| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
1 Research Group in Human Reproductive Immunobiology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
2 Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK
Correspondence
A. Moffett
am485{at}cam.ac.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
Present address: Palmerston North Hospital, New Zealand. 
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). These human endogenous retroviruses (HERVs) arose from ancient germ-cell infections by exogenous retroviruses. During the course of evolution, most HERVs have accumulated mutations that have disrupted their protein-encoding potential. However, a small number of HERV genes have retained open reading frames (ORFs) and particular interest has been focused on the expression and functions of the corresponding products. From studies of the tissue distribution of various HERV transcripts, it has become apparent that the placenta is a preferential site of expression. It is the only normal tissue to have such a wide range and high level of HERV mRNAs (Taruscio & Mantovani, 1998; Muir et al., 2004).
The human placenta is made up of finger-like chorionic villi that are covered by a double layer of fetally derived trophoblast cells: the outer fused syncytiotrophoblast is formed from the underlying mononuclear cytotrophoblast cells. These two layers together comprise the villous trophoblast. Some of the cytotrophoblast cells grow through the syncytial layer to form columns of extravillous trophoblast cells. These extend towards the uterine wall, which they ultimately contact and invade, when several different subpopulations may be distinguished: endovascular cells lie within the uterine arteries and interstitial cells migrate through the decidua (pregnancy-transformed endometrium). These finally halt and form multinucleated, placental-bed giant cells (Loke & King, 1995; Moffett-King, 2002).
Placental expression of HERV-W env has attracted particular attention. HERV-W is a multi-copy family with an estimated 70 gag, 100 pro and 30 env genes in the genome (Voisset et al., 2000). Although most of these sequences are defective, an ORF has been reported for an env gene, now known as syncytin, on chromosome 7 and for single gag and pro sequences on chromosomes 3 and 6, respectively (Blond et al., 1999; Mi et al., 2000; Voisset et al., 2000). The putative Env product was predicted to have features typical of a retroviral envelope protein with surface and transmembrane portions, the latter containing regions with potential fusogenic and immunosuppressive activities (Blond et al., 1999; de Parseval et al., 2003). Interestingly, this gene appears to be expressed at high levels only in the placenta. Multiple-tissue Northern analysis detected substantial transcription only in the placenta, with weaker expression in the testis (Mi et al., 2000). A later study using quantitative PCR confirmed the uniquely high placental expression and revealed lower levels of transcription in several normal tissues (de Parseval et al., 2003). HERV-W env RNA was localized to the syncytiotrophoblast of term villi by in situ hybridization (Mi et al., 2000). However, in vitro, isolated primary cytotrophoblasts from both early and term placentae expressed the transcripts (Frendo et al., 2003). A recent study also demonstrated syncytin expression throughout gestation (Okahara et al., 2004). The translation of a corresponding protein has now been confirmed. HERV-W Env was detected in a placental extract and in differentiating primary cytotrophoblast cells by Western blot (Voisset et al., 2000; Frendo et al., 2003). Immunohistochemical staining of villi with a range of gestational ages from first trimester to term has consistently shown preferential expression in the syncytiotrophoblast compared with the underlying cytotrophoblast (Blond et al., 2000; Lee et al., 2001; Frendo et al., 2003; Smallwood et al., 2003). However, the extravillous trophoblast populations have been little studied. There had been only a single report of syncytin expression in the cytotrophoblast cell columns (Smallwood et al., 2003), but, since the submission of our manuscript, another report has appeared in the literature (Malassiné et al., 2005). Our present study using RT-PCR and immunohistochemical techniques confirms the expression of syncytin in extravillous trophoblast.
