The rescue of oral development of defective-micronucleate conjugants of Paramecium tetraurelia by normal gametic nuclei

0 Zoology Department, University of Hong Kong , Hong Knng of Paramecium tetraurelia by normal gametic nuclei - The present study further analyses the importance of postmeiotic divisional derivatives of the micronucleus in the development of the oral apparatus of Paramecium during sexual reproduction. Cell lines possessing defective micronuclei generated by laser microbeam irradiation of the micronucleus were employed. They exhibited anomalies in nuclear reorganization and stomatogenesis in the sexual cycle. During autogamy, in some cells the micronuclear cycle terminated shortly after meiosis, resulting in the loss of all postmeiotic micronuclear derivatives. Stomatogenesis became arrested at an early stage of assembly of the oral membranelles, but the old oral apparatus was resorbed as usual, leading to the production of astomatous cells at the end of The problem of the somatic function of the micronucleus of ciliated protozoa has recently been critically reexamined. The traditional view that the micronucleus possesses little or no somatic function and participates only in nuclear reorganization during sexual reproduction is no longer tenable, in view of clear-cut evidence demonstrating important somatic functions of the micronucleus (for review, see Ng, 1986). We have shown in Paramecium tetraurelia that the micronucleus is intimately involved in stomatogenesis (the development of the oral apparatus; Fig. 1) during both asexual and sexual reproduction (Ng & Mikami, 1981; Ng & Newman, 1984a,fc; Tarn & Ng, 1986; Chau & Ng, 1987). A viable amicronucleate cell line can be generated by removing the two micronuclei from the cell. Such amicronucleate cell lines, however, suffer a transient period of growth depression, for up to several dozen fissions, during which defective oral apparatuses are developed. These cell lines subJournal of Cell Science 90, 287-293 (1988) Printed in Great Britain The Company of Biologists Limited 1988 the sexual cycle. Conjugation of these cell lines with normal micronucleates rescued both nucleogenesis and stomatogenesis in the defectivemicronucleate conjugant, primarily as a result of transfer of the male gametic nucleus from the normal conjugant to the defective-micronucleate mate. These observations demonstrate the stomatogenic significance, in particular in the initiation of oral membranelle assembly, of the gametic nuclei during sexual reproduction. The present study also suggests the possibility of micronuclear activities in the early part of the sexual cycle affecting postzygotic nucleogenesis. sequently recover, and stomatogenesis returns to near normal. This shows that the micronucleus plays a significant role in the development of the oral apparatus during binary fission. This function of the micronucleus, however, is replaceable in its absence. On the other hand, when amicronucleates are induced to go through the sexual cycle (autogamy or conjugation), the pre-existing oral apparatus is resorbed, as usual, but development of the new oral apparatus invariably becomes arrested at an early stage of assembly of the oral membranelles, resulting in the formation of astomatous cells at the end of the sexual cycle. This shows that the micronucleus plays a unique, and also indispensable, role in the initiation of oral membranelle assembly in sexual reproduction. Our recent studies with cell lines possessing defective micronuclei have shown that the micronucleus also governs the development of a normal oral pattern during sexual reproduction (Tarn & Ng, 1986; Chau & Ng, 1988). The initiation of oral membranelle assembly in sexual reproduction is of interest, since it is obviously a crucial hurdle in the development of the oral apparatus. Temporally, this takes place shortly after micronuclear meiosis, when the pregametic, gametic and zygotic nuclei have appeared (Ng & Newman, 1984o). We have demonstrated, with cell lines possessing micronuclei that behave abnormally in nuclear reorganization in the sexual cycle, that the disappearance of the pregametic, gametic and zygotic nuclei during the sexual cycle prohibits oral membranelle assembly, leading to astomy (Tarn & Ng, 1986; Chau & Ng, 1987). The present study furnishes a positive demonstration of the importance of the gametic nucleus in mediating stomatogenesis in the sexual cycle. This can be achieved by introducing a gametic nucleus into an amicronucleate during the sexual cycle, by allowing it to conjugate with a normal micronucleate, and assessing the subsequent development of the oral apparatus in the exconjugants. This type of conjugation has been performed in other studies for the generation of haploid clones (Chau & Ng, 1987). Nevertheless, it remains difficult to induce conjugation with amicronucleates, especially to harvest a large number of pairs needed for the analysis of the oral apparatus in the exconjugants in the present study. This difficulty has been overcome by resorting to cell lines possessing defective micronuclei in which some of the cells will terminate nuclear reorganization after meiosis in the sexual cycle. These cell lines could be readily induced to conjugate with normal micronucleates, which contribute the male gametic nucleus. Materials and methods Stocks and culture Paramecium tetraureliti stock 51 (mating type VII) and stock d4-94, a derivative of stock 51 bearing the piiA1 gene (mating type VIII), were employed. Cells homozygous for the/rcu4' gene were unable to swim backwards when challenged with a solution of high Na+ concentration (20mM-Na+ in Dryl's solution) (Kung, 1971; Sonneborn, 1975). Cells were cultured in phosphate-buffered Cerophyl medium (2-5 g l ~ ' , pH7), inoculated with luiterobacter aerogenes and supplemented with 5 mgl~' stigniasterol. For culture and handling of paramecia the methods of Sonneborn (1950, 1970) were followed. Experiments were performed at 27C and back stocks were stored at 1315 CC. Generation of defective-micronucleate cell lines The generation of cell lines with cells possessing a defective micronucleus took two steps. P. tetraurelia is bimicronucleatcd, and unimicronucleate cells were first generated by removing one of the two micronuclei with a microinjection needle, using the micromanipulation system described by Ng (19.81). Two unimicronucleate cells of independent clonal origin thus generated were expanded into two cell lines. They were then employed for producing defective-micronucleate cell lines, at the clonal age of 40-50 fissions, by laser microbeam irradiation of the remaining micronucleus, as described by Ng (1980). The cells were kept in logarithmic growth phase, at 27C, for 1 day prior to irradiation. The micronucleus was irradiated for 0-5-1 s after staining with Acridine Orange (8 or 16/<gml~', 10-15 m). The operation was performed only when the micronucleus was free from the macronucleus and the oral apparatus, so as to minimize damage to these organelles. After irradiation, each cell was expand (...truncated)