THE EFFECTS OF NICOTINE ON

FERTILIZATION IN THE SEA URCHIN,

ARBACIA PUNCTULATA

308

FRANK J. LONGO and EVERETT ANDERSON

From the Department of Zoology, University of Massachusetts, Amherst, Massachusetts 01002,and the Marine Biological Laboratory, Woods Hole, Massachusetts 02543

ABSTRACT

The number of sperm incorporated into eggs made polyspermic with varying concentra-tions of nicotine (0 .025-0.25%, v/v) appears to be directly related to the concentrationsemployed . The cortical response is morphologically equivalent to that observed in controlpreparations. Shortly after their incorporation all of the spermatozoa undergo structuralevents normally associated with the development of the male pronucleus in monospermiceggs. During the reorganization of the spermatozoa, sperm asters are formed . The numberof male pronuclei that initially migrate to and encounter the female pronucleus is usuallyone to three . When pronuclei come into proximity to one another the surface of the femalepronucleus proximal to the advancing male pronuclei flattens and becomes highly con-voluted. Subsequently, the pronuclei contact each other and the outer and inner membranesof the pronuclear envelopes fuse, thereby producing the zygote nucleus . The male pro-nuclei remaining in the zygote after this initial series of pronuclear fusions continue todifferentiate, i .e . they enlarge, form nucleolus-like bodies, and undergo further chromatindispersion . In approximately 90% of the zygotes, all of the remaining male pronucleiprogressively migrate to the zygote nucleus and fuse to form one large nucleus by 80 minpostinsemination . Mitosis and cleavage of the polyspermic zygote occurs later than inmonospermic eggs.

INTRODUCTION

The pioneering studies of the Hertwigs (1887)and the investigations of Clark (1938) constitutemuch of what we know concerning agents whichinduce polyspermy, i .e ., the entrance of more thanone spermatozoon into an egg . The nature of themechanism which prevents polyspermy in seaurchins has been studied by numerous investiga-tors (Wilson, 1925 ; Rothschild and Swann, 1950 ;Hagström and Allen, 1956, Allen, 1958 ; Monroy,1965 ; Runnström, Hagström, and Perlmann,1959) . Many of the forementioned studies haveattempted to correlate the propagation of theblock to polyspermy with the morphological

events associated with the cortical reaction andthe formation of the activation calyx (see Mon-roy, 1965) . These investigators concluded that atthe time of insemination the sperm initiates achange in the cortex of the egg which renders itinaccessible to further insemination . Moreover, itis generally held that polyspermy is a result of analteration in the manner in which this change ispropagated over the surface of the egg (Lillie,1919) .Although polyspermy in the sea urchin is

pathological (Harvey, 1956), treatment of eggswith various chemical agents and the entrance of

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more than one sperm during insemination adds afurther dimension to the elucidation of the manyfacets of fertilization remaining unsolved . Boveri(1902) was one of the first investigators to takeadvantage of the additional parameter offered bythe polyspermic condition in his elegant experi-ments on multipolar mitosis in dispermic seaurchin eggs, whereby he conclusively demon-strated that chromosomes are qualitatively dis-tinct and individual in the determinants theycarry .

Most studies of polyspermy have been pri-marily concerned with the initial events of gameteinteraction and have paid relatively little atten-tion to its subcellular effects or consequencesduring later stages of development . The presentinvestigation was undertaken to study the intricatesequence of events involving the egg and thesperm during polyspermy and to compare thesewith those occurring during monospermy. In thismanner it may be possible to obtain more specificinformation about pronuclear development andfusion by exaggerating the mechanisms associatedwith these events, and by selecting factors thatare common to monospermy and polyspermy .This communication is concerned with an analysisof those events of polyspermy induced by nicotinetreatment in the sea urchin, Arbacia punctulata .

MATERIALS AND METHODSEggs and sperm of Arbacia punctulata, acquired fromthe Marine Biological Laboratory, Woods Hole,Massachusetts, during the months of June and July,were obtained according to procedures previouslydescribed (Longo and Anderson, 1968) . In an effort toobtain a high percentage of synchronously develop-ing embryos following nicotine treatment, we foundit necessary to employ eggs from a single female foreach experiment. A similar situation has also beenreported by Harvey (1936) . After they had beenwashed in sea water, the eggs were treated withnicotine in concentrations ranging from 0 .025 to0.25% (v/v) for 5 min, fertilized, and permitted todevelop at 20 °-23 °C with constant stirring. Controlpreparations consisting of eggs treated in the samemanner except for the incubation in nicotine wereinseminated with the same sperm dilution . Spermwere added to the nicotine-treated and untreatedeggs so that the final concentration was approxi-mately 2-5 X 106 spermatozoa/ml (Harvey, 1956) .For purposes of clarity, we have referred to insemina-tion as the introduction of the spermatozoa to the eggsuspension, and this marks the zero point from whichall timing measurements are made .

