fine-structural studies of the gametes and embryo …j. cell set. zo, 233-254 (1976 23) 3 printed in...

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. BRAWLEYDepartment 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. QUATRANODepartment 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.

SUMMARYIn 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 thepronucleus begins its migration through the cytoplasm of the egg. The highly condensedchromatin 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 con-figuration. The sperm eyespot and mitochondria follow the pronucleus through the cytoplasmtoward the egg pronucleus. The mitochondria of the sperm are distinguished from those ofthe 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 theadvancing sperm pronucleus. Immediately prior to pronuclear fusion, many egg mitochondriaaggregate in the vicinity of the sperm pronucleus. At this time, only the portion of the spermpronucleus facing the egg pronucleus is surrounded by an envelope. The egg mitochondriadisperse rapidly after pronuclear fusion. The sperm mitochondria and eyespot are still in theperinuclear region in 16-h-old embryos. At this time, the osmiophilia of the sperm eyespothas 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 fertilizationpatterns in other cryptogams and marine invertebrates and to polar axis determination in theFucaceae.

• Portions of these Results have been presented at the Annual Meetings of the PhycologicalSociety 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 thegreen 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 inwhich 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 themarine brown alga, Fucus vesiculosus L., is definitely oogamous, resulting from theunion of a large (70 /tm), non-motile egg and a small (6/tm), laterally biflagellatedsperm. It occurs outside the parent plant in the open sea. These characteristics ledus to ask whether fertilization in Fucus more closely resembles fertilization in marineinvertebrates with gametes similar to those of Fucus than fertilization in the greenalgae, which are taxonomically closer to Fucus. Fine-structural studies of fertilizationhave been reported in the annelid, Hydroides (Colwin & Colwin, 1961), the seaurchin, 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 culturePlants of the dioecious alga, Fucus vesiculosus L., were collected during July, 1973, at

Manomet, Massachusetts, and were prepared for microscopic examination at the MarineBiological Laboratory at Woods Hole (Mass.). The release of gametes, fertilization, andculture 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 wereremoved and fixed for light and electron microscopy in glutaraldehyde-paraformaldehydefollowed by osmium tetroxide as described by Wetherbee & Wynne (1973). Zygotes werecollected 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-BlumMT-2 ultramicrotome using glass knives, mounted on bare copper grids, and stained for 20 minin uranyl acetate and for 1 min in lead citrate (Venable & Coggeshall, 1965). Glass knives wereused because Fucus embry°s damage diamond knives. Sections were viewed in a SiemensElmiskop IA operating at 80 kV. One-micrometre sections were stained with toluidine blue Ofor light microscopy.

RESULTS

Early penetrat: m of the egg by the sperm

In the free-swimming sperm, the chromatin of the sperm pronucleus is highlycondensed, and it is surrounded by a nuclear envelope in which nuclear pores arenot observed (Fig. 1). The mitochondria of the sperm are distinguishable from thoseof 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 thesperm mitochondrion.

When mixed with eggs, many sperm are attracted to each egg by a pheromonalmechanism (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 eggfixed 10 min following gametic mixture shows the sperm within the peripheral eggcytoplasm (Fig. 2). The sperm pronucleus is still surrounded by a nuclear envelopeshortly after penetration (Figs. 2, 4). Egg mitochondria are observed close to thesperm mitochondria (Fig. 2). Flagellar microtubules in the 9 + 2 configuration areassociated closely with the other sperm organelles in the egg cytoplasm. Theseflagellar 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 stillclosely associated with it as they move deeper into the egg cytoplasm toward the eggpronucleus (Figs. 5, 7). The pronucleus of the sperm is no longer delimited by anenvelope (Figs. 5, 7). Microtubules are common in the cytoplasmic matrix close tothe sperm pronucleus as it moves toward the egg pronucleus (Figs. 5, 7, 8). Themicrotubules close to the sperm pronucleus are oriented in several groups withrespect to its longitudinal axis (Fig. 7). These microtubules do not appear to be theflagellar microtubules which remain close to the surface of the egg after movementof the other sperm organelles toward the egg pronucleus (Fig. 6).

