ARTIFICIAL PARTHENOGENESIS AND .FERTILISATION. 479

Artificial Parthenogenesis and Fertilisation:A Review.

ByThomas II. Bryce.

THIS article is an effort to gather together, in so far as theyrelate to the phenomena of fertilisation in the sea-urchinegg, the results obtained by experiments. It does not pre-tend to consider the problem of fertilisation as a whole; northe phenomena save in Echinoderms, and no attempt will bemade to establish comparisons with other forms in which thedetails may to some extent differ. The limitation to one formis in so far appropriate, that practically all the experimentshave been made on Echinoderm eggs.

I have personally studied fertilisation in the egg of Echinusesculent us—specially in sections,—and though I havenothing fresh to add to the description of the facts, thisarticle may in a measure be considered a sequel to a paperon maturation in the same form. In that paper my attentionwas chiefly directed to the chromosomes, and I did not followout the results of observers in the experimental field, butas some of the phenomena described are of interest in con-nection with these results, I shall take the opportunity ofreturning to them.

The two mitotic divisions characteristic of the maturationphases, differ markedly from those which take place in thesegmentation phases. In many respects there is a closeresemblance to phenomena observed in the eggs of Toxo-pneustes, which develop parthenogenetically under theinfluence of magnesium chloride solution (Wilson, 1901).

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480 THOMAS H. BEYOE.

On the dissolution of the nuclear membrane the site of thegerminal vesicle is occupied by a " kinoplasmic" mass,derived either entirely from the nuclear network, or alsopartly from protoplasm differentiated on the distribution ofthe nuclear substance into it. In fixed material this area hasa fibrillar appearance. This may be the result of the fixingreagents used, but in any case it indicates the accumulationat this part of protoplasm which has undergone some changein constitution physical or chemical. In the nuclear area,out of this material, asters are formed, and ultimately thefirst polar amphiaster. Besides the asters concerned inthe formation of the bipolar figure, there are secondaryasters, which seem to have only a temporary existence.In some few cases multipolar figures were observed. Nostructure recognisable as a centrosome or centiole was foundbefore the germinal vesicle broke down, and therefore thecentrosome was either derived from the nucleus, or formedde novo in the nuclear area. The astral radiations areconfined to a small and superficial part of the egg, and a veryunequal division results in the formation of the polar bodies.When the two divisions are over all the radiations and theremains of the kinoplasmic area disappear, the cytoplasmassumes its alveolar structure throughout, the nucleus retiresfrom the surface, and no centrosome can be recognisedin relation to it.

On the breaking down of the germinal vesicle the greaterpart of the nuclear material disappears as such, and not onlyis a change in the constitution and distribution of the proto-plasm to be recognised, but experiment proves that the egghas undergone a physiological chauge of state. Whereas aspermatozoon can neither fertilise an egg with the germinalvesicle intact, nor a fragment without the nucleus (Delage,1901), after the polar bodies are formed the egg becomescapable of fertilisation.1 This cytoplasmic maturation, de-pendent, probably (Delage, 1901), on the influence of the

1 For evidence and theories regarding the influence of the stage of matura-tion in Amphibian eggs see Bataillon (1901).

ARTIFICIAL PARTHENOGENESIS AND FERTILISATION. 481

nuclear sap set free from the germinal vesicle, is accompaniedby the conversion of the large vesicular nucleus related to themetabolic changes underlying the growth of the ovum, into asmall morphologically equivalent nucleus, possessing the samenumber of chromosomes as the sperm nucleus. This has beenproved by their enumeration when the nuclei undergo in-dependent transformation, and the number is one half thatfound in the segmentation divisions.

The eggs thus matured remain, in the case of the sea-urchin,for a considerable time quiescent within the ovaiy before theyare discharged—for the process of ripening in the ovaryis a gradual one. When discharged into sea water it seemsthat, like the eggs of some other forms (0. Hertwig, 1893,p. 239), after lying for- many hours unchanged, the sea-urchin eggs show spontaneously, karyokinetic transforma-tion; for instance, R. Hertwig (1896) observed in eggs whichhad been deposited- prematurely during transport, analogouschanges to those produced by treatment with strychnine.This phenomenon is one apparently of wide range. In aninteresting review entitled " Giebt es bei Wirbeltieren Par-thenogenesis " (1900), Bounet, after - examination of all theliterature up to that date, comes to the conclusion that,according to our present knowledge, the phenomena in verte-brates are due to degenerative divisions, and in meroblasticeggs to fragmentations, and the alleged parthenogeneticallydivided tuba], uterine or laid eggs, are either over-ripe, audtherefore badly fertilised, or are eggs normally fertilisedwith defective spermatozoa. In the light of the facts ofartificial parthenogenesis, it may be that this segmentationin unfertilised eggs, at least in certain invertebrates, is aneffort in the direction of true parthenogenesis which is abor-tive, the egg dying before the tardy process is accomplished.

In 1876 Greeff described parthenogenetic development inA.sterocanthion. The eggs were obtained from animalsearly in the season, before the spermatozoa were mobile, andthe blastulse formed differed from those produced in normalfertilisation. 0. Hertwig (1890) recorded some observations

482 THOMAS H. BRYCE.

on spontaneous parthenogenesis. In confirmation of Fol, hefound that eggs from fully-matured animals did not segmentspontaneously, but only after a considerable time underwentchanges considered pathological. The nucleus enlargedmore and more, and after ten to fifteen hours the eggsdied and fragmented. Only among hundreds of eggs hereand there one had divided into two. At Trieste, however,in a season when the animals were late in maturing, and at atime when males were rarely got, he observed in a limitednumber of cases (in Aster ias glacialis , and Asteropecten) that after the polar mitosis had occurred, the nucleusdid not come to rest, but continued to divide. There resultedan irregular division, but here and there a blastula was foundwhich had no vitelline membrane. Into the interestingobservations and suggestions regarding the failure of thesecond polar body extrusion, and the union of two vesicularnuclei in the egg, we cannot here enter. The main pointestablished was, that fully-matured eggs did not developparthenogenetically, but that in some few cases immatureeggs did divide irregularly, and in a small number of casesblastulte were formed. A number of observers havedescribed the occurrence of natural parthenogenesis inBchinoderms, and it is an open question; but apart from itspossible relation to immaturity of the ovum, the sources oferror in the matter of infection by spermatozoa are so many,and the causes which artificially start parthenogeneticdevelopment in certain cases are so slight, that all cases ofso-called " natural parthenogenesis " are open to suspicion,but even granting that it may occur, it is a matter of no greatmoment in the question of " artificial parthenogenesis." Itwould be only additional evidence of the fact, that there is inthese forms a tendency • to parthenogenetic development,which, however, does not normally occur.

