A 33 kDa molecular marker of sperm acrosome differentiation and maturation in the tammar wallaby (Macropus eugenii) M. A. Wade and M. Lin Cooperative Research Centre for the Conservation and Management of Marsupials, Department of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia This study was undertaken to identify potential molecular markers of acrosomal biogenesis and post-testicular maturation in marsupials, using the tammar wallaby as a model species. A two-step sperm extraction procedure yielded two protein extracts of apparent acrosomal origin and a tail extract. The extracts were analysed by SDS-PAGE under reducing conditions. Several prominent polypeptide bands (45, 38 and 33 kDa) appeared common to both acrosomal extracts. Antiserum raised against the 33 kDa polypeptide from the inner acrosomal membrane matrix (IAMM) extract showed immunoreactivity with 45, 38 and 33 kDa polypeptides in both acrosomal extracts, indicating that the 33 kDa polypeptide was related to the proteins in the 45 and 38 kDa bands. Therefore, the antiserum was used as a molecular probe. Indirect immuno- fluorescence indicated that the acrosome was the major location of the 33 kDa polypeptide. This contention was confirmed by ultrastructural study: immunogold labelling indicated that the 33 kDa polypeptide associated with acrosomal matrix components throughout acrosomal development in the testes and throughout post- testicular maturation in the epididymis. The label clearly delineated the changing morphology of the maturing marsupial acrosome. This is the first study to use immunocytochemical techniques to chart testicular and post-testicular development of any sperm organelle in a marsupial. As a result of this study, a 33 kDa molecular marker of marsupial acrosome differentiation and maturation has been identified. It may be possible to chart similar events in other marsupial species and identify opportunities for manipulating fertility. Introduction Previous studies have indicated that sperm acrosome differentiation and maturation in a number of Australian marsupials is different from that of other species (Cummins, 1976; Harding et ah, 1976a,b, 1982,1983,1984; Temple-Smith and Bedford, 1976; Kim et ah, 1987; Lin et ah, 1997; Setiadi et ah, 1997). Unlike the eutherian acrosome, which is derived from a dense granule from the Golgi and forms a cap-like structure over the nucleus at maturity (Bloom and Fawcett, 1975), the marsupial acrosome is derived from a sparsely granulated vacuole, also of Golgi origin. At maturity, the acrosome does not cap the nucleus but occupies a concavity on the anterior dorsal nuclear surface (Harding et ah, 1976a). In most eutherian species, acrosomal maturation is essentially complete at spermiation. However, in many marsupial species, extensive post-testicular processing is a significant feature of sperm acrosome maturation (Cummins, 1976; Temple-Smith and Bedford, 1976; Lin et ah, 1997; Setiadi et ah, 1997). In other marsupial species, the maturation process differs (Harding et ah, 1983) and is poorly understood. In two species, the brushtail possum (Trichosurus vulpécula) and the tammar wallaby (Macropus eugenii), extensive morphological reorganization of the acrosome has been reported during transit through the caput and corpus epididymidis (Cummins, 1976; Harding et ah, 1976a; Temple- Smith and Bedford, 1976; Lin et ah, 1997; Setiadi et ah, 1997). For example, at spermiation the tammar acrosome is a relatively immature structure. Ultrastructural studies reveal margins of the acrosome that extend both laterally and anteriorly from the nucleus, forming a concave disc or scoop structure. This is similar to the structure reported for the brushtail possum acrosome at spermiation (Harding et ah, 1976b). In tammar wallabies, the extensions move toward each other during epididymal transit. The margins of the extensions fuse during transit of the distal corpus epididymidis. Acrosomal condensation continues throughout epididymal transit and the compact button shape and anterior dorsal location of the mature tammar acrosome on the sperm nucleus is not achieved until the spermatozoon reaches the cauda epididymidis (Lin et ah, 1997; Setiadi et ah, 1997). Similar events occur in brushtail possums (Cummins, 1976; Harding et ah, 1976b; Temple-Smith and Bedford, 1976). Sperm head proteins from tammar wallabies have been isolated and a number of polypeptide bands, presumed to be Received 6 October 1998.