Date of Award
Doctor of Philosophy
Molecular, Cellular, and Developmental Biology
In order for fertilization to occur in the horseshoe crab, Limulus polyphemus, spermatozoa must become motile and the acrosome tips must readily adhere to the outer layer of the egg envelope. Shortly after this adherence or primary sperm attachment, the sperm acrosome reaction occurs, resulting in a stronger adhesion (secondary attachment). A sperm attachment assay that quantified the number of spermatozoa attaching to egg sections was utilized to identify components involved in primary sperm attachment. Egg sections treated with asparagus pea lectin (APL, 250 (mu)g/ml) bound significantly fewer spermatozoa as compared to other lectins. Furthermore, sperm attachment was also greatly reduced when egg sections were first incubated with the glycosidase, (alpha)-L-fucosidase ((LESSTHEQ) 5% of controls). Treatment of spermatozoa with (alpha)-L-fucose, fucoidan, or p-aminophenyl fucoside also reduced sperm attachment when compared to Millipore-filtered artificial seawater controls. This suggests that the methylpentose, (alpha)-L-fucose, plays an important role in primary sperm attachment in Limulus;Two major envelope proteins were identified with molecular weights of approximately 202,000 and 193,000. These proteins stained positive for the presence of saccharides using Periodic acid-Schiff staining of SDS gels. Biological activity was determined using the sperm attachment assay with spermatozoa that were treated with soluble envelope proteins;Sperm behavior in the presence of fucopyranoside and asparagus pea lectin-conjugated agarose beads confirmed that spermatozoa attach to fucose moieties. Surprisingly, spermatozoa tails adhered to asparagus pea lectin-conjugated beads. Fucose also was found to be a primary saccharide constituent of the seminal fluid and also suppressed sperm motility (seminal fluid had the same effect);Early postfertilization events and cleavage in Limulus have also been examined in the present study. Stage 1 which consists of nine embryonic surface events, Stage 2 (appearance of intralecithal nuclei), and Stages 3 and 4 which involve 'total' cleavage and blastula formation, respectively, were carefully described and a chronological sequence of developmental events was obtained.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Susan Ruttenberg Barnum
Barnum, Susan Ruttenberg, "Inhibition of primary sperm attachment, identification of egg envelope proteins, and early development of the horseshoe crab, Limulus polyphemus L " (1983). Retrospective Theses and Dissertations. 8407.