP-7 Epihealth
278418), and the Wilhelm Roux Programme of the Martin Luther University Faculty of Medicine.
Disclosure Summary: The authors have nothing to disclose.
16.
17.
18.
19.
20.
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REPRODUCTION
RESEARCH
Accumulation
of advanced glycation end products in the
rabbit blastocyst under maternal diabetes
Elisa Haucke, Alexander Navarrete Santos1, Andreas Simm1, Christian Henning2,
Marcus A Glomb2, Jacqueline Gürke, Maria Schindler, Bernd Fischer and Anne Navarrete Santos
Department of Anatomy and Cell Biology, Faculty of Medicine, Martin-Luther-University Halle-Wittenberg,
Grosse Steinstrasse 52, 06108 Halle (Saale), Germany, 1Department of Cardiothoracic Surgery, Martin-Luther-
University Halle-Wittenberg, Ernst Grube Strasse 40, 06120 Halle (Saale), Germany and 2Institute of Chemistry,
Food
Chemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes Strasse 2, 06120 Halle (Saale), Germany
Correspondence should be addressed to E Haucke; Email: [email protected]
Abstract
Diabetes mellitus (DM) during pregnancy is one of the leading causes of perinatal morbidity and birth defects. The mechanism by which
maternal hyperglycemia, the major teratogenic factor, induces embryonic malformations remains unclear. Advanced glycation end products
(AGEs) are known to accumulate during the course of DM and contribute to the development of diabetic complications. Employing a diabetic
rabbit model, we investigated the influence of maternal hyperglycemia during the preimplantation period on AGE formation (pentosidine,
argpyrimidine, and N3-carboxymethyllysine (CML)) in the reproductive tract and the embryo itself. As a consequence of type 1 DM, the AGE
levels in blood plasma increased up to 50%, correlating closely with an AGE accumulation in the endometrium of diabetic females. Embryos
from diabetic mothers had increased protein-bound CML levels and showed enhanced fluorescent signals for AGE-specific fluorescence in
the blastocyst cavity fluid (BCF). The quantification of CML by HPLC–mass spectrometry (MS/MS) showed a higher amount of soluble CML
in the BCF of blastocysts from diabetic rabbits (0.26G0.05 mmol/l) compared with controls (0.18G0.02 mmol/l). The high amount of AGEs in
blastocysts from diabetic mothers correlates positively with an increased AGER (receptor for AGE (RAGE)) mRNA expression. Our study gives
alarming insights into the consequences of poorly controlled maternal diabetes for AGE formation in the embryo. Maternal hyperglycemia
during the preimplantation period is correlated with an increase in AGE formation in the uterine environment and the embryo itself. This may
influence the development of the embryo through increased AGE-mediated cellular stress by RAGEs.
Reproduction (2014) 148 169–178
Introduction
Approximately 7% of pregnancies are complicated due
to diabetes mellitus (DM; American Diabetes Association 2013). Increasing obesity rates are a serious risk
factor for type 2 DM and gestational DM (American
Diabetes Association 2013). DM during pregnancy is of
great concern as it is a major cause of perinatal morbidity
and mortality (Combs & Kitzmiller 1991, Greene 1999).
Although our understanding and management of DM
have improved during the last decades, diabetic
pregnancies are still reported to have numerous adverse
effects (Combs & Kitzmiller 1991, Aberg et al. 2001,
Eriksson et al. 2003, Corrigan et al. 2009). Hyperglycemia is considered as a major teratogenic factor for
congenital malformation, although other associated
factors such as ketone bodies, branched amino acids,
and triglycerides have also been shown to exert adverse
effects on the developing embryo (Eriksson et al. 2000).
However, it is not yet clear in which way maternal
hyperglycemia affects prenatal embryo development.
q 2014 Society for Reproduction and Fertility
ISSN 1470–1626 (paper) 1741–7899 (online)
There is upcoming evidence that advanced glycation end
products (AGEs) might play a critical role in diabetic
pregnancies.
AGEs are a complex group of compounds formed via
non-enzymatic reactions between reducing sugars and
N-terminal amino groups on proteins, lipids, and nucleic
acids. End-stage products of the protein glycation can be
divided into fluorescent AGEs (such as argpyrimidine),
non-fluorescent AGEs (such as N3-carboxymethyllysine
(CML)), and cross-linking compounds (such as pentosidine). Owing to the intrinsic fluorescence of some
AGEs, plasma and tissue fluorescence can be used as
markers for AGE accumulation (Goh & Cooper 2008, Bos
et al. 2011). Formation and accumulation of AGEs are
related to aging as well as to prolonged hyperglycemia
and oxidative stress resulting from DM (Sell et al. 1991,
Lee & Cerami 1992, Dyer et al. 1993, Brownlee 1995).
