title: Binder of sperm 1 and epididymal sperm binding protein 1 are associated with different bull sperm subpopulations.
abstract: Previously, we showed that binder of sperm 1 (BSP1) and epididymal sperm binding protein 1 (ELSPBP1) proteins are more abundant in the immotile bovine sperm subpopulation following cryopreservation. In this study, we investigated the association of BSP1 and ELSPBP1 with sperm in relation to their ability to survive the cryopreservation process. Fresh and cryopreserved semen samples from the same ejaculate collected from nine Holstein bulls were incubated with a fixable viability probe, fixed and permeabilised and then immunolabelled with rabbit anti-BSP1, rabbit anti-ELSPBP1 or rabbit IgG as negative control. Spermatozoa were then incubated with Alexa 488-conjugated secondary antibody and Hoechst 33342. For each sample, 10 000 'Hoechst positive' events were analysed by flow cytometry. Alternatively, sperm populations were obtained by fluorescence-activated cell sorting. In freshly ejaculated live sperm, two distinct BSP1 detection patterns were revealed: a first population where BSP1 is present along the flagellar region (P1 subpopulation) and a second population where BSP1 is localised on both the flagellar and the acrosomal regions (P3 subpopulation). The dead population presented a BSP1 distribution similar to P3 but with a more intense fluorescence signal (P4 subpopulation). In the corresponding cryopreserved samples, all sperm in the P3 subpopulation were dead while only a small proportion of the P1 subpopulation was dead (P2 subpopulation). ELSPBP1 was detected only in dead spermatozoa and in comparable proportions in both freshly ejaculated and cryopreserved semen. These results show that the presence of BSP1 over the acrosomal region characterises spermatozoa sensitive to cryopreservation and that ELSPBP1 characterises spermatozoa that are already dead at ejaculation.
authors.affiliation: Département d'Obstétrique-Gynécologie, Faculté de Médecine, Centre de Recherche du Centre Hospitalier de l'Université Laval (CHUQ), Université Laval, 2705 Boulevard Laurier, Room T1-49, Québec City, Québec, Canada G1V 4G2.
authors.forename: Olivier
authors.initials: O
authors.lastname: D'Amours
authors.forename: Louis-Jean
authors.initials: LJ
authors.lastname: Bordeleau
authors.forename: Gilles
authors.initials: G
authors.lastname: Frenette
authors.forename: Patrick
authors.initials: P
authors.lastname: Blondin
authors.forename: Pierre
authors.initials: P
authors.lastname: Leclerc
authors.forename: Robert
authors.initials: R
authors.lastname: Sullivan
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D002417:Cattle / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D007150:Immunohistochemistry; D057230:Limit of Detection; D008297:Male; D011485:Protein Binding; D011817:Rabbits; D029607:Seminal Vesicle Secretory Proteins / Q000378:metabolism*; D013094:Spermatozoa / Q000166:cytology* / Q000378:metabolism*; D014018:Tissue Distribution
chemical_list: D029607:Seminal Vesicle Secretory Proteins