We isolated first-trimester trophoblast cells from routine vaginal terminations of pregnancy as described previously (Loke et al., 1997). Freshly purified cells consist primarily of villous trophoblast, defined phenotypically as HLA-G-negative (Chumbley et al., 1994). After plating on 20 µg human plasma fibronectin ml–1 (Stratech) for 72 h in Ham's F12 medium/20 % fetal calf serum, the cells have the phenotype of extravillous trophoblast: 80–90 % express the extravillous markers HLA-G, BC-1 and c-erbB2. Multinucleated, HLA-G-positive cells, which resemble placental-bed giant cells, also arise (Loke & Burland, 1988; Chumbley et al., 1991; Loke et al., 1992, 1997; Burrows et al., 1993). In addition to isolated trophoblast cells, we also obtained decidual tissues from decidua parietalis that did not contain trophoblast cells. Endometrial tissues from the follicular and secretory phases of the menstrual cycle were obtained during gynaecological laporoscopy. Term placental tissues were taken after delivery of normal pregnancies. Peripheral blood leukocytes were derived from buffy-coat blood samples. RT-PCR was performed on the above tissues. PCR used Taq DNA polymerase (Qiagen). Reactions (50 µl) contained 2 µl template (water, RT-negative control or cDNA), 1 µM each primer (Sigma-Genosys) and 0.2 mM each dNTP (Roche). Novel HERV-W env-specific primers were designed to detect a 1253 bp segment of the ORF: HERV-W env F, 5'-TGATGGGGGTGGAGTTCAAG-3'; HERV-W env R, 5'-TTGGGGCGTAGTAGAGGTTG-3'. The housekeeping gene 
-actin was amplified in parallel to generate a 520 bp product, which both verified the success of cDNA synthesis and allowed comparison of relative expression levels between tissues. Fig. 1 shows the results of HERV-W env RT-PCR performed on a number of different cell and tissue samples, including cells with both villous and extravillous phenotypes. One representative lane is shown for each triplicate set tested, but results were consistent for all three samples. Term placental tissue was positive for the 1253 bp HERV-W env mRNA (lane 6b). In addition, pooled first-trimester trophoblast cells, both of villous (lane 3b) and extravillous (lane 4b) phenotype, contained syncytin transcripts. The choriocarcinoma cell lines JAR (lane 1b) and JEG-3 (lane 2b), which are used as models of villous and extravillous cells, respectively, were also positive. The trophoblast specificity of expression was confirmed by the absence of transcripts in other uterine tissues, both decidua parietalis (lane 5b) and non-pregnant endometrium (lane 7b). Peripheral blood leukocytes (lane 8b) provided a further negative control.
|
shows staining with a rabbit polyclonal antibody towards syncytin and corresponding negative-control sections. Syncytin expression was observed in first-trimester villous trophoblast as early as 6 weeks gestation (Fig. 2a). Villi from second-trimester samples also stained with the antiserum, but staining intensity became weak at term (not shown). The syncytial layer stained diffusely with increased intensity at the apical membrane. However, in all first- and second-trimester tissues examined, syncytin was not confined to the syncytiotrophoblast, but was also detected strongly in the underlying cytotrophoblast cells. In each case, expression was maintained in the implanting columns (Fig. 2a) and in all populations of extravillous cells within the decidua: the invading interstitial trophoblast cells, endovascular trophoblast (Fig. 2c) and placental-bed giant cells (Fig. 2e). Interestingly, ectopic trophoblast in tubal pregnancies also expressed syncytin in both villous and extravillous cells (Fig. 2g). In ectopic syncytiotrophoblast, the same diffuse cytoplasmic staining and apical enhancement were observed as were found in normal trophoblast.
|
shows an immunoblot of untransfected 293T cells, 293T cells transiently transfected with the P-I-S construct and pooled, freshly isolated first-trimester trophoblast cells. An 80 kDa protein was detected in the P-I-S transfectants (Fig. 3b) and trophoblast cells (Fig. 3c). This was consistent with the size of the glycosylated HERV-W Env proteins detected by others in placental extracts, transfected cell lines and following in vitro transcription–translation (Blond et al., 1999, 2000; Voisset et al., 2000; Smallwood et al., 2003). This shows one representative blot; a band of the same size was detected in three further trophoblast samples analysed.
|
; Smallwood et al., 2003; Malassiné et al., 2005). This raises the question of the possible function of syncytin in these cells. There is much evidence to suggest that syncytin contributes to the cell-fusion events involved in generating syncytiotrophoblast (Blond et al., 2000; Mi et al., 2000; Frendo et al., 2003; Pötgens et al., 2004). The possibility that placental-bed giant cells might also arise by fusion is interesting, because there is still debate about whether the mechanism invloves fusion or endoreduplication (Al-Lamki et al., 1999). Besides HERV-W env (syncytin), two other complete coding envelope genes are transcribed at high levels in the placenta: ERV3 env and HERV-FRD env (de Parseval et al., 2003; Muir et al., 2004). Interestingly, HERV-FRD env also has fusogenic properties, so it appears that there might be a second potential HERV mediator of fusion within the placenta (Blaise et al., 2003).