In some experiments the eggs were washed once

in sea water following the nicotine treatment, andthen fertilized. To obtain a sequence of developmentfrom fertilization to cleavage, eggs were collectedand fixed at the following intervals subsequent toinsemination : every 15 sec for 2 min ; every 2 min for20 min followed by every 5 min for 70 min . Inaddition, unfertilized eggs incubated in various con-centrations of nicotine (ranging from 0.025 to 0 .25%)for periods from 2 to 10 min were collected andfixed . Some eggs were also washed and agitatedaccording to the methods of Lillie (1914) and Harvey(1914) for the removal of the jelly layer, treated withnicotine, and fertilized according to the methodsdescribed for jelly-intact eggs . Sperm suspensions,incubated for 5 min in each of the concentrations ofnicotine employed, were examined to be sure thatthey maintained their motility . Observations indi-cated that all fertilizable eggs were inseminated wellwithin this period .

Eggs and zygotes were prepared for light andelectron microscopy according to procedures pre-viously reported (Longo and Anderson, 1968) .Cultures, consisting of fertilized eggs, previouslytreated with various concentrations of nicotine, werealso examined with phase microscopy.

RESULTSGreater than 98% of all the eggs treated in thevarious concentrations of nicotine were observedto be polyspermic following insemination and toundergo similar morphological events of fertiliza-tion and development. In general, a correlationwas apparent between the degree of polyspermyand the concentration of nicotine, i.e . di- andtrispermy were prevalent at the lower concen-trations of nicotine, whereas at higher concen-trations fertilization normally involved 10-12 ormore sperm. A similar progression was also notedby Clark (1938) . The following observations per-tain primarily to eggs treated with 0 .15 % nicotinebut apply to all of the concentrations used . Minormorphological differences in zygotes from amongthe various concentrations of nicotine employedwill be noted where pertinent .

Control preparations, consisting of eggs treatedin the same manner as the experimentals but notincubated in nicotine, exhibited less than I %polyspermy and underwent the same structuralevents as previously described for normal devel-opment (Longo and Anderson, 1968 ; Anderson,1968). Unfertilized eggs incubated in various con-centrations of nicotine for 2-10 min appeared tobe morphologically equivalent to untreated eggsat light and electron microscopic levels of obser-vation .

FRANK J. LONGO AND EVERETT ANDERSON Nicotine-Induced Polyspermy

309

Within 15 sec after insemination, numerousspermatozoa were observed in contact with eggsthat had been treated with nicotine (Fig . 1 andinset) . Adherence of the gametes appeared to bemore stable than in the controls or irreversible,for fewer spermatozoa were dislodged from thesurface of the egg . The untreated eggs did notaccumulate supernumerary spermatozoa. Jelly-free eggs appeared to accumulate more sperma-tozoa at their surfaces than eggs with the jellylayer intact ; no other morphological differenceswere observed .When spermatozoa are exposed to nicotine

concentrations of 0 .15-0.25 % for periods in excessof 5 min, their motility eventually diminishesuntil they are no longer capable of fertilization .The spermatozoa recover their activity if they areput into fresh sea water (see also Hertwig andHertwig, 1887 ; Rothschild, 1953 ; Rothschild andSwann, 1950) . There was no indication that thenicotine treatment caused the eggs to becomemore adhesive to one another or to the glassware .

Since the block to polyspermy has been relatedto some component(s) of the cortical granules, itis necessary to give a brief account of the corticalreaction (Hagström and Allen, 1956 ; Rothschildand Swann, 1950; Monroy, 1965) . The corticalreaction and the formation of the activation calyxare well in progress 30 sec following insemination,i .e ., greater than 50% of the surface of all of theeggs that were activated (about 90%) containeddehiscing cortical granules (Fig. 2, "CG") . Therelease of the cortical granules and the formationof the activation calyx was not initiated at onelocus as normally observed in the monospermiccondition (see Anderson, 1968) but rather oc-curred at multiple loci which corresponded to thesites of gamete fusion (Fig . 2, inset) . At each siteof attachment of the sperm to the surface of the

egg the vitelline envelope becomes disjoined fromthe oolemma, producing an incomplete activationcalyx and the perivitelline space (Fig. 2 andinset) . Following the detachment of the vitellineenvelope there is a wavelike release of the corticalgranules beginning at each site of gamete fusion .The release of the cortical granules in such amanner produces areas of dehiscing granuleswhich eventually meet, thereby forming a con-tinuous perivitelline space and activation calyx(approximately 45 sec following insemination) .Following the cortical reaction there is also arelease of the rodlike structures from vesicularbodies into the perivitelline space as observed inmonospermy (Fig . 4) . The rodlike structures forma portion of the hyaline layer (see Anderson,1968) .The formation and organization of the activa-

