The Genetics and Cell Biology of Fertilization - Caenorhabditis Elegans: Molecular Genetics and Development - page 358

In contrast, newly fertilized oocytes from maternal or paternal egg-activation
mutants are expected to contain a visible sperm chromatin mass ( Kadandale et al.,
2005b; Maruyama et al.,2007;Parryet al.,2009 )( Fig. 7A ). Further, in egg-activation
mutants, a distinct endomitotic phenotype is seen. Rather than having a single large
polyploidy DNAmass, many smaller DNAmasses are formed ( Fig. 7D ). This is likely
due to the action of sperm-contributed centrosomes separating newly replicated DNA
with each endomitotic cycle. This difference in DNA morphology can be used to help
determine whether sperm entry has occurred.
Postfertilization defects in meiosis or subsequent cell divisions can be observed in
fixed embryos by staining with combinations of DAPI, the M-phase marker anti-
phosphohistone H3 (serine 10), and anti-ß -tubulin DM1A antibodies ( Golden,
2000 ). These defects can also be seen in live embryos by crossing two strains
containing either pie-1:Histone 2B:GFP ( Praitis et al., 2001 ) or pie-1:GFP:ß -tubulin
( Strome et al., 2001 ).
EGG-1 and EGG-2 are two proteins in the plasma membrane of the oocyte which
are necessary for fertilization ( Kadandale et al., 2005b ). These genes have proven to
be extremely useful diagnostic tools for analyzing egg-sterile mutants with defects in
fertilization ( Kadandale et al., 2005b ). Both genes encode type II transmembrane
molecules and their extracellular domains include eight low-density lipoprotein
(LDL)-receptor-repeats, which are known to function as receptors for a variety of
ligands and mediate multiple cellular responses ( Kadandale et al., 2005b; Nykjaer
and Willnow, 2002 ). Sperm are able to make contact with egg-1/2 deficient oocytes
but are unable to fuse with them ( Kadandale et al., 2005b ). The localization of
EGG-1 and EGG-2 can be visualized using GFP-tagged versions of the proteins.
GFP:EGG-1 ( Fig. 8G ) and EGG-2:GFP are localized to the surface of developing
oocytes but become undetectable after fertilization, presumably as a result of endo-
cytosis ( Kadandale et al., 2005b ).
Oocyte maturation, fertilization, and the completion of meiosis are overlapping
and dependent processes that are coordinated by a number of important regulatory
factors ( Marcello and Singson, 2010 ). Defects in any step can have deleterious
effects on subsequent steps and ultimately result in improper embryogenesis. To
understand how a specific gene or protein of interest functions in these processes,
mutants with defects in specific stages of meiosis or related GFP marked strains can
be used. For example, to analyze the relationship between cell cycle progression and
egg activation, the activity of the anaphase-promoting complex (APC/C) was
blocked using either temperature-sensitive mutants or RNAi. In oocytes depleted
of the APC/C subunit mat-1, fertilization occurs, but the fertilized oocytes remain
locked in a meiotic metaphase I state and subsequent events of the oocyte-to-embryo
transition do not occur ( Golden et al., 2000 ). In other studies, cyclin B:GFP levels
have been used to indicate meiotic progression. Degradation of maternally supplied
cyclin B by the APC/C was shown to drive the transition from metaphase I to
anaphase I ( Davis et al., 2002; Furuta et al., 2000; Golden et al., 2000; Rahman
and Kipreos, 2010 ); however, in the absence of fertilization, oocytes progress to
anaphase I but the drop in their cyclin B levels proved to be incomplete ( McNally and