Fine-Structural Studies of the Gametes and Embryo of Fucus Vesiculosus L. (Phaeophyta)

J. Cell Set. zo, 233-254 (1976) 233 Printed in Great Britain

FINE-STRUCTURAL STUDIES OF THE GAMETES AND EMBRYO OF FUCUS VESICULOSUS L. (PHAEOPHYTA)

I. FERTILIZATION AND PRONUCLEAR FUSION*

SUSAN H. BRAWLEY Department of Botany, University of California, Berkeley, California 94720, U.S.A.

RICHARD WETHERBEE School of Botany, University of Melbourne, Parkville 3052, Victoria, Australia AND RALPH S. QUATRANO Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331. U.S.A., AND The Marine Biological Laboratory, Woods Hole, Massachusetts 02543, U.S.A.

SUMMARY In the marine brown alga, Fucus vesiculosus L., the sperm pronucleus is delimited by an envelope following penetration of the egg by the sperm. This envelope disintegrates as the pronucleus begins its migration through the cytoplasm of the egg. The highly condensed chromatin of the sperm pronucleus disperses slightly following disintegration of the envelope. Microtubules of unknown origin are associated with the sperm pronucleus during its migration. The flagellar microtubules remain in the peripheral cytoplasm but lose their tight 9 + 2 configuration. The sperm eyespot and mitochondria follow the pronucleus through the cytoplasm toward the egg pronucleus. The mitochondria of the sperm are distinguished from those of the egg by their longitudinally oriented cristae and by electron-opaque material in the intra- cri8tal space. The pronucleus of the egg becomes convoluted along the surface nearest to the advancing sperm pronucleus. Immediately prior to pronuclear fusion, many egg mitochondria aggregate in the vicinity of the sperm pronucleus. At this time, only the portion of the sperm pronucleus facing the egg pronucleus is surrounded by an envelope. The egg mitochondria disperse rapidly after pronuclear fusion. The sperm mitochondria and eyespot are still in the perinuclear region in 16-h-old embryos. At this time, the osmiophilia of the sperm eyespot has increased, and the sperm mitochondrial membranes are less distinct than in earlier stages. The fine-structural features of fertilization in Fucus are discussed in relation to the fertilization patterns in other cryptogams and marine invertebrates and to polar axis determination in the Fucaceae.

• Portions of these Results have been presented at the Annual Meetings of the Phycological Society of America (1974) and (1975).

15 234 S- H. Brawley and others

INTRODUCTION Fine-structural analysis of fertilization in algae has been limited primarily to the green algae. The genera represented by these studies have a variety of gamete types: non-flagellated gametes (Fowke & Pickett-Heaps, 1971; Pickett-Heaps & Fowke, 1971); isogamous, flagellated gametes (Crawley, 1966; Brown, Johnson & Bold, 1968; Friedmann, Colwin & Colwin, 1968; Braten, 1971; Marchant & Pickett-Heaps, 1972); slightly anisogamous, flagellated gametes (Urban, 1969); and oogamous gametes in which the egg is non-motile and the sperm has 2 anterior flagella (Manton & Friedmann, i960) or many flagella (Hoffman, 1973 a, b, 1974). Fertilization in the marine brown alga, Fucus vesiculosus L., is definitely oogamous, resulting from the union of a large (70 /tm), non-motile egg and a small (6/tm), laterally biflagellated sperm. It occurs outside the parent plant in the open sea. These characteristics led us to ask whether fertilization in Fucus more closely resembles fertilization in marine invertebrates with gametes similar to those of Fucus than fertilization in the green algae, which are taxonomically closer to Fucus. Fine-structural studies of fertilization have been reported in the annelid, Hydroides (Colwin & Colwin, 1961), the sea urchin, Arbacia (e.g., Longo & Anderson, 1968), the lamellibranch, Mytilus (Longo & Anderson, 1969), and the surf clam, Spisula (Longo & Anderson, 1970).

