ISSN: 2577-4360
Authors: Subir K Nagdas*, Wallace S, Rahman R, Eaford D, and Raychoudhuri SS
Spermatozoa entering the epididymis have no capacity for protein synthesis, are incapable of forward motility, and have no fertilizing ability. During post-testicular maturation in the epididymis, the sperm surface undergoes extensive remodeling, and this physiological process plays a critical role in the development of the fertilization-competent spermatozoon. Previously, we identified farnesyltransferase (FTase), an enzyme which functions in post-translational protein lipidation in hamster epididymal spermatozoa. In addition, we have identified the signaling protein RAS, a FTase substrate, in a plasma membrane (PM) fraction. Immunoblot analysis of sperm plasma membrane fractions demonstrated the presence of PI3- Kinase, a downstream target of RAS. Coimmunoprecipitation (Co-IP) analysis demonstrated the interaction of PI3-Kinase with RAS. Does the protein lipidation play a significant role in the development of the fertilization-competent spermatozoon? The objectives of the present study included the biochemical localization of FTase and the identification and characterization of RAS upstream and downstream effectors in hamster spermatozoa. First, we examined if FTase is localized to soluble or particulate fractions of caput and cauda epididymal spermatozoa. Immunoblot analysis revealed that FTase is localized to the soluble fraction of both caput and cauda epididymal spermatozoa. Subsequent studies were performed to identify potential upstream and downstream effectors of RAS. Immunoblot analysis of sperm PM fractions demonstrated the presence of the upstream effector (GRB2) and the downstream effectors (RAF-Kinase). Immunofluorescence with anti-RAF-Kinase polyclonal antibody localized RAF- Kinase to the sperm connecting piece and flagellum. Coimmunoprecipitation analysis revealed the interaction of RAF-Kinase with RAS. The fate of RAS and its downstream effectors (PI3-Kinase and RAF-Kinase) was examined during capacitation of hamster cauda epididymal spermatozoa. RAS and its downstream effectors (PI3-Kinase and RAFKinase) remain anchored to the sperm pellet after the completion of capacitation. The interaction of RAS with RAF-Kinase suggests that RAS may regulate several signaling pathways in spermatozoa.
Keywords: RAS, Sperm; Signal Transduction; Sperm capacitation
