Cross-species insemination reveals mouse sperm ability to enter and cross the fish micropyle
- Sidra Medicine, Research Branch, Reproductive and Perinatal Health Division, Qatar
- Department of Biological Science, University of Tulsa, United States
- Department of Biomedical Sciences, Qatar University, Qatar
Figures
Figure 1 with 2 supplements
Mouse sperm do not bind to zebrafish zona proteins.
(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 µm2 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.
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Figure 1—source data 1
Original uncropped, unedited blot file.
- https://cdn.elifesciences.org/articles/106303/elife-106303-fig1-data1-v1.zip
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Figure 1—source data 2
Uncropped blot with molecular markers and zebrafish ZP2 and ZP3 protein bands labeled.
- https://cdn.elifesciences.org/articles/106303/elife-106303-fig1-data2-v1.zip
Figure 1—figure supplement 1
Zebrafish eggs in human tubal fluid/human serum albumin (HTF/HSA).
Zebrafish eggs maintained either in Hank’s solution at room temperature (top) or under mouse in vitro fertilization (IVF) conditions (bottom) for 240 min.
Figure 1—figure supplement 2
Cross-species insemination of ovulated mouse Metaphase II (MII) oocytes with zebrafish sperm.
(A) Left, Hoechst-stained zebrafish sperm (light blue) in the micropyle of freshly ovulated oocytes. Right, mouse MII oocytes inseminated with Hoechst-stained zebrafish sperm. (B) Quantification of the observations shown in A, presented as boxplots illustrating the median number of mouse sperm bound to mammalian/fish peptide-coated beads or control beads. Error bars represent the 10th and 90th percentiles, while the boxes indicate the interquartile range (25th to 75th percentiles). Superscript letters indicate statistically significant differences (p<0.05) as determined by one-way ANOVA followed by Tukey’s HSD post hoc test.
Figure 2
Fish sperm interaction with the micropyle.
(A) Confocal images of the micropyle stained by WGA-633 in zebrafish oocytes (n>15); arrows indicate the micropyle. (B) Zebrafish in vitro insemination: Hoechst-stained zebrafish sperm (light blue) that have approached or entered the micropyle in freshly ovulated oocytes (yellow, WGA-633-stained); samples were fixed in paraformaldehyde few seconds after insemination. (C) Zebrafish eggs, untreated (left), or treated with trypsin to eliminate the micropyle protein (right). (D) Fluorescence was measured (Fiji/ImageJ) within a 20 µm2 area; 0 indicates the micropyle opening position (yellow), 160 µm indicates the most distant position measured from micropyle opening. (E) Same as in (C); left, DIC, right, confocal images (maximum intensity projection) of zebrafish oocytes inseminated with zebrafish sperm. (F) Same as in Figure 1G, for the quantification of the number of zebrafish sperm approaching and entering the micropyle of oocytes treated/not treated (control) with trypsin (n=3). (G) Same as in Figure 1G, for fertilization rates (n=3).
Figure 3 with 5 supplements
Mouse sperm cross the fish micropyle.
(A) Cross-species insemination: mouse sperm (Hoechst-stained, light blue) in the zebrafish micropyle region of a chorion (60 min incubation) surrounding the oocyte (left) or of a chorion mechanically freed from the oocyte (right, Ghost). (B) Quantification of mouse sperm in the micropyle region of chorion with or without zebrafish oocyte: same as in Figure 1G; mouse eggs or two-cell embryos served as an internal positive and negative control for sperm binding (n=3). (C) X-Y plane confocal projection of zebrafish chorion encompassing the WGA-633 (yellow) micropyle region (~320 µm); bar with arrows indicates positions at which fluorescence was measured (Fiji/ImageJ) as in Figure 2D. (D) Quantification of mouse sperm across the micropyle region: boxplots represent the median (vertical line) raw integrated density ratio (RID ratio, left Y axis) measured on 10 zebrafish chorions; data points within the 10th and 90th percentiles (error bars). Boxes include the middle two quartiles, and dots indicate the outliers. A light blue line represents the number of sperm (right Y axis) found in the corresponding chorion position (X axis); error bars represent s.e.m.; statistical significance (p<0.05) across RID ratios or sperm numbers in different positions is defined by one-way ANOVA followed by Tukey’s HSD (honestly significant difference) post hoc test. (E) Time-lapse frames from Figure 3—video 1, showing the first sperm entering the micropyle of a freshly ovulated zebrafish egg. Green arrowheads indicate mouse sperm. Insets show the number of seconds (‘s’) after the first sperm appears in the field of view.
Figure 3—figure supplement 1
Trypsin treatment of zebrafish micropyle.
(A) Zebrafish eggs untreated (left) or treated with trypsin to eliminate the micropyle protein (MP) (right); top panels, differential interference contrast (DIC) images; bottom panels, confocal images. Yellow is the MP stained with WGA-633. (B) Same as in Figure 1G, with zebrafish trypsin-treated vs. untreated.
Figure 3—figure supplement 2
Mouse sperm bypassing the oocyte, swimming close without interacting with the micropyle or entering the micropyle canal.
Figure 3—figure supplement 3
Time-lapse confocal imaging of micropyle detachment under mouse in vitro fertilization (IVF) conditions.
Arrows indicate progressive separation of the micropyle from the egg cell. Seconds, ‘s’.
Figure 3—video 1
The entry of mouse sperm into the micropyle of an ovulated zebrafish egg.
