Pentoxifylline induced signalling events during capacitation of hamster spermatozoa: significance of protein tyrosine phosphorylation.
To fertilize the oocyte, mammalian spermatozoa must undergo capacitation and acrosome reaction. These events are believed to be associated with various biochemical changes primarily mediated by cAMP, Ca2+ and protein kinases. But the precise signaling mechanisms governing sperm function are not clear. To study this, we used pentoxifylline (PF), a sperm motility stimulant and a cAMP-phosphodiesterase inhibitor, during capacitation and acrosome reaction of hamster spermatozoa. PF induced an early onset of sperm capacitation and its action involved modulation of sperm cell signaling molecules viz, cAMP, [Ca2+]i and protein kinases. The PF-induced capacitation was associated with an early and increased total protein phosphorylation coupled with changes in the levels of reactive oxygen species. Protein kinase (PK)-A inhibitor (H-89) completely inhibited phosphorylation of a 29 kDa protein while PK-C inhibitor (staurosporine) did not inhibit phosphorylation. Interestingly, PF induced protein tyrosine phosphorylation of a set of proteins (Mr 45-80 K) and a greater proportion of PF-treated spermatozoa exhibited protein tyrosine phosphorylation, compared to untreated controls (82 + 9% vs 34 +/- 10%; p < 0.001); tyrosine-phosphorylated proteins were localized specifically to the mid-piece of the sperm. The profile of protein tyrosine phosphorylation was inhibitable by higher concentrations (> 0.5 mM) of tyrosine kinase inhibitor, tyrphostin A47. However, at lower (0.1-0.25 mM) concentrations, the compound interestingly induced early sperm capacitation and protein tyrosine phosphorylation, like PF. These results show that protein tyrosine phosphorylation in the mid-piece segment (mitochondrial sheath) appears to be an early and essential event during PF-induced capacitation and a regulated level of tyrosine phosphorylation of sperm proteins is critical for capacitation of hamster spermatozoa.
Seshagiri
P B
PB
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560 012, India. polani@mrdg.iisc.ernet.in
Thomas
M
M
Sreekumar
A
A
Ray
P
P
Mariappa
D
D
D020101:Acrosome Reaction / Q000187:drug effects*; D000818:Animals; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D007700:Kinetics; D008297:Male; D008647:Mesocricetus; D010431:Pentoxifylline / Q000494:pharmacology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013075:Sperm Capacitation / Q000187:drug effects*; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology; D020032:Tyrphostins / Q000494:pharmacology
D004791:Enzyme Inhibitors; D010726:Phosphodiesterase Inhibitors; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D020032:Tyrphostins; D000242:Cyclic AMP; D011494:Protein Kinases; D019311:Staurosporine; D010431:Pentoxifylline