Biology of Reproduction 67, 599-604 (2002)
© 2002 Society for the Study of Reproduction, Inc.
-Tocopherol Transfer Protein Is Specifically Localized at the Implantation Site of Pregnant Mouse Uterus
D.E. Kaempf-Rotzolla,b,
K. Igarashia,
J. Aokia,
K. Jishagec,
H. Suzukic,
H. Tamaid,
O. Linderkampb, and
H. Arai2,,a
a Graduate School of Pharmaceutical Sciences, Department of Health Chemistry, University of Tokyo, Bunkyo-ku, 113-0033 Tokyo, Japan
b Division of Neonatology, Department of Pediatrics, University of Heidelberg, D-69120 Heidelberg, Germany
c Pharmaceutical Technology Laboratory, Chugai Pharmaceutical Co., Ltd., Gotemba, 412-8513 Shizuoka, Japan
d Department of Pediatrics, Osaka Medical College, 569-8686 Takatsuki-shi, Japan
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ABSTRACT
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-Tocopherol transfer protein (-TTP) was first described to play a major role in maintaining -tocopherol levels in plasma, while -tocopherol was primarily reported to be a factor relevant for reproduction. Expression of -TTP is not only seen in the liver, from where it was first isolated, but also in mouse uterus, depending on its state of pregnancy, stressing the importance of -TTP for embryogenesis and fetal development. The cellular localization of -TTP in mouse uterus is reported here. By immunohistochemistry, -TTP could be localized in the secretory columnar epithelial cells of the pregnant uterus on Days 4.5 and 6.5 postcoitum as well as in the glandular epithelial cells and the inner decidual reaction zone surrounding the implantation site. On Days 8.5 and 10.5 postcoitum (midterm of mouse pregnancy), -TTP could still be detected in the uterine secretory columnar epithelial cells, while in -TTP knockout mice, no immunostaining was visible. It is suggested that -TTP plays a major role in supplying the placenta and consecutively the fetus with -tocopherol throughout pregnancy. We conclude that -tocopherol plays a role in the process of implantation and that -TTP may be necessary for adequate -tocopherol status of the fetus.
female reproductive tract, implantation, pregnancy, uterus
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INTRODUCTION
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Vitamin E (-tocopherol) was first discovered and recognized as a factor essential for reproduction by Evans and Bishop 80 years ago in 1922 [1]. Meanwhile, -tocopherol is recognized as a major lipid-soluble chain-breaking antioxidant found in cellular membranes and is known as an important factor in the protection of polyunsaturated fatty acids against peroxidative damage. Peroxidative damage is well known to take place in the placenta itself, the placental brush border being most vulnerable to oxidative stress [2]. Free radicals also play a role in the induction of fetal anomalies, such as impaired embryogenesis in diabetic pregnancy [3], suggesting that -tocopherol is of great importance in its function as the main naturally occurring antioxidant. -Tocopherol transfer protein (-TTP), with its high affinity for -tocopherol, plays a major role in maintaining adequate plasma -tocopherol levels by excreting -tocopherol from the hepatocyte into plasma [4]. The regulation of -tocopherol levels in the feto-maternal unit is little studied so far and the role of tocopherol-binding proteins is of great interest to elucidate how -tocopherol is transported from the maternal circulation to the uterus and placenta before uptake in fetal tissues can occur. -TTP, which has been cloned and chromosomally localized [5], is a 30-kDa protein identified as a product of the causative gene for ataxia with isolated vitamin E deficiency (AVED) [6]. Patients with AVED have practically undetectable serum vitamin E levels and show severe neurological symptoms and muscular weakness. While -TTP was primarily described as a cytosolic liver protein [6, 7], it has meanwhile been localized in many other tissues, namely in rat brain [8] and most recently in pregnant mouse uterus [9]. -TTP could by detected by northern blot analysis throughout mouse pregnancy (20 days of gestation) with a peak around 4.5 days postcoitum (dpc). This day coincides with the period of implantation in the mouse, suggesting a possible role of -tocopherol, the major ligand of -TTP, in this process. We therefore attempted to obtain more precise information on the localization of -TTP by immunohistochemistry in mouse uterus on the day of implantation (4.5 dpc), 6.5 and 8.5 dpc, as well as around midterm at 10.5 dpc and compared these results with nonpregnant, wild-type adult female mice as well as with -TTP knockout mice.
