PARENT SESSION
Follicular Development

CENTRAL ROLE OF MITOGEN-ACTIVATED PROTEIN KINASES IN THE PROLIFERATION OF GRANULOSA CELLS.

Ndjountche, Liliane¹, May, Jeffrey¹, Davis, John^1,2, ^{1 2}

ABSTRACT- Granulosa cell (GC) proliferation is necessary for the development of mammalian ovarian follicles. Epidermal growth factor (EGF) has been shown to be an important regulator of GC proliferation in vitro. The extracellular signal-regulated kinases (ERK) members of the mitogen-activated protein kinase family have been implicated in the regulation of cell proliferation and cell survival. We have shown previously that activation of the EGF receptor activates a Ras/Raf/MEK-1/ERK signaling cascade in porcine granulosa cells. In this report we provide evidence that ERKs may be a crucial signaling intermediate governing GC proliferation. Porcine GC were isolated from 1-4 mm antral follicles and allowed to attach to plastic dishes. The cultures were treated with control media +/- various growth factors (EGF, 10 ng/ml; IGF-I, 50 ng/ml), fetal calf serum (5% FCS), or the protein kinase C (PKC) activator PMA (10 nM). Proliferation was determined by counting cells after trypsin/EDTA treatment. ERK activation was measured by western blot using activated-state-specific antibodies and protein kinase assays. Treatment with EGF and FCS resulted in 10 fold increases in proliferation. Treatment with EGF and IGF-I or PMA resulted in 2-4 fold increases in proliferation. Treatment with growth factors, FCS and PMA acutely increased ERK activation. Furthermore, ERK signaling was evident during the active proliferation of GC. Treatment with the EGF receptor tyrosine kinase inhibitor AG1478 (100 nM) prevented EGF-induced ERK activation and blocked GC proliferation. We also observed that treatment of GC with the MEK-1 inhibitor PD98059 (0-25 M) inhibited ERK activation and caused concentration dependent reductions in GC proliferation (ED50 13 M). Treatment with PD98059 did not increase apoptosis of GC. In other studies incubation with the specific PKC inhibitor chelerythrine chloride (100 nM) blocked PMA-induced ERK activation and GC proliferation. These studies suggest that ERK activation is required for the proliferation of GC. Supported by the VA, USDA 99-03453, NIH HD38813.

KEY WORDS: proliferation, mitogen activated protein kinase, granulosa cells, EGF receptor tyrosine kinase