Endocrine Abstracts (2001) 2 OC26

PROLACTIN PROTECTS AGAINST CERAMIDE (C2)-INDUCED APOPTOSIS IN HUMAN OVARIAN GRANULOSA CELLS (GC)

CM Perks1, RJ Wright2, HD Mason2 & JMP Holly1


1Division of Surgery, Level 7, Bristol Royal Infirmary, Bristol, BS2 8HW, UK.; 2Departments of OB/Gyn and Physiology, St. George's Hospital Medical School, London, SW17 ORE, UK.


The role of prolactin in the regulation of ovarian folliculogenesis and in particular it's relationship to follicular atresia is as yet unclear; although a decline in prolactin concentrations in bovine follicular fluid appears to be related to morphological signs of atresia. In addition to the pituitary and endometrium sources of prolactin, there is also a local ovarian source of prolactin. Hence a potential autocrine mechanism of prolactin action may exist in the ovary. We investigated if prolactin could modulate C2-induced apoptosis in human ovarian GC. Cells were collected from IVF-flush and plated at 0.1-0.3 x 106 cells/well. Cells were incubated in growth media for 48 hrs, placed into serum-free media (SFM) for 24 hrs prior to dosing in SFM for 24 hrs. Dose responses to C2 and prolactin were performed and then cells were treated with an apoptotic dose of C2 alone, prolactin (100 ng/ml) alone or a combination of C2 and prolactin. Cell death was assessed by trypan blue cell counting and MTT assay and apoptosis confirmed by morphological assessment and flow cytometry. C2 (0-20 uM) induced a dose-dependent decrease in MTT activity, concomitant with a dose-dependent increase in cell death, whereas prolactin (0-200 ng/ml) had no effect on the metabolic activity or death of the cells. Morphologically cells exhibited the classical features of apoptosis including membrane blebbing and cell shrinkage. In the coincubation experiments, prolactin alone had no effect on cell death, whereas C2 induced an approximate 4 fold increase in apoptosis from 10 % up to 40%. This increase in cell death triggered by C2 was significantly (P<0.01) inhibited by 25 % in the presence of prolactin. We have determined that prolactin may fine-tune the process of folliculogenesis by acting as a potent survival factor for human ovarian GC. This work was supported by the Wellcome Trust.

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