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The development of the corpus luteum requires angiogenesis, and involves the complex interplay between factors such as vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). However, the relative role of these factors remains to be elucidated. This study used a new physiologically relevant mixed luteal cell culture system to test the hypotheses that: a) FGF2 and VEGFA are critical for bovine luteal angiogenesis; and b) local luteal PDGF signalling stimulates the formation of endothelial networks. Cells were treated with receptor tyrosine kinase inhibitors against VEGFA (SU1498), FGF2 (SU5402) or PDGF (AG1295) activity. After 9 days in culture, endothelial cells were immunostained for von Willebrand factor (VWF) and quantified by image analysis. Highly organised intricate endothelial networks were formed in the presence of exogenous VEGFA and FGF2. The inhibition of FGF2 activity reduced the total area of VWF staining versus controls (&gt;95%; P&lt;0.001). Inhibition of VEGF and PDGF activity reduced the endothelial network formation by more than 60 and 75% respectively (P&lt;0.05). Progesterone production increased in all treatments from day 1 to 7 (P&lt;0.001), and was unaffected by FGF2 or PDGF receptor kinase inhibition (P&gt;0.05), but was reduced by the VEGF receptor inhibitor on days 5 and 7 (P&lt;0.001). In conclusion, this study confirmed that VEGF signalling regulates both bovine luteal angiogenesis and progesterone production. However, FGF2 was crucial for luteal endothelial network formation. Also, for the first time, this study showed that local luteal PDGF activity regulates bovine luteal endothelial network formation in vitro.

FGF2 is crucial for the development of bovine luteal endothelial networks in vitro