AGEs are identified to play a role in the development of
diabetic complications such as diabetic nephropathy,
cardiomyopathy, atherosclerotic disease, peripheral neuropathy, and ocular disease (Ahmed & Thornalley 2007,
DOI: 10.1530/REP-14-0149
Online version via www.reproduction-online.org
E Haucke and others
Nass et al. 2007). The post-translational modification of
proteins by reducing sugars alters their biological
structure and function and leads not only to a loss of
molecular function but also to a reduced degradation
of these damaged proteins. An additional proposed
mechanism of AGE-induced damage is the release of
reactive oxygen species, particularly superoxide and
hydrogen peroxide by AGEs (Carubelli et al. 1995,
Ortwerth et al. 1998). AGEs are able to activate
intracellular cascades by binding specific receptors, for
example, the receptor for AGEs (RAGEs). AGE–RAGE
interactions induce a broad spectrum of signaling
mechanisms such as p21ras, Erk1/2 MAP kinases
(MAPKs), p38 and SAPK/JNK MAPKs, Rho GTPases,
PI3K, and the JAK/STAT pathway (Bierhaus et al. 2005,
Rouhiainen et al. 2013). Downstream consequence is
the activation of nuclear factor kB (NFkB) that results in
release of pro-inflammatory mediators such as free
the
radicals and cytokines (Berbaum et al. 2008). AGER
(RAGE) has been shown to be expressed in the rabbit
blastocyst, at mRNA and protein levels (Ott et al. 2014).
Women with gestational DM have significantly higher
serum AGE levels compared with healthy controls,
whereas women with types 1 and 2 DM, in good medical
supervision, show normal AGE levels (Buongiorno et al.
1997). There is a strong relationship between mothers and
neonates regarding AGEs. In women with gestational DM,
high levels of AGEs and advanced oxidation protein
products (AOPPs) are also detectable in the umbilical
blood of their neonates. Both, diabetic mother and
neonate, showed higher AGE and AOPP levels compared
with healthy controls (Boutzios et al. 2013). Elevated AGE
levels in women with gestational DM are associated with
pregnancy complications such as birth asphyxia, congenital malformations, or stillbirth (Guosheng et al. 2009).
The harmful effects of AGEs after implantation and
placentation are likely to threaten the embryo/fetus too,
as maternal hyperglycemic blood is connected to the
blood system of the embryo. However, this study
demonstrates that the preimplantation period is also of
great importance on AGE formation, especially in mothers
with poorly controlled preexisting DM.
The preimplantation period is a critical ontogenetic
stage in embryo development and highly vulnerable for
teratogenesis. At this period, the embryo is most sensitive
to its surrounding milieu, especially to deregulations by
external stimuli (Watkins et al. 2008). In this study, we
investigated the influence of a poorly controlled
maternal type 1 DM on AGE formation in preimplantation embryos and in the reproductive tract employing a
rabbit model. As the rabbit blastocyst implants at day 6.8
post coitum (p.c.), we recovered the blastocysts before,
i.e. at day 6.0 p.c. At this time, the rabbit blastocyst is
covered by an extraembryonic mucin layer (Fischer et al.
1991, Herrler et al. 2002). However, this layer does not
interfere with glucose uptake and metabolism in vivo
and in vitro (Fischer et al. 2010, Ramin et al. 2010,
Reproduction (2014) 148 169–178
Schindler et al. 2013). We show that DM not only is
critical for maternal metabolism but also affects the AGE
accumulation and AGER mRNA levels in the developing
embryo even before implantation.
Materials and methods
Alloxan treatment and allocation of samples
All animal experiments were performed in accordance with the
principles of laboratory animal care and the experimental
protocol was approved by the Local Ethical Commission of
the ‘Landesverwaltungsamt Dessau’ (reference number:
42502-2-812).
Experimental type 1 DM was induced by alloxan (Sigma–
Aldrich) treatment as described previously (Ramin et al. 2010).
Rabbits were allowed to eat ad libitum. The blood glucose level
of females with type 1 DM increased 1 day after alloxan
administration and was kept in a range between 20 and
30 mmol/l by insulin supplementation (Huminsulin, basal
(NPI), Lilly, Gießen, Germany; three times per day). The
duration for the poorly controlled type 1 DM was w10 days
before mating and during the 6 days of pregnancy. On average,
diabetic rabbits had a 4.5-fold higher blood glucose concentration in comparison to the normoglycemic reference group
(27.6G0.5 and 6.2G0.1 mmol/l, P!0.001; Fig. 1).
Follicle growth was stimulated by s.c. injection of 110 IU
pregnant mare’s serum gonadotropin (Intervet, Unterschleißheim, Germany) and ovulation was ensured by i.v.
injection of 75 IU human chorionic gonadotropin (Intervet)
after mating with fertile males. Samples from diabetic and
normoglycemic rabbits were obtained 6 days after mating (p.c.).
Rabbits were killed by an overdose of pentobarbital (Sigma–
Aldrich) and exsanguination. Later, we obtained maternal blood,
maternal tissues, and the blastocysts. The blastocysts were
flushed out of the uteri and washed three times with PBS to avoid
contamination of blastocyst samples with uterine tissue.
