Molecular Human Reproduction, Vol. 7, No. 12, 1159-1165,
December 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Expression and localization of thioredoxin during early implantation in the marmoset monkey
1 Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women's Hospital, Carlton, Victoria 3053, Australia, 2 Department of Cell Biology and Human Anatomy, University of California, Davis, 95616, USA, 3 School of Biomolecular and Biomedical Science, Griffith University, Nathan, Queensland 4111 and 4 School of Health Science, Griffith University, Southport, 4215, Australia
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Abstract |
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Thioredoxin is a powerful redox protein expressed in invasive cytotrophoblasts and essential for blastocyst implantation in mice. Isolated marmoset thioredoxin cDNA showed that the deduced amino acid sequence differed from the human sequence by four amino acids. The close homology of thioredoxin in the two species enabled us to use monoclonal antibodies against human thioredoxin to detect marmoset thioredoxin in implantation sites, blastocysts and culture medium. Immunocytochemistry on marmoset implantation sites, on pregnancy days 12 and 15, showed that thioredoxin is highly expressed in uterine luminal epithelium, glands and in some endometrial stromal cells. In attached blastocysts, thioredoxin staining was detected in mural and polar trophoblast cells and both visceral and parietal endoderm, whereas no staining was present in the inner cell mass. A similar pattern of thioredoxin expression was detected in hatched blastocysts attached to Matrigel in tissue culture. Trophoblastic vesicles derived from blastocysts expressed thioredoxin in inner endoderm-like cells and outer trophoblast-like cells and secreted thioredoxin into the culture medium. These experiments have demonstrated thioredoxin expression during early stages of embryo–maternal interaction. We propose that thioredoxin protects the early placenta from oxidative damage and that the marmoset is a valuable model for studying thioredoxin regulation and function during implantation and blastocyst differentiation.
blastocyst/implantation/marmoset monkey/thioredoxin/trophoblast
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Thioredoxin is a small (12 kDa) redox active protein with a highly conserved site of W-C-G-P-C-K at amino acid positions 31–36. The cysteine pair at positions 32 and 35 may oxidize to form a disulphide bond which is reduced by the NADPH-dependent enzyme thioredoxin reductase (Sahlin et al., 2000
). Reduced thioredoxin acts as a powerful protein disulphide reductase and through this redox activity it regulates many essential intracellular and extracellular functions and processes. Originally identified as a co-factor for ribonucleotide reductase in DNA synthesis (Laurent et al., 1964), the thioredoxin system (thioredoxin and thioredoxin reductase) has since been shown to play roles in the regulation of cell growth (Gasdaska et al., 1995), apoptosis (Baker et al., 1997), immune responses (Rosen et al., 1995), implantation (Matsui et al., 1996), HIV infection (Newman et al., 1994) and cellular responses to oxidative stress (Nakamura et al., 1997). Thioredoxin modulates gene regulation by acting directly on several transcription factors mediated by redox control of critical thiol groups. These transcription factors include NF-
B (Matthews et al., 1992), p53 (Ueno et al., 1999) and the oestrogen (Hayashi et al., 1997) and glucocorticoid receptors (Grippo et al., 1985).
Both normal and tumour cells secrete thioredoxin. This secreted thioredoxin has been found to act as an autocrine growth factor and also to express co-cytokine activity which enhances the action of other cytokines (Schenk et al., 1996). Secreted thioredoxin also binds to the external surface of the cell membrane where it may regulate cell–cell contact and contribute to redox regulation in the extracellular space (Sahaf et al., 1997; Stathakis et al., 1997).
Our studies have shown that thioredoxin plays a role in the `early pregnancy factor' system (Clarke et al., 1991; Tonissen et al., 1993). We (Perkins et al., 1995) and others (Kobayashi et al., 1995) have found very high and specific expression of thioredoxin by the cytotrophoblast cells of first trimester human placentae. During the establishment of the placenta, these cells aggressively invade the endometrium and share many characteristics with metastatic cancer cells (Strickland and Richards, 1992). Thioredoxin expression by these invasive cytotrophoblast cells during development of the placenta is striking while in contrast, the non-invasive syncytiotrophoblast cell layer surrounding the villi does not produce thioredoxin (Perkins et al., 1995). The stromal cells in the decidualized endometrium at this stage of pregnancy also strongly express thioredoxin (Perkins et al., 1995). Furthermore, gene knockout experiments in mice (Matsui et al., 1996) have shown the homozygote mutants develop to the blastocyst stage but then fail to implant into the uterine wall, suggesting that thioredoxin is required for normal development and implantation.
