Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2007) 14 OC7.4

ECE2007 Oral Communications Reproductive endocrinology I (7 abstracts)

Capacitation and acrosome reaction in human ejaculated spermatozoa involve activation of a novel SRC tyrosine kinase

Gabriele Varano , Adriana Lombardi , Gianni Forti , Elisabetta Baldi & Michaela Luconi

University of Florence, Dept. Clinical Physiopathology, Florence, Italy.

Tyrosine phosphorylation of proteins is one of the main processes associated with the development of some specific functions of ejaculated human spermatozoa. Although this process, as well as the identity of the phosphorylated targets, has been well characterized, only few tyrosine kinases (TKs) have been identified so far. Moreover, their roles in regulating sperm functions are still unknown.

In the present work, we report the presence and localization of Src kinase in ejaculated human spermatozoa and investigate the role played by this TK during capacitation. Immunoprecipitation and western blot analysis of protein lysates from human spermatozoa using specific anti-p60src antibodies identified a single band of about 70 kDa molecular weight. Immunofluorescence analysis of fixed and permeabilized sperm localized positivity mainly in the post-acrosomal region of sperm head and midpiece in over 80% of the sperm population. By both immunoprecipitation and immunofluorescence techniques with antibodies recognizing tyrosine phosphorylation of Src at 416 or at 527 position, which identify the active or inactive kinase respectively, we showed an increased phosphorylation in Y416 during sperm capacitation. Blocking Src activity with its inhibitor SU6656 resulted in a significant reduction in tyrosine phosphorylation of sperm proteins, in particular in the 80–115 kDa molecular weight range. Moreover, such inhibitor completely blocked progesterone-induced acrosome reaction and interfered with calcium response to progesterone evaluated in fura-2 loaded sperm. No effects on sperm motility and hyperactivation parameters resulted from incubation of sperm with SU6656. Finally, by the use of TK and PKA inhibitors (erbstatin A and H89, respectively), we demonstrated that Src activation during capacitation is dependent on tyrosine kinase but not on protein kinase A activity. In conclusion we identified a novel Src isoform in human spermatozoa and demonstrated its involvement in capacitation and acrosome reaction.

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