A single domain of the ZP2 zona pellucida protein mediates gamete recognition in mice and humans

Abstract

The extracellular zona pellucida surrounds ovulated eggs and mediates gamete recognition that is essential for mammalian fertilization. Zonae matrices contain three (mouse) or four (human) glycoproteins (ZP1-4), but which protein binds sperm remains controversial. A defining characteristic of an essential zona ligand is sterility after genetic ablation. We have established transgenic mice expressing human ZP4 that form zonae pellucidae in the absence of mouse or human ZP2. Neither mouse nor human sperm bound to these ovulated eggs, and these female mice were sterile after in vivo insemination or natural mating. The same phenotype was observed with truncated ZP2 that lacks a restricted domain within ZP2(51-149). Chimeric human/mouse ZP2 isoforms expressed in transgenic mice and recombinant peptide bead assays confirmed that this region accounts for the taxon specificity observed in human-mouse gamete recognition. These observations in transgenic mice document that the ZP2(51-149) sperm-binding domain is necessary for human and mouse gamete recognition and penetration through the zona pellucida.

Figure 1.

A zona pellucida is formed with ZP4 in the absence of ZP2, and female mice are sterile. (A) Phylogeny of mouse and human zona proteins indicate two clades; one composed of ZP1, ZP2, and ZP4 and the other of ZP3. There is no mouse ZP4 protein because of multiple stop and missense codons in the cognate gene. Mya, million years ago. (B) Schematic representation of the four zona pellucida proteins with 8 or 10 conserved cysteine residues. The resultant disulfide bonds differ in the zona domains of the ZP1/2/4 and ZP3 clades and are indicated as A and B, respectively. The postfertilization cleavage site is marked on ZP2, and both ZP1 and ZP4 contain trefoil domains. (C) Glutaraldehyde-fixed, plastic-embedded ovarian sections (3 µm) from 8–10-wk-old normal, Zp2^Null, moQuad^(huZP4), and moQuad-Zp2^Null mice were stained with periodic acid Schiff’s reagent to highlight the zona pellucida (arrows) and counterstained with hematoxylin. (D) Formaldehyde-fixed moQuad-Zp2^Null eggs stained with protein-specific monoclonal antibodies. Fluorescent and DIC images were merged and faux colored. (E) Mouse sperm binding to moQuad^(huZP4) and moQuad-Zp2^Null eggs. Inset, 2.5× magnification. Zp3^EGFP mouse eggs (green zona) and mouse two-cell embryos were positive and negative sperm-binding controls, respectively. Schematics to the left reflect protein composition of the zonae pellucidae with the source of sperm below.

Figure 2.

Truncated ZP2 does not support sperm binding, and female mice are sterile. (A) Representation of secreted ectodomains of normal mouse ZP2^35–633 and truncated ZP2 lacking ZP2^51–149. Cysteine residues, yellow. Monoclonal antibodies that bind N and C terminal to the postfertilization cleavage site (arrowhead) and zona domains are indicated above. (B) Ovarian histology of moZp2^Trunc and moQuad-Zp2^Trunc transgenic mice in Zp2^Null background as in Fig. 1 C. (C) moQuad^(huZP4) and moQuad-Zp2^Trunc eggs stained with domain-specific monoclonal antibodies as in Fig. 1 D. (D) Immunoblot of eggs (15) from moQuad^(huZP4) (1) and moQuad-Zp2^Trunc (2) mice stained with domain-specific monoclonal antibodies. Molecular masses are indicated on the left. (E) Mouse sperm binding to Zp2^Trunc and moQuad-Zp2^Trunc eggs as in Fig. 1 E.

Figure 3.

