Worm Breeder's Gazette 10(2): 64

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Treatment of Oocytes with Nocodazole in vivo, and Some Observations on Fertilization and Early Embryos, etc.

Tabitha Doniach

The following is a report of some of the observations I made during 
a short stay in Cynthia Kenyon's laboratory at UCSF, during the summer 
of 1987, to help get early embryo work started in her lab.
Nocodazole treatment of 
We were interested in determining how the polarity of the embryo is 
established.  We decided to ask whether microtubules are involved (for 
example, one could imagine that polarity is generated through a 
microtubule-organizing influence of the sperm centriole).
Strome and Wood ((1983) Cell 35, 15-25) showed that microtubules (
MTs) are not required for the anterior/posterior (A/P) polarity of the 
embryo that is generated during the first cell cycle, such as 
asymmetric distribution of P-granules.  In their experiments, MT 
depolymerizing drugs were applied after fertilization, during the 
pronuclear stage.  To test whether MTs are required for A/P polarity 
of the embryo any earlier than this (i.e.  before fertilization or 
very soon after) I applied nocodazole, a MT-depolymerizing drug, to 
oocytes while they were still in the ovary.
This was done by placing 2-3 decapitated adult hermaphrodites in 
fresh embryonic culture medium (e.g.  Priess (1987) Cell 48, 241-250) 
containing 20 g/ml nocodazole on a 2% agarose pad made up in isotonic 
medium (Priess, op.  cit., 1984: 0.1M NaCl, 3.7% sucrose) and 20 g/ml 
nocodazole.  The nocodazole (2mg/ml in DMSO) was added to the above 
solutions while they were warm (30 C) to avoid its precipitation.  The 
pad was trimmed flush with the coverslip and the edges sealed with 
mineral oil.
Under these conditions the oocytes were fertilized apparently 
normally, although the nocodazole gave the oocyte cytoplasm a 'cottage 
cheesey' texture.  The expected effects of nocodazole were observed: 
no polar body formation, no pronuclear migration, no spindle formation 
or cleavage.  However, the directly observable signs of polarity still 
occurred: the presumptive anterior (distal) end of the oocyte was more 
ruffled and furrowed than the future posterior (proximal) end, 
especially after successive cell cycles.  Eventually the irregular 
blobs of nuclei derived from the oocyte nucleus were swept (or 
migrated) to the 'posterior' end to meet their paternal counterparts, 
leaving a nuclei-free zone in the still blebbing 'anterior' end.  
Nocodazole was only considered to have entered the oocyte before 
fertilization if the previous oocyte also showed effects of the drug.  
I did not use antibodies to stain for P-granule distribution or the 
presence of intact microtubules; both would be interesting to 
The above method (without the drug) is also useful for observing 
several consecutive normal fertilizations, especially when using time-
lapse video recordings, because the mother is immobilized without 
using anesthetics.
The nuclear membrane of the oocyte breaks down a few minutes before 
the vigorous contractions of the ovary and spermatheca push it into 
the spermatheca for fertilization.  This is presumably a maturation 
event (i.e.  'germinal vesicle breakdown').  would be interesting to 
know whether maturation signals the ovary/spermatheca to contract, or 
vice versa.  It is also interesting to note that in the absence of 
sperm (e.g.  in females), oocytes tend to build up in the proximal arm 
without maturing, although those that pass through the spermatheca (
unfertilized) will undergo maturation and subsequent endoreplication 
of their DNA.  Whatever the mechanism, the breakdown event is a useful 
indicator of when fertilization or entry into the spermatheca is 
Preliminary observations indicate that although the oocyte nucleus 
is generally in the distal half of the oocyte at fertilization, it 
does not necessarily have to be in that position for normal A/P 
polarity.  I observed two cases (out of around 20) where the oocyte 
nucleus was central at fertilization.  In both, the polar bodies were 
extruded from the side of the egg rather than the distal end; however, 
first cleavage was positioned normally.  In another case two pronuclei 
and a polar body were observed at the proximal end of an egg; this end 
became the smaller blastomere, suggesting that it would give rise to 
the P lineage.  This kind of arrangement may give rise to the mythical 
'reversed embryos' that have been seen over the years, so-called 
because of the posterior polar body position.  At face value these 
embryos indicate that the oocyte nucleus does not determine the 
polarity of the embryo.  However, they do not say whether it is the 
sperm or the oocyte cytoplasm/cortex that determines the A/P polarity.
Germ cell and gamete (as well as other) nuclei of intact, living 
worms can be fluorescently stained by soaking them for 20-30 in 10 
g/ml Hoechst 33342 (in H2O).