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Variability of testis-specific proteins in Gasterosteus aculeatus L. and related species Lemke, Michael J.


Testis-specific protein (TSP) variability has been examined in the three-spined stickleback, Gasterosteus aculeatus and related species including Gasterosteus wheatlandi, Punqitius pungitius, and Aulorhynchus flavidus, in order to determine if such proteins can act as molecular markers for different species of Gasterosteiformes and for different populations of a single species. Cytochemistry of the sperm histones of Gasterosteus aculeatus revealed that these basic proteins can be classified as intermediate sperm histones according to Bloch's (1969 and 1976) categories. Electron microscopy indicates that the chromatin in the nucleus condenses in a granular pattern as the somatic histones of the spermatogonia are replaced by the sperm histones in the spermatid during spermiogenesis in this teleost. The testis specific proteins (TSP's) of G. aculeatus and related species were characterized by electrophoresis on polyacrylamide gels and by hydrolysis of the amidoblack stained bands from the gel and subsequent amino acid analysis. The compositional analysis revealed that all the TSP's of the fish in the order Gasterosteiformes were intermediate type containing histidine, lysine, and arginine amino acid residues. However, the TSP's of different species could be distinguished by their electrophoretic mobilities on polyacrylamide gels and by differences in the amino acid composition. Apparently TSP's can act as molecular markers to distinguish these particular teleosts. To establish the electrophoretic pattern for the TSP's of mature, breeding G. aculeatus, the developmental profile was investigated over the course of a season for a population of these fish in Jericho Pond, Vancouver, B.C. As the testis matures, somatic histones are replaced gradually by one or several rapidly moving TSP's. Some protein bands that are present in fish with immature testes are removed by the time the electrophoretic pattern typical for mature males occurs during the breeding season. This pattern shows only rapidly moving TSP's and very low levels of somatic histones. Experiments using alkaline phosphatase indicated that the electrophoretic profile of the multiplicity of TSP'.s of mature fish was not due to differences in charge because of phosphorylation of serine side chains in TSP bands. In addition to acting as molecular markers for different species, the TSP's showed an electrophoretic profile in anadromous G. aculeatus that differed from the profile in freshwater fish with respect to band morphology and also the number of bands seen on long gels. Here, too, the similarity of the freshwater and anadromous TSP profiles was reinforced by similar digestion patterns with cyanogen bromide, confirming the presence of methionine in these proteins. However, different populations of anadromous G. aculeatus either from British Columbia or from Quebec were indistinguishable by electrophoretic analysis. Moreover the electrophoretic and amino acid analysis could not distinguish between G. aculeatus from different freshwater populations. There was no discernable trend for the multiplicity of TSP's from such populations. However, in the fish from two freshwater lakes there was an indication that the benthic forms (bottom dwellers) might have reached sexual maturity later than the limnetic forms (top dwellers) as the limnetic animals lost their somatic histones before the benthics did. From these analyses, TSP's apparently can act as molecular markers between different species of Gasterosteiformes, and to a lesser extent between anadromous and freshwater forms of G. aculeatus. This agrees with the findings of Mann et. al. ( 1982) that the spermatid/sperm-specific proteins of the frog genus Xenopus can distinguish between different species of the genus and somewhat between different subspecies of Xenopus laevis. Both the Gasterosteiformes and anuran TSP's are of the intermediate type. In several instances, particular species of fish gave anomalous results. For example, an anadromous G. aculeatus showed the presence of a band typical for the TSP of G. wheatlandi on polyacrylamide gel electrophoresis. Perhaps this is due to hybridization between Gasterosteidae of different species. Finally, incubation of TSP preparations at 37 °C indicated the presence of endogenous protease at neutral pH. Such a protease was not active at acid pH and therefore did not interfere with the electrophoretic analysis.

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