Biology Reference
In-Depth Information
regulation or new protein synthesis; ribosomes, actin, and tubulin are all discarded
during the spermatid budding division.
It is important to distinguish between sperm function mutants (which affect
fertilization directly via defects in sperm-egg interaction) and sperm development
mutants (which affect the process only indirectly via defects in sperm morpho-
genesis or motility). Sperm development itself can be quickly assessed in micro-
scope preparations of dissected worms. To study early spermatogenesis, ''sperm
squashes'' are made: males are placed in sperm buffer and dissected at the
pharynx-intestine junction. A coverslip is used to press the gonad into a mono-
layer of spermatocytes and spermatids that are then examined using DIC micros-
copy for spermatid number, shape, and size. Lipid-soluble dyes such as Hoechst
33342 may be included to detect DNAvia epifluorescence, although UVexposure
causes the cells to deteriorate rapidly. For further analysis, one can quick freeze
these sperm squash preparations on dry ice, remove the coverslip, and prepare the
sample for immunofluorescence (for more details see the ''Immunofluorescence''
chapter in this volume by Shakes et al., 2009 ). Useful markers for the general
staging of spermatogenesis include combinations of DAPI (DNA) with antibodies
against
a -tubulin (Sigma FITC-labeled DM1A), phosphorylated histone H3
(Ser10)
(Upstate Biotechnology),
and/or MSP (Developmental Studies
Hybridoma Bank).
Spermatocytes/spermatids can be easily isolated from males, but naturally acti-
vated spermatozoa must usually be isolated from the gonads of mated hermaphro-
dites ( Chatterjee et al., 2005 ). The isolation of in vivo activated spermatozoa directly
from self-fertile hermaphrodites is substantially more challenging as hermaphrodite
spermatozoa are 50% smaller ( Geldziler et al., 2006; LaMunyon and Ward, 1998 )
and significantly less numerous.
Spermiogenesis may also be assessed in vitro via the addition of known activators
to sperm media before worm dissection. Pronase (a nonspecific collection of pro-
teases) can be used to examine activation using light microscopy, but other choices
such as monesin (a cationic ionophore) or triethanolamine (a compound affecting
intracellular pH) are more appropriate for studies using immunofluorescence
( Chatterjee et al., 2005; Roberts et al., 1986; Singaravelu et al., 2011; Zannoni
et al., 2003 ).
VII. Sperm Migratory Behavior/Tracking
For successful C. elegans fertilization, male-derived spermatozoa must first
migrate from the vulva (the site of insemination) to the spermatheca. Sperm (regard-
less of derivation) must then remain there, either by firmly attaching themselves to
the spermatheca wall or by crawling back if dislodged by passing oocytes. Defects in
hermaphrodite sperm retention can be assessed by comparing the number of sperm
in whole-mount, DAPI-stained young adult hermaphrodites immediately before
egg-laying with that of slightly older siblings that have already begun laying eggs
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