The Genetics and Cell Biology of Fertilization - Caenorhabditis Elegans: Molecular Genetics and Development - page 356

As oocytes progress closer to the spermatheca, they can be distinguished by their
temporal response to the MSP maturation signal provided by sperm in the sperma-
theca ( McCarter et al., 1999; Miller et al., 2003 ). Approximately every 23 min, the
most proximal oocyte in the gonad undergoes meiotic maturation in response toMSP
via VAB-1, an Eph receptor protein tyrosine kinase ( McCarter et al., 1999; Miller
et al., 2003 ). This progressive response can be monitored using antibodies against
activated MAPK (MAPK-YT) and Ser10 phosphohistone H3 ( Hsu et al., 2000;
Miller et al., 2001 ).
Maturation promoting factor (MPF) is necessary for allowing the oocyte to
progress from mitotic division to metaphase I of the first meiosis ( Burrows et al.,
2006; Schmitt and Nebreda, 2002 ). MPF is a complex of a cyclin-dependent kinase
(Cdk1) and cyclin B and the activity of this complex is inhibited by WEE-1.3
( Burrows et al., 2006; Doree and Hunt, 2002 ). MPF promotes a progression of
events beginning as early as in the -3 oocyte (the third oocyte proximal to the
spermatheca). One of the earliest events is the phosphorylation of serine 10 on
histone H3 ( Burrows et al., 2006 ). The subsequent sequence of events occur pri-
marily in the -1 oocytes and can be visualized using DIC microscopy. These include
the disappearance of the nucleolus, migration of the nucleus to the distal side of the
oocyte, nuclear envelope breakdown, and rounding of the oocyte ( McCarter et al.,
1999 ). Other events including entry into metaphase I of meiosis and formation of the
meiosis I spindle can be assessed in strains that include expression constructs with
GFP:His2B and GFP:tubulin to visualize the chromatin and microtubule structures
( McNally et al., 2006; Tenenhaus et al., 2001 ). The anaphase-promoting complex
subsequently promotes the rotation of the meiotic spindle and the metaphase to
anaphase transition ( McNally and McNally, 2005 ).
X. Assessing Fertilization and Egg Activation in Egg-Sterile
Mutants
The oocyte enters the spermatheca in metaphase I of the first meiotic division and
meiotic resumption and fertilization occur concurrently ( McNally and McNally,
2005; Ward and Carrel, 1979 ). As the oocyte enters the spermatheca, the meiosis
I spindle assembles into a pentagonal array of chromosomes. Next, the DNA is
translocated to the cortex, the spindle then rotates perpendicular to the cortex, and
the chromosomes begin to separate ( Albertson and Thomson, 1993; McNally and
McNally, 2005 ). If fertilization occurs, the cell will proceed through anaphase I
and half of the chromosomes will be deposited into the first polar body ( McNally
and McNally, 2005 ). The oocyte chromosomes subsequently undergo the second
meiotic division and a second polar body is extruded. Finally, pronuclei form
around both the maternal and paternal chromatin ( McNally and McNally, 2005;
Sadler and Shakes, 2000 ).
In addition to triggering meiotic resumption, the newly fertilized egg undergoes a
number of changes that are necessary for proper egg activation and embryo