The aim of our studies is to develop a system that can be utilized to define the role that thioredoxin plays during the early stages of implantation. Previous models for studying early implantation have utilized term cytotrophoblast cells, transformed cell cultures or mouse models, all of which have several limitations. Human transformed cell cultures or term placenta cytotrophoblasts do not represent the trophoblast cells at the time of implantation and the early stages of mouse implantation are different to those in humans (Salamonsen, 1999). An alternative model is to employ marmoset monkey peri-implantation blastocysts and their products to study the functions important for primate implantation (Summers et al., 1987; Lopata et al., 1995; Franek et al., 1999). The present study demonstrates that thioredoxin is highly expressed at the time of blastocyst attachment and implantation in the marmoset monkey and that an in-vitro trophoblast cell model is also suitable for further studies, since thioredoxin is not only highly expressed but is also actively secreted by these cells.
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Materials and methods |
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Isolation of marmoset sequences
Polyadenylated mRNA was isolated from 100 mg marmoset liver using an Oligotex mRNA isolation kit according to the manufacturer's instructions (Qiagen, Clifton Hill, Australia). The mRNA was reverse transcribed into cDNA using oligo dT to prime first strand synthesis with the SuperScript Preamplification System (Gibco-BRL, Melbourne, Australia). The marmoset thioredoxin sequence was obtained using polymerase chain reaction (PCR). Oligonucleotides (5' dGGA TCC ATT TCC ATC GGT CC) and (5' dTAG CCA ATG GCT GGT TAT GT) were designed based on human thioredoxin sequences to bind to 5' and 3' untranslated sequences respectively to amplify the complete coding sequences of 330 bp. The PCR procedure utilized a denaturation step at 94°C for 1 min followed by an annealing step at 37°C for 1 min and then a 1 min extension at 72°C. This was repeated a further five times. The samples were then subjected to a further 30 cycles where the annealing temperature was increased to 50°C. PCR reactions contained 0.2 mmol/l of each dNTP, 2.5 mmol/l magnesium chloride, reaction buffer (Stratagene, Austin, TX, USA), 2 units Pfu Polymerase (Stratagene) and 100 ng of each oligonucleotide. After completion, the reactions were spiked with 2 units Taq polymerase (Promega, Annandale, Australia) and 0.8 mmol/l dATP at 70°C for 30 min to ensure each DNA strand contained a 3' adenine residue prior to the cloning step. The fragments obtained from the PCR were purified on a 1% agarose gel run in TAE buffer (40 mmol/l Tris–acetate pH 8.2, 1 mmol/l EDTA) and ligated to the plasmid pGemTeasy (Promega) according to the manufacturer's protocol. Plasmids containing the desired length insert were sequenced using the automated cycle sequencing procedure (Perkin Elmer, Scoresby, Victoria, Australia) in the DNA Sequencing facility at Griffith University. The GenBank accession number for this sequence is AF353204.
Antibodies
The monoclonal antibodies used in these studies were raised against recombinant human thioredoxin prepared as described previously (Clarke et al., 1991) and purified in our laboratory. Cross-reaction between marmoset thioredoxin and monoclonal antibodies raised against human thioredoxin was checked by Western blotting procedures and by enzyme-linked immunosorbent assay (ELISA) as previously described (Di Trapani et al., 1998).