Mitotic division may be excited in unfertilised eggs in avariety of ways.

First, by increasing the degree of concentration of thesea water (Morgan, Hunter), or by increasing the osmotic

ARTIFICIAL rAETHENOGENESIS AND FERTILISATION. 483

pressure in various other ways (Loeb). This may bodone by adding various inorganic salts to the sea water,especially the salts of magnesium, potassium, sodium, andcalcium, in definite proportions (Loeb, Morgan, and others),or by adding sugar or urea (Loeb). Other salts have alsogiven results, e.g. chloride of manganese (Deluge). Theeffect is produced when the eggs, after being left fromhalf an hour to two hours in the solution, are transferred topure sea water, and is due to the disturbance of the osmoticpressure leading to loss of water by the egg, followed byrehydration (Bataillon, Loeb, G-iard, etc.), not to specificchemical stimulation.

The nuclear activity may also be roused by other cliemicalbodies, as strychnine (Hcrtwigs, Morgan), chloroform, ether,alcohol, by lack of oxygen (Mathews), by very dilute hydro-chloric acid (Loeb, Delage).

Further, purely physical agents may have the same effect—heat (Mathews, Bataillon, Delage, Viguier), cold (Morgan,Groeley), and, most important, agitation (Mathews). Mathewshad previously proved for Asterias, and Morgan for Arbaciaalso, that shaking of unripe eggs caused them to form thepolar bodies—the shaking presumably causing dissolution ofthe nuclear membrane. Ripe eggs of Asterias, but not of sea-urchin, act in the same way, but only after they have lainsome time in water; after two hours larvae begin to appearon shaking; after four hours, hard shaking produces a largeproportion of larvaa, and the mere transference of the eggsby a pipette from one vessel to another is sufficient to form afew larvae. A few hours later the slight amount of shockexperienced in the transference of the eggs, causes a largenumber to begin to develop, though they do not go beyondthe late segmentation stages. At this time shaking causesall to develop, but none reach the blastula stage. Loeb andFischer have extended this observation to the Annelids,Chaatopterus and Amphitrite.

The mitotic phenomena produced artificially are apt to beirregular, and the division of the cell body is often unequal

484 THOMAS H. Bit YOU.

when it occurs. Thus the nucleus may divide repeatedlywithout division of the cytoplasm, and then the eggmay break into as many segments as there are nuclei(Wilson and others). It is only in a relatively small pro-portion of eggs that division is regular enough to permit ofdevelopment to the larval stage. Further, the eggs of thesame species behave capriciously to the same agents underdifferent conditions (temperature, etc.), and the eggs of closelyallied species seem to react differently to the same agent.

There is no reasonable doubt, however, that true artificialparthenogenetic development has been demonstrated for theEchinoderms—sea-urchins and star-fish—and for at least tvvoAnnelids, though the same amount of independent testimonyis not available for the latter.

Actual development to a larval stage has been obtainedonly by certain of the agents enumerated above.

1. Increase of Osmotic Pressure.—The most successfulresults (Loeb, 1902) are obtained at a temperature about20° C, by the addition of the chlorides of potassium or sodiumto sea water, the optimum degree of concentration beingdetermined by experiment for each set of observations.1

After the eggs have remained in this for half an hour to twohours, the optimum being again tested by experiment, theyare restored to normal sea water.

Sea-urchins (Loeb, Wilson, Giard, Prowazek, Delage,Viguier, Hunter). Annelids : Cbsetopterus, Amphitrite,Nereis (Loeb, Fischer).

2. Agitation. — Asterias (Mathews), Chaetopterus andAmphitrite (Loeb, Fischer), but not sea-urchins (Mathews,Viguier).

3. Elevation of Temperature.—Asterias during maturation(Delage) ; not for ripe eggs (Greeley, Yignier).

4. Depression of Temperature.—Asterias (Greeley); notsea-urchins (Viguier).

1 Loeb (1902) uses a stock solution of 2J n. HC1, and adds tliis in differentproportions, 8, 10, 12, 14, 16, IS c.cm., to 100 c.c. of sea water in sixvessels to determine the best grade of concentration.

ARTIFICIAL PARTHENOGENESIS AND FERTILISATION. 4 8 5

5. Exposure to weak HC1 iu sea water, and subsequentrestoration to pure sea water. Asterias (Loeb, Delage).

6. Continuous exposure to a solution of a specific chemicalsubstance at the same osmotic pressure as normal sea water.

Potassium Chloride. Chaetopterus (Loeb).Calcium Chloride. Amphitrite (Fischer).The Ions of potassium and calcium are said to be specific

for these forms respectively.1

With regard to the influence of the state of maturation,Delage gives results to show that in A s t e r i a s glacialis,when the eggs are placed in sea water to which is added anequal quantity of a solution of HC1, raising the molecularconcentrations of the mixture to 0-660, different results aregot according to the stage of maturation. Among the eggsplaced in the liquid before maturation, 20 per cent, ofblastulse were got, at the appearance of the first polarbody 95 per cent., and after the appearance of the secondpolar body 5 per cent., while none of the controls showedany normal segmentation.

From all this it seems that changes in the osmoticpressure between the egg and its surrounding medium, andmechanical agitation, are the chief agents so far as yet

1 Delage (' Compt. llendus de 1'Acad. des Sciences,' October 13th and 20th,•1902) announces that lie has found an agent which is as certain and effectiveas the spermatozoon, in producing development to advanced larval stages, inAsterias. I t is sea water aerated by carbonic acid gas, and at the same osmoticpressure as ordinary sea water (or lower ?). When the eggs, at what liecalls the "critical stage"—i.e. when the nuclear membrane of the germinalvesicle is dissolved, up to the expulsion of the first polar body—are placed inthis, and after one hour transferred to pure sea waier, practically all the eggsdevelop. His view is, that the maturation is arrested temporarily, and onrestoration to pure sea water, the carbonic acid gas is quickly eliminated anddivision proceeds ; but it is not partial, as in the polar mitoses, but complete,and goes on to the formation of the normal larval forms. The result is notobtained at a stage after the polar bodies are extruded and the ovum has againcome to rest, nor is it applicable in sea-urchin, in which the maturation isover before the ova are shed. His theory as to the action of the gas is, thatit is a temporary poison which arrests maturation completely, and is quicklyremoved afterwards without altering the characters of the protoplasm.

