BOR - Papers in Press, published online ahead of print
July 13, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.043018
BIOLOGY OF REPRODUCTION 73, 983–987 (2005)
DOI: 10.1095/biolreprod.105.043018
© 2005 by the Society for the Study of Reproduction, Inc.
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, and
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
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
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ABSTRACT
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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-tocopherol 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 supplement of alpha-tocopherol to pregnant Ttpa–/– (homozygous null) mice was shown to be essential for maintenance of pregnancy from 6.5 to 13.5 days postcoitum but found not to be crucial before or after this time span, which corresponds to initial 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 syncytiotrophoblast cells, followed by necrosis of fetal blood vessel endothelial cells. When Ttpa–/–-fertilized eggs were transferred into Ttpa+/+ (wild-type) recipients, plasma alpha-tocopherol concentrations in the Ttpa–/– 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
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INTRODUCTION
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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 
-tocopherol), and that -tocopherol deficiency affects embryo survival [2–4]. Until recently, however, it was not clear when and where vitamin E is required during pregnancy, because it was not possible to control 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 -tocopherol, the most biologically active form of vitamin E, and is depleted in the patients of familial vitamin E deficiency [5]. TTPA plays a major role in maintaining adequate plasma -tocopherol levels by secreting -tocopherol from hepatocytes into plasma [6, 7] Previous studies demonstrated that Ttpa–/– mutant mice fed a commercial diet (45 mg -tocopherol/kg diet) show almost undetectable levels of -tocopherol in their plasma and that pregnant Ttpa–/ – mutant mice had severely impaired placentas with marked reduction of labyrinthine trophoblasts; their embryos died at midgestation[8]. Excess dietary supplementation of -tocopherol (about 700 mg/kg), however, resulted in a rise in plasma -tocopherol levels of Ttpa–/– females to levels equivalent to those of Ttpa+– (heterozygote) fed a normal diet and prevented placental failure, allowing full-term pregnancies [8]. In the present study, we examined the role of -tocopherol in pregnancy using Ttpa–/– mutant mice fed -tocopherol-supplemented diets.
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MATERIALS AND METHODS
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Animals
All mice were housed in a controlled environment of light/dark (lights-on from 0500 h to 1900 h), temperature (24°C), and humidity (50% ± 10%). Sexually mature female Ttpa–/– 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.
-Tocopherol Dietary Supplementation
Ttpa–/– mutant mice were fed a commercial diet (CE-2 containing 45 mg/kg of d--tocopherol; CLEA Japan). After mating, mice were provided with -tocopherol-supplemented diet (CE-2 with 819 mg/kg supplementary d--tocopherol) following the protocols shown in Figure 1.
Analysis of Plasma--Tocopherol Concentrations in Ttpa Mutant Mice under -Tocopherol Supplementation
Prior to the initiation of a supplemented diet, Ttpa–/– mutant mice were fed commercial CE-2 chow. For the study, the animals were given -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 the CE-2 diet, blood samples were taken at 4, 8, 24, 48, and 96 h. To determine the concentration of -tocopherol, each plasma sample (25 µl) was diluted with 975 µl of PBS and then mixed with 1 ml of 6% pyrogallol in ethanol. Next, 0.5 µg of 
-tocopherol was 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 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 temperature. The hexane layer was saved and the hexane extract was evaporated 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 µm, 
4.6 x 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 -tocopherol used as the internal standard and 11.24 min for -tocopherol.
Analysis of Plasma -Tocopherol Concentrations in the Mouse Fetus
Ttpa+/+ and Ttpa–/– embryos at the 2-cell stage were transferred to the oviduct of Jcl:ICR (Ttpa+/+) recipients on Day 0.5 dpc, as described previously [9]. The recipients were fed a diet of the CE-2 chow. Blood samples were collected from the fetuses at 18.5 dpc.
Placental Histology
For analysis of the role of -tocopherol in the formation of the placenta, Ttpa–/– pregnant mice received -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 segments were subsequently processed into paraffin sections and deparaffinized for staining with hematoxylin/eosin.
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 Pharmaceuticals, Shizuoka, Japan.
Statistical Analysis
All data are expressed as mean ± 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.
