Human Reproduction, Vol. 18, No. 12, 2672-2677,
December 2003
© 2003 European Society of Human Reproduction and Embryology
Prolactin acts as a potent survival factor against C2-ceramide-induced apoptosis in human granulosa cells
1 Division of Surgery, Department of Hospital Medicine, Bristol Royal Infirmary, Bristol BS2 8HW, 2 Centre for Cardiovascular Genetics, BHF Laboratories, Department of Medicine, The Rayne Building, 5 University Street, London WC1E 6JF and 3 Departments of Basic Medical Sciences and Clinical Developmental Sciences, St George’s Hospital Medical School, London SW17 0RE, UK
4 To whom correspondence should be addressed. e-mail: Claire.M.Perks{at}bristol.ac.uk
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 0.1–0.3x106 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 µmol/l) induced a dose-dependent increase in cell death (63.8% increase at 40 µmol/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 
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.
Key words: apoptosis/C2-ceramide/granulosa cells/prolactin
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