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  • J. Cell Sci. 3a, S5-66 (1978) 55Printed in Great Britain Company of Biologists Limited 197S

    A SCANNING ELECTRON-MICROSCOPIC

    STUDY OF THE LOCAL DEGENERATION

    OF CILIA DURING SEXUAL REPRODUCTION

    IN PARAMECIUM

    TSUYOSHI WATANABEBiological Institute, Tohoku University, Sendai 980, Japan

    SUMMARY

    The location and extent of local degeneration of cilia during sexual reproduction of Para-mecium was studied using scanning electron microscopy to examine cells undergoing con-jugation and autogamy. At some time during the mating reaction, but prior to conjugantpair formation, ciliary degeneration begins at the antero-ventral tip of cells and proceed*posteriorly along the suture. In the anterior part of the cell, degeneration occurs on both sidesof the suture, but in the posterior part it is restricted to the right side of the suture. In 5 speciesof Paramedum examined, degeneration occurred in nearly the same region. No degenerationof cilia is observed in natural autogamy of P. tetranrelia, whereas in chemically induced auto-gamy of P. caiidatum degeneration occurs as in ordinary conjugation. Conjugant pairs neverexpose any deciliated cell surface except at the postero-ventral tip. The maximum extent ofciliary degeneration is best seen in the chemically induced autogamous cells: 7 kineties (rowsof unit teritories) at the anterior-left, 4 kineties at the anterior-right, 10 or more kineties at theposterior-right and the right wall of the vestibule of the mouth. Before complete disappearanceof the cilia, many short cilia are observed. This suggests that ciliary degeneration is due toresorption. Degeneration extends more rapidly in cells with stronger mating reactivity. Therelations between mating reactivity, ciliary degeneration and nuclear activation are discussed.

    INTRODUCTION

    In Paramedum and other ciliates, interesting studies of inheritance and morpho-genesis of cortical pattern have been possible because of local differentiation instructure and function of the cell surface (Sonneborn, 1963, 1970a). One notablecase is the set of events in conjugation restricted to the ventral surface of the cell.In the conjugation process of Paramedum, cell contact is established in 3 steps:mating reaction, holdfast union, and paroral union. In the mating reaction, cells ofcomplementary mating type stick together by 'mating reactive' cilia located on theventral surface of the cell (Hiwatashi, 1961). As the mating reaction proceeds, ciliaand trichocysts at the anterior tip and on the ventral surface just behind the tipdisappear, and pairs of cells unite at the antero-ventral surface in a 'holdfast union'.Cilia continue to disappear on the ventral surface, and the cells unite more firmly,especially in a region just posterior to the mouth, in a 'paroral union'. Details of theholdfast and paroral unions may be found in Wichterman (1946), Hiwatashi (19556),Vivier & Andre" (1961) and Miyake (1966). Hiwatashi (1961) suggested that there issome relation between the contact region of the mating reaction and that of the

  • 56 T. Watanabe

    holdfast union. Moreover, the works of Hiwatashi (19556) and Miyake (1966,1968 a, b) strongly indicate that the loss of cilia during conjugation is essential notonly for the formation of holdfast and paroral union but also for nuclear activation.

    Thus the loss of cilia is an important phenomenon, not only from the viewpointof morphogenetic problems but also from that of fertilization (Miyake, 1974). How-ever, the exact details and controlling mechanisms of the degeneration of cilia havenever been reported. To clarify these points, scanning electron-microscopic obser-vations were made on cells undergoing conjugation and autogamy.

    MATERIALS AND METHODS

    Animals and culture methods

    Stocks Kt, dKKi4a, 27aG3, dNi4a and di2-3~4 of Paramecium caudatum, syngen 3, wereused throughout the study. For conjugation via mating type, the cells were cultured withlettuce juice medium (Hiwatashi, 1968) in which 1 vol. of fresh lettuce juice was diluted with40 vol. of Dryl's solution (Dryl, 1959) and bacterized with Klebsiella aerogenes. For chemicalinduction of autogamy, the cells were cultured with a Ca-poor medium in which lettuce juicewas diluted in 2 i m sodium phosphate buffer (pH 7-0) instead of Dryl's solution. For com-parison, conjugating cells of P. tetraurelia (stock 51), P. multitnicronucleatum (CH-312 andCH-313,) P. bwsaria (TK-i and TK-3) and P. trichium (PC-2 and PC-5) were used. Cells ofP. tetraurelia, stock 51, were also used for studies of natural autogamy.