486 THOMAS H. BRYOE.

known, which tend to the production of artificial partheno-genesis, but that in the case of the Annelids there is evidenceto show that certain Ions may have a specific effect.

According to Loeb (1902) the solutions must act, first, byfavouring the solution or dissolution of the nuclear mem-brane; and second, by changing, in some sense, the physicalproperties of the protoplasm (viscidity, etc.).

Mathews (1900), as a conclusion from his experiments onAi'bacia eggs, pointed out that the known methods of causingliquefaction in protoplasm will induce karyokiuesis in theseeggs, and also shows that loss of water has a liquefyingaction.

Before considering further the bearing of the physiologicaland physico-chemical conceptions regarding fertilisation, Ishall proceed to the morphological changes which have beendescribed in unfertilised eggs which undergo parthenogeneticdevelopment.

R. Hertwig (1896) studied the changes in the egg aftertreatment with strychnine. On the breaking down of thenucleus, half spindles, aud in a few cases whole spindles,supposed to arise from the fan spindles, were formed. Thefan spindle fibres he regarded as derived from the achro-matic network of the nucleus. The chromosomes derivedfrom the nucleoli became attached to the primary rays.Later, protoplasmic rays also appeared, centering on thefocal point of fclie half spindle. At this central point, andderived from the central parts of the rays, there appeared arounded body resembling in every way a centrosome, though,none such was to be found before the nucleus broke down.The body was an ovocentrum, formed from the achromaticportion of the nucleus, and, according to Hertwig, the

•individualised cenfcrosome is ultimately a derivative of thenucleus—is, in'fact, an achromatic nucleus.

Doflein (1897), contrariwise, examined the phenomenaof karyokinesis of the sperm nucleus in eggs which, afterfertilisation, had been treated by chloral solution after themanner of the experiment of 0. and R. Hertwig. The nuclei

AttTTPICrAI PARTHENOGENESIS AND FERTILISATION. 487

did not unite, but underwent independent transformation.Doflein, like R. Hertwig, considered the middle piece of thespermatozoon as equivalent to the centrosome, and from theexperiments concluded, that from the centrosome a completespindle could form, and out of this, again, the achromaticnuclear network. Thus, compared with Hertwig's results, theripe sperm nucleus contains all the parts, even as the ripeegg nucleus, which are necessary for a further development.

In Hertwig's results we have evidence of a centrosomearising from the nucleus de novo. Morgan, in 1896,described the formation of artificial astrospheres in thecytoplasm of the eggs of Arbacia treated by salt solutions,and from his further observations published in 1899 and1900 he decided, that in spite of certain differences theseartificial astrospheres corresponded to the normal sphereswhich occur at the apices of the spindles in the segmentationstages; further (1900), that both artificial and normal spheresaredue to accumulation of a specific substance,and that theyolkspheres are excluded from- the substance of the astrosphores.

His view of the astral radiations is that they serve totransport the chromosomes, but are not concerned in thedivision of the cytoplasm.

Evidence of free formation of the centrosomes is foundalso in the appearance of asters in the cytoplasm in variousforms, Echinus among them, on the breaking down of thegerminal vesicle in maturation.

Boveri (1901) in essence accepted Hertwig's definition ofthe structure described by him as an ovocentrum, and itsorigin apparently de novo. He argued that phylogeneticallythe centrosome is an individualised cytocentrum, derivedfrom a centro-nucleus in which the centrosome or its

-equivalent is not differentiated from the chromatin nucleus.To the nucleus of the sea-urchin egg must necessarilybe attributed the properties of a centro-nucleus, with thecapacity of producing out of itself, under the action ofcertain stimuli, individualised centrosomes, when such fail tobe supplied in the normal way in fertilisation. If even under

488 THOMAS H. BR.YCE.

similar conditions, the sperm centrosome be present, the cyto-centrum remains latent. The centrosome looked at in thisway, is not a specific cell organ in the sense that it mustconsist of a specific chemical substance, but that parts of asubstance contained in the nucleus, undergoing certainchanges, and grouping themselves together, are organisedinto a centrosome.

Thus the ovocentrum of the sea-urchin egg is not to beconsidered an individualised centrosome, but an intra-nuclear latent cytocentrum, and the nucleus is a centro-nucleus. Thus the centrosome in such a case is not some-thing strictly new, but arises by the transformation in adefinite manner of a cytocentrum already present. It is acase not of new formation, but of " reparation." "GervisseOentronuclei sind im stande unter bestimmten BedingungenCcntrosomen zu reparieren."

Morgan's artificial astrospheres lie did not admit to havetrue centrosomes—the essential character of capacity fordivision was not proved for them.

This bring3 me to Wilson's very interesting and importantpaper on the morphological phenomena in parthenogeneticeggs.

The main results are that under the influence of themagnesium chloride solution, not only are asters producedde novo in connection with the nucleus, but also in thecytoplasm. " Not only the asters connected with chromo-somes (nuclear asters), but also the supernumerary astersunconnected with nuclear matter (cytasters), may multiplyby division; the cytasters contain deeply staining centralgranules indistinguishable from centrosomes, that divideto form the centres of the daughter asters. These astersoperate with greater or less energy as centres of cyto-plasmic division. Typical cytasters, often containing deeplystaining central granules resembling centrosomes, are formedin the magnesium solution in enucleated egg fragmentsproduced by shaking the unfertilised eggs to pieces, andthese asbers likewise may multiply by division, though

ARTIFICIAL, PAUTIIENOGKNISSIS AND I1 JS UTILISATION. 4 8 9

no cytoplasmic cleavage takes place. The cleavage centro-somes first make their appearance outside the nucleus, butdirectly on the nuclear membrane, and the evidence rendersit nearly certain that they arise by the division of a singleprimary egg centrosome that is formed de novo. All theevidence goes to show that the cleavage centrosomes are ofthe same general nature as the central bodies of the oytasters."

