Hamster eggs appear unique among known mammalian eggs in that nearly 80% were found to undergo spontaneous activation, extruding the second polar body, within 24 hr. of ovulation. Often the eggs showed spontaneous early parthenogenesis the development of one or occasionally two nuclei and the formation of a haploid first cleavage spindle took place in a manner that closely resembled the corresponding stages in fertilization. Time relations, too, were much the same. Rarely, however, does the cleavage spindle pass into mitosis, but instead it breaks up and the chromosomes become scattered. Only one normal-looking 2-cell egg was found.

Rat and hamster eggs were very resistant to activation by hypothermia while still in the ovary. After ovulation, however, 100% of rat eggs were activated by body temperatures between 0 and 1° C. and about 60-70% of hamster eggs by temperatures between 0 and 16° C. In view of the high incidence of spontaneous activation in hamster eggs, the effect of chilling in this animal can be regarded as merely that of hastening the change.

Hypoxia alone activated 36% of rat eggs. Relative tissue anoxia is therefore probably the immediate effective agent in activation by anaesthetics and is partly responsible for activation by low temperatures. Cold-shock itself, however, is evidently a more potent activator. Hamster eggs were not activated by hypoxia, probably because a less severe tissue anoxia was induced.

Rat eggs activated by hypothermia showed less tendency to early parthenogenesis than those activated by local chilling of the Fallopian tube.

Nucleus and spindle formation was rather better in artificially than in spontaneously activated hamster eggs, but the prospects of extensive parthenogenetic development did not seem in any way improved. Only two 2-cell eggs resembling fertilized eggs were seen.

Nuclei in activated hamster eggs achieved a volume similar to that of male or female pronuclei at full development. Total nucleolar volume was equal to the sum of the nucleolar volumes of male and female pronuclei.

Previous work on the activation of mammalian eggs by cold has involved treatment of the eggs in vitro, followed by replacement in the Fallopian tube (Chang, 1954), or chilling of the eggs in vivo, by the application of cold water or ice to the tube (Pincus & Shapiro, 1940; Thibault, 1949; Austin & Braden, 1954a, b). In the rat, only the latter method has been employed. As a result of the cold-shock, rat eggs regularly extruded the second polar body and occasionally showed reformation of a nucleus and early cleavage. Since a variety of anaesthetics also induced activation of eggs in rats, it was suggested that the effect was due, in part at least, to the development of a tissue anoxia (Austin & Braden, 1954a).

No observations appear to have been reported on the artificial activation of the hamster egg, though some instances of spontaneous activation have been seen (Austin, 1956).

The development of methods for inducing profound hypothermia in rats and hamsters provides an opportunity for investigating the activating influence of cold without interference from anaesthesia or surgical operation. The observations described in the present communication show that eggs may be activated at temperatures well within the range that can be tolerated by the animal body as a whole.

Rats were selected for experiment when they provided fully cornified vaginal smears on examination between 9.0 and 10.0 a.m., and hamsters when they gave smears of nucleated epithelial cells at this time. Thus, in both species, freshly ovulated eggs were present in the Fallopian tubes when hypothermia was induced. Some rats and hamsters were kept under controlled illumination, so that ovulation occurred chiefly between 12.0 noon and 4.0 p.m. (Austin & Braden, 1954c; Austin, 1956)—these animals were used in experiments involving chilling before ovulation. For the other animals it was assumed from the results of earlier work (Graves, 1945 ; Ward, 1946; Runner, 1947; Everett, 1948; Pederson, 1951; Austin & Braden, 1954 c) that ovulation took place chiefly between 12.0 midnight and 4.0 a.m.

