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. 2016 Aug 18;6:31816. doi: 10.1038/srep31816

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Copyright © 2016, The Author(s)

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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(A–H) Zona-free oocytes fuse with immotile immature epididymal sperm in the presence of Sendai virus fusion extract, though the oocytes are typically polyspermic. The caput sperm were first treated with ionomycin to assist with membrane fusion with the zona-denuded oocytes. (A–D) Monospermic Insemination With Caput Sperm. E-H Dispermic Insemination With Caput Sperm. By 6.5 hrs post-insemination, two decondensing sperm heads (A, E: blue, DNA) with GFP-CETN2 centrioles are detectable (A, E green, arrowheads) though microtubules do not assemble adjacent to the incorporated sperm (A, E: left insets: red, microtubules). Oocytes remain at metaphase-II arrest (A, E: red), suggesting caput sperm, even after ionomycin, cannot initiate oocyte activation. Acetylated α-tubulin–labeled axonemes (red) are detached (A: short arrow; right inset: red) or adherent (E and right inset: red). A, E (long arrows): Bound, unincorporated sperm at the oocyte surfaces. Cauda Sperm: (I–L) Polyspermic Insemination With Cauda Sperm. Inseminated with cauda GFP-CETN2-sperm. By 6.5 hrs post-insemination, three decondensed male pronuclei are visible (I: MPn; blue, DNA) and 2^nd polar body extrusion demonstrates oocyte activation (I: FPn, female pronucleus; red). Neither GFP-CETN2 (I: green) nor microtubule assembly (I: red) near the male pronuclei are observed. (I: A rare GFP-CETN2–expressing cauda sperm early in decondensation (green, arrowheads; right inset: arrowheads; red: acetylated α-tubulin) is detected in a region devoid of assembled microtubules (left inset: red, microtubules). Maternal cytoplasmic MTOCs assemble microtubule asters (left inset: arrows). (B,C,F,G,J,K) rotation renderings of z-stacked image discriminate incorporated sperm (blue, arrowheads) vs unincorporated ones (long arrows), together with GFP-CETN2 centrioles (green, arrowheads) and axonemes (red, short arrows) in lateral (B,F,J) and en face (C,G,K; directionality, figure bottom). Sperm heads/male pronuclei ((J,K): MPn, arrowheads), female pronuclei ((J,K): FPn). Quadruple-labeled for GFP-CETN2 (green), acetylated α-tubulin (red and right insets) or YOL 134 microtubules (left insets, red), and Hoechst (blue). Bars: μm.