Reproduction Abstracts

In dairy cows, post-partum uterine inflammation caused by Gram negative bacteria (e.g. E. coli) suppresses follicular function and reduces fertility. LPS is an endotoxin that is present on outer membrane of Gram-negative bacteria. Furthermore, LPS is detected in follicular fluid (0.04–0.88 μg/ml) of cows with endometritis. This study tested the hypothesis that LPS would decrease the formation of luteal endothelial cell (EC) network and progesterone production in vitro. Luteal cells (EC, steroidogenic luteal cells, and pericytes) were enzymatically dispersed from abattoir-derived bovine corpora lutea (early or mid-luteal phase) and incubated at 39 °C in 5% CO₂. On day 1 and thereafter, cells were treated with LPS (0, 0.01, 0.1, 1 and 10 μg/ml) under basal and angiogenic-stimulated conditions (FGF2 plus VEGFA; both 1 ng/ml). Spent media was analysed for progesterone by ELISA and replaced every 2 days. On day 9, ECs were immunostained for von Willebrand factor and EC networks quantified by image analysis. In control wells, EC formed highly organised tubule-like networks, which was dramatically increased under angiogenic stimulated conditions. LPS dose-dependently decreased the total area of EC network, the number of EC clusters and branch points as well as the degree of branching per EC cluster (P<0.001), even detectably lower at 0.01 μg/ml (P<0.05). This pattern was observed under both basal and angiogenic-stimulated conditions. Surprisingly, in vitro progesterone production was unaffected by LPS but was increased under angiogenic stimulated conditions (P<0.001). In conclusion, LPS dramatically inhibited luteal endothelial cell network formation in a dose dependent manner in vitro.