MATERIALS AND METHODS Collection and culture Plants of the dioecious alga, Fucus vesiculosus L., were collected during July, 1973, at Manomet, Massachusetts, and were prepared for microscopic examination at the Marine Biological Laboratory at Woods Hole (Mass.). The release of gametes, fertilization, and culture of synchronously developing zygotes were induced as described by Quatrano (1974). Microscopic procedures At 5-min intervals between o and 30 min after mixture of eggs and sperm, zygotes were removed and fixed for light and electron microscopy in glutaraldehyde-paraformaldehyde followed by osmium tetroxide as described by Wetherbee & Wynne (1973). Zygotes were collected by gentle centrifugation after each step of the fixation and dehydration procedures, and they were embedded in Epon (Luft, 1961). Silver sections were cut with a Porter-Blum MT-2 ultramicrotome using glass knives, mounted on bare copper grids, and stained for 20 min in uranyl acetate and for 1 min in lead citrate (Venable & Coggeshall, 1965). Glass knives were used because Fucus embry°s damage diamond knives. Sections were viewed in a Siemens Elmiskop IA operating at 80 kV. One-micrometre sections were stained with toluidine blue O for light microscopy.

RESULTS Early penetrat: m of the egg by the sperm In the free-swimming sperm, the chromatin of the sperm pronucleus is highly condensed, and it is surrounded by a nuclear envelope in which nuclear pores are not observed (Fig. 1). The mitochondria of the sperm are distinguishable from those of the egg by electron-opaque material in the intracristal space of the sperm Fertilization in Fucus 235 mitochondrion (Figs. 1-3) and by the longitudinal orientation of the cristae of the sperm mitochondrion. When mixed with eggs, many sperm are attracted to each egg by a pheromonal mechanism (Cook & Elvidge, 1951; Miiller, 1973). After attachment to the egg surface, the beating of the sperm flagella causes the egg to rotate for several minutes. An egg fixed 10 min following gametic mixture shows the sperm within the peripheral egg cytoplasm (Fig. 2). The sperm pronucleus is still surrounded by a nuclear envelope shortly after penetration (Figs. 2, 4). Egg mitochondria are observed close to the sperm mitochondria (Fig. 2). Flagellar microtubules in the 9 + 2 configuration are associated closely with the other sperm organelles in the egg cytoplasm. These flagellar profiles are not membrane bound (Figs. 2, 6).

Fate of the sperm organelles during pronuclear migration The eyespot and mitochondria of the sperm trail the pronucleus but are still closely associated with it as they move deeper into the egg cytoplasm toward the egg pronucleus (Figs. 5, 7). The pronucleus of the sperm is no longer delimited by an envelope (Figs. 5, 7). Microtubules are common in the cytoplasmic matrix close to the sperm pronucleus as it moves toward the egg pronucleus (Figs. 5, 7, 8). The microtubules close to the sperm pronucleus are oriented in several groups with respect to its longitudinal axis (Fig. 7). These microtubules do not appear to be the flagellar microtubules which remain close to the surface of the egg after movement of the other sperm organelles toward the egg pronucleus (Fig. 6). Pronuclear fusion As the sperm pronucleus approaches the egg pronucleus, the contour of the pronuclear envelope of the egg becomes highly convoluted on the side closest to the approaching pronucleus of the sperm, while the surface away from the sperm pronucleus is smooth (Figs. 9, 11). The envelope of the egg pronucleus has well defined pores throughout its surface (Fig. 9). Numerous egg mitochondria are asymmetrically distributed around the egg pronucleus, being concentrated around the sperm pronucleus (Figs. 9, 11, 12). The sperm pronucleus is easily distinguished from the egg pronucleus at this stage because of its much larger amount of heterochromatin. The portion of the sperm pronucleus closest to the egg pronucleus has regenerated a nuclear envelope, while the surface closest to the plasmalemma is not yet membrane-bound (Fig. 10). Elements of endoplasmic reticulum are common near the pronuclei (Fig. 10). Following the fusion of the sperm and egg pronuclei (Fig. 13), the asymmetrically concentrated mitochondria disperse. Microtubules are observed near the 'sperm chromatin' of the zygote (Fig. 14). Portions of the envelope which enclose the protruding 'sperm chromatin' do not have clearly defined pores as does the 'egg pronuclear membrane' (Fig. 14). In 16-h-old embryos, the sperm mitochondria and eyespot are still present in the perinuclear region (Fig. 15). The osmiophilia of the eyespot pigments increases con- siderably between o and 16 h while the sperm mitochondrial membranes are not as clearly delineated at the end of this period.

