Worm Breeder's Gazette 3(2): 14b

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

A Vital Dye for Observing Sperm Transfer

S. Ward

In order to understand the mechanism of preferential fertilization 
by male sperm (Gazette 2, 2), I sought a vital dye to label male sperm 
to distinguish them from hermaphrodite sperm after mating.  All the 
known vital dyes and fluorescent compounds available at Carnegie were 
fed to worms.  Most labeled only the gut, but one, the lipid stain 
Nile Blue A, was taken up into all the tissues.  The dye fluoresces in 
the red with green illumination and individual sperm can be readily 
seen.  Nile Blue A labelled males are as fertile as unlabeled males 
and the sperm are utilized preferentially.  Observation of matings by 
dye labelled males revealed that the sperm migrate up the uterus among 
the eggs and pass through the spermathecal valve.  In the spermatheca 
they appear to accumulate preferentially against the spermathecal wall,
displacing the hermaphrodite sperm to the center of the spermathecal 
cavity.  This displacement may be the mechanism of preferential 
fertilization.  The dye transfers rapidly from the sperm to the walls 
of the spermatheca, so it is not possible to follow the sperm all the 
way to fertilization.  This presumably reflects its lipid solubility.
Males are labelled by placing larvae on seeded plates to which 0.4 
ml of 0.65% Nile Blue A was added and allowed to soak in.  The dye is 
dissolved initially at 5% in 95% ETOH and diluted With M-9 buffer 
prior to use.