ACCELERATED PUBLICATION
-Tocopherol Transfer Protein Is Important for the Normal
Development of Placental Labyrinthine Trophoblasts in Mice*
Kou-ichi
Jishage
,
Makoto
Arita§,
Keiji
Igarashi§,
Takamitsu
Iwata
,
Miho
Watanabe
,
Masako
Ogawa§,
Otoya
Ueda
,
Nobuo
Kamada
,
Keizo
Inoue§,
Hiroyuki
Arai§, and
Hiroshi
Suzuki
¶
From the
Pharmaceutical Technology Laboratory, Chugai
Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka, 412-8513 Japan and the § Department of Health Chemistry, Graduate
School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan
Received for publication, September 27, 2000, and in revised form, November 10, 2000
 |
ABSTRACT |
-Tocopherol transfer protein (
-TTP), a
cytosolic protein that specifically binds
-tocopherol, is known as a
product of the causative gene in patients with ataxia that is
associated with vitamin E deficiency. Targeted disruption of the
-TTP gene revealed that
-tocopherol
concentration in the circulation was regulated by
-TTP
expression levels. Male
-TTP
/
mice were fertile;
however, placentas of pregnant
-TTP
/
females were
severely impaired with marked reduction of labyrinthine trophoblasts,
and the embryos died at mid-gestation even when fertilized eggs of
-TTP+/+ mice were transferred into
-TTP
/
recipients. The use of excess
-tocopherol
or a synthetic antioxidant (BO-653) dietary supplement by
-TTP
/
females prevented placental failure and
allowed full-term pregnancies. In
-TTP+/+ animals,
-TTP gene expression was observed in the uterus, and its
level transiently increased after implantation (4.5 days postcoitum). Our results suggest that oxidative stress in the labyrinth region of
the placenta is protected by vitamin E during development and that in
addition to the hepatic
-TTP, which governs plasma
-tocopherol level, the uterine
-TTP may also play an important role in supplying this vitamin.
 |
INTRODUCTION |
Vitamin E (
-tocopherol) is the most potent lipid-soluble
antioxidant in biological membranes, where it contributes to membrane stability. Patients with ataxia and isolated vitamin E deficiency (AVED)1 have low or
undetectable serum vitamin E concentrations and exhibit neurological
dysfunction and muscular weakness. It is now established that
-tocopherol transfer protein (
-TTP), a cytosolic liver protein
known to specifically bind to
-tocopherol (1), is defective in AVED
patients (2), indicating that
-TTP is a major determinant of plasma
-tocopherol level. Although
-tocopherol was initially identified
as an anti-sterility factor to prevent abortion (3), the mechanism of
action and the molecules responsible for its anti-sterility effect
remain unknown. One of the reasons for this is that vitamin E is
difficult to deplete from tissues and requires elaborate manipulations
to cause deficiency symptoms to occur in experimental animals. In this
study, we established a mouse model lacking
-TTP by targeted
mutagenesis. This animal model for human AVED patients is suitable for
examination of the complex pathophysiology of diseases associated with
vitamin E deficiency and/or caused by oxidative stress. Here we
examined the role of
-TTP in pregnancy and embryogenesis using our
new animal model.
 |
MATERIALS AND METHODS |
Generation of
-TTP Knockout Mice--
An
-TTP targeting
vector was constructed from an 8.8-kb
-TTP genome
fragment encompassing exon 1. We inserted a fragment of PGK-neo
cassette into the SmaI-SmaI site positioned 5'
and 3' to exon 1 and flanked a 1.8-kb fragment of HSV-tk gene
downstream of exon 2. AB2.2-Prime ES cells (Lexicon Genetics) or A3-1
ES (4) cells were transfected by electroporation with a linearized targeting vector. G418/gancyclovir-resistant clones were screened by
PCR, and then ES cells containing the disrupted allele were injected
into C57BL/6J (CLEA, Japan) blastocysts as described previously (5). To
obtain
-TTP+/
mutants, chimeras were mated with
C57BL/6J females.
