Worm Breeder's Gazette 8(1): 24a
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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.