David A. Chapman

Proteomics of cauda epididymal fluid from mature Holstein bulls.

The proteome of cauda epididymal fluid (CEF) from Holstein bulls was defined. Fluid was collected from the vas deferens, subjected to 2-D SDS-PAGE and spots identified by CapLC-MS/MS and MALDI-ToF/ToF. Because albumin accounted for 21.1% of all spot intensities in the gels examined by PDQuest, samples were subjected to albumin depletion and then analyzed again as before. Original CEF gels had 114 ± 3 spots, including as the most abundant: albumin, epididymal secretory protein E1, prostaglandin d-synthase and gelsolin. Epididymal fluid also expressed: clusterin, transferrin, N-acetyl-β-glucosaminidase, cauxin, glutathione peroxidase, acidic seminal fluid protein (aSFP), aldehyde reductase, α-l-fucosidase, α-1-β-glycoprotein, apolipoprotein A-1, β actin, calmodulin, cathepsin D, cystatin E/M, enolase, galectin 3-binding protein, leucine amino-peptidase and nucleobindin. Albumin depletion decreased that very spot to 10% of its original intensity and the resulting gels had, on average, 137 ± 4 spots. Spots identified as dipeptidyl-peptidase 7, angiotensin-converting enzyme, arylsulfatase A, aspartylglucosaminidase, serine protease inhibitors, new isoforms of calmodulin, cystatin E/M and a 17-kDa nucleobindin appeared only in depleted maps. This study is the first to report nucleobindin and aSFP as epididymal components. We suggest that CEF proteins act to facilitate membrane remodeling, transport of lipophilic substances, protect sperm and prevent premature acrosome reaction.

Detection of osteopontin on Holstein bull spermatozoa, in cauda epididymal fluid and testis homogenates, and its potential role in bovine fertilization.

Osteopontin (OPN) is a secreted extracellular matrix phosphoprotein identified in various tissues and fluids including those of the male and female reproductive tracts. OPN was previously identified as a 55 kDa high fertility marker in Holstein bull seminal plasma, produced by the ampulla and the vesicular gland. The objectives of this study were to characterize OPN on ejaculated and cauda epididymal sperm using immunofluorescence and western blot analysis, and to assess the role of sperm OPN in fertilization. Solubilized sperm membrane proteins from ejaculated and cauda epididymal sperm were separated by 1D SDS-PAGE, transferred to nitrocellulose, and probed with an antibody to bovine milk OPN. A 35 kDa protein was detected by this antibody in both ejaculated and cauda epididymal sperm membranes. Analyses also recognized OPN at 55 and 25 kDa in cauda epididymal fluid and testicular parenchyma homogenates respectively. Immunofluorescent analysis of ejaculated and cauda epididymal sperm showed OPN localization in a well-defined band in the postacrosomal region of the sperm head and also on the midpiece. Results of in vitro fertilization experiments showed that sperm treated with an antibody to OPN fertilized fewer oocytes than sperm treated with control medium while increasing incidence of polyspermy, suggesting a role of sperm-associated OPN in fertilization and a block to polyspermy. These studies demonstrate that OPN exists at multiple molecular weight forms in the bull reproductive tract and its presence on ejaculated sperm may signal its importance in fertilization by interacting with integrins or other proteins on the oocyte plasma membrane.

Induction of the acrosome reaction in bull spermatozoa with plasmin

Proteolytic enzymes appear to have an essential role in multiple phases of mammalian fertilization. Plasmin, the active enzyme of the plasminogen activation system that stimulates fibrinolysis and proteolysis has a less well‐documented role in reproduction. The current study was conducted to investigate the effect of the active protease, plasmin, on the ability of bovine sperm to undergo the acrosome reaction. Aliquots of freshly ejaculated bull sperm were incubated in capacitating conditions with 10 μg ml−1 of heparin for 4 h. Every 2 h an aliquot of spermatozoa was exposed to lysophosphatidylcholine (100 μg ml−1) or 0, 0.1, 1, 10 and 100 mU of plasmin to induce the acrosome reaction in capacitated spermatozoa. Plasmin increased the percentage of live acrosome reacted sperm after 4 h of incubation in the capacitation medium. Viability was not affected by any of the treatments. This study provides new information on bovine acrosome reaction during in vitro incubation with plasmin and indicates that this protease may participate in the proteolytic events that accompany fertilization.