Among many interesting details I will refer here only tothe changes described for eggs which underwent segmenta-tion, and were capable of developing into swimming embryos,because in certain particulars they are reminiscent of whattakes place in the formation of the first polar amphiaster.

I may summarise as follows :—(1)' The first change thatoccurs is a coarsening in the appearance of the protoplasm,

.better marked in eggs treated by stronger solutions. (2) Aprimary radiation appears centering on the nucleus, bettermarked in eggs treated with weaker solutions. (3) A varyingnumber of secondary radiations appear in eggs especiallytreated with stronger solutions. The extent of the primaryradiations is inversely in proportion to the number of thesecondary radiations. These latter appear as vague clearspots in the cytoplasm, which gradually become surroundedwith radiations, and finally assume the form of asters. Theyalways appear in situ, and do not change their position till a,later period. (4)'Coincident with the appearance of theradiations there is a gradual growth of the nucleus. (5)Round the nucleus appears a clear perinuclear zone of hyalo-plasm. (6) The nuclear membrane fades out, and a vagueirregular clear space is left, to which the hyaline zone con-tributes. (7) The rays then diminish, and, indeed, almostdisappear.

The eggs at this point were restored to pure sea water, andafter a pause the radiations reappear and advance centri-fugally towards the periphery. In eggs capable of develop-ment the principal rays are now focussed on two centres atopposite poles of the nuclear area, which now forms a spindleconnecting the two asters. If the amphiaster is typical,

490 THOMAS H. BIIYCE.

division proceeds as in normal fertilisation. If more thantwo asters are formed from the nuclear area, multipolar figuresform, and irregular cleavage results. If there is only a singleradiation which does not resolve itself into a bipolar figure,the egg never properly segments, but there are regularlyalternating phases of nuclear transformation.

Analysing the meaning of the phenomena, Wilson says, " Wemay therefore state that the first general effect of the stimulus,whether the magnesium solution or the spermatozoon, is to

• arouse an activity of the cytoplasm, one result of which is theestablishment of a centripetal movement of the hyaloplasmtowards one or more points at which the hyaloplasm accumu-lates." The rays in this view are the expression, in part atany rate, of centripetal enrrents, and the substance flowingin, is the hyaloplasm or interalveolar substance. The hyalo-plasm spheres at the centres oC the asters are local accumula-tions of this hyaloplasm. In fixed material, studied insections, the radiations are fibrillar in appearance, and asthey stain much more deeply than the general networkthe hyaloplasm in the rays must probably have undergonesome physical or chemical change. The centrosome is awell-defined body of considerable size and of spongy con-sistence, composed of intensely staining granules, which oftengive the centrosome the appearance of a minute nucleuscontaining a chromatin reticulum. The hyaloplasm spheresin the living egg correspond to the centrosome, the clear arearound it, and the innermost darkly staining radiated zone ofthe aster taken together.

Thus Wilson lias proved that structures which cannot bedistinguished morphologically from " true centrosomes"appear in the cytoplasm de n o v o ; and further/ that theydivide to form the apices of bipolar figures, even in enucleatedfragments.

In a recent paper Meves (1902), using Boveri's nomen-clature, expresses the view that the centrosome is only themantle of the centriole, and is only present in rapidly-dividingcells like the blastomeres. The " Doppelkorchen" of the

ARTIFICIAL PARTHENOGENESIS AND FERTILISATION. 4 9 1

tissue-cells are to be considered, as centrioles, and " nur vonden Centriolen nicht aber von den Centrosomen, kann dahergelten, dass sie allgemeine und dauernde Zellorgane sind."The results of Morgan and Wilson can only then be held toprove that centrioles under certain conditions may, by theaction of salt solutions, be excited to form centrosomes andradiations round them, for their results might be explained bya multiplication of the two centrioles which the egg hasderived from the last division of the division period, and thedistribution of these centrioles through the cell. Even inenucleated fragments there is no proof that the fragment didnot contain the centriole of the cell.

Such a supposition admits of neither proof nor disproof,and the presence of a free "centriole" in the unfertilisedsea-urchin egg has not been demonstrated. I have seen inyoung oocytes minute bodies, stained black with ironhsematoxylin—sometimes double bodies,—but I have not beenable to' convince myself that they are more than accidents ofstaining and fixing.

Turning now to the phenomena of fertilisation in the sea-urchin, there is to be recognised (1) a local stimulation atthe place of contact of the chosen spermatozoon,1 manifestedby the streaming out of the protoplasm to form the entrancecone. (2) A general stimulation, manifested by the throw-ing off of the vitelline membrane, and by a change in theconstitution of the protoplasm. It becomes more viscid for atime (Morgan); a funnel-shaped area of darkly staining sub-stance follows the path of the sperm head (Wilson). (3)A protoplasmic movement focussed on the situation of themiddle piece giving rise to the sperm aster. This appearssoon after the entrance of the spermatozoon, when the headhas begun a movement of rotation. The rotation goes on

Buller (1902) has studied tlie question of the bearing of chemolaxis onfertilisation in Echinodfirms. His conclusion is that cliemolaxis plays norole in bringing the sexual elements together. The meeting is a matter ofchance. The passage through the gelatinous coat is radial in direction, andprobably purely mechanical, though possibly due to stereotaxis.

492 THOMAS H. BltYCH.

through 180° till the base of the conical sperm head isdirected inwards. The rays of the aster now extend widely,and at their centre is a clear area. Meantime the sperm headbecomes converted into a small round nucleus. The move-ment of the sperm head is, at first, radial; then there is achange, and it assumes a new direction towards a point notquite in the centre of the egg; when this change of path istaken up the egg nucleus begins to move towards the pointwhere the nuclei ultimately meet (Wilson and G-iardina).The aster now comes in contact with the egg nucleus, andas the nuclei approach, the clear area at its centre spreadsout over its side. The aster then divides and the nucleiconjugate. The radiations now die down during a pause inwhich the nucleus grows in size (Wilson), to redevelop againfocussed at the poles of the nucleus.