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MATERIALS AND METHODS
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Animal Breeding and Tissue Preparation
Ten C57BL/6Cr wild-type female mice (SLC, Hamamatsu, Japan) and eight -TTP knockout female mice, of which the generation is described elsewhere [9], were included in the study. Two nonpregnant wild-type and two nonpregnant -TTP knockout mice were killed at 8 wk of age. Two mice each at the following stages of 4.5, 6.5, and 8.5 dpc gestation, wild-type vs. knockout, were included, as were two wild-type mice at 10.5 dpc. -TTP knockout mice at 10.5 dpc were not included in the study due to expected morphological changes in placenta and fetus as well as the fact that no additional information was expected from these animals. The tissues were immediately dissected free from surrounding tissues. Dissected tissues were rinsed in buffer (250 mM sucrose/1 mM EDTA/10 mM Tris-HCl, pH 7.4) and fixed overnight in 4% paraformaldehyde at 4°C followed by subsequent dehydration and paraffin embedding. Sections (5 µm) were cut on a Microm HM 400R microtome and adhered to polylysine-coated microscope slides (Matsunami, Tokyo, Japan).
Preparation of Mouse -TTP-Specific Rat Monoclonal Antibodies
The coding region of mouse -TTP cDNA was inserted into the SalI/EcoRI sites of the pET21a vector (pET system, Novagen, Madison, WI). After the plasmid was introduced into Escherichia coli strain BL21 (DE3) (Novagen), the protein was expressed as a His-tagged protein by induction with 1 mM isopropyl-ß-D-thiogalactopyranoside. The protein was purified using nickel column chromatography (Novagen) according to the manufacturer's protocol. Rats (WKY strains, female, 6-wk; SLC) were immunized by injecting the protein into the hind foot pads using Freund complex adjuvant. At 3-wk intervals after the initial injection, the rats were injected twice with the purified protein mixed with Freund complex adjuvant. Three weeks after the second booster injection, the enlarged medial iliac lymph nodes from the rats were used for cell fusion with mouse myeloma cells, line PA1. Several monoclonal antibody-producing hybridoma cell lines were established. From the various antibodies isolated, controls were not only performed by Western blotting but also by testing the immunohistochemical usefulness of these by checking mouse liver sections for -TTP localization in cytoplasm of hepatocytes. Only the clones positive in Western blotting and immunohistochemistry were selected. In this study, the monoclonal antibody from clone A8-F1 (rat IgG2a) was used for immunohistochemistry.
Immunohistochemistry
Immunohistochemistry was performed based on avidin-biotin amplification and oxidation with 3,3'-diaminobenzidine tetrahydrochloride [10]. Tissue sections adhered to polylysine-coated slides (Matsunami) were deparaffinized in xylene and rehydrated in a graded series of ethanol. The endogenous, tissue-specific peroxidase was blocked with 3% H2O2 in methanol for 20 min followed by washing in 0.05 M Tris-HCl/0.15 M NaCl, pH 7.6 (TBS). Antigen retrieval was performed using a microwave oven: samples were microwaved at 750 W and boiled five times consecutively for 5 min in 0.01 M citrate buffer, pH 6.0, followed by brief washing in tap water and TBS prior to blocking with 10% rabbit serum (Vector Laboratories, Burlingame, CA) in TBS for 30 min at room temperature. Excess fluid was allowed to drain from the sections, which were then covered with 50 µl of a 1:20 dilution of the mouse -TTP-specific rat monoclonal antibodies. Control sections were left at the blocking stage and not covered with the primary antibodies. Incubation of all slides was carried out overnight at room temperature in humidified air-tight chambers. After three 5-min washes in TBS, the sections were covered with a 1:1000 dilution of rabbit biotinylated anti-rat IgG (Vector Laboratories) for 60 min at room temperature. After three 5-min washes in TBS, the slides were covered with avidin-biotin-complex elite (Vectastain ABC Kit, Vector Laboratories) for 30 min and washed again three times for 5 min in TBS. After oxidation with 3,3'-diaminobenzidine tetrahydrochloride for 5 min and brief washing in tap water, light counterstaining was performed with Mayers hematoxylin. The slides were then cleared and mounted with 60% HSR solution (Kokusai Shiyaku, Kobe, Japan). Each staining procedure was repeated three times, consisting of 20 consecutive sections. The investigations were conducted in accordance with the Guide for Care and Use of Laboratory Animals.
