Regulation of tyrosine kinase activity during capacitation in goat sperm.
Protein tyrosine phosphorylation is a key event accompanying sperm capacitation. Although this signaling cascade generates an array of tyrosine-phosphorylated polypeptides, their molecular characterization is still limited. It is necessary to differentiate the localization of the tyrosine-phosphorylated proteins in spermatozoa to understand the link between the different phosphorylated proteins and the corresponding regulated sperm function. cAMP plays a pivotal role in the regulation of tyrosine phosphorylation. The intracellular cAMP levels were raised in goat spermatozoa by the addition of the phosphodiesterase inhibitor, IBMX in conjugation with caffeine. Tyrosine phosphorylation was significantly up-regulated following treatment with these two reagents. Treatment of caudal spermatozoa with IBMX and caffeine, time dependent up-regulated phosphorylation of the protein of molecular weights 50 and 200 kDa was observed. Increased phosphorylation was observed with a combination of IBMX and caffeine treatment. Tyrosine phosphorylation in caput spermatozoa was not affected significantly under these conditions. The expression level of tyrosine kinase in sperm was examined with specific inhibitors and with anti-phosphotyrosine antibody. The indirect immunofluorescence staining was carried out on ethanol permeabilized sperm using anti-phosphotyrosine antibody. Western blot analysis was done using two separate PKA antibodies: anti-PKA catalytic and anti-PKA RIalpha. Almost no difference was found in the intracellular presence of the PKA RIalpha and RIIalpha subunits in caput and caudal epididymal spermatozoa. However, the catalytic subunit seemed to be present in higher amount in caudal spermatozoa. The results show that caprine sperm displays an enhancement of phosphorylation in the tyrosine residues of specific proteins under in vitro capacitation conditions.
Division of Molecular Medicine, Bose Institute, P-1/12, C.I.T. Scheme, VII-M, Kolakata, 700054, India.
Madhumouli
M
Chatterjee
Pinki
P
Nandi
Swatilekha
S
Ghosh
Parimal C
PC
Sen
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D045163:Antibodies, Phospho-Specific; D002110:Caffeine / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004822:Epididymis / Q000166:cytology; D006041:Goats; D008297:Male; D009928:Organ Specificity; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D013075:Sperm Capacitation / Q000187:drug effects / Q000502:physiology*; D013077:Sperm Head / Q000187:drug effects / Q000378:metabolism; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism*
D045163:Antibodies, Phospho-Specific; D010726:Phosphodiesterase Inhibitors; D010750:Phosphoproteins; D021122:Protein Subunits; D002110:Caffeine; D014443:Tyrosine; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D015056:1-Methyl-3-isobutylxanthine