Pronuclear fusion

As the sperm pronucleus approaches the egg pronucleus, the contour of thepronuclear envelope of the egg becomes highly convoluted on the side closest tothe approaching pronucleus of the sperm, while the surface away from the spermpronucleus is smooth (Figs. 9, 11). The envelope of the egg pronucleus has welldefined pores throughout its surface (Fig. 9). Numerous egg mitochondria areasymmetrically distributed around the egg pronucleus, being concentrated aroundthe sperm pronucleus (Figs. 9, 11, 12). The sperm pronucleus is easily distinguishedfrom the egg pronucleus at this stage because of its much larger amount ofheterochromatin. The portion of the sperm pronucleus closest to the egg pronucleushas regenerated a nuclear envelope, while the surface closest to the plasmalemmais not yet membrane-bound (Fig. 10). Elements of endoplasmic reticulum arecommon near the pronuclei (Fig. 10).

Following the fusion of the sperm and egg pronuclei (Fig. 13), the asymmetricallyconcentrated mitochondria disperse. Microtubules are observed near the 'spermchromatin' of the zygote (Fig. 14). Portions of the envelope which enclose theprotruding 'sperm chromatin' do not have clearly defined pores as does the 'eggpronuclear membrane' (Fig. 14).

In 16-h-old embryos, the sperm mitochondria and eyespot are still present in theperinuclear region (Fig. 15). The osmiophilia of the eyespot pigments increases con-siderably between o and 16 h while the sperm mitochondrial membranes are not asclearly delineated at the end of this period.

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236 S. H. Brawley and others

DISCUSSION

Pronuclear morphogenesis

The nuclear envelope of the sperm in Fucus was shown to disintegrate after gameticfusion 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; Friedmannet al. 1968; Urban, 1969; Braten, 1971; Fowke & Pickett-Heaps, 1971; Pickett-Heaps& Fowke, 1971; Marchant & Pickett-Heaps, 1972; Hoffman, 1973a, 1974) no suchbreakdown of the sperm nuclear envelope following syngamy has been described.In Ascaris (Foor, 1968), Marsilea (Myles, 1973) and Pteridium (Bell, 1975) thechromatin of the free-swimming sperm is not envelope-bound; however, in Marsileaand Pteridium a pronuclear envelope forms around the sperm chromatin after gameticfusion. The initial generation of a new nuclear envelope on only the surface of thesperm pronucleus facing the egg pronucleus represents a unique feature of fertilizationin Fucus. The accumulation of elements of endoplasmic reticulum around thepronuclei shortly before pronuclei fusion suggests that they are responsible for thenew portion of the sperm pronuclear envelope in Fucus. Endoplasmic reticulum isimportant 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, onlyBryopsis gametes lack nuclear pores (Urban, 1969). Longo & Anderson (1968, 1969,1970) found that sperm pronuclear envelopes also possess nuclear pores prior topronuclear fusion in the invertebrates which they studied, but they did not detectpores in the nuclei of the free-swimming sperm. In Fucus we observed nuclear poresin the eggs but not in the free-swimming sperm or in the initial portion of the envelopegenerated by the sperm pronucleus just before pronuclear fusion.

The high degree of convolution of the Fucus egg pronucleus, localized in the zonefacing the advancing sperm pronucleus, resembles that observed in the invertebrates,Arbacia (Longo & Anderson, 1968) and Mytilus (Longo & Anderson, 1969), and inthe cryptogams, Marsilea (Myles, 1973) and Pteridium (Bell, 1975). This is in con-trast, 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 modeof fertilization, does not become convoluted along the nuclear surface (Hoffman,1973 a, 1974). The absence of egg pronuclear convolution in Oedogonium whencompared with marine invertebrates, Marsilea, Pteridium and Fucus may be relatedto the higher euchromatin: heterochromatin ratio of the sperm pronucleus inOedogonium prior to gametic fusion as well as to its internal rather than external modeof fertilization.


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 presentbetween the gametic nuclei in Oedogonium prior to fusion (Hoffman, 1974). Theorigin of the microtubules observed in association with the sperm pronucleusfollowing fertilization in Fucus eggs is not clear. They may be from the sperm cyto-plasm where Bouck (1970) found microtubules associated with the eyespot. Anothersource 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 axonemecan 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 spermorganelles through the egg cytoplasm (Longo & Anderson, 1968). Finally, the micro-tubules associated with the sperm pronucleus may originate from the egg. Micro-tubules are rarely encountered in the cytoplasm of the mature egg or zygote untilmitosis 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. Similarmitochondrial 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 otherorganelles, we did not observe the direct apposition preceding the degeneration ofsperm mitochondria which Anderson (1968) found in the sea urchin, Paracentrotus.

