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Vertebrate reproductive science and technology

Cyclic-AMP initiates protein tyrosine phosphorylation independent of cholesterol efflux during ram sperm capacitation

Carmen Colas A , Peter James B , Liz Howes B , Roy Jones B , José A. Cebrian-Perez A and Teresa Muiño-Blanco A C
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A Department of Biochemistry and Molecular and Cellular Biology, School of Veterinary Medicine, University of Zaragoza, Miguel Servet, 177, 50013-Zaragoza, Spain.

B Laboratory of Molecular Signalling, Babraham Institute, Babraham, Cambridge CB22 3AT, United Kingdom.

C Corresponding author. Email:

Reproduction, Fertility and Development 20(6) 649-658
Submitted: 8 February 2008  Accepted: 21 April 2008   Published: 9 July 2008


Unlike most other species, ram spermatozoa are difficult to capacitate in vitro. Bicarbonate and Ca2+ are necessary, whereas bovine serum albumin does not appear to be obligatory. In the present investigation we have assessed (1) the ability of the cholesterol-sequestering agent, methyl-β-cyclodextrin (M-β-CD), to initiate protein tyrosine phosphorylation, and (2) the importance of phosphodiesterases (PDEs) in controlling the levels of cAMP. Results show that despite removing significant amounts of membrane cholesterol, as assessed by filipin staining, M-β-CD treatment did not stimulate major increases in protein tyrosine phosphorylation. Addition of a cocktail of PDE inhibitors (theophylline and caffeine), a phosphatase inhibitor (okadaic acid) and dibutyryl-cAMP (db-cAMP), however, stimulated specific tyrosine phosphorylation of several proteins between 30 and 120 kDa. On their own, none of the above reagents were effective but a combination of db-cAMP + PDE inhibitors was sufficient to achieve a maximal response. H-89, a protein kinase-A inhibitor, suppressed tyrosine phosphorylation significantly. Immunofluorescence revealed that the newly-phosphorylated proteins localised mainly in the sperm tail. These findings suggest that in ram spermatozoa cAMP levels are too low to initiate tyrosine phosphorylation of flagellar proteins that are indicative of the capacitation state and that this is caused by unusually high levels of intracellular PDEs.

Additional keywords: cAMP agonists, immunocytochemistry, methyl-β-cyclodextrin.


The present study was supported by the BBSRC (UK), and grants CICYT-FEDER AGL 2005–02614, CICYT-FEDER AGL 2007–061229, and DGA 2007-A26 to the University of Zaragoza, Spain. Carmen Colás was financed by a fellowship of Diputación General de Aragón (B085/2003) and CAI-UNIZAR CM12/05. The authors thank ANGRA for supplying the sires, S. Morales (Zaragoza) and members of the large animal facility at Babraham Institute for the collection of semen samples, and M. Cebrián (Zaragoza) for technical assistance with illustrations.


Aitken, R. J. , Paterson, M. , Fisher, H. , Buckingham, D. W. , and van Duin, M. (1995). Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function. J. Cell Sci. 108((Pt 5)), 2017–2025.
PubMed | White I. G., and Darin-Bennett A. (1976). The lipids of sperm in relation to cold shock. In ‘Proceedings of the 8th International Congress of Animal Reproduction & A.I., Krakow’. (Ed. Kirk-Bonagura.) pp. 951–954.

Yanagimachi R. (1994). Mammalian fertilization. In ‘The Physiology of Reproduction’. (Eds E. Knobil and J. D. Neill.) pp. 189–317. (Raven Press Ltd.: New York.)

Yancey, P. G. , Rodrigueza, W. V. , Kilsdonk, E. P. , Stoudt, G. W. , Johnson, W. J. , Phillips, M. C. , and Rothblat, G. H. (1996). Cellular cholesterol efflux mediated by cyclodextrins. Demonstration of kinetic pools and mechanism of efflux. J. Biol. Chem. 271, 16026–16034.
Crossref | GoogleScholarGoogle Scholar | PubMed |