Studies on protamine biosynthesis in herring testes

Abstract

Protamines are small basic proteins found in association with nuclear DNA in the sperm of certain species. The biosynthesis of protamine has been studied in testes material from the European herring (Clupea harengus). The herring testes were classified by the Hjort maturity scale into arbitrary maturity stages as immature (stage I), intermediate (stages II-IV) and mature (stages V-VI). Bulk protamine was purified from acid-soluble nuclear proteins of mature sperm by successive chromatography on ion-exchange and gel exclusion columns. Characterisation of this protamine by further ion-exchange chromatography showed the presence of three components. However, difficulties were encountered in extension of these methods to analysis of the relative rates of protamine component synthesis as a function of development. Enzymatic dephosphorylation of newly-synthesised protamine was found necessary prior to radioactive component analysis, since the presence of phosphate groups made interpretation of the results difficult. Treatment with alkaline phosphatase changed the elution profiles previously obtained with mature protamine and so a method for analysis of radioactive protamine components could not be developed. Using analytical electrophoresis on 20% polyacrylamide gels, protamine was first detected at stage II, increased during subsequent stages and reached a maximum at stages V and VI. Greatest protamine synthesis occurred at stage III. Testes from stages II-IV were therefore used as a source for mRNA isolation and characterisation. Microsomal RNA was separated into high and low molecular weight RNA by differential salt precipitation and these fractions tested, for mRNA activity in a cell-free System. Both fractions exhibited activity, over 95% of the total being in the high molecular weight RNA. The latter was separated into poly A (+) and poly A (-) fractions by chromatography on oligo (dT) cellulose. All fractions exhibited messenger activity, the poly A (-) RNA comprising over 80% of the total. Sucrose gradient analysis of protamine mRNA under denaturing conditions showed that its apparent large size was artifactual. However, the presence of the messenger in poly A (+) and poly A (-) forms was shown to be real by elimination of possible RNA degradation and overloading of the oligo (dT) cellulose column.