Fine-Structural Aspects of Fertilization in Chlamydomonas Reinhardi

New York 0 U.S.A. 0 0 Department of Biology, Queens College of the City University of New York , Flushing SUMMARY Gametes of C. reinhardi lack the cell wall which vegetative cells possess. Just below the cell apex gametes form a. fertilization tubule which is up to 2 fi long and 0-2 /* in diameter; its plasma membrane and that of the apex have slender tubular projections. At the base of the fertilization tubule regularly lies the choanoid body, a collar-shaped cytoplasmic organelle; the plasma membrane overlying the body appears as an electron-dense ring. Gametes possess two 'free' basal bodies in addition to the basal bodies of the two flagella. In the initial stage of union the conjugating cells are connected by the fertilization tubule whose plasma membrane is continuous with that of both copulants. At one end of the tubule lies a conspicuous choanoid body, but at the other end is a small structure which possibly is a homologue of the choanoid body. Subsequently, the fertilization tubule shortens and widens until finally no tubule exists and the apical ends of the two protoplasts adjoin. The merging cells then bend like a jack-knife and lateral alignment of the protoplasts occurs. This fourflagellated zygote becomes motile at about the time when the flagellar bases of the former gametes seem to approach each other and when fibrillar elements of the flagellar roots come into contact. In the motile zygote the nuclei do not fuse but remain ensheathed in the cup-shaped plastids of the two gametes. A mating of plus (+ ) and minus ( -) strains cultured, respectively, for high and low starch content suggested that gametes of only the plus ( +) mating type contain the choanoid body. Since it appears that the gamete containing the choanoid body also produces the fertilization tubule, it is inferred that gametes of only the plus (+ ) mating type produce the fertilization tubule. Should further investigation support this inference, it would be established that there is a structural basis for designating the plus ( +) mating type as male and the minus ( -) type as female. FINE-STRUCTURAL ASPECTS OF FERTILIZATION IN I. FRIEDMANN,* A. L. COLWIN AND LAURA H. COLWIN Department of Biology, Queens College of the City University of New York, Flushing, New York, U.S.A. SUMMARY Fine-structural details of the fertilization process in algae are to a considerable extent still unknown. The process in the green alga Prasiola stipitata has been studied in thin sections by Manton & Friedmann (i960). In this species the plasma membranes of the two gametes coalesce and then the internal cellular contents merge. In the beginning the membrane of one of the two spermatozoid flagella coalesces with the membrane of the egg, and the fibrillar core of that flagellum becomes the first part to be incorporated into the egg protoplast. However, the coming together of the gametes by means of such a flagellum is not characteristic of all types of algal fertilization. It was suggested (Friedmann, 1964) that fertilization in algae might follow two different patterns, the one being exemplified by Prasiola and the brown algae in which the gametes join by means of one of the male flagella, and the other by Chlamydomonas in which the junction involves rather the apical regions of the gametes. It is therefore of interest to investigate details of the pattern in Chlamydomonas by electron microscopy. Among the Chlamydomonas species generally used in laboratory investigations, C. moewusii and C. etigametos have motile zygotes, termed 'vis-d-vis pairs' by Lewin (1954 a), which seem to represent a type found only in a relatively small number of algae. In contrast, the four-flagellated motile zygote of C. reinhardi seems to be a characteristic type occurring in the great majority of isogamous green algae. This species, therefore, was selected for the present investigation. In C. etigametos, early electron-microscopic studies by Lewin & Meinhart (1953) showed the protoplasmic bridge described by Moewus (1933) connecting the two gametes in the motile zygote (vis-a-vis pair) and Gibbs, Lewin & Philpott (1958) in a study of thin sections reported that the bridge, delimited by a membrane, is first filled with homogeneous cytoplasm, and that later an electron-dense core deriving from the blepharoplasts appears to grow across the bridge. Johnson (1964) reported having studied by electron microscopy the sexual reproduction of C. moewusii. Fertilization in Chlamydomonas reinhardi was observed in the light microscope and recorded by phase-contrast cinemicrography and the results will be treated in a forthcoming publication. In the present study an attempt was made to match the stages in fusion of the protoplasts seen in thin sections with the stages seen in cinemicrographic records. A short preliminary report of the present study was published earlier (Friedmann, Colwin & Colwin, 1966). MATERIALS AND METHODS Cultures of C. reinhardi were obtained from the Culture Collection of Algae at Indiana University (strains 89 and 90) and from Dr Ruth Sager, Columbia University, New York (strains 21 gr and 6145 c). These have been used with similar results. However, strain 21 gr, considered as plus ( + ) , crosses with strain 89, also considered as plus ( + ) ; and conversely, strain 6145 c, considered as minus (), crosses with 90, also considered as minus ( ). These strains were subsequently tested against strains 11/32 a ( + ) and 11/32 b ( - ) obtained from the Culture Collection of Algae and Protozoa at Cambridge University; the latter strains represent the original isolates of Dr G. M. Smith sent to Cambridge in 1950. It was found that strains 21 gr and 90 are of the plus ( + ) mating type and 6145 c and 89 are of the minus ( ) mating type. Cultures were grown on agar plates under standard culture conditions (14-h light cycle) using Bold's (1949) modified Bristol medium or nutrient medium I of Sager & Granick (1953). Gametes were harvested in distilled water and suspensions of mating gametes werefixedin 1 % phosphate-buffered osmium tetroxide or in 4 % phosphatebuffered glutaraldehyde at pH 7-2. After fixation for 2-2^ h at room temperature, the preparations were washed, dehydrated and embedded in Epon according to the procedures of Luft (1961). Sections were cut with a DuPont diamond knife, stained with lead citrate (Venable & Coggeshall, 1965) and uranyl acetate (saturated aqueous solution diluted before use with an equal volume of absolute ethanol). For the present study the osmic-fixed material proved more suitable and all the electron micrographs show such Fig. 1. Diagrammatic representation of the apical region of a gamete with a fertilization tubule. The four lateral ridges lying below the apical papilla of the cell are somewhat exaggerated in the diagram. At the base of the fertilization tubule lies the choanoid body. Slender tubular projections rise from the fertilization tubule and the cell apex. material. Sections w (...truncated)