J. Cell Sd. 39, 189-195 (1978)Printed in Great Britain © Company of Biologists Limited igj8

A MICROTUBULE-NUCLEAR ENVELOPE

COMPLEX IN THE SPERMATOZOID OF

PTERIDIUM

PETER R. BELLDepartment of Botany and Microbiology, University College,London WCiE 6BT, England

SUMMARYThe microtubules on the outside of the nucleus of the mature spermatozoid of Pteridium

form a complex with the envelope, and the perinuclear space is eliminated. These dissimilarcomponents are held firmly together, possibly by hydrophobic bonding. The outer part of thechromatin is also attached to the inner boundary of the envelope, and pulls away when theenvelope is detached.

INTRODUCTION

The nuclear envelope typically consists of 2 membranes, each yielding a 'unitmembrane' profile (Robinson, 1964), separated by a space, the perinuclear space(Watson, 1955), commonly 10-30 nm wide. Although the contours of the nucleusmay sometimes be highly irregular, the space between the 2 membranes is usuallyretained. An exception to this rule is in motile male gametes where the perinuclearspace is often eliminated (Fawcett, 1970), a feature well shown in the spermatozoidsof ferns. In Pteridium, as in other ferns, the nucleus of the mature gamete is helical,and a ribbon of microtubules runs along the outside of the helix (Bell & Duckett,1976). Electron micrographs of this region reveal no spaces lying between the micro-tubules, the nuclear envelope and the condensed chromatin, despite a clear gap, some20 nm wide, between the microtubules and the nuclear envelope in differentiatingspermatids (Figs. 1, 2).

Spermatozoids lying in the venter of the archegonium above fertilized eggs ofPteridium are often partially dismembered. It appeared that dismemberment undermore controlled conditions might give a clue to the structural relationships of themicrotubular ribbon and nuclear envelope in the motile gamete. In the followinginvestigation Pteridium spermatozoids have been immobilized in Marsilea mucilage.Their flexing movements in this viscous medium generated shearing forces whichgave the limited disintegration required.

MATERIALS AND METHODS

Gametophytes of Pteridium aquilinum were grown in pure culture as described previously(Bell, 1972). Sporocarps of Marsilea vestita (from a plant of Californian origin in cultivation)were scored at the margin and placed in sterile tapwater. After 4-5 h the sporocarps had

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190 P. R. Bell

opened and the megaspores, now mostly free of sporangial membranes, had generated theirmucilaginous envelopes (Machlis & Rawitscher-Kunkel, 1967).

A dense suspension of Pteridium spermatozoids was prepared by scraping a mass of younggametophytes into a few millilitres of water. About 50 Marsilea megaspores (taken from theopened sporocarps well before the liberation of the Marsilea spermatozoids) were plunged intothe sperm suspension and left submerged for 2 h. The megaspores with their adhering mucilagewere then removed and transferred immediately to 2-5 % glutaraldehyde (TAAB Laboratories,Reading, U.K.) in 0-025 M phosphate buffer (pH 6-9). Fixation was continued for 2 h at roomtemperature. Following overnight washing in ice-cold buffer, the megaspores and mucilagewere then osmicated for 2 h (1 % aqueous), dehydrated with acetone, and finally embedded inDurcupan ACM (Fluka AG, Buchs, Switzerland).

Sections were first cut at 4 fim, using a glass knife, and those containing groups of spermato-zoids trapped in the mucilage remounted for thin-sectioning (Woodcock & Bell, 1967). Stainingof thin sections was with uranyl acetate (7 % aqueous) alone (10 min at 35 °C), uranyl acetatefollowed by lead citrate (Reynolds, 1963) (both at room temperature), or with potassiumpermanganate (1 % aqueous at room temperature for 1 min). Sections were viewed in anElmiskop 1.

Microdensitometry

To obtain objective estimates of the width of the nuclear envelope traverses were madeacross negatives, photographed at x 40000 magnification, with a Joyce-Loebl recording micro-densitometer (Mark III C). The final aperture width (1 mm) and height (5 mm) correspondedto a rectangle 43 by 215 fim (the long side parallel to the plane of the envelope) on the negative.The tables were linked to give a 50-fold amplification.

RESULTS

Numerous spermatozoids were encountered in the mucilage, particularly above thesingle archegonium of the megaspore (Fig. 1, inset). In the electron microscope they

