Evidence for a mating inducer molecule necessary for cell fusion and zygote formation in Didymium iridis

Abstract

Haploid myxamoeboid mating types, CR5-11A11 and HON 1-2A1, of the acellular myxomycete didymium iridis, were isolated and grown in liquid culture. Previously it has been postulated that a molecule, possibly a hormone, is responsible for the induction of mating competency through receptor site synthesis. The goal of this study was to isolate and characterize the molecule and to determine its mode of action. Experiments were designed in order to learn whether it acts only upon the cells which produce it (self induction) or does it have an effect only on opposite mating types (cross induction). In order to do this, separate experiments, one involving a membrane spin culture and the other a separate spin culture, were maintained. The membrane separated culture contained a .45um filter which allowed any diffusable molecule to freely pass through, but not cells, which would allow cross induction to occur while the separate cultures would allow self induction only. Three daily samples were taken, two of which acted as controls of the two clones while the third sample was a cross of the two clones. The latter contained induced cells which should produce zygotes, or fused cells. These samples were subjected to proper fixation, collection, and staining utilizing the Feulgen reaction. A microspectrophotometric analysis was then performed to quantitatively measure cellular nuclear DNA content, and to provide a means for the detection of cellular fusion. Additionally, experimentally crossed compatible mating types were plated to solid growth media in order to detect any diploid plasmodia formed from fused cells. Plasmodia were similarly prepared and analyzed. Interestingly,it was found that plasmodia can only form on solid media, although zygotes are readily formed in liquid media. Also, competency of cells to fuse, or receptor formation, required that thy first undergo a period of maturation. The use of High Pressure Liquid Chromatography (HPLC) techniques was also employed in order that the inducer molecule be studied. Samples were taken from separate liquid cultures of the two clones, frozen and subsequently analyzed. Also, a control sample containing only the media was run and analyzed. It was demonstrated that the inducer molecule is similarly produced by both mating types, its production is paralleled by cell numbers, and it is important in cellular maturation and subsequent fusion through receptor site formation. It was also found that mating competency it self induced.