ZP2 peptide beads select human sperm in vitro, decoy mouse sperm in vivo, and provide reversible contraception

Abstract

Gamete recognition in the female reproductive tract occurs at the surface of the zona pellucida surrounding ovulated eggs. The acellular zona matrix is composed of three (mouse) or four (human) proteins (ZP1 to ZP4), and the amino terminus of ZP2 is the primary sperm-binding ligand. Mouse and human sperm bind, respectively, to recombinant moZP2 and huZP2 39-154 peptides attached to agarose beads. Mouse ZP2 peptide beads markedly inhibited fertilization of ovulated mouse eggs inseminated in vitro and incubated overnight. Similarly, human ZP2 peptide beads prevented sperm binding and penetration of transgenic ZP2Rescue zonae pellucidae, in which human ZP2 replaced mouse ZP2. When mouse ZP2 peptide beads were transcervically deposited into the uterus, there was no change in mating behavior and copulatory plugs were present, but bound sperm did not progress into the oviduct and female mice were infertile. On average, contraception lasted >10 estrus cycles but was reversible with no detectable pathology in the reproductive tract. Despite the long-term contraceptive effect, initial sperm binding to the peptide beads was reversible in vitro. We exploited this observation to select human sperm that were better able to penetrate the zonae of human ZP2 Rescue eggs, and the approach holds promise for identifying superior sperm for human assisted reproductive technologies (ART). We conclude that the amino-terminal ZP2 peptide supports sperm binding, which is initially reversible but, with time, becomes irreversible. Short-term, reversible binding may be useful in selecting sperm for ART, and long-term binding decoys sperm and results in effective contraception in mice.

The molecular mechanisms mediating mammalian fertilization

“…In addition, time-lapse imaging of Acr GFP mouse sperm shows that acrosome-reacted sperm penetrate the zona with higher efficiency than acrosome-intact sperm (Jin et al, 2011). These observations are consistent with recent findings showing that both mouse and human sperm can remain bound to the N terminus of ZP2 after acrosome exocytosis (Avella et al, 2016). However, this model does not fully explain the acrosomal shrouds (residues of the vesiculated acrosomal caps) that are found attached to the zonae surrounding fertilized eggs in different mammalian species (VandeVoort et al, 1997;Wakayama et al, 1996;Yanagimachi and Phillips, 1984).…”

“…Together, this evidence indicates that the putative sperm binding site should not reside on human ZP3. Moreover, recombinant mouse or human ZP2 N termini directly interact with mouse or human sperm in peptide-bead binding assays (Avella et al, 2014(Avella et al, , 2016Baibakov et al, 2012). From all these data, we hypothesize a direct interaction between sperm and the N terminus of ZP2 and we conclude that, for successful fertilization, sperm bind to the zona via the N terminus of ZP2.…”

Cytoplasmic cleavage of DPPA3 is required for intracellular trafficking and cleavage-stage development in mice

“…Ovulated eggs isolated from wild-type and Dppa3 KO female mice were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and processed for electron microscopy 71 .…”

“…To stain 5hmC, in vivo mated zygotes were isolated and incubated (30 min, RT) in 4 N HCl and then incubated (30 min, RT) in 0.1 M EDTA. Fixed samples were imaged by LSM 780 (Carl Zeiss) confocal microscopy 71 and Manders’ co-localization coefficients 72 were calculated by LSM Image Examiner.…”

Chemoenzymatic Synthesis of Asymmetrical Multi‐Antennary N‐Glycans to Dissect Glycan‐Mediated Interactions between Human Sperm and Oocytes

“…Human ZP is composed of 4 glycoproteins termed as ZP1, ZP2, ZP3 and ZP4 which are organized into long cross‐linked fibrils. Both ZP2 and ZP3 have been implicated as binding partners for sperm to induce spermatozoa exocytosis …”