    Conjugation via mating type

    Mating-reactive cells of complementary mating types were mixed in depression slides. Thecells agglutinated soon after mixing, and holdfast pairs were formed about 60 min after thebeginning of the mating reaction. Although the exact time of the onset of paroral union wasuncertain, firmly united pairs which had undergone paroral union were present after 90 min.Such pairs were identified when drawing them into and expelling them from a pipette failedto separate the 2 cells. Cells of P. caudatum were fixed at various times from 15 min to 5 h afteronset of the mating reaction. The cells of other Paramecium species were fixed at the time ofholdfast pair formation.

    Strength of mating reactivity was assessed by noting if many clumps are formed quicklyafter mixing mating types ('strong' or 'high' reactivity) or if few cells form clumps soon aftermixing ('weak' or 'low' reactivity). No method is currently available for determining theamount of mating substance on each cell.

    Chemical induction of autogamy

    Chemical induction of autogamy was performed by a modification (Tsukii, in preparation)of the method of Miyake (1968 a, b). The cells of a single mating type of P. caudatum werecultured in Ca-poor medium and washed once in a modified Miyake's (1958) physiologicalbalanced solution called K-PBS-II (i-smMNaCl, r 8 mM KC1, o-irriMMgCl,, o-oi mMCaClj, 18 mM KH2PO4 and 0-2 mM K,HPO4, pH 60). Then the cells were treated with theautogamy-inducing medium (6 mM KC1, 50 mM methyl urea, and 40-80 /tg/ml ficin or 5-10/*g/ml papain in K-PBS-II). Partially purified ficin was prepared from crude ficin (WakoPure Chemicals Co., Ltd.) according to the method of Hammond & Gutfreund (1959). Papainused was a crystalline preparation (Sigma, 2 x crystallized). When the cells were treated withthe autogamy-inducing medium, neither agglutination nor pair formation was observed. At20 h after the beginning of induction of autogamy, the occurrence of autogamy was ascertainedby looking for macronuclear fragmentation, a characteristic of sexual reproduction. Details ofthe chemical induction of autogamy in P. caudatum will be described elsewhere (Tsukii, inpreparation).

  • Ciliary degeneration in Paramecium mNatural autogamy

    Natural autogamy was induced in P. tetraurelia by starving sufficiently old cells (Sonneborn,19706) at 27 CC. It is difficult to induce autogamy synchronously or to distinguish cells under-going autogamy by external appearance. Therefore, to obtain a population of cells in variousstages of autogamy including early stages, cells were fixed when 10 % of the cells showedmacronuclear fragmentation. In unfixed controls, the proportion of cells with fragmentedmacronuclei rose to 50 % 7 h after this time of fixation. In conjugation of P. aurelia, separationof conjugants and macronuclear fragmentation occur 6-7 h after the initiation of conjugation(Jurand & Selman, 1969).

    Scanning electron microscopy

    Cells were fixed in Parducz solution (Parducz, 1967) for 30 min at room temperature.After washing in deionized water, the cells were dehydrated in a series of ethanol and isoamylacetate. The cells in isoamyl acetate were put on coverglasses or aluminium disks and air-dried. The specimens were coated with gold and examined with an Hitach-Akashi MSM-4scanning electron microscope.

    Fig. 1. Silver preparation of P. caudatum (ventral view), al, anterior left field; ar,anterior right field; as, anterior suture; pi, posterior left field; pr, posterior rightfield; ps, posterior suture; v, vestibule, x 480.

  • T. Watanabe

  • Ciliary degeneration in Paramecium 59

    RESULTS

    Morphology of vegetative cells

    The cortical structure of Paramecium has been described by many investigatorsusing the silver impregnation technique. The ventral morphology of the vegetativecell will be described briefly. The most characteristic organelle of the ventral surfaceis the mouth. The position of the opening of the mouth is somewhat different fromspecies to species. In P. caudatum, the mouth is located slightly posterior to thecentre of the cell. The suture runs longitudinally across the mouth. Therefore, theventral surface of Paramecium is divided into 4 parts by the suture and the mouth:anterior-right, anterior-left, posterior-right and posterior-left (Figs. 1, 2). Theanterior-left corresponds to the oral groove, which extends from the anterior end tothe vestibule of the mouth and is apparent in living cells.

    Order and location of ciliary degeneration in early stages of the conjugating process

    The location of degenerating cilia and the order of their disappearance was examinedin cells of P. caudatum undergoing conjugation after the mating reaction. At 15 minafter the beginning of the mating reaction, no ciliary degeneration was observed.At 30 min, when cells are still in the mating clumps, small numbers of cells showshort cilia or no cilia at their anterior tips (Fig. 3). Ciliary degeneration proceedsposteriorly along the suture at about the time of holdfast pair formation. At this time,some cells have short cilia of various lengths (Figs. 4, 5). This suggests that thedegeneration of cilia is due to resorption. The degeneration of cilia extends to thesides and to the po

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