Abstract
Acrosomal exocytosis is a calcium-regulated exocytosis that can be triggered by PKC activators. The involvement of PKC in acrosomal exocytosis has not been fully elucidated, and it is unknown if MARCKS, the major substrate for PKC, participates in this exocytosis. Here, we report that MARCKS is expressed in human spermatozoa and localizes to the sperm head and the tail. Calcium- and phorbol ester-triggered acrosomal exocytosis in permeabilized sperm was abrogated by different anti-MARCKS antibodies raised against two different domains, indicating that the protein participates in acrosomal exocytosis. Interestingly, an anti-phosphorylated MARCKS antibody was not able to inhibit secretion. Similar results were obtained using recombinant proteins and phospho-mutants of MARCKS effector domain (ED), indicating that phosphorylation regulates MARCKS function in acrosomal exocytosis. It is known that unphosphorylated MARCKS sequesters PIP2. This phospholipid is the precursor for IP3, which in turn triggers release of calcium from the acrosome during acrosomal exocytosis. We found that PIP2 and adenophostin, a potent IP3-receptor agonist, rescued MARCKS inhibition in permeabilized sperm, suggesting that MARCKS inhibits acrosomal exocytosis by sequestering PIP2 and, indirectly, MARCKS regulates the intracellular calcium mobilization. In non-permeabilized sperm, a permeable peptide of MARCKS ED also inhibited acrosomal exocytosis when stimulated by a natural agonist such as progesterone, and pharmacological inducers such as calcium ionophore and phorbol ester. The preincubation of human sperm with the permeable MARCKS ED abolished the increase in calcium levels caused by progesterone, demonstrating that MARCKS regulates calcium mobilization. In addition, the phosphorylation of MARCKS increased during acrosomal exocytosis stimulated by the same activators. Altogether, these results show that MARCKS is a negative modulator of the acrosomal exocytosis, probably by sequestering PIP2, and that it is phosphorylated during acrosomal exocytosis.
Highlights
The acrosomal exocytosis is a calcium-regulated secretion required for physiological fertilization in mammalian sperm [1]
In living sperm, using a permeable peptide corresponding to Myristoylated alanine-rich C kinase substrate (MARCKS) effector domain (ED), we show that MARCKS inhibits exocytosis and, in addition, abrogates calcium mobilization induced by progesterone
Our results show that MARCKS is a negative modulator in acrosomal exocytosis, which is phosphorylated during acrosomal exocytosis, and regulates calcium mobilization
Summary
The acrosomal exocytosis is a calcium-regulated secretion required for physiological fertilization in mammalian sperm [1]. During this synchronized and tightly regulated process, the outer membrane of the acrosome and the plasma membrane fuse at multiple sites in the anterior region of the sperm head and, as a result, the acrosome content is released. It has been documented that phorbol esters, known PKC activators, trigger acrosome exocytosis in different species [3,4,5,6], and that levels of DAG, a natural agonist of PKC, increase when human sperm are stimulated by progesterone or the calcium ionophore A23187 [7]. The expression of MARCKS in mature sperm is unclear
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