BACKGROUND: The role of prolactin in the regulation of ovarian folliculogenesis and corpus luteal function and in particular its relationship to atresia in these structures is as yet unclear. We established a model of apoptosis in which to examine the actions of prolactin. METHOD: Granulosa cells collected from IVF-flush were cultured at 0.1-0.3 x 10(6) cells/well in growth media for 48 h, placed into serum-free media for 24 h prior to dosing for 24 h. Dose responses to C2-ceramide and prolactin were performed. Cells were then treated with an apoptotic dose of C2-ceramide alone, prolactin (100 ng/ml) alone or a combination of the two. Cell death was assessed by Trypan Blue cell counting and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl Blue] assay and apoptosis confirmed by morphological assessment and flow cytometry. RESULTS: C2-ceramide (0-40 micro mol/l) induced a dose-dependent increase in cell death (63.8% increase at 40 micro mol/l) and, morphologically, cells exhibited classical features of apoptosis. Prolactin alone had no effect on metabolic activity or total cell number. On co- incubation, prolactin alone had no effect on cell death, whereas C2-ceramide induced an approximately 62.6% increase in apoptosis, which was inhibited in the presence of prolactin. CONCLUSIONS: Prolactin may contribute significantly to early corpus luteum formation and survival by acting as a potent antiapoptotic factor for human granulosa cells.
BACKGROUND: The role of prolactin in the regulation of ovarian folliculogenesis and corpus luteal function and in particular its relationship to atresia in these structures is as yet unclear. We established a model of apoptosis in which to examine the actions of prolactin. METHOD: Granulosa cells collected from IVF-flush were cultured at 0.1-0.3 x 10(6) cells/well in growth media for 48 h, placed into serum-free media for 24 h prior to dosing for 24 h. Dose responses to C2-ceramide and prolactin were performed. Cells were then treated with an apoptotic dose of C2-ceramide alone, prolactin (100 ng/ml) alone or a combination of the two. Cell death was assessed by Trypan Blue cell counting and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl Blue] assay and apoptosis confirmed by morphological assessment and flow cytometry. RESULTS:C2-ceramide (0-40 micro mol/l) induced a dose-dependent increase in cell death (63.8% increase at 40 micro mol/l) and, morphologically, cells exhibited classical features of apoptosis. Prolactin alone had no effect on metabolic activity or total cell number. On co- incubation, prolactin alone had no effect on cell death, whereas C2-ceramide induced an approximately 62.6% increase in apoptosis, which was inhibited in the presence of prolactin. CONCLUSIONS:Prolactin may contribute significantly to early corpus luteum formation and survival by acting as a potent antiapoptotic factor for human granulosa cells.