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RESULTS
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Controls
Control histological sections incubated with solely 10% rabbit serum followed by anti-rat biotinylated antiserum showed no staining whatsoever. A number of representative controls are displayed in Figures 2B, 3A, 4A, 5A, 6B, and 7B (insets), showing various stages of early and midpregnancy.To verify the specificity of -TTP staining in pregnancy, mouse uteri of nonpregnant mice (8 wk old) were stained identically as the sections above, showing no -TTP reaction (Fig. 1 with inset A) when treated with -TTP monoclonal antibodies. Negative controls hereof (identical immunohistochemical procedure except that -TTP monoclonal antibodies were not used) also showed no staining (data not shown).
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FIG. 2. Transverse section (5 µm) through gravid mouse uterus, 4.5 dpc (magnification x4). Immunohistochemical binding of -TTP-specific monoclonal antibody to secretory endometrial columnar epithelium (arrowhead) is evident. Magnification x60 hereof is shown in inset A; inset B shows negative control. SM, Two layers of smooth muscle cells; G, glandular epithelium; OD, outer decidual reaction zone; ID, inner decidual reaction zone
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FIG. 3. Transverse section (5 µm) through gravid mouse uterus, 4.5 dpc, at the site of implantation (magnification x4). Besides the staining of the secretory endometrial columnar epithelial cells (SE), staining of the inner decidual reaction zone (ID) and the glandular epithelial cells (G) in the outer decidual reaction zone (OD) can be seen. E, Embryonal cavity. Inset A shows negative control
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FIG. 4. Immunohistochemical localization of -TTP-specific monoclonal antibody binding to secretory endometrial columnar epithelium (upper arrowhead) as well as to the glandular epithelial cells (lower arrow) in the inner decidual reaction zone (ID) and binding to the inner decidual reaction zone itself (lower arrowhead), pregnant uterus at 6.5 dpc (magnification x4). Upper arrow shows nonstained glandular epithelium in the outer decidual reaction zone (OD). E, Embryonal cavity with embryonal tissue; SM, two layers of smooth muscle cells. Inset A shows negative control
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FIG. 5. Immunohistochemical localization of -TTP-specific monoclonal antibody binding to secretory endometrial columnar epithelium (arrowhead) at 8.5 dpc. Note enlarged embryonal cavity with embryo (E) and enhanced decidualization around embryonal cavity. Inset A shows negative control of secretory endometrial columnar epithelium, magnification x60
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FIG. 6. -TTP-specific monoclonal antibody binding to secretory endometrial columnar epithelium (arrowhead) of the pregnant uterus at midterm, 10.5 dpc, magnification x4. Secretory endometrial columnar epithelium shows weaker binding than at prior gestational stages. E, Embryonal tissue; YS, visceral yolk sac; PM, placental membrane; LT, labyrinthine trophoblast; ST, spongiotrophoblast; TG, trophoblast giant cells; BL, basal layer of placenta; MV, dilated maternal blood vessels. Inset A shows magnification x60 of secretory endometrial columnar epithelium marked by arrowhead; inset B is negative control
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FIG. 1. Transverse section (5 µm) through nonpregnant mouse uterus (magnification x4). No staining with -TTP-specific monoclonal antibody can be seen. Arrowhead marks secretory endometrial columnar epithelium, of which magnification x60 is shown in inset A. SM, Two layers of smooth muscle cells; G, glandular epithelium; UL, uterine lumen
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Early Pregnancy (4.5 and 6.5 dpc)
