Worm Breeder's Gazette 8(1): 24a
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.
We have examined a number of monoclonal and polyclonal antisera generously provided to us by other labs in order to assess their binding to sperm antigens. The antisera include the following: two rabbit sera and eight mouse monoclonals raised against muscle proteins by Ross Francis: five anti-sperm mouse monoclonals prepared by Susi Strome and obtained initially from Michael Klass; eight anti-sperm mouse monoclonals prepared by Fred Pavalko and Tom Roberts; and a rabbit anti-glyceraldehyde phosphate dehydrogenase (GAPDH) serum obtained from Patrice Yarborough and Ralph Hecht. Our interest in these sera is to use them to identify new sperm antigens whose cellular localization and developmental history can be compared with the sperm specific antigens we have alrealy examined. All the antisera were first assayed for tissue specificity by a solid phase immunoassay with proteins from sperm, eggs, larvae or fem- 1( hc17) hemaphrodites which have no sperm. Those antisera that showed significant binding to sperm proteins were then characterized further by immunostaining of sperm or fem-1 proteins transferred from 1-D or 2- D gels to nitrocellulose ('Western transfers'). In addition the cellular localization of antigens was determined by immunofluorescence. All the anti-muscle protein antibodies showed much less binding to sperm proteins than to other tissues. Most of them had too little binding to test further. However, Ross's anti-alpha actinin serum does bind to a protein in sperm that comigrates on 1-D and 2-D gels with worm muscle alpha actinin. What is alpha actinin doing in a cell with almost no actin? GAPDH is also present in sperm as Ralph and Patrice knew already. We have been unable to detect these proteins in the spermatozoa by immunofluorescence. Several of the anti-sperm monoclonals reacted with both sperm and other tissues, but seven of them were strictly sperm specific by both the solid phase assay and by Westerns. By adding dilutions of sperm proteins to the other tissue proteins it was found that the sensitivity of detection of the solid phase assay is between 10+E3 and 10+E4 of the amount of antigen present in sperm. Thus other tissues have less than 1/1000th as much of the sperm antigens as do sperm. In addition, no staining is detected in other tissues by immunofluorescence. One of these monoclonals is directed toward the major sperm protein. Three others (two from Susi, one from Fred) give an identical pattern on Westerns: they bind to eight distinct bands. The major band has Mw about 100,000. On 2-D gels it is a multiple band with its major component of pI<4. higher molecular weight bands are not gel artifacts and the lower ones are present whether or not heroic efforts are made to block protease activity. The antigenicity of all bands is destroyed by any of six added proteases and is unaffected by periodate oxidation or treatment with carbohydrate removing enzymes. Thus we think that although these proteins appear to have some post-translational modifications, the modification is not the antigen so the proteins may share some antigenic domain in common. The antigens first appear in late primary spermatocytes at the same time that other sperm-specific antigens appear. They are localized in both the fibrous body membranous organelle complex and on the plasma membrane. We are continuing to characterize these and other antisera and would be happy to test other interesting monoclonals for binding to sperm antigens.