A FINE STRUCTURAL INVESTIGATION

OF SURFACE SPECIALIZATIONS AND

THE CORTICAL REACTION IN EGGS OF

THE CNIDARIAN BUNODOSOMA CAVERNATA

WILLIAM C . DEWEL and WALLIS H . CLARK, JR .

From the Department of Biology, Appalachian State University, Boone, North Carolina 28608and the Department of Biology, University of Houston, Houston, Texas 77004

ABSTRACT

Developing oocytes of the cnidarian Bunodosoma cavernata are located within the meso-glea of the mesenteries of the gastrovascular cavity. The cortex of the more maturevitellogenic oocytes contains numerous, electron-dense, membrane-bound, corticalgranules. The surface of these oocytes possesses prominent radially projecting structurestermed cytospines. Each cytospine has a core of microfilaments, 50-70 A. in diameter,that extends basally as a rootlet through the cortical layer . During spawning, ova lackingany extraneous investments are released from the enclosing gastrodermis . As a conse-quence of fertilization or events associated with the earliest stages of development the ovaundergo a massive cortical reaction. This reaction, which occurs during or just after re-lease of the ova, involves extensive reorganization of the cortical layer. The corticalgranule membranes and egg surface membrane fuse and vesiculate resulting in the mas-

sive discharge of granule contents. This event is accompanied by the loss of vesicular

remnants of cortical ooplasm and the disruption of cytospine organization . Light andelectron microscope comparisons of unreacted and reacted eggs show that the reactionresults in a significant decrease in egg diameter with the oolemma of the reacted egg re-organizing in a position centripetal to its original location .

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INTRODUCTION

Many excellent fine structural investigations cen-tered on the final stages of gamete developmentand/or gamete interaction during fertilizationhave been presented in recent years . With theexception of a few, these investigations are con-fined to those invertebrate groups in which thespermatozoon has a well-defined acrosome andthe egg possesses an investment(s) that must betraversed before gametic union (see reference 6for review) .

In view of this it is interesting that severalrecent fine structural studies on acoelomate in-vertebrates reveal that the spermatozoa charac-teristically lack well-defined acrosomes . Theseinclude one poriferan (34) and several memberseach of the hydrozoans (2, 18, 19, 23, 31-33, 37),scyphozoans (3, 19), and anthozoans (13, 19) .Although it is reported that the eggs of the porifer-ans and cnidarians in general lack egg invest-ments other than the egg plasma membrane (27),

THE JOURNAL OF CELL BioLoaY • VOLUME 60, 1974 . pages 78-91

there is only preliminary ultrastructural evidenceon the egg surface and cortex in these groups(12, 29, 32) .

Furthermore, no studies deal specifically withthe cortical reaction in eggs that lack investments .In those organisms having eggs with investmentsas well as cortical granules which undergo a dis-tinct cortical reaction, the investments are usuallyaffected in some way by the reaction. For example,the release of cortical material, whether during acortical reaction in response to fertilization (5, 6,9, 17, 20, 35, 38) or to other factors such as exposureto sea water (25, 26), is often associated with theelevation and/or structural and chemical changeof the investment . These are probably severalfunctions involved in this interaction between theinvestment and the released cortical material .Whatever functions are proposed, however, theyshould be considered in light of those animalswhose eggs are limited solely by an oolemma .For this reason the examination of a more primitivecondition, such as that exhibited by the cnidarianBunodosoma cavernata, may lead to a clearer under-standing of the significance of the cortical reactionin other organisms .

MATERIALS AND METHODS

Adult animals were collected near Port Bolivar on theTexas Gulf Coast and maintained in the laboratoryin shallow aquaria. Preparations of ovary were ob-tained by dissecting the animal and transferring por-tions of the mesentery containing ovary to fixative .Fixation was carried out for 1 h at room temperaturein the paraformaldehyde-glutaraldehyde (pH 7 .4)mixture recommended by Karnovsky (21) . Subse-quently, the preparations were washed with sea waterand postfixed in 1 .0% osmium tetroxide in sea waterfor I h at 4 °C. The tissue was then rapidly dehydratedin a graded acetone series, embedded in a low viscosityepoxy resin (30), and sectioned on a Sorvall MT-2ultramicrotome. Thin sections were mounted on un-coated grids, stained with alcoholic uranyl acetateand lead citrate (36), and observed with an AEI EM-6B, Hitachi HS-8, or Hitachi HU-11B electronmicroscope . Thick sections (1 µm) for general lightmicroscopy were stained with 0 .25% aqueous tolui-dine blue in 0.15% sodium borate.

