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itamin E Is Essential for Mouse Placentation but Not for Embryonic

Sunday, April 19, 2009

BIOLOGY OF REPRODUCTION 73, 983–987 (2005)
Published online before print 13 July 2005.
DOI 10.1095/biolreprod.105.043018
Vitamin E Is Essential for Mouse Placentation but Not for Embryonic
Development Itself

Kou-ichi Jishage,
2 Takanori Tachibe,
2 Tsuneo Ito,
2 Norihito Shibata,
3 Shigeo Suzuki,
2 Toshio Mori,
2
Toshio Hani,
2 Hiroyuki Arai,
3 Hiroshi Suzuki
1,4,5
Pharmacology and Pathology Research Center,
2 Chugai Research Institute for Medical Science, Inc., Gotemba,
Shizuoka, 412-8513, Japan
Department of Health Chemistry, Graduate School of Pharmaceutical Sciences,
3 and Department of Developmental
and Medical Technology, Graduate School of Medicine,
4 University of Tokyo, 113-0033 Tokyo, Japan
National Research Centre for Protozoan Diseases,
5 Obihiro University of Agriculture and Veterinary Medicine, Obihiro,
Hokkaido, 080-8555, Japan
ABSTRACT
Vitamin E (alpha-tocopherol) was discovered 80 years ago to
be an indispensable nutrient for reproduction in the female.
However, it has not been clarified when or where vitamin E is
required during pregnancy. We examined the role of alpha-to-
copherol in pregnancy using alpha-tocopherol transfer protein
(Ttpa)-deficient mice fed specific alpha-tocopherol diets that led
to daily, measurable change in plasma alpha-tocopherol levels
from nearly normal to almost undetectable levels. A dietary sup-
plement of alpha-tocopherol to pregnant Ttpa2/2 (homozygous
null) mice was shown to be essential for maintenance of preg-
nancy from 6.5 to 13.5 days postcoitum but found not to be
crucial before or after this time span, which corresponds to ini-
tial development and maturation of the placenta. In addition,
exposure to a low alpha-tocopherol environment after initiation
of placental formation might result in necrosis of placental syn-
cytiotrophoblast cells, followed by necrosis of fetal blood vessel
endothelial cells. When Ttpa2/2-fertilized eggs were transferred
into Ttpa1/1 (wild-type) recipients, plasma alpha-tocopherol
concentrations in the Ttpa2/2 fetuses were below the detection
limit but the fetuses grew normally. These results indicate that
alpha-tocopherol is indispensable for the proliferation and/or
function of the placenta but not necessary for development of
the embryo itself.
embryo, placenta, syncytiotrophoblast, trophoblast
INTRODUCTION
Vitamin E (tocopherol) is an essential lipid component
of biological membranes that interacts with peroxyl radicals
and thereby interrupts the propagation of lipid peroxidation.
Tocopherol was first discovered as an anti-infertility factor
in 1922 [1]. It is recognized that the development of both
placenta and embryos requires alpha-tocopherol (known
throughout as a-tocopherol), and that a-tocopherol defi-
ciency affects embryo survival [2–4]. Until recently, how-
ever, it was not clear when and where vitamin E is required
during pregnancy, because it was not possible to control
1Correspondence: Hiroshi Suzuki, National Research Center for Proto-
zoan Diseases, Obihiro University of Agriculture and Veterinary Medi-
cine, Nishi 2-13, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.
FAX: 81 155 49 5643; e-mail: hisuzuki@obihiro.ac.jp
Received: 19 April 2005.
First decision: 12 May 2005.
Accepted: 13 Jul 2005.
Q 2005 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
maternal and fetal vitamin E concentrations on a day-to-
day basis, and because it was difficult to regulate vitamin
E concentrations of the embryo and the mother separately.
