SEQUENCE OF A RABBIT SPERM ZONA-PELLUCIDA BINDING-PROTEIN AND LOCALIZATION DURING THE ACROSOME REACTION

The interaction of the mammalian spermatozoon with the oocyte's extrac ellular matrix or zona pellucida is a critical first step leading to s uccessful fertilization. In this cell-extracellular matrix interaction it is the carbohydrate of the zona pellucida which serves as the sper m receptor and the surface of the spermatozoon which provides the lect in-like adhesion molecules. To better understand sperm-zona pellucida binding we have analyzed one specific zona binding protein (ZBP). This study has determined the mRNA sequence encoding a mammalian testis an d sperm specific protein of 16,891 Da, which we have designated Sp17. Analysis of Sp17 revealed that the mRNA is present in rabbit, mouse, a nd human testes but not in any somatic tissue tested. In the rabbit, S p17 is the 17-kDa member of the rabbit sperm autoantigen family of spe rm specific autoantigens and is encoded by two mRNAs of 0.9 and 1.1 kb . Each mRNA has a unique 5' untranslated region but both have identica l coding regions. The deduced amino acid sequence of the Sp17 ZBP show ed several interesting features, including a similarity to the N-termi nal of human testis cAMP-dependent protein kinase. Localization of Sp1 7 on live spermatozoa using antibodies to recombinant Sp17 or to the S p17 peptide, G22C, revealed that the peptide backbone of Sp17 is inacc essible until the acrosome reaction begins. However, on paraformaldehy de fixed, acrosome intact spermatozoa, the peptide backbone is accessi ble to the antibodies which localize Sp17 to the apical surface. In th e rabbit as well as other similar species in which the corona radiata (granulosa) cells adhere tightly to the zona pellucida and synthesize zona glycoproteins, the fertilizing spermatozoon may have already begu n the acrosome reaction within the cumulus oophorus. Thus, the rabbit sperm surface would be modified to expose the Sp17 polypeptide during the final phase of cumulus passage and consequently Sp17 would be avai lable for initial zona binding. The present study has also demonstrate d that recombinant Sp17 can bind zona pellucida, dextran, and dextran sulfate. (C) 1994 Academic Press, Inc.