title: Characterization and identification of epididymal factors that protect ejaculated bovine sperm during in vitro storage.
abstract: The role of secretory epididymal factors on sperm survival and storage in bovine cauda epididymides is poorly understood. Thus, the effects of bovine epididymal epithelium fluid (BEEF) on frozen-thawed bovine sperm motility have been evaluated in vitro. Sperm motion parameters were assessed by computer-assisted sperm analysis. Compared with serum bovine proteins, BEEF efficiently sustained bovine sperm motility after a 6-h incubation period. The positive effect of BEEF on sperm motility was even more apparent using a fractionated BEEF extract (>10 kDa, 2 mg/ml). This beneficial effect was abolished when the BEEF active fraction was heat treated before incubation. A minimal 2-h BEEF preincubation period was necessary to maintain sperm motility activity and to protect sperm against oxidative injury caused by 150 microM hydrogen peroxide. The proteins from the BEEF >10-kDa fractions were biotinylated to identify the proteins that bind to the sperm surface. Five specific sperm-surface-binding proteins were revealed by Western blot analysis probed with avidin-horseradish peroxidase conjugate. These proteins were digested with trypsin for identification by matrix-assisted laser desorption ionization time-of-flight peptide mass spectrometric analyzer. Under reducing conditions, 5 bovine proteins were identified: the beta (36-kDa spot) and alpha (38-kDa spot) chains of clusterin, the beta-adrenergic receptor kinase 2 (48-kDa spot), and the antithrombin-III and the fibrinogen gamma-B chains, both corresponding to a doublet of about 50-52 kDa. These proteins are known to be present at the sperm surface in other species and could play a role in sperm protection in vivo. These results provide new insights to explain how secretory epididymal proteins sustain sperm motility during storage in vitro.
authors.affiliation: Centre de Recherche en Biologie de la Reproduction (CRBR), Université Laval, Sainte-Foy, Québec G1K 7P4, Canada.
authors.forename: Carlos
authors.initials: C
authors.lastname: Reyes-Moreno
authors.forename: Mathieu
authors.initials: M
authors.lastname: Boilard
authors.forename: Robert
authors.initials: R
authors.lastname: Sullivan
authors.forename: Marc-André
authors.initials: MA
authors.lastname: Sirard
mesh_terms: D000818:Animals; D001685:Biological Factors / Q000502:physiology*; D001710:Biotin; D002417:Cattle; D051152:Clusterin; D015925:Cryopreservation; D004822:Epididymis / Q000737:chemistry*; D006023:Glycoproteins / Q000378:metabolism; D006728:Hormones / Q000737:chemistry; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007525:Isoelectric Focusing; D008297:Male; D018832:Molecular Chaperones / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology; D011485:Protein Binding; D012662:Semen Preservation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013081:Sperm Motility / Q000502:physiology; D013094:Spermatozoa / Q000502:physiology*
chemical_list: D001685:Biological Factors; D051152:Clusterin; D006023:Glycoproteins; D006728:Hormones; D007202:Indicators and Reagents; D018832:Molecular Chaperones; D001710:Biotin