ELISA
The concentration of thioredoxin in culture medium was determined using a dual-antibody sandwich ELISA as previously described (Di Trapani et al., 1998). Monoclonal antibody 1B3 was used as the capture antibody and the biotinylated monoclonal antibody 2B1 was used for detection. Briefly, wells of a 96-well plate (ICN, Seven Hills, NSW, Australia) were coated with the capture antibody 1B3 at a concentration of 40 µg/ml in 0.05 mol/l sodium bicarbonate buffer pH 9.6 for 1 h at 37°C. Plates were washed with TBST (0.05 mol/l Tris pH 7.5, 0.3 mol/l NaCl, 0.05% Tween 20) and non-specific sites were blocked by incubation with Blotto (5% w/v milk protein in TBS) at 37°C for 1 h. A standard curve using known concentrations of pure recombinant thioredoxin was prepared. Duplicates of trophoblastic vesicle culture media samples, prepared as previously described (Franek et al., 1999), were loaded into remaining wells and the plates were incubated overnight at 4°C. Plates were then washed three times with TBST and incubated with biotinylated 2B1 at a concentration of 2.97 µg/ml in Blotto for 2 h at 37°C. Plates were then washed with TBST and incubated with an avidin horseradish peroxidase-conjugated anti-mouse IgG antibody (Bio-Rad, Regents Park NSW, Australia) for 1 h at 37°C. Plates were washed three times in TBST and incubated in TMB light-up solution [0.1 mol/l sodium acetate, 0.001% hydrogen peroxide, 200 µg/ml TMB (3',3',5',5'-tetramethylbenzidine)] for 15 min at room temperature in the dark. This reaction was stopped with 1 mol/l sulphuric acid and the plate was read at 450 nm.
Preparation of marmoset implantation sites
Marmoset monkeys (Callithrix jacchus) were housed in pairs at an approved quarantine facility at the Royal Women's Hospital (Melbourne, Australia) and all appropriate animal experimentation was approved by the hospital's and the University of Melbourne Animal Experimentation Ethics Committees. The methods used for timing of the pregnancies have been described previously (Lopata et al., 1995). A detailed description of the collection of tissues for the immunocytochemistry experiments performed in this paper has also been published previously (Enders and Lopata, 1999). Briefly, two marmoset monkeys, estimated to be at day 12 and day 15 of pregnancy, were perfused in situ with 4% paraformaldehyde, and the entire genital tract was removed. The uterus of each animal was then trimmed and opened carefully to identify implantation sites. Each implantation site was carefully trimmed without disturbing its integrity, and then processed and embedded in paraffin. The blocks were serially sectioned and placed individually on slides for immunocytochemistry. Representative sections were stained with haematoxylin and eosin to determine the position and stage of development of the contained embryos and implantation sites (Enders and Lopata, 1999).
Immunocytochemistry
Sections were deparaffinized by treating with histolene and rehydrated by step-wise reducing the ethanol levels in the solution. Endogenous peroxidases were blocked by application of 3% (v/v) hydrogen peroxide in phosphate-buffered saline (PBS) for 5 min. Slides were rinsed and the blocking solution (20% normal rabbit serum in PBS) was applied for 20 min. Excess serum was tapped off and diluted primary antibody (monoclonal 6F3 anti-thioredoxin, 1/2000 dilution) was applied to the sections and incubated for 1 h. The slides were rinsed in PBS and the secondary antibody (biotinylated rabbit anti-mouse IgM diluted 1:400 in PBS) was applied and incubated at room temperature for 30 min. Sections were then rinsed in PBS before the prepared ABC solution [avidin- and peroxidase-linked biotin (Vector Laboratories Inc., Burlingame, CA, USA)] was applied. Following 30 min incubation and subsequent washing, the peroxidase was visualized by application of 0.05% (w/v) DAB (3,3'-diaminobenzidine) (Research Organics Inc, Cleveland, OH, USA) and 0.09% hydrogen peroxide in PBS. The sections were rinsed and counterstained in Mayer's haematoxylin (Sigma, Castle Hill, Australia), dehydrated in ethanol, cleared with xylene and mounted with DePeX (Merck Pty Ltd, Kilsyth, Australia) and after application of coverslips they were allowed to dry at 37°C overnight. A negative control for each batch of slides was included using mouse isotype serum to replace the primary antibody.