Human sperm binding to the zona pellucida requires human ZP2. (A) Ovarian histology from huQuad^(huZP1–4) and huQuad-ZP2^Null mice as in Fig. 1 C. (B) Eggs from huQuad-ZP2^Null stained with monoclonal antibodies as in Fig. 1 D. (C) Human sperm binding to huQuad^(huZP1–4), huQuad-ZP2^Null, and huQuad-ZP2^Null; Zp2^Mo eggs (as in Fig. 1 E) using noninseminated human oocytes and mouse Zp3^EGFP eggs (green zona) as positive and negative controls, respectively. (D) Litter sizes after transcervical insemination of control (Cd9^+/−) mice compared with natural mating (top). Sperm in the perivitelline space (PVS) of Cd9^−/− eggs after transcervical insemination with mouse sperm (bottom). Recovered eggs (left) and the number of sperm in PVS (right). Arrows indicate sperm. (E) As in D (bottom) but with huZP2^Rescue (top) and huZP3^Rescue (bottom) eggs after transcervical insemination with human sperm. (F) In vivo oviduct transfer of human sperm (2.3 × 10³ sperm in 0.5 µl) to hormonally stimulated, anesthetized huZP2^Rescue and huZP3^Rescue female mice. (G) As in E, but after in vivo oviductal transfer. (H) Normal mouse, huZP2^Rescue, huQuad eggs, and human oocyte stained with antibody to the sialyl-Lewis^X antigen.

Figure 4.

Taxon-specific sperm recognition of the N terminus of chimeric ZP2. (A) Ectodomains of huZP2, chimeric hu/moZP2, and chimeric mo/huZP2 proteins. Red and green, human and mouse protein, respectively. Yellow, conserved cysteine residues. Postfertilization cleavage site (arrowhead) and zona domains are indicated above. (B) Ovarian histology of hu/moZp2^Rescue and mo/huZP2^Rescue as in Fig. 1 C. (C) huZP2^Rescue, hu/moZp2^Rescue, and mo/huZP2^Rescue eggs stained with domain-specific monoclonal antibodies as in Fig. 2 C. (D) Human sperm binding to huZP2^Rescue, hu/moZp2^Rescue, and mo/huZP2^Rescue eggs as in Fig. 3 C. (E) Schematic of human (red) and mouse (green) recombinant peptides in which mouse ZP2^52–83, ZP2^85–101, or ZP2^103–133 replace the corresponding human sequence. The green bar under the huZP2 protein was deleted in the mouse ZP2^Trunc. Cysteine residues, yellow bars. Predicted N-glycosylation sites, blue bars with asterisks. Arrowhead, di-acidic residues and potential ovastacin cleavage sites. (F) Capacitated human sperm binding to chimeric ZP2 peptide beads. huZP2^39–154 and moZP2^35–149 peptides were positive and negative controls, respectively. DIC (top) and confocal z projection (bottom) images after staining with Hoechst. (G) Box plots reflect the median (vertical line) number of human sperm binding to peptide beads (left) and data points within the 10th and 90th percentiles (error bars). Boxes include the middle two quartiles and outliers are indicated by dots.

Figure 5.

Model of gamete recognition on the surface of the zona pellucida. The mouse zona pellucida (aquamarine) is composed of ZP1, ZP2, and ZP3, and surrounds ovulated eggs and early embryos. Sperm, capacitated by passage through the female reproductive tract, bind on the surface of the zona pellucida to an N-terminal domain of ZP2 in unfertilized eggs. After sperm acrosome exocytosis and penetration of the zona matrix, gametes fuse at fertilization and activate the egg. This triggers egg cortical granule migration and fusion with the plasma membrane, which releases ovastacin, a zinc metalloendoprotease that cleaves ZP2 at ¹⁶⁶LA↓DE¹⁶⁹. The immediate postfertilization block to polyspermy prevents additional sperm from fusing with eggs or penetrating through the zona pellucida matrix. The most definitive block is secondary to the proteolytic destruction of the sperm binding domain at the N terminus of ZP2. If sperm do not bind, they will not penetrate nor fuse with the egg’s plasma membrane.