Animals collected in the late spring, when spawn-ing occurs in natural populations, would occasionallyexhibit a spawning reaction in the laboratory within24 h after collection. For this study spawned eggs wereobtained : (1) from the bottom of aquaria or from theoral cavity of spawning females which were (a) sepa-rated from males or (b) with males that failed tospawn and, (2) from the oral cavity of spawning fe-

males in the same aquaria with spawning males . Eggsso obtained were fixed according to the techniques al-ready described . Those eggs used for determining anypossible changes in egg diameter as a consequence ofthe cortical reaction were obtained from the same fe-male, one that released both reacted and unreactedeggs (see Results) . Measurements were made on wholeeggs embedded in flat embedding molds .

RESULTS

The sexes are separate in B. cavernata . In femalesthe developing oocytes are located within themesoglea of the incomplete mesenteries just radialto the septal filaments (Fig. 1) . The loose layer ofmesoglea that separates the oocytes from the over-lying gastrodermis also extends between the de-veloping oocytes throughout the gametic region .The vitellogenic oocyte has a very large germinalvesicle with a single, discrete, electron-dense nu-cleolus (Figs . 1, 2) . Mitochondria, lipid-like in-clusions, annulate lamellae, and both membrane-and nonmembrane-bound fibrous bodies are com-monly present (Figs . 2, 3) . With continued growthyolk bodies fill the cytoplasm eventually occupyingall but the cortical region of the oocyte (Figs . 2,3). Compared to the cortical granules the yolkbodies are generally larger, more electron denseand less homogeneous as a result of electron-lucent inclusions . The cortical granules arespherical, homogeneous, and membrane-bound(Figs . 3-5) . Although their apparent abundanceat the oocyte surface depends upon the angle ofsection and the degree of maturation, they fill theoutermost cytoplasm to a depth several times theirdiameter in the nearly mature oocyte and egg . Re-gardless of the state of maturity some corticalgranules discharge their contents to the outside(Figs . 3, 5) . Golgi elements involved in the packag-ing of the cortical granules, as well as other oocyteconstituents such as yolk, are visible (Fig. 4) .

The surface of the nearly mature oocyte andegg possesses striking spike-shaped projections(Figs . 3, 6, 8) . These structures, perhaps bestdescribed as cytospines (see Discussion) form ag-gregates of 20-30 members whether the cell isconfined within the ovary or free, after spawning(Figs . 3, 6, 8, 10) . Individual cytospines are 15 µmin length (Figs. 6, 9, 21) and possess a core offilaments, 50-70 A in diameter (Figs . 7, 11) .This core extends basally as rootlet through thecortical layer (Figs . 6, 7, 11). In describing anaggregate of cytospines the term "spine" is occa-sionally employed since it appears both in early

W. C. DEWEL AND W. H. CLARK, JR . Cnidarian Cortical Reaction 79

FIGURE 1 A light micrograph showing oocytes lying within the gastrodermis of the incomplete mesen-teries of the gastrovascular cavity . Mesoglea separates the oocytes from one another and from the over-lying gastrodermis. g, gastrodermis ; go, germinal vesicle ; m, mesoglea ; arrow, nucleolus . X 1,200 .

and recent light microscope studies in referenceto their appearance at the magnification level ofthe light microscope (11, 15, 16, 28) .

