A study of nucleo-cytoplasmic interactions in pre-embryonic development, at maturation stages in particular, is of great interest. During this period ooplasmic segregation takes place, specific properties of the cortex arise, and the cytoplasm obtains information from the oöcyte nucleus which promotes the development of the embryo during all the changes related to fertilization, cleavage and blastulation (Wilson, 1925; Raven, 1961; Brachet, 1957, 1960; Briggs & King, 1959; Neyfakh, 1962).

The first data on the rôle of the oöcyte nucleus (germinal vesicle or GV) in cytoplasmic maturation were obtained by Delage (1899, 1901) on the starfish Asterias glacialis, and by Wilson (1903) on the nemertine Cerebratulus lacteus. In both species oöcytes are expelled by the female before the GV dissolves, and mature in sea water. This allowed Delage and Wilson to experiment with oöcytes at different maturation stages. They showed that anuclear fragments obtained by shaking the oöcytes at the stage before GV dissolution did not reach maturity and could not be fertilized, although nucleated fragments could. All parts of oöcytes fragmented immediately after GV dissolution mature, both those containing the nucleus (at the stage of the metaphase of the first cleavage division), and those lacking it. The same differences in the stage of the cytoplasm of Cerebratulus oöcytes were found before and after GV dissolution with respect to the ability of the cytoplasm to form cytasters under the action of salts (Yatsu, 1905). From his experiments Delage drew the very important conclusion that cytoplasmic maturation requires the passage of the nuclear sap from the germinal vesicle to the cytoplasm. Delage made an attempt to elucidate the nature of the effect of the nuclear sap upon cytoplasmic maturation but at that time the level of knowledge in this field was inadequate for the solution of this problem.

Modern achievements in the study of the realization of genetic information, of the mechanisms of protein synthesis and, especially, in the study of the composition and changes of karyoplasm of the germinal vesicle during the growth and maturation (cf. Gall, 1955,1958,1963; Brachet, 1957,1960; Vincent, 1957; Briggs & King, 1959; Raven, 1961; Ficq, 1961, 1962; Macgregor, 1963), make a return to the problems raised by Delage seem promising. However, a study of these problems encounters considerable technical difficulties, since in the majority of animals, including all the vertebrates, oöcyte maturation proceeds under the influence of gonadotropic hormones within the body of the female. In this connexion the fact that in some animals, including many amphibian species, hypophyseal preparations can induce oöcyte maturation in vitro, in saline solution, is of particular interest (Heilbrunn, Daugherty & Wilbur, 1939; Wright, 1945; Nadamitsu, 1953; Tchou-Su & Wang Yu-lan, 1956, 1958; and others). This discovery makes possible an experimental study of maturation in vertebrates including the use of surgical techniques hitherto impracticable (Dettlaff, 1961).

This paper describes the first results of such experiments on toad oöcytes. The rôle of the GV in oöcyte maturation and in the changes in the properties of the oöcyte cortical layer was studied by removal of the GV at successive stages of maturation, artificial disruption of the GV membrane at early maturation stages, and injection of the GV contents into other oöcytes. In order to differentiate between the rôles played by the nuclear sap and by the chromosomes, and to elucidate the specificity of GV action, experiments were carried out on substitution for the GV by nuclei from blastulae or early gastrulae. These latter have been shown to be an adequate substitute for the chromosomes of the mature egg in several amphibian species (Briggs & King, 1952, 1953, 1957, 1960; Fishberg, Gurdon & Elsdale, 1958; Stroeva & Nikitina, 1960; Subtelny & Bradt, 1961; Sambuichi, 1961; Signoret, Briggs & Humphrey, 1962; Signoret & Picheral, 1962; Nikitina, 1964). By transplanting nuclei into oöcytes at various stages, nucleo-cytoplasm interactions at different stages of cytoplasmic maturation were studied by testing the ability of the oöcytes to undergo division.

Experiments were carried out on the oöcytes of two toad species: Bufo bufo (asiaticus) and B. viridis. B. bufo females were obtained in winter in a state of dormancy and kept for a long time (2–3 and more months) at 4–6° C. B. viridis were caught after they had left their hibernation sites and were also kept at 4–6° C. for 2–3 weeks before the experiment. The oöcytes of both species were at the GV stage (a large GV was located at some distance from the surface of the animal region, and no passage of karyoplasm to the cytoplasm was observed); this stage will be called simply ‘the initial’ one below (Plate 1, Fig. 8; Plate 3, Fig. la).

With the aim of inducing maturation in vivo a suspension of three, later of two, freshly obtained hypophyses was injected into each female, B. bufo and B. viridis hypophysis being interchangeable. The toad oöcytes did not respond to chorionic gonadotropin, while hypophyses of Rana temporaria caused only a very slight effect.

In order to obtain oöcyte maturation in vitro we used the method developed in detail by Tshou-Su & Wang Yu-lan (1958) on B. bufo asiaticus. Hypophyses were homogenized and diluted in Ringer solution, two to three hypophyses in 25 ml. of solution. If a large sized piece of ovary was placed into this solution in B. viridis, unlike B. bufo, only a few oöcytes matured and ovulated, but when the ovary was minced and pieces containing only a few oöcytes were used, then, as shown by Zuichenko (unpublished), all the oöcytes underwent maturation and ovulation. In many experiments the wound through which the ovarian pieces had been taken was sewn up, and if these females had not received a hypophyseal injection, they were injected with a suspension of two to three hypophyses in order to bring their oöcytes to later maturation stages and to obtain data on the duration of maturation of their oöcytes in vivo. Injected females and males were kept at the same temperature as the experimental oöcytes.

The oöcytes were used in the experiments either at the initial stage or at different times after the beginning of the action of the hypophyseal hormone (in vitro or in vivo), i.e. at different maturation stages. In order to know the stage achieved by the oöcytes used in the experiments, some were fixed for 1-2 min. in boiling water, cut in the plane of the animal-vegetative axis, and the section studied under a low powered microscope. The stage was determined by the position of the GV with respect to the surface of the animal region, and by GV dimensions which are sufficiently distinguishable after such a simple treatment (Plate 1, Figs. 8, 10; Plate 2, Figs. 1, 3, 7). Concurrently, the oöcytes were also fixed with San Felice, Bouin’s fluid and with formalin (1:9) for microscopic study. Sections 7 p thick were stained by the Heidenhain azan method to reveal acidophilic karyoplasm and to find the transplanted nuclei more easily.