There have been reports of spatially abnormal expression of syncytin in diseases of pregnancy such as pre-eclampsia, but it is unclear whether these observations reflect a cause or effect of the placental abnormalities (Lee et al., 2001; Knerr et al., 2002). This is further complicated by the fact that there is still much confusion regarding the subcellular distribution of syncytin in the layer of syncytiotrophoblast in normal pregnancy. Observations have varied between studies, even when using the same antibody. We have found diffuse cytoplasmic labelling of syncytiotrophoblast with enhancement at the apical membrane. This is in accord with the observation of Frendo et al. (2003), but contrasts with the findings of Lee et al. (2001), who showed syncytin to be located predominantly on the basal aspect of syncytiotrophoblast in normal pregnancies, but apically in pre-eclamptic placentae.
As our laboratory has a special focus on pregnancy immunology, we are particularly interested in a possible immunological function for syncytin in the uterus. The invading trophoblast cells are in close proximity to large numbers of maternal leukocytes at the implantation site, primarily uterine natural killer (NK) cells (Moffett-King, 2002). The nature and functional importance of trophoblast leukocyte interactions are poorly understood, but syncytin may have a modulatory role via a region of amino acids that is highly conserved between the envelope proteins of many retroviruses, both endogenous and exogenous (Bénit et al., 2001). CKS-17, a synthetic peptide derived from this sequence, has numerous immunological effects in vitro, for example influencing the cytokine production, proliferation, chemotaxis and killing activity of various immune-cell types (Cianciolo et al., 1985; Nakagawa & Harrison, 1996). Interestingly, CKS-17 inhibited killing by peripheral-blood NK cells (Harris et al., 1987).
The available data are presently insufficient to designate a definitive function for syncytin in the placenta. Whether the expression of this protein is vital to the success of pregnancy is also unknown. The fact that some mammals lack a syncytin homologue, yet undergo normal pregnancies, implies redundancy (Stoye & Coffin, 2000). However, our findings do suggest that, in addition to its probable contribution to cell fusion within the placenta, syncytin may have other important effects in the extravillous cells.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Bénit, L., Dessen, P. & Heidmann, T. (2001). Identification, phylogeny, and evolution of retroviral elements based on their envelope genes. J Virol 75, 11709–11719.
Blaise, S., de Parseval, N., Bénit, L. & Heidmann, T. (2003). Genomewide screening for fusogenic human endogenous retrovirus envelopes identifies syncytin 2, a gene conserved on primate evolution. Proc Natl Acad Sci U S A 100, 13013–13018.
Blond, J.-L., Besème, F., Duret, L., Bouton, O., Bedin, F., Perron, H., Mandrand, B. & Mallet, F. (1999). Molecular characterization and placental expression of HERV-W, a new human endogenous retrovirus family. J Virol 73, 1175–1185.
Blond, J.-L., LaVillette, D., Cheynet, V., Bouton, O., Oriol, G., Chapel-Fernandes, S., Mandrand, B., Mallet, F. & Cosset, F.-L. (2000). An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor. J Virol 74, 3321–3329.
Burrows, T. D., King, A. & Loke, Y. W. (1993). Expression of integrins by human trophoblast and differential adhesion to laminin or fibronectin. Hum Reprod 8, 475–484.
Chumbley, G., Hawley, S., Carter, N. P. & Loke, Y. W. (1991). Human extravillous trophoblast MHC class I expression is resistant to regulation by interferon-
. J Reprod Immunol 20, 289–296.[CrossRef][Medline]
Chumbley, G., King, A., Gardner, L., Howlett, S., Holmes, N. & Loke, Y. W. (1994). Generation of an antibody to HLA-G in transgenic mice and demonstration of the tissue reactivity of this antibody. J Reprod Immunol 27, 173–186.[CrossRef][Medline]
Cianciolo, G., Copeland, T. D., Oroszlan, S. & Snyderman, R. (1985). Inhibition of lymphocyte proliferation by a synthetic peptide homologous to retroviral envelope proteins. Science 230, 453–455.
de Parseval, N., Lazar, V., Casella, J.-F., Benit, L. & Heidmann, T. (2003). Survey of human genes of retroviral origin: identification and transcriptome of the genes with coding capacity for complete envelope proteins. J Virol 77, 10414–10422.
Frendo, J.-L., Olivier, D., Cheynet, V., Blond, J.-L., Bouton, O., Vidaud, M., Rabreau, M., Evain-Brion, D. & Mallet, F. (2003). Direct involvement of HERV-W Env glycoprotein in human trophoblast cell fusion and differentiation. Mol Cell Biol 23, 3566–3574.
