Characterization of the biologic activities of a recombinant human zona pellucida protein 3 expressed in human ovarian teratocarcinoma (PA-1) cells.
OBJECTIVE
This study was undertaken to clone and express a recombinant human zona pellucida protein 3 and to characterize its biologic activities as a sperm ligand and an inducer of the acrosome reaction.


METHODS
Human ovarian teratocarcinoma (PA-1) cells were transfected with an expression vector containing human zona pellucida protein 3 complementary deoxyribonucleic acid with a sequence coding for a 6-histidine tail introduced into its 3' end. Purification of the secreted glycoprotein was performed by sequential affinity (lectin and nickel--nitrilotriacetic acid) and ion-exchange chromatography.


RESULTS
Western blot analysis confirmed a molecular weight of approximately 65 kd for the purified product. A cell-free translation system revealed a correctly sized protein backbone of 47 kd. The recombinant human zona pellucida protein 3 demonstrated specific, potent, and dose-dependent competitive inhibition of sperm-zona pellucida binding in vitro under hemizona assay conditions. Recombinant human zona pellucida protein 3 also stimulated the acrosome reaction of live sperm. This effect was fast, dose dependent, and capacitation time dependent. Furthermore, advance incubation with pertussis toxin, an inactivator of heterotrimeric G proteins, blocked recombinant human zona pellucida protein 3--induced acrosomal exocytosis.


CONCLUSIONS
The recombinant human zona pellucida protein 3 expressed in PA-1 cells manifested the full spectrum of expected biologic activities. It therefore represents a valuable tool for examination of human fertilization and the design of new strategies in diagnosis of male factor infertility and in contraception.
Dong
K W
KW
Department of Obstetrics and Gynecology, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, VA 23507, USA.
Chi
T F
TF
Juan
Y W
YW
Chen
C W
CW
Lin
Z
Z
Xiang
X Q
XQ
Mahony
M
M
Gibbons
W E
WE
Oehninger
S
S
D020101:Acrosome Reaction / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004527:Egg Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D005260:Female; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D010051:Ovarian Neoplasms; D010053:Ovary / Q000378:metabolism / Q000502:physiology*; D037342:Pertussis Toxin; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013075:Sperm Capacitation / Q000187:drug effects / Q000502:physiology; D013081:Sperm Motility / Q000187:drug effects; D013084:Sperm-Ovum Interactions / Q000187:drug effects* / Q000502:physiology; D013094:Spermatozoa / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000072216:Zona Pellucida Glycoproteins
D018076:DNA, Complementary; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D000072216:Zona Pellucida Glycoproteins; D037342:Pertussis Toxin