Research ArticleINFERTILITY AND CONTRACEPTION

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

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Science Translational Medicine  27 Apr 2016:
Vol. 8, Issue 336, pp. 336ra60
DOI: 10.1126/scitranslmed.aad9946
  • Fig. 1. Mouse and human sperm bind to the N terminus of ZP2.

    (A) Schematic of the transgene used to establish the AcrmCherry transgenic mice. SV40 poly(A), SV40 simian virus polyadenylation signal. (B) Epididymal sperm isolated from AcrmCherry mice were fixed on polylysine-coated slides, stained with Hoechst, and imaged by confocal alone (left) or merged with differential interference contrast (DIC) microscopy (center). Two frames, 1.5-s apart, of a video before (top right) and after (bottom right) induction of acrosome exocytosis with calcium ionophore A23187. Arrows, acrosome; asterisk, nucleus. Scale bars, 10 μm. (C) Binding of sperm to cumulus-free mouse eggs using AcrEGFP sperm freshly released from the epididymis (green, left) or AcrmCherry sperm after 1 hour of incubation in HTF/BSA (red, middle) alone or mixed (1:1) with the freshly released sperm (right). DIC (upper) and confocal Z projection (lower) images were obtained after fixation and staining with Hoechst (three biological replicates). Scale bar, 20 μm. (D) Schematic of moZP235–149 (blue) and huZP239–154 (gray) peptides at the N terminus of ZP2 (tan) that mediate sperm-egg binding in mice and humans, respectively. Inverted triangle, moZP2 postfertilization cleavage site (167LADE170); zona domain, ZP2365–630; yellow vertical bars in lower flyout, cysteine residues. (E) Model of moZP235–149 or huZP239–154 peptide beads interacting with sperm freshly released from the epididymis (green) and incubated for 1 hour (red) in HTF/BSA before insemination. (F) Mouse sperm freshly released from the epididymis (left) and thawed human sperm (right) bound to moZP235–149 and huZP239–154 peptide beads, respectively, in comparable numbers when assayed immediately or after 1 hour (hr) of incubation in HTF/BSA before insemination. Neither human nor mouse sperm bound to beads alone control (three biological replicates) (fig. S1E). Box plots reflect the median (horizontal line) number of mouse or human sperm binding to peptide beads and data points within the 10th and 90th percentiles (error bars). Boxes include the middle two quartiles, and outliers are indicated by dots.

  • Fig. 2. The N terminus of moZP2 decoys sperm in vitro and prevents mouse fertilization.

    (A) Schematic of in vitro fertilization, in which mouse eggs in cumulus mass were incubated overnight with 1 × 105 progressive motile mouse sperm in HTF/BSA (500 μl) alone, or in the presence of beads alone or moZP235–154 peptide beads (100 μl). (B) In vitro fertilization (%) after co-incubation of eggs in cumulus mass with medium (no beads) (1), beads alone (2), or ZP2 peptide beads (3) (three biological replicates). Statistical difference from control is indicated by a letter different than a; (b) Ρ = 0.027; (c) Ρ < 0.001. (C) Sperm from AcrmCherry (red acrosome) mice were incubated with moZP235–149 peptide before (left) or after (right) acrosome exocytosis, cross-linked with formaldehyde, immunostained with a monoclonal antibody (mAb) to the N-terminal ZP2 peptide (green), DNA-stained with Hoechst (blue), and imaged by confocal microscopy and DIC. Asterisk, acrosome; Eq, equatorial segment; arrows, postacrosomal region. Scale bar, 5 μm. (D) Progressive motility of unbound mouse sperm over 8 hours. Sperm were isolated either immediately after release from the epididymis or after 1 hour of incubation in HTF/BSA before incubation with ZP2 peptide beads. Control sperm were incubated with beads alone (three biological replicates). Statistical difference from control is indicated by a letter different than a (details in table S3). (E) Sperm were prepared as in (D), and the percentage of acrosome-intact sperm that remained bound to ZP2 peptide beads was determined over 8 hours (three biological replicates). (F) Electron microscopy of acrosome-intact (left panel) and acrosome-reacted (right panel) sperm that remain bound to the ZP2 peptide beads 8 hours after insemination. IAM, inner acrosomal membrane. OAM, outer acrosomal membrane; PAR, postacrosomal region; P, perforatorium. Scale bar, 0.5 μm.

