Recombinant Porcine Zona Pellucida Glycoproteins Expressed in Sf9 Cells Bind to Bovine Sperm but Not to Porcine Sperm

Abstract

The zona pellucida, which surrounds the mammalian oocyte, consists of the ZPA, ZPB, and ZPC glycoproteins and plays roles in species-selective sperm-egg interactions via its carbohydrate moieties. In the pig, this activity is conferred by tri-and tetraantennary complex type chains; in cattle, it is conferred by a chain of 5 mannose residues. In this study, porcine zona glycoproteins were expressed as secreted forms, using the baculovirus-Sf9 insect cell system. The sperm binding activities of the recombinant proteins were examined in three different assays. The assays clearly demonstrated that recombinant ZPB bound bovine sperm weakly but did not bind porcine sperm; when recombinant ZPC was also present, bovine sperm binding activity was greatly increased, but porcine sperm still was not bound. The major sugar chains of ZPB were pauci and high mannose type chains that were similar in structure to the major neutral N-linked chain of the bovine zona. In fact, the nonreducing terminal ␣-mannose residues were necessary for the sperm binding activity. These results show that the carbohydrate moieties of zona glycoproteins, but not the polypeptide moieties, play an essential role in species-selective recognition of porcine and bovine sperm. Moreover, Asn to Asp mutations at either of two of the N-glycosylation sites of ZPB, residue 203 or 220, significantly reduced the sperm binding activity of the ZPB/ ZPC mixture, whereas a similar mutation at the third N-glycosylation site, Asn-333, had no effect on binding. These results suggest that the N-glycans located in the N-terminal half of the ZP domain of porcine ZPB are involved in sperm-zona binding.Mammalian oocytes are surrounded by a transparent envelope called the zona pellucida, which is involved in several critical aspects of fertilization. Its functions include speciesselective recognition of sperm, blocking of polyspermy, and protection of the oocyte and embryo until implantation (1-3).The zona consists of three glycoproteins (ZPGs) 1 that are designated ZPA, ZPB, and ZPC in order of the sizes of their respective cDNAs (4). In the pig, these glycoproteins were formerly known as ZP1, ZP3␣, and ZP3␤, respectively (4).Studies of the murine zona pellucida have established that the carbohydrate moieties of the ZPGs play an essential role in sperm binding. Mouse sperm were proposed to bind to the O-linked carbohydrate chains linked to Ser-332 and Ser-334 of ZPC (5, 6); the nonreducing terminal residues, such as ␣-Gal (7), ␤-GlcNAc (8, 9), ␣-Fuc (10), ␣-Man (11), and ␤-Gal (12), are thought to mediate mouse sperm binding. Nevertheless, the suspected level of importance of the carbohydrate moieties in mouse sperm binding has declined because of the results of recent studies using genetically engineered mice that lack a glycosyltransferase, recent structural data on N-and O-linked carbohydrate chains of the mouse zona pellucida, and the determination of glycosylation sites on the mouse ZPGs, as outlined in recent reviews (13, 14) and discussed in recent papers (15)(16...

Zona pellucida glycoprotein ZP3 and fertilization in mammals

“…Differences in glycosylation patterns may be responsible for these different behaviors with mouse gametes as seen with hZP3 from transgenic mice (inhibitory) versus EC-and CHO-hZP3 (not inhibitory). A similar situation has been reported for native and recombinant porcine ZP glycoproteins and their interaction with porcine and bovine sperm (Yonezawa et al, 2005). It is likely that mouse oocytes modify hZP3 polypeptide with oligosaccharides recognized by mouse sperm, whereas EC and CHO cells do not.…”

Mammalian fertilization:the eggs multifunctional zona pellucida

“…These experiments were possible because, although hamster ZP3 purified from hamster egg ZP inhibits binding of mouse sperm to eggs in vitro, recombinant hamster ZP3 made by EC cells has no effect on binding of mouse gametes (Kinloch et al, 1991;Litscher and Wassarman, 1996b). A similar situation (i.e., a shift in sperm binding specificity) has been described by Yonezawa et al (2005) Shown is the average percent inhibition ± SD of sperm binding to eggs for wild-type and hybrid ZP3. In a control sample sperm were pre-incubated in the presence of M199-M alone (0 % inhibition).…”

Effect of N-Acetyl-D-Glucosamine on Bovine Sperm-Oocyte Interactions

“…The lower limit of the GlcNAc concentration that affects sperm-ZP primary attachment remains to be determined. In bovine, the carbohydrate chain on the surface of the ZP is a high mannose-type glycan that contains GlcNAc [2], and binding between lectin and this specific glycan is mediated by hydrogen bound among these molecules, which impacts their three-dimensional structure [21]. In addition, when GlcNAc was added to the sperm incubation medium, sperm-sperm aggregation was increased just after GlcNAc supplementation (data not shown).…”

“…The acrosomereacted, penetrated sperm fuses with the egg plasma membrane, which enables the egg to complete meiosis, and initiates mechanisms to prevent polyspermic fertilization by the exocytosis of cortical granules (CG) [1]. Oligosaccharides on the ZP include some monosaccharides, such as N-acetyl-D-glucosamine (GlcNAc), D-mannose and D-fucose [2], and these monosaccharides are involved in many sperm-oocyte interactions. For example, in sperm-oocyte recognition, N-acetyl-D-glucosaminidase (NAG) inhibits sperm binding to the ZP in the human [3,4].…”