Operations were carried out under aseptic conditions; penicillin and strepto-mycin being added to the Ringer solution. Prior to operation the follicular membrane was carefully removed from oöcytes with well sharpened watch-makers’ forceps so as not to injure the oöcyte itself. Oöcytes devoid of follicle cells mature successfully in vitro. Such oöcytes were exposed to various operations (described in the appropriate sections of the paper) and put into Ringer solution with hypophyses or without them. In control runs some small ovarian fragments (ten to fifteen large oöcytes) were placed into Ringer solution with or without hypophysis. The behaviour of the oöcytes of each female used for experiment was judged by changes in control oöcytes in Ringer solution with hypophysis. Some time after ovulation of the control oöcytes, nuclei from blastulae or early gastrulae were transplanted into experimental oöcytes in all experimental series to elucidate the degree of maturation of their cytoplasm and its ability to become activated and to cleave under the influence of the centrioles introduced into the egg when somatic nuclei are injected (Subtelny & Bradt, 1960,1961). Insemination could not be used for this purpose since mature amphibian oöcytes lacking the jelly envelope are not fertilizable (Ryan & Grant, 1940; Nadamitsu, 1957; Tchou-Su & Wang Yu-lan, 1956). In some experiments nuclear transplantation was replaced by pricking as a means of activation.

Transplantation of nuclei was carried out by the Briggs & King method (1952, 1953, 1960). In different experimental series nuclei of ectoderm cells from the inner layer of blastula roof and nuclei of endoderm cells from the region of blastocoel floor taken from blastulae or early gastrulae were used. Pieces of donor embryo tissues were dissociated to single cells in Niu-Twitty solution devoid of calcium and magnesium ions, and then placed into normal Niu-Twitty solution. Directly before transplantation the cells were put together with oöcytes into a dish with Ringer solution, and transplantation of nuclei was carried out here. This modification of the Briggs and King method is due to the fact that Niu-Twitty solution, which is the most suitable medium for the transplantation of nuclei into mature eggs (Briggs & King, 1953), is hypotonic for oöcytes (Tchou-Su & Wang Yu-lan, 1958) which are rapidly injured in it. In order to increase the probability of transplanting nuclei that would be able to ensure cleavage, several nuclei were injected into one oöcyte in most experiments. Upon nuclear transplantation into intact oöcytes, the pipette was introduced somewhat above the equator so as not to injure the GV. In transplantation into oöcytes from which the GV had been removed, the pipette was introduced through the wound left after GV removal.

In all experiments the properties of the oöcyte cortical layer were noted. The state of the cortical layer was judged by its contractility in response to the cut and its ability to participate in cytokinesis. The degree of cytoplasmic maturity was determined by the behaviour of the cortical layer, and the ability of the eggs to undergo activation and cleavage. The ability of the eggs to be activated, i.e. to form a fertilization groove and perivitelline space, was also noted in the oöcytes whose GV had not been removed. In all, 1390 oöcytes from sixteen B. bufo and fourteen B. viridis females were used. In some cases oöcytes were taken repeatedly from one female at different times after the beginning of the action of the hypophyseal hormone. The results of experiments are compared in different series and within each series, in various experiments differing in the stage of oöcyte maturation. The data obtained are presented below.

I. Behaviour of oöcytes, taken at the initial stage, in Ringer solution with or without hypophysis

IA. In Ringer solution without hypophysis no B. bufo and B. viridis oöcytes taken at the initial stage either matured or changed. After 2 or 3 days they began to degenerate. The maturation of the oöcytes removed at different times after the onset of the hypophyseal action but before dissolution of the GV and then placed into Ringer solution without hypophysis did not go further than the stage they had reached at the moment of their transfer. However, at the stage directly before GV dissolution and at its very beginning, the oöcytes already displayed maturation inertia because after transfer from solution with hypophysis to one without it they completed their maturation and ovulated.

IB. In Ringer solution with hypophysis all the experimental oöcytes ovulated and matured whether placed into Ringer at the initial stage or later. In the first case after 15–20 hr. (at 16–17°C.) the oöcytes were usually activated by pricking. In B. bufo eggs matured in vitro and activated by pricking one or two atypical and rudimentary furrows appeared only rarely (Plate 1, Fig. 6). These oöcytes were of the same appearance as those shown in the photographs of Tchou-Su & Wang Yu-lan (1959, Table 4, Figs. 23–30). More regular cleavage, as shown in their Figs. 31-33, was not obtained. Perhaps these differences are due to a deterioration of B. bufo gonads during transportation, or to the smaller number of experiments carried out, or to the fact that in our experiments oöcytes were not activated at the optimal time. When B. viridis oöcytes matured in vitro a greater number of the eggs cleaved after activation; they reached the stage of many blastomeres but perished later.

Tchou-Su & Wang Yu-lan (1958,1959) showed on B. bufo that oöcyte maturation, both in vivo and in vitro, proceeded in the same manner. Some time after the injection of hypophysis into a female or in vitro the GV starts to move slowly towards the surface of the animal region; later, some of the karyoplasm in the more vegetative region of the GV begins to pass out to the cytoplasm through the GV membrane (Plate 3 Fig. lb); soon this membrane dissolves and the entire karyoplasm moves into the cytoplasm (Plate 3, Fig. lc).

We found that at the dissolution of the GV membrane the properties of the oöcyte cortical layer underwent drastic changes followed by changes in the osmotic properties of the endoplasm. At the initial stage and a few hours later no signs of contractility could be found in the cortical layer of the cytoplasm. After a cut the edges of the wound had no tendency either to stretch or to contract. The wound persisted for some time, its edges closing very slowly. In Ringer solution the endoplasm does not exude from the cut. With the approach of the moment of GV dissolution the edges of the wound begin to close somewhat more actively.

When the GV dissolves the cortical layer acquires strong contractility. The edges of the wound now close immediately after a cut; and if the surface of the oöcyte is slightly pressed at this time, some endoplasm covered with cortical layer protrudes through the cut; on ending the pressure the outgrowth is with-drawn under the membrane again. This kind of response, however, disappears, rapidly being replaced by an active formation of exovates. This does not seem to be related to a further change in the properties of the cortical layer but to change in the osmotic properties of the cytoplasm (cf. Tchou-Su & Yen Pai-Hu, 1950). The appearance of contractility in the cortical layer and dissolution of the GV are followed by a sharp increase in the osmotic pressure of the cytoplasm. When, at a stage close to ovulation, a cut is made in the cortical layer similar to one performed at earlier stages, a mixture of karyoplasm and cytoplasm is actively expelled through it in the same Ringer solution. Thereafter the edges of the wound close rapidly and protrusions of different size are later formed through the cut in the egg membrane. The stalk connecting these outgrowths with the egg narrows gradually and they finally separate and become rounded.

