The Genetics and Cell Biology of Fertilization - Caenorhabditis Elegans: Molecular Genetics and Development - page 355

hermaphrodite sperm within the spermatheca than sperm from another male
( LaMunyon and Ward, 1999 ).
The competitive ability of male-derived sperm has useful implications for the
study of sterile mutants in C. elegans. To be competitive, the males themselves must
successfully mate and transfer sperm to the hermaphrodite while their sperm must
have successfully completed in vivo spermiogenesis, become motile, and migrated to
the spermatheca. Sperm competition assays are therefore valuable tools for the
characterization of sterile mutations in C. elegans, but these assumptions should
be confirmed via other direct means (such as the sperm tracking methods described
above). In a typical experiment, morphologically marked L4 hermaphrodites (e.g.
dpy-5) are crossed with unmarked mutant males. The numbers of outcrossed (Non-
Dpy) and selfed (Dpy) progeny are counted, and the percentage self-progeny is
recorded ( Chatterjee et al., 2005; Geldziler et al., 2005; Kroft et al., 2005;

Singson et al., 1999; Xu and Sternberg, 2003 ).
In addition to unmated hermaphrodite and wild-type male controls, only healthy
and age-matched worms should be used as male mating behavior and efficiency may
be affected by age and overall health. To minimize variations in the microenviron-
ment, all crosses should be done concurrently using the same food batch and
incubator.

IX. Oogenesis and Oocyte Maturation

The earliest stages of oocyte and sperm development are indistinguishable in
C. elegans hermaphrodites; a common pool of germ cells gives rise to both gamete
types ( Fig. 1 ). During oogenesis, the most proximal pachytene nuclei are induced to
exit their arrest by a localized MAPK-mediated signal ( Church et al., 1995 ). They
then undergo programmed cell death or begin the late stage of oocyte growth and
differentiation ( Gumienny et al., 1999 ). This developmental switch is partly regu-
lated by GLD-1, which localizes to the pachytene region of the germ line where it
actively represses the expression of late-stage oocyte differentiation markers ( Lee
and Schedl, 2001 ). As nonapoptotic presumptive oocytes then pass through the loop
region, they progress from diplotene to diakinesis. During this phase, they rapidly
enlarge while remaining connected to a progressively narrowing rachis. In wild-type
hermaphrodites only the most proximal oocytes are fully cellularized, and only the
most proximal oocyte undergoes oocyte maturation.
A variety of cell cycle and differentiation markers are available to check the
developmental progression of proximal oocytes for those egg-sterile mutants that
may have defects in late-stage oocyte differentiation. In the early stages of this post-
loop differentiation process, wild-type oocytes begin to express two proteins whose
levels continuously increase during oocyte development - OMA-1 ( Detwiler et al.,
2001; Lin, 2003 ) and the yolk protein receptor RME-2 ( Grant and Hirsh, 1999 ). The
uptake of intestinally synthesized yolk proteins can also be visualized using the
vitellegenin GFP strain YP170::GFP ( Grant and Hirsh, 1999 ).