Gamete Biology

Reactive Oxygen Species and Protein Kinases Modulate the Level of Phospho-MEK-Like Proteins During Human Sperm Capacitation

Abstract
Capacitation is an essential process by which spermatozoa acquire fertilizing ability. Reactive oxygen species (ROS), protein kinase A (PKA), C (PKC), protein tyrosine kinases (PTK) 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 3 protein bands (55, 94 and 115 kDa) increased in spermatozoa treated with fetal cord serum ultrafiltrate (FCSu), bovine serum albumin (BSA) or isobutylmethylxanthine+dibutyryl cAMP (IBMX+dbcAMP) as capacitating agents. These phospho-MEK-like proteins are localized along the sperm flagellum. PD98059 and U126 (MEK inhibitors) prevented this phosphorylation, suggesting that these proteins are MEK-like proteins. ROS scavengers prevented, and the addition of hydrogen peroxide (H₂O₂) 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, H₂O₂ 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.

Key words: Gamete Biology • Kinases • Nitric oxide • Signal transduction • Sperm capacitation

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