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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. 1 — 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