15-2 236 S. H. Brawley and others

DISCUSSION Pronuclear morphogenesis The nuclear envelope of the sperm in Fucus was shown to disintegrate after gametic fusion in a fashion similar to that in a number of vertebrates and invertebrates (Bedford, 1972; Longo & Anderson, 1968, 1969, 1970; Yanagimachi & Noda, 1970). However, in the nematode, Ascaris (Foor, 1968), the cryptogams, Marsilea (Myles, 1973) and Pteridium (Bell, 1975), and in various algae (Brown et al. 1968; Friedmann et al. 1968; Urban, 1969; Braten, 1971; Fowke & Pickett-Heaps, 1971; Pickett-Heaps & Fowke, 1971; Marchant & Pickett-Heaps, 1972; Hoffman, 1973a, 1974) no such breakdown of the sperm nuclear envelope following syngamy has been described. In Ascaris (Foor, 1968), Marsilea (Myles, 1973) and Pteridium (Bell, 1975) the chromatin of the free-swimming sperm is not envelope-bound; however, in Marsilea and Pteridium a pronuclear envelope forms around the sperm chromatin after gametic fusion. The initial generation of a new nuclear envelope on only the surface of the sperm pronucleus facing the egg pronucleus represents a unique feature of fertilization in Fucus. The accumulation of elements of endoplasmic reticulum around the pronuclei shortly before pronuclei fusion suggests that they are responsible for the new portion of the sperm pronuclear envelope in Fucus. Endoplasmic reticulum is important in the formation of pronuclear envelopes in rabbits (Zamboni & Mastroianni, 1966) and Marsilea (Myles, 1973). The sperm and egg nuclear envelopes of most algae studied possess nuclear pores (Manton & Friedmann, i960; Crawley, 1966; Friedmann et al. 1968; Brown et al. 1968; Fowke & Pickett-Heaps, 1971; Pickett-Heaps & Fowke, 1971; Marchant & Pickett-Heaps, 1972; Hoffman, 1973 a). Of the algal gametes described to date, only Bryopsis gametes lack nuclear pores (Urban, 1969). Longo & Anderson (1968, 1969, 1970) found that sperm pronuclear envelopes also possess nuclear pores prior to pronuclear fusion in the invertebrates which they studied, but they did not detect pores in the nuclei of the free-swimming sperm. In Fucus we observed nuclear pores in the eggs but not in the free-swimming sperm or in the initial portion of the envelope generated by the sperm pronucleus just before pronuclear fusion. The high degree of convolution of the Fucus egg pronucleus, localized in the zone facing the advancing sperm pronucleus, resembles that observed in the invertebrates, Arbacia (Longo & Anderson, 1968) and Mytilus (Longo & Anderson, 1969), and in the cryptogams, Marsilea (Myles, 1973) and Pteridium (Bell, 1975). This is in contrast, however, to most algae. For example, the egg of the green alga, Oedogonium, whose degree of anisogamy approaches that of Fucus but which has an internal mode of fertilization, does not become convoluted along the nuclear surface (Hoffman, 1973 a, 1974). The absence of egg pronuclear convolution in Oedogonium when compared with marine invertebrates, Marsilea, Pteridium and Fucus may be related to the higher euchromatin: heterochromatin ratio of the sperm pronucleus in Oedogonium prior to gametic fusion as well as to its internal rather than external mode of fertilization. Fertilization in Fucus 237

Microtubules associated with the pronucleus The importance of microtubules in effecting pronuclear fusion has been demon- strated by high pressure and colcemid treatment of Arbacia eggs (Zimmerman & Silberman, 1964; Zimmerman & Zimmerman, 1967). Microtubules are present between the gametic nuclei in Oedogonium prior to fusion (Hoffman, 1974). The origin of the microtubules observed in association with the sperm pronucleus following fertilization in Fucus eggs is not clear. They may be from the sperm cytoplasm where Bouck (1970) found microtubules associated with the eyespot. Another source of the microtubules may be the flagella of the sperm. In Marsilea (Myles, 1973) and Mytilus (Longo & Anderson, 1969), the 9 + 2 configuration of the axoneme can be observed immediately after gametic fusion but it is lost a short time later. However, in some invertebrates (e.g. Arbacia) the flagella migrate with the other sperm organelles through the egg cytoplasm (Longo & Anderson, 1968). Finally, the microtubules associated with the sperm pronucleus may originate from the egg. Micro- tubules are rarely encountered in the cytoplasm of the mature egg or zygote until mitosis occurs at about 16 h after fertilization (Brawley, Wetherbee & Quatrano, 1976 a, b).

