The sperm serine protease BSp66 is included in membrane rafts

CESARI A; KATUNAR MR; FORNÉS MW

Belo Horizonte, MG, Brasil

Simposio; International Symposium on Animal Biology of Reproduction.; 2006

We have partially purified and characterized a serine proteinase named BSp66, that is located on the surface of the acrosomal region of various mammalian spermatozoa (Cesari et al. 2004a; Cesari et al. 2004b). As other sperm serine proteases, BSp66 has been shown to have an important role in early steps of fertilization in bovine and hamster in vitro fertilization systems (Cesari et al. 2005; Cesari et al. 2004c). BSp66 depicted an abnormal electrophoretic migration in capacitated and cryopreserved sperm (Cesari et al. 2003; Cesari et al. 2005). Several attempts to isolate this protease in a soluble way have failed, and we also failed in the obtention of its amino acid sequence by both Edman and mass spectrometry methods. The evidences obtained prompt us to hypothesize that BSp66 might be included in membrane insoluble microdomains called rafts, based on the reported data for other proteins with similar behavior. In this work we wanted to investigate if our protease was also included in this microdomains in bovine and mouse sperm. Materials and methods Bovine cryopreserved sperm were obtained from INTA-Balcarce. Mouse sperm were obtained from cauda epididymis as described (Cesari et al. 2004b). After extensive washing, sperm membrane raft were prepared as reported by Sleight et al. (Susan B. Sleight and Heidi Scrable 2005) for both species. Proteins from each fraction were electrophoresed in 12% SDS-PAGE under non-reducing conditions.  Fractions containing rafts were monitored by WB with anti-cav 1. The presence of BSp66 was evaluated by WB polyclonal anti-BSp66 (Cesari et al. 2004a). Results and discussion Membrane rafts are formed by polymerization of caveolins or caveolin related integral membrane proteins which tightly bind cholesterol and are implicated in signaling events related to sperm capacitation (Susan B. Sleight and Heidi Scrable 2005). In this work, sperm membrane rafts were separated from soluble proteins by sacarose gradient in both bovine and mouse sperm. Caveolin signal was distributed from fraction 4 to 6 in mouse sperm and fraction 6-8 in bull sperm, indicating the presence of the light buoyant density of raft domains. When anti-BSp66 was used, a 66 kDa immunoreactive protein was detected in the same fractions were caveolin was present in bull sperm, indicating that BSp66 is included in rafts. However in mouse, a 66 kDa signal was detected in fraction 6 and higher molecular mass immunoreactive proteins were distributed from fraction 8 to 10 (bottom). This result suggest that one isoform of BSp66 in included in mouse sperm rafts, while protein covalent aggregates exist in the soluble fraction. Because of the methodology used, the possibility that BSp66 is localized to the inner or outer acrosomal membranes cannot be discarded (Susan B. Sleight and Heidi Scrable 2005). The discrepancies between both species can be attributed to different sperm architectures or to changes in membrane composition accomplished to capacitation (Susan B. Sleight and Heidi Scrable 2005). This hyphotesis is based on the crycapacitation phenomena described for cryopreserved sperm, since bovine sperm were frozen-thawed while mouse sperm were fresh. The identification of proteins included in mouse sperm rafts performed by Sleight and co-workers (Susan B. Sleight and Heidi Scrable 2005), revealed four serine proteases after bioinformatic search in a “Expressed Sequence Tags” (EST) database: Testis Serine Protease 1 (TESP1), Testis Specific Tes101rp (TEX101) , Testis Serine Protease 2 (TESP2), Serine Protease-Like 1 (1700036D21RIK, kallikrein).  This information suggests that BSp66 may be one of this annotated proteins.