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alpha -Tocopherol Transfer Protein Is Important for the Normal Development of Placental Labyrinthine Trophoblasts in Mice*

Kou-ichi JishageDagger , Makoto Arita§, Keiji Igarashi§, Takamitsu IwataDagger , Miho WatanabeDagger , Masako Ogawa§, Otoya UedaDagger , Nobuo KamadaDagger , Keizo Inoue§, Hiroyuki Arai§, and Hiroshi SuzukiDagger

From the Dagger  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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

alpha -Tocopherol transfer protein (alpha -TTP), a cytosolic protein that specifically binds alpha -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 alpha -TTP gene revealed that alpha -tocopherol concentration in the circulation was regulated by alpha -TTP expression levels. Male alpha -TTP-/- mice were fertile; however, placentas of pregnant alpha -TTP-/- females were severely impaired with marked reduction of labyrinthine trophoblasts, and the embryos died at mid-gestation even when fertilized eggs of alpha -TTP+/+ mice were transferred into alpha -TTP-/- recipients. The use of excess alpha -tocopherol or a synthetic antioxidant (BO-653) dietary supplement by alpha -TTP-/- females prevented placental failure and allowed full-term pregnancies. In alpha -TTP+/+ animals, alpha -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 alpha -TTP, which governs plasma alpha -tocopherol level, the uterine alpha -TTP may also play an important role in supplying this vitamin.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Vitamin E (alpha -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 alpha -tocopherol transfer protein (alpha -TTP), a cytosolic liver protein known to specifically bind to alpha -tocopherol (1), is defective in AVED patients (2), indicating that alpha -TTP is a major determinant of plasma alpha -tocopherol level. Although alpha -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 alpha -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 alpha -TTP in pregnancy and embryogenesis using our new animal model.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of alpha -TTP Knockout Mice-- An alpha -TTP targeting vector was constructed from an 8.8-kb alpha -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 alpha -TTP+/- mutants, chimeras were mated with C57BL/6J females. alpha -TTP-/- mutant mice were produced from alpha -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 alpha -TTP mutant allele. PCR primer pairs (ot198 and TTPN17, 5'-TCTCTGCAATGCCCGCCGTGCTGTCCCG-3') were used to detect the alpha -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 alpha -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 alpha -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 alpha -TTP Expression by Northern Blotting-- Total RNA was extracted from the liver of each adult mouse genotype and from uterus, placentas, and embryos of alpha -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 alpha -Tocopherol Concentrations-- Mice were fed a normal (36 mg of alpha -tocopherol/kg diet) or alpha -tocopherol-supplemented diet (600 mg of alpha -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-alpha tocopherol. D-alpha -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 alpha -tocopherol as described previously (6).

Embryo Transfer-- alpha -TTP+/+ and alpha -TTP-/- embryos at the 2-cell stage were transferred to the oviduct of alpha -TTP-/- or alpha -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 alpha -TTP Mutant Mice-- To determine the time of death, alpha -TTP+/+ and alpha -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.

alpha -Tocopherol and Synthetic Antioxidant Dietary Supplementation-- Mice were fed a commercial diet (CE-2, CLEA Japan, containing 45 mg/kg of D-alpha -tocopherol) after weaning. The alpha -TTP+/+ and alpha -TTP-/- mutant females were mated with C57BL/6J males. At 0.5 dpc after mating, mice were fed alpha -tocopherol supplementation (CE-2 with supplementary D-alpha -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

alpha -TTP Mutant Mice-- To delete the initiation codon for alpha -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 alpha -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 alpha -TTP-/- mutants as expected for a recessive mutation (alpha -TTP+/+:alpha -TTP+/-:alpha -TTP-/- = 63:105:74; Fig. 1C). Both alpha -TTP+/- and alpha -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 alpha -TTP null mice. A, mouse alpha -TTP locus, the targeting vector, and the predicted structure of the alpha -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 alpha -TTP mutant and wild-type alleles, respectively. B, Southern blot analysis of EcoRI-digested genomic DNA. Probe A including exon 1 or a mouse alpha -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 alpha -TTP was analyzed by Northern blot using total RNA from the liver. alpha -TTP-/- and alpha -TTP+/- mice had undetectable or half-levels of alpha -TTP mRNA in the liver compared with alpha -TTP+/+ mice, respectively. The blot was reprobed for cholesterol 7alpha -hydroxylase, which was used as loading control.