-TTP
/
mutant mice were produced
from
-TTP+/
crosses. Genotypes were determined by PCR
and confirmed by Southern blot analysis of DNA from tail tissue. The
PCR primer pairs (ot198, 5'-AGCCCACACAAAAATGAAAAACGTCTCCAAG-3' and
PGK-1, 5'-GCTAAAGCGCATGCTCCAGACTGCCTTG-3') were used to detect the
-TTP mutant allele. PCR primer pairs (ot198 and TTPN17,
5'-TCTCTGCAATGCCCGCCGTGCTGTCCCG-3') were used to detect the
-TTP wild-type allele. After an initial hot start at
94 °C for 1 min, 35 cycles (94 °C for 30 s, 62 °C for 1 min, and 72 °C for 1 min and 20 s) were run using Takara EX
Taq (TaKaRa, Japan). The expected PCR products of wild-type
and mutant alleles were 990 and 950 bp, respectively. Genomic DNA from
mutant mice were analyzed by Southern blotting using probe A including
exon 1 and mouse
-TTP cDNA (open-reading frame) probe, after
digestion with EcoRI. The resultant two fragments, which had
approximately the same number of nucleotides, were mixed and used for
probe A. Mouse cDNA probe for
-TTP was prepared by RT-PCR with
mouse liver total RNA. In the next step, 15 µg of genomic DNA was
electrophoresed on a 0.7% agarose gel and transferred onto a Hybond N+
membrane (Amersham Pharmacia Biotech). The membranes were hybridized
overnight at 42 °C in a buffer containing 50% formamide, 5× SSPE,
0.5% SDS, 5× Denhardt's solution, and 250 µg/ml denatured salmon
sperm DNA with 32P-labeled probe. The membranes were washed
for 30 min in 2× SSC, 0.2% SDS, and then in 0.5× SSC, 0.2% SDS at
65 °C for 30 min. The 3.75-kb EcoRI fragment represents
the wild-type allele.
Analysis of
-TTP Expression by Northern Blotting--
Total
RNA was extracted from the liver of each adult mouse genotype and from
uterus, placentas, and embryos of
-TTP+/+ mice using
ISOGEN (Nippon Gene, Japan). 10 µg of total RNA from liver and 20 µg of total RNA from uterus, placenta, and embryo were
electrophoresed on a 1% agarose gel and transferred onto a Hybond N+
membrane. The membranes were hybridized and washed using the method
described above for Southern blotting.
Determination of Plasma
-Tocopherol Concentrations--
Mice
were fed a normal (36 mg of
-tocopherol/kg diet) or
-tocopherol-supplemented diet (600 mg of
-tocopherol/kg diet) after weaning. These diets were prepared from a vitamin E-deficient diet (Funabashi Farm, Chiba, Japan) supplemented with 5.0% (w/w) stripped corn oil (Tama Biochemical, Tokyo, Japan) and
D-
tocopherol. D-
-Tocopherol was kindly
provided by Eisai Co. Ltd. (Tokyo, Japan). Blood samples were collected
from overnight fasted animals, and plasma was separated from whole
blood by centrifugation. Plasma (50 µl) was diluted with 950 µl of
phosphate buffered saline and was used for the following procedure.
Diluted plasma was mixed with 1 ml of 6% pyrogallol in ethanol, and
2.0 µg of tocol was subsequently added as an internal standard and
mixed vigorously. After incubation at 70 °C for 2 min, 0.2 ml of
60% KOH was added, and the mixture was incubated at 70 °C for 30 min. In the next step, 5 ml of n-hexane and 2.5 ml of water
were added, and the mixture was mixed vigorously and then centrifuged
at room temperature. The hexane layer was saved and the hexane extracts
were evaporated under nitrogen. The residue was redissolved in 100 ml
of ethanol and subjected to HPLC analysis and electrochemical
detection. The HPLC system was an IRIKA P-530 (IRIKA, Kyoto) with an
IRIKA RP-18 column (4 × 250 mm). The eluent was
methanol/water/NaClO4 at a ratio of 1000:2:7 (v/v/w) and a
flow rate of 10 ml/min. Detection was performed with an IRIKA
Amperometric E-520 detector. The retention time was 6.88 min for tocol,
which was used as an internal standard, and 10.88 min for
-tocopherol as described previously (6).
Embryo Transfer--
-TTP+/+ and
-TTP
/
embryos at the 2-cell stage were transferred
to the oviduct of
-TTP
/
or
-TTP+/+
recipients on day 0.5 of pseudopregnancy, respectively, as described previously (7). The recipients were sacrificed on 18.5 days postcoitum
(dpc).
