Sphingosine-1-phosphate, regulated by FSH and VEGF, stimulates granulosa cell proliferation

Abstract Sphingosine-1-phosphate (S1P) is a bioactive polar sphingolipid which stimulates proliferation, growth and survival in various cell types. In the ovary S1P has been shown protect the granulosa cells and oocytes from insults such as oxidative stress and radiotherapy, and S1P concentrations are greater in healthy than atretic large follicles. Hence, we postulate that S1P is fundamental in follicle development and that it is activated in ovarian granulosa cells in response to FSH and VEGF. To test this hypothesis we set out: i) to evaluate the effect of FSH and VEGF on S1P synthesis in cultured bovine granulosa cells and ii) to analyse the effect of S1P on proliferation and survival of bovine granulosa cells in vitro . Seventy five thousand bovine granulosa cells from healthy medium-sized (4–7 mm) follicles were cultured in 96-well plates in McCoy’s 5a medium containing 10 ng/mL of insulin and 1 ng/mL of LR-IGF-I at 37 °C in a 5% CO 2 /air atmosphere at 37 °C. Granulosa cell production of S1P was tested in response to treatment with FSH (0, 0.1, 1 and 10 ng/mL) and VEGF (0, 0.01, 0.1, 1, 10 and 100 ng/mL) and measured by HPLC. Granulosa cells produced S1P at 48 and 96 h, with the maximum production observed with 1 ng/mL of FSH. Likewise, 0.01 ng/mL of VEGF stimulated S1P production at 48, but not 96 h of culture. Further, the granulosa cell expression of sphingosine kinase-1 (SK1), responsible for S1P synthesis, was demonstrated by Western blot after 48 h of culture. FSH increased the expression of phosphorylated SK1 (P