Embryonal carcinoma cells transfected with ZP3 genes differentially glycosylate similar polypeptides and secrete active mouse sperm receptor.
Mouse and hamster sperm receptors, called mZP3 (approximately 83,000 Mr) and hZP3 (approximately 56,000 Mr), respectively, are glycoproteins located in the ovulated egg zona pellucida. Certain of the glycoprotein O-linked oligosaccharides are essential for sperm receptor activity. Here, we transfected mouse embryonal carcinoma (EC) cells with mZP3 and hZP3 genes placed under control of a constitutive promoter. Transfected cells synthesized and secreted large amounts of the glycoproteins, called EC-mZP3 and EC-hZP3. Although the primary structures of mZP3 and hZP3 polypeptides (44,000 Mr) are very similar to one another, EC-mZP3 (approximately 83,000 Mr) and EC-hZP3 (approximately 49,000 Mr) were glycosylated to very different extents, such that they resembled their egg counterparts. Like egg mZP3, EC-mZP3 inhibited binding of sperm to ovulated eggs and induced sperm to acrosome-react in vitro. In addition, large numbers of sperm bound to aggregates of mZP3-transfected EC cells in vitro. On the other hand, unlike egg hZP3, EC-hZP3 did not exhibit either sperm receptor or acrosome reaction-inducing activity, and sperm failed to bind to aggregates of hZP3-transfected EC cells. Thus, transfected EC cells not only express sperm receptor genes, but also discriminate between very similar polypeptides with respect to glycosylation and, in the case of mZP3, add specific oligosaccharides essential for biological activity. In addition, the results demonstrate that EC cells can serve as a source for large amounts of functional mouse sperm receptor.
Kinloch
R A
RA
Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Mortillo
S
S
Stewart
C L
CL
Wassarman
P M
PM
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D005260:Female; D006023:Glycoproteins / Q000235:genetics*; D006031:Glycosylation; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010063:Ovum / Q000166:cytology / Q000502:physiology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D013075:Sperm Capacitation; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology; D013724:Teratoma; D014162:Transfection
D006023:Glycoproteins; D009838:Oligodeoxyribonucleotides; D011956:Receptors, Cell Surface; C035027:egg surface sperm receptor