II. Transplantation of nuclei from blastula or early gastrula cells into intact oöcytes at different maturation stages

The nuclei were transplanted into oöcytes at the initial stage, at different times after the onset of maturation, and into mature oöcytes (see Table 1). In some experiments the latter were first activated by pricking with a glass needle and enucleated by the Porter (1939) method, at the metaphase of the 2nd maturation division. The oöcytes used before the onset of maturation inertia were put into Ringer solution with hypophysis after the transplantation of nuclei into them, while those at later stages were put into Ringer without hypophysis. At a time when female oöcyte donors were ovipositing (or had eggs in their oviducts) somatic nuclei were repeatedly transplanted into experimental oöcytes, or the oöcytes were activated by pricking.

Table 1.

Transplantation of nuclei from blastulae and early gastrulae into oöcytes of different stages with and without the GV (or a part of the karyoplasm and cytoplasm)

Transplantation of nuclei from blastulae and early gastrulae into oöcytes of different stages with and without the GV (or a part of the karyoplasm and cytoplasm)
Transplantation of nuclei from blastulae and early gastrulae into oöcytes of different stages with and without the GV (or a part of the karyoplasm and cytoplasm)

In the latter case it was found that the nuclei transplanted into the immature cytoplasm waited for it to mature and preserved viability for several hours, i.e. they were able to start division and to promote the egg to cytokinesis. But this requires, apart from cytoplasmic maturation, activation of the egg as well. Matured oöcytes of B. viridis containing blastula nuclei never started division unless activated by pricking. B. bufo oöcytes were often activated spontaneously, sometimes at earlier stages, when their cytoplasm had not achieved complete maturation.

After activation in good time, the furrows in some eggs were arranged very regularly and many morulae formed. Eggs from one experiment, in which somatic nuclei were transplanted into oöcytes at the initial stage, are shown in Plate 1, Figs, la, lb, 2. After maturation in Ringer solution with hypophysis these oöcytes were activated by pricking, some of them 3 hr. earlier than others. The former oöcytes (Fig. la, b) began to cleave earlier than the latter ones (Fig. 2). An early gastrula developed from an oöcyte into the immature cytoplasm of which a blastula cell nucleus had been transplanted; this oöcyte was activated by pricking after maturation in Ringer solution with hypophysis.

In B. bufo oöcytes that were activated before complete maturation the nuclei changed to the active state and atypical cytokinesis started, accompanied by characteristic disturbances (constriction of cortex and exposure of the endoplasm, formation of very small blastomeres in the region of the constricted cortex, Plate 1, Fig, 4a, b). Sometimes, the cortex broke along the line of the incipient furrow, and the oöcyte perished. More often such cases of pathological cleavage occurred when nuclei were transplanted into oöcytes at the stage of GV dissolution or somewhat later (Plate 2, Fig. 8a, b).

The rôle of cytoplasmic hydration for the events of cytokinesis is clearly demonstrated by the oöcytes that underwent an earlier activation after the transplantation of nuclei into them. In Ringer solution furrows often appear in such oöcytes but then smooth down and disappear again very rapidly. If left in Ringer solution such oöcytes do not divide, but when transferred into Niu-Twitty solution or into diluted Ringer, even for a short time, the furrows rapidly reappear on their surface and deepen. Oöcytes that have been activated at the wrong time are, as a rule, injured. Somewhat older oöcytes are not injured either in diluted Ringer or Niu-Twitty solutions or in water, and cleave better than in undiluted Ringer solution which becomes hypertonic to them.

Thus, after transplantation into oöcytes and their activation, the nuclei of somatic cells can engage the cytoplasm in a process of cytokinesis whose character depends upon the extent of cytoplasmic maturation (of the cortical layer and endoplasm) and hydration. The oöcyte cytoplasm, in its turn, affects the behaviour of somatic nuclei transplanted into it. Immature cytoplasm inhibits nuclear division, the time of inhibition being the longer, the more immature are the oöcytes. Cytoplasmic maturation and cortical reaction stimulate nuclear division. However, nuclear stimulation is possible at somewhat earlier stages of cytoplasmic maturation than those at which typical cytokinesis can occur. It seems that maturation of the cortical layer and the possibility of the cortical reaction are sufficient for the stimulation of nuclei, while normal cytokinesis requires harmonious maturation of the entire cytoplasm.

The participation of the egg chromosomes and of those of the transplanted somatic nuclei in the process of cleavage has not been studied in the present work. However, in parallel experiments in which ectodermal and endodermal nuclei were transplanted into enucleated eggs of B. bufo, normal tadpoles developed with the help of the transplanted nuclei (Nikitina, 1964; Stroeva, unpubl.).

Finally, one more observation deserves to be noted. Upon the transplantation of nuclei of somatic cells into exovates devoid of egg membranes these nuclei divide, but the blastomeres arising from them round off and soon lose connexion with each other. Such an exovate photographed during the separation of one blastomere is shown in Plate 1, Fig. 7. It seems that the connexion between blastomeres (as in the case of lamprey blastomeres lacking the egg membrane— Yamamoto, 1956) is not sufficiently strong to hold them together in the absence of the egg membrane.

III. Removal of the GV (at later stages—removal of a part of karyoplasm and cytoplasm) and transplantation of somatic nuclei

III A. Removal of the GV (or of karyoplasm and cytoplasm) without simultaneous transplantation of somatic nuclei

The GV was removed by the method applied earlier to the oöcytes of sturgeons (Dettlaff, 1961). After the removal of the follicle cells at the animal pole of the oöcyte, a very small knife cut in the surface layer was made above the GV. In some cases this was sufficient, and a semi-transparent vesicle appeared in the cut after several minutes; it moved the edges of the wound apart forcing its way out and, after a few more minutes, separated in the form of a rounded bubble. When the GV lay further from the cut it was displaced by a slight pressure upon the oöcyte surface at the edges of the cut or by slightly pushing aside the cytoplasmic layer covering the GV. After the extrusion of the GV the edges of the wound gradually came closer, and in successful experiments only a small trace of the wound, marked by several yolk granules, remained (Plate 2, Fig. 2a). The removal of the entire GV in B. bufo usually succeeds if performed during the first 7 – 8 56 hr. after the introduction of hypophyses in vivo (at 16 – 17°C.). If the GV is removed at this time, only that part of it which has earlier passed through the GV membrane into the perinuclear zone remains in the cytoplasm (Plate 2, Fig. 3; Plate 3, Figs. 2a, 3).