tion calyx and the contents of the perivitellinespace are the same as found in monospermic eggs ;however, there are differences with respect to thetime at which certain structures are formed (seeDiscussion) . 1 min after insemination, the activa-tion calyx is fully extended from the surface ofeggs treated in nicotine . About 2 min after insem-ination various areas become thicker and lami-nated so that the calyx appears to be a more rigidstructure (Fig . 3, AC) . Approximately 6 minafter insemination much of the activation calyxappears to be a tough, laminated layer. Approxi-mately 2 min after insemination the materialswithin the perivitelline space become organizedinto the hyaline layer (Fig . 3, HL) .Incorporation of the Spermatozoa andFormation of the Male Pronucleus andSperm AsterAt the site of gamete fusion a confluence of theegg and sperm is established and a fertilization

FIGURE 1 Electron micrograph and photomicrograph (inset) depiciting the initial contact of the gametesand the aggregation of sperm at the surface of nicotine-treated eggs (inset) . Note that the cortical reac-tion has not been initiated. The acrosome (A) is associated with some flocculent material at the egg'ssurface (arrow) . CG, cortical granule ; M, mitochondria ; SM, sperm mitochondrion ; SN, sperm nucleus ;C, centriole . Fig. 1, X 25,500 ; inset, X 400, Epon embedded, toluidine blue stained .

FIGURE 2 Electron micrograph and photomicrograph (inset) of the cortical reaction and the formationof the activation calyx (AC) . Two spermatozoa (S) are attached to the activation calyx by their acro-somes (A) . The inset shows an egg undergoing the breakdown of cortical granules to either side of at-tached spermatozoa (arrows), CG, cortical granule ; V, vesicles formed by the fusion of cortical granules("CG") with the oolemma ; *, contents of cortical granules which have been discharged into the peri-vitelline space (PS) . Fig . 2, X 25,500; inset, X 400, Epon embedded, toluidine blue stained .

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FIGURES 3 and 4 Small portions of the surface of activated eggs, ~t and 8 min after insemination, re-spectively, showing the hyaline layer (HL) and the perivitelline space (PS) . Arrows indicate rodlikeelements which have been discharged into the perivitelline space from the rod containing vesicles (RV) .*, indicate regions of the activation calyx (AC) with a laminated architecture . F, filaments located withinmicrovilli (MV) ; CG, cortical granule . Figs . 3 and 4, X 225 .500 .

cone is produced . Although the fertilization coneappears to be larger in polyspermic eggs, its mor-phology and ontogeny is similar to that previouslydescribed for monospermic eggs (Longo andAnderson, 1968) . Gamete attachment and fusiontakes place within the first 45 sec following in-semination, for sperm incorporation was notobserved at any time following this period .

Usually the spermatozoon rotates soon afterentering the egg ; however, occasionally sperma-tozoa either fail to rotate or remain for extendedperiods (2-4 min) in the fertilization cone . In thelatter cases, the spermatozoa appear to be in-volved in various phases of morphogenesis nor-mally associated with the reorganization of thesperm nucleus, i .e ., the vesiculation of the spermnuclear envelope and dispersion of the spermchromatin . Sperm which failed to rotate or com-

3 12 THE JOURNAL OF CELL BIOLOGY . VOLUME 46, 1970

menced pronuclear development within the fer-tilization cone were at the same stage of develop-ment as those which appeared to be incorporatedin the normal fashion (Longo and Anderson,1968) .Development of the male pronucleus in poly-

spermic eggs follows the same course as observedin monospermic eggs (Longo and Anderson, 1968) .All of the male pronuclei that developed withinpolyspermic eggs appeared to be associated witha sperm aster, a sperm mitochondrion, andflagellum throughout the later stages of develop-ment (Fig . 5 and inset) .

Migration and Fusion of the Pronuclei

Nicotine treatment does not alter the morphologyof the female pronucleus (Figs. I and 5, insets),

FIGURE 5 Two male pronuclei (a PN) associated with a female pronucleus (4 PN) . The inset is aphotomicrograph of two male pronuclei (d' pn) migrating to the female pronucleus (4 pn). NLB9,nucleolus-like body located within the female pronucleus ; SM, sperm mitochondrion . Fig. 5, X 13,000 ;inset, X 400. Epon embedded, toluiditw blue stained .