The role of sperm and egg mitochondria in zygote formation The sperm mitochondria degenerate by 16 h after fertilization in Fucus. Similar mitochondrial degeneration occurs in a variety of animals and cryptogams (Szollosi, 1965; Stefanini, Oura & Zamboni, 1969; Longo & Anderson, 1969; Myles, 1973). Although egg mitochondria are present close to the sperm mitochondria and other organelles, we did not observe the direct apposition preceding the degeneration of sperm mitochondria which Anderson (1968) found in the sea urchin, Paracentrotus. The localization of electron-opaque material in the intracristal space of Fucus sperm mitochondria differs from that observed in liver (Behnke, 1965; Svoboda & Higginson, 1964; Takada, Porta & Hartroft, 1966; Bart6k, Viragh & Menyhdrt, 1973), in ameloblasts (Jessen, 1968), in neuroglial cells (Mugnaini, 1963) and in skeletal muscle (Bonilla, Schotland, DiMauro & Aldover, 1975) in that, in Fucus sperm, all cristae possess the material rather than only a few cristae as in the animal cells. Also, no periodic pattern or helical filaments were found in the intracristal material of Fucus sperm. Manton & Clarke (1956) found no distinguishing features between the mitochondria of the egg and sperm in Fucus. Their figure (no. 24) does show the opaque material in the sperm cristae, although due to the preparative methods then available, the mitochondria were not well fixed. Biochemical studies of Fucus sperm mitochondria are needed to determine whether this morphological property is related to a specific physiological function of the sperm. The asymmetrical distribution of egg mitochondria in Fucus just prior to pronuclear fusion is quite unique, and it may be related to the orientation of the polar axis, since it reflects the site of sperm penetration which can orient the polar axis in the absence of any external gradient (Knapp, 1931; Quatrano, 1974). The observation of mitochondrial concentration just before pronuclear fusion is also interesting in 238 S. H. Brawley and others light of the events which occur in the perinuclear region at the time of polar axis formation approximately 12 h after fertilization. Quatrano (1972) found that the first sign of polarity is an accumulation of mitochondria in the perinuclear region closest to the site of rhizoid formation. These mitochondria were aligned with their longitudinal axes parallel to the plane of polarity. No such accumulation was detected between 1 and 12 h. If one assumes that the site of sperm penetration is marked at 16 h by the presence of the sperm mitochondria and eyespot in the perinuclear region, one should be able to associate this cytological marker with the site of mitochondrial localization during polar axis determination in zygotes grown in the absence of any external gradient.

Fertilization pattern Important features of fertilization in Fucus which contrast sharply with those in other algae include the pattern of highly condensed chromatin in the sperm nucleus, the breakdown and reformation of an envelope around the sperm chromatin, and the asymmetric convolution of the egg pronuclear envelope. These characteristics ally Fucus with representatives of several invertebrate phyla and with the cryptogams, Pteridium and Marsilea. Favard & Andre (1970) have commented that, 'the characteristics of sperm mitochondria are less an image of the Systematics than that of the fertilization procedures. Sperm are very highly specialized cells for which survival in the medium, where they are released or stored, and potency to reach the egg have played the main role in their modelling during evolution'. Their comments seem to apply equally well to the features of fertilization in Fucus which more closely resemble those of Marsilea, Pteridium, Mytilus, Arbacia and Spisula than those of other algae. A fertilization pattern of adaptive significance to oogamous gametes - particularly to those of marine organisms with external fertilization - seems to be emerging. It contrasts with features of fertilization found in flagellated, isogamous and non- flagellated, algal gametes, and it is clearly independent of taxonomic relationships.

