Cellular origin of the Bufo arenarum sperm receptor gp75, a ZP2 family member: its proteolysis after fertilization.
BACKGROUND
The egg envelope is an extracellular matrix that surrounds oocytes. In frogs and mammals, a prominent feature of envelope modification following fertilization is the N-terminal proteolysis of the envelope glycoproteins, ZPA [ZP (zona pellucida) A]. It was proposed that ZPA N-terminal proteolysis leads to a conformational change in egg envelope glycoproteins, resulting in the prevention of polyspermy. Bufo arenarum VE (vitelline envelope) is made up of at least four glycoproteins: gp120 (glycoprotein 120), gp75, gp41 and gp38. The aim of the present study was to identify and characterize the baZPA (B. arenarum ZPA homologue). Also, our aim was to evaluate its integrity and functional significance during fertilization.


RESULTS
VE components were labelled with FITC in order to study their sperm-binding capacity. The assay showed that gp75, gp41 and gp38 possess sperm-binding activity. We obtained a full-length cDNA of 2062 bp containing one ORF (open reading frame) with a sequence for 687 amino acids. The predicted amino acid sequence had close similarity to that of mammalian ZPA. This result indicates that gp75 is the baZPA. Antibodies raised against an N-terminal sequence recognized baZPA and inhibited sperm-baZPA extracted from VE binding. This protein does not induce the acrosome reaction in homologue sperm. Northern-blot studies indicated that the transcript is exclusively expressed in the ovary. In situ hybridization studies confirmed this and pointed to previtellogenic oocytes and follicle cells surrounding the oocyte as the source of the transcript. baZPA was cleaved during fertilization and the N-terminal peptide fragment remained disulfide bonded to the glycoprotein moiety following proteolysis.


CONCLUSIONS
From the sequence analysis, it was possible to consider that gp75 is the baZPA. It is expressed by previtellogenic oocytes and follicle cells. Also, it can be considered as a sperm receptor that undergoes N-terminal proteolysis during fertilization. The N-terminal peptide could be necessary for sperm binding.
Scarpeci
Sonia L
SL
División Biología del Desarrollo, IBR (CONICET-UNR) and Area Biología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario S2002RLK, Republic of Argentina.
Sanchez
Mercedes L
ML
Cabada
Marcelo O
MO
D020101:Acrosome Reaction / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001483:Base Sequence; D002022:Bufo arenarum / Q000235:genetics / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D003001:Cloning, Molecular; D004527:Egg Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D005306:Fertilization; D005455:Fluorescent Antibody Technique; D017403:In Situ Hybridization; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D012333:RNA, Messenger / Q000032:analysis; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017385:Sequence Homology; D013084:Sperm-Ovum Interactions / Q000187:drug effects; D014817:Vitelline Membrane / Q000737:chemistry / Q000378:metabolism; D000072216:Zona Pellucida Glycoproteins
D000906:Antibodies; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D010455:Peptides; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins; C035027:egg surface sperm receptor