The Ultrastructure of Fertilization and Zygote Formation in the Green Alga Ulva Mutabilis Føyn

T. Brdten 0 0 T. BRATEN Electron Microscopical Unit for Biological Sciences, University of Oslo , Blindern, Oslo 3 , Norway - SUMMARY Fertilization and zygote formation in the multicellular green alga Ulva mutabilis Foyn were studied with the aid of transmission and scanning electron microscopes. The growth mutant 'slender' was used in this investigation. The initial contact between mating gametes is made by theflagella,whereupon cytoplasmic contact is established within a few seconds. Fusion of the cytoplasm of the 2 cells is completed in 5 min and the nuclei can be seen to fuse 30 min after the onset of copulation. The settling of the young zygotes initiates a process during which the 4flagellaare absorbed into the cell. Cell wall formation also starts when the zygotes settle. Observations indicate disintegration of one of the 2 chloroplasts in the young zygote. Few studies have been made of the ultrastructure of the fertilization process in algae. The first study of this kind was done by Manton & Friedmann (i960) on the anisogamous green alga Prasiola stipitata. In spite of the limited techniques available at that time, the work of Manton & Friedmann furnished a wealth of information about this important phase of the algal life-cycle. Subsequent publications have dealt with the fine structure of the fertilization of Chlamydomonas moewusii (Brown, Johnson & Bold, 1968) and C. reinhardii (Friedmann, Colwin & Colwin, 1968). Crawley (1966) describes the ultrastructure of the zygote of Acetabularia, but due to technical difficulties he was not able to observe the early stages of copulating gametes. No ultrastructural studies have been published on the fertilization process in Ulva, but Levring (1955) gives a short account of the process in Ulva lactuca as seen in the light microscope. The details of the fertilization process in algae, as observed with the aid of the electron microscope, show great variations even in closely related species. Thus the process in Chlamydomonas reinhardii as described by Friedmann et al. (1968) differs considerably from that of C. moewusii (Brown et al. 1968). In C. reinhardii the first cytoplasmic contact is made via a fertilization tubule possessed by the plus gamete. This species thus turns out to be anisogamous. In C. moewusii it was found that successful cytoplasmic contact depended on a precisely timed formation of the so-called plasma papilla in both sexes. The fate of the flagella after fertilization also differs in algae. In Chlamydomonas theflagellaare abscissed from the zygote (Brown et al. 1968), while in Prasiola the male flagella are taken into the zygote (Manton & Friedmann, i960). In the present investigation study of the behaviour of the flagella during the fertilization process with the aid of the scanning electron microscope was found to be most Observations reported in this paper were made on the mutant strain ' slender' of the green alga Ulva mutabilh Foyn. No morphological differences can be found between the gametes of this mutant and those of the wild type. Algae were grown in a light-dark cycle of 17 h light and 7 h dark at 19 C in Nordby's modification of Provasoli's medium (Levlie, 1969). Almost synchronous release of gametes was obtained by changing to fresh medium in a culture of mature gametophytes. Large numbers of gametes could then be harvested on the side of the culture vessel exposed to the light. Copulation was achieved by mixing approximately equal numbers of plus and minus gametes, which were then fixed at intervals ranging from a few seconds to several days after copulation. The initial stages were fixed while still motile and kept in suspension throughout the preparation procedure. Zygotes older than 10 min to be studied in the transmission electron microscope were allowed to settle on agar before being fixed. Zygotes to be studied in the scanning electron microscope were made to settle on platinum disks. The following stock solution of fixative was used: 4% glutaraldehyde, 4% formaldehyde (prepared from paraformaldehyde as described by Robertson, Bodenheimer & Stage, 1963) and 4% polyvinylpyrrolidone (PVP, Taab Laboratories, Reading, England; approx. mol. wt. 40000) in 0-2 M cacodylate buffer. This stock solution was diluted with an equal volume of seawater before use. The total osmolarity of the fixative was approximately 1300 m-osmol, which is slightly hypertonic to seawater. For transmission electron microscopy the material was fixed for 35 h and then rinsed for 15 min in a solution composed of equal volumes of seawater and 0-2 M cacodylate buffer containing 4% PVP. Postfixation was carried out overnight in 2 % OsO4 made up in 0-2 M cacodylate buffer/seawater 1:1. All solutions had a pH of 7-5 and the fixation and rinsing were carried out at room temperature. This was also the case for the subsequent dehydration in graded ethanols followed by propylene oxide before the final embedding in Araldite (Fluka). Silvery sections were cut with a diamond or glass knife and double-stained in uranyl acetate and lead citrate, using the method described by Venable & Coggeshall (1965). For scanning electron microscopy the same fixation procedures were performed except that the initial fixation was extended to 24 h. After postfixation in OsO4 the specimens were rinsed several times in 30% acetone and transferred to a desiccator, where they were placed over 100% acetone in a slight vacuum. The desiccator also contained CaCl, as a water absorbent (Sitte, 1962). The specimens were left overnight in the desiccator, then air-dried and coated with a thin film (approx. 30 nm) of gold/palladium. Ulva mutabilis is isogamous and even at the ultrastructural level no morphological differences can be found between the 2 sexes. The internal morphology of an Ulva gamete as it appears in a median longitudinal section is shown in Fig. 2. The mixing of plus and minus gametes results in the formation of clusters held together by the tips of their flagella (Fig. 3). The clusters break up almost immediately into mating pairs, still held together by the flagella (Fig. 4). Direct cytoplasmic contact is, however, established between the cell bodies within a few seconds of the onset of copulation. The flagella then separate from each other and the mating pair swims away from the light source. The initial cytoplasmic contact between the 2 gametes takes place at the anterior end of the gamete slightly posterior to the flagellar base (Fig. 5). No specific organelle can be found to be associated with the first cytoplasmic contact. The plasma membranes of the copulating gametes simply fuse at the point of contact. The fusion of the 2 gametes is a rapid process and stages like the one shown in Fig. 6 can be found only 30 s after the initial mixing of the 2 sexes. Increasingly larger areas of the 2 gametes come into contact as the two fold together in a jack-knife manner; 3 min after the onset of copulation the cytoplasm of the 2 g (...truncated)