Protein preparation of blastocysts and AGE detection by
slot blot analyses
For protein extraction, a group of eight to ten blastocysts from at
least three mothers were randomly pooled, dissolved in RIPA
30
35
30
***
25
Diabetic
25
20
20
mmol/l
170
mmol/l
15
15
10
10
5
5
0
0
0
2
4
6
8
10 12 14 16 18
Control
Diabetic
Days
Figure 1 (A) Blood glucose levels after alloxan injection (day 0) in
rabbits. The blood glucose level was monitored using commercially
available blood glucose test strips three times a day. Data are shown as
an average of nine animals with three measured data each per day
(meanGS.E.M., ***P!0.001).
www.reproduction-online.org
AGE accumulation in diabetic blastocysts
lysis
buffer (PBS, 1% vol/vol NP-40, 0.5% wt/vol sodium
deoxycholate, and 0.1% wt/vol SDS), and homogenized with
syringe (Omnifix 40 Duo, Braun, Melsungen, Germany).
aAfter
incubation on ice for 30 min, the samples were
centrifuged at 13 000 g for 20 min. The supernatant was stored
at K80 8C until use for slot blot analysis.
Slot blot analyses were performed with 25 mg protein.
Denatured protein samples (heated for 10 min at 80 8C) were
spotted onto a nylon membrane (GE Healthcare, München,
Germany) using a slot blot apparatus (Biostep, Rabenau,
Germany). The protein load was determined by Ponceau S
staining. After blocking with 5% milk/TBS–T for 1 h, the
membrane was incubated with monoclonal mouse antibodies
against CML, argpyrimidine, or pentosidine (1:100, Biologo,
Kassel, Germany), respectively, overnight at 4 8C. Samples were
rinsed three times with TBS–T for 5 min and subsequently
incubated with a secondary goat anti-mouse IgG for 1 h (Dianova,
Hamburg, Germany). The immunoreactive signal was visualized
by ECL detection (Millipore, Schwalbach, Germany) and
quantified by Fusion FX7 and the corresponding software Fusion
15.18. Protein modification rate was calculated as the ratio of
protein load (Ponceau) and slot intensity by antibody reaction.
Protein preparation of maternal tissues and AGE
171
incubating the slides with 3% H2O2 in methanol. After
blocking in 10% goat serum for at least 1 h, the sections were
incubated with the mouse MABs against CML, argpyrimidine,
or pentosidine (1:100, Biologo) at 4 8C overnight. Samples
were washed with TBS–T and incubated with the HRPconjugated secondary goat anti-mouse IgG (Dako, Hamburg,
Germany). The AGE-modified proteins were visualized by the
peroxidase–diaminobenzidine reaction. The nuclei were
counterstained with hematoxylin. Analysis was performed as
described for blastocysts.
Quantification of AGER mRNA in the rabbit blastocyst
mRNA of single blastocysts was extracted using Dynabeads Oligo
(dT) 25 (Invitrogen) and subsequently used for cDNA synthesis.
The nucleotide sequence for rabbit AGER was determined using
human primers for amplification of rabbit lung cDNA. The
obtained rabbit AGER primers are as follows: sense, GCTACTGCTCCACCTTCTGG and antisense, GCAGTCAGAGCTGATGGTGA
(ref. LOC100343142). The amount of AGER transcripts was
determined by real-time quantitative PCR (RT-qPCR) using the
Applied Biosystems StepOnePlus System (Applied Biosystems).
The entire protocol for mRNA quantification and RT-qPCR has
been described previously (Schindler et al. 2013).
detection by slot blot analyses
After flushing out the blastocysts, we dissected the uterine tissue.
Protein isolation was carried out either with the entire uteri or
with separated endometria. For the latter, the endometrium was
scraped mechanically from the myometrium using sharp
scalpels. Grinded uteri and scraped endometria were mixed
with RIPA and homogenized with precellys (Peqlab, Erlangen,
Germany). Slot blot analyses were performed in the same way as
for blastocysts. AGE accumulation in blood was determined
using EDTA–plasma. Slot blot analysis with uterine proteins was
performed in the same way as for blastocysts.
Immunohistochemical localization of AGEs
in blastocysts
The immunohistochemical analysis was performed with single
blastocysts as described previously (Schindler et al. 2014).
Embryonic discs were incubated with mouse MABs against
CML, argpyrimidine, and pentosidine (1:100, Biologo). The
nuclei were counterstained with hematoxylin. Embryonic
discs were assessed using a light microscope (BZ 8100E,
Keyence, Neu-Isenburg, Germany). The specificity of immuno staining of the secondary antibody reaction was proven by
the absence of signals in sections processed after omission of
the primary antibody.
Immunohistochemical localization of AGEs in ovary
and uterus
Ovarian and uterine tissues were fixed in Bouin’s solution,
embedded in paraffin, and sectioned at 5 mm. Sections were
mounted on silanized slides and deparaffinized at 60 8C
overnight. Later, sections were rehydrated through a series of
graded alcohols. Endogenous peroxidases were inhibited by
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AGE fluorescence in the blastocyst cavity fluid
To obtain the blastocyst cavity fluid (BCF), single blastocysts
were washed twice with ice-cold PBS and placed on a Petri dish.