for it remains unchanged from the time of insem-ination until its encounter with male pronuclei(see Longo and Anderson, 1968) .About 10 min after insemination some of the

male pronuclei have migrated to the vicinity ofthe female pronucleus which, in most cases, hasmoved to the center of the zygote . The associationof the male and female pronuclei involves theintermixing of the constituents of the spermaster(s) with cytoplasmic matrix surrounding thefemale pronucleus . As in monospermic eggs theregions of the female pronucleus facing the ad-vancing male pronuclei become flattened andhighly irregular (Fig . 5) . Subsequently, the outerand the inner lamina of the male and femalepronuclear envelopes contact and fuse, therebyforming a zygote nucleus (Fig. 6, ZN) (Longoand Anderson, 1968) .Initially, the zygote nucleus has a number of

internuclear bridges which reflect the number ofmale pronuclei that have fused with the femalepronucleus over a given period (Fig . 8, inset) .The diameter of the internuclear bridges are ini-tially small ; however, they gradually increase indiameter, thereby forming a spheroidal or ellip-soidal zygote nucleus . Subsequently, the paternalchromatin is recognized as dense fibrillar massesat the various sites of fusion (Figs . 6, 8, and 11 ;PC) . Later, this material diffuses throughout thewhole of the zygote nucleus (Fig . 11, inset) .The number of male pronuclei which initially

fuse with the female pronucleus is usually one tothree. While the initial series of pronuclear fusionsis occurring the remaining male pronuclei arelocated within the peripheral cytoplasm or are invarious stages of migration towards the zygotenucleus (Fig . 7 and inset) .

Differentiation of the Male Pronuclei

Subsequent to the initial series of pronuclearfusions the remaining male pronuclei continue todevelop . This development yields male pronucleiwhich, in time, resemble a female pronucleus

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(Fig . 9) . Moreover, there appears to be a directrelation between the period a male pronucleusremains in the zygote cytoplasm and its resem-blance to a female pronucleus (see Discussion) .Male pronuclei, having structural characteristicssimilar to female pronuclei, have been observed ineggs treated with 0 .15 0/0 nicotine as late as 90 minfollowing insemination .

During the differentiation of the male pro-nucleus there is a continued dispersion of thechromatin which appears as a reduction in itselectron opacity and a further dissipation of itsfibrillar matrix (Figs. 9 and 10) . Concomitantly,the male pronucleus increases in volume and mayeventually become larger than a female pro-nucleus, i .e ., approximately 12 µ in diameter(Fig . 9, inset B) .Enlargement of the male pronucleus involves

an increase in the pronuclear envelope . Theincrease of the male pronuclear envelope appearsto occur throught the aggregation and fusion ofvesicles (see Longo and Anderson, 1968) .

Prior to the initial series of pronuclear fusions,male pronuclei were not observed to containnucleolus-like bodies; however, subsequent tothis phase of fertilization these intranuclearstructures appear (approximately 14 min post-insemination) (Figs. 7, 9, and 10) . At first thenucleolus-like bodies are rather small and sparse ;later they become larger and more numerousStructurally, the nucleolus-like bodies possess afine texture material and appear to be similar tothose found in the female pronucleus or the zygotenucleus. Nucleolus-like bodies have not beenobserved in the male pronucleus of monospermiceggs (Longo and Anderson, 1968) . However, theyhave also been observed in eggs made polysper-mic by chemical agents such as urethane andchloral hydrate (F. J. Longo and E . Anderson,unpublished observations) .

A continual migration of male pronuclei towardthe zygote nucleus occurs from approximately 15to 75 min postinsemination in cultures treated

FIGURE 6 Zygote nucleus (ZN) containing nucleolus-like bodies (NLBz) and a region occupied bypaternal chromatin (PC). Fig . 6, X 17,500 .

FIGURE 7 Electron micrograph and photomicrograph (inset) of a male pronucleus (d' pn) and a spermasters (a) migrating to a zygote nucleus (zn) 16 min following insemination . NLB d, nucleolus-like bodyof the male pronucleus ; C, centriole ; M, mitochondrion . Fig . 7, X 25,500 ; inset, X 400, Epon embedded,toluidine blue stained.

THE JOURNAL OF CELL BIOLOGY • VOLUME 46, 1970

FIGURE 8 Male pronucleus (d' PN) prior to its fusion with the zygote nucleus (ZN) . M, mitochondria ;AL, annulate lamellae ; C, centriole; SM, sperm mitochondrion ; NLBz, nucleolus-like body of the zygotenucleus ; PC, paternal chromatin . The inset is a photomicrograph depicting two male pronuclei (A and B)which are associated with the zygote nucleus (zn) via intranuclear bridges (inb) . Fig. 8, X 17,500 ; inset,X 400. Epon embedded, toluidine blue stained .

with 0.15% nicotine. During this period many

male pronuclei are closely associated with one

another (Fig . 9, inset A) . Although male pronucleiwere not observed fusing with one another, theyhave been observed in various stages of fusionwith regions of the zygote nucleus that were for-merly portions of male pronuclei (Fig . 10) .

Eggs treated with 0.15 0/0 nicotine undergosynchronous development for about the first 60min following insemination. Subsequent to thisperiod, many zygotes enter mitosis and cyto-kinesis while others continue to undergo morpho-genic events reminiscent of the later stages of fer-tilization, i.e ., pronuclear migration and fusion .