According to Hertwig, Doflein, Erlanger, and Wilson'searlier account, the centrosome corresponds to the wholemiddle piece, but later Wilson described the middle piece ascast aside, and in the centre of the aster is a small darkly-staining granule. Boveri (1901) represents the spermcentrosome as a spherical body smaller than the middle piece,and containing within it two centrioles shortly after itsentrance into the egg.

Various other observers have represented a dark-staininggrannie at the centre of the aster. My own observationsare inconclusive, and do not warrant me in expressing anopinion.1

1 The character of the fully-formed ceul.rosomc in the sea-urchin egg isstill subject to difference of opinion. The form in which I see it in osniicacid material is that of a largish sphere of very Gnely alveolar structure. InWilson's papers on magnesium and etherised eggs, "it appears as a well-defined body of considerable size, consisting of intensely stained granules,wliicli often give the centrosome exactly the appearance of a miuute nucleuscontaining a curomatin network." This becomes in the antiphases more homo-geneous, and flattens down into a plate-form, which in the telopliases oftenlies directly on the membrane of the newly-formed nucleus precisely as Boveri(1901) has described for Echinus. Boveri (1901) represents it in severalforms. In one set of preparations it is a largish sphere of very finely alveolar

ARTIFICIAL PARTHENOGENESIS AND FERTILISATION. 493

The essential difference between the processes seen inmagnesium eggs and normal fertilisation is that whereas infertilisation there is only one, and that a definitely localisedpoint of astral activity, in the magnesium eggs there are anumber of foci, and development in large measure dependson the accident of their number in the nuclear area.

There is the same want of unity of purpose that is seen inpolyspermic eggs, in which the number of points oE astralactivity depends on the number of spermatozoa which gainan entrance.

It has long been recognised that the union of the nucleiand the initiation of division are co-ordinated, but in ameasure independent factors in fertilisation. Partheno-genetic development under artificial agents is the latestproof of this. The possibility of the development ofenucleated egg fragments when entered by a spermatozoon,as described by Boveri, and afterwards named merogony byDelage, is another. Either nucleus is sufficient in itself.

With the problems underlying the nuclear conjugationthis article is not concerned. It starts from the assumptionthat the union of equivalent nuclei is the end of fertilisation,but not the means (Boveri).

The cause of the nuclear conjugation is not as yet under-stood. The first possibility is that the aster is concerned inbringing them together. Giardina (October, 1902) bringsthe latest suggestion on this line. Starting from the basisof the alveolar structure of protoplasm, he suggests that theaster is the expression of both centripetal and centrifugalcurrents. The centrosome is concerned in the diffusion ofchemotrophic substances into the egg, while at the same timestructure. In auother set, in which the centrosome had reacted differently,there is a centriole within tlie centrosome, which divides before the centrosome,so that it is double in tlie metaphase. In Wilson's earlier account there wasno central body, but in later descriptions there was a mass of granules in awell-defined sphere, which succeeded a single granule of earlier stages. Inmy previous paper, I regret that I misrepresented Professor Wilson's nomen-clature by referring to this as his centrosome. The sphere, as a whole, isnamed the centrosome. See note to page 314, "Tlie cell, etc.," 1900.

494 THOMAS H. BR.YCK.

the hyaloplasm flows in towards the centre. He points outthat the germ nucleus does not move till the rays of the asterhave reached it, and the aster has assumed a position ofequilibrium towards the centre of the egg. The union isthus the result of the chemotactic forces of which the asteris the expression.

Wilson (1901 B) shows, however, that the nuclei mayunite in the entire absence of an aster. When eggs, im-mediately after fertilisation, are placed in a weak solution ofchloi'al (0. and R. Hertwig), or ether (Wilson), no aster isdeveloped, but when replaced in sea water the rays reappearand the nuclei unite. In a certain proportion of cases,which will be referred to later, the nuclei remain apart andundergo independent transformation ; but in some instances,also while the eggs are still in ether, the nuclei enlarge, andlater conjugate in the entire absence of an aster. Thishappens, however, only when the spermatozoon has enteredat a point not too far from the egg nucleus. Griardina holdsthat this fact, and the other—that the nuclei quickly unitewhenever the eggs are put in pure sea water, and the asterdevelops,—makes Wilson's observation insufficient to excludehis hypothesis. Other explanations, such as mass attractionand direct chemical attraction, both obsei'vers reject.Wilson thinks the latter improbable. Again, the idea ofprotoplasmic currents such as suggested by Bntschli,Erlanger, aud Conklin, is not proved by actual evidence innormal conditions in the sea-urchin egg (Wilson). Thechanges of shape of the germ nucleus might suggestamoeboid movement on its part ; but, again, this does notapply to the sperm nucleus, which travels through a longerpath (Wilson). The changes in form might be due to theexercise of chemotactic forces on the nucleus (Giardina).

The phenomenon described by Boveri (1888) under thename of " Partial Fertilisation," has recently been workedout in detail in Boveri's fixed preparations by Teichmann(1902). The method by which the results were obtainedwas that eggs which had lain fourteen hours in unrenewed

ARTIFICIAL PARTHENOGENESIS AND FERTILISATION. 495

sea water were fertilised with spermatozoa, which weretreated with a "05 per cent, solution of potassium hydrateuntil only a few were mobile. While polyspermy occurred inmore than half the eggs, the remainder were fertilised by asingle spermatozoon. In these cases, however, the spermnucleus did not unite with the germ nucleus, but the asterbecame detached from it, and advanced alone to the germnucleus, a bipolar figure was formed and division proceeded.The sperm nucleus took no share in the process, but passed un-altered into one of the blastomeres. Later, however, either inthe two- or the four-cell space, it broke up into its chromosomes,which entered into the equatorial plate of the cell in which itwas included, and which now divided like its neighbours.Such eggs were capable of developing to the blastula stage.

The question presented itself: Was this aster and theamphiaster the result of the activity of an ovocentrum, orwere they the product of the sperm aster ?