Harris, D. T., Cianciolo, G. J., Snyderman, R., Argov, S. & Koren, H. S. (1987). Inhibition of human natural killer cell activity by a synthetic peptide homologous to a conserved region in the retroviral protein, p15E. J Immunol 138, 889–894.[Abstract]
International Human Genome Sequencing Consortium (2001). Initial sequencing and analysis of the human genome. Nature 409, 860–921.[CrossRef][Medline]
Knerr, I., Beinder, E. & Rascher, W. (2002). Syncytin, a novel human endogenous retroviral gene in human placenta: evidence for its dysregulation in preeclampsia and HELLP syndrome. Am J Obstet Gynecol 186, 210–213.[CrossRef][Medline]
Lee, X., Keith, J. C., Jr, Stumm, N. & 9 other authors (2001). Downregulation of placental syncytin expression and abnormal protein localization in pre-eclampsia. Placenta 22, 808–812.[CrossRef][Medline]
Loke, Y. W. & Burland, K. (1988). Human trophoblast cells cultured in modified medium and supported by extracellular matrix. Placenta 9, 173–182.[Medline]
Loke, Y. W. & King, A. (1995). Human Implantation: Cell Biology and Immunology. Cambridge: Cambridge University Press.
Loke, Y. W., Hsi, B.-L., Bulmer, J., Grivaux, C., Hawley, S., Gardner, L., King, A. & Carter, N. P. (1992). Evaluation of a monoclonal antibody, BC-1, which identifies an antigen expressed on the surface membrane of human extravillous trophoblast. Am J Reprod Immunol 27, 77–81.
Loke, Y. W., King, A., Burrows, T., Gardner, L., Bowen, M., Hiby, S., Howlett, S., Holmes, N. & Jacobs, D. (1997). Evaluation of trophoblast HLA-G antigen with a specific monoclonal antibody. Tissue Antigens 50, 135–146.[Medline]
Malassiné, A., Handschuh, K., Tsatsaris, V., Gerbaud, P., Cheynet, V., Oriol, G., Mallet, F. & Evain-Brion, D. (2005). Expression of HERV-W Env glycoprotein (syncytin) in the extravillous trophoblast of first trimester human placenta. Placenta 26, 556–562.[Medline]
Mi, S., Lee, X., Li, X.-P. & 9 other authors (2000). Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature 403, 785–789.[CrossRef][Medline]
Moffett-King, A. (2002). Natural killer cells and pregnancy. Nat Rev Immunol 2, 656–663.[CrossRef][Medline]
Muir, A., Lever, A. & Moffett, A. (2004). Expression and functions of human endogenous retroviruses in the placenta: an update. Placenta 25 (Suppl. A), S16–S25.
Nakagawa, K. & Harrison, L. C. (1996). The potential roles of endogenous retroviruses in autoimmunity. Immunol Rev 152, 193–236.[CrossRef][Medline]
Okahara, G., Matsubara, S., Oda, T., Sugimoto, J., Jinno, Y. & Kanaya, F. (2004). Expression analyses of human endogenous retroviruses (HERVs): tissue-specific and developmental stage-dependent expression of HERVs. Genomics 84, 982–990.[CrossRef][Medline]
Pötgens, A. J. G., Drewlo, S., Kokozidou, M. & Kaufmann, P. (2004). Syncytin: the major regulator of trophoblast fusion? Recent developments and hypotheses on its action. Hum Reprod Update 10, 487–496.
Smallwood, A., Papageorghiou, A., Nicolaides, K., Alley, M. K. R., Jim, A., Nargund, G., Ojha, K., Campbell, S. & Banerjee, S. (2003). Temporal regulation of the expression of syncytin (HERV-W), maternally imprinted PEG10, and SGCE in human placenta. Biol Reprod 69, 286–293.
Stoye, J. P. & Coffin, J. M. (2000). A provirus put to work. Nature 403, 715–717.[CrossRef][Medline]
Taruscio, D. & Mantovani, A. (1998). Human endogenous retroviral sequences: possible roles in reproductive physiopathology. Biol Reprod 59, 713–724.
Voisset, C., Bouton, O., Bedin, F., Duret, L., Mandrand, B., Mallet, F. & Paranhos-Baccalà, G. (2000). Chromosomal distribution and coding capacity of the human endogenous retrovirus HERV-W family. AIDS Res Hum Retroviruses 16, 731–740.[CrossRef][Medline]
Received 7 August 2005;
accepted 5 March 2006.
This article has been cited by other articles:
|
|
 |
|
  M. Yoshie, H. Kashima, T. Bessho, M. Takeichi, K. Isaka, and K. Tamura Expression of stathmin, a microtubule regulatory protein, is associated with the migration and differentiation of cultured early trophoblasts Hum. Reprod., December 1, 2008; 23(12): 2766 - 2774. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
