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Requirement of protein tyrosine kinase and phosphatase activities for human sperm exocytosis
Authors:Tomes C N  Roggero C M  De Blas G  Saling P M  Mayorga L S
Institution:Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, CC56, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina. ctomes@fcm.uncu.edu.ar
Abstract:The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli, sperm undergo calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Protein tyrosine phosphorylation and dephosphorylation are a mechanisms by which multiple cellular events are regulated. Here we report that calcium induces tyrosine phosphorylation in streptolysin O (SLO)-permeabilized human sperm. As expected, pretreatment with tyrphostin A47-a tyrosine kinase inhibitor-abolishes the calcium effect. Interestingly, the calcium-induced increase in tyrosine phosphorylation has a functional correlate in sperm exocytosis. Masking of phosphotyrosyl groups with a specific antibody or inhibition of tyrosine kinases with genistein, tyrphostin A47, and tyrphostin A51 prevent the acrosome reaction. By reversibly sequestering intra-acrosomal calcium with a photo-inhibitable chelator, we show a requirement for protein tyrosine phosphorylation late in the exocytotic pathway, after the efflux of intra-acrosomal calcium. Both mouse and human sperm contain highly active tyrosine phosphatases. Importantly, this activity declines when sperm are incubated under capacitating conditions. Inhibition of tyrosine phosphatases with pervanadate, bis(N,N-dimethylhydroxoamido)hydroxovanadate, ethyl-3,4-dephostatin, and phenylarsine oxide prevents the acrosome reaction. Our results show that both tyrosine kinases and phosphatases play a central role in sperm exocytosis.
Keywords:Acrosome reaction  Calcium  Capacitation  Exocytosis  Fusion  Kinase  Phosphatase  Sperm  Tyrosine phosphorylation
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