Develop. Growth & Differ., zyxwvuts 36 (6), 615-620 (1994) zyxwvu Development zy Growth & Differentiation Oviductal Localization of the Cortical Granule Lectin Ligand Involved in the Block to Polyspermy of Xenopus Laevis (CGL/ polyspermy/fertilization /Xenopus/oviduct) Timothy A. Quill' and Jerry L. Hedrick' Sectton zyxwvutsrqp of Molecular and Cellular Biology University of Cal/fornia, Davis Davis, California 95676, USA 'present address Howard Hughes Medical Institute, Univ of Texas, Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd ~ Dallas, Texas 75235-9050 'reprint requests ~ ~~ The fertilization layer of Xenopus laevis is formed upon egg activation by the binding of the cortical granule lectin (CGL) to its ligand in the egg extracellular matrix. Using Western blotting methods with biotinylated CGL as a probe, oviductal tissue extracts were examined to determine the site of origin of the CGL ligand. Three glycoprotein ligands of Mrs= >250,000, 160,000, and 90,000 (reduced samples) were localized to the pars convoluta oviduct immediately posterior to the pars recta oviduct. The binding of CGL to these glycoproteins was inhibited in the presence of 200 mM galactose, but not with 200 mM mannose indicating a specific lectin interaction. The Mrs= >250,000 and 90,000 glycoproteins were linked by disulfide bonds. In addition, these ligands were secreted from a more anterior region of the pars convoluta oviduct than the Mr=160,000 ligand. No binding of CGL was detected to pars recta secretory granule lysate components. The highest molecular weight CGL ligand seen in the pars convoluta corresponded to the CGL ligand in isolated fertilization envelopes. Thus, the CGL ligand involved in the formation of the fertilization layer is a product of the pars convoluta oviduct. Introduction The exocytosis of cortical granules of Xenopus laevis eggs alters the surrounding extracellular matrix to produce a permanent block to potysper- my. In particular, the cortical granule lectin, CGL, is released from the cortical granules and diffuses into the extracellular matrix of the egg. At the interface between the vitelline envelope and the jelly, CGL binds to its ligand forming the fertilization layer which is impenetrable to sperm (3). The binding of CGL to its ligand is galactose specific and calcium dependent. Initially, the CGL ligand was proposed to be located in the innermost jelly layer, J1, due to the precipitation reaction between CGL and J1 as seen by double diffusion experiments (13). Sub- sequent electron microscopy studies demons- trated the presence of a distinct structure, the prefertilization layer, located between the vitelline envelope and J1, which is morphologically similar to the fertilization layer (15, 16). Using immuno- localization methodology, the prefertilization layer CGL; cortical granule lectin, JI; innermost jelly layer, PC; pars convoluta oviduct, PR; pars recta oviduct 615 was shown to be a secretion of the posterior pars recta oviduct separate from the pars convoluta oviduct which secretes the jelly layers. In addi- tion, prefertilization layer antigens were identified in zy the isolated fertilization layer. On the basis of these observations, the prefertilization layer was hypothesized to be the CGL ligand. To test this hypothesis, extracts were pre- pared from sections of the oviduct, and analyzed for CGL ligand using Western blotting methods with biotinylated CGL as a probe. The CGL ligand was found only in extracts from the pars convoluta oviduct immediately following the pars recta oviduct. Thus, the CGL ligand which partici- pates in the formation of the fertilization layer is a secretory product of the pars convoluta oviduct. Materials and Methods lsolation of envelopes : Fertilization envelopes were isolated from dejellied oviposited eggs as previously described (2). Biotinylation of CGL: CGL was purified as described by Nishihara et al. (7), and biotinylated using the procedure of Yolken et al. (14). Briefly, Copyright zyxwv 0 by The Japanese Society of Developmental Biologists 1994 All rights of reproduction in any form reSeNed