Cytological studies of marsupial and monotreme cells in tissue culture

Recent advances in cytological techniques have made it possible to determine chromosome numbers of animals whose karyotype had not been known with certainty because of large chromosome numbers and small size of some of the chromosomal complement. The discovery by Nowellithat phytohaemagglutinin (a protein with a mucopolysaccharide prosthetic group, obtained readily as a crude extract from the common bean) can initiate mitotic division in blood leucocytes cultured in vitro has made it possible to determine the karyotype of an animal from as little as a few drops of blood. Hypotonic treatment produces excellent spreads of chromosomes without the use of squash techniques, and the addition of colchicine for the final two hours of the culture period increases the number of cells arrested at metaphase and further facilitates the determination of chromosome numbers and morphology. In placental mammals, chromosome numbers vary over a wide range (from 18 to 86 for the diploid number), although the DNA content has been found to be relatively constant for all species. Marsupials are characteriZed by large chromosomes and small numbers (2n = 10 to 22), but the DNA content and volume accord with those of eutherian mammals. Little information has been recorded about the chromosomes of the monotremes. The karyotype of the platypus has been investigated by Matthey (1949) although with the techniques available at that time the chromosome number (70 ±10) could not be ascertained with accuracy. Matthey remarked that the karyotype in the monotrines shows a division into macro and micro elements and is similar to that in birds and reptiles. Van Brink (1959) published a report on the karyotype in Echidna giving numbers for the male between 62 and 64. She recorded one large chromosome as being unpaired, but the scarcity of divisions in the material available did not allow for a decision between an X0 and XY heterogamety. In most mammalian species studied there is a typical XX-XY sex chromosome mechanism, and the male is the heterogametic sex. However, there is an XX-XY1 Y2 mechanism in two macropod marsupials, Potorous tridactylus and Wallabia bicolor, and at least one eutherian mammal, Sorex araneus. A, more complex sex-determining system, X1X1X2X2 in the female andX 1X2Yin the male, has been found in two placental mammals and recently in an Australian marsupial, the hare wallaby. The sex-determining system in the Monotremata is completely unknown. In this work an attempt has been made to resolve the sex-determining system of the monotremes and to determine their karyotype. Metaphases from colchicine-treated short-term cultures of leucocytes from the peripheral blood of the male and female echidna and platypus gave suitable preparations from which the chromosome number could be determined without ambiguity. In addition, chromosome spreads from two male echidnas were obtained by subjecting testis material to trypsinization and hypotonic treatment for examination of mitosis and meiosis in spermatogenesis. The testes of the male platypus available for this study were small and undeveloped and no divisions were found. Good chromosome preparations were obtained, however, from the spleen of the female platypus. Both the monotremes are highly protected species. Although the platypus and echidna are much more common in Tasmania than in other Australian states, the platypus is rarely seen, and presents a further problem since it often dies of shock after a few hours in captivity. The breeding season, about which little information is available, is of short duration; and the scarcity of divisions in the gonads, which often regress for considerable periods, make the study of testicular meiosis extremely difficult without sacrificing large numbers of animals.