The Cell in Development and Inheritance

as in the ordinary mitosis of tissue-cells, and its mode of origin there- fore involves the same questions as those already discussed at page 72. It is quite otherwise with the centrosomes at the astral centres, the oridn of which still remains one of the most difficult, as it is one of the most interesting, problems relating to fertilization. After the formation of the polar bodies, the egg-nucleus is recon- stituted near the upper iK)le of the (^'-^g, and the entire polar mitotic apparatus disappears. In the meantime a new astral system (sperm-

Fig. 93. — Fertilization of the egg of tlie gasteropod, Pterotrachea. \ BOVERI.] A. The egg-nucleus {E) and sperm-nucleus {S) approaching after formation of the polar bodies; the latter shown above {P. B.)\ each germ-nucleus contains sixteen chromosomes; the sperm-amphiaster fully developed. D. The mitotic figure for the first cleavage nearly established; the nuclear membranes have disappeared, leaving the maternal group of chromosomes above the spindle, the paternal below it.

aster or amphiaster) is developed in the neighbourhood of the sperm- nucleus, and this in a large number of cases gives rise or is definitely related to the cleavao:e- amphiaster (coelente- rates, flat-worms, echi- noderms, nematodes, annelids, arthropods, mollusks, tunicates, ver- tebrates). In many of these cases the sperm- aster, which by divi- sion gives rise to the amphiaster, has been found to arise in inti- mate relation with the middle -piece of the spermatozoon ; e.g. in echinoderms(Flemming, Hertwig, Boveri, Wil- son, Mathews, Hill, etc.), in the axolotl(Fick) and salamander (Michaelis), in the tunicates (Hill), annelids (Foot, Vejdov- sky), insects (Henking), nematodes (Meyer, Er- langer), and mollusks (Henking, Kostanecki, and Wierzejski). The agreement between forms so diverse is very strong evidence that this is a very general phe- nomenon, and it is one of great interest, owing to the fact that the middle- piece is itself derived from or contains the centrosome of the sper- matid.^

The facts may be il- lustrated by a brief description of the phe- Fig. 94. — Entrance and rotation of the sperm-head and formation of tlie sperm-aster in the sea-urchin, loxopneustes A. Sperm-head before entrance; n. nucleus; w. mid- dle-piece and part of the flagellum. />'. C. Immediately after entrance, showing entrance-cone. />». Rotation of the sperm-head, formation of the sperm-aster about the middle- piece. £. Casting off of middle-piece; centrosome at focus of the ravs (r/C Fig. 12). The changes figured occupy about eight minutes. F. G. Approach of the germ-nuclei ; growth of the aster.

nomena in the sea-urchin Toxop)icustcs (Fig. 94). As described at page 197, the tail is in this case left outside, and only the head and middle-piece enter the egg. Within a few minutes after its entrance, and while still very near the periphery, the lance-shaped sperm-head, carrying the niiddle-})iece at its base, rotates through nearly or cpiite 180°, so that the pointed end is directed outward and the middle- piece is turned inward ( P^ig. 94, A-F)} During or shortly after the rotation appears a minute aster centring in or very near the middle- piece. As it enlarges, the middle-piece itself is thrown to one side (Fig. 12), where it soon degenerates, while in the centre of the aster a minute intensely staining centrosome may be seen, l^oth sperm- nucleus and aster now rapidly advance toward the centre of the egg, the aster leading the way and its rays extending far out into the cytoplasm and finally traversing nearly an entire hemisphere. The central mass of the aster comes in contact with the egg-nucleus, divides into two, and the daughter-asters pass to opposite poles of the egg-nucleus, while the sperm-nucleus flattens against the latter and assumes the form of a biconvex lens (Fig. 95). The nuclei now fuse to form the cleavage-nucleus. Shortly afterward the nuclear membrane fades away, a spindle is developed between the asters, and a group of chromosomes arises from the cleavage-nucleus. These are 36 or 38 in number ; and although their relation to the paternal and maternal chromatin cannot in this case be accurately traced, owing to the apparent fusion of the nuclei, there can be no doubt on general grounds that one-half have been derived from each germ- nucleus. The egg then divides into two, four, etc., by ordinary mitosis (Figs. 4, 52).

