The Genetics and Cell Biology of Fertilization - Caenorhabditis Elegans: Molecular Genetics and Development - page 349

or can be generated by appropriate screening strategies ( Kadandale et al., 2005a;
L ' Hernault et al., 1988; Putiri et al., 2004 ). Although, by definition, ts sterility alleles
produce progeny at the permissive temperature, the fertility of these strains may not
be as robust as in wild-type animals; they are sometimes leaky, giving low brood
sizes at the permissive temperature.
In some instances, sterile mutants can be stably maintained as homozygotes
through mating because their mutant phenotype is sex-specific. All members of the
spe-8 class of genes, for example, are specifically required for hermaphrodite sper-
miogenesis ( Geldziler et al., 2005; L ' Hernault, 1997; Shakes and Ward, 1989 ).
However because the mutant males are fertile, homozygous populations can be
maintained simply by crossing mutant males to mutant hermaphrodites. Mutants with
male-specific spermatogenesis-defects also exist ( Stanfield and Villeneuve, 2006 ).
Although ideal for maintaining mutant populations, these types of sex-specific sterile
mutations are rare.
IV. Fecundity Analysis
Once mutant strains of interest have been isolated, outcrossed, and genetically
stabilized, they can be characterized in detail. Quantifying fertility using brood
analysis, for example, is of fundamental importance for determining a mutation ' s
functional severity. In hermaphrodites, this is done by allowing individually plated
wild-type and mutant L4 animals to self-fertilize and then counting, averaging, and
comparing the numbers of eggs/oocytes laid. In practice, eggs/oocytes are usually
counted daily while the hermaphrodite is moved to a new plate to facilitate counting
the next day ' s totals. Animals are typically transferred to new plates until no new
eggs/oocytes are laid within a 24 h period. Alternatively, the eggs/oocytes may be
removed each day to avoid damaging delicate mutants via unnecessary manipula-
tions. Brood sizes may also be examined over shorter time periods (an approach that
is less traumatic for the worm and less labor intensive for the investigator). The total
number of lifetime progeny can be assessed using the ''mate to death'' assay ( Kimble
and Ward, 1988 ); the daily introduction of new males constantly replenishes the
experimental hermaphrodite ' s sperm supply. Brood results are most usefully
expressed as the percentage of wild-type numbers; absolute numbers are less infor-
mative due to the wide variation in actual brood sizes among animals resulting from
unknown or difficult-to-control factors. In studies of maternal-effect lethal mutants,
quantitative assessments of fertility should include the percentage hatched as well as
distinct categories for non/weak-shelled ''oocytes'' (oocyte or egg steriles), hard-
shelled dead embryos, and viable larvae ( Fig. 2 ).
Male fertility is typically assessed by crossing mutant males to marked strains and
counting the number of outcrossed (wild-type looking) progeny. dpy-5 is often used
as a marker as it is easily scorable, but any easily identified recessive morphological
marker will suffice. Another method obviates marked strains; one simply counts the
number of F 1 males and multiplies by two, since males sire 50% male progeny.