From 60 to 75 min postinsemination male pro-nuclei are not observed in mitotic embryos whereasnonmitotic zygotes invariably contain at least onemale pronucleus. From 75 to 90 min postinsemina-tion the number of mitotic and cleaving embryosincreases (up to 90-95%), and concomitantly

there is a decrease in the number of zygotespossessing male pronuclei. By 85 to 90 min post-insemination almost all of the zygotes are in thelatter stages of mitosis or cleaving, while in a fewembryos (5-10%) the zygote nucleus and severalremaining male pronuclei are in prophase (seealso Hertwig and Hertwig, 1887 ; Wilson, 1902and 1925) . As far as we were able to determine,in approximately 90% of the eggs treated withnicotine all male pronuclei that develop in thecytoplasm eventually fuse with the female pro-nucleus or zygote nucleus by 80 min postinsemina-tion. There is no indication that male pronucleidegenerate, i .e ., all sperm that enter the eggdifferentiate into male pronuclei .

Differentiation of the Sperm AsterDuring the period in which the male pronucleidifferentiate and migrate to the zygote nucleus,the sperm asters enlarge presumably by theacquisition of microtubules, endoplasmic reticu-lum, and annulate lamellae (Fig . 7, inset) . Thisenlargement appears to be proportional to theconcentration of nicotine used or more specificallythe number of incorporated spermatozoa (seebelow). Frequently, two or three pronuclei arelocated at the periphery of the zygote associatedwith one large sperm aster (Fig . 9, inset A) . Thepresence of such an association suggests thatseveral sperm asters have presumably coalescedsince all male pronuclei possessed sperm astersprior to the initial series of pronuclear fusions .

Fusion of Male Pronuclei with theZygote Nucleus

Subsequent to the initial series of pronuclearfusions, in approximately 90% of the embryos,all of the remaining male pronuclei progressivelymigrate to the center of the zygote and fuse withthe zygote nucleus. In cultures treated with highconcentrations of nicotine (0 .15-0.25%), as manyas six male pronuclei have been observed in thevicinity of the zygote nucleus. The fusion of malepronuclei with the zygote nucleus during theearly stages of development is structurally similarto that previously described for the male andfemale pronuclei (Fig. 8) . However, during laterstages of development, and presumably aftermany male pronuclei have fused, the zygotenucleus no longer exhibits the flattening and theextension of nucleoplasmic projections in the

direction of the advancing male pronucleus (Fig .9). Nevertheless, the zygote nucleus (and also thefemale pronucleus) appears to be receptive forfusion with male pronuclei along any portion ofits envelope in either a sequential or simultaneousfashion .

Fusion of the male pronuclei and the zygotenucleus brings about the incorporation of themale pronuclear envelope into the zygote nuclearenvelope, including those specialized portionsthat were derived from the sperm nuclear en-velope, e.g., the apical end and centriolar fossaregion (Fig. 11, arrow) .

With continued development the zygote nucleusincreases in size until it is a large spheroid struc-ture, presumably due to the fusion of many malepronuclei (Fig . 11 and inset) . The size of the

zygote nucleus following the fusion of all of the

male pronuclei appears to vary directly with the

concentration of nicotine employed . For example,

in eggs treated with 0.15 11/0 nicotine the zygote

nucleus attains a diameter of about 30 µ. Thezygote nucleus of monospermic fertilization is

approximately 13 µ in diameter.

Concomitantly, the size and the number of

nucleolus-like bodies increases as does the cyto-

plasmic area surrounding the zygote nucleus .

Within this cytoplasmic area are microtubules,

annulate lamellae, and endoplasmic reticulum,much of which is apparently derived from the

sperm asters (Fig . 11). A number of centrioles (as

many as six have been observed) and sperm

FRANK J. LONGO AND EVERETT ANDERSON Nicotine-Induced Polyspermy

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FIGURE 10 Electron micrograph of a male pronucleus (d' PN) fusing with the zygote nuclear en-velope (ZNE) at a region (arrows) that once was a portion of a male pronuclear envelope (*) . PC, pa-ternal chromatin within the zygote nucleus (ZN) ; SF, portion of a sperm flagellum . Fig. 10, X 17,500 .

mitochondria and flagella are also located withinthis region (Figs. 10 and 11) .

DISCUSSION

The data presented in this paper have demon-strated that nicotine induces the eggs of the seaurchin Arbacia punctulata to become polyspermic,and that this effect appears to be primarily aresult of the agent's action on the female gamete .It has been generally held that polyspermy-inducing agents act by retarding the rate at whichthe cortical change is propagated over the surface

of the egg from the site of sperm entry (Lillie,1919). Although this may be the simplest andmost easily visualized mechanism to explain theobserved effects, results from the present studyand from others (Clark, 1938 ; Rothschild, 1953 ;Hagström and Allen, 1956) are difficult to recon-cile with this idea and suggest that the action ofnicotine may have an entirely different basis .

