Fig 2: Phosphatidylserine on live sperm is important for in vitro fertilization. a Depiction of the mouse testis and epididymis. b Sperm from different regions of the epididymis were allowed to swim/disperse in TYH + BSA medium, stained with Annexin V and Hoechst, and evaluated by microscopy. c, d Annexin V staining of sperm. Asterisks denote sperm heads, and arrows midpiece. Scale bar, 20 µm. e Percentage of Annexin V + sperm from the caput (n = 9 mice), corpus (n = 8 mice), and cauda (n = 15 mice) epididymis, with each dot representing one mouse (six independent experiments). *p < 0.05, ***p < 0.001 (one-way non parametric ANOVA was followed by Kruskal–Wallis test for multiple comparisons). f, g, Sperm from cauda epididymis were incubated with 50 µg/ml GST only f or GST-BAI1-TSR g, washed, fixed and visualized by GST immunofluorescence. Scale bar, 20 µm. h Snap shots of a movie depicting motility of live Annexin V + (green) sperm (t: time in min). The trajectory of a single sperm is traced by a white dotted line. Scale bar, 30 µm. i Schematic of the in vitro fertilization assay: cumulus oocyte complexes isolated from wt super-ovulated females were incubated with caudal sperm previously capacitated, in the presence or absence of 10 µg/ml Annexin V, 50 µg/ml GST, or 50 µg/ml GST-BAI1-TSR. The percentage of fertilized eggs (two-cell embryos) was evaluated after 24 h. j Multiple two-cell embryos fertilization with control sperm (left panel, arrows), whereas fewer fertilized eggs were observed with Annexin V (right panel, arrows). Scale bar, 100 µm. k, l Annexin V masking of PtdSer on sperm (k, n = 4 experiments) or GST-BAI1-TSR (l, n = 4 experiments) reduces fertilization. The total number of eggs analyzed is shown in parentheses. Each line represents one experiment and the matching experiments are shown (shape and color). Error bars are s.e.m. *p < 0.05, **p < 0.01 (Two-tailed unpaired Student’s t test). m, n Greater unfertilized oocytes (asterisks) seen after competition with O-Phospho-l-Serine compared with O-Phospho-d-Serine (top). Scale bar, 100 µm. Each line represents one experiment and matching experiments are shown with the same shape and color (n = 4 experiments). Error bars are s.e.m. **p < 0.01 (Two-tailed unpaired Student’s t test). Source Data are provided in the Source Data File

Fig 3: Genetic testing of PtdSer receptors and cytoplasmic signaling in oocytes. a Schematic of PtdSer receptors BAI1/3, the downstream ELMO-DOCK-RAC1 signaling pathway, and other receptors on oocytes. b, c The PtdSer receptors Tim-4, BAI1 and Mer-TK participate in fertilization. ZP-intact oocytes from wt or Tim-4-/- mice b Mer-tk + / + Bai1 + / + , Mer-tk-/- Bai1 + / + , or Mer-tk-/- Bai1 + /- or Mer-tk-/- Bai1-/- c were mixed with wt sperm, and two-cell embryos evaluated at 24 h. Fertilization index is the percentage of fertilized eggs from the experimental group divided by percentage of fertilized eggs from the control group (wt mice). Each dot represents one mouse (b, n = 6 experiments including 17 wt mice and 9 Tim-4-/- mice, c: n = 5 experiments including 15 wt mice, 6 Mer-tk-/- Bai1 + / + and 6 Mer-tk-/- Bai1-/-), total number of eggs (parentheses). *p < 0.05 (b, two-tailed unpaired Student t test), **p < 0.01 (c one-way ANOVA followed by Dunnet’s multiple comparisons test). d Elmo1 and Elmo2 mRNA on cumulus-free Metaphase II oocytes. e Intracellular ELMO1 in isolated Metaphase II (MII) ZP-free oocytes. Scale bar, 20 µm. f Schematic for generation of oocyte-specific Elmo1-deficient mice. g Percentage of fertilized eggs after incubation of control (Elmo1fl/fl) or Elmo1-deficient (Ddx4-Cre/Elmo1fl/fl) oocytes with wt sperm (n = 6 independent experiments including 15 Elmo1fl/fl mice and 12 Ddx4-Cre/Elmo1fl/fl mice). Each dot represents 1 or 2 pooled mice. *p < 0.05 (Two-tailed unpaired Student’s t test). h Schematic for the evaluation early sperm entry into oocytes via DAPI staining of decondensed sperm DNA. ZP-free wt oocytes were incubated with RAC1 inhibitor (EHT-1864, 80 µm) and loaded with DAPI. After several washes, sperm were added, and the presence of internalized sperm with decondensed nuclei was evaluated after 1 h. i Control oocytes displaying the decondensation of the sperm DNA incorporated into the oocyte (arrow), whereas the sperm tail has not yet been internalized. DAPI also highlights the oocyte chromosomes in anaphase II indicating the resumption of meiosis II. Scale bar, 20 µm. j, Decreased percentage of oocytes with decondensed sperm DNA after RAC1 inhibition (n = 4 independent experiments). Numbers in parentheses reflect total number of eggs scored. **p < 0.01 (Two-tailed unpaired Student’s t test). k Sperm motility was not affected by RAC1 inhibition p > 0.05 (two-tailed unpaired Student’s t test). Data are presented as mean ± s.e.m. Source Data are provided in the Source Data File

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