In the type of fertilization just described, the polar bodies are formed long before the entrance of the spermatozoon and the germ- nuclei conjugate immediately upon entrance of the spermatozoon, fusing to form a true cleavage-nucleus. In a second and more frequent type {Ascaris, Fig. 90; PJiysa, Fig. 89; Nereis, Fig. 97; Cyclops, Fig. 98) the sperm-nucleus penetrates for a certain distance, often to the centre of the egg, and then pauses while the polar bodies are formed. It then conjugates with the re-formed egg- nucleus. In this case the sperm-aster always divides to form an amphiaster before conjugation of the nuclei, while in the first case the aster may be still undivided at the time of union. This difference is doubtless due merely to a difference in the time elapsing between entrance of the spermatozoon and conjugation of the nuclei, the amphiaster having, in the second case, time to

^ The first, as far as I know, to observe the rotation of the sperm-head was Flemming in the echinoderm-egg ('8i, pp. 17-19). It has since been clearly observed in several other cases, and is probably a phenomenon of very general occurrence. form during extrusion of the polar bodies. The two types just described (Fig. 96) are connected by various gradations. Thus, in the lamprey, the frog, the rabbit, and in An.pln.xus one polar body is expelled before, and one after, the entrance of the sper- matozoon ; in the annelid Othryotrocha, entrance takes place when the first polar spindle is in the stage of the equatorial plate ;

A. Union of the nuclei ; extension egg-nucleus ; division of the aster. while in CJuctopterus and Pi en's the first polar spindle has advanced into the anaphase.^ It is an interesting and signiftcant fact that the aster or amphiaster always leads the way in the march toward the egg-nucleus ; and in many cases it may be far in advance of the sperm-nucleus.- Boveri ('87, I) has observed in sea-urchins that the sperm-nucleus may indeed be left entirely behind, the aster alone conjugating with the egg-

Fig. 96. — Diagrams of two principal types of fertilization. /. Polar bodies formed after the entrance of the spermatozoa (annelids, mollusks, flat-worms). //. Polar bodies formed before entrance (echinoderms). A. Sperm-nucleus and centrosome at c^ ; first polar body forming at 9 . B. Polar bodies formed ; approach of the nuclei. C. Union of the nuclei. D. Approach of the nuclei. E. Union of the nuclei. F. Cleavage-nucleus. nucleus and causing division of the egg ivitJiout union of tJic gcnn- nuclci, though the sperm-nucleus afterward conjugates with one of the nuclei of the two-cell stage. This process, known as " partial fer- tilization," is undoubtedly to be regarded as abnormal. It affords, however, a beautiful illustration of the view that it is the centro- some alone that incites division of the egg, and is tJierefore the fer- tilizing element proper (Boveri, '87, 2).

The foregoing facts lead us to a consideration of Boveri's theory of fertilization, which has for several years formed a central point of discussion. The ground for this theory had been prepared by Oscar Hertwig and Fol. The latter ('73) early reached the conclusion that the asters represented " centres of attraction " lying outside and independent of the nucleus. Oscar Hertwig showed, in 1^75, that in the sea-urchin egg, the amphiaster arises by the division of a single aster that first appears near the sperm-nucleus and accom,ianies it in its progress toward the egg-nucleus. A similar observation was soon afterward made by Fol ('79) i" the eggs of Asl.nas and Sagiita, and in the latter case he determined the fact that the astral

rays do not centre in the nucleus, as Hertwig described, but at a point in advance of it — a fact afterward confirmed by Hertwig himself and by l^overi {^'^'S), i ). Hertwig and Fol afterward found that in cases of polyspermy, when several spermatozoa enter the <iz^, each sperm-nucleus is accompanied by an aster, and Hertwig proved that each of these might give rise to an amphiaster (Fig. loi). In 1886-87 \^ejdovsky brought forward strong evidence to show that in the fresh-water annelid RJiynchchnis the cleavage-amphiaster arises directly from the sperm-amphiaster, itself derive'd by the division of a '* periplast " (attraction-sphere) imported into the (t^^^ by the sj^ermatozoon, while the polar amphiaster entirely disappears. It was Boveri i^'^J, 2) who first carefully studied the facts with reference to the centrosome, reaching the conclusion (in the case of Ascaris and the sea-urchin) that a single centrosome is brought in by the spermatozoon, and that it divides to form two centres about which are developed the two asters of the cleavage-figure. He was thus led to the following conclusion, which has received the sup- port of many later investigators : The ripe egg possesses all of the organs and qualities necessary for division excepting the centrosome, by which division is initiated. The spermatozoon, on the other Juitid, is provided zvith a centivsome, but lacks the substance in ivhich this organ of division may exert its activity. TJirough the union of the tiuo cells in fertilization, all of the essential organs necessary for division are brought together ; the egg 7iozv contains a centrosome ivJiich by its oivn division leads the zcay in the embryonic develop- ment} Very numerous observations, supporting this conclusion, have been made by later observers.