Induction of Polyspermy

Studies have indicated that the induction of poly-spermy by nicotine may be brought about by one

FIGURE 9 Two male pronuclei (d PN) adjacent to the zygote nucleus (ZN) approximately 80 minpostinsemination. NLBz, and NLBä, nucleolus-like bodies of the zygote nucleus and male pronuclei,respectively. Inset A is a photomicrograph of two male pronuclei located within one large sperm aster(sa) . Inset B is a photomicrograph depicting the large size attained by many male pronuclei (d' pn)during the latter stages of fertilization (approximately 40 min postinsemination) . zn, zygote nucleus.Fig . 9, X 17,500 ; insets A and B, X 400, Epon embedded, toluidine blue stained .

FRANK J. LONGO AND EVERETT ANDERSON Nicotine-Induced Polyspermy

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or more of the following : (a) a decrease in therate of the cortical reaction (Rothschild andSwann, 1950) or its failure (Hagström and Allen,1956) ; (b) partial fertilization of the eggs (Hag-ström and Allen, 1956) ; (c) abnormal hyalinelayer development (Hagström and Allen, 1956) ;(d) an elimination or retardation of the fast partialblock to polyspermy (Rothschild, 1953 ; 1954) ; and(e) an alteration of the gamete's surface in such amanner as to increase the probability of a success-ful sperm-egg collision, e .g ., the development of amore stable adherence of the sperm to the egg(Rothschild and Swann, 1950; Hagström andAllen, 1956) .Observations made in our study are most

easily reconciled, although not entirely, with thesuggestion that nicotine promotes a more stableadhesion of the gametes either in a qualitative orquantitative manner and therefore allows moresperm to fuse with the egg than normal. Severalfacts tend to support such a contention : (a) nico-tine appears to cause an irreversible adhesion ofthe sperm to the egg's surface, i .e . when thespermatozoa contact the egg many do not appearto be dislodged, and (b) experiments by Werleand Schievelbein (1965) have shown that rabbitblood platelets aggregate in the presence of nico-tine and that this aggregation may be due to achange in the electrical properties of the plateletplasma membrane. We recognize the fact thatplatelets are portions of megakaryocytes ; however,a similar alteration of the electrical or otherproperties of the oolemma may also occur upontreatment of the egg with nicotine which wouldfacilitate gamete adhesion. Morphological altera-tions of the oolemma and of material comprisingthe vitelline layer in nicotine-treated eggs havenot been observed with the electron microscope .Although the mechanisms of gamete plasma

membrane fusion are unknown, it is likely thatthis process requires a rather stable adhesion ofthe two gametes. Once the gametes firmly adhereto one another, they would then be free to fuse .Various factors, such as sperm-sperm interference,

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electrostatic forces, Brownian movements, etc .(see Hultin and Hagström, 1956 ; Curtis, 1967 ;Colwin and Colwin, 1967 a and b ; Allen andHagström, 1955 ; Hultin, 1956 ; Hagström andAllen, 1956), may impede or completely destroythe attainment of the proper state of adhesionfor gamete fusion. Therefore, not all of the spermthat contact the egg fuse or even adhere, and inorder for sperm incorporation to take place thevarious parameters associated with adhesion andfusion must be fulfilled . With so many and specificconstraints on the adhesion and fusion processes,the likelihood of polyspermy is reduced .

Under normal conditions, i .e. monospermy, at45 sec following insemination approximately 50%of the egg's surface has undergone the corticalreaction . By 60 sec the cortical reaction is com-pleted. The formation of the activation calyx andthe presence of dehiscing cortical granules nodoubt disturb the association of the sperm andegg, thereby reducing the probability of manygamete fusions . Micrographs demonstrating sper-matozoa apparently adhering to the activationcalyx as it is separating from the surface of the eggsupport this suggestion .

The Cortical Reaction and Formation of theActivation Calyx

Temporally there does not appear to be a decreasein the rate at which the cortical response is propa-gated and the way the activation calyx and hya-line layer are formed in nicotine-treated eggs .However, these processes are completed beforeequivalent events in untreated eggs. This decreasein the duration of the cortical response may be adirect consequence of the nicotine itself and/or ofthe fusion of more than one sperm to the egg'ssurface, thereby initiating the cortical reactionfrom many loci .

