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

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Copyright © 2016, The Author(s)

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(A) GFP-CETN2 is detected at the centriole pair at the implantation fossa (green, arrowhead) of a caput spermatozoon one hour after fertilization by ICSI. Microtubules are not detected assembling at the sperm head (red, microtubules; blue, DNA) as might be predicted if a sperm aster were to form. The sperm axoneme (red; long arrow) remains detectable, attached to the still condensed sperm head (blue). The oocyte remains arrested at metaphase-II and cytoplasmic microtubules, assembled into small cytasters, are found throughout the cytoplasm (red, short arrows). Top inset: GFP-CETN2 caput spermatozoa centrioles, green; microtubules, red; and DNA, blue. Lower inset: DIC showing sperm head (arrowhead). (B,C) By 1.5–2 hrs post-ICSI, the microinjected sperm head initiates decondensation to form the male pronucleus (M, male pronucleus; blue, DNA) as the female pronucleus forms (F, female pronucleus; blue, DNA) and 2nd polar body extrusion (2nd PB) within the oocyte’s cytoplasm was activated, probably by the microinjection event itself. The GFP-CETN2 centrioles remain at the implantation fossa (green, arrowheads) in a region devoid of assembling microtubules (red; microtubules, short arrows), even though many cytoplasmic MTOCs nucleate microtubules. Top inset: sperm decondensation (blue), GFP-CETN2 centrioles (green, arrowheads) and microtubules (red) at the sperm heads. Lower inset: DIC showing ICSI sperm (arrowheads) with attached axoneme (long arrows), female pronucleus (F) and 2nd polar body. The dividing 1st polar body is identified in B. (D,E) Two zygotes 3 hrs post-ICSI. Early male pronuclear decondensation (D: M; blue, DNA) shows caput spermatozoon GFP-CETN2 centrioles adjacent to the sperm head (D: green, arrowhead). As male pronucleus nears completion (E: M; blue, DNA), the GFP-CETN2 signal appears to disperse into punctate foci (E: green, arrowhead). Numerous microtubule asters assemble from cytoplasmic MTOCs (D, E: red; microtubules, short arrows), and one is near the male pronucleus (D, E: red, short double arrows), but it does not emanate from the GFP-CETN2 centrioles (D, E: green, arrowheads) in the activated zygotes with female pronucleus (D, E: F female pronucleus) and 2nd polar bodies. Insets: (B,C): sperm axoneme (short arrow). Triple-labeled for GFP-CETN2 centrioles (green), microtubules (red) and DNA (blue). DIC: differential interference contrast. Bar: 20 μm.