Bbhm could find in Petromyzon ('88) and the trout ('91) no radiations near the egg-nucleus after the formation of the polar-bodies, while a beautiful sperm-aster is devel- oped near the sperm-nucleus and divides to form the amphiaster. Platner ('86) had already made similar observations in the snail Arion, and the same result was soon afterward reached by Brauer ('92) in the case of Branchipus, and by Julin ('93) in Styleopsis. Pick's careful study of fertilization of the axolotl ('93) proved in a very convincing manner not only that the amphiaster is a product of the sperm-aster, but also that the latter is developed about the middle-piece as a centre. The same result was indicated by Foot's observations on the earthworm ('94), and it was soon afterward conclusively demonstrated in echinoderms through the independent and nearly simultaneous researches of myself on the Qgg of Toxo- pneustes, of Mathews on Arbacia, and of Boveri on EcJiinus. Nearly at the same time a careful study was made by Mead ('95, '98, i) of the annelid Chcetoptei'us, and of the starfish Asterias by Mathews,

both observers independently showing that the polar spindle contains distinct centrosomes, which, however, degenerate after the formation of the polar bodies, their place being taken by the sperm-centrosome, which divides to form an amphiaster before union of the nuclei, as in RJiyiicJielmis. Exactly the same result has since been reached by Hill ('95) and Reinke ('95) in SpJicerecJiiims, by Hill in the tunicate Phallusia, by Kostanecki and Wierzejski ('96) in Physa (Fig. 89), and by Van der Stricht ('98) in Thysanozodn ; and in all of these the centrosome is likewise shown to arise from the middle-piece or in its immediate neighbourhood. Among others who have produced

Fig. 98. — Fertilization of the egg in the copepod, Cyclops strenuus. [RUCKERT.] A. Sperm-nucleus soon after entrance, the sperm-aster dividing. B. The germ-nuclei ap- proaching; cf, the enlarged sperm-nucleus with a large aster at each pole; 9, the egg-nucleus re-formed after formation of the second polar body, shown at the right. C. The apposed reticular germ-nuclei, now of equal size ; the spindle is immediately afterward developed between the two enormous sperm-asters ; polar body at the left.

evidence that the cleavage-centrosome stands in definite relation to the spermatozoon, may be mentioned Oppel ('92) in reptiles, Hrauor ('92) in Branchipus, Henking (92) in insects, Riickert ('95.2) in Cyclops, Sobotta ('95) in the mouse and ('98) Ampliioxus, Ziegler (95) in Diplogaster and Rhabditis, Castle ('96) in Ciomu Korschelt ('95) in OpJiryotrocha, Meyer (95) in Strougylus, Griffin ('96, "99) in Thalassema, and Coe ('98) in Ccrcbratulns. Beside the foregoing evidence may be placed the following addi- tional data based on experiment and the study of pathological fer- tilization, (i) In the case of sea-urchin eggs, Hertwig, Boveri, and o

several later observers have shown that egg-fragments, obtained by shaking eggs to pieces, are readily penetrated by the spermatozoa, and that such fragments, though containing no nuclear matter from the egg. may segment and give rise to perfect larvae.^ (2) Hoveri ('88) has observed that in ordinary fertiUzation the sperm-aster may separate from the sperm-nucleus, travel through the cytoplasm to the egg-nucleus and cause cleavage, the sperm-nucleus afterward fusing with one of the nuclei of the two-cell stage ("partial fertiHzation "). (3) Most remaikable of all, l^overi, confirmed by Ziegler (98), has recently observed that during the first cleavage the whole of the chromatin may pass to one pole, so that upon division one of the halves of the ^%^ receives only a centrosome without a nucleus. In the nucleated half cleavage proceeds as usual. In the enucleated half the centrosomes and asters continue for a considerable period to multiply at the same rate as the cleavage of the nucleated half, though the cell-body does not itself divide.^ Putting these facts together we must conclude ( i ) that something is introduced into the egg by the middle-piece of each spermatozoon entering it that is either a centrosome or has the power to incite the formation of one ; (2) that the centrosome thus arising is structurally independent of both nuclei and may divide independently of them ; (3) that indepen- dently of the division of the nucleus or cell-body there is some kind of historical continuity between the centrosomes of successive genera- tions.