Hypothermia was induced by the methods described by Andjus & Lovelock Activation of eggs by hypothermia in rats and hamsters 339 (1955) and Andjus & Smith (1955) for the rat, and by Smith, Lovelock & Parkes (1954) and Smith & Lovelock (1955) for the hamster. By these means body temperatures down to—6° C. in hamsters and o° C. in rats can be obtained with subsequent recovery. For body temperatures above 0° C. the chilling procedure was curtailed and the temperature maintained at the desired level for such a period that the duration of the experiment was similar to that involved by the unabridged method. In this way all animals experienced subnormal temperatures for approximately the same time, although the depth of hypothermia differed.

The effect of hypoxia alone was investigated by keeping the animal in a closed jar of about 800 ml. capacity, at room temperature, until respiration almost ceased (about 15-20 min.). On removal from the jar, the animals, particularly the hamsters, were found to recover rapidly and without assistance.

Eggs were obtained by dissecting the Fallopian tubes under normal saline solution and examined with a phase-contrast microscope. For study of chromosomes and spindle morphology the eggs were fixed, while still compressed under a cover-slip, with a 5 % solution of acetic acid in absolute alcohol and stained with an aqueous solution of toluidine blue. Estimates of nuclear and nucleolar volumes were made on living eggs by the means previously described (Austin, 1952).

(1) Rats

Five rats, that had been kept under controlled illumination, were chilled to body temperatures between o and 1° C. about 0-4 hr. before the time of ovulation (Table 1). When killed the following day these rats yielded 45 eggs, of which only 2 (4 %) showed evidence of activation : a polar body, presumably the second, for the first rarely persists (Austin & Braden, 1954a), had been extruded but the chromosomes had become scattered.

Table 1.

The incidence of activation in rat eggs as a result of induced hypoxia or hypothermia

The incidence of activation in rat eggs as a result of induced hypoxia or hypothermia
The incidence of activation in rat eggs as a result of induced hypoxia or hypothermia

All the remaining rats were kept under normal lighting conditions, and the experimental treatments were applied about 8-12 hr. after ovulation.

Twelve rats were subjected to a body temperature of between 0·0 and 1·0 ° C., and six of them were killed about 3 hr. later. These provided 75 eggs, in all of which the second polar body had been extruded : all the eggs were therefore classed as activated (Table 1). The other six rats were killed the following day; 65 eggs were recovered, none of which exhibited well-formed nuclei. Eight eggs had undergone fragmentation, and 2 of these superficially resembled fertilized 2-cell eggs, but they possessed only scattered subnuclei in the cytoplasm in place of normal nuclei.

In eight rats body temperatures between 17 and 31° C. were induced; of 101 eggs recovered later 25 % were found to be activated. Finally, eight rats were subjected to hypoxia alone, the body temperature remaining within 3° C. of the normal. These animals yielded 109 eggs of which 36 % were activated.

(2) Hamsters

(a) Spontaneous activation

Six untreated hamsters, killed 8—12 hr. after ovulation, provided 60 eggs in which the second maturation division was in metaphase and only one polar body was visible. In none of these eggs, therefore, was there any evidence of activation (Table 2).

Table 2.

Hamster eggs showing spontaneous activation when recovered at different times after ovulation

Hamster eggs showing spontaneous activation when recovered at different times after ovulation
Hamster eggs showing spontaneous activation when recovered at different times after ovulation

From six hamsters killed 13-17 hr. after ovulation, 58 eggs were recovered, of which 10 (17%) showed evidence of activation; 5 eggs exhibited phases of the second meiotic division and 5 had two polar bodies and nuclei similar to those shown in Pl. 4, figs. 1-3.

A much higher incidence of activation, namely 77 %, was found among the eggs recovered 18-22 hr. after ovulation. Most of the eggs had a nucleus and two polar bodies, but there were 2 eggs that still displayed the second meiotic division. The nuclei were usually well formed and resembled those shown in Pl. 4, figs. 3 and 4, but in some eggs they did not look quite normal in that the nuclear boundary was indistinct and the nucleoli were gathered at the centre of the nucleus.

