Figure 5

Mitotic spindle assembly and first cleavage patterns differ between IVF- and ICSI-Fertilized NHP Zygotes. Rhesus IVF- (A–E) or ICSI- (F–J) fertilized zygotes. (A,F) Interphase zygotes with radially oriented (IVF: A) versus para-tangentially (ICSI: F) aligned apposed male and female pronuclei. White asterisks: duplicated centrosome positions; white bars: spindle equator chromosome alignment and anaphase cleavage plane direction. (B,G) fixed immunostained zygotes with duplicated, split centrosomes (arrowheads) juxtaposed between the apposed male and female pronuclei (blue) and nucleating astral microtubules (green; (G) inset, total tubulin [red] and DNA [blue]; incorporated sperm axoneme [small arrows]). (C,H) fixed metaphase spindles immunostained for microtubules (green) and DNA (blue), showing eccentric and tangentially-oriented IVF spindles, are distinct from radially oriented, cortically distal ICSI spindles (H: sperm axoneme, small arrows). White bars: the orthogonal anaphase chromosome separation plane. (D,I) A fixed, immunostained IVF anaphase spindle with enhanced spindle microtubules (D: green) signaling cortical cleavage furrow initiation (arrow) compared to an ICSI anaphase spindle lacking cortical microtubule interaction at the furrow (I: green, arrow). Both cleavage planes are orthogonal to the separating sister chromosomes (blue). (E,J) Equal IVF telophase (E) cleavage versus unequal ICSI telophase cleavage (J) despite asymmetric cleavage furrows (arrows) progressing orthogonally to midbodies (green, arrowheads; blue, DNA; small arrowhead: centrosome; small arrows: sperm axoneme). (K1-K4, L1-L4). TLVM images of two ICSI rhesus zygotes, both with eccentric, radially aligned, apposed male and female pronuclei (K1, L1). The cleavage furrow begins near the previously intact MPn (K2, L2: arrows), progressing asymmetrically (K3, L3: arrows) but not through the second polar bodies (Pb). (K4,L4) anti-tubulin (green) and Hoechst DNA (blue) immunostaining of (K3,L3) zygotes fixed 2 min post-live imaging. Central microtubule midbodies (green, arrowheads) lie orthogonal to the progressing cleavage furrows (arrows). Polar bodies are excluded from the cleavage planes (K4: Pb; L4: * marks lost polar bodies). HMC images: (A,F,K1–K3,L1–L3); B: triple-labeled for microtubules (green), γ-tubulin (red), and DNA (blue); (G) quadruple-labeled for glutamylated-microtubules/γ-tubulin (green), total tubulin (red), and DNA (blue); (K4,L4) microtubules (green) and DNA (blue). Pb: polar bodies. Bars = 20 μm.