In the case of echinoderm-eggs this continuity is not yet known to be effected by actual persistence of the centrosomes.^ There are, however, a number of cases in which the division of the primary cleavage-centrosomes and the persistence of their descendants as those of the daughter-cells seem to have been conclusively shown — for example on Ascaris (Van Beneden, Boveri, Kostanecki, and Sied- lecki), in the trout (Henneguy, '96), in Thalasscma (Griffin, '96, '99), in ClicetoptcrHs{Vi<i'3.Ci, '95, '98), in Pliysa (Kostanecki and Wierzejski, '96), in Ccrcbmtulus (Coe, '98), and in Rhynchchnis (Vejdovsky and Mrazek, 98). In Thalasscma and Ccrcbratulus (Figs. 99, 155) the centrosome is a minute granule at the focus of the sperm-aster, which divides to form an amphiaster soon after the entrance of the spermatozoon. During the early anaphase of the first cleavage, each centrosome divides into two, passes to the outer periphery of the centrosphere, and there forms a minute amphiaster for the second

3 Erlangcr's statement ('98) that the centrosomes persist through the first cleavage in echinoderm-eggs is not supported by his figures ; and I am convinced from my own long- continued studies of these eggs, as well as by an examination of Erlanger's preparations, kindly placed in my hands by Professor Biitschli, that these difficult olijects are very unfavour- able for a decision of the question. cleavage before the first cleavage takes place. The minute centro- somes of the second cleavage are therefore the direct descendants of the sperm-centrosome ; and there is good reason to believe that the continuity is not broken in later stages. The facts are nearly similar

Fig. 99. — Fertilization in an annelid (armed Gephyrean), Thalasscma. [CiRlFFlN.] A. Second polar body forming; sperm-nucleus and centrosome below. />. Apjiroach of the egg-nucleus and sperm-nucleus, the latter accompanied by the sperm-amphiaster. C. Union of the nuclei. D. Later stage of last. E. Prophase of cleavage-spindle. /'. Anaphasr of the same; centrosome divided. G. H. I. Successive stages in the nuclear reconstitution and lormation of the daughter-amphiasters for the second cleavage. J. Two-cell stage.

in the trout, in Chcetoptcrus, and in PJiysa. In Ascaris division of the centrosome first occurs at a somewhat later period ( Figs. 90, 176). If now the centrosomes were indeed permanent cell-organs, wc should thus reach the following result: During cleavage the cytoplasm of the blastomeres is derived from that of the egg, the centrosomes from the sptnnatozodn, ivJiilc the nuelei {chromatin) are equally derived from There is very strong reason to accept the first part of this con- clusion (applying to nucleus and cytoplasm), but the question of the centrosomes remains an open one. The array of evidence given above, derived from the study of so many diverse groups, seems to place Boveri's lucid and enticing hypothesis upon a strong foundation. Two essential points still remain, however, to be determined : first, whether the facts observed in Ascaris, l^xhinoderms, PJiysa, Thalas- sema, and the like, are typical of all forms of fertilization ; and, second, whether, if so, the primary cleavage-centrosome is actually imported into the ^gg by the spermatozoon or is only formed under its influence out of the egg-substance. Both these questions have been raised by recent inves^tigators, apparently on good evidence, and some of this evidence is directly opposed to both of the principal assumptions of Boveri's theory. Thus, Wheeler ('97) has found that in Mycostoma both centrosomes are derived from the egg ; Carnoy and Le Brun ('97) maintain that in Ascaris one centrosome is derived from each of the germ-nuclei; in some moUusks, according to MacFarland ('97) and Lillie ('97), both egg-cehtrosomes and sperm-centrosomcs dis- appear, to be replaced by two centrosomes of unknown origin ; while recent botanical workers are unable to find any centrosomes in fertili- zation. These and other divergent results will be critically considered bevond (p. 208) in connection with a more detailed examination of the general subject. It may be pointed out here, however, that recent researches on spermatogenesis (p. 170) render it nearly certain that the centrosome of the sperm-aster cannot be the unmodified cen- trosome of the spermatid, since the latter, in some cases, enlarges to form a " middle-piece " or analogous structure that is far larger than the sperm-centrosome.