A similar incidence (76 %) of activated eggs was noted at 24-28 hr. after ovulation ; of these eggs about half had formed what appeared to be the metaphase of a first cleavage spindle, the remainder still having nuclei. No anaphase or telophase stages were seen. While a number of the spindles were well formed, like that shown in Pl. 5, fig. 6, most were poorly developed, and in some eggs the chromosomes were already somewhat scattered (Pl. 5, fig. 10). In some of the unactivated eggs also, the maturation spindles were beginning to break up (Pl. 5, fig. 9). Disorganization of spindles was more common in the eggs recovered at the next period of observation, 30-34 hr. after ovulation, and in many instances the chromosomes were so widely scattered that it was uncertain whether they were derived from maturation or cleavage spindles. Consequently, the figure given for the incidence of activation in this group, 81 %, may be too high.

In the next two groups of hamsters, killed 36-40 and 58-62 hr. after ovulation, chromosome scatter was much more common and in none of the eggs could a cleavage spindle be positively identified. Many of the eggs, indeed, apparently contained no chromatic material at all. Nuclei, when present, were abnormal, grossly so in the eggs seen at 58-62 hr. Amongst all this evidence of abnormality there was one 2-cell egg (Pl. 5, fig. 8) which closely resembled a fertilized egg in its general form and in the appearance of its nuclei.

Among the 68 spontaneously activated eggs containing nuclei, and excluding those recovered at 58—62 hr. with grossly abnormal nuclei (Table 2), there were 15 binucleate eggs (Pl. 4, fig. 5), the rest being mononucleate. It was noteworthy that the binucleate eggs with the best-formed nuclei were those that had a single polar body—often the two nuclei were very similar to male and female pronuclei. In binucleate eggs with two polar bodies the nuclei were small and ill-formed.

Many of the eggs recovered from untreated hamsters, including the activated eggs, were characterized by the presence of one or more cytoplasmic vacuoles, more especially at the later periods. Vacuoles are seldom seen in fertilized eggs. In none of the eggs from untreated hamsters was there evidence of fragmentation, such as commonly occurs in unfertilized rat eggs (Austin, 1949).

(b) Induced activation

Six hamsters were chilled before ovulation; they had been maintained under controlled illumination, and the chilling procedure was begun at 9.30-10.0 a.m. on the day of ovulation. Minimum temperatures were reached about noon to 12.30 p.m. and rewarming was virtually complete by 1·0-1·30 p.m. Minimum temperatures varied between o and 10° C. (two hamsters at 0·5° C., two at5° C., and two at 10° C.). The animals were killed at 5.0 p.m. (1-5 hr. after ovulation); they yielded a total of 46 eggs, all of which had a single polar body and the second maturation spindle in metaphase (Table 3). None of the eggs, therefore, had been activated by the chilling.

Table 3.

Eggs recovered at different times from hamsters subjected to hypothermia or hypoxia

Eggs recovered at different times from hamsters subjected to hypothermia or hypoxia
Eggs recovered at different times from hamsters subjected to hypothermia or hypoxia

Hamsters with freshly ovulated eggs in the Fallopian tube were chilled to body temperatures of o-1° C., 3-5° C., 8-16° C. or 20-25° C. The chilling procedure began about 10.o a.m. and rewarming was in progress by 1.0 p.m. The animals were killed at 5.0 p.m. (13-17 hr. after ovulation). A high incidence of activation was found after chilling to o-i° C. (80% activation), to 3-5° C. (81 % activation) and to 8-16°C. (86% activation) (Table 3). Body temperatures of 20-25° C., however, gave rise to only 34% activation. Hypoxia at room temperature, which was associated with a negligible fall in body temperature (to 36-38° C.), did not lead to activation in any of the eggs.

The activated eggs seen at 13-17 hr. after ovulation included a few showing stages of the second maturation division, but the majority had nuclei and two polar bodies. All stages of nuclear development were seen (Pl. 4, figs. 1-4), and at each the induced nucleus closely resembled, both in size and form, a normal female pronucleus. Dimensions of six of the larger nuclei were measured: the mean nuclear volume was 2790 µ3 (range 2261 to 3222 μ3), total nucleolar volume 327 μ3 (309-440 μ3) and number of nucleoli 7·5 per nucleus (2-12).

