Fig. 4. Disruption of the cortical cytoplasm associated with the position of the sperm entry disturbs spatial patterning of the blastocyst. (A) Fertilisation cone (fc) of freshly fertilised eggs was marked by attaching a fluorescent bead at the time of polar body (pb) extrusion. (B) 2-3 hours later, after the male pronucleus (mp) started to migrate toward the female pronucleus (fp) at the egg centre, the cortex and the associated cytoplasm marked by the bead was removed. (C) Fragment of excised cytoplasm. Next, the site of the operation was re-labelled with another fluorescent bead (not shown). Blastomeres at the 2-cell stage were labelled with different coloured dyes and the distribution of cells was examined by the confocal sectioning at the blastocyst stage. (D) An individual section of such a blastocyst. The clonal border of the 2-cell stage progeny (marked by yellow dashed line) is tilted with respect to the boundary zone between the embryonic and abembryonic parts (white dashed lines). A fluorescent bead (pale green) is visible. (E) A control experiment in which another region of the cortex of the zygote, approximately 90° from the fertilisation cone, was removed instead. (F) Fragment of excised cytoplasm. Resulting eggs from such a manipulation (G) were labelled at the 2-cell stage as before and cultured to the blastocyst stage. (H) An individual section of such a blastocyst showing distribution of labelled cells. Scale bar: 25 µm (in A for A,B,C,E,F,G and In D for D,H).

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