Characterization of Proteins in the Bovine Epididymal and Seminal Fluid and Proteins Attached to Epididymal and Ejaculated Sperm.

During final maturation sperm lose their ability to biosynthesize, repair, grow, and divide. They have minimal metabolic function, and thus become completely dependent on their environment. In the epididymis, sperm are stored for a long period of time, but upon ejaculation motility is increased and lifespan is decreased from several wks to hrs. The objective of this experiment was to identify differences in proteins that are both in the environment (fluid) and attached to the sperm in both the epididymis and following ejaculation by LCMS-MS. Ejaculated and epididymal (after slaughter) sperm were collected from each of 9 bulls. Following collection, spermatozoa were washed with a high ionic solution to remove any proteins attached to the sperm, samples were then pooled and proteins were identified by LCMS-MS at the University of Minnesota Mass Spectrometry facility. Data were analyzed using the Scaffold software package with a false discovery rate set at 1%, a minimum of 1 peptide to identify a protein, and minimum of a 50% confidence in the identity of the protein. Total unique spectra count was then used to determine proteins found in one sample but not the other or found in both samples. In the fluid (epididymal and ejaculated), there were 208 proteins identified in epididymal fluid that were not identified in ejaculated fluid, and 75 proteins identified in ejaculated fluid that were not identified in epididymal fluid, and 103 proteins identified in both; for a total of 386 proteins identified in the fluid samples. Among proteins stripped from the sperm there were 113 proteins identified from the epididymal samples there were not identified in the ejaculated samples, 77 samples identified from the ejaculated samples that were not identified in epididymal samples, and 221 proteins identified in both; for a total of 411 proteins identified that were attached to sperm. There were 69 proteins identified attached to sperm that were not identified in fluid samples, and 49 proteins identified in fluid samples that were not identified in the samples attached to sperm. Thus a large number of proteins change between the epididymis and ejaculation, further investigation of proteins attached to the sperm and in the fluid environment that were not found in ejaculated samples may provide insight into the processes that allow sperm to be stored for extended periods of time in the epididymis but not after ejaculation. USDA is an equal opportunity provider and employer.