Six hamsters that had been chilled to body temperatures between o and i6° C. were killed the following day. Of the 73 eggs recovered, 82% were activated (Table 3), the majority displaying the metaphase of a first cleavage spindle, as in Pl. 5, fig. 6. Again, no anaphase or telophase stages were seen. The spindles were nearly all normal in appearance, although they presumably had a haploid chromosome complement, since two polar bodies had been extruded. In one egg (Pl. 5, fig. 7) the chromosomes were conveniently displayed, and twenty to twenty-two chromosomes could readily be counted ; this is approximately the haploid number (2n = 44). Two eggs were nucleated and one still showed the second meiosis. There were also two 2-cell eggs, such as that in Pl. 5, fig. 8, which closely resembled fertilized 2-cell eggs. In addition to the activated eggs there were 4 eggs in which activation had not apparently occurred, and 9 eggs that consisted only of the zona pellucida containing a small amount of granular debris (Pl. 5, fig. 11) ; these are referred to in Table 3 as ‘zonas’. The cytoplasm had evidently been lost through a break in the zona.

A further six hamsters, similarly treated, were killed 54-62 hr. after ovulation (Table 3), and these yielded 43 eggs, most of which showed varying degrees of degeneration ; three had undergone fragmentation. There were 28 eggs that showed scattered chromosomes, which could have originated from a cleavage spindle, but the disarrangement was often so bad that they could equally well have come from the second maturation spindle. In 3 eggs no chromatic material at all could be discerned. There were also 12 eggs that were classed as ‘zonas’, since all or most of the cytoplasm had been lost.

Of the 94 nucleated eggs recovered from chilled hamsters, there were only 3 binucleate eggs, resembling the egg shown in Pl. 4, fig. 5, the rest being mononucleate. All the nucleated eggs seen at 13-17 hr. after ovulation had two polar bodies ; no decision could be made on the two eggs recovered at 30-40 hr. owing to the presence of much granular debris in the perivitelline space.

The hamster egg evidently has a strong tendency towards spontaneous parthenogenesis. In the present study nearly 80 % of eggs from untreated hamsters were observed to have undergone activation within 24 hr. of ovulation. Moreover, the progress of the meiosis, and generally also the development of the nuclei, took place in a manner closely similar to that of the corresponding stages in fertilization. The time relations, too, were much the same. Comparison of the present data with data published previously on fertilization in hamsters (Austin, 1956) shows that the times required for the egg to pass from the stage of spermatozoon penetration or of spontaneous activation to the formation of a cleavage spindle are both about 13-17 hr.

The occurrence of spontaneous early parthenogenesis appears to be unknown in other mammals, except as a rare event. Unfertilized rat eggs were found sometimes to undergo fragmentation in such a way as superficially to resemble cleaving fertilized eggs, but both 1-cell and divided eggs nearly always showed decided abnormalities of nuclear and cytoplasmic structure (Austin, 1949). In a later communication (Austin & Braden, 1954d) it was reported that among several thousand eggs from rats, mice and rabbits there were only two (rat) eggs that exhibited spontaneous nucleus formation. The striking feature of the hamster eggs is that so many of them displayed a normal appearance in nuclear and cytoplasmic components at all stages up to and sometimes including the cleavage spindle.

At 24-28 hr. after ovulation and later, eggs showing apparent parthenogenesis became less common in the untreated hamsters. Most of the cleavage spindles seem to break up instead of passing into the mitosis, and only one 2-cell egg was seen. The tendency towards parthenogenetic development, then, ceases at this stage, or else some item normally contributed by the spermatozoon is required for further progress.