The remaining PBS was removed and the blastocyst was
punctured using a syringe. The escaping BCF was taken in an
Eppendorf tube and stored at K80 8C. The AGE fluorescence
was determined in BCF using a Synergy MX 200 microplate
reader (BioTek, Bad Friedrichshall, Germany). Then, 3 ml of the
undiluted BCF were analyzed in a black Take 3 Micro-Volume
Plate (BioTek). PBS was used as a control. Fluorescence emission
spectra were recorded at excitation wavelengths of 330 and
360 nm. The maximum emission for the tested excitation
wavelengths were found at 405 and 440 nm respectively.
CML quantification in the BCF by HPLC/mass
spectrometry
CML was synthesized according to the literature (Glomb &
Monnier 1995). The identity of the reference compound was
verified by nuclear magnetic resonance experiments. Furthermore, the elemental composition was confirmed by accurate
mass determination. Up to seven BCF of at least two animals were
pooled. The pooled BCF was diluted 1:10 with ultrapure water.
The HPLC apparatus (Jasco, Gross-Umstadt, Germany) consisted
of a pump (PU-2080 Plus) with a degasser (LG-2080-02) and a
quaternary gradient mixer (LG-2080-04), a column oven (Jasco
Jetstream II), and an Autosampler (AS-2057 Plus). Chromatographic separations were performed on a stainless steel column
packed with RP-18 material (VYDAC CRT, no. 218TP54, 250!
4.6 mm, RP 18, 5 mm, Hesperia, CA, USA) using a flow rate of
1.0 ml/min. The mobile phase used was water (solvent A) and
methanol/water (7:3 (v/v), solvent B). To both solvents (A and B),
1.2 ml/l heptafluorobutyric acid was added. Analysis was
Reproduction (2014) 148 169–178
E Haucke and others
performed at 35 8C column temperature using isocratic elution at
98% of A/2% of solvent B. Mass spectrometric detection was
on a API 4000 Q Trap LC/mass spectrometry (MS/MS)
conducted
system (AB Sciex, Darmstadt, Germany) equipped with a turbo
ionspray source using electrospray ionization in the positive
mode: sprayer capillary voltage, 2.5 kV; nebulizing gas flow,
50 ml/min; heating gas, 60 ml/min at 550 8C; and curtain gas,
40 ml/min. The multiple reaction monitoring mode was used,
utilizing collision-induced dissociation of the protonated
molecule with compound-specific orifice potential (50 V) and
fragment-specific collision energies (CEs). CML (m/z 205.1/
130.2 (CE 17), 84.1 (CE 46), and 159.1 (CE 15)) was detected
at the retention time of tRZ6.0 min.
Quantification was performed by the standard addition
method. Briefly, increasing concentrations of an authentic
reference compound by factors of 0.5, 1, 2, and 3!the
concentration of the analyte in the sample were added to
separate aliquots of the sample after workup procedure. The
aliquots were analyzed, and a regression of response vs
concentration was used to determine the concentration of the
analyte in the sample. Calibration using this method resolves
potential matrix interferences.
All samples were analyzed in a single batch to exclude interassay variations. Intra-assay coefficient of variation values
(CVZ9%) were determined by repeated analyses of a BCF
sample (nZ5). In addition, the limit of detection (LODZ
4.8 pmol/ml) and the limit of quantification (LOQZ
14.5 pmol/ml) with all steps of the analysis included were
estimated according to the German standard method DIN
32645: 2008-11 (nZ5, confidence level PZ0.95, and kZ3).
Determination of glyoxal and methylglyoxal in plasma
and blastocysts
The quantification of glyoxal (GO) and methylglyoxal (MGO)
was achieved mainly as described by Espinosa-Mansilla et al.
Standard curves of GO and MGO (40% aqueous
(2007).
solution, Sigma–Aldrich) were obtained by preparing serial
dilution of GO and MGO (2700, 900, 300, 100, and 0 nM) in
HPLC-grade water (Millipore). The rabbit plasma proteins and
cell lysate proteins of the embryos were precipitated using
trifluoroacetic acid (1/10 of the sample volume). After 10 min of
incubation on ice, the samples were centrifuged at 13 000 g for
10 min. To 100 ml of the supernatant, HPLC-grade water was
added (final volume, 1 ml) followed by the addition of 0.125 ml
ammonium chloride (0.5 M; pH 10.0) and 2.5 ml of 5,6diamino-2,4-hydroxypyrimidine sulfate (0.75 mM). The mixture was incubated for 90 min at 60 8C with constant shaking.