For their stimulating discussions throughout this work as well as for their review of the manuscript, we wish to express our appreciation to Dr Darlene Southworth (Berkeley), Dr John A. West (Berkeley), Dr Helen A. Padykula (Wellesley College) and Eleanor Crump (Berkeley). We thank Dr Mary M. Allen (Wellesley College) and Dr Padykula for the use of their laboratories during a portion of this work and Marea H. Grant (Berkeley) for proofreading the manuscript. Our work was supported by a Sigma Xi Grant-in-Aid-of-Research (to SHB) and by grants (to RSQ) from the NSF (GB37149) and PHS (GM19247). Dr Wetherbee was the recipient of a postdoctoral appointment with Dr West (NSF GB40550) during a portion of this study.

REFERENCES ANDERSON, W. A. (1968). Structure and fate of the paternal mitochondrion during early embryogenesis of Paracentrotus lividus.J. Ultrastruct. Res. 24, 311-321. BART6K, I., VIRACH, Sz. & MENYHART, J. (1973). Prompt divisions and peculiar transformation of cristae in liver mitochondria of rats rehydrated after prolonged water deprivation. J. Ultrastruct. Res. 44, 49-51. BEDFORD, J. M. (1972). An electron microscope study of sperm penetration into the rabbit egg after natural mating. Am. J. Anat. 133, 213-254. Fertilization in Fucus 239 BEHNKE, O. (1965). Helical filaments in rat liver mitochondria. Expl Cell Res. 37, 687-689. BELL, P. R. (1975). Observations on the male nucleus during fertilization in the fern Pteridium aquilinum. J. Cell Sci. 17, 141-154. BONILLA, E., SCHOTLAND, D. L., DIMAURO, S. & ALDOVER, B. (1975). Electron cytochemistry of crystalline inclusions in human skeletal muscle mitochondria. J. Ultrastruct. Res. 51, 404-408. BOUCK, G. B. (1970). The development and postfertilization fate of the eyespot and the apparent photoreceptor in Fucus sperm. Ann. N.Y. Acad. Sci. 175, 673-685. BRATEN, T. (1971). The ultrastructure of fertilization and zygote formation in the green alga, Ulva mutabilis Fayn.J. Cell Sci. 9, 621-635. BRAWLEY, S. H., WETHERBEE, R. & QUATRANO, R. S. (1976a). Fine-structural studies of the gametes and embryo of Fucus vesiculosus L. (Phaeophyta). II. The cytoplasm of the egg and young zygote. J. Cell Sci. 20, 255-271. BRAWLEY, S. H., WETHERBEE, R. & QUATRANO, R. S. (19766). Fine-structural studies of the gametes and embryo of Fucus vesiculosus (Phaeophyta). III. Cell division. J. Cell Sci. (submitted). BROWN, R. M., JR., JOHNSON, C. & BOLD, H. C. (1968). Electron and phase contrast microscopy of sexual reproduction in Chlamydomonas moezvusii. J. Phycol. 4, 100—120. COLWIN, A. L. & COLWIN, L. H. (1961). Changes in the spermatozoon during fertilization in Hydroides hexagonus (Annelida). II. Incorporation with the egg. J. biophys. biochem. Cytol. 10, 255-274. COOK, H. A. & ELVIDGE, J. A. (1951). Fertilization in the Fucaceae: investigations in the nature of the chemotactic substance produced by eggs of Fucus serratus and Fucus vesiculosus. Proc. R. Soc. B 138, 97-114. CRAWLEY, J. C. W. (1966). Some observations on the fine structure of the gametes and zygotes of Acetabidaria. Planta 69, 363-376. FAVARD, P. & ANDRE, J. (1970). The mitochondria of spermatozoa. In Comparative Sperma- tology (ed. B. Baccetti), pp. 415-430. New York and London: Academic Press. FOOR, W. E. (1968). Zygote formation in Ascaris lumbricoides (Nematoda). J. Cell Biol. 39, H9-I34- FOWKE, L. C. & PICKETT-HEAPS, J. D. (1971). Conjugation in Spirogyra.J. Phycol. 7, 285-294. FRIEDMANN, I., COLWIN, A. L. & COLWIN, L. H. (1968). Fine structural aspects of fertilization in Chlamydomonas reinhardi. J. Cell Sci. 3, 115-128. HOFFMAN, L. R. (1973 a). Fertilization in Oedogonium. I. Plasmogamy. J. Phycol. 9, 62-84. HOFFMAN, L. R. (19736). Fertilization in Oedogonium. II. Polyspermy. J. Phycol. 9, 296-301. HOFFMAN, L. R. (1974). Fertilization in Oedogonium. III. Karyogamy. Am.J.Bot.61,1076-1090. JESSEN, H. (1968). The morphology and distribution of mitochondria in ameloblasts with special reference to a helix-containing type. J'. Ultrastruct. Res. 22, 120-135. KNAPP, E. (1931). Entwicklungsphysiologische Untersuchungen an Fucaceen Eiern. Planta 14,

