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Following fertilization, the Xenopus egg cortex rotates relative to the cytoplasm by 30 degrees about a horizontal axis. The direction of rotation, and as a result the orientation of the embryonic body axes, is normally specified by the position of sperm entry. The mechanism of rotation appears to involve an array of aligned microtubules in the vegetal cortex (Elinson and Rowning, 1988, Devl Biol. 128, 185-197). We performed anti-tubulin immunofluorescence on sections to follow the formation of this array. Microtubules disappear rapidly from the egg following fertilization, and reappear first in the sperm aster. Surprisingly, astral microtubules then extend radially through both the animal and vegetal cytoplasm. The cortical array arises as they reach the vegetal cell surface. The eccentric position of the sperm aster gives asymmetry to the formation of the array and may explain its alignment since microtubules reaching the cortex tend to bend away from the sperm entry side. The radial polymerization of cytoplasmic microtubules is not dependent on the sperm aster or on the female pronucleus: similar but more symmetric patterns arise in artificially activated and enucleate eggs, slightly later than in fertilized eggs. These observations suggest that the cortical microtubule array forms as a result of asymmetric microtubule growth outward from cytoplasm to cortex and, since cortical and cytoplasmic microtubules remain connected throughout the period of the rotation, that the microtubules of the array rotate with the cytoplasm.
Fig. 1. Microtubule staining (with anti-/3-tubulin) on sections of unfertilized eggs. Many microtubules are seen in both the
animal (A) and vegetal (B) cytoplasm. (C) A region around the brightly stained metaphase spindle has fewer microtubules,
as is evident in this section cut adjacent to the spindle. (D) Microtubules extend into the cytoplasm from the spindle poles,
indicating that the spindle is not truly anastral. (E) Another spindle at higher magnification with clear astral fibers. Bars
20 Um.
Fig. 2. Microtubule staining (anti-a--tubulin: A-I, anti-/3-tubulin: J-K) on sections of fertilized eggs fixed between 0.4 and
0.5 NT. (A, B) In this egg fixed at 0.41 NT, microtubules are absent in the vegetal cytoplasm (A) except in a region on the
sperm entry side of the egg (B) where microtubules extend down from the sperm aster. (C-G) Higher magnification of
different regions from another section. The sperm aster has not yet extended into the animal cytoplasm on the non-sperm
side (C), but astral microtubules extend to the animal cortex on the sperm side (D). In the vegetal half, no microtubules
are visible on the non-sperm side (E), but on the sperm side, they extend through the vegetal yolk mass, reaching the
cortex at an angle and bending to run toward the vegetal pole (F). (G) Close examination of the vegetal cortex on the nonsperm-
side confirms the absence of microtubules. (H,I) As the aster grows, more microtubules extend into vegetal
cytoplasm (H) but they are still absent from the vegetal cytoplasm on the non-sperm side (I). (J, K) Closer examination
reveals that astral microtubules have turned into the cortex and started to form an array beneath the egg surface on the
sperm (J), but not the non-sperm side (K). The approximate position of these regions is indicated in Fig. 5A. Bars 20,um.
Fig. 3. Microtubule staining
(anti-^-tubulin) on sections of
fertilized eggs fixed at 0.76 NT
(A,C) and surface views of
whole eggs fixed at 0.70 NT
(B,D). In the animal half,
microtubules appear to stop
when they reach the cortex
(A) and few microtubules are
seen running parallel to the
animal surface (B). In the
vegetal half, microtubules
running through the cytoplasm
appear to link in with a dense
cortical array of microtubules
(C) which show characteristic
alignment when viewed from
the surface (D). Bar 10UM.
Fig. 4. (A-C) Vegetal microtubule staining (anti-^3-tubuhn) from a single section of a fertilized egg fixed at 0 76 NT. The
approximate position of these regions is indicated in Fig. 5A. (A) Sperm side showing many microtubules running through
the cytoplasm to bend into the cortical region (B) At the vegetal pole a similar pattern is seen. (C) Non-sperm side
showing less organized microtubules in the cytoplasm, although the cortical array has formed. In all areas some cytoplasmic
microtubules (arrowed) can be seen linking in to the cortical array (ca) Bar 25/an. (D, E) Higher magnification of
microtubules linking into the cortex in eggs fixed at 0.59 NT and stained with anti-a-tubulin Bars 20 Um.
Fig. 5. Diagram summarizing
the general patterns of
microtubule polymerization
through the first cell cycle in
(A) fertilized, (B) artificially
activated eggs and (C)
enucleate activated eggs.
Pronuclear positions are from
Stewart-Savage and Grey, 1982
and Manes and Barbien, 1977.
Note the new position of the
cortex * relative to the vegetal
yolk mass following its
rotation. Numbers indicate
areas shown in Fig. 2 and 4.
Fig. 6. Microtubule staining (anti-o--tubuhn A-F, anti-^-tubulin' G-H) on sections of activated eggs. (A-D) Regions of
an electrically activated egg fixed at 0.45 NT Radially arranged microtubules extend to the animal surface (A) and into a
vegetal region just below the pigment boundary (B), but not to the vegetal cortex (C) (D) Occasional short microtubules
can found in the vegetal cortex at this time. (E,F) By 0.59 NT, radial microtubules in some eggs are found through the
marginal region (E) and linking to an array of cortical microtubules (F) (G) Vegetal region of a prick-activated egg fixed
at 0.50 NT showing both radial cytoplasmic microtubules and a dense cortical array. (H) An electrically activated egg fixed
at 0.76 NT showing microtubules in the vegetal cortex linked with ones in the cytoplasm Bars 20 Um
Fig. 7. Microtubule staining (anti-/3-
tububn) on sections of enucleated
activated eggs. (A) An egg fixed at 0 50
NT has microtubules running through
the vegetal cytoplasm and a dense
cortical array of microtubules (B) Both
of these eggs were activated by
pricking, but the one on the left was
enucleated, as indicated by the lack of
abortive furrows (C) A section from
such an enucleated egg fixed at 1.58
NT, a time when all control prickactivated
eggs had undergone
furrowing. Many microtubules run
through the vegetal cytoplasm. Bars in
A and C, 20/m\. Egg diameters in B
are approximately 1.2 mm