The germinal vesicle probably breaks down be-fore egg release since it is never present in spawnedeggs, whether or not they have undergone acortical reaction . We have not yet observed polarbodies associated with the surface of the egg . Per-haps the mechanical disturbance incurred duringthe release of the eggs from the fibrous mesoglearesults in the loss of these bodies from the eggsurface . With the exception of the nonmembrane-bound fibrous bodies all the organelles and inclu-sions characteristic of the ovarian oocyte are alsofound in the spawned egg (compare Figs. 2, 3with Fig . 8) . Notably, however, the cortical gran-ules in the spawned egg have a less sphericalcontour than those in the ovarian oocytes (com-pare Fig . 4 with Figs . 8, 11) .

Eggs that had undergone a cortical reactionwere obtainable only when both sexes spawnedsimultaneously. For this study the reacted eggswere collected by inserting a Pasteur pipette intothe mouth opening of the female and withdrawing

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the eggs as they were released into the gastrcvascular cavity. Some of the eggs thus obtainedwere still loosely bound by ovarian tissue (W .C. Dewel, unpublished data) . Of these, severalshowed a cortical reaction over only a portion oftheir surfaces (Fig . 12) . It is important to notethat only those eggs which displayed a corticalreaction developed normally. From samples takenbefore fixation we found that normal cleavageoccurred in greater than 90% of those sampled(three replicates, 10 eggs per replicate). Manyof those embryos not fixed developed to the planulastage (W. C. Dewel, unpublished data) . Eggswhich had not undergone a cortical reaction werecollected both in the manner described above andfrom the bottom of aquaria either when femalesspawned before males or when males failed tospawn. If a female began spawning before anymales it was possible to obtain both unreacted(before male's spawning) and reacted (aftermale's spawning) eggs from the same animal . Ofthe eggs released at spawning unreacted onesnever developed and all attempts at in vitrofertilization were unsuccessful even when they

FIGURE 2 An electron micrograph of the nuclear region of an oocyte . gv, germinal vesicle ; ne, nucleolus;y, yolk ; 1, lipid-like body ; m, mitochondrion ; f, fibrous inclusion ; arrows, membrane-bound fibrous bodies .X 9,200.

FIGURE 8 An electron micrograph showing the cortical region of a maturing oocyte . y, yolk ; m, mito-chondrion; g, Golgi ; 1, lipid-like body ; al, annulate lamellae ; es, cytospines ; arrows, mesoglea ; cg, corticalgranule ; double arrow, discharging cortical granule. X 9,600.

FIGURE 4 A section showing Golgi elements associated with developing yolk and cortical granules . y,yolk ; cg, cortical granule ; g, Golgi ; m, mesoglea . X 14,800 .

FIGURE 5 The oocyte surface showing cortical granule discharge . The cortical granule at the surface hasundergone fusion and vesiculation with oolemma while those subjacent to it have fused and vesiculatedwith the peripheral cortical granule . cg, cortical granule ; dcg, discharging cortical granule ; m, mesoglea ;arrows, vesicles . X 28,900 .

FIGURE 6 A low magnification electron micrograph of the basal region of the cytospines present on theovarian oocyte. cs, cytospines . X 8,800.

FIGURE 7 A higher magnification of the cytospine . Note the core of microfilaments which extend as arootlet into the ooplasm,mf, microfilaments ; r, rootlet . X 26,200 .

were exposed to increasing concentrations of

Except for the cortical region, the cytoplasm ofsperm from males responsible for successful fertili- the reacted egg is similar to that of the unreactedzation (i .e ., sperm from males in adjoining aquaria egg with respect to density and presence and dis-where eggs underwent cleavage and developed to position of cellular organelles and inclusions (Figs .the planula stage) .

