Biology Reference
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II. Finding Sterile Mutants
A major advantage of using C. elegans for fertilization study is the ability to use
forward genetic screens to isolate fertility mutants. Mutant screening in C. elegans is
typically carried out using ethyl methane sulphonate (EMS) to induce mutations in
the germ line of wild-type hermaphrodites. In the subsequent F2 generation, homo-
zygous mutants are screened for the phenotype of interest ( L ' Hernault et al., 1988 ).
Using standard concentrations of EMS, investigators can expect to find one null
mutation per 2000 gene copies examined ( Jorgensen and Mango, 2002 ) and approx-
imately one in every 30 F2 generation animals will display a Spe phenotype
(S. L ' Hernault, personal communication).
The power of these genetic screens lies in the ability to isolate mutations in sperm-
specific genes by simply selecting hermaphrodites that are self-sterile but whose
oocytes can be still be fertilized by wild-type male sperm ( Fig. 5 ). More than 60
sperm-specific mutants have been identified this way, enabling the genetic delinea-
tion of sperm development and function pathways ( L ' Hernault et al., 1988;
L ' Hernault, 1997, 2006; Nishimura and L ' Hernault, 2010 ). C. elegans is particularly
amenable to such sperm-specific screens because they can be carried out with
hermaphrodites (rather than in males as in gonochoristic systems such as mice or
flies) and thus remain independent of confounding issues such as male mating
behavior or secondary sex characteristics ( L ' Hernault et al., 1988; Lessard et al.,
2004; Wakimoto et al., 2004 ).
Genetic screens for conditional egg-sterile mutants have likewise yielded many
interesting candidate genes (G. Singaravelu, D. Shakes, and A. Singson, unpub-
lished). These conditional screens are especially useful for the isolation of fertility
mutants as they allow the propagation of homozygous mutations in fertility genes.
Isolating egg-sterile mutants in standard nonconditional F 2 screens requires consid-
erably more work since progeny cannot be recovered from homozygous egg-sterile
mutants. As a result, the mutant chromosome must be recovered from heterozygous
siblings.
Conditional fertility screens are similar to the conditional maternal effect lethal
(mel) screens conducted in C. elegans ( Jorgensen and Mango, 2002; O ' Connell
et al., 1998 ). In a typical mel screen, worms with a pre-existing mutation that blocks
normal egg-laying are mutagenized, and the uteri of the resulting F 2 hermaphrodites
are examined for the presence of dead F 3 progeny. The screen is first carried out at
25 C, and any F 2 animals that are filled with dead F 3 embryos are subsequently
shifted to 16 C to recover homozygous temperature-sensitive (ts) mutants. This
same screen can be modified to isolate fertility mutants simply by looking for worms
containing unfertilized oocytes rather than dead embryos. Oocytes that have been
fertilized and have successfully completed egg-activation encase themselves in a
rigid eggshell ( Fig. 2A, B ). Oocytes that remain unfertilized or that have defects in
egg-activation lack this shell and are visibly distinct (the phenotype known as
''squashy,'' ''mushy,'' or ''ugly brown mels'' ) ( Fig. 2C, D ). Because such ''squashy''
oocytes were routinely discarded in most large-scale mel screens, it is likely that new
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