The Genetics and Cell Biology of Fertilization - Caenorhabditis Elegans: Molecular Genetics and Development - page 354

( L ' Hernault et al., 1988 ). The morphology of DAPI-stained sperm nuclei is quite
distinctive and can be easily scored within the spermatheca and uterus. Alternatively,
the hermaphrodite gonad can be directly observed using DIC microscopy and time-
lapse photography. Animals can be dissected on glass microscope slides or anesthe-
tized on agar pads to study sperm number and morphology. It can be challenging,
however, to follow the four-dimensional movements of an individual spermatozoon
using DIC optics alone.
Fluorescent methods for sperm-tracking experiments include the use of Nile Blue
( Ward and Carrel, 1979 ), SYTO17 ( Hill and L ' Hernault, 2001; Singson et al., 1998 ),
or MITO tracker ( Kubagawa et al., 2006; Stanfield and Villeneuve, 2006 ). Males are
labeled by soaking them in a dye-containing solution and are then mated to unlabeled
hermaphrodites. These hermaphrodites are subsequently anesthetized and examined
via fluorescent microscopy for the presence and location of sperm.
Care must be taken to ensure that the fluorescent dye used does not adversely
affect the process/function under study. The use of mutant hermaphrodites with
reduced gut autofluorescence (such as daf-4 or glo-1) facilitates the identification
of male-derived sperm within the female reproductive tract ( Artal-Sanz et al., 2003;
Kroft et al., 2005; Hill and L ' Hernault, 2001 ).

VIII. Sperm Competition

Although hermaphrodite self-fertilization is the primary mode of C. elegans
reproduction, mated hermaphrodites produce predominantly outcrossed progeny
because of the competitive superiority of male-derived sperm (termed ''sperm
competition''). This phenomenon is a primary reason that C. elegans is such a
powerful genetic system. The transition towards outcross progeny typically occurs
within 1 day after male-introduction; by day two, progeny are almost exclusively
outcrossed ( LaMunyon and Ward, 1995, 1997, 1998, 1999; Singson et al., 1999;
Ward and Carrel, 1979 ). Sperm competition is independent of the ability to fertilize;
the sperm of fertilization-defective C. elegans mutants can effectively outcompete
hermaphrodite sperm, reducing the self-fertility of these mated hermaphrodites
( Singson et al., 1999 ). The dominance of male-derived sperm may be partially
due to size; C. elegans male-derived sperm are approximately 50% larger than
hermaphrodite-derived sperm, move faster, and are thought to displace them from
the distal end of the spermatheca ( LaMunyon and Ward, 1998 ). However, size per se
cannot be the complete explanation, as C. remanei male-derived sperm do not reduce
self-fertility in mated C. elegans hermaphrodites, despite being two times larger in
diameter ( Hill and L ' Hernault, 2001 ). C. elegans hermaphrodites may also have an
independent mechanism for actively selecting functional sperm ( Kadandale and
Singson, 2004 ).
Sequential mating experiments with multiple males suggest sperm competition in
C. elegans is limited to males versus hermaphrodites. This is perhaps not surprising
given that in wild populations male sperm are more likely to encounter