When the GV is removed 9 – 10 hr. after the introduction of hypophysis its membrane easily breaks down and some of the contents flow out through the wound to form an opalescing opaque cloud at the oöcyte surface. The GV membrane with the remaining karyoplasm within it, together with a small amount of the cytoplasm fused with the membrane, has to be removed with forceps. Some of the GV karyoplasm may remain in the cytoplasm. Later, after dissolution of the GV membrane, a mixture of karyoplasm and cytoplasm was removed by pressing out of the cut a volume which was not less than that of the GV.

After the operation the oöcytes were placed in Ringer solution with hypo-physis, while some of the oöcytes that had been used at stages when they already possessed some maturation inertia were placed in fresh Ringer solution. When control oöcytes attained maturity, blastula nuclei were transplanted into the experimental ones.

The results of experiments are presented in Table 1. Whenever the removal of the whole GV could be managed the oöcytes did not mature in Ringer solution with hypophysis. The majority of them looked healthy but did not cleave after receiving somatic nuclei (Plate 2, Figs. 4, 6). In some of the oöcytes the animal pole flattened and in this way something like a perivitelline space arose between the animal pole and the egg membrane (Plate 2, Fig. 5), but it lacked colloid. The endoplasm preserved its initial properties. In the oöcytes from which the GV had been removed shortly before the development of inertia a slight contractility seemed to appear in the cortical layer in Ringer solution with hypo-physis. This phenomenon requires further investigation.

If the GV was removed at the stage of the appearance of maturation inertia, single oöcytes whose GV could be completely removed did not cleave in Ringer solution, neither did they in Ringer solution with hypophysis. However, on operation at this stage the GV membrane broke down in most of the oöcytes, and some karyoplasm remained in the oöcyte. A considerable percentage of such oöcytes matured in Ringer solution even in the absence of hypophysis. The oöcytes of B. viridis cleaved after activation by pricking, while those of B. bufo cleaved after the transplantation of somatic nuclei. B. bufo and B. viridis oöcytes also cleaved when a considerable amount of the mixture of karyoplasm and cytoplasm was removed from their animal region at the stage after dissolution of the GV (Plate 2, Fig. 9a, b).

III B. Removal of the GV and simultaneous transplantation of somatic nuclei

The GV was removed as before at stages from the initial one up to the onset of dissolution of the GV, and somatic cell nuclei were then transplanted into oöcytes which were placed in Ringer solution with hypophysis. After 16-24 hr. somatic cell nuclei were transplanted for a second time and the oöcytes were again placed into Ringer solution with hypophysis. In this experiment oöcytes were exposed to the action of gonadotropic hormones at the state when oöcyte nuclei were replaced by those of somatic cells. Nevertheless, in this experiment, as well as in experiment II (see Table 1), oöcytes did not mature; the properties of their cortical layer did not change as in the preceding experiment, in fact, they did not respond at all to the presence of blastula nuclei (Plate 2, Fig. 6a, b). Only in three oöcytes out of 100 did the cortical layer show changes which bore witness to a partial maturation; their GV, however, was removed just prior to its dissolution and may have been somewhat injured during removal. It is also possible that before dissolution, when the GV can still be completely removed, the permeability of its membrane changes to such an extent that some of the GV material can pass through it into the cytoplasm.

In sections through oöcytes of experiments IIIA and IIIB after the removal of the GV, the anilinophilous karyoplasm of the GV was either absent or present in small amounts in the cytoplasm of the animal region (Plate 3, Figs. 2, 3); in this latter case the karyoplasm formed restricted lacunae and sometimes marked the path of the GV through the cytoplasm. This is the karyoplasm which passed from the GV into the cytoplasm before GV removal. In the cytoplasm of the animal region, blastula cells with spherical nuclei, and also free nuclei, can be seen (Plate 3, Figs. 2a, b, 3). These nuclei stain with aniline blue and have an appearance of small vesicles which can be easily distinguished at the background of the orange-rose yolk (Plate 3, Figs. 2b, 3). It seems that during nuclear transplantation some blastula cells preserved their membranes while in others it broke down and their nuclei found themselves in direct contact with the oöcyte cytoplasm. In the oöcytes devoid of their GV these nuclei did not divide and remained at the site of transplantation. A small amount of the nuclear sap that passed into the cytoplasm before dissolution of the GV did not cause an effect in the absence of the GV. In the oöcytes whose GV was not removed from our experiment II (Plate 3, Fig. 4) only cells with intact membranes were seen in the region of nuclear transplantation. A large number of free cleaving nuclei were dispersed over the cytoplasm and between them traces of incipient cleavage furrows were seen.

Thus, in the absence of the GV the cytoplasm of the oöcyte placed into Ringer solution with hypophysis did not mature either when containing blastula nuclei or when without them (IIIA and IIIB). The presence of the GV prior to its dissolution did not affect the ability of the cytoplasm to cleave in Ringer solution with hypophysis; after the removal of the GV at these stages (experiment IIIA) the oöcyte did not cleave either, as in case of the GV removal at the initial state. At the same time the passage of a small amount of karyoplasm to the cytoplasm at the stage of GV dissolution allowed cytoplasmic maturation and later cleavage in Ringer solution without further action of gonadotropic hormones.

IV. Rupture of the GV membrane without GV removal

In order to obtain direct contact between karyoplasm and cytoplasm before the normal time, the GV membrane was artificially ruptured within the oöcyte. As in experiments on sturgeon oöcytes (Dettlaff, 1961), a glass needle was intro-duced into the oöcyte at the initial stage through the centre of the animal region and was moved upwards, downwards and sidewards without being withdrawn. To prevent loss of GV contents through the wound, the needle was not removed from the oöcyte for several minutes. The oöcytes were then left in Ringer solution without hypophysis. Simultaneously with the maturation of control oöcytes, nuclei of somatic cells were transplanted into such experimental oöcytes of B. bufo (fourteen oöcytes), while B. viridis oöcytes (twenty) were activated by pricking. None of them showed any sign of maturation, they were not activated and they did not cleave, their cortical layer did not acquire contractility. In all the oöcytes fixed in boiling water the GV bearing traces of pricking were observed in sections. On microscopic preparations of these oöcytes (as in sturgeon ones), karyoplasm can be seen to come into direct contact with the cytoplasm at the sites of the membrane breakdown, although they do not mix together as they do after normal dissolution of the GV. A comparison of this experiment with the preceding ones suggests that during the period from the onset of gonadotropic hormone activity up to the stage of GV dissolution the properties of the karyo-plasm undergo considerable changes necessary for its subsequent action upon cytoplasmic maturation.

