Immunoelectrophoretic analysis of the porcine zona pellucida

Sacco, Anthony G.; Yurewicz, Edward C.; Zhang, S.

doi:10.1530/jrf.0.0680021

Cited by 15 publications

(5 citation statements)

References 11 publications

(23 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The complexity of the antigens associated with the mammalian ZP has been previously described in studies using immunoelectrophoresis and immunoblotting techniques (Dunbar and Raynor, 1980;Sacco et al, 1981Sacco et al, , 1983Sacco et al, , 1986Wood and Dunbar, 1981;Surneva-Nakova and Nakov, 1982;Yurewicz et al, 1983;Skinner et al, 1984;Surneva-Nakova et al, 1984; see also reviews by Dunbar, 1983b;Dunbar and Wolgemuth, 1984;Skinner and Dunbar, 1986). To better characterize ZP antigens, the present studies have been carried out using both monoclonal and polyclonal antibodies in conjunction with limited proteolysis and/or deglycosylation of glycoproteins, and identification by immunoblotting using highresolution 2D-PAGE.…”

Section: Discussionmentioning

confidence: 82%

Use of Specific Monoclonal and Polyclonal Antibodies to Define Distinct Antigens of the Porcine Zonae Pellucidae1

Timmons

Maresh

Bundman

et al. 1987

Biology of Reproduction

View full text Add to dashboard Cite

Specific monoclonal and polyclonal antibodies to solubilized porcine and rabbit zonae pellucidae (ZP) and to purified ZP glycoprotein components have been used to define distinct ZP antigens. These studies demonstrate that the individual ZP glycoproteins contain both unique and shared determinants. One monoclonal antibody (R5) has been used to demonstrate that the major porcine ZP glycoprotein, which has multiple charge species ranging in molecular weight from 42,000 to 120,000, is composed of two distinct polypeptide antigens unique to this glycoprotein class. These distinct antigens can be differentiated by immunoblotting after high-resolution two-dimensional polyacrylamide gel electrophoretic separation of trypsin-treated or deglycosylated glycoproteins. The two polypeptides also differ in their staining properties with the silver-based color stain and in their susceptibility to proteolysis. A second monoclonal antibody (PSI) has been used to define a determinant shared by all three major porcine ZP glycoprotein classes. This determinant appears to involve either a carbohydrate moiety or some other molecular feature related to post-translational modification, since the antibody recognizes only the acidic species of each glycoprotein class, and does not recognize the deglycosylated forms of the proteins. This work demonstrates that there are both unique and shared antigenic determinants present in the individual components of the ZP, but that the immunodominant determinants appear to be unique to each glycoprotein.

show abstract

Section: Discussionmentioning

confidence: 82%

Use of Specific Monoclonal and Polyclonal Antibodies to Define Distinct Antigens of the Porcine Zonae Pellucidae1

Timmons

Maresh

Bundman

et al. 1987

Biology of Reproduction

View full text Add to dashboard Cite

show abstract

“…5. Reac¬ tion of ZP3 into anti-ZP3 serum resulted in the overlapping 'stacked' pattern of precipitin arcs which is characteristic of the pig zona pellucida system (Wood & Dunbar, 1981;Sacco et al, 1983). In this reaction 6-7 precipitin arcs were detected (Fig.…”

Section: Lmm Unochemical Proceduresmentioning

confidence: 93%

“…All antisera were produced using a multi-site, intradermal injection regimen (Vaitukaitis, Robbins, Nieschlag & Ross, 1971), in conjunction with Freund's adjuvant. Details regarding production and characterizations of antisera to isolated zonae (IZP), purified pig zona antigen (PPZA), and heat-solubilized isolated zonae (SIZP) have been described previously (Sacco & Palm, 1977;Subramanian et al, 1981;Sacco, Yurewicz & Zhang, 1983). For this investigation, two additional antisera were prepared in rabbits against ZP3 and deglycosylated ZP3.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Carbohydrate influences the immunogenic and antigenic characteristics of the ZP3 macromolecule (Mr 55 000) of the pig zona pellucida

Sacco¹,

Yurewicz²,

Subramanian³

1986

Reproduction

Self Cite

View full text Add to dashboard Cite

Summary. The glycoprotein molecule ZP3 of the pig zona pellucida (Mr 55 000) was deglycosylated by trifluoromethane\p=n-\sulphonic acid. While gas chromatography established that deglycosylation resulted in >91% carbohydrate removal, deglycosylated ZP3 (DG-ZP3) still retained significant immunogenic potential as shown by its ability to elicit antibody production in the rabbit. Several lines of evidence, however, demonstrated that deglycosylation produced significant modifications in the ZP3 macromolecule which profoundly altered its antigenicity: (1) 125I-radiolabelling procedures using chloramine-T consistently resulted in 125I-labelled ZP3 with a higher specific activity than 125I-labelled DG-ZP3; (2) in competitive binding radioimmunoassays (RIAs) using an 125I-labelled-ZP3 vs anti-ZP3 system, unlabelled DG-ZP3 successfully displaced ZP3 in a dose-related manner; (3) in direct binding RIAs comparing the reaction of labelled ZP3 and DG-ZP3 versus 5 different zona antisera, higher titres were consistently achieved with 125I-labelled ZP3; (4) as compared to ZP3, 2-dimensional immunoelectrophoresis using DG-ZP3 as antigen yielded significantly modified precipitin arc patterns; (5) with electrophoretic blotting procedures, antisera to ZP3 or DG-ZP3 cross-reacted with the heterologous antigen; (6) treatment of pig zonae with anti-DG-ZP3 serum produced a dense precipitation layer on the zona surface. When evaluated collectively, these results provide grounds for an important role for protein and carbohydrate in establishing the immunological characteristics of the Mr 55 000 macromolecule of the pig zona pellucida.

show abstract

“…Progress has been made in identifying the egg's sperm receptor. In the zona pellucida of the mouse three glycoproteins have been identified, ZP1,ZP2, and ZP3 of which ZP3 (M, = 83 kD) has been identified as responsible for sperm binding and the induction of the acrosome reaction (Bleil and Wassarman, 1980, 19831. More recently ZP2 (M, = 120 kD) has been reported to also bind spermatozoa, but perhaps only after the acrosome reaction [Bleil and Wassarman, 19861. In the pig, ZP3 of M, = 55 kD (55 kD-60 kD range) has been identified as the boar sperm receptor [Sacco et al, 1983[Sacco et al, , 1984 In the human both ZP3 (M, = 57 kD-73 kD) and ZP1-2 (M, = 92 kD-120 kD) in their nonreduced forms bind spermatozoa [Shabanowitz and O'Rand, 19871, while in the rabbit a M, = 87 kD component has been identified which is probably a combination of ZP2 and ZP3 as assessed by two-dimensional (2-D) SDS-PAGE [Fisher et al, 19871. In additional studies on the mouse zona, Florman and Wassarman [1985] identified a glycopeptide from the ZP3 molecule with an 0-linked carbohydrate moiety which was responsible for the binding of the sperm to the zona. The peptide portion of ZP3 was shown to be necessary for inducing the acrosome reaction [Wassarman et al, 19861.…”

Section: Zona Pellucida Binding Proteins Of Spermatozoamentioning

confidence: 99%