The Genetics and Cell Biology of Fertilization - Caenorhabditis Elegans: Molecular Genetics and Development - page 360

events likely result in a multitude of branching cellular pathways ( Marcello and
Singson, 2010; Singson et al., 2008 ).
Egg-activation mutants are likely to fall into different classes, and appropriate
GFP strains and disruption of gene function by RNAi or mutation can be used to
analyze genes or proteins of interest in egg activation. Sperm entry promotes the
establishment of embryonic polarity and triggers egg activation ( Goldstein and Hird,
1996; Sadler and Shakes, 2000 ). One example of a paternal egg-activation mutant is
spe-11; when spe-11 sperm fertilize wild-type oocytes, the oocytes fail to either
complete meiosis or secrete an eggshell, but instead form multinucleate one-cell
embryos ( Browning and Strome, 1996; Hill et al., 1989 ). Other egg-activation
mutants may arise from mutations in maternally required genes that regulate and
coordinate egg-activation events ( Cheng et al., 2009; Maruyama et al., 2007; Parry
et al., 2009; Stitzel et al., 2007 ). Such genes would include regulatory proteins at the
oocyte cortex that couple fertilization with cell-cycle progression through the second
meiotic division and the events of egg activation ( Govindan and Greenstein, 2007;
Maruyama et al., 2007; Parry et al., 2009; Parry and Singson, 2011 ). For example,
EGG-3, EGG-4, and EGG-5 are pseudophosphatases located in the oocyte cortex
that coordinate fertilization with cell-cycle resumption by regulating the activity
of minibrain kinase-2 (MBK-2) ( Cheng et al., 2009; Maruyama et al., 2007; Parry
et al., 2009; Stitzel et al., 2007; Stitzel and Seydoux, 2007 ). MBK-2 helps regulate
early embryonic development after fertilization by phosphorylating maternal
substrates for degradation. These MBK-2 targets include the katanin subunit
MEI-1, the RNA/TAF-4 binding proteins OMA-1 and OMA-2, and the polarity
factors MEX-5 and MEX-6 ( Detwiler et al., 2001; Guven-Ozkan et al., 2008;
Nishi and Lin, 2005; Pang et al., 2004; Pellettieri et al., 2003; Quintin et al.,
2003 ). Appropriate GFP strains can be used to assess whether other egg-activation
mutants exhibit defects in the localization, sorting, or degradation of any of these
known components.
XI. Eggshell Production
In response to fertilization and proper egg activation, a multilayered eggshell is
formed around the developing embryo to allow for completion of meiosis, polar
body extrusion, embryo polarity, and formation of an osmotic barrier. The devel-
oping oocyte is covered by a thin vitelline layer that can be observed by electron
microscopy or staining with Malcura pomifera agglutinin (MPA) or Griffonia
simplicifolia lectin I (GSL I). At the time of fertilization this vitelline layer begins
to separate from the plasma membrane and becomes the outermost layer of the
mature eggshell ( Bembenek et al., 2007; Rappleye et al., 1999 ). The second
(middle) layer is formed when the oocyte membrane protein CHS-1 catalyses
UDP-N-acteylglucosamine polymerization to produce chitin, the material that
gives eggshells their mechanical strength. Chitin deposition can be assayed by
staining with a rhodamine-conjugated chitin-binding probe. The proteolipid inner