Protein kinase C plays an important role in the human zona pellucida- induced acrosome reaction

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Abstract
To investigate the involvement of protein kinases in signal transduction in the human zona pellucida (ZP)-induced acrosome reaction (AR), the effects of protein kinase (PK) activators, dibutyryl cAMP (PKA) and cGMP (PKG), phorbol 12-myristate 13-acetate (PMA, PKC), and the PKC inhibitor, staurosporine were studied. Sperm samples were obtained from normozoospermic men with normal sperm-ZP binding. Oocytes were obtained from other patients with failure of fertilization in vitro. Motile spermatozoa selected by a swim-up technique were pre-incubated with 2.5-20 microM PMA, 1 mM dibutyryl cAMP or cGMP, 3 mM pentoxifylline or 0.125-2.0 microM staurosporine for 30 min and then incubated with four oocytes for 2 h in human tubal fluid supplemented with bovine serum albumin. The spermatozoa bound to the ZP were dislodged by repeatedly aspirating the oocytes with a small bore pipette and the state of the AR was determined by fluorescein-labelled Pisum sativum agglutinin. Motility and movement characteristics were assessed by computer-assisted sperm analysis (CASA) after incubation of spermatozoa with PMA for 30 min and 2 h. The dibutyryl cAMP and cGMP analogues had a small positive effect (P < 0.05) but pentoxifylline had no effect on stimulating the ZP-induced AR (P > 0.05). In contrast, PMA stimulated ZP-induced AR in a marked dose-dependent manner. Only the highest concentrations (15-20 microM) of PMA significantly decreased percentage motility (P < 0.001). Doses of 2.5-15 microM of PMA significantly stimulated ZP-induced AR without decreasing motility (P < 0.001). The PKC inhibitor, staurosporine (0.125-0.25 microM) significantly inhibited ZP-induced AR without affecting motility (P < 0.001). Sperm samples from 33 normozoospermic men were used for studies of the ZP-induced AR augmented with 15 microM PMA. One sample did not show a response to PMA stimulation. Among the 14 men with low ZP-induced AR, half had normal responses to PMA and other half had low responses to PMA. In conclusion, activation or inhibition of PKC significantly increases or decreases human ZP-induced AR suggesting that PKC plays a important role in the signal transduction pathway for the physiological AR.

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