The localization of electron-opaque material in the intracristal space of Fucussperm 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 inskeletal muscle (Bonilla, Schotland, DiMauro & Aldover, 1975) in that, in Fucussperm, all cristae possess the material rather than only a few cristae as in the animalcells. Also, no periodic pattern or helical filaments were found in the intracristalmaterial of Fucus sperm. Manton & Clarke (1956) found no distinguishing featuresbetween the mitochondria of the egg and sperm in Fucus. Their figure (no. 24) doesshow the opaque material in the sperm cristae, although due to the preparativemethods then available, the mitochondria were not well fixed. Biochemical studies ofFucus sperm mitochondria are needed to determine whether this morphologicalproperty is related to a specific physiological function of the sperm.

The asymmetrical distribution of egg mitochondria in Fucus just prior to pro-nuclear fusion is quite unique, and it may be related to the orientation of the polaraxis, since it reflects the site of sperm penetration which can orient the polar axis inthe absence of any external gradient (Knapp, 1931; Quatrano, 1974). The observationof mitochondrial concentration just before pronuclear fusion is also interesting in


light of the events which occur in the perinuclear region at the time of polar axisformation approximately 12 h after fertilization. Quatrano (1972) found that the firstsign of polarity is an accumulation of mitochondria in the perinuclear region closestto the site of rhizoid formation. These mitochondria were aligned with their longi-tudinal axes parallel to the plane of polarity. No such accumulation was detectedbetween 1 and 12 h. If one assumes that the site of sperm penetration is marked at16 h by the presence of the sperm mitochondria and eyespot in the perinuclearregion, one should be able to associate this cytological marker with the site ofmitochondrial localization during polar axis determination in zygotes grown in theabsence of any external gradient.

Fertilization pattern

Important features of fertilization in Fucus which contrast sharply with those inother algae include the pattern of highly condensed chromatin in the sperm nucleus,the breakdown and reformation of an envelope around the sperm chromatin, andthe asymmetric convolution of the egg pronuclear envelope. These characteristicsally Fucus with representatives of several invertebrate phyla and with the cryptogams,Pteridium and Marsilea. Favard & Andre (1970) have commented that, 'the charac-teristics of sperm mitochondria are less an image of the Systematics than that of thefertilization procedures. Sperm are very highly specialized cells for which survivalin the medium, where they are released or stored, and potency to reach the egg haveplayed the main role in their modelling during evolution'. Their comments seem toapply equally well to the features of fertilization in Fucus which more closely resemblethose of Marsilea, Pteridium, Mytilus, Arbacia and Spisula than those of other algae.A fertilization pattern of adaptive significance to oogamous gametes - particularly tothose of marine organisms with external fertilization - seems to be emerging. Itcontrasts 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 themanuscript, 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 oftheir laboratories during a portion of this work and Marea H. Grant (Berkeley) for proofreadingthe 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 wasthe recipient of a postdoctoral appointment with Dr West (NSF GB40550) during a portionof this study.

REFERENCES

ANDERSON, W. A. (1968). Structure and fate of the paternal mitochondrion during earlyembryogenesis of Paracentrotus lividus.J. Ultrastruct. Res. 24, 311-321.

BART6K, I., VIRACH, Sz. & MENYHART, J. (1973). Prompt divisions and peculiar transformationof 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 eggafter natural mating. Am. J. Anat. 133, 213-254.


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 theapparent 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 thegametes and embryo of Fucus vesiculosus L. (Phaeophyta). II. The cytoplasm of the egg andyoung zygote. J. Cell Sci. 20, 255-271.

BRAWLEY, S. H., WETHERBEE, R. & QUATRANO, R. S. (19766). Fine-structural studies of thegametes 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 micro-scopy of sexual reproduction in Chlamydomonas moezvusii. J. Phycol. 4, 100—120.

COLWIN, A. L. & COLWIN, L. H. (1961). Changes in the spermatozoon during fertilization inHydroides 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 natureof 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 zygotesof 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 andfusion 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 maternallyand paternally derived chromosomes. J. exp. Zool. 172, 97-120.

