Biology Reference
In-Depth Information
Markers/mutations may affect male mating behavior/efficiency; some him mutants,
for example, have smaller broods than wild-type animals due to aneuploidy, and
almost all morphologically marked males have at least partially compromised mat-
ing efficiencies (
Hodgkin, 1997
). Mating behavior/efficiency issues must therefore
be distinguished from fertility issues when assessing overall fecundity.
Minimizing brood size variability due to environmental and genetic factors is
important. Because fertility is age dependent, age-matched wild-type controls are
essential. Nutritional effects can be minimized by using growth plates made at the
same time and seeded from the same bacterial culture. Any plate contamination will
complicate oocyte counts. For consistency of measurements, a single investigator
should collect all data and proper statistical analysis must be performed to determine
the significance of observed differences between groups. When assessing transgenic
lines, it is important to remember that transgenic worms frequently produce smaller
broods due to the unrelated effects of germ line silencing (
Putiri et al., 2004
). The
brood sizes of several different lines should be assessed as individual transgenic
arrays frequently exhibit distinct levels of expression.
Directionality to sperm migration has been observed; sperm sometimes move to a
single spermatheca rather than both, resulting in a mated Spe hermaphrodite laying a
mix of eggs and unfertilized oocytes. If this is seen, the hermaphrodite should be
examined to determine whether sperm are differentially localized in this manner.
V. Ovulation/Ovulation Rates
Ovulation levels/rates are closely related to overall brood sizes and can be used to
differentiate Spe mutant classes. Meiotic maturation/ovulation is stimulated by a
sperm-derived signal: the major sperm protein (MSP) (
Kosinski et al., 2005; Miller
et al., 2001
). On an average, sperm-containing wild-type hermaphrodites ovulate
approximately 2.5 times per gonad arm per hour, while fog-2 females (which lack
sperm) ovulate only 0.09 times per gonad arm per hour (
McCarter et al., 1999; Miller
et al., 2003
). Consequently, ovulation rates can be used to indirectly assess the
presence of spermatids/spermatozoa. The ovulation rates of spermatocyte-arrest
Spe mutants are significantly lower than those of Spe mutants whose genes affect
spermiogenesis/sperm function (
Kadandale and Singson, 2004; Singson et al.,
1998
). Presumably only the spermatid/spermatozoa-producing Spe mutants have
the ability to produce and respond to the MSP signal and ovulation rates have been
used to distinguish spe mutant classes (
Chatterjee et al., 2005; Singson et al., 1998,
2008
).
Measurement of ovulation rates can be made in several ways. Paralyzed or anes-
thetized animals can be directly observed under a compound microscope using time-
lapse video, for example (
McCarter et al., 1999; Ward and Carrel, 1979
), or one can
use a dissecting scope to count oocytes on single worm plates at set time intervals
(
Kadandale and Singson, 2004; Miller et al., 2003
). Reproducible plate counts
require fresh uncontaminated bacterial lawns but are less traumatic for the worms
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