12, 19) . However, in the cortical region the major

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portion of the cortical granules undergoes amarked change as the reaction takes place. Themembranes of those granules adjoining the eggsurface fuse and vesiculate with the egg plasmamembrane (Fig . 12) . Concomitantly the mem-branes of subjacent granules fuse and vesiculatewith the membranes of each other and/or thoseof already discharging granules (Figs. 12-14) . Asa result several cortical granules may share a com-mon matrix interrupted only by plate-like vesicu-lar areas marking the original membranousboundary (Fig . 13) . Fusion between granules doesnot necessarily result in structural changes in theenclosed contents (Fig. 13) . Apparently contactof this cortical granule material with the externalmilieu results in its morphological alteration .Initially the material loses its homogeneous ap-pearance and exhibits a granularity of two differentdensities (Figs . 12, 14) . Subsequently, this ma-terial becomes a dispersed flocculent that movesapically between the cytospines (Figs . 12, 15) .This flocculent does not immediately dissipate butforms a layer of material over the egg surface(Figs . 17-19) .

Significantly, somewhat comparable events alsooccur in the unreacted egg, as well as in the ovarianoocyte (Figs . 3, 5, 8, 11), but involve only periph-eral granules, usually on an individual basis andseemingly always without previous fusion . Evi-dently, the discharge is at such a reduced ratethat the relative number of granules remains es-sentially unchanged for several hours (W . C .Dewel, unpublished data) .

The extensive fusion and vesiculation of corticalgranule membrane with egg plasma membraneas well as with subjacent cortical granule mem-brane establishes a honeycombed collection ofchambers or channels over the cortical region ofthe egg (Fig . 14) . The channels thus formed opento the outside between the cytospines (Figs . 12,15). Excluding the membrane of the spines, thelimiting membrane of the egg is now a highly con-voluted mosaic of both egg plasma membrane andcortical granule membrane . Qualitatively, thecortical granule membrane is by far the largestcontributor to this mosaic . At first the developingsurface appears extremely irregular (Figs . 16, 20)but later it exhibits a smoother profile (Figs . 18, 19) .Confined outside this surface are numerousvesicular remnants of the original oolemma andcortical granule membranes (Figs . 12, 16, 19, 20) .There is considerable variability in vesicle size .Even membrane-enclosed portions of the cortical

cytoplasm with characteristic cellular constituents(e .g. mitochondria, lipid-like inclusions, endo-plasmic reticulum, and ribosomes) are occasionallypresent (Figs . 16, 18, 20) .

As a result of the reaction the newly organizedoolemma lies centripetal to the former oolemma .In other words, the diameter, when it is measuredfrom the surface of the egg (48 reacted and 50unreacted) is significantly less (P

of the individual members . In addition, the termdoes not reflect the cytoplasmic nature of thesespecializations. To overcome these limitations wepropose adoption of the term "cytospine ." Theuse of microvillus as a possible alternative is lessappropriate since these structures are not suffi-ciently similar to microvilli in respect to size,arrangement, and rich microfilamentous content .However, adopting the term cytospine not onlywould allow for the continuation of differentiationbetween spiny anthozoan eggs and those with asmoother microvillous surface but also would ef-fectively designate the unit structure and thusfacilitate the description of individual cytospinevariability among all spiny eggs in terms of theirsize, arrangement, and fine structure .

Many investigators have described corticalreaction in the eggs of marine invertebrates (1, 4-6,22, 24) . In the case of B . cavernata we have notdefinitely established what event initiates corticalgranule discharge . Nevertheless, we do know thatspawning females that are either isolated or withnonspawning males produce eggs that (a) do notdisplay a cortical reaction, (b) cannot (at least todate) be fertilized in vitro, and (c) never undergodevelopment . In contrast, spawning females heldin the same aquaria with simultaneously spawningmales produce eggs which display a cortical reac-tion and which undergo normal cleavage . Further-more, these reacted eggs are obtainable from thegastrovascular cavity of the female and in manycases they are still in clusters held together bylayers of mesoglea . These considerations lead usto conclude tentatively that (a) the cortical reac-tion can take place internally on or just subse-

8 4

quent to egg release and (b) the cortical reaction isin response to fertilization or as yet unknownevents related to the earliest stages of development,but not in response to release or exposure to seawater as in certain other species (25, 26) .The cortical reaction in B. cavernata involves

the multiple fusion and vesiculation of corticalgranule membranes with each other as well aswith the egg plasma membrane . The mechanismof this reaction is not unusual ; it is apparentlysimilar to that described by Barros et al . (8) in theacrosome reaction of a mammal, namely "theoccurrence of multiple unions between two cellularmembranes lying in close apposition, with theformation first of a double-walled fenestrated layerand ultimately of an array of separate membranebounded vesicles ." The basic process was alsofound in the cortical reaction of the egg of thesea urchin Arbacia punctulata (5) .