Culture of blastocysts and trophoblast cells
Preimplantation marmoset monkey embryos were collected from uterine flushes and cultured to the hatched blastocyst stage as previously described (Lopata et al., 1995). The blastocysts were then placed on a Matrigel (Collaborative Biomedical Products, Becton-Dickson, NJ, USA)-coated tissue culture insert inside a 4-well culture plate (Nunc, Roskilde, Denmark) in alpha-MEM supplemented with 10% FCS, 0.1% insulin (Sigma) and 0.1% transferrin (Sigma) for a further 5–7 days. They were then fixed in 4% paraformaldehyde for processing and embedded in paraffin wax for the immunocytochemistry experiments. Long-term trophoblast cultures were established by subdividing highly expanded blastocysts into ~20 fragments. These fragments were propagated by subculture in the media described above. Each fragment formed a new and separate vesicle that can grow to a size of 5 mm in diameter consisting of an outer layer of trophoblast–like cells (identified by cytokeratin immunocytochemistry) and an inner layer of endoderm-like cells (identified by vimentin immunocytochemistry and electron microscopy). The procedure for trophoblastic vesicle propagation has been described in detail in other studies (Lopata et al., 1995; Franek et al., 1999). Vesicles were processed and embedded in paraffin for immunocytochemistry. For secretion studies, newly dissected fragments from a vesicle were placed in a 96-well culture plate (Falcon; Becton–Dickson, NJ, USA), one fragment per well in 250 µl of media. Over an 8 day period, 220 µl of media were collected from each well each day so that duplicate samples could be analysed for thioredoxin protein using the capture ELISA method.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Isolation of marmoset thioredoxin sequences
A cDNA clone containing the coding region for marmoset thioredoxin was isolated from marmoset liver using a reverse transcription–PCR protocol. Primers were designed to be complementary to human 5' and 3' untranslated sequences so that the complete full-length coding region sequences could be obtained for marmoset thioredoxin. The cDNA sequence (GenBank accession no. AF353204) was found to be 96% homologous to human cDNA sequences while the deduced protein sequence differed from the human sequence by four amino acids. The marmoset thioredoxin amino acid sequence also differed from the published rhesus monkey thioredoxin sequence (An and Wu, 1992
) in four positions, whereas the rhesus monkey sequence differed from the human sequence by three amino acids (shown in Figure 1). All active site residues were conserved and the other three cysteine residues common to all mammalian thioredoxin sequences published to date were also conserved in the marmoset sequence. These minimal sequence differences encouraged us to apply the human specific antibodies for detection of the marmoset thioredoxin protein. The marmoset predicted thioredoxin amino acid sequence differed from the rat sequence at 11 positions and to mouse thioredoxin at 13 positions, including amino acid number 46 which is an additional sixth cysteine residue found only in rat and mouse thioredoxin sequences (Figure 1).
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Expression of thioredoxin at in-vivo implantation sites
Monoclonal antibodies raised against purified human recombinant thioredoxin were utilized in immunocytochemistry studies performed on sections of implantation sites and the results are shown in Figure 2
. A morphological description of these sections using light and electron microscopy has been previously published (Enders and Lopata, 1999). Staining of marmoset sections using the thioredoxin-specific monoclonal antibody 6F3 was optimized using kidney sections as controls. Other studies have shown using immunocytochemistry that thioredoxin is expressed at significant levels in the tubules of the kidney but is almost undetectable in the glomeruli (Fujii et al., 1991; Oberley et al., 2001). Figure 2A is a section of marmoset kidney cortex and clearly shows thioredoxin staining in the tubules but none in the glomerulus, as reported by other investigators. In addition, a negative control slide in which a standard mouse isotype serum was used exhibited no staining in the tubules (data not shown). The 6F3 antibody that was effective in detecting marmoset thioredoxin expression in the kidney was then utilized for immunocytochemical analysis of marmoset embryo attachment sites.
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Figure 2B
shows a longitudinal section through the uterus taken from a marmoset 12 days after ovulation. The blastocyst was attached to the uterine epithelium in the region of the inner cell mass and in regions beyond the polar trophoblast as shown in Figure 2B. There was strong staining for thioredoxin in the uterine glands and in the surface epithelium. Thioredoxin expression was not detectable in the inner cell mass but some staining appeared to be present in mural trophoblast.
Figure 2C and D show sections of a marmoset uterus on day 15 after ovulation near an implantation site where the trophoblast has not attached. Both panels show a high power view of the mural trophectoderm lying in close proximity to the uterine epithelium. The surface epithelium expressed high levels of thioredoxin, particularly along the luminal surface. The uterine glands were also highly stained and this level of staining decreased in glands situated further from the uterine cavity (data not shown). The decidual and other endometrial stromal cells also contained thioredoxin activity. Patchy positive staining was also evident in the trophectoderm at the higher magnification (Figure 2C and D). Figure 2E is a section from the same implantation site and shows a region of the inner cell mass in the day 15 blastocyst. Thioredoxin staining was evident in both the parietal and visceral endoderm tissues, but the inner cell mass of the embryo was negative for the protein.