Anderson (1968) has observed that the devel-opment of the laminar structure of the activationcalyx occurs at the time of pronuclear fusion(approximately 10-12 min following insemina-

FIGURE 11 Electron micrograph and photomicrograph (inset) of zygote nuclei following multiplepronuclear fusions . PC,, PC2, and PCs indicate regions of the zygote nucleus where three male pronucleihave fused . The arrow indicates a region of the zygote nuclear envelope that was formerly a portionof the sperm nuclear envelope that was associated with the centriolar fossa . SM, sperm mitochondrion ;C, centriole ; AL, annulate lamellae ; L, lipid ; Y, yolk ; M, mitochondria, ER, endoplasmic reticulum ;GC, Golgi complex . Fig. 11, X 13,000 ; inset, X 400, Epon embedded, toludine blue stained .

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tion), and as the embryo continues to develop thestratum increases in thickness . In eggs treatedwith nicotine, differentiation of the activation

calyx occurs at about 2-4 min after inseminationand continues as the polyspermic embryos devel-oped .

The Male Pronucleus

As previously stated, all incorporated sperm

appear to differentiate into male pronuclei andwere never observed in cleaving zygotes or inblastomeres . This situation is unlike that found inthe pig where the majority of sperm nuclei inhighly polyspermic eggs experience little if anyswelling (Hunter, 1967) . Hunter (1967) suggeststhat differentiation of the sperm nucleus is ini-tiated by an interaction between elements of thespermatozoon and the egg which are present inlimited amounts. Thus, failure of the large pro-portion of sperm nuclei to undergo developmentmay be due to a deficiency in some specific com-ponent(s) in the cytoplasm, which is exhaustedduring the formation of a small number of malepronuclei. The existence of a similar ooplasmiccomponent(s), limited in quantity, which inter-acts with the sperm to bring about its differentia-

tion is not so readily evident as in the case ofpolyspermic Arbacia eggs, since all incorporated

spermatozoa differentiate into male pronuclei .

However, spermatozoa which have inseminatedoocytes (germinal vesicle stage) of Arbacia do notdevelop into pronuclei and form sperm asters(Franklin, 1965) . This lack of development hasbeen attributed to the absence of a specific cyto-plasmic component(s) necessary for the morpho-genesis of the male pronucleus (Lillie, 1914 ;Longo and Anderson, 1968) . Therefore, a cyto-plasmic component(s) exists in the mature eggwhich interacts with the incorporated sperm andis responsible, at least in part, for the developmentof the male pronucleus and the sperm aster .Experiments with polyspermic eggs, however,demonstrate that this cytoplasmic component(s) isnot present in limited amounts, at least accordingto the methods we have employed .

Development of male pronuclei in eggs madepolyspermic with nicotine is fairly well syn-chronized, indicating that sperm incorporationoccurs during a given time period . In connectionwith this, Anderson (1969) has found that there isa simultaneous incorporation of thymidine-3Hinto DNA in all of the male pronuclei observed inzygotes made polyspermic with nicotine .

Increase in the size of the male pronuclei and

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dispersion of the sperm chromatin persists through-out all stages of development. Similar changeshave also been noted by Gurdon (1967, 1968) innuclei transplanted into Xenopus eggs. In nucleartransplantation experiments it is uncertain towhat extent the increase in volume is necessaryfor subsequent changes in nucleic acid synthesis(Gurdon, 1967) . Gurdon (1968) states that :" . . . nuclear swelling does not itself induce anyone kind of nuclear change, but should be re-garded as a process of derepression, the result ofwhich is to make chromosomes more reactive tothe particular cytoplasmic environment in whichthey happen to lie" (see also Gurdon and Wood-land, 1968) .

Enlargement, further chromatin dispersion, and

the acquisition of nucleolus-like bodies yield malepronuclei which possess many characteristicsobserved in the female pronucleus . An example ofa naturally occurring situation where the male

pronucleus attains the same or similar structuralcharacteristics as the female pronucleus is foundin the Ascaris type of fertilization (Wilson, 1925 ;

Longo and Anderson, 1969 a and b) . Wilson(1925) suggested that fertilization may take essen-tially two forms, depending upon the morphologyof the male and female pronuclei when they

become associated . The two forms are: (a) Thesea urchin type of fertilization and (b) the Ascaristype of fertilization .

One of the characteristics of the Ascaris type of

fertilization is the large size attained by the malepronucleus, in many cases becoming as large asthe female pronucleus (see Longo and Anderson,1969 a and b) . The sea urchin type of fertilizationis characterized by the dramatic inequality of thepronuclei at the time of fusion (see Longo andAnderson, 1968) . This difference in the size of themale pronuclei is closely correlated with themeiotic state of the egg and the time it is normallyfertilized (Wilson, 1925) . Artificial prolongationof the interval between the entrance of the spermand the fusion of the pronuclei in the sea urchinegg may cause the phenomena of the sea urchintype of fertilization to take on, more or less com-pletely, the character of the Ascaris type of fer-tilization (Wilson, 1902) . The observations pre-

sented here confirm this suggestion.

