Porcine Zona Pellucida: Association of Sperm Receptor Activity with the α-Glycoprotein Component of the Mr = 55,000 Family1

Abstract

The immunogenicity and sperm receptor activity of five preparations of the major porcine zona pellucida glycoprotein family ZP3 (Mr = 55,000) were investigated. These included (1) ZP3, a chromatographically purified preparation of the 55,000 family; (2) ZP3 alpha, and (3) ZP3 beta, the two-component glycoproteins of the ZP3 family; (4) ZP3-EBGD, a partially deglycosylated preparation of ZP3 obtained by enzymatic treatment; and (5) ZP3-DG, a chemically deglycosylated preparation of ZP3. Titer studies using mouse and rabbit antisera prepared against each preparation yielded the following order of immunogenicity: ZP3 and ZP3 beta greater than ZP3-EBGD and ZP3 alpha greater than ZP3-DG, indicating that ZP3 becomes less immunogenic as more carbohydrate is removed. Pretreatment of intact zona with the various antisera prior to zona exposure to sperm resulted in an inhibition of sperm attachment to those zona treated with antibodies to ZP3, ZP3-EBGD, and ZP3 alpha. Pretreatment of zona with antibodies to ZP3 beta and ZP3-DG had no effect on sperm attachment. Studies involving pretreatment of boar sperm with the various ZP3 preparations prior to their use in a sperm-zona attachment assay and investigations involving displacement of the radiolabeled ZP3 preparations from sperm by unlabeled ZP3 preparations also yielded findings similar to the antibody studies. Collectively, these data indicate that ZP3 alpha probably functions as a zona receptor for boar sperm and that carbohydrate has an important role in maintaining the functional integrity of the ZP3 alpha glycoprotein.

The polypeptide backbone of recombinant human zona pellucida glycoprotein-3 initiates acrosomal exocytosis in human spermatozoa in vitro

“…In a number of non-human mammals such as mice and pigs, it is generally held that glycosylation of ZP3 is required for successful gamete interaction to occur [2,7]. However, contrasting data concerning the role of carbohydrate in spermatozoa-ZP3 (or ZP3α) binding in both mice and pigs have been presented.…”

“…However, contrasting data concerning the role of carbohydrate in spermatozoa-ZP3 (or ZP3α) binding in both mice and pigs have been presented. For example, it has been documented that carbohydrates associated with mouse ZP3 or pig ZP3α mediate spermatozoa-zona binding in these mammals in itro [2,7] yet fertilization in both mice and pigs can be blocked by specific monoclonal antisera directed against synthetic peptides based on the native ZP3 primary amino acid sequences. However, although such observations indicate a central role for the polypeptide backbone of ZP3 in the binding of spermatozoa to the zona in these species [29,30], one should exercise caution with such interpretations.…”

“…In mammals such as mice and pigs, it is generally accepted that carbohydrate epitopes displayed by zona pellucida glycoprotein 3 (ZP3) play a pivotal role in spermatozoa-ZP3 interaction [2,7]. However, although both native human zona pellucida and purified, glycosylated recombinant human ZP3 (rhuZP3) secreted by Chinese Hamster Ovary (CHO) cells have been shown to stimulate acrosomal exocytosis in human spermatozoa [8][9][10][11], it has yet to be firmly established whether the ability Abbreviations used : IPTG, isopropyl-β-D-thiogalactopyranoside ; OD 595 , optical density at 595 nm ; rhuZP3, recombinant human ZP3 ; GST-HuZP3, glutathione S-transferase-recombinant human ZP3 fusion protein ; TTB, Towbin Transfer Buffer.…”

“…One method to examine the role of the protein backbone in gamete interaction has been to deglycosylate zona proteins either enzymatically or chemically [2,7]. Although both approaches successfully remove carbohydrate from the polypeptide, there are a number of limitations in such experiments.…”

Carbohydrates and fertilization in animals

“…LAGS accounted for the charge heterogeneity seen with this porcine zona pellucida antigen and the antigen was actually composed of two unrelated LAG-containing proteins. The cr-glycoprotein of the 55-kDa antigen complex possessed receptor activity for porcine sperm and was shown to induce acrosome reactions in porcine sperm, an activity lost with deglycosylation (Sacco et al, 1984;Sacco et al, 1989;Berger et al, 1989b) or treatment with P-N-acetylglucosaminidase (Berger et al, 1989a), suggesting the importance of terminal Nacetylglucosamine on LAGS and the potential role of sperm GT, which recognizes terminal N-acetylglucosamine.…”