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Increased Activity of the Human Sperm Tyrosine Kinase SRC by the cAMP-Dependent Pathway in the Presence of Calcium¹

Département d'Obstétrique/Gynécologie, Centre de Recherche en Biologie de la Reproduction, Université Laval and Ontogénie et Reproduction, Centre de recherche du Centre Hospitalier Universitaire de Québec-Centre Hospitalier Université Laval (CHUQ-CHUL), Québec, Québec, Canada G1V 4G2

ABSTRACT

SRC-related tyrosine kinases are suggested to play a role in the increase of sperm protein phosphotyrosine content that occurs during capacitation. In our laboratory, we previously demonstrated that the SRC-related tyrosine kinase YES1 (also known as c-YES) is present in human spermatozoa. However, since it is negatively regulated by Ca²⁺, whose intracellular concentration increases during capacitation, another kinase would most likely be involved in the capacitation-related increase in sperm protein tyrosine phosphorylation. The present study represents the first direct assessment of SRC tyrosine kinase activity in ejaculated mammalian sperm. By immunohistochemistry on human testis sections, it is clearly shown that SRC is expressed during spermatogenesis, mainly in round and elongating spermatids. Using an indirect immunofluorescence approach, SRC is detected in the acrosomal region of the head and in the sperm flagellum of ejaculated sperm. This tyrosine kinase is associated with the plasma membrane and with cytoskeletal elements, as suggested by its partial solubility in nonionic detergents. Despite its partial solubility, SRC kinase activity was assayed after immunoprecipitation using acid-denatured enolase as a substrate. It is clearly demonstrated that SRC activity is inhibited by SU6656 and PP1, selective SRC family tyrosine kinase inhibitors, and activated in a Ca²⁺-dependent manner. Furthermore, it is shown that SRC is activated in a cAMP/PRKA-dependent manner; SRC coimmunoprecipitates with the catalytic subunit of the cAMP-dependent protein kinase (PRKAC) and is phosphorylated by this latter kinase, resulting in an increase in enolase phosphorylation. All these results support the involvement of the tyrosine kinase SRC in the increase in sperm protein phosphotyrosine content observed during capacitation.

calcium, cAMP, gamete biology, kinases, PRKAC, sperm capacitation, spermatozoa, SRC, tyrosine phoshorylation

¹Supported by grants from the Canadian Institutes for Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada (NSERC) to P.L. C.L. is supported by a studentship from the CIHR. P.L. holds a senior chercheur-boursier scholarship from the Fonds de la Recherche en Santé du Québec (FRSQ).

Correspondence: ²Pierre Leclerc, Ontogénie et Reproduction, Centre de recherche du CHUL, 2705 Boul. Laurier, Sainte-Foy T1-49, PQ, Canada G1V 4G2. FAX: 418 654 2765; e-mail: pierre.leclerc{at}crchul.ulaval.ca