Viability of Embryos in the Uterus of
-TTP Mutant
Mice--
To determine the time of death,
-TTP+/+ and
-TTP
/
mutant females were mated with C57BL/6J males,
and then the pregnant females were sacrificed between 9.5 and 14.5 dpc.
The death of embryos was confirmed by the absence of a heartbeat.
Morphological Appearance and Histology--
Embryos and
placentas with and/or without the uterine horns were fixed with 10%
neutral-buffered formalin for up to 24 h. Embryos and uterine horn
segments were subsequently processed into paraffin sections and
deparaffinized for staining with hematoxylin/eosin before microscopic analysis.
-Tocopherol and Synthetic Antioxidant Dietary
Supplementation--
Mice were fed a commercial diet (CE-2, CLEA
Japan, containing 45 mg/kg of D-
-tocopherol) after
weaning. The
-TTP+/+ and
-TTP
/
mutant
females were mated with C57BL/6J males. At 0.5 dpc after mating, mice
were fed
-tocopherol supplementation (CE-2 with supplementary
D-
-tocopherol, 567 mg/kg), synthetic antioxidant (CE-2
with 0.65% BO-653) diet, or CE-2 as a control. Mothers were sacrificed
at 18.5 dpc to examine the site of implantation and fetuses.
All experiments described in the present study were conducted in
accordance with the Guiding Principles for the Care and Use of Research
Animals promulgated by Chugai Pharmaceutical, Shizuoka, Japan.
 |
RESULTS |
-TTP Mutant Mice--
To delete the initiation codon for
-TTP, a targeting vector was designed in which the entire exon 1 was
replaced by a neomycin-resistance cassette (Fig.
1A). This targeting vector was
introduced into ES cells by electroporation, and then the ES cells were
used to introduce vector into the mouse germline. We obtained three
independent mutant mouse lines. Two lines (clone nos. L236 and L254)
were derived from AB2.2-Prime ES cells and one line (clone no. C229) was derived from A3-1 ES cells. The chimeras of these lines were bred
with C57BL/6J to produce heterozygous mice for
-TTP. Mice from the
L236 and C229 lines were bred and used for further analysis. When
heterozygous mice were interbred, approximately one-fourth of the
offspring were
-TTP
/
mutants as expected for a
recessive mutation
(
-TTP+/+:
-TTP+/
:
-TTP
/
= 63:105:74; Fig. 1C). Both
-TTP+/
and
-TTP
/
mice were normal in appearance and growth for
at least 6 months. There were no significant differences among the
genotypes in the plasma levels of VLDL, LDL, and HDL cholesterol as
measured by HPLC (data not shown).

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Fig. 1.
Generation of -TTP
null mice. A, mouse -TTP locus, the
targeting vector, and the predicted structure of the -TTP locus
after homologous recombination. The neomycin cassette was inserted into
the SmaI-SmaI restriction site positioned 5' and
3' to exon 1. The PCR primer pairs ot198 and PGK-1 and ot198 and TTPN17
were used to detect -TTP mutant and wild-type alleles,
respectively. B, Southern blot analysis of
EcoRI-digested genomic DNA. Probe A including exon 1 or a
mouse -TTP cDNA probe did not hybridize to a 3.75-kb fragment in
homozygous mutant mouse genomes. C, genotyping of offspring
from heterozygous F1 intercrosses were analyzed by PCR. D,
expression of -TTP was analyzed by Northern blot using total RNA
from the liver. -TTP / and -TTP+/
mice had undetectable or half-levels of -TTP mRNA in the liver
compared with -TTP+/+ mice, respectively. The blot was
reprobed for cholesterol 7 -hydroxylase, which was used as loading
control.
|
|
Plasma
-Tocopherol Concentrations in
-TTP Mutant
Mice--
When mice were fed a normal diet (36 mg of
-tocopherol/kg
diet), plasma
-tocopherol concentrations were about 400 µg/dl in
-TTP+/+ mice, half of this level in
-TTP+/
mice, and undetectable in
-TTP
/
mice (Fig. 2).
These results indicate that
-TTP activity in the liver is a
determinant of plasma
-tocopherol levels.

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Fig. 2.
