Requirement of protein tyrosine kinase and phosphatase activities for human sperm exocytosis.
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.
Tomes
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CN
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
Roggero
C M
CM
De Blas
G
G
Saling
P M
PM
Mayorga
L S
LS
D000177:Acrosome / Q000378:metabolism; D020101:Acrosome Reaction / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D013094:Spermatozoa / Q000187:drug effects / Q000201:enzymology*; D014638:Vanadates / Q000494:pharmacology; D020826:rab3A GTP-Binding Protein / Q000378:metabolism
C076715:pervanadate; D014638:Vanadates; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases; D020826:rab3A GTP-Binding Protein; D002118:Calcium