It does not lie within the scope of this work to consider the innumerable modes by which the germ-cells are brought together, further than to recall the fact that their union may take place inside the body of the mother or outside, and that in the latter case both eggs and spermatozoa are as a rule discharged into the water, where fertilization and development take place. The spermatozoa may live for a long period, either before or after their discharge, without losing their fertilizing power, and their movements may continue throughout this period. In many cases they are motionless when first discharged, and only begin their characteristic swimming move- ments after coming in contact with the water. There is clear evi-

dence of a definite attraction between the germ-cells, which is in some cases so marked (for example in the polyp Roiilla) that when spermatozoa and ova are mixed in a small vessel, each ovum becomes in a few moments surrounded by a dense fringe of sperma- tozoa attached to its periphery by their heads and by their move- ments actually causing the ovum to move about. The nature of the attraction is not positively known, but Pfeffer's researches on the spermatozoids of plants leave little doubt that it is of a chemical nature, since he found the spermatozoids of ferns and of Sclni^iuc/ia to be as actively attracted by solutions of malic acid or malates (con- tained in capillary tubes) as by the substance extruded from the

Fig. ioo< — Entrance of the spermatozoon into the egg. A-G. In tlie sea-urchin. Toxopneustei. H. In tlie medusa, Mitrocoma. [MetSCHNIKOFF.] /. In the star-fish Astcrias. [FOL.] A. Spermatozoon of Toxopneustes, X 2000; a. the apical body, w. nucleus, w. middlo-pu-ci-. f. flagellum. B. Contact with the egg-periphery. C. D. Entrance of the head, formation of tlie entrance-cone and of the vitelline membrane {v), leaving the tail outside. E. F. I^ter st.iges. G. Appearance of the sperm-aster (j) about 3-5 minutes after first contact; entrance-cone break- ing up. H. Entrance of the spermatozoon into a preformed depression. /. .Approach of the spermatozoon, showing the preformed attraction-cone.

neck of the archegonium. Those of mosses, on the other hand, are indifferent to malic acid, but are attracted by cane-sugar. These experiments indicate that the specific attraction between the germ- cells of the same species is owing to the presence of specific chemical substances in each case. There is clear evidence, furthermore, that the attractive force is not exerted by the egg-nucleus alone, but by the egg-cytoplasm; for, as the Hertwigs and others have shown, spermatozoa will readily enter egg-fragments entirely devoid ot a

In naked eggs, such as those of some echinoderms, and coelen- terates, the spermatozoon may enter at any pcnnt ; but there are some cases in which the point of entrance is predetermined by the presence of special structures through which the spermatozoon enters (F'ig. lOO). Thus, the starhsh-egg, according to Fol, pos- sesses before fertilization a pecuHar protoplasmic "attraction-cone" to which the head of the spermatozoon becomes attached, and through which it enters the *^'^^^. In some of the hydromedusas, on the other hand, the entrance point is marked by a funnel-shaped depression at the egg-periphery ( Metschnikoff ). When no preformed attraction- cone is i)resent, an " entrance-cone " is sometimes formed by a rush of protoplasm toward the point at which the spermatozoon strikes the Q.^^ and there forming a conical elevation into which the sperm- head passes. In the sea-urchin (Fig. lOO) this structure persists only a short time after the spermatozoon enters, soon assuming a ragged flame-shape and breaking up into slender rays. In some cases the o.^^^ remains naked, even after fertilization, as appears to be the case in many ccelenterates. More commonly a vitelline mem- brane is quickly formed after contact of the spermatozoon, — e.g. in AnipJiioxus, in the echinoderms, and in many plants, — and by means of this the entrance of other spermatozoa is prevented. In eggs surrounded by a membrane before fertilization, the spermato- zoon either bores its way through the membrane at any point, as is probably the case with mammals and Amphibia, or may make its entrance through a micropyle.

