Poster Sessions

DEV-2

Gagandeep Gahlay

G. Gahlay, L. Gauthier, B. Baibakov, O. Epifano, J. Dean

Dependence of Sperm-egg Recognition on the Cleavage Status of ZP2, a Major Component of the Mouse Zona Pellucida

The zona pellucida (ZP), an extracellular matrix surrounding mammalian eggs, is composed of three glycoproteins (ZP1, ZP2, ZP3) in mice. It mediates fertilization being recognized by sperm prior to, but not after, fertilization. We evaluate two extant models for sperm-egg recognition. The “glycan release” model postulates that O-glycans attached to Ser332 and Ser334 on ZP3 act as ligands for a sperm receptor and mediate sperm-egg recognition. Following fertilization, a glycosidase released by egg cortical granule (CG) exocytosis removes these O-glycans and accounts for the inability of sperm to bind to two-cell embryos. This model predicts that the absence of these O-glycans would preclude sperm binding and female mice would be sterile. The “ZP2 cleavage” model proposes that the three zona proteins form a supramolecular structure that is permissive for sperm-egg recognition when ZP2 is intact (pre-fertilization), but not when cleaved (post-fertilization) by a CG protease. This model predicts that if ZP2 is not cleaved, sperm will continue to bind to the zona surrounding early embryos despite fertilization and CG exocytosis. We have begun to test these predictions with mouse transgenesis. Using site-directed mutagenesis and DNA recombineering, we have isolated two transgenes and established transgenic mouse lines expressing either mutant ZP2 in which the biochemcially defined cut site has been modified to prevent cleavage or expressing mutant ZP3 which lacks O-glycans at Ser332 and Ser334. Each transgene is expressed in the ovary which has otherwise normal histology and the mutant proteins are incorporated into the ZP surrounding ovulated eggs. As anticipated, mutant ZP2 protein is not cleaved following fertilization and mutant ZP3 has a shift in molecular mass due to the loss of an N-glycan not implicated in sperm-egg recognition. Founder lines are fertile and transmit the transgenes through the germline. The mutant ZP2 and ZP3 transgenic lines are being crossed into previously established Zp2 null and Zp3 null mice, respectively, to ensure that the endogenous protein is replaced by the mutant isoform in Zp2Mut rescue and Zp3Mut rescue mouse lines. Based on preliminary observations, we anticipate the Zp2Mut and Zp3Mut rescue females to be fertile and the Zp2Mut to bind sperm independent of fertilization and CG exocytosis (i.e., sperm will bind to the zona surrounding 2-cell embryos), thus supporting the “ZP2 cleavage” model of sperm-egg recognition.