Expression of thioredoxin in blastocysts
Thioredoxin expression was also detected in marmoset blastocysts that were obtained by in-vitro culture of 8-cell to morula stages. These cleavage stage embryos were flushed from the uterus on days 4–5 of pregnancy, and then grown for several days in culture before incubation on Matrigel, an extracellular matrix resembling basement membrane. The attached blastocysts were then processed and sectioned as described in Materials and methods, and subjected to immunocytochemistry with the thioredoxin antibody 6F3. A representative blastocyst is shown in Figure 2F and G. As was observed in the in-vivo blastocyst, thioredoxin staining was observed in the trophectoderm and visceral and parietal endoderm layers, whereas the inner cell mass was negative.
Expression and secretion of thioredoxin by marmoset trophoblastic vesicles
As described in the Materials and methods, the trophoblastic vesicles, obtained by culturing fragments of subdivided hatched blastocysts, consisted of an outer layer of trophoblast cells and an inner layer of endoderm cells. Figure 2H shows immunocytochemical staining of a representative trophoblastic vesicle showing thioredoxin expression in these structures. In all sections prepared, thioredoxin expression was always present in the trophoblast and endoderm. A high power section of the wall of a typical trophoblastic vesicle shows the presence of thioredoxin staining in both the outer trophoblast cells and the inner endoderm cells, with the highest concentration of reactive thioredoxin located at the interface between the two layers (Figure 2I). Thioredoxin appeared to be present in both the nucleus and cytoplasm of the cells comprising the vesicle wall. Control sections (Figure 2J) utilizing standard mouse isotype antibody were negative.
The secretion of thioredoxin from the vesicles was investigated using a capture ELISA. Culture medium in which individual vesicles were grown was collected daily for 8 days and the levels of released thioredoxin in seven experiments are shown in Figure 3. The levels of thioredoxin secreted into the medium increased with increasing growth of the vesicles.
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Discussion |
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In this study we have shown that marmoset monkey thioredoxin is closely homologous to the thioredoxin proteins of higher primates including the human. We have also demonstrated that a monoclonal antibody to human thioredoxin reacted with a variety of marmoset tissues, localizing the redox protein at these sites. The observation that the monoclonal antibody reacted with renal tubules but not the glomeruli, with endoderm and trophoblast but not with the inner cell mass, and with only some of the endometrial stromal cells, suggests that the antibody identified a specific protein, thioredoxin. Based on such findings, this is the first study that has shown the localization of thioredoxin at the embryo–maternal interface comprising early implantation sites, as well as in cultured blastocysts and related tissues, in a primate species.
Our findings revealed prominent expression of thioredoxin in the marmoset uterine luminal and glandular epithelium, and in the endometrial stroma, during early pregnancy. These results are consistent with the intense thioredoxin staining in human uterine glands and endometrial stroma, particularly during the secretory phase of the menstrual cycle (Maruyama et al., 1997). Similar findings on thioredoxin expression in the uterine lining and glands during the reproductive cycle were reported in the mouse (Osborne et al., 2001). Since thioredoxin is an important cellular disulphide reducing protein which is probably secreted into the extracellular space and uterine fluid, it may act to protect cleavage stage embryos (Natsuyama et al., 1992; Kuribayashi and Gagnon, 1996) and possibly peri-implantation stage blastocysts, against damage from oxidative stress.
Thioredoxin was expressed both in the polar trophoblast of attaching blastocysts and in the mural trophoblast cells. The polar region is known to be involved in the invasive process in primates. Although the mural trophoblast appears to have a less active role, in the marmoset, it is involved with the progressive expansion of the implantation site and in the localized attachment of the peripheral blastocyst wall to the uterine epithelium (Enders and Lopata, 1999). Our findings on the expression of thioredoxin in trophoblast cells are consistent with reports that this redox protein is expressed in cytotrophoblast cells involved in the invasive process (Di Trapani et al., 1998) and in human placental tissues (Perkins et al., 1995; Ejima et al., 1999). The present observations suggest that thioredoxin is involved in some of the earliest stages of differentiation of the primate placenta.