TTPA is a cytosolic protein that specifically binds a-to-
copherol, the most biologically active form of vitamin E,
and is depleted in the patients of familial vitamin E defi-
ciency [5]. TTPA plays a major role in maintaining ade-
quate plasma a-tocopherol levels by secreting a-tocopherol
from hepatocytes into plasma [6, 7] Previous studies dem-
onstrated that Ttpa2/2 mutant mice fed a commercial diet
(45 mg a-tocopherol/kg diet) show almost undetectable lev-
els of a-tocopherol in their plasma and that pregnant Ttpa2/
2 mutant mice had severely impaired placentas with marked
reduction of labyrinthine trophoblasts; their embryos died
at midgestation[8]. Excess dietary supplementation of a-
tocopherol (about 700 mg/kg), however, resulted in a rise
in plasma a-tocopherol levels of Ttpa2/2 females to levels
equivalent to those of Ttpa12 (heterozygote) fed a normal
diet and prevented placental failure, allowing full-term
pregnancies [8]. In the present study, we examined the role
of a-tocopherol in pregnancy using Ttpa2/2 mutant mice
fed a-tocopherol-supplemented diets.
MATERIALS AND METHODS
Animals
All mice were housed in a controlled environment of light/dark (lights-
on from 0500 h to 1900 h), temperature (248C), and humidity (50% 6
10%). Sexually mature female Ttpa2/2 mutant mice (B6; 129S7 Ttpatm1Hsz)
[8] were mated with Jcl:ICR (CLEA Japan, Tokyo, Japan) male mice in
the afternoon. The morning after mating, the females were checked for
the presence of a copulation plug in the vagina. Mothers were killed at
18.5 days postcoitum (dpc) (plug date, 0.5 dpc), to examine the sites of
implantation; fetuses were examined, and all fetuses were weighed.
a-Tocopherol Dietary Supplementation
Ttpa2/2 mutant mice were fed a commercial diet (CE-2 containing 45
mg/kg of d-a-tocopherol; CLEA Japan). After mating, mice were provided
with a-tocopherol-supplemented diet (CE-2 with 819 mg/kg supplemen-
tary d-a-tocopherol) following the protocols shown in Figure 1.
Analysis of Plasma-a-Tocopherol Concentrations in Ttpa
Mutant Mice under a-Tocopherol Supplementation
Prior to the initiation of a supplemented diet, Ttpa2/2 mutant mice were
fed commercial CE-2 chow. For the study, the animals were given a-
tocopherol supplementation. After the supplemented diet was begun, blood
samples were taken at 4, 8, 12, 16, 20, and 24 h and at 2 wk. At the end
of 2 wk, the diet was changed back to CE-2 chow. After the return to the984 JISHAGE ET AL.
FIG. 1. Protocols of a-tocopherol dietary supplementation during the
pregnancy in Ttpa2/2 mutant mice. Ttpa2/2 and Ttpa1/1 mice were fed
commercial diet CE-2 (yellow fill indicates CE-2 feeding). After mating,
Ttpa2/2 mutant mice were provided with a-tocopherol-supplemented
chow (blue fill indicates a-tocopherol supplementation) following the pro-
tocol shown in each experimental group. For example, in Group 7, Ttpa2/
2 pregnant mice were provided CE-2 chow between 0.5 and 6.5 dpc.
Between 6.5 and 13.5dpc, their diet was changed to a-tocopherol-sup-
plemented chow. Then, between 13.5 and 18.5 dpc, they were again
provided normal CE-2 chow. The Ttpa1/1 mice were fed CE-2 diet
throughout pregnancy.
FIG. 2. Plasma a-tocopherol concentration in Ttpa homozygous mutant
mice. Food was changed to a-tocopherol-supplementation diet from CE-
2. Blood samples were taken at 4, 8, 12, 16, 20, and 24 h and at 2 wk
after feeding a-tocopherol-supplementation diet. Then, food was changed
to CE-2 from a-tocopherol-supplementation diet and blood samples were
collected at 4, 8, 24, 48, and 96 h after feeding CE-2. Data are expressed
as mean 6 SD.