In monospermic eggs Teichmann found the early stagesvery scarce, and, though very suggestive, too few for absoluteproof, and the phenomena seen in dyspermic eggs aredescribed to fill the gap. It may be admitted that in theseeggs, in spite of the apparent inactivity of the sperm nucleus,the sperm aster with its centrum is the operative factor instarting the developmental process. The appearances arevery similar to those in the etherised eggs described byWilson (1901). In that form, as in Echinus, the nuclei con-jugate when they are very unequal in size, and before thedivision of the aster. In Asterias and other forms an amphi-aster is developed before conjugation, and the nuclei arenearly equal in size. In the experiments the union wasdelayed, as in "partial fertilisation," and the amphiaster wasformed before the conjugation.

Among Teichmann's observations I shall refer only to thoseof monospermic eggs. The main feature is the detachmentof the sperm aster from its nucleus, its application to the eggnucleus, aud its normal division, followed by normal segmen-tation. The fate of the sperm nucleus depends on the

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496 THOMAS H. BEYOB.

position it assumes in the egg, relative to the cleavageplane. If it lies outside the equatorial plate of the spindleit passes unchanged into one of the blastcmeres; if itlies within the field of the first spindle, it does notactually unite -with the chromatin of the female nucleus, butits chromatin undergoes a mai'ked relaxation. Though itshows a marked resistance to the tractive forces, it is drawnout and torn into several shreds. It thus passes undividedinto one of the blastomeres, and no chromatin elementsderived from it are found at the poles of the spindle. Inseveral cases where the nucleus lay exactly at the equator,and the traction of the poles was nearly equal, it wasobserved that the chromatin mass was much broken up, andwas torn into two parts. The cleavage of the cell body mayhave helped to complete the division. The loosening of thesperm chromatin mass in the first spiudle seems to havebroken its power of resistance, for when the next division isinitiated, the two nuclei lying side by side in one of theblastomeres unite in the equatorial plate stage, and thechromatin of both is equally distributed in the next division.The number of chromosomes is now different in the blasto-meres, sometimes double, perhaps quadruple in some cases(though an accurate count was not possible), as if the chromo-somes of the sperm nucleus emerged in double number,though the first division was suppressed.

The sperm nucleus in the case where it has not lain withinthe power of the spindle in the first division, may now, inanalogous fashion, be caught in the second division, to unitelater with the chromatin of one of the blastomeres of thefour-cell stage, or it may even pass over into the eight-cellstage, as seen in living eggs by Boveri.

Teichmann concludes that the radiations are derived fromthe sperm centrosome as their starting point, and on thesupposition that the centrosome is introduced by the sperma-tozoon into the egg, that it has suffered less from the chemi-cal reagent than the nucleus. The centrosome behaves asin ordinary fertilisation, the sperm nucleus is passive, and

498 THOMAS H. BltYCE.

Regarding the main point, it may be admitted that theaster and its centvosome here concerned is that belonging tothe sperm nucleus, and that in its behaviour we have abeautiful demonstration of the independence of the twofactors in fertilisation, or, in other words, of the two func-tions of the spermatozoon, and that the two functions havebeen disturbed in uuequal degree. At the same time theegg protoplasm, after the fourteen hours' sojourn inunrenewed sea water, was approaching to that stage inwhich it acquires spontaneously the tendency to developastral activities, and it might be held thut the conditions arethe same as in magnesium eggs in which, as a result of ageneral stimulation, asters and ceutrosomes which areunconnected with the nucleus appear de novo in thecytoplasm. While the results are of interest in connectionwith the apparent independence of the factors in fertilisation,they also show how they are co-ordinated together. Thesperm nucleus becomes dissociated from the aster, and fails ofunion, because it has not undergone the transformation whichproperly corresponds to the phase reached in the cycle of thecentrosomal changes. Further, while the nuclei may be re-solved into chromosomes before union, and yet unite in theequatorial plate stage, a certain stage in the transformationof the dense mass of chromatin of the sperm head intoa nucleus with distinct chromatin network, must be reachedbefore union can take place. This seems to show that severalco-ordinated factors are at work in the nuclear conjugation.

Further insight into the behaviour of the factors infertilisation is given by an experiment described by Zieglor(1898). This consisted in carrying newly-fertilised eggs bya gentle current of water in his compressorium againstthreads of cotton wool. The egg was caught on a threadand nearly cut through, leaving only a slender bridge ofprotoplasm between the two portions of the egg. The onecontained the sperm nucleus, the other the germ nucleus.While the sperm nucleus regularly divided, followed bydivision of the cytoplasm, the egg nucleus merely underwent

ARTIFICIAL PAKTH1SN0GENESIS ANII FEJi'l J1.1SAT1ON. 499

alternate changes of disintegration and reconstruction with-out division of the cytoplasm. Radiations appeared and dis-appeared, and after three cycles, owing to the segmentationof tlie portions containing the sperm nucleus, the egg-nuclearportion became detached and disintegrated. In anotherexperiment the egg-nuclear portion underwent changes ofform suggesting abortive attempts at cleavage. The mitotictransformation of the egg nucleus was not synchronous withthat of the sperm nucleus, but always a little behind.

These observations show that under the conditions of theexperiments the egg nucleus is excited to division withoutdirect contact witli the sperm nucleus or usler, but tliafc tliumitotic phenomena are ineffective to produce cytoplastniccleavage. Ziegler refers this to the general stimulation ofthe egg by the spermatozoon, manifested also by the throw-ing off of the vitelliue membrane. Boveri has shown thatthe same phenomena occur in egg fragments produced byslinking some minutes after fertilisation, and he (1902) refersto cases of this kind in which he has observed divisions ofthe nucleus followed by cell cleavage. The division wasrepeated a second time, and thus the four-cell stage wasreached, but development then ceased. Another example ofthe effect of this general stimulation is to be seen (Boveri,1902) in the cases in which the egg is incited to throw off Lhepolar bodies by the entrauce of the spermatozoon.

