Expression of HERV-W env mRNA and glycoprotein is colinear with primary cytotrophoblast differentiation. (A) Desmoplakin immunodetection after 24, 48, and 72 h of culture of trophoblast cells isolated from a term placenta. Positive immunofluorescent staining is observed only in aggregated cytotrophoblasts (24 h) or in cytotrophoblasts in contact with the syncytiotrophoblast (48 h); staining has disappeared in the fused syncytiotrophoblast characterized by multiple nuclei (72 h). Nuclei were labeled with DAPI (blue fluorescence). (B) Levels of hCG (expressed in milli-international units per milliliter per 10⁶ cellules) secreted into the culture medium at the indicated times and characterizing the observed differentiation. Since cells were plated in triplicate (see Materials and Methods), hCG levels were determined for each plate. Results are expressed as the mean and SEM of the three culture dishes. (C) Env-W and hCG-mRNA expression in cytotrophoblasts (CT0) and during their in vitro differentiation for 3 days. mRNA data are expressed as the level of each marker mRNA normalized by RPLPo mRNA levels (Po). Two culture dishes were pooled for each determination, and transcripts were assayed in duplicate. (D) Western blot analysis of Env glycoprotein detected by the anti-SU polyclonal antibody during trophoblast differentiation (0 to 72 h). Fibroblast protein extract was used as a negative control (Fib), and a BeWo cell line (B30) and env-transfected TE 671 cells (phCMV-Env) were used as positive controls. The whole figure (A through D) illustrates an experiment which is representative of four.

Expression of the HERV-W Env glycoprotein at the surface membrane of primary trophoblasts. The cocultivation of primary cytotrophoblasts (T) with XC (top) indicator cells induced the formation of homotypic syncytia (f-T) after 72 h of culture. These syncytia contained only cytotrophoblast nuclei; no XC nuclei were associated with the fusion event (nf-XC). Conversely, the cocultivation of primary cytotrophoblasts with XC-RDR⁺ (bottom) indicator cells induced the formation of heterotypic syncytia (f-T/XC) containing both cytotrophoblast and XC nuclei, showing that the RDR-dependent fusogenic HERV-W Env glycoprotein was expressed at the surface membrane of primary cytotrophoblasts. Anti-β-galactosidase antibody specifically indicated the XC cell nuclei and antidesmoplakin antibody specifically indicated the syncytiotrophoblasts. Nuclei of both cell types were labeled by DAPI (blue immunofluorescence).

Specific inhibition of HERV-W env expression reduces syncytium formation and hCG secretion. After 5 h of culture, the primary cytotrophoblasts were transfected with random (Scrambled) or HERV-W specific (Antisense) oligonucleotides. (A) Western blot analysis of Env glycoprotein in the cell lysates in the absence (Scrambled) or presence (Antisense) of HERV-W- specific oligonucleotides. Detection was done with the anti-SU polyclonal antibody and standardization using an anti-actin monoclonal antibody. (B) hCG titration in the absence (Scrambled) or presence (Antisense) of HERV-W-specific oligonucleotides. (C) After 48 h of culture, the fusion activity of the envelope glycoproteins was determined. The cells were fixed, immunostained with anti-desmoplakin monoclonal antibody, and counterstained with DAPI. Mononuclear cells were counted, and the fusion index (7) was determined as (N − S)/T, where N is the number of nuclei in the syncytia, S is the number of syncytia, and T is the total number of nuclei counted. Results are expressed as percentages of the fusion indices. (D) Large syncytia were observed using random oligonucleotide (scrambled), and smaller ones were observed using HERV-W-specific oligonucleotides (antisense). The nucleus distribution was evaluated as followed: 100 syncytia were scored after staining, and the nuclei were counted in each syncytium. Data from one representative experiment are expressed as the distribution of syncytia as a function of the number of nuclei per syncytium. Three coverslips incubated in separate wells of a six-well microplate were observed (means and SEM) for each incubation condition. *, P < 0.05.

Expression of the HERV-W Env glycoprotein at the apical syncytiotrophoblast microvillus membrane. (A) Detection of the Env glycoprotein with the 6A2B2 monoclonal antibody on the external face of the syncytiotrophoblast (brown arrows) on term and 13-week normal placenta sections. (B) Double labeling on a serial section of a 10-week-gestation normal placenta with anti-Env (upper and lower image, brown arrows) and antidesmoplakin (lower image, red arrow) antibodies. Desmoplakin, a protein of the desmosomiale plaque involved in intercellular junctions, is absent from the syncytiotrophoblast fused tissue and lines the plasmatic membranes of the cytotrophoblasts. Note the presence of maternal red blood cells (Mrbc) in the intervillous space.

A factor regulating primary trophoblast differentiation also regulates HERV-W env mRNA and protein expression. (A) After 24 h of culture, the primary cytotrophoblasts were incubated for 48 h in the presence of 0.1 mM 8-bromo-cAMP. At 72 h, the cells were fixed and immunostained with anti-desmoplakin monoclonal antibody (green fluorescence). Nuclei were labeled with DAPI (blue fluorescence). Large syncytia were observed in the control and with the cAMP treatment. (B) HERV-W and hCGβ mRNA expression after 72 h of cytotrophoblast culture. mRNA data are expressed as the level of each marker mRNA normalized by Po mRNA levels. Three culture dishes were pooled for each determination, and transcripts were assayed in duplicate. (C) Levels of hCG (expressed in milli-international units per milliliter) secreted at the indicated time into the culture medium of control cells or cells treated with 0.1 mM 8-bromo-cAMP. (D) Western blot analysis of HERV-W Env expression in cell lysates of cells treated with cAMP or left untreated (detection by the anti-SU polyclonal antibody). **, P < 0.01; ***, P < 0.001.

(A) Oligonucleotide uptake by primary cytotrophoblasts. After 5 h of culture, cytotrophoblasts were incubated with FITC-labeled scrambled oligonucleotide for 1, 2, 24, and 48 h. For each time point, cells were washed three times in PBS, fixed, and analyzed by fluorescence microscopy. Nuclei were stained in blue by DAPI. Intensely fluorescent green cells have internalized the scrambled antisense oligonucleotide (FITC). (B) Evaluation of Env-W antisense oligonucleotide. After 5 h of culture, cytotrophoblasts were incubated with three specific env antisense oligonucleotides (Env-W 744-766, Env-W 750-770, and Env-W 758-777) or a random oligonucleotide (scrambled) or without oligonucleotide (control). After 48 h of culture, proteins were extracted and Env glycoprotein expression was analyzed by Western blotting using an anti-SU polyclonal antibody and standardized using an antiactin monoclonal antibody. The most efficient reduction of Env expression was obtained with the Env-W 758-777 antisense oligonucleotide.