Prolactin acts as a potent survival factor against C2-ceramide-induced apoptosis in 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.


METHODS
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.
Perks
C M
CM
Department of Hospital Medicine, Bristol Royal Infirmary, Bristol BS2 8HW, UK. Claire.M.Perks@bristol.ac.uk
Newcomb
P V
PV
Grohmann
M
M
Wright
R J
RJ
Mason
H D
HD
Holly
J M P
JM
D017209:Apoptosis / Q000187:drug effects*; D002452:Cell Count; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003338:Corpus Luteum / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D005260:Female; D005434:Flow Cytometry; D006107:Granulosa Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D011374:Progesterone / Q000032:analysis / Q000096:biosynthesis; D011388:Prolactin / Q000008:administration & dosage* / Q000032:analysis / Q000502:physiology; D013110:Sphingosine / Q000008:administration & dosage* / Q000031:analogs & derivatives*
C064769:N-acetylsphingosine; D011374:Progesterone; D011388:Prolactin; D013110:Sphingosine