CE-2 diet, blood samples were taken at 4, 8, 24, 48, and 96 h. To deter-
mine the concentration of a-tocopherol, each plasma sample (25 ml) was
diluted with 975 ml of PBS and then mixed with 1 ml of 6% pyrogallol
in ethanol. Next, 0.5 mgof d-tocopherol was added as an internal standard
and mixed vigorously. After incubation at 708C for 2 min, 0.2 ml of 60%
KOH was added, and the mixture was incubated at 708C for another 30
min. For the next step, 5 ml of n-hexane and 3 ml of water were added,
and the components were mixed vigorously and centrifuged at room tem-
perature. The hexane layer was saved and the hexane extract was evapo-
rated under nitrogen. The residue was redissolved in 100 ml of ethanol
and subjected to HPLC analysis and electrochemical detection. The Gilson
UniPoint system (Gilson, WI) with a Capcell Pak C18 (5 mm, F 4.6 3
250 mm; Shiseido, Tokyo, Japan) was used with the HPLC system. 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 electrochemical
detector (ECD-300; EiCOM, Kyoto, Japan). The retention time was 8.62
min for d-tocopherol used as the internal standard and 11.24 min for a-
tocopherol.
Analysis of Plasma a-Tocopherol Concentrations
in the Mouse Fetus
Ttpa1/1 and Ttpa2/2 embryos at the 2-cell stage were transferred to the
oviduct of Jcl:ICR (Ttpa1/1) recipients on Day 0.5 dpc, as described pre-
viously [9]. The recipients were fed a diet of the CE-2 chow. Blood sam-
ples were collected from the fetuses at 18.5 dpc.
Placental Histology
For analysis of the role of a-tocopherol in the formation of the pla-
centa, Ttpa2/2 pregnant mice received a-tocopherol supplementation until
Day 11.5 dpc and were then fed the CE-2 diet until 14.5 dpc, at which
time the mice were killed. Placentas with uterine horns were fixed with
20% neutral-buffered formalin for up to 24 h, and the uterine horn seg-
ments were subsequently processed into paraffin sections and deparaffin-
ized for staining with hematoxylin/eosin.
All experiments described in the present study were conducted in ac-
cordance with the Guiding Principles for the Care and Use of Research
Animals promulgated by Chugai Pharmaceuticals, Shizuoka, Japan.
Statistical Analysis
All data are expressed as mean 6 SD. Differences between groups
were examined for statistical significance using the Student t-test. A P
value of less than 0.05 denotes the presence of a statistically significant
difference.
RESULTS
Plasma a-Tocopherol Concentration in Ttpa2/2 Mutant
Adults under a-Tocopherol Supplementation
Within 24 h after beginning a-tocopherol supplementa-
tion, plasma a-tocopherol levels rose from almost unde-
tectable levels to values of about 150 mg/dl (Fig. 2), cor-
responding to equivalent levels seen in Ttpa1/2 mice and
half the level seen in wild-type mice under a commercial
diet [8]. Upon changing from an a-tocopherol-supplement-
ed diet to a commercial diet, a decrease of around 50% in
plasma a-tocopherol concentrations could be seen within 8
hinthe Ttpa2/2 mutant adult mice.
Indispensable Period of a-Tocopherol in Maintenance
of Pregnancy
To analyze the requirement of a-tocopherol in mainte-
nance of pregnancy, Ttpa2/2 mutant mice were provided
with a-tocopherol supplements at various times during
pregnancy, as shown in Figure 1 and Table 1. a-Tocopherol
dietary supplementation in pregnant Ttpa2/2 mutant mice
from 6.5 dpc to 13.5 dpc was indispensable for continuation
of pregnancy but showed no effect when provided before
6.5 dpc or after 13.5 dpc (Group 7; Fig. 1 and Table 1). In
pregnant Ttpa2/2 mutant mice not receiving a-tocopherol985 VITAMIN E ISN’T ESSENTIAL FOR EMBRYO ITSELF
TABLE 1. Effect of dietary supplementation of a-tocopherol on embryonic development in Ttpa2/2 mice during gestation.