Subsequently, citrate buffer (10 mM; pH 6.0) was added to a
final volume of 25 ml. For the analysis, 10 ml of the samples were
injected (ZORBAX, Eclipse XDB-C18 4.6!150 mm 5-micron
column; Agilent, Oberhaching, Germany) and then separated
by gradient elution with solvents A (3% acetonitrile) and
B (97% citrate buffer) at a flow rate of 0.8 ml/min.
Statistical analysis
To
obtain statistically funded data, we repeated the animal
experiment at least three times (nZ3). In each animal
Reproduction (2014) 148 169–178
experiment, we had nine diabetic and six healthy rabbits. We
pooled tissue samples and blastocysts from each individual
experiment from at least three animals. Levels of significance
between groups were calculated using Student’s t-test after
proving normal distribution (SigmaPlot v. 11.0). The Mann–
Whitney U rank sum test was used when a normal distribution
was not guaranteed. Data are expressed as meanGS.E.M.
The levels of statistical significance were *P!0.05,
**P!0.01, and ***P!0.001.
Results
Determination of a-dicarbonyls and AGEs in the plasma
of female rabbits with type 1 DM
As the majority of AGEs in vivo appear to be formed from
a-dicarbonyls (Brownlee 1995, Rabbani & Thornalley
2012), we measured the plasma level of GO and
MGO by HPLC. The AGE precursor MGO was not
altered, whereas GO (control 398G31 nM and
diabetic 522G59 nM) was tendentially enhanced
(Fig. 2A). To determine the AGE status of the
diabetic rabbits, we investigated various AGEs by slot
blot analysis with specific antibodies against
pentosidine, CML, and argpyrimidine respectively.
All of the analyzed protein modifications showed
a considerable increase in the plasma probes of diabetic
rabbits (Fig. 2B and C).
Determination of AGE modifications in the
reproductive tract of female rabbits with type 1 DM
The constitution of the uterus tissue is crucial for the
course of pregnancy. The endometrium, a dynamic
mucosa adjacent to the myometrium of the uterus, is
important for the implantation process. Argpyrimidine,
A
B
250
1000
800
P =0.055
600
400
200
Intensity relative
to control (100%)
172
nM
Diabetic
**
200
*
150
*
Control
100
50
0
0
Control
Diabetic
Pentosidine
CML Argpyrimidine
C
Control
Diabetic
Figure 2 (A) Concentration of glyoxal (GO) in diabetic and non-diabetic
rabbits. GO was quantified by HPLC (meanGS.E.M.; NZ3, nZ3;
PZas indicated). (B) Relative amount of protein-bound pentosidine,
N3-carboxymethyllysine (CML), and argpyrimidine in the plasma of
rabbits after being diabetic for w2 weeks compared with the nondiabetic control (set 100%). The quantification was performed by slot
blot analysis (meanGS.E.M.; NZ3, nZ9; *P!0.05 and **P!0.01).
The AGE amount is related to the protein load (Ponceau S staining).
(C) A representative slot blot is shown for pentosidine.
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AGE accumulation in diabetic blastocysts
A
Argpyrimidine
Negative control
CML
173
Pentosidine
Control
M
M
E
E
70 μm
Diabetic
M
M
E
40 μm
E
40 μm
40 μmv
E
E
E
E
M
70 μm
40 μm
M
M
20 μmv
20 μmv
B
140
Diabetic
120
100
Control
80
60
40
20
Intensity relative
to control (100%)
Intensity relative
to control (100%)
20 μmv
Uterus
140
20 μmv
Diabetic
20 μmv
40 μmv
Control
Epithelial
endometrium
M
40 μm
20 μmv
Endometrium
Diabetic
120
100
Control
80
60
40
20
0
0
Argpyrimidine CML
Pentosidine
Argpyrimidine CML
Pentosidine
Figure 3 (A) Immunohistochemical analysis of
argpyrimidine, N3-carboxymethyllysine (CML), and
pentosidine in the pregnant uterus of rabbits with
type 1 diabetes mellitus and in healthy controls.
The AGE-modified proteins were visualized by the
peroxidase–diaminobenzidine reaction (brown color).
The nucleus was counterstained with hematoxylin
(blue colour). The negative control is the control
reaction of the HRP-conjugated secondary goat antimouse IgG without a primary antibody. CML and
argpyrimidine show an exclusive cytosolic staining,
whereas pentosidine is localized to both the cytosol
and the nucleus. The epithelial endometrium (E) of
diabetic rabbits shows a stronger staining, whereas the
staining of the myometrium (M) seems to be
unchanged. (B) Relative amounts of protein-bound
argpyrimidine, CML, and pentosidine in the entire
uterus and the separated endometrium from diabetic
and healthy rabbits measured by slot blot analysis
(meanGS.E.M.; NZ3, nZ9). The amounts of AGE are
related to the protein load (Ponceau S staining).
CML, and pentosidine are present in the endometrium
and myometrium. AGEs are localized to smooth muscle
cells, not only in the myometrium but also in the
endothelium of vessels. In the endometrium, CML and
pentosidine are exclusively present in the epithelium;
argpyrimidine is also slightly present in the stroma. CML
and argpyrimidine were mainly localized to the
cytoplasm. Pentosidine showed a cytosolic staining and
stained nuclei. CML, pentosidine, and argpyrimidine
showed a strongly stained endometrial epithelium in
diabetic rabbits (Fig. 3A).
