Extracellular signal-regulated kinases (ERKs) pathway and reactive oxygen species regulate tyrosine phosphorylation in capacitating boar spermatozoa.
The extracellular signal-regulated kinase (ERK) family of the mitogen-activated protein kinase (MAPK) pathway is identified for the first time in boar sperm and is associated with capacitation and tyrosine phosphorylation (tyr-P). Reactive oxygen species (ROS) modulate this signal transduction. Western immunoblotting detected the ERK pathway components RAF1, MEK1/2, and ERK1/2 in extracts from fresh boar spermatozoa and determined that their phosphoprotein profiles differed in a capacitation-dependent fashion. Capacitation was accompanied by appearance of two new ERKs (158 and 161 kDa) and disappearance of others. Capacitation was verified with increased tyr-P, which was inhibited by a 30-min pre-exposure of fresh boar sperm to a xanthine/xanthine oxidase ROS-generating system prior to the capacitating incubation; ROS pre-exposure also affected the phosphorylation of RAF1, MEK1/2, and ERK1/2. Preincubating sperm with inhibitors of the ERK components with or without the ROS generator affected subsequent capacitation. Inhibiting ERK1/2 inhibited tyr-P of capacitated boar spermatozoa proteins of 172, 97, and 66 kDa (P ≤ 0.04); with ROS, this inhibition increased (P < 0.002) and tyr-P of 111 kDa declined (P < 0.028). Pre-exposure to ROS plus MEK1/2 inhibitor prevented capacitation-induced tyr-P of proteins of 187 (P < 0.01) and 112 kDa (P < 0.04) versus capacitation with or without ROS. Therefore, ERK1/2 components of the MAPK pathway significantly regulate boar sperm capacitation, and RAF1 and MEK1/2 may have some lesser influence through crosstalk with different pathways. ROS affect RAF1, MEK1/2, and ERK1/2 and could influence the sequential events of boar sperm capacitation.
Awda
Basim J
BJ
Department of Animal & Poultry Science, University of Guelph, Guelph, Ontario, Canada.
Buhr
Mary M
MM
D000818:Animals; D015153:Blotting, Western; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008297:Male; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism* / Q000494:pharmacology; D013075:Sperm Capacitation / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D034421:Sus scrofa / Q000502:physiology*; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*
D010750:Phosphoproteins; D020033:Protein Isoforms; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D014443:Tyrosine; D048049:Extracellular Signal-Regulated MAP Kinases