V. Transplantation of the G V contents or of a mixture of karyoplasm and cytoplasm into oöcytes at the initial stage

In this experiment the karyoplasm (with some cytoplasm) was taken from oöcytes possessing maturation inertia and injected into oöcytes at the initial stage (Plate 1, Fig. 8). After injection the oöcytes were left in Ringer solution without hypophysis. Experiments were carried out on the oöcytes of the same B. viridis female as in the preceding experiment (see Table 2). Donor oöcytes were kept in Ringer solution with hypophysis until the onset of dissolution of the GV membrane. Then a small cut was made in the animal region, and, as soon as the GV appeared in the cut, a micropipette was introduced into it. The bright, slightly opalescent, karyoplasm and, if possible, the GV membrane were sucked into the pipette. Care was taken to suck up as little cytoplasm and Ringer solution as possible. The karyoplasm, with some cytoplasm and Ringer unavoidably present, was injected into oöcytes at the initial stage. At later stages of the donor oöcytes, just after dissolution of the GV, a mixture of karyoplasm and cytoplasm was taken from the animal region and a small amount of it injected into oöcytes at the initial stage. In control experiments Ringer solution was injected into oöcytes at this stage. The micropipette was introduced from one side, near the equator, so as to prevent an injury to the GV. Operated oöcytes were left in Ringer solution with hypophysis, experimental and control oöcytes were activated by pricking.

Table 2.

Injection of oöcyte karyoplasm and cytoplasm at or after the stage of GV dissolution into oöcytes in the initial stage

Injection of oöcyte karyoplasm and cytoplasm at or after the stage of GV dissolution into oöcytes in the initial stage
Injection of oöcyte karyoplasm and cytoplasm at or after the stage of GV dissolution into oöcytes in the initial stage

The results are presented in Table 2. They show that a small amount of karyoplasm with some cytoplasm (taken at the onset of GV dissolution), or a small amount of the mixture of karyoplasm and cytoplasm (after GV dissolution), injected into oöcytes at the initial stage promote oöcyte maturation in Ringer solution without hypophysis. A high percentage of oöcytes react and about one-third of them reach such a degree of maturity that they undergo irregular cleavage after artificial activation (Plate 1, Figs. 9, 10).

The results of the first two series are compared in the first and second lines of Table 2. They differ in that the oöcyte donors of karyoplasm of the first line were taken 2 hr. earlier than those of the second line. Of the control oöcytes of the younger group placed simultaneously into Ringer solution without hypophysis, only a few possessed maturation inertia, while after 2 hr. all of them possessed it, although their GV was at the stage when its membrane was dissolving. In both experiments karyoplasm was injected into the oöcytes of the same females. Despite the small number of oöcytes in the experiment, the difference between the results is evident. The oöcytes did not normally respond to the presence of the karyoplasm taken before the appearance of maturation inertia (the weak response of a small number of oöcytes probably being due to the use of rather older donors). On the other hand, the oöcytes at the initial stage showed a clear reaction to the injection of karyoplasm taken from the oöcytes of the same females after the appearance of maturation inertia in the donors (Table 2). This suggests that the active principle involved is a karyoplasmic substance or substances formed or activated when the oöcyte acquires maturation inertia rather than hypophyseal hormone as such. Hormone would have been present in the substances injected both before or after the appearance of maturation inertia. The participation of the cytoplasmic part of the injected material is also unlikely since it remains inactive in the absence of the GV material. However, the possibility cannot be ruled out that as karyoplasm passes into the cytoplasm the former’s activity may increase.

In control experiments oöcytes did not change after an injection of Ringer (Plate 1, Fig. Ila, b). Only in one experiment when, by mistake, Ringer was injected with the same micropipette with which the mixture of karyoplasm and cytoplasm had been injected before, in two oöcytes out of eight the GV dissolved.

This suggests that active substances inducing cytoplasmic maturation can act in considerable dilutions. At the same time, since, in the case of the injection of karyoplasm and cytoplasm, the oöcytes fixed at the same time are at different maturation stages, there are grounds for thinking that the amount of active substances injected into them affects the rate of maturation.

The data obtained reveal that in the oöcytes of amphibians, like those of Asterias glacialis (Delage, 1901) and of Cerebratulus lacteus (Wilson, 1903), cytoplasmic maturation is possible only if karyoplasm passes to the cytoplasm, and that maturation has started by the time of dissolution of the GV membrane. It was found that unknown changes in nuclear properties precede changes in the cytoplasm. They occur before the onset of the GV dissolution as a result of the continuous action of gonadotropic hormones at this time. In the oöcytes at the initial stage artificial disruption of the GV membrane, which brings the karyo-plasm into direct contact with the cytoplasm, did not result in maturation. At this stage karyoplasm neither dissolved nor mixed with the cytoplasm, as normally occurs when the GV dissolves. At the onset of dissolution of the GV membrane, when the oöcytes acquire maturation inertia and do not need further action of gonadotropic hormones, the properties of the karypolasm undergo drastic changes. At this time the karyoplasm actively affects the state of the cytoplasm. At the time of dissolution of the GV the properties of the cortical layer change, it acquires strong contractility, and somewhat later, after the movement of karyoplasm into the cytoplasm, the osmotic properties of the endoplasm undergo drastic changes in their turn. In the following hours the cytoplasm acquires the ability to undertake a cortical reaction, to stimulate blastula nuclei transplanted into it to division, and to undergo cytokinesis. Passage of a small amount of karyoplasm into the cytoplasm is sufficient to start all these changes. More-over, karyoplasm (with cytoplasm) taken from the oöcyte at the onset of GV dissolution can stimulate maturation of oöcytes at the initial stage, i.e. it substitutes for the action of gonadotropic hormones. At the same time the cytoplasm lacking the GV does not respond to the action of these hormones. All these facts show that the gonadotropic hormones affect oöcyte maturation through the oöcyte nucleus, for which blastula and gastrula cell nuclei cannot be a substitute. Since after the dissolution of the GV membrane and the formation of the spindle of the 1st maturation division the chromosomal apparatus of the oöcyte nucleus can successfully be replaced by blastula nuclei (Briggs & King, 1959), it would be natural to suggest that specificity of the oöcyte nucleus at the initial stage is determined first of all by its nuclear sap prepared during the period of growth by preceding activity of the chromosomal apparatus of the oöcyte (cf. Mulnard, 1954; Gall, 1955, 1958, 1963; Vincent, 1957; Brachet, 1957; Mac-gregor, 1963).