Both visceral and parietal endoderm was found to contain thioredoxin reactivity in blastocysts that developed in vivo or in vitro. It may be postulated that the appearance of thioredoxin in the differentiating endoderm layer foreshadows oxidative stress at the feto-maternal interface and the need for a cellular protective mechanism, particularly in the sensitive endothelial cells (Fernando et al., 1992) of the capillaries of the developing haemo-monochorial marmoset placenta. The role of thioredoxin in implantation stage embryos was recently evaluated in mice carrying a targeted disruption of the thioredoxin gene (Matsui et al., 1996). In these studies, homozygous mutants died shortly after implantation, indicating that thioredoxin was essential for survival of the mouse blastocyst during early pregnancy. Whether thioredoxin plays such a critical role in primate implantation requires further experimentation.
The early pregnancy factor (EPF) phenomenon becomes identifiable within 24 h after fertilization in the mouse (Clarke, 1992) and probably some primate species. The EPF system becomes demonstrable by means of the rosette inhibition assay after the appearance of a specific moiety of thioredoxin in the serum that is associated with the establishment of early pregnancy (Clarke et al., 1991). Our finding that thioredoxin was expressed in blastocyst cells destined to become extra-embryonic tissues (trophectoderm and endoderm), but not in the inner cell mass, raises a number of important questions that could be resolved using marmoset embryos. First, how soon after fertilization is thioredoxin expressed in the embryo? Second, if thioredoxin appears in the fertilized oocyte or early cleavage stages, does its expression reflect the appearance of EPF? Third, if it is expressed in cleaving embryos then does the down-regulation of thioredoxin in the inner cells of the morula foreshadow blastocyst formation and the differentiation of the inner cell mass? Alternatively, in such studies the demonstration of thioredoxin activity only after blastocyst differentiation would suggest that the EPF phenomenon was not associated with thioredoxin of embryonic origin.
The trophoblastic vesicles used in the present studies were derived from highly expanded marmoset blastocysts (5–6 mm in diameter) that were cultured from cleavage stage uterine embryos. Nearly all of the vesicles originated from the blastocyst wall, cut into ~20 fragments, and cultured for several days until they established cellular spheres comprising an outer trophectoderm and inner endoderm layer. Immunohistochemistry experiments indicated that thioredoxin was expressed in both cell layers, as in the intact blastocyst. Moreover, it was demonstrated that trophoblastic vesicles released thioredoxin into the culture medium, suggesting that blastocysts also secrete this protein. The secreted thioredoxin may, therefore, exert autocrine and/or paracrine effects during blastocyst differentiation and implantation.
The importance of thioredoxin expression during cellular invasion is being widely recognized. Invasive cancer cells overexpress thioredoxin and the thioredoxin/thioredoxin reductase system has become a potential target for chemotherapeutic drugs (Kirkpatrick et al., 1998). We have demonstrated thioredoxin expression in invading cytotrophoblast cells during human implantation and the establishment of pregnancy, suggesting an important role for this redox protein at the feto-maternal interface (Perkins et al., 1995). Recently we have observed decreased production of thioredoxin in placentas from patients with pre-eclampsia, a condition where trophoblast invasion is limited and the placental bed becomes hypoxic and suffers from oxidative stress (A.V.Perkins et al., unpublished data). The role of thioredoxin in the establishment of pregnancy and the pathogenesis of pre-eclampsia is subject to further study and may include regulation of specific transcription factors such as NF-B, control of apoptosis and protection from oxidative stress.
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Acknowledgements |
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We wish to thank Janet Zan and Angela Nelson for their expert care of the marmoset colony, Judy Borg for processing some of the tissues, Karen Oliva for the progesterone assays for tracking marmoset cycles and the Royal Women's Hospital, University of Melbourne and Griffith University for financial support. Kelly Bloomfield is a recipient of an Australian Postgraduate Award.
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Notes |
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5 Present address: The Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, 3052, Australia
6 To whom correspondence should be addressed. E-mail: K.Tonissen{at}sct.gu.edu.au
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References |
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Submitted on May 31, 2001; accepted on September 13, 2001.
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