Experimental groupsa Wi
b 1234 5 678 9101112
No. of litters
No. of implantations
No. of live fetuses
% of live fetuses
3
19
18
95
2
13
12
92
1
4
0
0
2
16
0
0
2
20
19
95
3
26
26
100
3
33
0
0
3
19
16
84
3
24
0
0
1
10
10
100
3
30
23
77
2
11
11
100
4
27
26
96
a Experimental groups are the same as in Figure 1.
b Wi, wild-type mice.
FIG. 3. The weight of live fetuses at 18.5 dpc. The numbers of the groups
correspond to those in Figure 1. Mothers were killed at 18.5 dpc and all
fetuses weighed. Data are expressed as mean 6 SD. *P , 0.05 by Student
t-test. Wi, Wild-type mice.
supplementation either before 12.5 dpc (Group 6) or after
7.5 dpc (Group 8), the embryo could not be rescued. The
delivered pups in Group 7 showed normal body weight
(Fig. 3), growth, and fertility at adulthood. The placental
weights at 18.5 dpc showed no difference between Group
7 (121 6 14 mg, mean 6 SD) and wild-type (118 6 17
mg, mean 6 SD). After establishing that 6.5–13.5 dpc of
mouse gestation is a crucial window for maintenance of
pregnancy dependent on a-tocopherol dietary supplemen-
tation, we focused on the influence of short-term interrup-
tion of supplementation during this time period. When a-
tocopherol supplementation was withheld for 2 days, spe-
cifically on Days 9.5 and 10.5 dpc (Group 12), the body
weights of fetuses at 18.5 dpc were significantly lower than
those of fetuses of wild-type or rescued fetuses of other
experimental groups (Fig. 3).
Plasma a-Tocopherol Levels of Fetus
To clarify how a-tocopherol affects fetal viability and
growth, we transferred Ttpa2/2 fertilized eggs into Ttpa1/1
recipients and determined plasma a-tocopherol levels and
growth of Ttpa2/2 fetuses in Ttpa1/1 wild-type pregnant
mice fed on the commercial diet. Although plasma a-to-
copherol in Ttpa2/2 fetuses was below the detection limit
and therefore significantly lower than in wild-type fetuses
(Fig. 4), Ttpa2/2 fetuses in Ttpa1/1 uteri grew normally.
Furthermore, plasma a-tocopherol concentrations of Ttpa1/
1 fetuses were significantly (about 5%) lower than those of
Ttpa1/1 adults.
Influence of a Low a-Tocopherol Environment after the
Formation of Placenta
To analyze the effects of a-tocopherol deficiency after
the formation of the placenta, but before its complete mat-
uration, three Ttpa2/2 pregnant mice received a-tocopherol
supplementation from 6.5 dpc to 11.5 dpc for normal pla-
centation and then received normal diet until 14.5 dpc. His-
tological examination did not show any uterine abnormality
in all pregnant mice (Fig. 5, A, C, and G). However, ab-
normalities were evident in the labyrinthine region of the
placenta even in viable embryos. In the placentas of the
living embryos, necrosis was observed in some cells of
layers 2–3 of the labyrinthine region (syncytiotrophoblasts)
located near fetal blood (Fig. 5, D and F). In the placenta
of dead embryos, necrosis was observed in most syncytio-
trophoblasts and endothelial cells of fetal blood vessels
(Fig. 5H). The nuclei of trophoblast cells in layer 1 of the
labyrinthine region close to maternal blood vessels re-
mained unaffected; however, narrowing of the still intact
maternal blood vessels was noted. No abnormalities were
observed in chorionic trophoblast cells, spongiotrophob-
lasts, or trophoblast giant cells (Fig. 5E).986 JISHAGE ET AL.
