Figure 2
cAMP via PKA inhibits PKC-dependent activation of ERK1/2 in human spermatozoa.
(A) cAMP has no effect on ERK1/2 activation in human spermatozoa. Cells were incubated with 1 mM 8-Br-cAMP for the indicated times, while PMA (25 nM, 15 min) served as a positive control. Cells were then lysed and analyzed for ERK2 activity by western blotting using an antibody for phospho-ERK1/2 (pERK). Total ERK1/2 (gERK) was detected with polyclonal antibodies as a control for sample loading. In this and subsequent figures representative blots are shown and bars are mean ± SEM from three experiments. (B) cAMP and IBMX have no effect on ERK2 activity in human sperm. Human spermatozoa were preincubated with or without 100 μM IBMX for 30 min. 8-Br-cAMP (1 mM) was then added for the indicated times (in minutes) and ERK2 activity was determined as above. (C) cAMP inhibits PMA-induced ERK1/2 activation in human spermatozoa. Human spermatozoa were incubated with or without 0.1 mM, 0.5 mM or 1 mM 8-Br-cAMP for 10 min followed by 25 nM PMA for additional 15 minutes and ERK2 activity was determined as above. (D) IBMX inhibits PMA-induced ERK1/2 activation in human spermatozoa. Human spermatozoa were preincubated with or without 100 μM IBMX for 30 minutes and PMA (25 nM) was then added for 15 min and ERK2 activity was determined as above. (E) PKA mediates the cAMP inhibition of PMA-induced ERK2 activation in human spermatozoa. Human spermatozoa were washed and preincubated with the PKA inhibitor PKI (20 μM) for 1 h, with or without 8-Br-cAMP (1 mM) for 10 min. Thereafter PMA (25 nM) was added for another 15 min. Cell lysates were analyzed for ERK2 activity by Western blotting as above. A representative blot is shown and bars are mean ± SEM. from three experiments. Means designated by * are significantly different (p < 0.05).