Development of the Sperm Aster andPronuclear Migration

The increase in size of the sperm asters with con-

tinued development appears to be due to the

accumulation of large quantities of microtubules,endoplasmic reticulum, and annulate lamellae .Although the egg is richly endowed with a randomdispersal of annulate lamellae and endoplasmicreticulum, very few microtubules are found otherthan those associated with the sperm aster . Theorigin and method of assembly of these lamellar-tubular structures is unknown.Concomitant with the enlargement of the

sperm aster, the male pronuclei appear to beconfined to the peripheral aspect of the zygote .The peripheral displacement of the male pro-nuclei may be due to the impedence of their mi-gration which is a result of a physical hindranceby the enlarged sperm asters . Such an associationwould account for the asynchrony that occurs atthe later stages of development and the delay inmitosis observed in nicotine-treated embryos .Studies of polyspermy in frog eggs have indicatedthat pronuclei may be restricted during theirmovements due to the physical obstruction andgrowth of adjacent sperm asters (see Morgan,1927 ; Rothschild, 1956), and lend support to thissuggestion .

Our findings indicate that in most cases the

female pronucleus moves from a peripheral posi-tion to a more central one following insemination .Concomitantly, the male pronuclei appear tomigrate to the center of the zygote followingtheir development. It is possible that the pro-nuclei are primarily "attracted" to the center ofthe zygote and secondarily to each other . Themovement of the female pronucleus from aperipheral to a central location in the egg follow-ing activation with sperm or chemical agents (M .Sachs and E. Anderson, unpublished observations)tends to support this suggestion (see also Wilson,1925; Tyler, 1955) . How pronuclear movement isaffected is unknown ; however, agents whichinhibit microtubule development (e .g . colcemid)delay pronuclear migration and fusion (F. J .Longo and E . Anderson, unpublished observa-tions; Zimmerman and Zimmerman, 1967) .

Multiple Pronuclear FusionMultiple nuclear fusions normally occur in plantswhere there is (a) a fusion of a male nucleuswith the female nucleus and (b) the fusion of asecond male nucleus with the fused polar nuclei,yielding the triploid nucleus of the endosperm(Jensen, 1964) . The fusion of more than twohaploid nuclei in the case of the sea urchin Arbaciapunctulata is pathological .

Although we could not determine unequivocally

whether or not male pronuclei fuse with one

another, they appeared to fuse sequentially orsimultaneously at any locus along the femalepronucleus or zygote nucleus and along regions ofthe zygote nucleus that were once portions of themale pronuclear envelope . These observationstestify to the lack of structural or spatial specificityof the fusion process. Austin (1961) has observedthat in the polyandrous rat egg the male pro-nuclei may approach and contact each other asfrequently as a male and a female pronucleus,therefore indicating the lack of specificity in theforces that draw the pronuclei together .

In contrast to the situation that exists in manyurodeles, reptiles, birds, and insects where the

eggs are normally polyspermic (Rothschild,1954), in most instances all pronuclei fuse to-gether to form one zygote nucleus in nicotine-treated Arbacia eggs. Fankhauser (1948), investi-gating polyspermy in salamanders, showed that

the male pronucleus closest to the female pro-nucleus following meiosis becomes associated withit to produce the diploid state of the embryo .

Subsequently, the supernumerary male pronucleidegenerate at various times according to theirdistance from the associated male and femalepronuclei.

Although our findings suggest that the prox-imity of the male pronucleus and its associatedstructures induces morphological alterations ofthe female pronucleus, there appears to be agradual decrease in the response of the zygotenucleus as greater numbers of male pronuclei fusewith it (see also Longo and Anderson, 1969 a and

b) .

Mitosis and CleavageThe delay of mitosis and cleavage may be due toan "inhibitory effect" of the male pronuclei . The

concomitant increase in the number of sperm in-corporated and the delay of cleavage supportssuch a suggestion . These results are in direct con-trast to those reported by Rothschild (1953) whostates that polyspermic sea urchin eggs (Paracen-trotus lividus) undergo their first cleavage markedlyearlier than monospermic eggs . The reason forthis discrepancy is not clear ; however, it shouldbe pointed out that the experimental design andorganisms employed in both cases differed . One

cannot exclude the possibility that nicotine iscausing the delay in cleavage directly ; however,this appears to be unlikely since other poly-spermy-inducing agents (e .g., urethane andchloral hydrate) also delay cleavage in a similar

FRANK J. LONGO AND EVERETT ANDERSON Nicotine-Induced Polyspermy

323

manner (F . J . Longo and E . Anderson, unpub-lished observations) .

Despite the treatment and the conditions im-posed by polyspermy, the sperm and egg organelles

continue to undergo specific morphological proc-esses normally observed during monospermy . Theresults of this study indicate the high degree ofindependence of the cellular operations andorganelles which allow for their individual modi-fication and modulation with only a temporary

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