Plasma -tocopherol
levels. Mice aged 4 and 11 weeks were maintained on normal and
-tocopherol supplemented diets. After overnight fasting, blood
samples were collected from -TTP+/+,
-TTP+/ , and -TTP / mice for
determination of -tocopherol. Data are expressed as mean ± S.D. from six mice. Statistical analysis used Student's
t-test. *, significantly different at p < 0.05.
|
|
Infertility of Female
-TTP
/
Mice--
As shown
in Table I,
-TTP
/
males were fertile. The
-TTP
/
females became
pregnant after mating, but none of the four or five tested delivered
offspring (Table I). Because
-TTP
/
mutants were
obtained from mating
-TTP+/
males and females in a
Mendelian fashion, the
-TTP
/
zygotes could develop
to full-term. On the other hand, although fertilized eggs from
-TTP+/+ mice could be successfully implanted into
-TTP
/
recipients, they failed to develop to
full-term (Table II). The number of live
embryos (as determined by the presence of a heartbeat) of
-TTP
/
mice markedly decreased between 11.5 and 14.5 dpc (Fig. 3).
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Table II
Number of resorption sites and fetuses observed on 18.5 dpc after
transfer of embryos into the oviduct of recipient mice
|
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Fig. 3.
Viability of the embryos in the uterus
of -TTP mutant mice. Over 70% of the
embryos died on 11.5 dpc. The proportion of live embryos in the uteri
of -TTP / mice markedly decreased between 11.5 and
14.5 dpc. Data are expressed as mean number of embryos in three
pregnancies.
|
|
The placentas and embryos of various maternal genotypes were not
morphologically different at 9.5 dpc (data not shown). However, the
embryos in the uteri of
-TTP
/
mutants showed
developmental failure from 10.5 dpc, and the majority of these embryos
showed neural tube malformations (Fig.
4E). In normal pregnancy, the
labyrinth region of the placenta starts development from around 9-9.5
dpc and then functions as a nutrient transport unit (8). Under normal
embryogenesis, the allantoic vessels are seen by about 10 dpc where
they penetrate the chorionic plate, and the ectoplacental plate is
transformed into the labyrinthine part of the placenta (8). At this
stage, the placenta could be divided into several well defined layers
such as the spongiotrophoblast layer and the labyrinth region.
Histological examination showed a specific abnormality limited to the
labyrinth region of the
-TTP
/
mutant at 10.5 dpc
(Fig. 4, C and F). In these mice, there was a
marked reduction in the number of trophoblast cells, resulting in an
abnormally small labyrinth. Furthermore, embryonic blood vessels were
virtually absent in the trophoblast.

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Fig. 4.
Morphological and histological appearance of
embryos and placentas in uteri of homozygous mice.
A-C, embryos and placentas in uteri of
-TTP+/+ mice. D-F, embryos and
placentas in uteri of -TTP / uterus. A,
B, D, and E, embryos at 10.5 dpc (× 25). Live embryos at 10.5 dpc in -TTP / uteri were
generally abnormal in appearance. C and F,
placentas at 10.5 dpc (× 40). S, spongiotrophoblast region;
L, labyrinth region. The development of labyrinth region in
-TTP / uterus was noted in less than 50% of those in
-TTP+/+.
|
|
Expression of the
-TTP Gene in the Uterus--
In
-TTP+/+ mice, expression of the
-TTP gene
was observed in the uterus throughout pregnancy (Fig.
5), and the expression level of the
-TTP gene increased transiently after implantation on 4.5 dpc and gradually decreased by parturition. Because
-TTP expression
did not increase in pseudopregnant mice at 4.5 dpc (data not shown),
implantation of embryos or the development of embryos seems to have
stimulated
-TTP gene expression. After about 4.5 dpc, the
polar trophectoderm gives rise to extraembryonic ectoderm of the
chorion, which later contributes to the trophoblast component of the
labyrinth region, and the ectoplacental cone, which later produces the
spongiotrophoblast layer (9).
-TTP was not expressed in the placenta
at any time during development (Fig. 5). Although expression of the
-TTP gene in embryos was moderate, expression of this
gene does not seem to be essential for embryonic development because
-TTP
/
eggs developed to full-term. These results
suggest that
-TTP acts as a uterine factor and plays an important
role in placental development.

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Fig. 5.