Slot blot analyses revealed no differences in protein bound CML, pentosidine, and argpyrimidine between
diabetic and normoglycemic rabbits for the entire uterus
and for the endometrium in particular (Fig. 3B).
Immunohistochemical staining of the ovary revealed
no differences between normoglycemic and diabetic
females. Figure 4 shows the AGE distribution in the ovary
from normoglycemic controls. All determined AGEs
were detectable in the ovary. CML and argpyrimidine
showed an exclusive cytosolic staining, whereas pentosidine was localized to both the cytoplasm and nuclei.
The oocyte showed positive staining for CML, pentosidine, and argpyrimidine in the cytoplasm, but not in the
nucleus in the investigated follicle stages (primary,
secondary, and tertiary follicle).
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Determination of AGE modifications in 6-day-old rabbit
blastocysts developed under diabetic conditions
Immunohistochemical detection of pentosidine, CML,
and argpyrimidine showed strong staining in the
embryoblast (EB) and well-stained trophoblast (TB)
cells (Fig. 5A). All detected AGEs were exclusively
present in the cytoplasm. Slot blot analysis revealed a
significantly higher level of protein-bound CML in
blastocysts from diabetic mothers (Fig. 5B). Similarly,
argpyrimidine level was tendentially increased. The
detection of the reactive a-dicarbonyls MGO showed
no differences between blastocysts from diabetic and
normoglycemic rabbits (Fig. 5C). GO was under the
detection limit.
The BCF is known to be an important reservoir for
nutrients during preimplantation. We used the fluorescent properties of AGEs to determine AGE accumulation in the BCF. Specific peaks for the known AGE
fluorescence with excitation and emission at wavelengths of 330/405 and 360/440 nm, respectively, were
detectable. Both were significantly increased in the BCF
of blastocysts developed under diabetic conditions
(Fig. 6A). The protein content of the BCF was equal in
both groups with 0.43G0.012 mg/ml. Besides fluorescent
AGEs, the non-fluorescent CML as a free adduct was
identified by HPLC/MS in the BCF. The quantification of
Reproduction (2014) 148 169–178
174
E Haucke and others
Negative control
Primary follicle
Secondary follicle
Tertiary follicle
A
200 μm
20 μm
30 μm
50 μm
B
*
C
*
200 μm
20 μm
30 μm
50 μm
200 μm
20 μm
30 μm
70 μm
Figure 4 Immunohistochemical analysis of
N3-carboxymethyllysine (A), pentosidine (B), and
argpyrimidine (C) in the ovary of healthy rabbits.
Follicle growth was stimulated by s.c. injection of
pregnant mare’s serum gonadotropin. The AGEmodified proteins were visualized by the peroxidase–
diaminobenzidine reaction (brown color). The nuclei
were counterstained with hematoxylin (blue color).
The negative control is the control reaction of the
HRP-conjugated secondary goat anti-mouse IgG
without a primary antibody. CML and argpyrimidine
show an exclusive cytosolic staining, whereas pentosidine was localized to both the cytoplasm and the
nuclei, except for nuclei of the oocytes*.
CML showed a higher amount of soluble CML in the BCF
of blastocysts from diabetic mothers with 0.26G
0.05 mmol/l compared with controls with 0.18G
0.02 mmol/l (Fig. 6B).
AGER mRNA amount in rabbit blastocysts
AGER mRNA was detectable from the early blastocyst
stage (day 4 p.c.) onwards (Ott et al. 2014). At day 6 p.c.,
the amount of AGER mRNA was significantly increased
under diabetic conditions (Fig. 7).
Discussion
An intrauterine exposure to hyperglycemia has been
shown to cause alterations in pre- and postnatal growth
patterns. The underlying metabolic disorder may predis-
pose offspring to develop metabolic diseases in later life
(Silveira et al. 2007). Several animal studies demonstrated a developmental delay for embryos recovered
from diabetic mothers (Giavini et al. 1986, Moley et al.
1991, Ramin et al. 2010). Our study provides new
insights into the effects of a maternal DM during the
preimplantation period by analyzing the AGE formation
in both, the mother and the developing embryo. Thus far,
AGE formation is mostly associated with aging and
diseases. By contrast, Ling et al. (2001) had already
reported AGE modifications in fetal rats from day 10 p.c.
onwards. A recently published study has revealed a high
rate of glycated and oxidized proteins in undifferentiated
mouse embryonic stem cells (ESCs) and in mouse
blastocysts at day 3.5 p.c. HSPA8 (HSC70) was identified
as the major protein modified by CML in undifferentiated
mouse ESCs (Hernebring et al. 2006). Similar to the
Reproduction (2014) 148 169–178
findings of our study on day 6 rabbit blastocysts,
AGE-modified proteins were mainly present in the EB,
and strongly stained cells were also found in the TB
(Fig. 5A). The protein modifications by pentosidine,
CML, and argpyrimidine were almost exclusively
observed in the cytoplasm.