The problem of whether the gonadotropic hormones act directly on the nuclear sap, or indirectly through the chromosomal apparatus, remains un-solved. Estrogens are thought to act upon the mechanism of protein synthesis by accelerating the synthesis of RNA (Mueller, Gorski & Aizawa, 1961; Aizawa & Mueller, 1961; Hamilton, 1963; Epifanova, 1964). In newt oöcytes (Macgregor 1963), under the action of gonadotropic hormones, specific changes take place in the structure of certain chromosomal loci (lampbrush chromosomes) resembling the changes in giant chromosomes of insects under the action of ecdyson (Clever, 1961, 1962). Macgregor (1963) also describes changes in the composition of the nuclear sap of the GV under the action of estrogens. Kroeger (1963) advanced a suggestion that the nuclear sap was an intermediate system relating the hormonal stimulus with a certain locus in the chromosomes. The specificity of the action of the chromosomes in the GV should also be seen in terms of their interaction with the nuclear sap accumulated in the GV of the mature oöcyte.

It is known that an active passage of substances takes place through the GV membrane during the period of growth and maturation and that the perinuclear zone is the site where active cytoplasmic synthetic processes occur (cf. Gall, 1955, 1958, 1963; Brachet & Ficq, 1956; Vincent, 1957; Ficq, 1961, 1962; Raven, 1961). At the same time substances promoting cytoplasmic maturation (and especially those governing the ability to cleave) enter it only with the dissolution of the GV membrane as shown by our data. It might be supposed that these substances accumulate gradually in the karyoplasm but cannot pass through the GV membrane. It is more likely, however, that substances inducing cyto-plasmic maturation are formed in the karyoplasm only just prior to dissolution of the GV membrane. This is supported by the results of our experiment V : karyoplasm taken at the stages before the beginning of dissolution of the GV membrane and injected into oöcytes at the initial stage, does not induce their maturation. This is also in agreement with the data obtained by Delage, Wilson & Yatsu (see also Briggs & King, 1959).

There are grounds for believing (Briggs & King, 1959) that ooplasmic segregation of the cytoplasm is also closely related to the GV materials. This problem is of paramount importance but difficult to experiment with. Experiments on the injection of GV material into older embryos undertaken with various aims in view (Lopashov, 1946; Kriegel, 1956, 1961; Huff, 1962) have not solved this problem. At the same time operations on oöcytes under the conditions permitting their maturation in vitro seem to open vast prospects in this field.

Substances which are expelled from the GV into the cytoplasm and make it mature are directly related to the process of activation and to cytokinesis. It should be borne in mind that the main criteria of cytoplasmic maturity are ability to be activated, to be fertilized and to undergo cytokinesis. In the process of egg activation GV substances are related to the appearance of hydrophilous colloid expelled by the egg under the vitelline membrane (Wintrebert, 1933; Dettlaff, 1962). In toad oöcytes, after GV removal, the volume of the animal region diminishes, it flattens, and a space sometimes arises between the oöcyte surface and the membrane, but without the colloid. As to cleavage, the papers of Delage (1901) and Yatsu (1905) have already shown that the ability to form a cytaster appears in the cytoplasm only provided that the nuclear sap has passed into it. Multiple formation of cytasters in the cytoplasm at the moment of dissolution of the GV at the sites of mixing up of karyoplasm and cytoplasm was described by various authors (e.g. in sea urchins, Ephrussi, 1933; in sturgeon, Dettlaff, in press). Cleavage of enucleated eggs is explained by the presence in their cytoplasm of GV materials required for the formation and functioning of asters (Briggs, Green & King, 1951).

It can be supposed that of both nuclear and cytoplasmic (β-granules) material participating, according to Zimmerman & Marsland (1956) and Marsland, Zimmerman & Auclair (1960), in the mechanism of furrow formation during cleavage, β-granules are related to the nuclear sap of the GV. If it is so, both the ‘cytoplasmic’ and ‘nuclear’ material are of the nuclear origin. The former passes to the cytoplasm at the GV dissolution, during maturation period, while the latter is ejected into the cytoplasm at each cleavage division.

It is shown that at the beginning of dissolution of the GV membrane the properties of the cortical layer underwent a very rapid change manifested in the appearance of a strong contractility. In the absence of the GV the properties of the cortical layer change very slowly, if at all. At the same time, karyoplasm, when taken at the dissolution of the GV membrane, can induce changes in the properties of the cortical layer of the intact oöcyte. This agrees well with the data of Mulnard (1954) who had shown that in the beetle Acanthoscelides at the moment of the breakdown of the GV wall, RNA- and SH-group rich cytoplasm streamed under the surface layer of the oöcytes, and the cortex was formed. Whether some structural and physiological peculiarities of the cortical layer that make it such a responsible participant in morphogenesis (cf. Curtis, 1960, 1962, 1963; Raven, 1961) arise simultaneously with the appearance of contractility cannot be decided for the time being. It is without doubt, however, that the appearance of the special organization of the cortical layer is causally related to the effect of substances arising in the GV under the action of gonadotropic hormones.

  1. Oöcyte maturation in Bufo bufo (asiaticus) and B. viridis, the rôle of the germinal vesicle (GV) in this process, the interrelationship of the GV and the cytoplasm and the action of gonadotropic hormones on oöcyte maturation were studied. Removal of the GV, the injection of its contents into oöcytes and the transplantation of somatic nuclei into them at different stages of the action of hypophyseal hormones, and a replacement of the GV by somatic nuclei, were the methods used. The main criterion of cytoplasmic maturation was ability to cleave. Changes in the properties of the oöcyte cortical layer and endoplasm at different stages of cytoplasmic maturation were also considered.

  2. The method of oöcyte maturation in vitro (Tchou-Su & Wang Yu-lan, 1958) was applied in the experiments. Oöcyte maturation in both species requires the action of gonadotropic hormones only during a certain period before the beginning of dissolution of the GV membrane. Thereafter the process of maturation can be completed in Ringer solution in the absence of hormones.

  3. The passage of karyoplasm into the cytoplasm during dissolution of the GV membrane is accompanied by a marked change in the properties of the oöcyte cortical layer which acquires a strong contractility, and by a sharp increase in the osmotic pressure of the entire cytoplasm. The change in the cortical layer is preceded by one in the endoplasm.

  4. Nuclei of blastula cells, when transplanted into the oöcyte cytoplasm, do not divide before the beginning of maturation but preserve their ability to divide and to promote cleavage for a long time. Apart from cytoplasmic maturation, the entry of transplanted nuclei into division requires activation of the egg.

  5. After transplantation of nuclei from ectoderm or endoderm cells of blastulae and early gastrulae into oöcytes the percentage of mature eggs undergoing cleavage after activation is similar (about 65 per cent.). The nuclei of blastula cells revert to synchronous divisions during the cleavage of the eggs into which they have been transplanted.

