Reproduction Abstracts

Introduction: The formation of a functional corpus luteum (CL) is integral to the establishment and maintenance of pregnancy. The development of the CL requires tightly regulated angiogenesis, which is controlled by a plethora of pro and anti-angiogenic factors. Transforming growth factor B (TGFB) has been identified as a potential mediator of this process. This study tested the hypothesis that TGFB would adversely impact on endothelial cell (EC) development and reduce the steroidogenic capacity of bovine luteal cells in vitro.

Methods: Bovine luteal cells from early CL (n=4) were cultured in a physiologically relevant system and treated with TGFB (0, 1, 10 ng/ml). Treatment commenced on day 1 or 5, cells were fixed 4 days later. Von Willebrand factor, VE-cadherin and smooth muscle actin immunohistochemistry were utilised to identify areas of EC and mural cell growth. Total area and perimeter of EC networks as well as the number of individual EC clusters were quantified by image analysis. Spent culture media was collected for measurement of progesterone and fibroblast growth factor 2 (FGF2) concentrations by ELISA.

Results and discussion: TGFB reduced (P<0.001) the area, perimeter and number of EC networks formed at both time points. Indeed, these were completely abolished by TGFB at 10 ng/ml. Similar effects of TGFB were observed on VE-cadherin EC networks, with a clearly visible reduction in the number and size of networks throughout culture at both TGFB doses. TGFB appeared to increase mural cell growth in a dose dependent manner at both time points, however this was not quantified. Spent media progesterone concentrations were decreased tenfold by TGFB on both day 5 and 9 (P<0.001). However, TGFB had no impact on FGF2 concentrations on days 5 and 9. In conclusion, TGFB inhibited luteal angiogenesis and progesterone production whilst promoting mural cell development in vitro.