It is known that AGEs accumulate intracellularly
(Goldin et al. 2006). Besides the physiological appearance of AGEs in preimplantation embryos, we observed
pathological AGE accumulations in blastocysts from
diabetic mothers. Blastocysts are obviously susceptible
for AGE formation while growing up in a diabetic uterine
milieu. A 6-day-old rabbit blastocyst developed in a
diabetic uterine milieu showed tendentially more AGEs
intracellularly and in the BCF compared with age- and
stage-matched control blastocysts (Figs 5 and 6). Proteinbound CML and argpyrimidine levels were elevated,
whereas protein-bound pentosidine was unchanged.
CML and argpyrimidine are AGEs resulting from
reactions of a-dicarbonyls (GO and MGO) and amino
groups that are known to be highly reactive. Although
glucose and fructose are present in significant concentrations in uterine secretions, the fact that they react
slowly with proteins to form AGEs must be taken into
consideration. Pentosidine is sourced mostly from ribose
but also from glucose (Dyer et al. 1991, Grandhee &
Monnier 1991). Previous investigations had demonstrated an unchanged glucose uptake in rabbit blastocysts developed under normoglycemic and diabetic
conditions (Schindler et al. 2013). These findings might
explain the unchanged protein-bound pentosidine
concentration. The intracellular glucose concentration
has not been determined so far. We found measurable,
but unaltered, concentrations of MGO in blastocysts
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Negative control
Pentosidine
TB
TB
EB
EB
200 μm
200 μm
CML
Argpyrimidine
TB
TB
EB
EB
200 μm
200 μm
*
Control
Diabetic
1.5
(100%)
2.0
B
Intensity relative to control
1.0
0.5
0.0
Argpyrimidine
CML
Pentosidine
C
1200
Methylglyoxal (nM)
1000
800
600
400
200
175
from diabetic and normoglycemic rabbits (Fig. 5C). The
quantification of GO in blastocysts failed at the detection
limit of 300 nM. Recent estimates of the cellular
concentrations are 1–5 mM for MGO and 0.1–1 mM for
GO (Dobler et al. 2006). Formation of MGO arises from
a decreased activity of the reductive pentose phosphate
pathway and formation of GO arises from lipid
peroxidation (Fu et al. 1996, Januszewski et al. 2003,
Thornalley & Rabbani 2009). We have new evidence
that the lipid metabolism is altered in the rabbit
blastocyst in case of a maternal DM. Besides noticeable
lipid accumulations, the transport protein for fatty acids
and other lipophilic substances, fatty acid-binding
protein, is upregulated (Schindler et al. 2014). The two
pathways – altered fatty acid metabolism and AGE
formation – may interact and contribute to specific
aspects of maternal subfertility in DM. In the rabbit
model, maternal DM is associated with a reduced
ovulation rate and lower number of blastocysts.
Blastocyst development is retarded (Ramin et al. 2010).
A recently published study has demonstrated that in ART
patients an increased AGE concentration in human
follicular fluid is associated with diminished follicle
growth, a lower fertilization rate, and delayed embryo
development (Jinno et al. 2011).
A major reason for AGE-mediated damage is the
activation of RAGEs (Bierhaus et al. 2005, Nass et al.
2007, Berbaum et al. 2008, Rouhiainen et al. 2013).
RAGE–ligand interaction leads to an increase in the
expression of AGER. This type of positive feedback loop
results in prolonged NFkB activation (Bierhaus &
Nawroth 2009, Fritz 2011). In our study, we found a
similar positive correlation between AGE accumulation
and an increased AGER mRNA expression in blastocysts
from diabetic mothers, confirming the view that
AGE–RAGE interaction leads to an upregulation of
RAGEs. This finding could be a proof for an active
AGE–RAGE system in the blastocyst, which may
negatively affect the embryo quality in diabetic mothers.
From which source the observed AGEs in the early
embryo originate is uncertain. As AGE formation,
especially due to glucose reactions, is an exceedingly
0
Control
Diabetic
Figure 5 (A) Immunohistochemical analysis of argpyrimidine,
N3-carboxymethyllysine (CML), and pentosidine in embryoblast (EB) and
trophoblast (TB) cells of 6-day-old rabbit blastocysts. The AGE-modified
proteins were visualized by the peroxidase–diaminobenzidine reaction
(brown color). The used antibodies show an exclusive cytosolic staining.
The EB has tendentially more AGE modifications than the TB. The
nucleus was counterstained with hematoxylin (blue colour). The negative
is the control reaction of the HRP-conjugated secondary goat
control
anti-mouse IgG without a primary antibody. (B) Relative amounts of
protein-bound argpyrimidine, CML, and pentosidine in 6-day-old rabbit
from diabetic and healthy (control) rabbits analyzed by slot
blastocysts
blot analysis (meanGS.E.M.; NZ6, nZ8–10; *P!0.05). The amount of
AGE is related to the protein load (Ponceau S staining). (C) Methylglyoxal
quantification in 6-day-old rabbit blastocyst by HPLC (meanGS.E.M.;
NZ3, nZ8–10).
