title: A flow cytometric assay for global estimation of tyrosine phosphorylation associated with capacitation of spermatozoa from two marsupial species, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula).
abstract: The phosphorylation of tyrosine residues in cellular proteins is a major signal transduction event during sperm capacitation. In this study protein phosphorylation was monitored using a fluorescein isothiocyanate (FITC)-labeled antiphosphotyrosine monoclonal antibody and a flow cytometric procedure optimized for sperm. Using this technique, the correlation between tyrosine phosphorylation and sperm capacitation was examined in two marsupial species, the brushtail possum and the tammar wallaby and compared with that of ram spermatozoa. The levels of tyrosine phosphorylation in sperm from all three species were increased by the addition of cyclic AMP (cAMP) and vandate, a phosphotyrosine phosphatase inhibitor and were decreased by the addition of the phosphotyrosine kinase inhibitor, staurosporine. Oviductal conditioned media (CM) induced a progressive increase in tyrosine phosphorylation in both marsupial species and also induced morphological transition from a streamlined to a 'T'-shape configuration in brushtail possum spermatozoa but not in tammar wallaby spermatozoa. Transition to the 'T'-shape orientation associated with capacitation in marsupial spermatozoa was observed by 2 h of incubation in both species when tyrosine phosphorylation was increased by higher levels of cAMP i.e. 5 mM dibutyryl cAMP plus 3 mM pentoxyphylline. Thus the tyrosine phosphorylation trigger with CM may differ in these two marsupial species. Ram sperm tyrosine phosphorylation could be increased by addition of lower levels of cAMP (1 mM). These results support the finding that tyrosine phosphorylation is associated with sperm capacitation in marsupials. Similar results were obtained by using SDS PAGE/Western blot analysis of tyrosine phosphorylation in the brushtail possum spermatozoa. The specificity, efficiency and sensitivity of the procedure described here make it applicable for routine assessment of capacitation in large numbers of samples and in other species.
authors.affiliation: Cooperative Research Center for Conservation and Management of Marsupials, Macquarie University, NSW 2109, Australia. k.sidhu@unsw.edu.au
authors.forename: K S
authors.initials: KS
authors.lastname: Sidhu
authors.forename: K E
authors.initials: KE
authors.lastname: Mate
authors.forename: T
authors.initials: T
authors.lastname: Gunasekera
authors.forename: D
authors.initials: D
authors.lastname: Veal
authors.forename: L
authors.initials: L
authors.lastname: Hetherington
authors.forename: M A
authors.initials: MA
authors.lastname: Baker
authors.forename: R J
authors.initials: RJ
authors.lastname: Aitken
authors.forename: J C
authors.initials: JC
authors.lastname: Rodger
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018920:Coculture Techniques; D000242:Cyclic AMP / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D007614:Macropodidae; D008297:Male; D008394:Marsupialia / Q000378:metabolism*; D009893:Opossums; D010057:Oviducts / Q000378:metabolism; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D012756:Sheep; D015398:Signal Transduction / Q000502:physiology*; D013075:Sperm Capacitation / Q000502:physiology*; D013094:Spermatozoa / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D014638:Vanadates; D014443:Tyrosine; D000242:Cyclic AMP; D017027:Protein Tyrosine Phosphatases; D019311:Staurosporine