During this pregnancy stage, immunoreactive -TTP was detected in three specific localizations of mouse uterus. Firstly, marked binding of the monoclonal antibody specifically to the secretory endometrial columnar epithelium was noted (Figs. 2 with inset A, 3, and 4). Second, the glandular epithelial cells partly showed immunoreactivity, namely when they were located near the implantation site in the inner decidual reaction zone. It should be noted that the endometrium forms the mucosal lining of the uterine cavity and consists of columnar secretory epithelial cells that are bipolar with respect to their histologic orientation within this tissue. Their nucleus is displaced toward the basal side of the cell, and at the opposite side, the luminal surface forms the place of secretion. The epithelial cell population is comprised of the secretory luminal columnar epithelium and of glandular epithelial cells [11], which are found in the inner and outer decidual reaction zone. In Figure 2, the section farthest away from two neighboring implantation sites was chosen, and in this section, the glandular epithelium was not stained. In Figure 3, the embryonic cavity is seen, and directly around this site, the glandular epithelium showed immunoreactivity. This phenomenon was documented again at 6.5 dpc, where, in Figure 4, the upper arrow marks glandular epithelium in the outer decidual reaction zone showing no immunostaining, while the lower arrow marks glandular epithelium in direct vicinity to the embryonic cavity in the inner decidual reaction zone, showing immunoreactivity. Third, the inner decidual reaction zone itself, as shown in Figure 3 (arrowhead) and Figure 4 (lower arrowhead), consisting of reticular connective tissue filled abundantly with lymphocytes and granulocytes and surrounding the implantation site, showed staining as well.
Midpregnancy (8.5 and 10.5 dpc)
The staining pattern during this period of pregnancy now focused mainly on the secretory luminal endometrial epithelium. Reactivity of glandular epithelium could hardly be verified anymore due to the enlarging embryo and the concomitant thinning of uterine lumen. The decidual reaction in the inner and outer zones became more and more striking, and maternal vessel dilation with developing maturation of the placenta was seen (Figs. 5 and 6). It could be noted that the -TTP immunoreactive staining of the luminal epithelium seemed to decrease compared with prior pregnancy stages. There was, however, one striking change at 10.5 dpc concerning the localization of -TTP in the luminal epithelium: The staining was mainly located in the apical, supranuclear region of these cells.
-TTP Knockout Mice
As stated elsewhere [9], the availability of -TTP knockout mice made it possible to verify the specificity of the antibodies used in this study to show -TTP immunoreactivity. Here we give one example, in Figure 7, showing no -TTP staining of the luminal epithelium of -TTP knockout mice at 8.5 dpc (data of nonpregnant knockout mice and mice from 4.5 and 6.5 dpc are not shown).
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DISCUSSION
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Vitamin E was initially identified as a factor required for animals to have offspring and to prevent miscarriage [1, 12]. In this study, the secretory epithelium of the pregnant uterus, consisting of both the secretory luminal and glandular epithelium as well as the inner decidual reaction zone directly surrounding the embryonal implantation area, could be identified as localization sites of -TTP, the major transporter for -tocopherol, by monoclonal antibody studies. These findings are consistent with the Northern blot analysis results published earlier [9], showing -TTP gene expression in the uterus throughout pregnancy with a transient increase at implantation (4.5 dpc) followed by a gradual decrease until parturition on Day 20 of pregnancy. It has been shown that the secretory epithelial cells of the pregnant uterus are involved in transport of ions, sugars, free amino acids, and other substances into the uterine lumen [13]. The endometrium-derived secretions have been shown to play a role in influencing the growth and development of the embryo [11, 13], and the presence of -TTP in the secretory epithelial cells suggests a major role of -tocopherol in early pregnancy but also in the supply of the placenta with -tocopherol throughout pregnancy.