Plasma alpha -Tocopherol Concentrations in alpha -TTP Mutant Mice-- When mice were fed a normal diet (36 mg of alpha -tocopherol/kg diet), plasma alpha -tocopherol concentrations were about 400 µg/dl in alpha -TTP+/+ mice, half of this level in alpha -TTP+/- mice, and undetectable in alpha -TTP-/- mice (Fig. 2). These results indicate that alpha -TTP activity in the liver is a determinant of plasma alpha -tocopherol levels.



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Fig. 2.   Plasma alpha -tocopherol levels. Mice aged 4 and 11 weeks were maintained on normal and alpha -tocopherol supplemented diets. After overnight fasting, blood samples were collected from alpha -TTP+/+, alpha -TTP+/-, and alpha -TTP-/- mice for determination of alpha -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 alpha -TTP-/- Mice-- As shown in Table I, alpha -TTP-/- males were fertile. The alpha -TTP-/- females became pregnant after mating, but none of the four or five tested delivered offspring (Table I). Because alpha -TTP-/- mutants were obtained from mating alpha -TTP+/- males and females in a Mendelian fashion, the alpha -TTP-/- zygotes could develop to full-term. On the other hand, although fertilized eggs from alpha -TTP+/+ mice could be successfully implanted into alpha -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 alpha -TTP-/- mice markedly decreased between 11.5 and 14.5 dpc (Fig. 3).


                              
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Table I
Reproductive rates of alpha -TTP mutant mice


                              
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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 alpha -TTP mutant mice. Over 70% of the embryos died on 11.5 dpc. The proportion of live embryos in the uteri of alpha -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 alpha -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 alpha -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 alpha -TTP+/+ mice. D-F, embryos and placentas in uteri of alpha -TTP-/- uterus. A, B, D, and E, embryos at 10.5 dpc (× 25). Live embryos at 10.5 dpc in alpha -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 alpha -TTP-/- uterus was noted in less than 50% of those in alpha -TTP+/+.

Expression of the alpha -TTP Gene in the Uterus-- In alpha -TTP+/+ mice, expression of the alpha -TTP gene was observed in the uterus throughout pregnancy (Fig. 5), and the expression level of the alpha -TTP gene increased transiently after implantation on 4.5 dpc and gradually decreased by parturition. Because alpha -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 alpha -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). alpha -TTP was not expressed in the placenta at any time during development (Fig. 5). Although expression of the alpha -TTP gene in embryos was moderate, expression of this gene does not seem to be essential for embryonic development because alpha -TTP-/- eggs developed to full-term. These results suggest that alpha -TTP acts as a uterine factor and plays an important role in placental development.



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Fig. 5.   Northern blot analysis of alpha -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 beta -actin, which was used as the loading control.

Rescue of Embryos in Uteri of alpha  -TTP-/- Mutants by Diet Containing Excess Amounts of alpha -Tocopherol or Synthetic Antioxidant-- To examine the effect of alpha -tocopherol dietary supplementation on the development of the placenta and embryos in uteri of alpha -TTP-/- mutants, the diet was supplemented with alpha -tocopherol (567 mg/kg diet) either starting at 0.5 dpc after mating or throughout the experiment. With this diet, plasma alpha -tocopherol levels in alpha -TTP-/- mice were maintained within the normal range, which were close to the levels in alpha -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 alpha -TTP-/- mutants (Table III). The delivered pups showed normal growth and behavior and were fertile at adulthood.


                              
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Table III
The effect of dietary supplementation of alpha -tocopherol for the gestation of alpha -TTP-/- mothers



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Vitamin E was identified in the 1920s as a substance required for animals to have offspring (3). In this study, we generated alpha -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 alpha -TTP-/- female mice have defective labyrinthine trophoblast formation during embryogenesis. The placental failure was effectively abrogated by alpha -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 alpha -tocopherol content in the placental membrane increased as gestation progressed.

In this context, it is interesting to note that the expression of the alpha -TTP gene in the uterus of normal mice transiently increased around 4.5 dpc, possibly leading to an increase in alpha -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-alpha -tocopherol (best ligand for alpha -TTP), crossed most efficiently (19). alpha -TTP expressed in the uterus may explain stereospecific transport of tocopherols to the placenta, and up-regulation of alpha -TTP expression may result in the increase in the transport of alpha -tocopherol to the placenta during embryogenesis. In addition to the hepatic alpha -TTP, which governs plasma alpha -tocopherol levels, the uterine alpha -TTP may also be the important factor for feto-placental development. We have established alpha -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; alpha -TTP, alpha -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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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