Both in rats and hamsters the eggs were found to be highly resistant to artificial activation whilst still in the ovary. Evidently the second meiotic division is precluded in some way from following immediately upon the first. On the other hand, a high incidence of activation in rat and hamster eggs was obtained by chilling the whole animal after ovulation, though there were several points of difference between the two species. In the rat a body temperature of 0-1° C. was just as effective as the application of ice to the Fallopian tube (Austin & Braden, 1954a, b), both methods giving 100% activation. But whereas in the earlier experience about 10% of eggs developed nuclei or, at later stages, showed apparently normal cleavage, in the present experiments no such examples of early parthenogenesis were seen. Perhaps this was because with hypothermia a reduced temperature is maintained for a much longer time than is involved when local chilling is effected by placing ice against the Fallopian tube.

In the hamster, only about 80% of eggs were found to be activated, but this occurred with a wide range of temperatures (0-16° C.). The incidence of activation referable to chilling is actually less than 80 %, for at least 17 % of eggs would have undergone spontaneous activation. The true figure would be between 60 and 70%. In view of the large proportion of eggs that ultimately become activated spontaneously the action of hypothermia may therefore be regarded simply as the hastening of a change that is inevitable. Nucleus and spindle formation were in general better with artificial activation, but the prospects of more extensive parthenogenetic development through this means do not appear to have been improved at all. Indeed, signs of degeneration—the frequency of fragmented eggs and ‘zonas’—are more in evidence than with spontaneous activation.

Hypoxia alone produced activation in 36% of rat eggs, and this supports the contention that a relative tissue anoxia is probably the immediate effective agent in the activation by anaesthetics and to some degree in the activation by cold also. However, the fact that chilling to o-1° C. had a greater influence than hypoxia alone means that the low temperature itself was responsible for much of the effect. The activation obtained (25 %) with body temperatures between 17 and 31° C. is presumably referable entirely to the hypoxia induced in the early phases of the chilling procedure.

Hamster eggs were unaffected by hypoxia as brought about by restricted air supply. This may well be due to the development of a less intense tissue anoxia in these animals. Certainly the hamsters recovered much more rapidly than the rats.

In general the nuclei developed in artificially activated hamster eggs more often resembled pronuclei in appearance than did the nuclei in spontaneously activated eggs. The nuclear and nucleolar dimensions may be compared with those of pronuclei at full development, as previously determined (Austin, 1955). Thus, the mean nuclear volume of induced nuclei was about 2800 μ3, while that of larger (probably male) pronuclei was about 3100μ3 and of smaller pronuclei about 2700 μ3. On the other hand, the mean total nucleolar volumes were about 330 μ3 for induced nuclei, and about 180 μ3 and 150 μ3 for larger and smaller pronuclei respectively. It has been proposed for rat and mouse eggs that the male and female pronuclei compete for limited amounts of formative material available in the egg cytoplasm, that the male pronucleus has the greater affinity for this material, and that there is also an innate restriction on nuclear size (Austin & Braden, 1955). In the hamster egg the pronuclei evidently have much the same affinity for formative material and the innate restriction, which only becomes operative when a single nucleus develops in an egg, is such as to maintain nuclear size and form within the range exhibited by pronuclei. As in the eggs of rats and mice the restriction on total nucleolar volume is less than on nuclear volume. Indeed, it is possible that the innate restriction on total nucleolar volume is not effective in the hamster egg, since the volume attained by the nucleoli in induced nuclei approached the theoretical maximum, namely about the sum of the corresponding values of male and female pronuclei. These conclusions are supported by unpublished data obtained by the author on three hamster eggs that were undergoing apparent early gynogenesis. Each egg contained a single large nucleus and a spermatozoon, the head of which was still in an early phase of its metamorphosis. Mean nuclear volume was about 3050 μ3 and total nucleolar volume about 340 μ3. Here again, nuclear volume is about that of a single male or female pronucleus, while nucleolar volume is approximately equal to the sum of the nucleolar volumes of the two pronuclei. It seems likely that the gynogenetic eggs originated with the occurrence of spermatozoon penetration after spontaneous activation. Probably, then, by the time the spermatozoon head was ready for conversion into a male pronucleus the egg nucleus had completed most of its growth and in so doing had taken up the available formative material from the vitellus. This would apply particularly to the material required for the growth of nucleoli, and consequently the potential male pronucleus would be handicapped for nucleus formation and precluded from developing nucleoli. Consistently, in the gynogenetic eggs seen, the sperm head had been transformed into a small vesicular structure devoid of nucleoli.