-TTP in the hepatocyte fulfills the role of transporting -tocopherol after uptake from the chylomicron fraction and facilitating its secretion into plasma [4, 14, 15]. It is plausible that the secretory columnar epithelial cells can be compared with the hepatocyte regarding their expression of -TTP, facilitating the secretion of maternal -tocopherol to the placenta and consecutively supplying the embryo and fetus with this antioxidative substance. The cellular localization of -TTP is also of interest: While during implantation, -TTP has no specific localization in the secretory columnar epithelial cell, -TTP seems to be more abundant at the apical border of these cells facing the placenta at midpregnancy.
The fact that -TTP is expressed in the uterine secretory epithelium throughout pregnancy stresses the fact that the fetus is not able to accumulate large amounts of -tocopherol during pregnancy and is therefore dependent on a continuous supply by the mother [16]. Fetal tissue concentrations are extremely low and -TTP in fetal rat liver is practically not detectable [17]. This is of great clinical importance with respect to the nutritional requirements in premature infants and their supplementation with vitamin E [18, 19]. Premature infants without major clinical symptoms are born with adequate vitamin E levels with respect to their gestational ages [19] but deplete very quickly if a sufficient supply with vitamin E is not feasible. In utero, the continuous supply of vitamin E, namely -tocopherol, supplied via -TTP in the uterine secretory epithelial cells to the placenta and fetus, is maintained. By premature birth, this supply is cut off and deficiency states can occur in sick, premature infants.
It has been reported that a 15-kDa transport protein for tocopherols exists in many tissues, including the human placenta [20, 21]. So far, the presence of the 30-kDa -TTP could not be verified in the mouse placenta, but the necessity of this -TTP and its transport of -tocopherol for the normal development of the labyrinthine portion of the mouse placenta could be documented in our -TTP-disrupted mouse model [9]. Due to the fact that the labyrinth of the mouse and the chorionic villi in the human placenta are homologous but not identical structures [22] and that the interhemal barrier in the mouse is hemotrichorial versus hemomonochorial in humans, the necessity of -TTP for the development of the human placenta can be postulated. Furthermore, it could also be documented that the embryos in the -TTP-disrupted mouse model showed developmental failure from 10.5 dpc, showing mainly neural tube malformations [9]. This, together with the observations made in premature infants, suggests that -tocopherol also plays an essential role in normal fetal development in midterm and later pregnancy.
It is well known that the feto-placental unit is exposed to oxidative stress and that the placental membrane is very susceptible to peroxidation [2]. Therefore, the efficiency of enzymatic and nonenzymatic reactive oxygen scavengers must be ensured throughout pregnancy for normal fetal growth and development, not only in early but also in late pregnancy. The transport of vitamin E, mainly -tocopherol as a major chain-breaking antioxidant, to the fetus throughout pregnancy must be sustained and this must, at least in part, be managed by the presence of -TTP in the uterine secretory luminal and glandular epithelial cells as well as of the inner decidual reaction zone.
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FOOTNOTES
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First decision: 15 January 2002.
1 The first author thanks the Novartis Foundation (Japan) for the Promotion of Science for the grant of Japan-Europe Scientists Exchange Program 2001. 
2 Correspondence: Hiroyuki Arai, University of Tokyo, Graduate School of Pharmaceutical Sciences, Department of Health Chemistry, Hongo 7-3-1, Bunkyo-ku, 113-0033 Tokyo, Japan. FAX: 81 3 3818 3173; harai{at}mol.f.u-tokyo.ac.jp
Accepted: March 18, 2002.
Received: December 19, 2001.
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ABSTRACT
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