Male gamete development in the heterosporous water fern Marsilea vestita is a highly synchronous process which is completed within 5 to 6 hours at 30°C. The morphogenetic and ultrastructural changes which take place during spermatogenesis have been well characterised in light and electron microscope investigations (Sharp 1914; Hepler 1976) but few studies exist on any biochemical aspects of spermatogenesis in this system. To obtain a more comprehensive overview of the molecular events occurring during microspore development, we have investigated the pattern of some of the biochemical processes by a variety of methods.
Results from experiments in which inhibitors of RNA, DNA and protein synthesis and microtubule function were applied to developing microspores at various times during the 6 hour period have been described previously (Hyams et al. 1983). They have enabled us, with certain limitations, to determine at what times during spermatogenesis specific molecular syntheses or functions are important. Protein synthesis has been studied further in some detail. Polyacrylamide gel electrophoresis of extracts of Marsilea microspores during development reveals that there is an overall increase in the amount of protein present as spermatogenesis proceeds. In addition to tubulin, the major flagellar protein and constituent of the microtubule ribbon, major polypeptides are detected at 15 k, 26 k, 31 k and 90 k on Coomassie Blue stained gels. Few qualitative changes over the 6 hour period can be detected, even when very sensitive staining methods are used to detect minor proteins. This would appear to show that few proteins are synthesised specifically at particular stages of microspore development. We have investigated this question further by labelling the microspores with 14C leucine and 35S methionine, and performing fluorography of SDS polyacrylamide gels run with the radiolabelled samples. Fluorographs reveal more detail than it is possible to resolve with ordinary staining methods, and several polypeptides have been detected which appear to be heavily labelled at the onset of spermatogenesis but which incorporate progressively less radioactive amino acid as development proceeds. As yet, the identity of these proteins is unknown.
We have initiated a study into the presence of individual proteins during spermatogenesis, using specific antibodies reacted against Western blots of polyacrylamide gels. A monoclonal antibody against tubulin (Kilmartin et al. 1982) reveals that this protein is present in small amounts from the onset of microspore development but that the amount increases greatly between 3 and 4 hours, the time when sperm flagella are observed to assemble. We have recently raised a polyclonal serum in rabbit which cross reacts with a high molecular weight polypeptide on Western blots of Marsilea microspore extracts. This protein appears to be present in large amounts at the beginning of the 6 hour period but decreases slightly as development proceeds. The identity of this protein is currently under investigation.