Stimulatory effect of luteinizing hormone, insulin-like growth factor-1, and epidermal growth factor on vascular endothelial growth factor production in cultured bubaline luteal cells

Abstract

The purpose of this study was to evaluate the temporal (24, 48, and 72 hours) and dose-dependent (0, 5, 10, and 100 ng/mL of LH, insulin-like growth factor 1 [IGF-1], and EGF) in vitro expression and secretion patterns of vascular endothelial growth factor (VEGF) in luteal cell culture during different stages of estrous cycle in water buffaloes. Corpus luteum samples from ovaries of early luteal phase (ELP; Days 1-4), midluteal phase (Days 5-10), and late luteal phase (Days 11-16) were collected from a local slaughterhouse. The samples were then processed and cultured in (serum containing) appropriate cell culture medium and incubated separately with three factors (LH, IGF-1, or EGF) at the previously mentioned three dose-duration combinations. At the end of the respective incubation periods, VEGF was assayed in the spent culture medium by ELISA, whereas the cultured cells were used for VEGF mRNA expression by quantitative real-time polymerase chain reaction. The results of the present study disclosed dose- and time-dependent stimulatory effects of LH, IGF-1, and EGF on VEGF production in bubaline luteal cells. The VEGF expression and secretion from the cultured luteal cells were highest during the ELP, intermediate in the midluteal phase, and lowest in the late luteal phase of the estrous cycle for all the three tested factors. Comparison of the results of the three treatments depicted EGF as the most potent stimulating factor followed by IGF-1 and LH. Immunocytochemistry findings in luteal cell culture of ELP agreed with the VEGF expression and secretion. In conclusion, mRNA expression, protein secretion, and immunolocalization of VEGF data clearly indicated for the first time that LH, IGF-1, and EGF play an important role in stimulating luteal angiogenesis in buffalo CL. The highest expression and secretion of VEGF in the ELP might be associated with the development of blood vessels in early growth of CL, which in turn gets augmented by the aforementioned factors emphasizing their regulatory role in luteal angiogenesis. Further studies are however necessary to divulge more information on other factors which regulate VEGF secretion in bubaline CL and the synergistic effects existing among such growth factors.

Keywords: Buffalo; CL; EGF; Insulin-like growth factor 1; LH; Vascular endothelial growth factor.