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layer is formed immediately before the first zygotic cell division and provides a
permeability and osmotic barrier. The fidelity of this osmotic/permeability barrier
can be determined by staining with lipophilic fluorescent plasma membrane dye
FM 4-64, which can only penetrate and stain embryos that have not yet formed an
intact barrier or eggshell.
Unfertilized oocytes and activation-defective eggs are sensitive to osmotic
strength due to the lack of a protective eggshell and must be handled with
special care. Intact and dissected hermaphrodites should at a minimum be
handled in osmotic egg buffers ( Edgar, 1995 ). Otherwise, unfertilized oocytes
may burst under osmotic pressure and even intact embryos may swell within a
weak eggshell and cause an embryo to be mistakenly scored as cytokinesis
defective. In some cases, oocytes are so fragile that dissections from the uterus
are impossible. These oocytes must be examined carefully in intact animals
( Parry et al., 2009 ).
CG exocytosis is necessary for proper eggshell formation ( Bembenek et al.,
2007 ). CGs transport chondroitin proteoglycans to the extracellular space sur-
rounding the embryo, and can be detected by wheat germ aggultinin (WGA) or
the Golgi marker UGTP-1 ( Bembenek et al., 2007 ). CAV-1 is prominent but
nonessential component of CGs ( Sato et al., 2008 ). CG exocytosis, which occurs
during anaphase I, does not require fertilization. However, it does require a
variety of cell-cycle components including the APC/C and separase (sep-1)as
well as the small GTPase RAB-11 and the target-SNARE SYN-4
( Bembenek et al.,2007 ). Additional exocyotic events must be required for proper
eggshell formation; however, the exact nature of these events remains unclear
( Bembenek et al., 2007 ).
Although CG exocytosis is known to contribute to the membrane-based poly-
spermy block in other organisms, the connection between CG exocytosis and
polyspermy in C. elegans is not well understood ( We s s e l et al.,2001 ). In
C. elegans, polyspermy has been observed after the depletion of chs-1, gna-2,or
egg-4/5 ( Parry et al., 2009; Johnston et al., 2010 ). gna-2 encodes a GLD-regulated
glucosamine-6-P N acetyltransferase that supplies UDP-N-acteyl glucosamine for
chitin biosynthesis ( Johnston et al., 2006 ). The deposition of chitin is independent
of CG exocytosis and the presence of chitin seems to play a role in the membrane-
based polyspermy block ( Johnston et al., 2010; Parry et al., 2009; Sato et al., 2006,
2008 ). Potential defects in the polyspermy block can be assessed by DAPI staining
intact hermaphrodites or dissected embryos and looking for evidence of multiple
sperm chromatin masses within recently fertilized, meiotic-stage oocytes
( Johnston et al., 2010; Parry et al., 2009 ). However, in intact or poorly dissected
animals, it can be difficult to distinguish nuclei within the embryo from those in
the surrounding periphery ( Johnston et al., 2010; Parry et al.,2009 ). When stain-
ing dissected embryos that do not have eggshells, a grouping of embryos can lack
clear demarcation of each embryo boundary. The use of GFP:PH (Plextrin
Homology) construct can be used to help visualize the plasma membrane
( Audhya et al., 2005 ) and minimize this problem.
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