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1 October 2002 Identification of Ras and Its Downstream Signaling Elements and Their Potential Role in Hamster Sperm Motility
Subir K. NagDas, Virginia P. Winfrey, Gary E. Olson
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Ras, a member of the small G-protein family, regulates multiple signaling pathways in somatic cells. The objectives of the present study included the characterization and localization of Ras and the identification of its downstream effectors in hamster spermatozoa. Immunoblot analysis with a pan-Ras monoclonal antibody localized Ras to the particulate fraction of sonicated testicular and caput and cauda epididymal spermatozoa. However, Ras was present in both the particulate and soluble fractions of spermatocytes and round spermatids, suggesting that its membrane recruitment is completed during spermiogenesis. Immunoblots of plasma membrane fractions demonstrated that hamster spermatozoa express both N-Ras and K-Ras. Indirect immunofluorescence with pan-Ras antibody localized Ras to the flagellum. Immunoblot analysis of sperm plasma membrane fractions demonstrated the presence of phosphatidylinositol 3-kinase (PI3-kinase) and protein kinase C ζ (PKCζ), the downstream targets of Ras, and coimmunoprecipitation analysis demonstrated their interaction with Ras. Inhibitors of PI3-kinase (wortmannin and 2-(4- morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) and PKCζ (staurosporine) inhibited the hyperactivation of sperm motility during capacitation in a dose-dependent manner, indicating that both PI3-kinase and PKCζ are associated with development of this motility pattern. The interaction of Ras with both PI3-kinase and PKCζ suggests that Ras may regulate several signaling pathways in spermatozoa.

Subir K. NagDas, Virginia P. Winfrey, and Gary E. Olson "Identification of Ras and Its Downstream Signaling Elements and Their Potential Role in Hamster Sperm Motility," Biology of Reproduction 67(4), 1058-1066, (1 October 2002).
Received: 9 January 2002; Accepted: 1 April 2002; Published: 1 October 2002
gamete biology
signal transduction
sperm maturation
sperm motility and transport
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