Ward S et al. (1983) Worm Breeder's Gazette "Monoclonal and Polyclonal Antibodies that Bind to Sperm Antigens"

Hogan E, Ward S

[Worm Breeder's Gazette, 1983]

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.

Pavalko FM et al. (1983) Worm Breeder's Gazette "amonoclonal and polyclonal antibodies that bind to sperm antigens."

Roberts TM, Pavalko FM

[Worm Breeder's Gazette, 1983]

We have been applying hybridoma technology to our research by generating monoclonal antibodies (MAb's) against C. elegans sperm. We have produced 10 mAb's in our lab and have obtained 2 others from Susie Strome at Boulder. Seven of these mAb's target antigens present on the cell surface. We have been purifying monoclonal antibodies from ascites fluid by DEAE Affi-Gel blue chromatography (supplied by Bio-Rad). Although yields have been lower than ideal (40-60%), these antibodies are free of all contaminants except mouse transferrin. Membrane lipid flow and directed movement of pseudopod surface components on sperm was previously reported by Roberts and Ward (J. Cell Bio. 92: 113-120). We have evidence that the movement of antigen-antibody complexes may be due to a continual flow of membrane lipid (see Bretscher, 1976, Nature 260: 21-23) from the tip of the pseudopod back toward the cell body. Bretscher's lipid flow model of membrane movement predicts that membrane components which diffuse slowly are displaced by the lipid stream whereas those which diffuse rapidly effectively escape lipid flow. Because the rate of diffusion is related to molecular weight, large complexes (e.g. antibody- crosslinked membrane proteins) would be swept along more rapidly than individual molecules (e.g.- unbound membrane proteins). We have tested this prediction by comparing clearance rates of various sized complexes form the cell pseudopod. On sperm, relatively small antigen- mAb complexes clear from the pseudopod surface slowly (2-3 min) while larger, cross-linked antigen-mAb-2 antibody complexes are cleared more rapidly (30-45 seconds). Using mAb's conjugated directly to colloidal gold particles (CGP), we have obtained evidence that new surface antigens are preferentially inserted at the tips of pseudopodial projections and then move rearward toward the cell body pseudopod junction (Pavalko and Roberts, C. elegans Meeting Abstracts, 1983). This observation predicts that there must be a pool of antigen in the cytoplasm which would be available for insertion into the membrane. Labelling thin sections of spermatozoa with CGP conjugates of mAb's 11, 63, and 56 (each of which bind to the cell surface in indirect immunofluorescence assays) reveals antigen on the surface (plasma membrane, exposed face of fused membranous organelles and MO contents). In addition, these antibodies label the cytoplasmic laminar membranes and the pseudopod cytoplasm. Many of the gold particles in the cytoplasm lie just beneath the plasma membrane suggesting that they are bound to antigens that were destined for insertion onto the surface. Because there are no vesicles in the cytoplasm, the mechanism which transports these antigens is unknown. We will be trying to characterize the antigens targeted by the mAb's and to solve the question of how they are shuttled around the cell.

Shakes DC et al. (1988) Worm Breeder's Gazette "analysis of two loci required for the normal morphology and cellular localization of a unique organelle in sperm."

Shakes DC, Ward S

[Worm Breeder's Gazette, 1988]