FIG. 4. Plasma a-tocopherol levels of the fetuses in uteri of wild-type
mice. The recipient mothers (Jcl:ICR, Ttpa1/1) were maintained on a CE-
2 diet. Blood samples were collected from the fetuses at 18.5 dpc. Data
are expressed as mean 6 SD. * P , 0.05, by Student t-test. Data for adult
mice are those reported by Jishage et al.[8] for comparison. Ho, Ttpa-
homozygous mutant mice; Wi, wild-type mice.
FIG. 5. Histological examination of placentas at 14.5 dpc in mice with
low concentrations of a-tocopherol. Ttpa2/2 pregnant mice received a-
tocopherol supplementation from 6.5 to 11.5 dpc and were then fed reg-
ular CE-2 chow until 14.5 dpc. A and B) Placenta of Ttpa1/1 pregnant
mice. C–F) Placenta of living embryo in Ttpa2/2 pregnant mice. G and H)
Placenta of dead embryo in Ttpa2/2 pregnant mouse. B, D, F, and G)
Labyrinthine region of placenta. D) In some places, pyknosis of tropho-
blastic nuclei was apparent and eosinophilic necrosis was evident in the
cytoplasm. E) Spongy and basal region of placenta. F) The necrosis region
(marked with box in D) is shown at a higher magnification. H) Note the
disappearance of the trophoblastic nuclei in layers 2 and 3 of the laby-
rinthine region and the eosinophilic necrosis in the cytoplasm. The tro-
phoblasts in layer 1 of the labyrinthine region are still intact. Note also
the necrosis of fetal blood vessels and the narrowing of maternal blood
vessels. (em) Embryo, (pl) placenta, (U) uterus, (1) Layer 1 of the labyrin-
thine region, (2–3) Layers 2 and 3 of the labyrinthine region (syncytiotro-
phoblasts), (N) necrosis, (M) maternal blood vessels, (f) fetal blood vessels,
(P) pyknosis of nucleus. (S) spongiotrophoblast, (g) trophoblast giant cell,
(b) basal layer. Magnification 32(A, C, and G), 320 (B, D, E, H), 360
(F).
DISCUSSION
It was concluded that in pregnant Ttpa2/2 mutant mice,
day-to-day change of a-tocopherol levels in plasma with or
without a-tocopherol supplementation in the diet results in
a 24-h increase to normal levels with supplementation or
an 8-h decrease to low a-tocopherol levels when supple-
mentation is withheld (Fig. 2). Therefore, this dietary sup-
plementation model is an excellent tool to investigate day-
by-day necessity of an adequate supply of a-tocopherol in
mouse gestation.
A dietary supplement of a-tocopherol to pregnant
Ttpa2/2 mice was shown to be essential for maintenance of
pregnancy from 6.5 to 13.5 dpc but not crucial before or
after this time span (Table 1). Around 7.0 dpc, the concep-
tus divides into the ectoplacenta, which probably is trans-987 VITAMIN E ISN’T ESSENTIAL FOR EMBRYO ITSELF
formed into the labyrinthine region, amniotic cavity, and
exocoelomic cavity [10]. At Day 13.5 dpc, placentation is
completed, with the total number of cells per placenta
reaching a plateau on Day 14 dpc [11]. Interestingly, the
period during which a-tocopherol is required for mainte-
nance of mouse pregnancy corresponds exactly to the time
of placentation. In addition, it could be concluded that plas-
ma a-tocopherol levels in the fetus are not responsible for
fetal viability and growth (Fig. 4). On the other hand, ma-
ternal plasma a-tocopherol seems to guarantee normal fetal
viability and growth. Although it is believed that a-tocoph-
erol is essential for both placental and embryonic devel-
opment [2–4], our results suggest that a-tocopherol is nec-
essary for placentation, but not essential for the develop-
ment of the embryo itself. Then, which cell type in placenta
requires a-tocopherol? Our data seem to suggest that a de-
crease in plasma a-tocopherol after the formation of the
placenta results in necrosis of layers 2 and 3 of the syn-
cytiotrophoblast cells in the labyrinthine part of the placen-
ta, followed by necrosis of endothelial cells of fetal blood
vessels (Fig. 5). However, further functional studies would
be required to determine the involvement of a-tocopherol
in placental physiology. We concluded that a-tocopherol
seems to be important for the viability of syncytiotropho-
blast cells in the labyrinthine region of the mouse placenta.