  6. Oöcytes from which the GV was removed before the beginning of its dissolution do not mature in Ringer solution to which has been added a source of gonadotropic hormone (pituitary gland), and do not cleave after the transplantation of somatic nuclei into them. Oöcytes at the onset of GV dissolution, in which some GV materials remain after GV removal, mature in Ringer solution even in the absence of hypophysis, and cleave after the transplantation of nuclei.

  7. Oöcytes in which nuclei of ectoderm or endoderm blastula cells replace the GV neither mature nor cleave in Ringer solution with added hypophysis. Such nuclei cannot substitute for the GV in its promotion of cytoplasmic maturation.

  8. Artificial rupture of the GV membrane at the initial stage, unlike natural dissolution of the GV membrane, does not induce cytoplasmic maturation in Ringer solution without added hypophysis.

  9. From the beginning of the action of gonadotropic hormone up to dissolution of the GV membrane, the properties of the karyoplasm undergo profound changes which are responsible for its action upon cytoplasmic maturation at subsequent stages.

  10. The injection of a mixture of karyoplasm and cytoplasm taken from oöcytes during or after the dissolution of the GV membrane into oöcytes at the initial stage makes them mature in Ringer solution without hypophyses, i.e. is a substitute for the action of gonadotropic hormones.

  11. Gonadotropic hormones may affect oöcyte maturation through the oöcyte nucleus (GV).

The authors would like to thank Professor G. V. Lopashov for his vivid interest in this work and for his valuable advice in preparing the manuscript for press. Thanks are also due to Mrs S. I. Zuichenko and Miss T. B. Sidorova for their help in the experiments and in the histological preparation of experimental material.

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Abbreviations used: cl.—cleavage furrow; g.v.—germinal vesicle; g.v.a.—germinal vesicle absent; w.—wound through which the g.v. was removed; m.bl.—microblastomeres; k.— karyoplasm; n.s.—nuclear sap; n.s.c.—nuclei of somatic cells; s.c.—somatic cells. All photographs of whole oöcytes and embryos were taken after their fixation with formalin (1:9); sections through oöcytes were made after fixation with boiling water.

Plate 1

Figs. 1 – 7. Transplantation of nuclei of somatic cells into intact oöcytes of Bufo bufo.

Fig. la, b; Fig. 2. Oöcytes taken from a ♀ 7 – 30 hr. after injection of two hypophyses (t° 17 – 18°C.); oöcytes were placed into Ringer solution + hypophysis after the transplantation of nuclei of ectodermal cells from the roof of early gastrula. Oöcytes la, lb activated by pricking with a glass needle 16 hr. after nuclear transplantation. The oöcyte in Fig. 2 after 19 hr. Oöcytes cleaved in Niu-Twitty solution.

Fig. 3. The embryo at the stage of early gastrula formed from the oöcyte into which at the initial stage, before hypophyseal injection, nuclei of ectodermal blastula cells were transplanted. After 14 hr. stay in Ringer+hypophysis the oöcyte was activated by pricking and left in water.

Fig. 4. Oöcytes after a transplantation at the initial stage of endoderm blastula cells maturated in Ringer solution+hypophysis activated spontaneously and cleaved atypically.

Fig. 5. Morula from an oöcyte into which nuclei of endodermal early gastrula cells were transplanted after its maturation in vitro.

Fig. 6. Artificial parthenogenesis. Oöcytes after 24 hr. in Ringer+hypophysis, activated by pricking and transferred to Niu-Twitty solution.

Fig. 7. Cleaving exovates. Separation of a blastomere.

Figs. 8 – 11. Experiment with an injection of either karyoplasm+cytoplasm or Ringer solution into intact Bufo viridis oöcytes.

Fig. 8. Oöcyte at the initial stage, a large GV.

Fig. 9. Oöcytes into which at the initial stage a mixture of karyoplasm+cytoplasm was injected from the oöcytes taken just after dissolution of their GV. Oöcytes matured in Ringer solution without hypophysis and cleaved abnormally when activated by pricking (Fig 9a, b, c).

Fig. 10. The same oöcytes as in Fig. 9 form exovates (Fig. 10a, appearance; 10b, in section, GV dissolved).

Fig. 11. Oöcytes which were given an injection of Ringer at the initial stage and placed into Ringer solution without hypophysis. They did not mature, cleave or become activated after pricking.

Plate 1

Figs. 1 – 7. Transplantation of nuclei of somatic cells into intact oöcytes of Bufo bufo.

Fig. la, b; Fig. 2. Oöcytes taken from a ♀ 7 – 30 hr. after injection of two hypophyses (t° 17 – 18°C.); oöcytes were placed into Ringer solution + hypophysis after the transplantation of nuclei of ectodermal cells from the roof of early gastrula. Oöcytes la, lb activated by pricking with a glass needle 16 hr. after nuclear transplantation. The oöcyte in Fig. 2 after 19 hr. Oöcytes cleaved in Niu-Twitty solution.

Fig. 3. The embryo at the stage of early gastrula formed from the oöcyte into which at the initial stage, before hypophyseal injection, nuclei of ectodermal blastula cells were transplanted. After 14 hr. stay in Ringer+hypophysis the oöcyte was activated by pricking and left in water.

Fig. 4. Oöcytes after a transplantation at the initial stage of endoderm blastula cells maturated in Ringer solution+hypophysis activated spontaneously and cleaved atypically.

Fig. 5. Morula from an oöcyte into which nuclei of endodermal early gastrula cells were transplanted after its maturation in vitro.

Fig. 6. Artificial parthenogenesis. Oöcytes after 24 hr. in Ringer+hypophysis, activated by pricking and transferred to Niu-Twitty solution.

Fig. 7. Cleaving exovates. Separation of a blastomere.

Figs. 8 – 11. Experiment with an injection of either karyoplasm+cytoplasm or Ringer solution into intact Bufo viridis oöcytes.

Fig. 8. Oöcyte at the initial stage, a large GV.

Fig. 9. Oöcytes into which at the initial stage a mixture of karyoplasm+cytoplasm was injected from the oöcytes taken just after dissolution of their GV. Oöcytes matured in Ringer solution without hypophysis and cleaved abnormally when activated by pricking (Fig 9a, b, c).

Fig. 10. The same oöcytes as in Fig. 9 form exovates (Fig. 10a, appearance; 10b, in section, GV dissolved).

Fig. 11. Oöcytes which were given an injection of Ringer at the initial stage and placed into Ringer solution without hypophysis. They did not mature, cleave or become activated after pricking.