Northern blot analysis of
-TTP in uteri, placentas, and embryos. Total
RNA was isolated from uteri (on 0.5, 4.5, 8.5, 10.5, 15.5, and 20 dpc),
placentas (on 8.5, 10.5, and 15.5 dpc), and embryos (on 8.5, 10.5, and
15.5 dpc) of wild-type mice. Blots were reprobed for -actin, which
was used as the loading control.
|
|
Rescue of Embryos in Uteri of
-TTP
/
Mutants by
Diet Containing Excess Amounts of
-Tocopherol or Synthetic
Antioxidant--
To examine the effect of
-tocopherol dietary
supplementation on the development of the placenta and embryos in uteri
of
-TTP
/
mutants, the diet was supplemented
with
-tocopherol (567 mg/kg diet) either starting at 0.5 dpc after
mating or throughout the experiment. With this diet, plasma
-tocopherol levels in
-TTP
/
mice were maintained
within the normal range, which were close to the levels in
-TTP+/
mice fed a normal diet (Fig. 2). This therapy,
as well as supplementation of a synthetic antioxidant, BO-653 (10), had
a pronounced effect on full-term development of embryos in the uteri of
-TTP
/
mutants (Table
III). The delivered pups showed normal
growth and behavior and were fertile at adulthood.
 |
DISCUSSION |
Vitamin E was identified in the 1920s as a substance required for
animals to have offspring (3). In this study, we generated
-TTP
/
mice with undetectable levels of plasma
vitamin E even upon feeding with normal diet. Using these mice, we
analyzed the infertility caused by vitamin E deficiency and found that
the
-TTP
/
female mice have defective labyrinthine
trophoblast formation during embryogenesis. The placental failure was
effectively abrogated by
-tocopherol or synthetic antioxidant
dietary supplement, indicating that vitamin E or other antioxidants are
essential for the formation of labyrinthine trophoblasts. It is well
known that the feto-placental system is prone to the attack of oxidants
and that placental brush border membrane is most susceptible to
peroxidation (11, 12). Oxygen-free radicals are also involved in the
induction of fetal anomalies. For example, excess oxygen radical
activity has been reported to be associated with disturbed
embryogenesis in diabetic pregnancy (13). Other studies have also shown
a reduction in the severity of these diseases with administration of
vitamin E during early pregnancy (14, 15). These findings, together with the present results, suggest that embryogenesis, especially the
formation of the placental labyrinthine trophoblasts, is more susceptible to oxidative stress. Efficient functioning of the enzymic
and nonenzymic reactive oxygen species scavengers ensures a normal
intrauterine fetal growth and development (12, 16). Mukherjea and
co-workers (17, 18) demonstrated that
-tocopherol content in the
placental membrane increased as gestation progressed.
In this context, it is interesting to note that the expression of the
-TTP gene in the uterus of normal mice transiently increased around 4.5 dpc, possibly leading to an increase in
-tocopherol levels supplied to the embryo. On the other hand, it was
also demonstrated that vitamin E crosses the placenta from the mother to the embryo, and interestingly, of the various forms of vitamin E
transferred, the RRR-
-tocopherol (best ligand for
-TTP), crossed most efficiently (19).
-TTP expressed in the uterus may explain stereospecific transport of tocopherols to the placenta, and
up-regulation of
-TTP expression may result in the increase in the
transport of
-tocopherol to the placenta during embryogenesis. In
addition to the hepatic
-TTP, which governs plasma
-tocopherol
levels, the uterine
-TTP may also be the important factor for
feto-placental development. We have established
-TTP-disrupted mice
as a model for vitamin E deficiency. This model should be a useful tool
for the study of diseases caused by oxidation stress.
 |
ACKNOWLEDGEMENTS |
We thank Y. Toyoda for
encouragement, H. Tamai and M. Kobayashi (Osaka Medical College) for
tocopherol determination, and Y. Kawase and S. Uchida for technical assistance.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.: 81 550 87-6741; Fax: 81 550 87-5387; E-mail:
suzukihirs@gt.chugai-pharm.co.jp.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.C000676200
 |
ABBREVIATIONS |
The abbreviations used are:
AVED, ataxia and
isolated vitamin E deficiency;
ES, embryonic stem cell;
-TTP,
-tocopherol transfer protein;
PCR, polymerase chain reaction;
RT-PCR, reverse transcription-PCR;
bp, base pair(s);
kb, kilobase;
SSPE, saline/sodium phosphate/EDTA;
dpc, days postcoitum;
HPLC, high
performance liquid chromatography;
LDL, low density lipoprotein;
HDL, high density lipoprotein.
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.