Article Figures & Data
Figures
- FIG. 1.
Intracellular trafficking of ZP3-EGFP reporter protein lacking a zona domain. (A) Expression plasmid ZP31-31aa-EGFP-ZP3342-424aa, in which EGFP replaced the zona domain while the N-terminal signal peptide (amino acids 1 to 31) and the carboxyl terminus (amino acids 342 to 424) of ZP3 were preserved, including the potential proprotein convertase (furin) site. (B) The ZP3-EGFP reporter construct was placed downstream of a CMV promoter within a circular plasmid and injected into the nucleus of growing oocytes. After 20 h of incubation, lacy EGFP signals were observed as a lacy pattern in the perinuclear region, in the periphery, and in large (up to 3 μm in diameter) circular structures. (C) Light microscopic image of (B) by using differential interference optics to visualize the nucleus and extracellular zona pellucida matrix. Bars, 10μ m.
- FIG. 2.
Linker-scanning mutations of the carboxyl terminus of ZP3. (A) A second ZP3-EGFP expression vector (top) was used to construct four linker-scanning mutations (MutA, MutB, MutC, and MutD) with a FLAG epitope (DYKDDDDK) to sequentially replace 8-amino-acid domains between the potential proprotein convertase (Furin) cleavage site and the transmembrane domain. Nine amino acid sequences of ZP3 (mouse [Mo], rat, hamster [Ha], human [Hu], macaque [Mac], marmoset [Mar], pig, cow, and dog) were aligned from the terminal, conserved cysteine residue of the zona domain to the predicted transmembrane domain. Residues that are identical in all sequences are indicated as white on a black background; those that were conservative substitutions in all sequences are indicated as black on a gray background. The sequence included in the mouse ZP31-31-EGFP-ZP3342-424aa construct (Fig. 1A) was enclosed in a box. The monoclonal antibody binding site on mouse ZP3 (amino acids 336 to 342), the potential proprotein convertase (furin) recognition site (amino acids 350 to 354), and the hydrophobic patch (amino acids 361 to 369) are indicated above the alignment. (B) Western blot of ZP3-EGFP mutants expressed in heterologous somatic cells. At 3 days after transfection with plasmid vectors encoding either normal or MutA, MutB, MutC, or MutD ZP3-EGFP, 10T[1/2] cell supernatants (left) and pellets (right) were harvested. Proteins in each were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with a monoclonal antibody specific to mouse ZP3. Molecular mass markers are indicated on the left in kilodaltons.
- FIG. 3.
Confocal microscopy of ZP3-EGFP mutants expressed in heterologous somatic cells. Cells transfected with plasmid expression vectors were incubated with BFA overnight, allowed to recover for 30 min, and fixed for imaging. Cells expressing normal (A1 to 4 and B1 to 4), MutA (A5 to 8 and B5 to 8), MutB (A9 to 12 and B9 to 12), MutC (A13 to 16 and B13 to 16), or MutD (A17 to 20 and B17 to 20) ZP3-EGFP were incubated with antibodies to PDI (A) or α-ManII (B) and imaged to detect antibody binding with a Cy5-conjugated secondary antibody (A1, 5, 9, 13, and 17 and B1, 5, 9, 13, and 17), ZP3-EGFP (A2, 6, 10, 14, and 18 and B2, 6, 10, 14, and 18) or both (A3, 7, 11, 15, and 19 and B3, 7, 11, 15, and 19). Differential interference optic images were obtained for each transfected cell line (A4, 8, 12, 16, and 20 and B4, 8, 12, 16, and 20). ManII, α-ManII, DIC, differential interference optics. Scale bar, 2μ m.
- FIG. 4.
Confocal microscopy of mutant ZP3-EGFP expressed in growing oocytes. Plasmid vectors expressing normal, MutA, MutB, MutC, or MutD ZP3-EGFP were injected into the nucleus of growing oocytes and cultured for 40 h. Oocytes were incubated with a lipid membrane stain (PM-R18) before (A1, 4, 7, 10, and 13) or after (B1, 4, 7, 10, and 13) freeze-thawing in the presence of 0.5 M NaCl and 1% NP-40. PM-18 (A1, 4, 7, 10, and 13 and B1, 4, 7, 10, and 13) and ZP3-EGFP (A2, 5, 8, 11, and 14 and B2, 5, 8, 11, and 14) were viewed by individually and as a composite (A3, 6, 9, 12, and 16 and B3, 6, 9, 12, and 15) after superimposition on a light microscopic image. Scale bar, 20μ m.
- FIG. 5.
Confocal microscopic imaging of ZP3-EGFP mutants expressed in transgenic mice. Mouse lines with transgenes expressing either normal, MutA, or MutB ZP3-EGFP were established and compared to normal, nontransgenic mice. Almost fully grown oocytes or eggs were isolated and imaged by confocal microscopy to detect EGFP alone (A, C, G, I, and K) or superimposed on images obtained by differential interference contrast optics (B, D, H, J, and L). No background signal was detected in the cytoplasm or zona pellucida of normal mice (A and B), and the strongest signal was observed in fully grown oocytes from normal ZP3-EGFP transgenic mice (C and D). In smaller growing oocytes, normal ZP3-EGFP also was observed in large circular structures (E) that costained with BODIPY-TR ceramide (F). (G and H) A diminished, although significant signal, was observed in MutA ZP3-EGFP mice. (I and J) No EGFP was observed in the zona pellucida of MutB ZP3-EGFP mice, but reporter protein was present in the cytoplasm and incorporated in the circular structures. (K and L) EGFP was not observed in the cytoplasm or in the zona pellucida of ovulated eggs isolated from MutB ZP3-EGFP transgenic mice. Scale bar, 20μ m.
- FIG. 6.
MS defines C terminus of ZP3. (A) Microscale electrospray QTOF-MS analysis of an AspN digest of deglycosylated zona pellucida proteins detected quadruply (upper) and triply (lower) charged ions of 330DSSSSQFQIHGPRQWSKLVSRN351 at 40.3 min by reversed-phase liquid chromatography. (B) The triply and quadruply charged ions of the carboxyl terminus peptide at m/z 848.71 and 636.78. (C) The identity of the quadruply charged ion of 330DSSSSQFQIHGPRQWSKLVSRN351 was confirmed by MS/MS.
- FIG. 7.
Zona pellucida proteins. Mouse ZP1 (623 amino acids), ZP2 (713 amino acids), and ZP3 (424 amino acids) each has a signal peptide (blue oval) to direct it into a secretory pathway, an ∼260-amino-acid zona domain that contains eight conserved cysteine residues (yellow oval) and a transmembrane domain (red oval) near the carboxyl termini followed by a short cytoplasmic tail. The amino acid sequences of the three mouse zona proteins are aligned between a ZP3-specific monoclonal antibody binding site (darkened box, amino acids 336 to 342) and the ZP3 transmembrane domain (amino acids 387 to 410). Each zona protein has a conserved cleavage site (arrow) for proprotein convertase/furin (R-X-R/K-R) and a hydrophobic patch that are 36 to 53 and 25 to 40 amino acids, respectively, N terminal to the transmembrane domain. The carboxyl terminus of ZP3 defined by MS is shown in red letters. The binding site of the monoclonal antibody that recognizes ZP3 in the extracellular zona pellucida is underlined.