This time-lapse video captures the moment the first sperm enters the micropyle of a freshly ovulated zebrafish egg. At the 1 s mark, two sperm are visible in the field of view. One sperm displays an abnormal structure, with a partially coiled tail, while the other sperm is attracted to the micropyle. Within 6 s, the structurally normal sperm successfully navigates to and enters the micropyle canal.
Figure 3—video 2
Simultaneous entry of two sperm into the micropyle canal of a zebrafish egg.
Simultaneous entry of two sperm into the micropyle of a freshly ovulated zebrafish egg. Initially, both sperm interact with the wall of the micropyle. After 12 s, they move together into the micropyle canal opening.
Figure 4 with 5 supplements
Localization and dynamics of sperm interactions with the micropyle and inter-chorion space (ICS) in zebrafish oocytes.
(A) Hoechst-stained sperm (blue) which has crossed the micropyle. Differential interference contrast (DIC) (left) and confocal (mid-panel) projection of sperm accumulated in the ICS of a zebrafish oocyte imaged from the top; in the left and middle/top panels, the 633 channel is turned off to visualize the sperm accumulated in the ICS around the micropyle region (yellow circled); inset shows a longitudinal section of the same oocyte, showing the Hoechst-stained sperm (blue) under the WGA-633-stained micropyle region (yellow). Right, Hoechst-stained sperm (blue) is included in the ICS. (B) Quantification as in Figure 1D of mouse sperm in the ICS below (left) or away (right) from the micropyle region. Error bars represent s.e.m., statistical significance (p<0.05) is defined by one-way ANOVA followed by Tukey’s HSD (honestly significant difference) (n=3). (C) Electron microscopy of mouse sperm in the zebrafish micropyle region and within the micropyle canal, 1 hr after insemination. Left, scanning electron microscopy (SEM), mid and right panels, transmission electron microscopy (TEM); IAM, inner acrosomal membrane; OAM, outer acrosomal membrane; ES, equatorial segment. (D) AcrTg sperm in the micropyle region. Left, DIC; right, confocal projection. The yellow arrow indicates the micropyle opening. (E) As in Figure 1G, with AcrTg sperm (intact vs. reacted). (F) Acrosome-intact (red arrows) and reacted (light-blue arrows) mouse sperm in zebrafish ICS. Inset represents a ×2 magnification of an acrosome-intact sperm in the ICS. (G) Left panel, micropyle structure after the entry of multiple mouse sperm. Right panel, acrosome-intact sperm from the ICS of the oocyte, the micropyle of which is shown in the left panel. Black arrows, mouse sperm.
Figure 4—figure supplement 1
Orthogonal view generated using ZEN Lite software (Zeiss, Germany), showing z-stack sections at three focal planes.
Axes: bottom panels=XY, top panel = XZ. (A) Surface of the WGA-stained chorion; (B) mid-region of the inter-chorion space (ICS); (C) oocyte plasma membrane (PM). Sperm nuclei stained with Hoechst appear in light blue.
Figure 4—figure supplement 2
Interaction of conserved mouse and fish sperm trimmers with mammalian or fish egg proteins.
(A) Heatmaps showing the predicted aligned error (PAE) plots for the top scoring model in AlphaFold2-Multimer of the interaction between the fish or mouse trimers (Izumo1+Spaca6+Tmem81) and the zebrafish Bouncer or mouse Izumo1R. Blue indicates low, red high PAE scores. (B) Predicted 3D structure of the interactions in (A). (C) Box plots showing the distribution of interface-predicted template modeling (ipTM) scores. Asterisks denote statistically significant differences (p<0.001) as determined by a two-sided t-test.
Figure 4—video 1
Mouse sperm interaction with zebrafish egg’s inter-chorion space (ICS).
Motility pattern of mouse sperm within the zebrafish egg’s ICS (×10 magnification).
Figure 4—video 2
Mouse sperm in the zebrafish inter-chorion space (ICS).
Same as in Supplementary video 3, ×20 magnification.
Figure 4—video 3
High-resolution view of mouse sperm zebrafish inter-chorion space (ICS) area.
Same as in Supplementary video 3, ×40 magnification.
Figure 5
CatSper is necessary for mouse sperm crossing the zebrafish micropyle.
(A) Hoechst-stained fertile CatsperdHet (control, left) and CatsperdNull sperm binding to ovulated mouse eggs. (B) Same as in Figure 1D, per mouse egg. (C) Fertile CatsperdHet (control, left) and CatsperdNull sperm in the perivitelline space (PVS) of Cd9Null female eggs upon in vivo mating. (D) Same as in Figure 1D, number of PVS sperm per Cd9Null mouse egg. (E) Fertile CatsperdHet (control, left) and CatsperdNull (right) Hoechst-stained sperm in the micropyle region of zebrafish eggs; top, differential interference contrast (DIC) image; bottom, confocal. (F) Same as in Figure 1G, with CatsperdHet vs. CatsperdNull sperm. B, D, and F (n=3).
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/106303/elife-106303-mdarchecklist1-v1.docx
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Source data 1
Source data for quantitative analyses, immunoblots, and microscopy measurements supporting all main and figure supplements.
- https://cdn.elifesciences.org/articles/106303/elife-106303-data1-v1.xlsx
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Cross-species insemination reveals mouse sperm ability to enter and cross the fish micropyle
eLife 14:RP106303.
https://doi.org/10.7554/eLife.106303.3
