Characterization and Identification of Epididymal Factors That Protect Ejaculated Bovine Sperm During In Vitro Storage

• Published in: Biology of reproduction 2002-01, Vol.66 (1), p.159-166
• Main Authors: Reyes-Moreno, Carlos, Boilard, Mathieu, Sullivan, Robert, Sirard, Marc-André
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Biological Factors - physiology; Biotin; Cattle; Clusterin; Cryopreservation; Epididymis - chemistry; Glycoproteins - metabolism; Hormones - chemistry; In Vitro Techniques; Indicators and Reagents; Isoelectric Focusing; Male; Molecular Chaperones - metabolism; Oxidative Stress - physiology; Protein Binding; Semen Preservation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sperm Motility - physiology; Spermatozoa - physiology
• ISSN: 0006-3363; 1529-7268
• DOI: 10.1095/biolreprod66.1.159

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 μM 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 β-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.