A Minimum Essential Medium for Cultivation of Schistosoma japonicum Eggs

Abstract

In schistosomiasis japonica, eggs of the schistosome play a predominant role in its pathology (Warren, 1978, Nature 273: 609-612). Therefore, knowledge of the metabolism of the eggs is essential for better understanding of pathogenesis. The eggs are deposited within the mesenteric blood vessels in an unembryonated state and embryonate in the tissue of the mammalian host (Vogel, 1942, Dtsch. Trop. Z. 46: 57-69, 81-91). Cultivation of the schistosome egg from a single egg cell into a miracidium in a chemically defined medium is a prerequisite for strict investigation of its metabolism. In order to improve or simplify the composition of culture media for schistosome eggs, it is important to know the nutritional requirements of the eggs. We previously reported that the eggs require 14 L-amino acids (arginine, cystine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine), two vitamins (choline chloride and nicotinamide), and glucose for full development (Kawanaka et al., 1983, J. Parasitol. 69: 857). These essential nutrients had been determined by the deletion of single components from Medium RPMI 1640 that supports development of the eggs and contains 20 amino acids, glutathione, 11 vitamins, and glucose in a balanced salt solution. However, in the medium composed of all these essential 14 amino acids, two vitamins, glucose, and electrolytes, S. japonicum eggs did not mature. The electrolytes were those in Earle's balanced salt solution. This medium was tentatively designated as the basic one and the minimum necessary supplement to the basic medium was searched for among six amino acids, the remaining amino acid components in Medium RPMI 1640. The six amino acids tested were asparagine, aspartic acid, proline, hydroxyproline, glutamic acid, and glutamine. Here we report that glutamine is an essential and sufficient supplement to the basic medium. Preparation of the minimum essential medium for cultivation of Schistosoma japonicum eggs is presented. Adult schistosomes (8 wk old) were recovered under aseptic condition from the dd strain of mice that had been infected intraperitoneally with 50 cercariae of a Japanese strain of Schistosoma japonicum. Techniques relating to the collection of newly laid eggs of schistosome and their subsequent maintenance in culture have been described, as have the method for the assessment of maturation of the eggs (Kawanaka et al., loc. cit.). The concentrations of amino acids, vitamins, and glucose in test media for the present experiment are the same as those in Medium RPMI 1640. Table I shows results of the addition of supplementary amino acids alone or in combination to the basic medium in Earle's balanced salt solution. The single addition of asparagine, aspartic acid, proline, hydroxyproline, or glutamic acid elicited no effect on the development of eggs.