N OCIETY' - the Society for Reproductive Biology

EXISTENCE OF EXTRACELLULAR SIGNAL-RELATED KINASE (ERK) AND p38 MITOGENACTIVATED PROTEIN KINASE (MAPK) IN THE RAT CORPUS LUTEUM (CL).M.A. Abdo, M.A. Bogoyevitch l and A.M. DharmarajanDepartment of Anatomy & Human Biology, and lDepartment ofBiochemistry,University of Western Australia, Perth, Western Australia 6907..IntroductionThe MAPK signalling cascade has· been proposed ascritical to the events of cell survival and cell death,chiefly apoptosis, in a number of biological systems.Current evidence suggests that the selective activationof MAPK family members may be important in'determining a cell's fate. MAPK activation occurs inresponse to growth factors, heat shock, and cytokines(1). Tumor necrosis factor - alpha (TNFa) is acytokine that has been shown to activate both theapoptotic (p38 and c-JUN NH2-terminal protein kinase(JNK)), and anti-apoptotic (ERK) MAPK pathways indifferent cell systems (2). As reported previously,TNFa has been implicated in the induction ofapoptosis within the CL (3). Although themechanisms of signal transduction in TNFa-mediatedapoptosis are largely unknown. Thus our aim in thispresent study was to determine whether MAP Kinases(p38, JNK, ERK) are present in the rat CL and whetherchanges in their expression correlate with the onset ofspontaneous apoptosis and TNFa-induced apoptosis.Materials & MethodsTotal protein and DNA was isolated from pregnant rat(Day 16, Wistar) CL cultured under two experimentalconditions. CL were either incubated without trophicsupport for up to 8h in minimal essential medium(MEM: Gibco BRL) to induce spontaneous apoptosis,or 6h in MEM supplemented with 30% fetal bovineserum (FBS: Trace Biosciences) and increasingconcentrations of recombinant rat TNFa (Genzyme).Proteins were resolved by SDS-PAGE and transferredto nitrocellulose membrane (0.45Ilm). Membraneswere then incubated with the primary antibodies forERKI (Santa Cruz) and ERK2 (Santa Cruz)simultaneously, JNKI (Santa Cruz), or p38 (SantaCruz) diluted 1:1000 for Ih at room temperature. Thiswas followed by incubation with sheep anti-goat IgGsecondary antibody (Sanofi Diagnostics) for Ih atroom temperature. The reaction was visualized usingECL detection (Pierce). All washes were in Trisbuffered saline supplemented with 0.1 % Tween 20. 3'end DNA labelling was performed as describedpreviously (4). A minimum of 3 animals was used foreach experimental variable.ResultsWestern blot analysis revealed the appropriatemolecular weight bands corresponding to ERKl&2(44 & 42 kDa) and p38 MAPK (38 kDa) (Fig. 1),~nder both culture conditions. No significant changeIn the amount of protein for either p38 MAPK orERKI&2 was detected in response92to a 7-fold increase in DNA fragmentation after 8hincubation (Fig. 2) or to increasing concentrations of TNFa(2-fold increase in DNA fragmentation at 125ng/mL) (Fig.3). We were unable to demonstrate the presence of JNKthrough Western blot analysis under either of theexperimental conditions.Time.__---AFig 2 (left)Autoradiographo C'l '