Role of Osteopontin in Bovine Sperm Capacitation and Fertilization

Open Access
Erikson, David William
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
August 04, 2006
Committee Members:
  • Gary Joseph Killian, Committee Chair
  • Ronald Scott Kensinger, Committee Member
  • Daniel R Hagen, Committee Member
  • Guy Barbato, Committee Member
  • Ramesh Ramachandran, Committee Member
  • capacitation
  • spermatozoa
  • fertilization
  • osteopontin
Osteopontin (OPN) is an acidic, secreted phosphoprotein found in various tissues including bone, milk, kidney and the male and female reproductive tracts. Bovine OPN has a calcium binding site, a thrombin cleavage site and binds to various integrins through which it promotes cell adhesion and intracellular signaling. OPN has also been positively correlated to fertility in the seminal plasma of Holstein bulls and has been identified in bovine oviductal fluid. The purpose of this research was to investigate the presence of OPN on Holstein bull sperm, the role of OPN in bovine fertilization and the effects of OPN on sperm capacitation, intracellular calcium content, mitochondrial activity and viability to gain a better understanding of its role in fertility. Semen was collected by artificial vagina and solubilized sperm membranes were subjected to SDS-PAGE and Western blot analysis. A polyclonal rabbit antibody to purified bovine OPN (anti-OPN) detected OPN on sperm membranes, in testis homogenates and in cauda epididymal fluid. Results indicated that ejaculated sperm had approximately 50% more OPN than epididymal sperm and the protein was localized to the post-acrosomal region of the sperm head as well as the midpiece. Fertilization rates were reduced and incidence of polyspermy increased when sperm were incubated in anti-OPN prior to exposure to oocytes. OPN was localized to the same postacrosomal region on sperm in which the acrosome reaction had been induced. Integrin subunits known to associate with OPN were identified on bovine sperm and oocytes using SDS-PAGE and Western blot analysis. Other studies have shown that antibodies to these integrins decreased fertilization rates in the same manner as anti-OPN. These results suggest that OPN interacts with integrins during bovine fertilization, and may be involved in a block to polyspermy. OPN was also able to induce sperm capacitation and positively influenced sperm viability, but had no effect on sperm intracellular calcium content or mitochondrial activity. Taken together these results suggest exciting new roles for OPN in male reproductive physiology.