Mean s

Mean s.e.m. levels of zinc affect forward motility of sperm to prevent their passage through the zona matrix. This transient, post-fertilization block to sperm penetration provides a temporal windows to total the cleavage of ZP2 which prevents sperm binding to ensure monospermy. INTRODUCTION The successful onset of development depends on RU.521 (RU320521) the ability of sperm to bind and penetrate the extracellular zona pellucida surrounding eggs, but not embryos, which ensures monospermic fertilization (Wong and Wessel, 2006; Avella et al., 2013; Okabe, 2013). Mouse and human zonae pellucidae contain three and four glycoproteins (ZP1C4), respectively (Bleil and Wassarman, 1980b; Bauskin et al., 1999) and zona glycans have been implicated in gamete acknowledgement (for review, observe Yonezawa, 2014). A single genetic locus encodes each zona protein in mouse and human genomes. Genetic ablation of decreases fecundity, but female mice form a zona matrix and are fertile (Rankin et al., 1999). Mouse is usually a pseudogene (Lefivre et al., 2004) and human ZP4 is not sufficient to support human sperm binding in transgenic mice (Yauger et al., 2011). Thus, neither RU.521 (RU320521) ZP1 nor ZP4 appear essential for sperm-egg interactions and fertility. ZP2 and ZP3 are common to all vertebrate zonae and each has been proposed as a zona ligand for sperm binding (Bleil and Wassarman, 1980a; Tian et al., 1997). However, no zona matrix is present surrounding ovulated eggs after genetic ablation of or which in the beginning precluded meaningful evaluation of either as a sperm binding ligand (Liu et al., 1996; Rankin et al., 1996; Rankin et al., 2001). More recent gain-of-function and loss-of-function assays in genetically engineered mice, have implicated ZP2 as the primary ligand for human and mouse sperm binding to the zona pellucida (Baibakov et al., 2012; Avella et al., 2014). Following fertilization, mature ZP235?633 is cleaved near its N-terminus by ovastacin, an egg cortical granule astacin-like metalloendopeptidase encoded by maintains ZP2 intact and mouse sperm bind to the zona pellucida even after fertilization and cortical granule exocytosis (Gahlay et al., 2010; Burkart et al., 2012). In loss-of-function assays, mice lacking ZP251?149 form a zona matrix, but are infertile after natural mating (Avella et al., 2014). The interpretation of these observations has been controversial and whether the loss of fertility in the absence of the N-terminus of ZP2 was a direct or indirect effect on gamete recognition was not determined. Nor did these investigations experimentally address an essential role for glycans in sperm-zona interactions which long has been a central tenet of the molecular basis of gamete interactions (Abou-Haila et al., 2014; Chiu et al., 2014; Clark, 2014). Using mouse transgenesis, we now report that moZP235?149 or moZP235?262 fused to the N-terminus of huZP4 is sufficient for sperm binding and fertility in RU.521 (RU320521) the absence of native ZP2. Oglycans are not detected in native mouse ZP2 (Boja et al., 2003) and mutant mice lacking the single N-glycan in this region are fertile. Thus, neither N- nor O-glycans are required for sperm binding to the N-terminus of ZP2 and for fertility. We further document that following fertilization, there is a rapid block to sperm penetration of the zona matrix that is transient, dependent on the enzymatic activity of ovastacin and is associated with release of cortical granule zinc that affects sperm motility. Subsequently, ovastacin cleavage of ZP2 provides a permanent block to sperm binding and ensures monospermic fertilization. RESULTS Sperm bind to moZP235?149/huZP4 and moZP235?262/huZP4 fusion proteins in the absence of native ZP2 Mouse ZP2 contains 713 amino acids, including a signal peptide (1C34 aa), a zona module (364C630 aa), a transmembrane domain (684C703 aa) and a short cytoplasmic tail (704C713 aa). After processing and PTGIS secretion, mature ZP2 (35C633 aa) associates with ZP1 and ZP3 in forming the extracellular zona pellucida. DNA recombineering was used to clone transgenes that contained genomic DNA encoding either mouse ZP235?149 (moexons 2C5) or mouse ZP235?262 (moexons 2C9) inserted into huexon 1 (Figures 1A, S1A and S1B). Pronuclear injection of.