Among the spontaneously activated hamster eggs with nuclei there were more with two nuclei than among the artificially activated eggs : 15 eggs out of 68 (22 %) as compared with 3 eggs out of 94 (3 %). This is probably because spontaneous activation occurred later than artificial activation, allowing the second maturation spindle time to move inwards away from the surface of the egg. If this happened, two nuclei would be formed after the completion of meiosis, as the spindle would be too far from the surface to permit extrusion of the second polar body. The formation of two female pronuclei in Triturus eggs after hot-shock treatment has been shown to occur by such a mechanism (Fankhauser & Godwin, 1948).

The author’s thanks are due to Dr A. U. Smith for advice and help in the chilling of hamsters and to Dr S. Goldsveig for reviving the rats taken to 0-1° C.

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The illustrations are of hamster eggs recovered from unmated animals. The photographs were all taken with a phase-contrast microscope, except for fig. 7. The eggs shown in figs. 6, 7, 9 and 10 were fixed and stained, whereas the others were photographed in the fresh state.

Plate 4

Figs. 1-4. Stages of the development of nuclei in eggs from hamsters that had been chilled, × 1000.

Fig. 5. A binucleate egg, with one polar body, from an untreated hamster, × 400.

Figs. 1-4. Stages of the development of nuclei in eggs from hamsters that had been chilled, × 1000.

Fig. 5. A binucleate egg, with one polar body, from an untreated hamster, × 400.

Plate 5

Fig. 6. A normal-looking first cleavage spindle in an egg recovered from a chilled hamster killed 30-40 hr. after ovulation, × 1500.

Fig. 7. First cleavage metaphase chromosome group in an artificially activated egg showing twenty to twenty-two chromosomes (2n = 44). × 1000.

Fig. 8. A 2-cell egg recovered 36-40 hr. after ovulation from an untreated hamster. In outward appearance and nuclear form it closely resembled a fertilized 2-cell egg. × 400.

Fig. 9. A scattered group of chromosomes from the break-up of a second maturation spindle. The egg came from an untreated hamster 24-28 hr. after ovulation, × 1000.

Fig. 10. The disintegrating cleavage spindle from an untreated hamster 30-34 hr. after ovulation, × 1500.

Fig. 11. Two eggs that consisted only of the zona pellucida with a small amount of granular debris inside. The rupture through which the greater part of the vitellus was lost is visible. These ‘zonas’ were recovered 54-62 hr. after ovulation from chilled hamsters. × 100.

Fig. 6. A normal-looking first cleavage spindle in an egg recovered from a chilled hamster killed 30-40 hr. after ovulation, × 1500.

Fig. 7. First cleavage metaphase chromosome group in an artificially activated egg showing twenty to twenty-two chromosomes (2n = 44). × 1000.

Fig. 8. A 2-cell egg recovered 36-40 hr. after ovulation from an untreated hamster. In outward appearance and nuclear form it closely resembled a fertilized 2-cell egg. × 400.

Fig. 9. A scattered group of chromosomes from the break-up of a second maturation spindle. The egg came from an untreated hamster 24-28 hr. after ovulation, × 1000.

Fig. 10. The disintegrating cleavage spindle from an untreated hamster 30-34 hr. after ovulation, × 1500.

Fig. 11. Two eggs that consisted only of the zona pellucida with a small amount of granular debris inside. The rupture through which the greater part of the vitellus was lost is visible. These ‘zonas’ were recovered 54-62 hr. after ovulation from chilled hamsters. × 100.