Results

When eggs (R. pipiens) are simply pricked with a clean glass needle they rotate, form the ephemeral “black dots” localizing the second maturation division spindle, and within a few hours show puckering of the surface or abortive and irregular cleavage furrows. There is no genuine cleavage or blastula formation. In our experiments 99% (2831 out of 2853) of the pricked eggs behaved in this way. By contrast, eggs which are pricked and enucleated fail to give any signs of cleavage. When observed a few hours after activation these eggs show none of the puckerings, etc., which are present in practically all of the pricked eggs at this time. Thus, 631 out of 638 enucleated eggs behaved in this way, indicating that 99% of the operations for removal of the egg nucleus were successful. Actually, the 7 eggs which did show puckers were ones in which the exovate, which forms when the egg in enucleated and which contains the egg nucleus, still retained a connection with the egg. In these the egg proper may have been enucleated, the puckers forming through the influence of the nucleus in the attached exovate.

In order to check further on the success of the operation for enucleation we always enucleated some normally inseminated eggs at the end of each experiment. From these we should obtain androgenetic haploids when the operation is successful, and normal diploids when it is not. Out of 358 operations, regarded at the time as successful, we obtained 337 embryos, all of them haploids. An additional 169 operations, regarded as not absolutely certain, gave 161 embryos of which 160 were haploids.

From these results we can say that eggs which are pricked and then enucleated will practically never retain the egg nucleus by mistake, and will never develop. When these eggs are now each injected with a diploid blastula cell nucleus more than half of them cleave, giving rise to partial and complete blastulae as summarized in table 1. Some of these blastulae were fixed. The remainder developed as shown in table 2. Of the complete blastulae the majority (74%) gastrulated normally and formed complete embryos. Approximately half of these were normal in appearance when fixed at stages ranging from stage 19 (5-mm. embryo) to the tadpole stage. The remainder formed complete neurulae but later on displayed slight to moderate abnormalities such as growth retardation, microcephaly in some cases, larger than normal heads in others and so on. Of the 15 abnormal embryos listed in table 2, 14 were diploids and 1 was polyploid as judged from the ectodermal cell size. Among the 15 normal embryos there were 7 diploids and 8 polyploids. The only other embryos that developed far enough to give clear indications of chromosome number were 4 of the 5 abnormal neurulae listed in the table. These also were diploid (2 cases) or polyploid (2 cases). Thus, among the 35 embryos obtained there were no haploids. Had they occurred they would have been hard to account for in these experiments, since the injected nucleus was diploid. The polyploids, on the other hand, may be explained. The blastula cell nuclei are undoubtedly in various stages of mitosis when transplanted, and there should be ample opportunity for a doubling of chromosomes to occur after the nucleus has been transplanted and before the egg cytoplasm is prepared to cleave.

In order to get cytological evidence of the removal of the egg nucleus 22 of the injected eggs were fixed at stages ranging from morula to gastrula, and were sectioned and stained with the Feulgen reagent. In 21 of these eggs the exovate could be seen trapped in the inner jelly layer but separated from the egg proper. In 9 cases the egg nucleus was found in the exovate, while the egg proper consisted of cells containing normal nuclei which could only have been derived from the transplanted nucleus. In 6 additional cases Feulgen positive material was seen in the exovate, but it could not be definitely recognized as the egg nucleus. The remaining 6 cases showed no Feulgen positive material. In these the egg nucleus may have been lost from the exovate, or it could have been obscured by the pigment of the surface coat surrounding the exovate.

The evidence summarized above shows quite conclusively that living nuclei can be transplanted from blastula cells into enucleated eggs. However, in order to get an additional proof of the success of the technique we have transplanted R. catesbeiana nuclei into R. pipiens enucleated eggs. It is known from hybridization experiments^{8, 12, 13} that this combination is lethal. The diploid hybrid is arrested in late blastula or early gastrula stage and dies in about 3 days, while the androgenetic haploid hybrid is always arrested in late blastula stage but dies later—at about 4 to 5 days. Thus, if the transplantation of the foreign nucleus is successful we should obtain a uniform arrest of development followed at the appropriate interval by the death of the embryo.

