Expression and localization of locally produced growth factors regulating lymphangiogenesis during different stages of the estrous cycle in corpus luteum of buffalo (Bubalus bubalis)

Recent experiments using expression, immunolocalization, and cell culture approaches have provided leading insights into regulation of luteal angiogenesis by different growth factor systems and its role in the function of corpus luteum (CL) in buffalo. On the contrary, lymphangiogenesis and its regulation in the CL are still poorly understood. The aim of this study was to evaluate the expression and localization of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGFD), their receptor (VEGFR3), and lymphatic endothelial marker (LYVE1) in bubaline CL during different stages of the estrous cycle and to investigate functional role of VEGFC and VEGFD in luteal lymphangeogenesis. The mRNA and protein expression of VEGFC, VEGFD, and VEGFR3 was significantly greater in mid and late luteal phases, which correlated well with the expression of LYVE1. The lymphangiogenic factors were localized in luteal cells, exclusively in the cytoplasm. Immunoreactivity of VEGFC was greater during midluteal phase and that of VEGFD was greater during the mid and late luteal phases. Luteal cells were cultured in vitro and treated for different time duration (24, 48, and 72 hours) with VEGFC and VEGFD each at 50, 100, and 150 ng/mL concentration and VEGFC with VEGFD at 100 ng/mL concentration. The temporal increase in LYVE1 mRNA expression was significant (P < 0.05) in VEGFC and VEGFC with VEGFD treatment and no significant change was seen in VEGFD treatment. Thus, it seems likely that VEGFD itself has little role in lymphangiogenesis but along with VEGFC it might have a synergistic effect on VEGFR3 receptors for inducing lymphangiogenesis. In summary, the present study provided evidence that VEGFC and VEGFD, and their receptor VEGFR3, are expressed in bubaline CL and are localized exclusively in the cell cytoplasm, suggesting that these factors have a functional role in lymphangiogenesis of CL in buffalo.