Fig. 1. Transverse section of nucleus in second gyre of spermatozoid of Pteridium aqui-linum trapped in Marsilea mucilage, m, microtubular ribbon; n, nucleus. The darkline between the ribbon and the nucleus is the site of the condensed nuclear envelope,x 75000. Inset: Spermatozoids trapped in mucilage. Resin section (4/im) sub-sequently remounted for fine-sectioning. Phase-contrast, x 1280.Fig. 2. The microtubular ribbon and nuclear envelope in a differentiating spermato-cyte, the coiling of the nucleus just beginning, n, nucleus, x 280000.Fig. 3. The complex of microtubules and nuclear envelope on the outside of a nucleusof a mature spermatozoid (as in Fig. 1). m, microtubular ribbon (indistinct becausecut obliquely); v, nucleus, p, plasmalemma. The nuclear envelope now lacks theperinuclear space and appears in profile as 3 dark lines, x 280000.Fig. 4. The complex and adjacent chromatin after staining with permanganate.Labelling as in Fig. 3. The arrow indicates a discontinuous clear layer adjacent to theinner boundary of the envelope, x 280000.Fig. 5. The complex being pulled away from the nucleus, stained with warm uronylacetate. Labelling as in Fig. 3. Note the layer with a strong affinity for uranyl ions whichis pulled away with the envelope, and the evidence of adhesion of chromatin to theenvelope, x 280000.Fig. 6. A portion of the complex pulled clear of the nucleus (n). p, m, as in Fig. 3. Thearrows point to connexions between microtubules and the plasmalemma, and betweenmicrotubules and the condensed nuclear envelope. Arms connecting the microtubulesare also visible, x 240000.'

Microtubule-nuclear envelope complex IQI

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10.2 P. R. Bell

were found to be in varying degrees of dismemberment, and occasionally their finestructure could not be clearly seen. It seems unlikely that this was a consequence ofpoor fixation, since the contents of the megaspore were regularly well preserved. It ispossible that the mucilage is rich in hydrolases which were beginning to producenotable effects on fragmented spermatozoids even within the 2-h period. The observa-tions below are representative of spermatozoids showing clearly defined structure.

The intact nuclear envelope and associated structures

In many preparations, particularly when sectioning was more or less parallel to thealignment of the microtubules, staining with uranyl acetate and lead revealed animage of 3 dark lines at the site of the nuclear envelope (Fig. 3). The middle line wasabout double the thickness of those on each side. The microtubules were very closelyappressed to the outside of the nuclear envelope and no gaps were visible. On theinside the chromatin appeared to be continuous right up to the dark line representingthe inner boundary of the envelope (Fig. 3).

• n i• uj

• c A /

• /

0

1%0

V m

B

Fig. 7. Representative microdensitometer traces across the condensed nuclear envelope.A, trace after staining with uranyl acetate and lead (from the negative yielding Fig. 3).B, trace after staining with permanganate (from the negative yielding Fig. 4). c,chromatin; i, inner boundary of nuclear envelope; o, outer boundary of envelope; m,microtubular ribbon. The intervals of the abscissa represent 10 nm. The ordinateis an arbitrary scale of transmission represented by darkness in the print. The arrowin B indicates the clear layer adjacent to the inner boundary of the envelopefollowing permanganate staining.

The plasmalemma showed the customary unit membrane profile (Fig. 3). Therewere indications of an irregular space, reaching about 5 nm in width, between theplasmalemma and the microtubules.

The microdensitometer trace of the field shown in Fig. 3 revealed a total thickenessof the envelope (measuring between the peaks representing its inner and outer

Microtubule-nuclear envelope complex 193

boundaries (Fig. 7 A, i and 0)) of the order of 10 nm. There was no significant dis-continuity between the chromatin and the inner boundary (Fig. 7, c).

Staining with permanganate gave a slightly different result (Fig. 4). In all instanceswhere the tripartite image was clearly observed there were indications of an electron-transparent layer between the inner boundary of the envelope and the chromatin(Fig. 5, arrow). This feature was also brought out by the microdensitometer trace, thesharp dip in the curve (Fig. jB, arrow) before the peak representing the inner boundaryof the envelope corresponding to the clear layer in the print. In this particular pre-paration the width of the envelope (measuring between the peaks) was of the order of11-5 nm.

The effects of disruption

The shearing forces set up in the flexing spermatozoids frequently caused theplasmalemma and microtubular ribbon to be detached from the nucleus. In everyinstance the plasmalemma still lay in position on the outside of the ribbon, and thenuclear envelope as a whole remained firmly attached to the inside. Further, the insideboundary of the envelope was frequently uneven and ill-defined, suggesting thatthe surface of the chromatin had also been torn away. Staining with uranyl acetatealone (Fig. 5) showed that there were indeed irregular adhesions between thedeparting envelopes and the chromatin. A thin layer with a strong affinity for uranylions was also associated with the envelope after this had been pulled clear (Fig. 5).

Microtubular ribbons were also observed dragged 50 nm or more from nuclei.In these, possibly as a result of the microtubules having been slightly separated bylateral tensions, arm-like bridges between the tubules were clearly evident (Fig. 6).Connexions between the tubules and the plasmalemma, and between the tubules andthe partially fragmented nuclear envelope (Fig. 6, arrows) could also be seen.

DISCUSSION

Elimination of the perinuclear space

Although the loss of the perinuclear space appears to be common in the malegametes of animals (Fawcett, 1970), the archegoniate plants are variable in thisrespect. In the ferns Pteridium and Dryopteris no perinuclear space is detectable inthe mature gamete, but in Equisetum (Duckett & Bell, 1977) the perinuclear space,although accumulating some electron-opaque material, remains clearly visible. In theliverwort Sphaerocarpos the 2 membranes of the envelope can be seen in maturegametes after fixation in aldehyde followed by osmication (Zimmerman, 1974), butnot following fixation in osmium tetroxide or potassium permanganate (Diers, 1967).That the absence of the space in Pteridium gametes is not a fixation artifact is clearlydemonstrated by various stages in the ordered reconstitution of the normal envelopefollowing syngamy (Bell, 1975).