In some forms only one spermatozoon normally enters the ovum, a? in echinoderms, mammals, many annelids, etc., while in others several may enter (insects, elasmobranchs, reptiles, the earthworm, Petroniyzon, etc.). In the former case more than one spermatozoon may accidentally enter (pathological polyspermy), but development is then always abnormal. In such cases each sperm-centrosome gives rise to an amphiaster, and the asters may then unite to form the most comj^lex polyasters, the nodes of w^hich are formed by the centrosomes (Fig. loi). Such eggs either do not divide at all or undergo an irregular multi])le cleavage and soon j^erish. If, how- ever, only two spermatozoa enter, the q,<^^j:^ may develop for a time. Thus Driesch has determined the interesting fact, which I have con- firmed, that sea-urchin eggs into which two spermatozoa have acci- dentally entered undergo a double cleavage, dividing into four at the first cleavage, and forming eight instead of four micromeres at the fourth cleavage. Such embryos develop as far as the blastula stage, but never form a gastrula.^ In cases where several spermatozoa normally enter the ^^^g (physiological polyspermy), only one of the sperm-nuclei normally unites with the egg-nucleus, the supernumer- ary sperm-nuclei either degenerating, or in rare cases — e.g. in elas- mobranchs and reptiles — living for a time and even dividing to form

"merocytes or accessory nuclei. The fate of the latter is still in doubt ; but they certainly take no part in fertilization. It is an interesting question how the entrance of supernumcrarv spermatozoa is prevented in normal monospermic fertilization. In the case of echinoderm-eggs Fol advanced the view that this is mechanically effected by means of the vitelline membrane formed instantly after the first spermatozoon touches the q^^. This is indi- cated by the following facts. Immature eggs, before the formation

A. Polyspermy in the egg of y^j^aWj ; below, the egg-nucleus ; above, three entire spermatozoa within the &gg. [Sala.] B. Polyspermy in sea-urchin egg treated with 0.0050'' nicotine solution; ton sperm-n" '••• shown, three of which have conjugated witli the egg-nucleus. C. Later stage of an egg sini: treated, showing polyasters formed by union of the sperm-amphiasters. [O. and K. Hkkiwkj.j of the polar bodies, have no power to form a vitelline membrane, and the spermatozoa always enter them in considerable numbers. Polyspermy also takes place, as O. and R. Hertwig's beautiful ex- periments showed {^'^J\ in ripe eggs whose vitality has been dimin- ished by the action of dilute poisons, such as nicotine, strychnine, and morphine, or by subjection to an abnormally high temperature

(31° C); and in these cases the vitelline membrane is only slowly formed, so that several spermatozoa have time to enter.^ Similar mechanical explanations have been given in various other cases. Thus Hoffman believes that in telcosts the micropyle is blocked by the polar bodies after the entrance of the first spermatozoon ; and Calberla suggested {Pcnvviycon) that the same result might be caused by the tail of the entering spermatozoon. It is, however, far from certain whether such rude mechanical explanations arc adequate ; and there is considerable reason to believe that the egg may possess a physiological power of exclusion called forth by the first spermatozoon. Thus Driesch found that spermatozoa did not enter fertilized sea-urchin eggs from which the membranes had been removed by shaking.- In some cases no membrane is formed (some ccelenterates), in others several spermatozoa are found inside the membrane (nemertines), in others the spermatozoon may penetrate the membrane at any point (mammals), yet monospermy is the rule.

The union of the germ-cells calls forth profound changes in both. {a) TJic SpcnnatozooJi. — Almost immediately after contact the tail ceases its movements. In some cases the tail is left outside, being carried away on the outer side of the vitelline membrane, and only the head and middle-piece enter the ^%g (echinoderms, Fig. 100). In other cases the entire spermatozoon enters (amphibia, earthworm, insects, etc., Fig. 89), but the tail always degenerates within the ovum and takes no part in fertilization. Within the ovum the sperm-nucleus rapidly grows, and both its structure and staining- capacity rapidly change (</. p. 182). The most important and signifi- cant result, however, is an iniincdiate rcsiiniptioi by the spcrm-nuclcus and spenn-ccntrosoinc of the power of division^ which has hitherto been suspended. This is not due to the union of the germ-nuclei ; for, as the Hertwigs and others have shown, the supernumerary sperm-nuclei in polyspermic eggs may divide freely without copu- lation with the egg-nucleus, and they divide as freely after entering enucleated egg-fragments. The stimulus to division must therefore be given by the egg-cytoplasm. It is a very interesting fact that in some cases the cytoplasm has this effect on the sperm-nucleus