However, in the case of B. cavernata a number ofunusual characteristics merit discussion . One isthe extraordinary massiveness of the reaction . Itis perhaps, with the exception of the eggs of Nereislimbata (14), unparalleled in terms of the amount ofcortex involved . The extensiveness is a result ofseveral factors including the considerable thicknessof the cortical layer and the high density ofgranules within the layer together with the seem-ingly simultaneous fusion and vesiculation ofgranule membranes with the oolemma and witheach other . Although it is difficult to determinethe precise sequence of fusion, there are examplesof completely fused granules which do not appearto have established any contact with the outerenvironment (Fig. 13) .

FIGURE 8 The surface of the released but unreacted egg. Note the thick layer of cortical granules . Thedouble arrow points to a cortical granule which is discharging its contents to the outside . Most of the corti-cal granules, however, remain undischarged. Compare this micrograph with the reacted egg shown in Fig .19. y, yolk : m, mitochondria ; 1, lipid-like body ; cg, cortical granule ; cs, cytospines ; arrows, membrane-bound fibrous bodies . X 7,400.

FIGURE 9 A light micrograph of a released but unreacted egg. Compare this micrograph with Fig . 17showing the reacted egg . pn, pronucleus ; cg, cortical granules ; s, spines . X 510 .

FIGURE 10 A cross section through a group of cytospines (ca) which taken together make up the spinesvisible at the light microscope level on certain anthozoan eggs . A coat of fibrillar material is visible on theouter surface of the cytospine membrane . X 45,000.

FIGURE 11 A longitudinal section through several cytospines on the surface of the released but unreactedegg . Note the rootlet extending through the cortical layer deep into the ooplasm . y, yolk ; cg, corticalgranule ; deg, discharging cortical granule ; mf, microfilament ; r, rootlet . X 91,300 .

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W. C. DEWEL AND W. H. CLARK, JR . Cnidarian Cortical Reaction

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FIGURE 12 A micrograph showing an egg in which the cortical reaction has proceeded over only a por-tion of its surface . The zone of junction between unreacted surface (lower) and reacted surface (upper) isapproximately in the middle of the micrograph . cg, cortical granule ; dcg, discharging cortical granule ; cs,cytospine ; v, vesicle ; y, yolk . X 11,700 .

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FIGURE 13 A section through a reacting egg showing the vesicular areas between fused but undischargedcortical granules . v, vesicles . X 46,100.

FIGURE 14 A tangential section through the egg surface showing its honeycombed appearance after thecortical reaction . cg, cortical granule ; dcg, discharging cortical granules. X 8,900 .

FIGURE 15 A micrograph showing the earlier stages of cortical granule discharge which is taking placebetween the cytospines. cg, cortical granule ; dcg, discharging cortical granules ; r, rootlet . X 16,300.

FIGURE 16 The surface of the reacted egg . Note the small membrane-enclosed vesicles which are abun-dant at the opening of a pocket formed by a discharging cortical granule . y, yolk ; v, small vesicles ; 1, lipid-like body . X 15,500 .

FIGURE 17 Alight micrograph of the reacted egg . Compare this micrograph with Fig . 9 . s, spines . X 810 .

FIGURE 18 A section through the surface of the reacted egg . Note the large organelle-containing vesiclesand the single rootlet (white arrow) visible in the cortical cytoplasm . At this stage the basal ends of thecytospines are swollen (arrow) . See also Fig. 19 . m, mitochondria . X 6,100 .

FIGURE 19 Another section of the egg surface after the cortical reaction . The arrows point to the swollenbases of the cytospines . Their microfilamentous core is disrupted and their former organization has beenlost . Compare this micrograph with the unreacted egg shown in Fig . 8 . cs, cytospines ; y, yolk, v, vesicles .X 4,600 .

