Reactive oxygen species and protein kinases modulate the level of phospho-MEK-like proteins during human sperm capacitation.
Capacitation is an essential process by which spermatozoa acquire fertilizing ability. Reactive oxygen species (ROS), protein kinase A (PKA), protein kinase C (PKC), protein tyrosine kinases (PTKs), and the extracellular signal-regulated protein kinase (ERK or mitogen-activated protein kinase [MAPK]) pathway regulate sperm capacitation. Our aim was to evaluate the phosphorylation of MEK (MAPK kinase or MAP2K) or MEK-like proteins in human sperm capacitation and its modulation by ROS and kinases. Immunoblotting using an anti-phospho-MEK antibody indicated that the phosphorylation of three protein bands (55, 94, and 115 kDa) increased in spermatozoa treated with fetal cord serum ultrafiltrate (FCSu), BSA, or isobutylmethylxanthine plus dibutyryl cAMP as capacitating agents. These phospho-MEK-like proteins are localized along the sperm flagellum. The MEK-inhibitors PD98059 and U126 prevented this phosphorylation, suggesting that these proteins are MEK-like proteins. The ROS scavengers prevented, and the addition of H(2)O(2) or spermine-NONOate (nitric oxide donor) triggered, the increase of phospho-MEK-like proteins. The capacitation-related increases in phospho-MEK-like proteins induced by FCSu, H(2)O(2), and spermine-NONOate were similarly modulated by PKA, PKC, and PTK, suggesting ROS as mediators in this phenomenon. These results indicate that phospho-MEK-like proteins are modulated by ROS and kinases and probably represent an intermediary step between the early events and the late tyrosine phosphorylation associated with capacitation.
O'Flaherty
Cristián
C
Urology Research Laboratory, Royal Victoria Hospital and Faculty of Medicine, McGill University, Montréal, Québec, Canada. coflaher@yahoo.com
de Lamirande
Eve
E
Gagnon
Claude
C
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D005312:Fetal Blood / Q000502:physiology; D005419:Flavonoids / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013075:Sperm Capacitation / Q000502:physiology*; D013094:Spermatozoa / Q000201:enzymology*
D005419:Flavonoids; D016166:Free Radical Scavengers; D020030:Nitric Oxide Donors; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D003994:Bucladesine; D011494:Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D015056:1-Methyl-3-isobutylxanthine