Expression and Transcriptional Regulation of Individual Pregnancy-specific Glycoprotein Genes in Differentiating Trophoblast Cells

Total PSG transcripts and proteins increase in JEG-3 cells undergoing differentiation. A) Immunostaining of fixed cells with anti-desmosomal protein (red) and nuclear counterstaining with Höechst (blue). Bar=10μm. Original magnifications: top panel ×200, bottom panel ×1000. B) Semi-quantitative RT-PCR of total PSG transcripts (tPSG). C) Western blot for total PSG proteins (100μg). The blots shown correspond to one representative experiment of three with similar results. Induction fold was calculated after dividing the density value obtained for PSG mRNA or protein expression by the value obtained for the corresponding internal control (GAPDH mRNA and α-tubulin, respectively). Basal expression was arbitrarily set as 1 and values represent the mean±SEM of three independent experiments. [For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.]

Total PSG transcripts and proteins increase in differentiated CTBs. A) Semi-quantitative RT-PCR of total PSG and βhCG in CTBs differentiated for the indicated hours. A representative experiment of three independent experiments performed from CTBs isolated from three placentas is shown. B) Western blot assay for total PSG proteins (0h, 100μg or 96h, 10μg). C and D) Immunofluorescence detection of PSG (C) or βhCG (D) proteins (green), desmosomal protein (red) and nuclei (blue) in CTBs cultured during 4, 16, 24, 48 and 96h. Asterisk, non-fused CTB expressing PSG proteins. Bar=10μm. Original magnification: ×1000. [For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.]

PSG1, PSG3, PSG5, PSG7, and PSG9 transcripts are up-regulated upon trophoblast differentiation. RT-PCR for the indicated PSG and GAPDH genes were performed with RNA from JEG-3 cells cultured (d) or not (u) in differentiating conditions for 96h, and of isolated CTBs cultured for 4h (u) or 96h (d).

Transcriptional activities of PSG constructs in JEG-3 and CTB cells. Reporter constructs with putative functional regulatory elements are shown on the left, CPE (○), RARE (□), FP1 (♢), FP3 (), FP4 (▵), (TG)n repeat (■) and GABP (⎔). Hatched symbols indicate subtle sequence differences with those previously identified in PSG5 . Mutated elements are in grey. A and B) JEG-3 cells transfected and treated (black bars) or not (grey bars) with 1μM Mtx. C, D and E) early (12h, grey bars) or late (48h, black bars) CTB cultures transfected with the indicated constructs. Results are expressed as relative luciferase activity respect to pGL3-basic (A and C) or respect to the PB3-luc activity in early CTBs (E). For easier comparison between the reporter activities of each construct in both differentiation conditions, results are expressed as activation fold respect to the undifferentiated cultures (B and D). Data from three to five experiments performed in triplicate are shown (mean±SEM). * Statistically significant difference (p<0.05) between the indicated constructs or between each construct in both cells conditions.