FIGURE 90 A higher magnification of the egg surface after the cortical reaction . Note the small vesicles(v) and the larger vesicles which contain cortical organelles . y, yolk ; m, mitochondrion . X 19,900.

FIGURE 21 A comparison of the diameter of the unreacted and reacted eggs showing the decrease in eggdiameter resulting from the cortical reaction (see Results) . X 310.

Perhaps the interaction of the granule mem-branes during the reaction is independent of thedevelopment of continuity of the granule contentswith the external milieu . Since fused granulesexhibiting unaltered contents have rarely beenobserved in oocytes or in released but unreactedeggs, the process may be largely restricted to thecortical reaction . Evidence of previous fusion is

also reported for the eggs of N . limbata (14) .

Another notable feature is the absence of "all-or-none" behavior in the cortical granules . As

previously indicated the granules occasionallydischarge their contents to the outside both indeveloping oocytes and in released but unreactedeggs . The manner in which this occurs is apparentlysimilar to that occurring during the cortical reac-tion. Perhaps it underlines an inherent lability orreactivity in the cortical granule and egg plasma

membranes. Unfortunately, the immediate affectsof fixation in possibly causing or augmenting anypremature release of cortical material cannot beruled out entirely. However, there is evidence that

released but unreacted eggs held for longer than18 h show a substantially reduced population ofcortical granules (W . C. Dewel, unpublisheddata) presumably due to this process of gradual

discharge. Regardless of whether the discharge isreal or artifactual it cannot be overemphasizedthat it is not at all comparable to the overwhelming

disruption of the cortex during the normal corticalreaction.

Another unusual characteristic of the reaction isthe formation and subsequent loss of vesicles largeenough to contain cellular organelles such as mito-chondria and rough endoplasmic reticulum . Pre-sumably these vesicles are derived from areas ofcortical cytoplasm interstitial to the corticalgranules. If there are multiple sites of fusion andif there is almost simultaneous interaction among

adjacent membranes, then the production of thesevesicles is easily visualized . Since the packing ofgranules within the cortex is not optimally close,it is apparent that significant amounts of ooplasmcould be lost during the reaction . A quantitatively

W. C. DEWEL AND W. H. CLARK, JR. Cnidarian Cortical Reaction 89

similar loss does not occur in the eggs of certain seaurchins (5, 7) nor in the eggs of N. limbata (14)although in the latter small vesicular remnants areconspicuous in the perivitelline space . Notably,before the cortical reaction in N. limbata, the alveoliseem to be very densely packed with the interven-ing cytoplasm-lacking organelles such as mito-chondria (14) . This factor together with the re-ported preformation of large coalesced alveolicould conceivably reduce the chances of largevesicles becoming isolated during the cortical re-action .

Possibly the most puzzling aspect of the corticalreaction in eggs of B. cavernata is the morphologicaland functional relationship between the cortex andthe cytospines . During the earliest stages of thereaction, when the cortical granules just begin todischarge between the cytospines, the integrity ofthe cytospines is apparently maintained . Never-theless, there are indications that the microfila-mentous rootlets are disrupted and disappear,that the cortical region becomes thoroughly dis-rupted as the reaction proceeds, and that thecontinuity of the cytospines with the egg is, at thevery least, obscured . It is clear that the high degreeof disruption and the unusual changes in cyto-spine structure, in particular the possibility of ac-tive participation by the microfilaments in surfaceand cortical reorganization, warrant further atten-tion .

The authors wish to express appreciation for theuse of the electron microscope laboratory in the De-partment of Zoology at the University of NorthCarolina, Chapel Hill. We are also grateful to Dr.John B. Morrill, New College, Sarasota, Floridaand Dr . Robert G. Summers, University of Maine,Orono, for reading the revised manuscript .This investigation was supported, in part, by a

National Aeronautics and Space AdministrationPredoctoral Traineeship, grant number NGT-44-005-004 no. 1/2 .Received for publication 6 October 1972, and in revised form22 August 1973.

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