Journal: eLife

Article Title: Cross-species insemination reveals mouse sperm ability to enter and cross the fish micropyle

doi: 10.7554/eLife.106303

Figure Lengend Snippet: ( A ) Schematic of zebrafish ZP2 and ZP3 peptides compared to mammalian homologs (Clustal Ω). Yellow bars represent cysteine residues. Percent values indicate identity between mouse and zebrafish amino acid sequences ( NP_035905.1 , mouse ZP2; AAK16578.1 , zebrafish ZP2, variant A; AAK16577.1 , zebrafish ZP2, variant B; AAK16579.1 , zebrafish ZP2, variant C; NP_035906.1 , mouse ZP3; NP_571406 , zebrafish ZP3). ( B ) SDS-PAGE of recombinant zebrafish ZP2, ZP3, or ZP2 and ZP3 peptides (6-His mAb, immunoblot) expressed in Sf9 cells after purification from agarose beads. Molecular mass is indicated on the left. ( C ) Capacitated mouse sperm binding to beads carrying zebrafish (zf) ZP2, zfZP3, or zebrafish zfZP2 and zfZP3. Beads carrying mouse ( m ) ZP2 N-termini and beads alone were used as positive and negative controls, respectively. Differential interference contrast (DIC) (top) and confocal z projection (bottom) images, sperm nuclei (blue) stained with Hoechst. ( D ) Boxplots represent the median (vertical line) number of mouse sperm binding to mammalian/fish peptide beads or beads alone and data points within the 10th and 90th percentiles (error bars). Boxes include the middle two quartiles, and dots indicate the outliers. Superscript letters show statistical significance (p<0.05) defined by one-way ANOVA followed by Tukey’s HSD (honestly significant difference) post hoc test. ( E ) Schematic of cross-species insemination assays using mouse sperm to inseminate zebrafish chorion (top) or eggs (bottom). ( F ) Representative pictures of zebrafish chorion or eggs inseminated with mouse sperm. Top-right panel: mouse sperm binding to normal mouse eggs after 60 min of incubation. Inset, ×2.0 magnification. Mouse two-cell embryos serve as a negative control for sperm binding. Bottom-right panel: mouse sperm binding to mouse two-cell embryos; mouse eggs serve as an internal positive control for mouse sperm binding. Top-left panel: mouse sperm incubated with zebrafish chorion without ovulated oocyte (60 min incubation). Bottom-left panel: mouse sperm incubated with zebrafish ovulated oocytes (60 min incubation). ( G ) Boxplots represent the median (vertical line) number of mouse sperm binding to mammalian/fish oocytes or embryos (sperm bound per 20 µm 2 projected surface area) and data points within the 10th and 90th percentiles (error bars). Boxes include the middle two quartiles, and dots indicate the outliers. Superscript letters show statistical significance (p<0.05) defined by one-way ANOVA followed by Tukey’s HSD post hoc test. D and G, three independent biological replicates. Figure 1—source data 1. Original uncropped, unedited blot file. Figure 1—source data 2. Uncropped blot with molecular markers and zebrafish ZP2 and ZP3 protein bands labeled.

Article Snippet: Recombinant peptides were purified on IMAC Sepharose High-Performance beads (Cytiva) per the manufacturer’s instructions and assayed on SDS-PAGE as previously reported ( ).

Techniques: Variant Assay, SDS Page, Recombinant, Western Blot, Purification, Binding Assay, Staining, Incubation, Negative Control, Positive Control, Labeling