01_ Form and function of Mammalian sperm and eggs

Eutherians

Work on sperm morphology of eutherian mammals has included a detailed comparative electron microscopical study of spermatozoa of Australian rodents. These investigations have demonstrated considerable interspecific diversity in sperm morphology in two of the Australian rodent genera, Pseudomys and Notomys, although in most species a falciform sperm head shape in which there are two processes that extend from the upper concave surface are present. These processes, which are more extensive in these Australasian rodents than any other species, contain filamentous actin (see Fig. 2 which is an illustration of a plains rat spermatozoon stained with propidium iodide for DNA and Bodipy phallacidin for F-actin) as well as various other proteins and are largely composed of a massive extension of the sperm head cytoskeleton. These are the most morphologically complex spermatozoa to have evolved in eutherian mammals. Studies are now under way to determine the chemical composition of the ventral processes and their functional significance.

Marsupials

Our studies on sperm form and function in marsupials have involved probing the structural organisation of the chromatin with atomic force microscopy and freeze fracture followed by transmission electron miscroscopy. In the dasyurid marsupial, Sminthopsis crassicaudata, we have found two types of chromatin in the mature spermatozoon; a central, electron-dense, region, and an outer region which has material that shows variable amounts of electron density. Immunofluorescent light microscopy and immunogold electron microscopy using antibodies to the histones has shown localisation of histones to this outer chromatin region which presumably explains its different morphological appearance. Further studies on marsupial sperm structural organisation are continuing at present with a particular emphasis on its cytoskeletal organisation.

Our studies on marsupial eggs have recently largely concentrated on isolation and characterisation of the egg coat glycoproteins. We have over the last couple of years isolated the cDNAs encoding two of the three zona pellucida glycoproteins ZPA and ZPB in marsupials and found that they both show a high degree of homology to the ZPA and ZPB in eutherian mammals in spite of separate evolution from this group for over 100 million years. We have also found that there are various oligosaccharides present within the zona and recent freeze substitution has shown that the zona has a honeycomb-like appearance. We are at present, exploring the distribution of proteins and sugars within this 3D meshwork of filaments.

Within our laboratory at the present time we are also characterising the molecules on the surface of the marsupial spermatozoon that are involved in the spermatozoon binding to the egg coat (the zona pellucida). At the present time, much of this work is being carried out on material obtained from the brush-tail possum. We have identified, and characterised, the peripheral and integral protein molecules on spermatozoa from the caput and cauda epididymides. SDS polyacrylamide gel electrophoresis of sperm plasma membrane extracts has indicated considerable differences in the proteins in the plasma membrane of the spermatozoa from these two regions of the duct. Freeze fracture studies of spermatozoa, and staining of caput and cauda epididymal sperm with FITC-labelled lectins, have indicated that significant changes take place to both the arrangement of the intramembranous protein molecules and in their oligosaccharide content during epididymal transit.

Using the second species of marsupial, Sminthopsis crassicaudata, we have demonstrated very efficient transport of spermatozoa to the isthmus of the oviduct where significant numbers are stored for up to three days. With the occurrence of ovulation reorientation of the sperm head on the tail occurs just prior to the sperm's final migration to the site of fertilisation.

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