The width of the profile of the fused membranes ranges from 8 to 12 nm. Theapposed faces evidently firmly adhere to each other while the gamete is motile. Itseems possible that the loss of fluid from the perinuclear space, which accompanies

194 P. R. Bellthe general dehydration of the nucleus as differentiation of the gamete is completed,results in the lipoproteins of the 2 faces becoming hydrophobically bonded. Suchbonds remain strong within a narrow range of reduced hydration (Franks, 1975), butcease to be effective as the amount of water in the organic material increases. A changeof this nature may account for the rapid reappearance of the male perinuclear spacein the female cytoplasm.

Attachment of chromatin to the inner nuclear membrane

The material which is pulled away from the nucleus with the envelope is indis-tinguishable, following uranyl staining, from the bulk of the chromatin. The indica-tions of a clear line beneath the envelope following permanganate staining suggest

Fig. 8. Reconstruction of the microtubule-nuclear envelope complex on the outsideof the mature spermatozoid of Pteridium. p, plasmalemma; m, microtubular ribbon;cmrn, outer membrane of nuclear envelope; inm, inner membrane of envelope closelyappressed to the outer, with elimination of the perinuclear space; c, chromatin. Theclear layer is that remaining unstained by permanganate, possibly chromatin richin nucleic acid and poor in protein, in contact with the membrane.

however, that the composition of the nucleus adjacent to the envelope is not homo-geneous. The clear line is not a space formed by separation since, when the envelopeis pulled away, the break does not always run along the line, but frequently dipsirregularly into the chromatin beneath. Since permanganate reacts preferentially withproteins (Davies, 1976), the staining behaviour of the chromatin at the periphery ofthe nucleus may indicate a thin layer adjacent to the envelope which is rich in nucleicacid and poor in protein. In some somatic nuclei, a layer of acidic protein (the' fibrouslamina'; see Wunderlich, Berezney & Kleinig, 1976) has been detected at the surfaceof the chromatin. Such a layer, being positively charged, would have an affinity foruranyl ions, but would also probably reduce permanganate, and thus seems ruled outin the present instance. The direct attachment of chromatin to the nuclear envelopeis well established in many somatic nuclei (see Wunderlich et al. 1976).

The attachment of the microtubular ribbon to the nuclear envelope and plasmalemma

The extensive lateral connexions between the microtubules and the, nuclear enve-lope revealed by the disruption experiments are a remarkable feature of the complex.When intact nuclei were sectioned it was regularly observed that the trilinear imageof the envelope was more evident when the plane of sectioning was parallel, or nearly

Microtubule-nuclear envelope complex 195

so, to the microtubules than when it was transverse. This suggests that in theundisturbed complex the microtubules are partially immersed in the outer surface ofthe outer membrane, so that in the transverse plane its boundary is corrugated andobscured (Fig. 8). The connexions between these 2 components do not seem to be ofthe same distinct form as the keels on the lower surface of the microtubules when theyare lying above the plates of the multilayered structure (Duckett, 1975). The con-nexions between the microtubules and the plasmalemma, however, are similar tothose frequently observed in somatic cells (for example, Cronshaw, 1967). Further,the profile of this membrane was quite clear whatever the plane of sectioning inrelation to the microtubules.

The financial support of the Science Research Council and the technical assistance of Mr J.Mackey are gratefully acknowledged. Dr Diana Myles kindly drew the author's attention to thechemotactic effect of Marsilea mucilage on Pteridium spermatozoids.

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BELL, P. R. (1975). Observations on the male nucleus during fertilization in the fern Pteridiumaqtiilinum. J. Cell Sci. iy, 141—153.

BELL, P. R. & DUCKETT, J. G. (1976). Gametogenesis and fertilization in Pteridium. Bot. J.Linn. Soc. 73, 47-78.

CRONSHAW, J. (1967). Tracheid differentiation in tobacco pith cultures. Planta 72, 78—90.DAVIES, H. G. (1976). Electron microscopy of interphase chromosomes in situ: binding of

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WATSON, M. L. (1955). The nuclear envelope. J. biophys. biochem. Cytol. 1, 257-270.WOODCOCK, C. L. F. & BELL, P. R. (1967). A method for mounting 4 /t resin sections routinely

for ultrathin sectioning. Jl R. microsc. Soc. 87, 485-487.WUNDERLICH, F., BEREZNEY, R. & KLEINIG, H. (1976). The nuclear envelope: an interdisci-

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ZIMMERMAN, H.-P. (1974). Elektronenmikroskopische Untersuchungen zur Spermiogenesevon Sphaerocarpos donnellii Aust. (Hepaticae). II. Der Kern. Cytobiologie 9, 144-161.

(Received 20 June 1977)