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RESULTS
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Plasma -Tocopherol Concentration in Ttpa–/– Mutant Adults under -Tocopherol Supplementation
Within 24 h after beginning -tocopherol supplementation, plasma -tocopherol levels rose from almost undetectable levels to values of about 150 µg/dl (Fig. 2), corresponding to equivalent levels seen in Ttpa+/– mice and half the level seen in wild-type mice under a commercial diet [8]. Upon changing from an -tocopherol-supplemented diet to a commercial diet, a decrease of around 50% in plasma -tocopherol concentrations could be seen within 8 h in the Ttpa–/– mutant adult mice.
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FIG. 2. Plasma -tocopherol concentration in Ttpa homozygous mutant mice. Food was changed to -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 -tocopherol-supplementation diet. Then, food was changed to CE-2 from -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 ± SD
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Indispensable Period of -Tocopherol in Maintenance of Pregnancy
To analyze the requirement of -tocopherol in maintenance of pregnancy, Ttpa–/– mutant mice were provided with -tocopherol supplements at various times during pregnancy, as shown in Figure 1 and Table 1. -Tocopherol dietary supplementation in pregnant Ttpa–/– 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 Ttpa–/– mutant mice not receiving -tocopherol 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 ± 14 mg, mean ± SD) and wild-type (118 ± 17 mg, mean ± SD). After establishing that 6.5–13.5 dpc of mouse gestation is a crucial window for maintenance of pregnancy dependent on -tocopherol dietary supplementation, we focused on the influence of short-term interruption of supplementation during this time period. When -tocopherol supplementation was withheld for 2 days, specifically 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).
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TABLE 1. Effect of dietary supplementation of -tocopherol on embryonic development in Ttpa–/– mice during gestation
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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 ± SD. *P < 0.05 by Student t-test. Wi, Wild-type mice
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Plasma -Tocopherol Levels of Fetus
To clarify how -tocopherol affects fetal viability and growth, we transferred Ttpa–/– fertilized eggs into Ttpa+/+ recipients and determined plasma -tocopherol levels and growth of Ttpa–/– fetuses in Ttpa+/+ wild-type pregnant mice fed on the commercial diet. Although plasma -tocopherol in Ttpa–/– fetuses was below the detection limit and therefore significantly lower than in wild-type fetuses (Fig. 4), Ttpa–/– fetuses in Ttpa+/+ uteri grew normally. Furthermore, plasma -tocopherol concentrations of Ttpa+/ + fetuses were significantly (about 5%) lower than those of Ttpa+/+ adults.
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FIG. 4. Plasma -tocopherol levels of the fetuses in uteri of wild-type mice. The recipient mothers (Jcl:ICR, Ttpa+/+) were maintained on a CE-2 diet. Blood samples were collected from the fetuses at 18.5 dpc. Data are expressed as mean ± 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
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Influence of a Low -Tocopherol Environment after the Formation of Placenta
To analyze the effects of -tocopherol deficiency after the formation of the placenta, but before its complete maturation, three Ttpa–/– pregnant mice received -tocopherol supplementation from 6.5 dpc to 11.5 dpc for normal placentation and then received normal diet until 14.5 dpc. Histological examination did not show any uterine abnormality in all pregnant mice (Fig. 5, A, C, and G). However, abnormalities 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 syncytiotrophoblasts 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 remained unaffected; however, narrowing of the still intact maternal blood vessels was noted. No abnormalities were observed in chorionic trophoblast cells, spongiotrophoblasts, or trophoblast giant cells (Fig. 5E).
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FIG. 5. Histological examination of placentas at 14.5 dpc in mice with low concentrations of -tocopherol. Ttpa–/– pregnant mice received -tocopherol supplementation from 6.5 to 11.5 dpc and were then fed regular CE-2 chow until 14.5 dpc. A and B) Placenta of Ttpa+/+ pregnant mice. C–F) Placenta of living embryo in Ttpa–/– pregnant mice. G and H) Placenta of dead embryo in Ttpa–/– pregnant mouse. B, D, F, and G) Labyrinthine region of placenta. D) In some places, pyknosis of trophoblastic 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 labyrinthine region and the eosinophilic necrosis in the cytoplasm. The trophoblasts 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 labyrinthine region, (2–3) Layers 2 and 3 of the labyrinthine region (syncytiotrophoblasts), (N) necrosis, (M) maternal blood vessels, (f) fetal blood vessels, (P) pyknosis of nucleus. (S) spongiotrophoblast, (g) trophoblast giant cell, (b) basal layer. Magnification x2 (A, C, and G), x20 (B, D, E, H), x60 (F)
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DISCUSSION
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It was concluded that in pregnant Ttpa–/– mutant mice, day-to-day change of -tocopherol levels in plasma with or without -tocopherol supplementation in the diet results in a 24-h increase to normal levels with supplementation or an 8-h decrease to low -tocopherol levels when supplementation is withheld (Fig. 2). Therefore, this dietary supplementation model is an excellent tool to investigate day-by-day necessity of an adequate supply of -tocopherol in mouse gestation.
