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. 2019 Oct 1;10:4456. doi: 10.1038/s41467-019-12406-z

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Sperm:myoblast fusion via PtdSer and the BAI3-ELMO2-RAC1 signaling axis. a Oocytes and myoblasts express similar molecules. Juno expression is readily detected on oocytes but not myoblasts. Bars represent mean ± s.e.m. c, Schematic of the sperm:myoblast fusion assay. Caudal epididymal sperm were labeled with Calcein-AM (red) and co-cultured with murine C2C12 myoblasts. After 4 h, myoblasts were washed, stained with Hoechst dye (to stain nuclei) and the percentage of Calcein-AM⁺ myoblasts was evaluated by microscopy. d Representative images depicting myoblasts that fuse with sperm to become Calcein-AM⁺ (3–10% per field) under control conditions (left panel), whereas this is greatly reduced when the sperm was pretreated with BAI1-TSR to mask PtdSer (middle panel) or after treatment of the myoblasts with the RAC1 inhibitor (right panel). Scale bar, 50 μm. e Detection of sperm inside the myoblasts. YFP⁺ sperm were co-incubated with C2C12 myoblasts for 4 h. Myoblasts were washed, fixed, and stained by immunofluorescence with antibodies to YFP/GFP (green) and Izumo1 (pink). Phalloidin (red) and Hoechst (blue) were used to stain the actin cytoskeleton and DNA, respectively. On the left panel, the dense sperm nucleus contained within the phalloidin⁺ cytoplasm is shown on the cross-sectional plane. White arrows: sperm nucleus; green arrow: sperm tail; dotted line: outline of the sperm head. Scale bar: 5 μm. f Reduced percentage of Calcein-AM⁺ myoblasts after masking of PtdSer on sperm using BAI1-TSR, antibody to BAI3, shRNA-mediated knockdown of Elmo2 in myoblasts, treatment of myoblasts with the RAC1 inhibitor EHT-1864 or after pretreatment of myoblasts with cytoskeletal disruption via cytochalasin D (Cyto. D) or paraformaldehyde (PFA) fixation. (n = 3 independent experiments for each condition except for RAC1 inhibitor, n = 4 independent experiments). Bar charts show mean ± s.e.m. *p < 0.05, **p < 0.001, ***p < 0.0001 (one-way ANOVA followed by Dunnet’s multiple comparisons test and two-tailed unpaired Student’s t test). Each dot represents one experiment. Source Data are provided in the Source Data File