LONGO, F. J. & ANDERSON, E. (1968). The fine structure of pronuclear development and fusion in the sea urchin, Arbacia punctulata. jf. Cell Biol. 39, 339-368. LONGO, F. J. & ANDERSON, E. (1969). Cytological aspects of fertilization in the lamellibranch, Mytilus edulis. II. Development of the male pronucleus and the association of the maternally and paternally derived chromosomes. J. exp. Zool. 172, 97-120. LONGO, F. J. & ANDERSON, E. (1970). An ultrastructural analysis of fertilization in the surf clam, Spisula solidissima. II. Development of the male pronucleus and the association of the maternally and paternally derived chromosomes. J. Ultrastruct. Res. 33, 515-527. LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. biophys. biochem. Cytol. 9, 409-414. MANTON, I. & CLARKE, B. (1956). Observations with the electron microscope on the internal structure of the spermatozoid of Fucus. J. exp. Bot. 7, 416—432. MANTON, I. & FRIEDMANN, I. (i960). Gametes, fertilization, and zygote formation in Prasiola stipitata Suhr. II. Electron microscopy. Nova Hedwegia 1, 443-462. MARCHANT, H. J. & PICKETT-HEAPS, J. D. (1972). Ultrastructure and differentiation of Hydro- dictyon reticulatum. IV. Conjugation of gametes and the development of zygospores and azygospores. Aust.J. biol. Sci. 25, 279-291. 240 S. H. Brawley and others MUGNAINI, E. (1963). Helical filaments in mitochondria of neuroglial cells in the rat corpus striatum. J. Ultrastruct. Res. 9, 398 (Abstr.). MOLLER, D. G. (1973). Fucoserraten, the female sex attractant otFucus serratus L. (Phaeophyta). FEBS Letters, Amsterdam 30, 137-139. MYLES, D. G. (1973). The Ultrastructure of Fertilization in Marsilea vestita. Ph.D. Dissertation in Botany, University of California (Berkeley). PICKETT-HEAPS, J. D. & FOWKE, L. C. (1971). Conjugation in the desmid, Closterium littorale. J. Phycol. 7, 37-50- QUATRANO, R. S. (1972). An ultrastnictural study of the determined site of rhizoid formation in Fucus zygotes. Expl Cell Res. 70, 1-12. QUATRANO R. S. (1974). Developmental biology: development in marine organisms. In Experimental Marine Biology (ed. R. Mariscal), pp. 303-346. New York and London: Academic Press. STEFANLNI, M., OURA, C. & ZAMBONI, L. (1969). Ultrastructure of fertilization in the mouse. II. Penetration of sperm into the ovum. J. submicrosc. Cytol. 1, 1-24. SVOBODA, D. J. & HIGGINSON, J. (1964). Ultrastructural changes produced by protein and related deficiencies in the rat liver. Am. J. Path. 45, 353-379. SZOLLOSI, D. G. (1965). The fate of sperm middle-piece mitochondria in the rat egg. J. exp. Zool. 159, 367-378. TAKADA, A., PORTA, F. A. & HARTROFT, W. S. (1966). Correlation of structural and functional recovery from cirrhosis in rats treated with lipotropic diets. Am. J. Path. 49, 841-869. URBAN, P. (1969). The fine structure of pronuclear fusion in the coenocytic marine alga, Bryopsis hypnoides Lamouroux. J. Cell Biol. 42, 606—611. VENABLE, J. H. & COGGESHALL, R. (1965). A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25, 407-408. WETHERBEE, R. & WYNNE, M. J. (1973). The fine structure of the nucleus and nuclear associ- ations of developing carposporangia in Polysiphonia novae-angliae (Rhodophyta). J. Phycol. 9, 402-407. YANAGIMACHI, R. & NODA, Y. D. (1970). Electron microscope studies of sperm incorporation into the golden hamster egg. Am. J. Anat. 138, 429-462. ZAMBONI, L. & MASTROIANNI, L., JR. (1966). Electron microscope studies on rabbit ova. II. The penetrated tubal ovum. J. Ultrastruct. Res. 14, 118—132. ZIMMERMAN, A. M. & SILBERMAN, L. (1964). Further studies on incorporation of H3-thymidine in Arbacia eggs under hydrostatic pressure. (Abstr.) Biol. Bull. mar. biol. Lab., Woods Hole 127, 355- ZIMMERMAN, A. M. & ZIMMERMAN, S. (1967). Action of colcemid in sea urchin eggs. J. Cell Biol. 34, 483-488. {Received 15 April 1975) Fertilization in Fucus 241