This brings me to a further reference to Wilson's observa-tions on etherised eggs (1901 B). AS lias already been said,under this treatment the sperm and germ nuclei remainapart and undergo independently karyokinetic transforma-tion. "The most striking fact is that, while the sperm asteroften gives rise to a perfect and symmetrical bipolar figure, theegg nucleus in a great number of cases produces a monaster,which seems at first incapable of resolving itself into abipolar figure." In typical cases the egg nucleus gives riseto a monaster such as described by Hertwig ('96), and suchas occurs in magnesium eggs. While the egg monaster doesnot at first give rise to a dicentric figure, it does so later, as

500 THOMAS H. BEYOE.

may be gathered from the description of an egg continuouslyobserved in the living state. At the height of its develop-ment the egg monaster lay at one side, the sperm amphiasterat the other, and no spindle was formed between them. Theogg divided into three cells, two larger and somewhatirregular containing two daughter sperm nuclei, and a smallone in which the single egg nucleus re-formed. At the seconddivision each of the sperm nuclei gave rise to a perfect amphi-aster, and divided into two, the accompanying cytoplasinicdivision resulting in tlie formation of two coniplete cells andone biuucleate cell. The single egg nucleus gave rise to atetraster, and divided into three cells, one binucleate, thenuclei of the latter quickly fusing together. The embryonow consisted, of six cells—three containing maternal, threepaternal nuclei. At the ensuing division fifteen cells wereformed, of which eight larger ones contained paternal nuclei,while seven much smaller ones containing maternal nuclei layin a definite group at one side. The egg observed afterwardsdied. Wilson has not seen an egg monaster become dicen-tric at the first division, but the above observations provethat it may operate as an effective division centre, withoutestablishing a spindle connection with either of the spermasters, and that it may divide later. A ceutrosome wasdemonstrated in the monaster, in the same form as in thesperm aster, and as in magnesium eggs. The possible actionof the chemical as the exciting agent of the karyokinetictransformation was excluded by control experiments, and itwas therefore concluded, that it was due to a stimulus effectedby the spermatozoon, as in Ziegler's experiment. Theseobservations, added to the results obtained in the magnesiumeggs, " demonstrate that under appropriate stimulus the eggmay give rise to a centrosome capable of progressive division,but the etherised eggs show in the clearest manner that th i scentrosome is less effective than the sperm centro-some."

I shall not venture on the general problem of the asters andccntrosoines. It will suffice for the present purpose if it be

502 THOMAS H. BUYCR.

to the egg, or by disturbing the general equilibrium in somesuch way as the loss of wafer does, when the normal osmoticrelations are disturbed ? Or does the spermatozoon carry intothe egg some specific chemical substance which produces alocal differentiation, of which the centrosome and the asterare Ihe expression ? Or does it import " a highly active centro-some or centroplasm about which the cytoplasmic energy isbrought to a focus ? "

Boveri (1902) holds that a general stimulation of the egg,with the sperm head as the point of predilection for theformation of the aster, as in magnesium eggs the egg nucleusis the point of predilection, is insufficient as an explanation.There is much rather something special present in the sperma-tozoon, which determines that the aster shall appear at thatpoint, and that point only; and thus he thinks that still theappearances may best be described as being due to the intro-duction of a centrosome. Even admitting—which, as has justbeen indicated, he does not—that the spermatozoon acts likeLoeb's agents, and in view of the demonstration by Morganand Wilson that their effect is to cause the egg to producecentrosomes de novo, only a modification of secondaryimportance would be required iu his theory of fertilisation,viz., that instead of saying that the spermatozoon brings acentrosome into the egg, it would be necessary to say that itcauses the formation of a centrosome in the egg, from thedivision of which the rest follows.

Taking the sperm aster as the manifestation of activitiesproduced by the spermatozoon, and looking to its sharplocalisation on the site of the middle piece, it seems reason-able to suppose that the localised excitement is the effect of anagent operative in fertilisation, and that it is probably relatedto the middle piece; but the actual continuity between thecentrosome of the spermatozoon and that in the aster has notbeen absolutely demonstrated, and the new facts in regard tothe centrosome put the matter in another light. Thus ibremains for the future to decide which of the two latteralternatives stated above shall be adopted, and perhaps after

AltTIKICTAL PARTHENOGENESIS AND FERTILISATION. 503

all there is only a formal difference, for the fundamentalproblem is the same when the question is raised how thecentrosome exercises its activities.

Loeb (1901), on the physico-chemical side, suggests that acatalytic substance is carried by the spermatozoon into theegg, that is one which accelerates physical or chemicalprocesses which would occur without it. The K ions act ascatalysers, and the loss of water acts also, though lessdirectly, in the same way, and it may be that it gives rise tosubstances which act catalytically. Inasmuch as in Chtetop-terus the normal development does not show the character-istics of a treatment of the eggs by K, it is probable thatnormal fertilisation is not brought about by K ions.

Delage (1901) considers that the egg is in an unstablestate of equilibrium, which is readily upset by variousagencies—loss of water, heat, etc., and he lays some weighton the specific action of the salts. He finds that thechloi'ide of manganese has, for Asterias, a specific actionsuperior to that of the alkaline salts. Together with his son,he showed that in the case of the sea-urchin there was lessmagnesium chloride in the sperm than in the eggs, by about1 per cent., so that this salt could not have a specific action.

Among other possible factors in the action of the sperma-tozoon, he gives prominence to its abstraction of water fromthe cytoplasm. During maturation the nuclear sap is shedinto the cytoplasm; until this is effected by the solution ofthe nuclear membrane, fertilisation is not possible; it is justat this " critical s tage" in Asterias that he finds artificialparthenogenesis most liable to occur. In the specialisationof the sexual elements, the egg thus becomes rich, while thespermatozoon has become poor, in water. After the spermhead has entered the ovum it increases in size by abstractionof fluid from the egg protoplasm, and this abstraction ofwater by the sperm nucleus has to be reckoned with as apossible factor in fertilisation.

Apart from the large assumptions involved in such anhypothesis, the facts of " pai'tial fertilisation, and the local-

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504 THOMAS H. ERTOU.

isation of the aster on the middle piece, are in oppositionto it.'5

It has been suggested that the centrosome is the seat offormation of a ferment. Mathews (1901), from the results ofhis experiments on the eggs of Arbacia, believes that " what-ever the details of the process may prove to be, the essentialbasis of karyokinetic.cell division is the production of localisedareas of liquefaction in the protoplasm." " Tlie centrosomemight be a liquefying enzyme."

