Worm Breeder's Gazette 8(3): 59

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.

Evidence That Calmodulin May Be Involved In Spermiogenesis

D. Shakes, S. Ward

Spermiogenesis in C.  elegans involves a rapid transition from a non-
motile, spherical spermatid to a motile, bipolar amoeboid spermatozoa. 
This activation can be induced in vitro by weak bases, monensin, and 
proteases (Ward, et.  al., Dev Biol 98:70-79, 1983).  Since calmodulin 
has been shown to play a major role in both motility systems and 
cytoskeletal organization in other organisms, it seemed logical that 
it might also play a role in sperm activation in C.  elegans.To test 
this proposal we have compared the effects of two structurally 
unrelated calmodulin inhibitors, trifluoperazine and W-7, on sperm 
activation.  Virgin E1490 males were hand dissected in buffer on acid-
washed slides and a slide chamber was created by supporting the 
coverslip on Vaseline.  Sperm activation was monitored using Nomarski 
optics on a Zeiss Universal microscope equipped with a time-lapse 
videotape recorder.
The cells respond to the drugs differently at different drug 
concentrations.  At the highest concentrations (100 m TFP and 500 m W7)
, the spermatids showed either no response or lysed.  There was no 
activation upon subsequent removal of the drug or the addition of 200 
m/ml Pronase.  At low concentrations (10 m - 20 m for both drugs), the 
spermatids showed no obvious drug response.  In contrast, the addition 
of low concentrations of the drugs to active spermatozoa caused the 
pseudopods to stop moving and smooth out as they slowly 'melted back'. 
This effect is rapidly and fully reversible by alternately washing 
the drug in and out with buffer.  The most interesting results were 
found in the intermediate range of 50 m for both drugs.  This 
concentration caused the spermatids to form long spiky pseudopods 
which thickened with time but which lacked projections and were 
nonmotile.  Subsequently, washing the drug out with buffer alone 
resulted in the formation of normal pseudopods.  Normal pseudopods 
formed in the presence of Pronase as well as those generated after 
washing out 50 m calmodulin inhibitors with buffer responded to 
further drug additions (at concentrations of 50 m or less) by halting 
motility and 'melting back'.  Thus these calmodulin inhibitors appear 
to initiate spermiogenesis although they also cause the 'melting back' 
of already formed normal pseudopods.  Similar results were obtained 
when using another known calmodulin inhibitor, chlorpromazine; and yet 
its inactive analog, chlorpromazine-sulfoxide, had no effect at 
similar concentrations.
One possible explanation for these results is that the differential 
drug effects reflect differences in binding constants of various 
calmodulin dependent enzymes in the cell.  Our preliminary results 
from gel-overlay studies using [125I]-labelled hog brain calmodulin to 
probe gels of sperms proteins (Palfrey, et.  al.  PNAS 79:3780-3784, 
1982) indicate that there may be several calmodulin binding proteins 
in sperm, some of which appear to be sperm-specific.
In parallel with these studies, we are using other techniques to 
study calmodulin.  As Marty Chalfie had mentioned to us earlier, we 
found that whole worms were also sensitive to calmodulin inhibitors.  
We hope to isolate calmodulin mutants by selecting animals which can 
survive and reproduce in the presence of these drugs.  In addition, we 
will try to isolate the gene for calmodulin from C.  elegans using a 
probe to electric eel calmodulin from A.  R.  Means (Lagace, et.  al., 
J.  Biol Chem 258:1684-1688, 1983).  Furthermore, we will use indirect 
immunofluorescence with commercially available anti-calmodulin 
antibody to see if the localization of calmodulin changes during