Fig. 1: Detection of secreted proteins in primary cultures of mouse trophoblast.

a Workflow for the detection of secreted proteins in primary cultures of trophoblast from mice at day 16 of pregnancy. b Visualization of EGFP (Tpbpa-Cre-EGFP reporter; green) by fluorescence microscopy to identify the Percoll gradient layer containing trophoblast endocrine cells. Cells in layers were counterstained with Hoechst dye (blue) and photographed at magnification ×10 (scale bar 3 mm). c Primary cell culture viability (determined by XTT; blue line), cell death (LDH release levels; red line), trophoblast density (Krt18 gene expression; black line), and apoptosis (p53 and Bax gene expression; purple and orange lines, respectively) from time 0 to 120 h. d Proportion of junctional zone (Tpbpa gene expression; dark pink bar) and labyrinthine zone (Mct4 gene expression; light blue bar) trophoblast at 0 and 48 h. e Relative abundance of the three junctional zone endocrine cell types, spongiotrophoblast, glycogen cells, and giant cells (gene expression of Prl8a8, Gjb3, and Hand1, respectively and indicated by purple, green, and light blue bars, respectively) at 0 and 48 h of culture. f Representative images of cells stained in situ using RNAscope probes against; Tpbpa, Prl8a8, Gjb3, and Hand1 to visualize trophoblast endocrine cells, spongiotrophoblast (SpT), glycogen cells (Gly), and giant cells (TGC), respectively. g Pipeline and results of the analysis of proteins detected by mass spectrometry in cultured trophoblast and their conditioned medium including conversion to RNA sequences and overlay with published RNA data for the mouse and human placenta. Secreted proteins identified using SignalP and combined gene ontology (GO) terms: extracellular region, extracellular exosome, extracellular region parts, and signal IP. h, i Pathway over-representation analysis using Reactome pathway by STRING V.11 for the 158 and 257 secreted placental proteins in cultured trophoblast and their conditioned medium, respectively that are expressed by both mouse and human placenta. XTT, LDH, and gene expression data relative to geometric mean of three housekeeping genes: Gapdh, RPII, and Hprt are presented as mean ± SEM and expressed relative to expression at time 0 h. Asterisks denote statistical significance versus time 0 h, using two-way ANOVA (b) or t-test (c, d), **p < 0.01, ****p < 0.001, n = 6–10 of four pooled litters.