Journal of Andrology
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Published-Ahead-of-Print March 4, 2010, DOI:10.2164/jandrol.109.008383

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Bicarbonate-Dependent Serine/Threonine Protein Dephosphorylation in Capacitating Boar Spermatozoa

Fahima A. Alnagar , Paul Brennan , and Ian A. Brewis *

* To whom correspondence should be addressed. E-mail: brewisia{at}cardiff.ac.uk.

This study investigates the dynamics of serine/threonine (S/T) protein phosphorylation in sperm incubated under capacitating conditions using the boar as a model system. For the first time, this approach has identified multiple dephosphorylation events that occur in a bicarbonate-dependent fashion. Different phospho-(S/T) kinase substrate antibodies were used and dephosphorylation of five S/T phosphoproteins was observed in capacitating (C) sperm compared with non-capacitated (N) cells. Specifically, dephosphorylation of p96, p90, p64 and p55kDa proteins were detected by immunoblotting using two phospho-Akt substrate antibodies and a phospho-PKA substrate antibody. In addition, dephosphorylation of a p105kD protein was detected using a phospho-ATM/ATR substrate antibody. In contrast, no dephosphorylation was observed using a phospho-PKC substrate antibody and increased tyrosine phosphorylation of p32 and p20kDa proteins was detected in C compared with N sperm. Immunolocalisation experiments revealed subtle changes in the pattern expression as well as a reduction of phosphorylation in C sperm. Whilst sperm incubated in N medium containing dibutyryl cAMP (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX) did not show protein dephosphorylation, incubation in C medium with dbcAMP/IBMX showed dephosphorylation as well as increased phosphorylation of other proteins (p68, p51 and p29kDa). Finally, calyculin A, a phosphatase inhibitor, prevented dephosphorylation of p96, p90, p64 and p55kDa but not p105. Based on this data, we propose two pathways of protein dephosphorylation that are active during capacitation and independent of cAMP. Together, this provides direct evidence for more complex S/T phosphorylation dynamics than has been previously described for sperm undergoing capacitation.


Key words: Sperm • S/T • capacitation • mammalian • phosphorylation • signalling




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