Tyrosine phosphorylation of a 38-kDa capacitation-associated buffalo (Bubalus bubalis) sperm protein is induced by L-arginine and regulated through a cAMP/PKA-independent pathway.
The aim of the present study was to determine the effect of L-arginine on nitric oxide (NO*) synthesis, capacitation and protein tyrosine phosphorylation in buffalo spermatozoa. Ejaculated buffalo spermatozoa were capacitated in the absence or presence of heparin, or L-arginine or N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS) for 6 h. Capacitating spermatozoa generated NO* both spontaneously and following stimulation with L-arginine and L-NAME quenched such L-arginine-induced NO* production. Immunolocalization of NOS suggested for existence of constitutive NOS in buffalo spermatozoa. L-Arginine (10 mm) was found to be a potent capacitating agent and addition of L-NAME to the incubation media attenuated both L-arginine and heparin-induced capacitation and suggested that NO* is involved in the capacitation of buffalo spermatozoa. Two sperm proteins of M(r) 38 000 (p38) and 20 000 (p20) were tyrosine phosphorylated extensively by both heparin and L-arginine. Of these, the tyrosine phosphorylation of p38 was insensitive to both induction by cAMP agonists as well as inhibition by a protein kinase A (PKA) inhibitor. Further, most of these L-arginine-induced tyrosine phosphorylated proteins were localized to the midpiece and principal piece regions of flagellum of capacitated spermatozoa and suggested that sperm flagellum takes active part during capacitation. These results indicated that L-arginine induces capacitation of buffalo spermatozoa through NO* synthesis and tyrosine phosphorylation of specific sperm proteins involving a pathway independent of cAMP/PKA.
Roy
S C
SC
Division of Animal Biochemistry, National Dairy Research Institute, Karnal 132 001, India. sudhircroy@rediffmail.com
Atreja
S K
SK
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D001120:Arginine / Q000008:administration & dosage / Q000494:pharmacology*; D003994:Bucladesine / Q000494:pharmacology; D002020:Buffaloes; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006493:Heparin / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis; D009994:Osmolar Concentration; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013075:Sperm Capacitation / Q000187:drug effects / Q000502:physiology*; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000378:metabolism / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007546:Isoquinolines; D010726:Phosphodiesterase Inhibitors; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D009569:Nitric Oxide; D014443:Tyrosine; D003994:Bucladesine; D006493:Heparin; D001120:Arginine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D015056:1-Methyl-3-isobutylxanthine; D019331:NG-Nitroarginine Methyl Ester