Fig 2: VEGFA mRNA and protein expression after in vitro culture of mid CL explants. Expression of (A) vascular endothelial growth factor A (VEGFA) mRNA, (B) VEGFA protein after 24 h culture: (i) no factor (C, negative control); (ii) Nodal (0.1 ng/mL); (iii) Nodal (1 ng/mL); (iv) Nodal (10 ng/mL) (n = 6). mRNA expression determined by real-time PCR, expression relative to β-2-microglobulin (B2MG) expression. Protein expression determined by western blot, upper panel: representative immunoblot; lower panel: densitometry of protein expression relative to β-actin expression. Values are expressed as means ± SEM in arbitrary units (AU). Statistical differences are marked with asterisks (*p < 0.05).

Fig 3: HIF1α mRNA and protein expression in fresh CL explants and after in vitro culture of mid CL explants. (A) Hypoxia inducible factor 1 α (HIF1α) mRNA and (B) HIF1α protein expression in early, mid and late CL explants (n = 6). Expression of (C) HIF1α mRNA and (D) HIF1α protein after 24 h culture: (i) no factor (C, negative control); (ii) Nodal (10 ng/mL); (iii) prostaglandin F2α (PGF2α, 10−7 M); and (iv) luteinizing hormone (LH, 10 ng/mL) (n = 6). (E) Nodal, HIF1α, and Alk7 protein level in early, mid and late CL explants. mRNA expression determined by real-time PCR, expression relative to β-2-microglobulin (B2MG) expression. Protein expression determined by western blot, upper panel: representative immunoblot; lower panel: densitometry of protein expression relative to β-actin expression. Values are expressed as means ± SEM in arbitrary units (AU). Statistical differences are marked with asterisks (*p < 0.05). (E) *Significant differences with regard to early CL; $significant differences with regard to mid CL (one symbol, p < 0.05; two symbols p < 0.01).

Fig 4: Nodal crosstalk with TSP1 and CD36 in mid CL explants cultured in vitro. Expression of (A) thrombospondin 1 (TSP1) mRNA, (B) TSP1 protein, (C) cluster of differentiation 36 (CD36) mRNA and (D) CD36 protein after 24 h culture: (i) no factor (C, negative control); (ii) Nodal (10 ng/mL); (iii) prostaglandin F2α (PGF2α 10−7 M); and (iv) luteinizing hormone (LH, 10 ng/mL) (n = 6). Expression of (E) TSP1 protein and (F) CD36 protein after 24 h culture: (i) no factor (C, negative control); (ii) SB (10 μM); (iii) PGF2α (10−7 M); and (iv) simultaneously SB (10 μM) and PGF2α (10−7 M) (n = 6). mRNA expression determined by real-time PCR, expression relative to b-2-microglobulin (B2MG) expression. Protein expression determined by western blot, upper panel: representative immunoblot; lower panel: densitometry of protein expression relative to β-actin expression. Values are expressed as means ± SEM in arbitrary units (AU). Statistical differences with regard to control are marked with asterisks (*p < 0.05; **p < 0.01; ***p < 0.001); Statistical differences with regard to PGF2α are marked with dollars ($p < 0.05).

Fig 5: Nodal signaling in mid CL explants cultured in vitro under hypoxia. Effect of hypoxia on (A) Nodal protein expression and (B) phosphorylation of Smad3 in mid CL explants cultured under 20% O2 or 5% O2 for 24 h (n = 4). Protein expression determined by western blot, upper panel: representative immunoblot; lower panel: densitometry of protein expression relative to β-actin expression. Values are expressed as means ± SEM in arbitrary units (AU). Statistical differences are marked with asterisks (*p < 0.05).