Based on the data for fetal weight from Group 12 (Fig.
3) and plasma a-tocopherol concentration in Ttpa2/2 mutant
mice (Fig. 2), it was concluded that maternal plasma a-
tocopherol concentrations should be maintained at an esti-
mated 100 mg/dl during the a-tocopherol-dependent pla-
centation phase (6.5–13.5 dpc) to enable full-term pregnan-
cy. Fetal weight was impaired when maternal a-tocopherol
concentration in Ttpa2/2 mutant mice was # 50 mg/dl for
2 days, from 9.5 dpc through 10.5 dpc, and thus this time
period was considered a vulnerable phase. A minimum con-
centration of 100 mg/dl, for the prevention of placental fail-
ure and fetal survival is equivalent to about one fourth of
the plasma a-tocopherol levels in normal wild-type mice.
However, when natural diet is the only source used for
maintaining the concentration of a-tocopherol in Ttpa2/2
pregnant mice, even if fed legumes with high tocopherol
content these mutants could consume quantities exceeding
30% of their body weight per day. Thus, if mice do not
have the Ttpa gene and cannot efficiently use tocopherol,
reproduction will be difficult. Interestingly, Ttpa gene ex-
pression has been detected in the uterus, and its level is
reported to increase transiently after implantation of the
embryo [8]. Such patterns of expression kinetics may in-
crease the availability of a-tocopherol to syncytiotropho-
blasts.
Vitamin E is recognized as the most potent lipid-soluble
biological antioxidant. It is well known that the fetoplacen-
tal system is prone to be attacked by oxidants [12, 13] and
that synthetic antioxidants have a pronounced effect on full-
term development of embryos in tocopherol-deficient rats
and mice [4, 8]. Oxidative stress is generated when gas
exchange between mother and fetuses occurs through the
placenta, and a-tocopherol seems to protect syncytiotro-
phoblasts against this oxidative stress. However, it is diffi-
cult to understand why a-tocopherol supplementation is not
necessary after 13.5 dpc in pregnant Ttpa2/2 mutant mice,
particularly because at this stage of gestation the fetus has
grown and gas exchange increases. As shown in Figure 5,
the syncytiotrophoblast cells became necrotic but the cells
of layer 1 of the labyrinthine and other regions of the pla-
centa remained intact in the presence of a low a-tocopherol
environment. It is presumed that the syncytiotrophoblast
cell is especially susceptible to oxidative stress, and/or that
the characteristic functions (e.g., trophic hormone produc-
tion) of this cell are themselves sources of oxidative stress
generation during placental formation. Because it is known
that densely clustered lipid droplets are amassed in syncy-
tiotrophoblasts [14], it can also be speculated that syncy-
tiotrophoblasts are especially susceptible to the lipid per-
oxides induced by a vitamin E deficiency in the mother. In
addition, in the relationship between the production of ste-
roid hormone and vitamin E, it is known that tocopherol
content in the adrenal gland is high and tocopherol defi-
ciency in the adrenals reduces corticosteroid production
[15].
In conclusion, our results demonstrated that a-tocopherol
is necessary for placentation, but is not necessary for em-
bryonic development itself. Our results also suggest that
hepatic and uterine Ttpa play an important role in supplying
a-tocopherol to the placenta.
ACKNOWLEDGMENTS
We thank S. Uchida for providing excellent technical assistance, M.
Miwa, H. Tanabe, and Y. Miura for breeding the mice, O. Ueda, Y. Ka-
wase, M. Suzuki, and M, Koto for their helpful comments, and D.E.
Kaempf-Rotzoll and Ms. F. Ford for proofreading the manuscript.
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