  • Fig. 3. The N terminus of huZP2 prevents zona matrix penetration of human sperm.

    (A) Same as Fig. 2A except that 1 × 105 progressive motile human sperm were added to huZP2Rescue eggs in cumulus with huZP239–154 peptide beads or beads alone. (B) Same as Fig. 2D except with human sperm (three biological replicates). Statistical difference from control is indicated by a letter different than a (details in table S3). (C) HuZP2Rescue eggs in cumulus mass were inseminated with capacitated human sperm, in the presence of medium, beads alone, moZP2 peptide beads, or huZP2 peptide beads, and incubated overnight. Eggs were fixed and stained with wheat germ agglutinin, Alexa Fluor 633 conjugate (WGA-633) (red) and Hoechst (blue) to detect zonae pellucidae and nuclei, respectively. Scale bar, 10 μm. The numbers of eggs (avg ± SEM) with 0, 1, 2, 3, or >3 sperm in the perivitelline space (PVS) were determined for each experimental group (lower). Representative confocal and DIC-merged images of huZP2Rescue eggs from each group are shown above the graphs. The total number of eggs analyzed in three independent biological replicates is indicated above each graph. Statistical differences from no sperm in the PVS is indicated by a letter different than a for each experimental condition: no beads, (b) P = 0.040 and (c) P = 0.019; beads alone, (d) P = 0.026; and huZP2 beads, (e) P < 0.001.

  • Fig. 4. Peptide beads select human sperm competent for binding and zona pellucida penetration.

    (A) In the representative merged confocal and DIC images, human sperm (Hoechst-stained) from fertile donors were unselected (medium control or beads alone) or selected on the basis of short-term reversible binding to moZP235–149 or huZP239–154 peptide beads and tested for their ability to bind to the surface of the zona pellucida (stained with WGA-633) surrounding huZP2Rescue eggs (see fig. S4, A and B, for individual donor data). Scale bar, 20 μm. (B) Box plots of sperm binding in (A) reflect the median (horizontal line) number of human sperm binding (cumulative data from five sperm donors) and data points within the 10th and 90th percentiles (error bars) (five biological replicates). Boxes include the middle two quartiles, and outliers are indicated by dots. (C) Human sperm were selected on the basis of short-term (30 min) reversible binding to ZP2 peptide beads (fig. S3C). The graphs show the number of huZP2Rescue eggs with 0, 1, 2, 3, or >3 sperm bound to the zona surface after incubation with medium, beads alone, moZP2 peptide beads, or huZP2 peptide beads using the sperm from five human donors. The total number of eggs analyzed in five biological replicates is indicated above each graph. (D to F) Same as (A) to (C) but assayed for sperm penetration into the perivitelline space (see fig. S4, C and D, for individual donor data). (G) Peptide bead selected human sperm (blue, Hoechst-stained nuclei) from fertile donors binding to zonae pellucidae surrounding premature human oocytes (left). Zp3EGFP mouse oocytes (green zona pellucida) served as a negative control. Scale bar, 100 μm; inset scale bar, 20 μm. The average number of selected human sperm (y axis) from three donors (x axis) binding to four or more zonae pellucidae (right) was analyzed as in (B).

  • Fig. 5. MoZP235–149 acts as a sperm decoy in vivo.

