Molecular Human Reproduction, Vol 3, 1037-1043, Copyright © 1997 by Oxford University Press
DY Liu and HW Baker
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.
JOURNAL ARTICLE
Protein kinase C plays an important role in the human zona pellucida- induced acrosome reaction
Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women's Hospital, Carlton, Victoria, Australia.
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