Plate 2

Removal of the GV from oöcytes of Bufo bufo and transplantation into them of somatic cell nuclei.

Plate 2

Fig. 1. Section through an oöcyte at the stage of operation (taken from the female 7 · 5 hr. after an injection of two hypophyses, t° 17°C.).

Fig. 2. Oöcyte appearance 20 min. after the removal of the GV.

Fig. 3. Section through the oöcyte after the removal of the GV.

Fig. 4a, b. Oöcytes after the removal of their GV placed into Ringer solution+hypophysis. After 12 hr. nuclei of endoderm cells of early gastrula were transplanted into these oöcytes, and after 12 hr. more oöcytes were transferred into diluted Ringer and pricked. The oöcytes did not mature.

Fig. 5. Oöcyte after the removal of the GV. Animal region flattened.

Fig. 6. Oöcytes from which GV were removed and nuclei of endoderm cells of early gastrula were transplanted simultaneously; 24 hr. later they were placed in Ringer solution + hypo-physis, the nuclei of endoderm cells were repeatedly transplanted into oöcytes, which were then transferred into diluted Ringer. Oöcytes did not mature.

Fig. 7. Section through oöcyte at the stage of GV dissolution.

Fig. 8a, b. oôcytés whose GV was removed at the onset of dissolution (10 hr. after the oöcytes were placed into Ringer solution+hypophysis, t° 14°); nuclei of blastula endoderm cells were transplanted to the oöcytes simultaneously. The oöcytes were left in Ringer+hy-pophysis and after 12 hr. nuclei were repeatedly transplanted into them. Partial maturation, cortex is pulled off (8a, b), small blastomeres are seen (8b).

Fig. 9a, b. Morulae are formed from oöcytes of Bufo bufo from which karyoplasm+cytoplasm was removed at the stage directly after dissolution of the GV, and nuclei of ectoderm blastula cells were transplanted. Oöcytes matured in Ringer solution without hypophysis and then cleaved well in Niu-Twitty solution, after a repeated nuclear transplantation.

Plate 2

Fig. 1. Section through an oöcyte at the stage of operation (taken from the female 7 · 5 hr. after an injection of two hypophyses, t° 17°C.).

Fig. 2. Oöcyte appearance 20 min. after the removal of the GV.

Fig. 3. Section through the oöcyte after the removal of the GV.

Fig. 4a, b. Oöcytes after the removal of their GV placed into Ringer solution+hypophysis. After 12 hr. nuclei of endoderm cells of early gastrula were transplanted into these oöcytes, and after 12 hr. more oöcytes were transferred into diluted Ringer and pricked. The oöcytes did not mature.

Fig. 5. Oöcyte after the removal of the GV. Animal region flattened.

Fig. 6. Oöcytes from which GV were removed and nuclei of endoderm cells of early gastrula were transplanted simultaneously; 24 hr. later they were placed in Ringer solution + hypo-physis, the nuclei of endoderm cells were repeatedly transplanted into oöcytes, which were then transferred into diluted Ringer. Oöcytes did not mature.

Fig. 7. Section through oöcyte at the stage of GV dissolution.

Fig. 8a, b. oôcytés whose GV was removed at the onset of dissolution (10 hr. after the oöcytes were placed into Ringer solution+hypophysis, t° 14°); nuclei of blastula endoderm cells were transplanted to the oöcytes simultaneously. The oöcytes were left in Ringer+hy-pophysis and after 12 hr. nuclei were repeatedly transplanted into them. Partial maturation, cortex is pulled off (8a, b), small blastomeres are seen (8b).

Fig. 9a, b. Morulae are formed from oöcytes of Bufo bufo from which karyoplasm+cytoplasm was removed at the stage directly after dissolution of the GV, and nuclei of ectoderm blastula cells were transplanted. Oöcytes matured in Ringer solution without hypophysis and then cleaved well in Niu-Twitty solution, after a repeated nuclear transplantation.

Plate 3

Sections through oöcytes of Bufo bufo and B. viridis (fixed with formalin 1:9 and stained by the Heidenhein azan method).

Plate 3

Fig. la. Section through a B. viridis oöcyte—a stage close to the initial one, oöcytes do not possess maturation inertia.

Fig. lb. Section through a B. viridis oöcyte; some oöcytes possesses maturation inertia at this time (9 hr. in Ringer solution + hypophysis, t° 17–18°C.).

Fig. 1c. Section through a B. bufo oöcyte after dissolution of the GV; nuclear sap has passed into the cytoplasm.

Fig. 2a. Section through a B. bufo oöcyte from which GV was removed at the initial stage and nuclei of endoderm blastula cells were transplanted at the same time. Bubble-shaped nucleus (2b) (n.s.c.) can be seen in the cytoplasm.

Fig. 3. Section through a B. bufo oöcyte. GV removed at the onset of maturation. Lacunae of karyoplasm remained in the cytoplasm that seem to have passed into cytoplasm from the GV prior to its removal. The oöcyte did not mature in Ringer + hypophysis. Intact transplanted blastula cells (s.c.) are seen in the cytoplasm.

Fig. 4. Section through a B. bufo oöcyte matured in vitro and, after transplantation of nuclei of endoderm blastula cells, was transferred into Niu-Twitty solution. Traces of incipient furrows are seen in the cytoplasm; intact blastula cells are found at the site of transplantation.

Plate 3

Fig. la. Section through a B. viridis oöcyte—a stage close to the initial one, oöcytes do not possess maturation inertia.

Fig. lb. Section through a B. viridis oöcyte; some oöcytes possesses maturation inertia at this time (9 hr. in Ringer solution + hypophysis, t° 17–18°C.).

Fig. 1c. Section through a B. bufo oöcyte after dissolution of the GV; nuclear sap has passed into the cytoplasm.

Fig. 2a. Section through a B. bufo oöcyte from which GV was removed at the initial stage and nuclei of endoderm blastula cells were transplanted at the same time. Bubble-shaped nucleus (2b) (n.s.c.) can be seen in the cytoplasm.

Fig. 3. Section through a B. bufo oöcyte. GV removed at the onset of maturation. Lacunae of karyoplasm remained in the cytoplasm that seem to have passed into cytoplasm from the GV prior to its removal. The oöcyte did not mature in Ringer + hypophysis. Intact transplanted blastula cells (s.c.) are seen in the cytoplasm.

Fig. 4. Section through a B. bufo oöcyte matured in vitro and, after transplantation of nuclei of endoderm blastula cells, was transferred into Niu-Twitty solution. Traces of incipient furrows are seen in the cytoplasm; intact blastula cells are found at the site of transplantation.