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.
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 activation.