LONGO, F. J. & ANDERSON, E. (1970). An ultrastructural analysis of fertilization in the surfclam, Spisula solidissima. II. Development of the male pronucleus and the association of thematernally 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 internalstructure of the spermatozoid of Fucus. J. exp. Bot. 7, 416—432.

MANTON, I. & FRIEDMANN, I. (i960). Gametes, fertilization, and zygote formation in Prasiolastipitata 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 andazygospores. Aust.J. biol. Sci. 25, 279-291.


MUGNAINI, E. (1963). Helical filaments in mitochondria of neuroglial cells in the rat corpusstriatum. 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. Dissertationin 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 formationin Fucus zygotes. Expl Cell Res. 70, 1-12.

QUATRANO R. S. (1974). Developmental biology: development in marine organisms. InExperimental 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.

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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 functionalrecovery from cirrhosis in rats treated with lipotropic diets. Am. J. Path. 49, 841-869.

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ZIMMERMAN, A. M. & SILBERMAN, L. (1964). Further studies on incorporation of H3-thymidinein Arbacia eggs under hydrostatic pressure. (Abstr.) Biol. Bull. mar. biol. Lab., Woods Hole127, 355-

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{Received 15 April 1975)


1Fig. 1. The sperm (<J) and egg (?) prior to fertilization. One of the sperm flagella (/)is against the egg plasmalemma. e, eyespot; m, mitochondria, n, nucleus; p, spermproboscis, x 27000.


Fig. 2. Sperm organelles within the egg cytoplasm immediately following fertilization.Egg mitochondria (?) do not contain intracristal material, and the orientation of thecristae with regard to the axis of the organelle is different from that of the spermmitochondria (<J). The sperm pronucleus (n) is envelope-bound. Note the grazingsection of the eyespot (e) and the obliquely cut section of one of the sperm flagella(arrow). The axoneme is not membrane-bound, x 26400.

Fig. 3. Intracristal electron-opaque material within a sperm mitochondrion. Notethe orderly arrangement of the cristae as seen in cross-section, x 75700.

Fig. 4. The envelope-bound (arrow) chromatin of the sperm pronucleus (n) shown inFig. 2. The chromatin is highly condensed except for small areas of dispersion (x).x 63000.

244 &• H. Brawley and others

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 islocated to the right of the micrograph). Microrubules (arrow) are associated withthe 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) show-ing axonemal microtubules (arrow). These remain close to the plasmalemma when theother sperm organelles have advanced deeper into the egg cytoplasm but lose theirtight 9 + 2 configuration. Note wall formation {w). x 29 800.

Fertilization in Fucus


Fig. 7. Sperm organelles advancing through the egg cytoplasm. The pronucleus (n)is not envelope-bound. Microtubules (arrows) occur in several regions of matrix closeto the sperm organelles. e, eyespot. Sperm mitochondria (<J) are distinct from eggmitochondria (£). The thick arrow indicates the direction of the sperm pronuclearmovement toward the egg pronucleus. x 37000.

Fig. 8. Area marked (X) in Fig. 7 showing cross-sections of 2 microtubules. x 96000.


Fig. 9. The sperm pronucleus ((J) and egg pronucleus (?) prior to fusion showing theasymmetric concentration of egg mitochondria (m) around the sperm pronucleus andthe 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 contrastswith the heterochromatin of the sperm pronucleus. The portion of the sperm pro-nucleus facing the egg pronucleus is membrane-bound (arrows). Endoplasmicreticulum (er) is common in the cytoplasmic matrix, x 35000.


Fig. 11. The egg pronucleus ($) and the asymmetric accumulation of egg mito-chondria. This is a serial section of the material in Fig. 9. The sperm pronucleus isclose to the location marked (X). x 6500.

Fig. 12. Toluidine blue O-stained section, serial to Figs. 9 and 11, illustrating the pro-nuclei (arrows). Note mitochondrial asymmetry, x 650.


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S. H. Brawley and others

Fig. 13. The pronuclei following fusion. The asymmetrically concentrated mito-chondria disperse rapidly. Nucleoli (n[) are present in the 'egg pronucleus'. x 4000.


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 inthe 'egg pronuclear membrane', x 36000.


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

fine-structural studies of the gametes and embryo …j. cell set. zo, 233-254 (1976 23) 3 printed in...

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