Fig. 1. The sperm (

Fig. 2. Sperm organelles within the egg cytoplasm immediately following fertilization. Egg mitochondria (?) do not contain intracristal material, and the orientation of the cristae with regard to the axis of the organelle is different from that of the sperm mitochondria (

Fig. 5. The sperm pronucleus (n) trailed by the eyespot (e) and sperm mitochondria (m) as it advances through the egg cytoplasm toward the egg pronucleus (which is located to the right of the micrograph). Microrubules (arrow) are associated with the sperm organelles. The sperm chromatin is not envelope-bound but little dis- persion occurs, x 29800. Fig. 6. A section serial to Fig. 5 (but 15 fim away from the area shown in Fig. 5) showing axonemal microtubules (arrow). These remain close to the plasmalemma when the other sperm organelles have advanced deeper into the egg cytoplasm but lose their tight 9 + 2 configuration. Note wall formation {w). x 29 800. Fertilization in Fucus 246 S. H. Brawley and others

Fig. 7. Sperm organelles advancing through the egg cytoplasm. The pronucleus (n) is not envelope-bound. Microtubules (arrows) occur in several regions of matrix close to the sperm organelles. e, eyespot. Sperm mitochondria (

Fig. 9. The sperm pronucleus ((J) and egg pronucleus (?) prior to fusion showing the asymmetric concentration of egg mitochondria (m) around the sperm pronucleus and the localized convolution of the egg pronucleus toward the sperm pronucleus. x 9 900. Fig. 10. The pronuclei of Fig. 9. The euchromatin of the egg pronucleus contrasts with the heterochromatin of the sperm pronucleus. The portion of the sperm pronucleus facing the egg pronucleus is membrane-bound (arrows). Endoplasmic reticulum (er) is common in the cytoplasmic matrix, x 35000. Fertilization in Fucus 249 250 S. H. Brawley and others

Fig. 11. The egg pronucleus ($) and the asymmetric accumulation of egg mitochondria. This is a serial section of the material in Fig. 9. The sperm pronucleus is close to the location marked (X). x 6500. Fig. 12. Toluidine blue O-stained section, serial to Figs. 9 and 11, illustrating the pronuclei (arrows). Note mitochondrial asymmetry, x 650. Fertilization in Fucus 351

16-2 S. H. Brawley and others

Fig. 13. The pronuclei following fusion. The asymmetrically concentrated mitochondria disperse rapidly. Nucleoli (n[) are present in the 'egg pronucleus'. x 4000. Fertilization in Fucus 253 K-

Fig. 14. The egg and sperm chromatin following fusion of the pronuclei. Microtubules (arrows) remain close to the 'sperm pronucleus'. Nuclear pores (x) are present in the 'egg pronuclear membrane', x 36000. 254 S. H. Brawley and others

Fig. 15. The sperm eyespot (e) and mitochondria (arrows) in a 16-h-old embryo just before division. Note the increased osmiophilia of the eyespot and the less distinct membranes of the mitochondria, n, .zygotic .nucleus, x 74000.