    (A) Schematic of female mouse reproductive tract with transcervical delivery of beads into the bilateral uterine horns. Interaction with moZP2 peptide beads in the uterus prevents normal sperm migration through the uterotubal junction (UTJ) into the oviduct, which results in female infertility. (B) MoZP2 peptide beads were stained with a monoclonal antibody to the N-terminal region of moZP2 and imaged by DIC (left) and confocal (right) microscopy. Scale bar, 50 μm. (C) MoZP2 peptide beads (top panel) and beads alone (bottom panel) were imaged in female uterine horns by confocal microscopy using monoclonal antibodies (green) to ZP2 and WGA-633 lectin (yellow), respectively, after cardiac perfusion and ScaleA2 clarification. Scale bars, 200 μm. (D) Spliced composite images of acrosome-intact (arrows) and acrosome-reacted (asterisks), Hoechst-stained AcrmCherry; Prm1EGFP; FiglaEGFP sperm. Scale bar, 10 μm. (E) Insemination of Zp3EGFP eggs in cumulus with AcrmCherry; Prm1EGFP; FiglaEGFP sperm. Mostly acrosome-intact (yellow) and fewer acrosome-reacted (green, arrows) sperm were visible through the cumulus mass after clarification. Scale bar, 50 μm. (F) Zp3EGFP; Cd9Null female mice were hormonally stimulated and mated with AcrmCherry; Prm1EGFP; FiglaEGFP male mice after transcervical administration of moZP2 peptide beads or beads alone (control) (three biological replicates). After clarification, confocal images (upper panels) were obtained to assay migration of fluorescently tagged sperm in the female reproductive tract. Outlines of organs (dotted lines) and the UTJ were apparent from background tissue autofluorescence. The flyout represents the higher magnification of sperm (green if acrosome-reacted; yellow if acrosome-intact) from the miduterine horn interacting with moZP2 peptide beads at single-cell resolution. In lower panels: arrows, eggs (green); arrowheads, sperm heads. Scale bar of panels, 200 μm; scale bar of the flyout, 20 μm.

  • Fig. 6. MoZP235–149 peptide beads provide long-term, reversible contraception.

    (A) Female mice were mated after transcervical administration of medium (control), beads alone, or moZP2 peptide beads, and fertilization was determined 40 hours later by the presence of two-cell embryos (as a percentage of total two-cell embryos plus unfertilized eggs in the oviduct; three biological replicates). Statistical difference from control is indicated by a letter different than a (Ρ < 0.001). (B) The number and time of live births from two litters each of five female mice continuously mated after transcervical administration of medium (control), beads alone, or moZP2 peptide beads. The numbers shown on the graph indicate the time of the first pups’ birth, expressed as the number of days (avg ± SEM) after the start of mating. (C) Light microscopic images of female reproductive tract 2 weeks after treatment with beads alone or moZP2 peptide beads. Left panels: scale bars, 150 μm; right panels: scale bars, 50 μm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/336/336ra60/DC1

    Fig. S1. Effect of incubation on sperm binding to ZP2 peptide beads.

    Fig. S2. Sperm binding and translocation of moZP2 peptide beads.

    Fig. S3. Selection of human sperm with huZP2 peptide beads.

    Fig. S4. Selection of superior human sperm from individual fertile donors.

    Fig. S5. Sperm binding to native moZP2 peptide beads and inhibition of fertilization.

    Table S1. Primers.

    Table S2. Source data and statistical summaries.

    Table S3. Statistical analyses.

  • Supplementary Material for:

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

    Matteo A. Avella, Boris A. Baibakov, Maria Jimenez-Movilla, Anna Burkart Sadusky, Jurrien Dean*

    *Corresponding author. Email: jurriend{at}helix.nih.gov

    Published 27 April 2016, Sci. Transl. Med. 8, 336ra60 (2016)
    DOI: 10.1126/scitranslmed.aad9946

    This PDF file includes:

    • Fig. S1. Effect of incubation on sperm binding to ZP2 peptide beads.
    • Fig. S2. Sperm binding and translocation of moZP2 peptide beads.
    • Fig. S3. Selection of human sperm with huZP2 peptide beads.
    • Fig. S4. Selection of superior human sperm from individual fertile donors.
    • Fig. S5. Sperm binding to native moZP2 peptide beads and inhibition of fertilization.
    • Table S1. Primers.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Source data and statistical summaries.
    • Table S3 (.pdf format). Statistical analyses.

    [Download Tables S2 and S3]

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