This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dubé, C. Articles by Bailey, J. L. Search for Related Content PubMed PubMed Citation Articles by Dubé, C. Articles by Bailey, J. L.

The Importance of Calcium in the Appearance of p32, a Boar Sperm Tyrosine Phosphoprotein, During In Vitro Capacitation

PIERRE LECLERC

From the Centre de Recherche en Biologie de la Reproduction, ^* Département des Sciences Animales and Département d'Obstétrique et Gynécologie, Université Laval, Sainte-Foy, Québec, Canada.

Correspondence to: Dr Janice L. Bailey, Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Pavillon Paul Comtois, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4 (e-mail: janice.bailey{at}crbr.ulaval.ca).

After ejaculation, mammalian sperm must undergo a preparation period known as "capacitation" to become capable of fertilizing the oocyte. Although physiological capacitation occurs in the female genital tract, the process can be reproduced in vitro by incubation in appropriate media. However, the signaling events regulating capacitation are poorly understood, especially in boar sperm. Calcium is thought to be of fundamental importance in capacitation. Our laboratory recently identified a tyrosine-phosphorylated protein of M_r 32 000 ("p32") from boar sperm, and its appearance is closely related to capacitation. The objective of this study was to understand the mechanism regulating the appearance of our p32 tyrosine phosphoprotein. Since calcium has been linked to sperm capacitation and protein tyrosine phosphorylation in other species, we hypothesized that extracellular calcium is involved in the appearance of the p32. Sperm were incubated in either noncapacitating medium (NCM) or capacitating medium (CM) for various times. Proteins were extracted with sodium dodecyl sulfate (SDS), separated by SDS-polyacrylamide gel electrophoresis (PAGE), and then immunoblotted with an antiphosphotyrosine antibody. To assess intracellular calcium levels, fresh sperm were loaded with the fluorescent calcium indicator indo-1, and relative fluorescence was measured by flow cytometry. Analysis demonstrated that relative intracellular calcium levels increased during incubation in capacitating conditions but not in NCM, which coincides with the appearance of the p32. The p32 tyrosinephosphorylated protein appeared only in the presence of calcium, and the calcium ionophore Br-A23187 accelerated its appearance. Consistent with our hypothesis, the appearance of the p32 was inhibited by extracellular calcium chelators (ethylene glycol-bis(2-aminoethylether)-N,N,N',N',-tetraacetic acid [EGTA], EDTA, and 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid potassium salt [BAPTA-K⁺]), showing the importance of calcium in protein tyrosine phosphorylation related to capacitation in boar sperm.

     Key words: Tyrosine phosphorylation, chelator, A23187, indo-1

This article has been cited by other articles:

				M.-F. Lusignan, A. Bergeron, M.-H. Crete, C. Lazure, and P. Manjunath Induction of Epididymal Boar Sperm Capacitation by pB1 and BSP-A1/-A2 Proteins, Members of the BSP Protein Family Biol Reprod, March 1, 2007; 76(3): 424 - 432. [Abstract] [Full Text] [PDF]

				C. Dube, P. Leclerc, T. Baba, C. Reyes-Moreno, and J. L. Bailey The Proacrosin Binding Protein, sp32, Is Tyrosine Phosphorylated During Capacitation of Pig Sperm J Androl, July 1, 2005; 26(4): 519 - 528. [Abstract] [Full Text] [PDF]

				J. C. Kirkman-Brown Editorial Commentary J Androl, September 1, 2003; 24(5): 734 - 735. [Full Text] [PDF]