The experiments were done as follows: Donor blastulae were produced by inseminating pipiens eggs with catesbeiana sperm. The egg nucleus was removed from some of these, giving us androgenetic haploid hybrids. The rest of the eggs were allowed to develop as diploid hybrids. After about 18 hours at 18° both types of hybrids had developed to stage 8 (mid to late blastula), and were usually used to provide nuclei for transplantation at this time. The hybrids are, of course, arrested shortly thereafter and we have evidence, not presented here, that nuclei taken from older blastulae (stage 9) generally will not cause enucleated eggs to cleave.

The results of the transplantations of nuclei from hybrid blastulae are summarized in table 3. About half of the enucleated eggs which were injected with the catesbeiana haploid nucleus cleaved and developed into partial or complete blastulae, as indicated in the table. The complete blastulae, it should be emphasized, looked perfectly normal. Yet none of them showed any signs of gastrulation. They were all arrested in late blastula stage, as were the androgenetic haploid hybrid controls, and later on they all died at approximately the same time as did the controls ( Fig. 1).

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Figure 1.

Exp. 1.—Mortality curves for (1) haploid hybrid controls, pipiens (♀) × catesbeiana ♂; (2) enucleated pipiens eggs injected with haploid catesbeiana nuclei; and (3) control diploid hybrids, pipiens ♀ × catesbeiana ♂.

Based on 55 haploid hybrid controls, 28 experimental haploid hybrids, and 147 diploid hybrid controls.

Exp. 2.—As for experiment 1, except for the experimental embryos which in this experiment are derived from enucleated pipiens eggs injected with diploid (pipiens ♀ × catesbeiana ♂) hybrid nuclei.

Based on 119 diploid hybrid controls, 21 experimental diploid hybrids, and 73 haploid hybrid controls.

Enucleated eggs injected with diploid hybrid nuclei also cleaved and gave rise to blastulae, as shown in table 3. These corresponded to the diploid hybrid controls in their development. That is, they were all arrested either in late blastula or early gastrula stages, and died when their controls did. ( Fig. 1.)

In order to see whether the nuclear phenomena associated with the arrest and death of the blastulae were occurring in the same way in the experimental and control blastulae we fixed several of each at ages of 42 to 72 hours. The controls consisted of 9 androgenetic haploid hybrids [pipiens (♀) × catesbeiana ♂]; the 17 experimental blastulae were all derived from enucleated pipiens eggs injected with haploid catesbeiana nuclei taken from androgenetic haploid hybrid blastulae.

A study of the arrested control blastulae revealed a variety of nuclear abnormalities. The majority of the nuclei were in an abnormal interphase condition characterized by an accumulation of chromatin around the periphery of the nucleus in the form of thick threads, leaving the central portion free of Feulgen-positive material. In addition there were arrested and abnormal metaphases, and a few anaphases which always showed bridges. In some of these figures the chromosomes were clumped. Finally, some cells contained small groups of chromosomes, or small pycnotic nuclei, presumably derived from irregular divisions that had occurred earlier.

In the majority (14 out of 17) of the experimental blastulae the nuclear abnormalities were exactly the same as those described above for the controls. In 3 cases, however, the condition was different, with the nuclei all in an abnormal interphase condition characterized by a rather diffuse Feulgen staining. We do not know yet how to account for these exceptional cases. It may be that in a larger series of cases the same type of nuclear phenomenon might appear among the controls, or occasionally there may be some effect of the transplantation on the subsequent behavior of the nuclei. In any case, in the large majority of the experimental blastulae the appearance of the nuclei is the same as it is in the controls. This fact, taken together with the fact that the extent of development and the time of death are also the same in the experimental and control blastulae provides convincing evidence of the successful transplantation of the foreign nucleus.