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A
2.4
2.0
1.6
B
Control
Diabetic
0.25
*
1.2
0.8
0.20
0.15
0.10
0.4
0.0
0.35
0.30
***
μmol/l
A
Relative FU
AGE accumulation in diabetic blastocysts
0.05
9
19
330/405
9
19
360/440
0.00
Control
Diabetic
Wavelength (nm)
Figure 6 (A) Relative AGE fluorescence in the blastocyst cavity fluid
(BCF) of blastocysts from diabetic and control rabbits (meanGS.E.M.,
NZ3, nZas indicated; *P!0.05 and ***P!0.001). (B) The free AGE
adduct N3-carboxymethyllysine (CML) in the BCF was quantified by
HPLC/mass spectrometry (NZ4, nZ15–18).
Reproduction (2014) 148 169–178
176
E Haucke and others
RAGE/1000 GAPDH molecules
1000
**
800
600
400
200
10
15
Control
Diabetic
0
Figure 7 RAGE mRNA amount in 6-day-old blastocysts from diabetic
and control rabbits. RAGE mRNA was related to the amount of GAPDH
mRNA molecules per blastocyst (meanGS.E.M.; NZ6, nZas indicated,
**P!0.01).
slow process and the degradation of AGEs in vivo is
negligible, it can be assumed that AGEs may descent
from germ cells. Matsumine et al. (2008) have demonstrated increased levels of pentosidine in the primordial,
primary, and atretic follicles in premenopausal women.
The reduced fertility and reduction in the follicle quality
during aging are supposed to be, besides other reasons,
due to AGE accumulation (Tatone & Amicarelli 2013).
Our rabbit model confirms the presence of AGEs in
oocytes (Fig. 4). As the experimentally induced type 1
DM had no effect on the AGE concentration in oocytes,
it is unlikely that the accumulated AGEs in diabetic
blastocysts are of oocyte origin in our model. However, it
is likely that AGEs found in the oocyte might be still
present in the developing embryo. This view may be
relevant to women of older age, as AGE accumulation
takes place for a longer term in this case, and to women
with a long-term poorly controlled DM.
The increased CML concentration in the diabetic
blastocyst (Fig. 5B) was additionally reflected by
increased CML concentrations in the BCF (Fig. 6B).
Furthermore, diabetic mothers showed increased
amounts of CML in the plasma (Fig. 2B) and, through
immunohistochemical detection, in the endometrium
(Fig. 3A). The disparity between the results of immunohistochemistry and slot blot analysis in uterine tissue
(Fig. 3) may be caused by the broad spectrum of AGE
modification in tissues. The used antibodies are able to
detect, besides protein-bound modifications, free AGEs.
It is known that the non-enzymatic reaction between
reducing sugars and amino groups also affects lipids and
nucleic acids. The immunohistochemical method might
capture more AGE modifications than slot blot analysis
that detects only protein-bound AGEs. As CML level is
increases in both, mother and blastocyst, it is possible that
the maternally formed CML are transferred to the embryo.
Little is known about the transport mechanism of AGEs.
Experiments with Caco-2 cells showed a low transepithelial flux of CML. However, there was no measurable
Reproduction (2014) 148 169–178
active transport for CML across the epithelial monolayer,
neither via PEPT1 (SLC15A1) nor by carriers for neutral
amino acids. The observation led to the conclusion that
the transport is based on simple diffusion (Grunwald et al.
2006). Further studies are necessary to clarify this
hypothesis.
For the first time, we demonstrate that one consequence of maternal DM is AGE formation in preimplantation embryos. AGEs do accumulate in blastocysts if the
maternal DM is poorly controlled. Although our results
do not provide a causative mechanism between embryo
toxicity and DM, it is likely that AGEs play a role as
stimuli for activating intracellular stress pathways and,
additionally, do affect the molecular function of
intracellular proteins. It is known that even moderate
changes in the preimplantation environment can
adversely affect the pre- and postnatal phenotypes
(Fleming et al. 2004, Sinclair & Singh 2007). A clear
consequence of these findings is the necessity for a strict
control of maternal blood glucose levels during
pregnancy from the day of conception onwards.
Declaration of interest
The authors declare that there is no conflict of interest that
could be perceived as prejudicing the impartiality of the
research reported.
Funding
This work was supported by the EU (FP-7 Epihealth Nr 278418)
and the Wilhelm Roux Programme of the MLU Faculty of
Medicine.
Acknowledgements
The authors thank Michaela Kirstein and Sabine Schrötter for
their excellent technical assistance.
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Received 17 March 2014
First decision 3 April 2014
Revised manuscript received 15 April 2014
Accepted 12 May 2014
www.reproduction-online.org
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