Experiments on this line have been tried, but withoutdefinite result. Pieri's results (1899), from which he sup-posed he had obtained a ferment " ovulase," have not beenconfirmed. Dnbois (1900) showed that there was noquestion of a ferment being obtained by Pieri's methods.He made various experiments on sperm and eggs, fromwhich he concluded that there was evidence of the existenceof a " zymase," which.he provisionally named " Spermase,"in the spermatozoa, and in the egg a substance, at leastmodifiable by " spermase," provisionally named " Ovulase."Spermase cannot enter the egg by diffusion or osmosis, butonly by a.mechanical means, which, is the raison d ' e t r e ofthe spermatozoon. Winkler's experiments (1900) are alsoinconclusive. He used sperm shaken for half an hour indistilled water and filtered five or six times through three-fold filter-paper. The filtrate was added to sea water, theprecaution being taken of keeping the mixture at the samedegree of concentration as the sea water. While the spermin heated sea water produced no results, tlie liquid caused inthe case of Sphserechinus and Arbacia eggs, though in a rela-tively small number, the beginnings of segmentation. Theseresults may have been due to osmotic influences.

Loeb (1900) states that up to that date he had found noenzyme save papain which had an effect in causing the eggto segment, and it was uncertain whether this was not dueto some accidental constituent of the enzyme preparation used.Gies (1901) made a complete study of the effects of extractsof sperm made by the ordinary methods for the preparation of

506 THOMAS H. BRYCE.

DELAGE, 1S99.—' Arcliiv. de Zoul. Exper.,' t. vii.

DELAGE, 1901.—'ltev. Gen. des Sciences,' 12 annee, No. 19.DEIAGE, 1901.—' Archiv. de Zool. Exp6r.,' t. ix, Nos. 2, 3.DOFLEIN, 1S97.—' Arch. f. mikroskopische Anat.,' Bd. I.

DUBOIS, 1900.—' C. JR. Soc. Bio].,' Paris, vol. Hi, p. 197.FisciiEit, 1902.—' Amer. Journ. of Physiology,'June 2nd, 1902.GIAUD, 1900.—'C. 11. Soc. Biol.,' Paris, vols. lii, liii.GIAUDJNA, 1902, a,—' Anat. Anzeiger,' Bd. xxi, No. 20.GIAUDINA, 1902, b.—'Anat. Anzeiger,' Bd. xxii, Nos. 22, 33.GJES, W. J., 1902.—' Amer. Joum. of Physiol.,' vol. vi.GUEEFE, K., 1876.—' Sitzungsber. der Ges. zur Baford. der geaainmlcn Natur-

wiss. zu Marburg,' No. 5, quoted from O. Hertwig, 1S90.GitEELEY, A. W., 1901.—'Amer. Joum. of Physiology,' vol. vi, p. 296.HEUTWIG, O. and R., 18S7.—' Jenaische Zeitschrift fiir Naturwissenschaft,'

Bd. xx.HERTWIG, O., 1890.—'Jenaische Zeitschrift fiir Naturwissenschaft,' Bd.xxivHEHTWIG, O., 1893.—' Die Zelle und die Geweue.'HEUTWIG, It., 1896.—' Uber die Entwick. des unbefurcliteten Seeigeleies

Leipzig, 1896.HUNTEK, S. J., 1901.—' Amer. Journ. of Pliysiology,' vol. vi, p. 177.LOEB, J., 1900.—' Amer. Journ. of Physiology,' vol. iii, No. 9.LOEB, J., 1900.—'Amer. Journ. of Physiology,' vol. iv, p. 178.LOEIS, J., 1901.—'Amer. Journ. of Physiology,' vol.iv, p. 423.LOEB, J., 1902.—' Arcliiv f. Entwickelungsmechanik,' vol. xiii, Heft 4.LOEB, J., 1902.—'Arcliiv f. Entwickelungsmechanik,' vol. xiv, Heft 4.MAAS, O., 1901.—'Sitzungsber. d. Ges. f. Morph. und Physiol. in Miinchen,'

Bd. xvii.MATIIEWS, A. P., 1900.—'Amer. Journ. of Physiology,' vol. iv, p. 343.MATIIEWS, A. P., 1901.—'Amer. Journ. of Physiology,' vol. vi, p. 142.MATHEWS, A. P., 1901.—'Amer. Journ. of Physiology,' vol. vi, p. 216.MEVES, F., 1902.—' Verhand. des Auat. Ges. Halle,' 1902, p. 132.MOKGAN, T. H., 1896.—'Arch. f. Entwickelungsmechanik,'Bd. iii Heft 3.MORGAN, T. H., 1899.—'Arch. f. EnUvickelungsmechanik,' Bd. viii, Heft 3.MOKGAN, T. H., 1900.—'Arch. f. Eiitwickelungsmechanik,' Bd. xii, Heft 2.NORMAN, VV. W., 1896.—'Arch. f. Enlwickelungsmechauik,' Bd. iii, Heft 1.PIEU.1, J. B., 1S99.—' Arcliiv. de Zool. Exper., Notes et Kevue,' vii, 3.PEOWAZEK, S., 1900.—' Zool. Anzeiger,' No. 618, p. 358.

ARTIFICIAL PARTHENOGENESIS AND FERTILISATION. 507

TEICHMANN, ERNST, 1902.—'Jen. Zeitschrift fiir Naturwiss.,' n. F., Bd. xxx,Heft 1.

VIGUIER, 1900.—' Compt. Rendus Acad. Sci.,' Paris, t. cxxxi, p. 118.VIGUIER, 1901.—' Compt, Rendus Acad. Sci.,' Paris, t. cxxxii, p. 1436.VIGUIER, 1902.—'Compt. Rendus Acad. Sci.,' Paris, t. cxxxv, p. GO.VIGUIER, 1902.—' Compt. Rendus Acad. Sci.,' Paris, t. cxxxv, p. 197.WEDEKIND, 1901.—' Ber. iib. der Verhand. d. 5 internat. Zool. Congress,'

Berlin, 1901.WILSON, ED. B., 1901.—' Arch. f. Entwickelungsmechanilc,' Bd. xii, Heft 4.WILSON, ED. B., 1901.—' Arch. f. Entwickelungjsmechanik,' Bd. xiii, Heft 1.WINKLEU, HANS, 1900.—' Naclir. K. Ges. Wiss. Gottingen, Math. Phys. Kl.,'

1900, Heft 2, p. 187.ZIEGLEE, H. E., 1898.—'Arch. f. Entwickelungsmechanik,' Bd. vi, Heft 2.

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