A dietary supplement of -tocopherol to pregnant Ttpa–/– 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 conceptus divides into the ectoplacenta, which probably is transformed 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 -tocopherol is required for maintenance of mouse pregnancy corresponds exactly to the time of placentation. In addition, it could be concluded that plasma -tocopherol levels in the fetus are not responsible for fetal viability and growth (Fig. 4). On the other hand, maternal plasma -tocopherol seems to guarantee normal fetal viability and growth. Although it is believed that -tocopherol is essential for both placental and embryonic development [2–4], our results suggest that -tocopherol is necessary for placentation, but not essential for the development of the embryo itself. Then, which cell type in placenta requires -tocopherol? Our data seem to suggest that a decrease in plasma -tocopherol after the formation of the placenta results in necrosis of layers 2 and 3 of the syncytiotrophoblast cells in the labyrinthine part of the placenta, followed by necrosis of endothelial cells of fetal blood vessels (Fig. 5). However, further functional studies would be required to determine the involvement of -tocopherol in placental physiology. We concluded that -tocopherol seems to be important for the viability of syncytiotrophoblast cells in the labyrinthine region of the mouse placenta.
Based on the data for fetal weight from Group 12 (Fig. 3) and plasma -tocopherol concentration in Ttpa–/– mutant mice (Fig. 2), it was concluded that maternal plasma -tocopherol concentrations should be maintained at an estimated 100 µg/dl during the -tocopherol-dependent placentation phase (6.5–13.5 dpc) to enable full-term pregnancy. Fetal weight was impaired when maternal -tocopherol concentration in Ttpa–/– mutant mice was 
50 µg/dl for 2 days, from 9.5 dpc through 10.5 dpc, and thus this time period was considered a vulnerable phase. A minimum concentration of 100 µg/dl, for the prevention of placental failure and fetal survival is equivalent to about one fourth of the plasma -tocopherol levels in normal wild-type mice. However, when natural diet is the only source used for maintaining the concentration of -tocopherol in Ttpa–/– 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 expression 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 increase the availability of -tocopherol to syncytiotrophoblasts.
Vitamin E is recognized as the most potent lipid-soluble biological antioxidant. It is well known that the fetoplacental 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 -tocopherol seems to protect syncytiotrophoblasts against this oxidative stress. However, it is difficult to understand why -tocopherol supplementation is not necessary after 13.5 dpc in pregnant Ttpa–/– 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 placenta remained intact in the presence of a low -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 production) 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 syncytiotrophoblasts [14], it can also be speculated that syncytiotrophoblasts are especially susceptible to the lipid peroxides induced by a vitamin E deficiency in the mother. In addition, in the relationship between the production of steroid hormone and vitamin E, it is known that tocopherol content in the adrenal gland is high and tocopherol deficiency in the adrenals reduces corticosteroid production [15].
In conclusion, our results demonstrated that -tocopherol is necessary for placentation, but is not necessary for embryonic development itself. Our results also suggest that hepatic and uterine Ttpa play an important role in supplying -tocopherol to the placenta.
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ACKNOWLEDGMENTS
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We thank S. Uchida for providing excellent technical assistance, M. Miwa, H. Tanabe, and Y. Miura for breeding the mice, O. Ueda, Y. Kawase, 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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FOOTNOTES
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1 Correspondence: Hiroshi Suzuki, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan. FAX: 81 155 49 5643; hisuzuki{at}obihiro.ac.jp 
Received: 19 April 2005.
First decision: 12 May 2005.
Accepted: 13 July 2005.
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ABSTRACT
INTRODUCTION
