Date of Award
Doctor of Philosophy (PhD)
Mark S. Elliot
Michael J. Solhaug
Mammalian fertilization involves interactions of sperm surface receptors with the ligands of the zona pellucida, an extracellular matrix surrounding the ovulated oocytes. In humans, the zona pellucida is composed of three major glycoproteins. One of them, ZP3, participates in the primary sperm binding and in the subsequent triggering of the spermatozoa's acrosome reaction. Studies on the role of this specific protein in the human fertilization process are hampered by the limited amount of available biologically functional proteins.
By use of a pcDNA 3.1(+) expression vector, a transfecting-vector was constructed containing a 1.3 kb histidine tagged hZP3 cDNA. This histidine tagged hZP3 cDNA containing vector was transfected to human ovarian teratocarcinoma (PA-1) cells, and a single-cell clone producing the recombinant human ZP3 glycoprotein (rhZP3) was generated. The rhZP3 glycoprotein were purified from supernatants of these cells by wheat germ agglutinin mediated glycoprotein chromatography isolation followed by the Ni-NTA mediated histidine tagged protein affinity purification. Western blotting analysis of the purified glycoproteins showed that the rhZP3 has a molecular mass ranging between 62-64 kDa which is similar to the profile of the native human ZP3. The rhZP3 triggered the acrosome reaction of spermatozoa and competitively inhibited the spermatozoa binding to the homologous zona pellucida in the hemizona assay, indicating that the recombinant glycoprotein is biologically active in both inducting the acrosome reaction and binding to its specific receptors on the surface of spermatozoa. Furthermore, the yield of the purified rhZP3 reaches the milligram level, therefore, this recombinant protein producing system can steadily supply sufficient biologically functional rhZP3 for studying the initial stages of the human fertilization process and may be adapted to clinical applications for male infertility.
"Expression, Isolation and Purification of Human Zona Pellucida Protein 3"
(1998). Doctor of Philosophy (PhD), dissertation, , Old Dominion University, DOI: 10.25777/bj4n-s171