The membranous organelle (MO) is an unusual sperm-specific organelle which plays a number of critical roles in the differentiation of spermatozoa. During premeiotic and meiotic development, the fibrous body/membranous organelle (FB-MO) complex functions both as the site of assembly of many of the newly synthesized gene products and as a structure for organizing these components for localization and transport to the budding haploid spermatids. Later, the fusion of the mature MO with the back end of the cell during spermiogenesis adds important, new components to the cell surface of the crawling spermatozoa (Roberts et al., JCB 102: 1787, 1986). In spe-17 mutant spermatocytes, the normally ribosome free FB-MO complexes are uniformly associated with numerous ribosomes, some of which are transported into the normally ribosome free, haploid spermatids. These ribosomes probably contribute to the abnormal appearance of the RNA-containing perinuclear halo in the spe-17 mutant spermatids. In addition, the spermatids are abnormally small, and the proper organization of their organelles is disrupted which suggests that a common cytoskeletal defect may be responsible for the various abnormalities. Despite these abnormalities, spe-17 spermatids form crawling spermatozoa which are capable of translocating across a surface. In spe-10 mutant spermatocytes, the FB-MO complexes disassemble prematurely during meiosis rather than in the newly budded spermatids. Membraneless FBs are commonly found left behind in the residual body, and in many cases, they bud off as small cytoplasts. The normal folding of the MOs into their compact spermatid morphology is also disrupted, and large, vaculated MOs are seen in more than 40% of the cells. These defects suggest that the FB must be associated with the MO membrane for proper segregation to the spermatids during meiosis and that proper MO folding may require structural or enzymatic cues from the FB. These vaculated MOs are segregated to the spe-10 spermatids. All spe-10 spermatids, regardless of the severity of their MO defects, are abnormal in their size and organelle organization. The spermatids with less severe MO defects can form spermatozoa, but these spermatozoa, like those of fer-1, fail to fuse their MOs and translocate.

Viney ME (1994) Worm Breeder's Gazette "The Genetics of Strongyloides"

Viney ME

[Worm Breeder's Gazette, 1994]

The beginning Strongyloides is an obligate parasitic nematode. It is the parasitic nematode taxonomically closest to Caenorhabditis. Its life-cycle (see below) consists of two distinct adult generations; one female and parasitic and the other dioecious and free-living. The free-living adult generation (known as heterogonic development) can be omitted and instead larvae can develop to infectiveL3 sdirectly (homogonic development). Cytological studies have concluded that the parasitic generation reproduces by mitotic parthenogenesis, and the free-living generation by pseudogamy (maternal inheritance only)(1). Here I describe a genetic analysis of the life-cycle of S. ratti. Doing genetics with Strongyloides Clones A rat can be infected with a singleL3 ,which gives rise to a single parasitic female. Larvae produced by this worm are able to develop both directly and indirectly. We refer to such infections (and the population derived from these) as clones, but they should probably be more correctly called isofemale lines. Controlled matings Virgin free-living males and females can be grown by collecting early stage larvae and maintaining them individually until they are mature. Virgin males and females from different clones can be brought together so that mating can occur. The progeny can be cloned back into rats or can be analyzed directly. Genetics of the free-living generation Crosses have been made between individual virgin males and virgin females of different clones (the parental clones). The resulting progeny of such crosses have also been cloned (the progeny clones). Parental and progeny clones have been analyzed by minisatellite fingerprinting (Jeffreys' probe 33.15)(2). The fingerprints showed that inheritance was bi-parental, and thus that pseudogamy did not occur. The inheritance patterns are fully consistent with the occurrence of "normal" sexual reproduction(3). These results were incompatible with the earlier cytological observations. Genetics of the parasitic generation Parthenogenesis can occur by a number of cytological mechanisms. In some cases, progeny are identical to each other and to their mother (mitotic and some forms of meiotic parthenogenesis). In other cases the progeny differ from each other and from their mother (other forms of meiotic parthenogenesis). In view of the quite different conclusions drawn from the cytological and genetic data, I have also analyzed the progeny of single parasitic females with respect to the functional difference (mitotic or meiotic) in parthenogenetic reproduction. Larval progeny of individual parasitic females have been analyzed for a RFLP in a PCR fragment of C. elegans actin 4 (4). Alleles of this locus segregated in a Mendelian manner in crosses of the free-living generation. Parasitic females that were heterozygous for this marker produced progeny all of which were heterozygous. Thus, it was concluded that the parasitic female was functionaIly mitotic. The genetic and cytological studies were not in conflict for this stage. The future Strongyloides is a parasitic nematode with a life-cycle that is amenable to genetic manipulation. Admittedly it isn't quite as easy to maintain or work with as C. elegans, but it is the most accessible of the parasitic nematodes. I would be happy to supply further details of the methods used or the work itself, if anyone is interested. References 1. Bolla & Roberts, 1968, J. Parasit., 54, 849-855. 2. Jeffreys, et al., 1985, Nature, 314, 67-73. 3. Viney, et al., 1993, Proc. R. Soc. Lond. B, 254, 213-219. 4. Krause, et al., 1989, J. Mol. Biol., 208, 381-392.