Biology Reference
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XII. Analysis of Fertilization-Specific Gene Products
Antibody-based approaches are useful for determining the subcellular localiza-
tion of fertilization-specific gene products. The standard approach is to generate
polyclonal antipeptide antibodies against two or more regions of the candidate
fertility protein since not all regions will be useful for generating antibodies
( Chatterjee et al., 2005; Parry et al., 2009; Zannoni et al., 2003 ). For these fertility
proteins, polyclonal, antipeptide antibodies have two distinct advantages over mono-
clonal antibodies: (1) they do not require the isolation of pure sperm and oocytes, (2)
polyclonal antibodies typically have higher binding efficiencies, which is useful
when attempting to detect cell-surface proteins that are typically expressed at low
levels. It is also important always to prescreen the animals used for antibody pro-
duction since many express unrelated nematode antibodies stemming from previous
nematode infections.
It is useful to understand the localization pattern of SPE proteins in both
spermatids and spermatozoa. Sperm squashes are used for staining spermatids,
and spermatozoa preparations can be obtained either from mated hermaphro-
dites or from males dissected in non-protease-based sperm activators. Samples
should be fixed separately in both paraformaldehyde and cold methanol as
membrane proteins can differ in their response to each. C. elegans spermatids
are small and possess only four major organelles (a central chromatin mass, an
associated inactive centriole, numerous mitochondria, and multiple MOs) sim-
plifying the analysis of localization patterns. MO localization may be confirmed
using the monoclonal antibody 1CB4 ( Okamoto and Thomson, 1985 )aswas
done for the proteins SPE-9 and SPE-38 ( Chatterjee et al., 2005; Zannoni et al.,
2003 )( Fig. 8A-F ).
Live cell staining (with antibodies added before fixation) can also be used to
assess protein localization on the external surface of the plasma membrane or in
fused MOs ( Chatterjee et al., 2005 ). fer-1 mutants can be used to confirm an initial
restriction to the MO since the fer-1 spermatozoa are specifically defective in MO
fusion despite their ability to form a small motile pseudopod ( Achanzar and Ward,
1997; Chatterjee et al., 2005; Xu and Sternberg, 2003 ).
To date, all fertilization-defective SPE proteins have at least partially localized to
the pseudopods of spermatozoa, now presumed to be the point of oocyte-sperm
contact ( Chatterjee et al., 2005; Xu and Sternberg, 2003; Zannoni et al., 2003 ). In
spermatids, some fertilization-defective SPE proteins such as SPE-9 localize to the
plasma membrane ( Zannoni et al., 2003 ) while others, such as SPE-38 and TRP-3/
SPE-41, localize to the unfused MOs of spermatids. Any patterns of localization
common to multiple members of the SPE-9 class remain to be determined ( Fig. 8 )
( Chatterjee et al., 2005 ).
Fusion proteins (e.g., GFP fusions) can be convenient tools for determining a
protein ' s distribution and dynamics in fixed or live cells. However, the expression of
oocyte and sperm fusion proteins is often repressed in the germ line ( Kelly et al.,
1997; Putiri
et al., 2004; Seydoux and Schedl, 2001 ). Oocyte fusion protein
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