/. Embryol. exp. Morph. Vol. 32, 3, pp. 557-571, 1974
Printed in Great Britain
557
Morbidity of aging non-incubated
chicken blastoderms: further cytological
evidence and interpretation
By T. KONISHI 1 AND I. L. KOSIN 2
From the Department of Animal Sciences,
Washington State University
SUMMARY
Cytological analysis was carried out on the blastodermal cells of White Leghorn eggs
subjected to several pre-incubation treatments. These treatments were storage, ranging from
0 days to 4 weeks, together with one or more of the following: (1) variation of ambient
temperature (5 °C, 15-5 °C, 24 °C), (2) variation of carbon dioxide level (normal air, 1-5%
CO2-enriched air), (3) increased humidity (wrapping in plastic bags). To facilitate analysis, the
chromosomal configuration of metaphase cells was classified into four types, I-IV, according
to their increasingly 'abnormal' appearance, which included condensation, dispersal and/or
clumping of chromosomes. In interphase cells, the degree of 'abnormality' was rated on the
staining capacity and shape of the nuclei.
The study yielded the following results.
(1) Temperature of storage was the most important single factor determining the state of
'normality' of the nuclei. The CO2 level in the storage chamber or the use of plastic bags (to
provide the eggs in storage with a special' mini-environment', believed largely due to increased
humidity) had little effect on the cytological picture of the affected blastoderms.
(2) As the blastoderms of eggs stored at 15-5 °C aged, the proportion of Type IV chromosomal configurations steadily rose. At 24 °C the aging process frequently followed a different
route: both metaphase and post-metaphase chromosomes often simply disintegrated; the
progression from Type I to Type IV was not in evidence. Aging at 5 °C resulted in an early
appearance of uniformly dark-stained, spherical or oval nuclei. These were similar to those
observed in the terminal stages of retrogression seen in the interphase nuclei.
(3) During extended storage at 15-5 °C, mitosis was shown to be blocked at metaphase. No
active mitoses were observed in the blastoderms of eggs stored at 5 °C. At 24 °C however,
limited mitotic activity was present, up to and including anaphase.
(4) The presence or absence of a high level of mitotic activity during pre-incubation storage
was not crucial to the survival of the blastoderm. However, an environment that permitted
limited mitosis was important if the cells were to have the best possible chance for remaining
alive during storage. The CO2 content of the air or the use of plastic bags played no role in
this respect.
(5) Two explanations, at the nuclear level, are suggested for the observed chain of events
in the blastoderm of a stored chicken egg.
1
Author's address: Kanebo Institute for Cancer Research, Kanebo Hospital, Hyogo-Ku,
Kobe, Japan.
2
Author's address: Department of Animal Sciences, Washington State University, Pullman,
Washington 99163, U.S.A.
36
EMB 32
558
T. KONISHI AND I. L. KOSIN
INTRODUCTION
Aging of the avian blastoderm during pre-incubation storage progressively
reduces its capability for normal embryogenesis (Lorkiewiezowa, 1960; Arora &
Kosin, 1966). Earlier work in this laboratory has shown that the higher morbidity of affected blastoderms and of older embryos is associated with (1) a
faster rate of intracellular oxidation (Kosin & Mun, 1965), (2) a higher incidence
of necrotic nuclei (Kosin & Arora, 1966), (3) a reduced affinity for [3H]thymidine
(Arora & Kosin, 1967) and (4) an increased frequency of mitoses blocked at
metaphase (Arora & Kosin, 1968).
More recently, we have examined the question of the reputed ameliorative
effect of carbon dioxide on the viability of blastoderms in stored chicken eggs.
On the whole, our results have proved to be negative with regard to the effectiveness of CO2 for preservation of the property to hatch (Kosin & Konishi,
1973). The present paper deals with a study of cytological changes and their
possible significance in the blastoderms of chicken eggs held in storage for
varying periods of time and under varying conditions.
MATERIALS AND METHODS
The experimental treatments were essentially as described in a previous
report (Kosin & Konishi, 1973). Briefly, these included storing White Leghorn
eggs up to four weeks at 15-5 °C in (a) normal air, (b) air in which the CO2 level
was raised to 1-5 % and (c) plastic bags (copolymer of polyvinylidene chloride trade name 'Cryovac'). In the latter treatment, the atmosphere outside the bags
was either normal or CO2-enriched air. Besides these treatments, the following
were added for the purposes of the present study: (1) storage for 2 weeks at 5 °C
and ca. 60 % relative humidity; (2) storage up to 4 weeks at 22-26 °C and
ca. 65% R.H. (subsequently this treatment will be referred to as the '24 ° C
storage) - some of these eggs were also kept in plastic bags; (3) storage for the
first 24 h or 48 h after laying at 24 °C, followed by storage at 15-5 °C, either in
normal or CO2-enriched air.
Appropriate non-stored eggs served as controls. Some of these were at the time
of fixation still warm to the touch (i.e. their internal temperature ranged between
38 and 40 °C). while others by then were already down to room temperature
(20-21 °C).
The blastoderms were fixed in situ in a mixture of formalin, 48 % ethyl
alcohol and acetic acid (2:6:1). Twenty-four hours later, they were excised
from the yolk, processed for embedding in paraffin, sectioned at 5-6 ju,m and
finally stained with Mayer's haemalum and eosin. The material was then ready
for cytological analysis.
Initial examination of a representative sample of the fixed material showed a
range of morphological conditions detectable in the chromosomal complexes of
Cytology of non-incubated chick blastoderm
559
the affected blastoderms, particularly in the specimens subjected to extended
storage either at 15 or 24 °C. Some of these complexes were obvious cases of
cytological abnormality. To facilitate the intended analysis, a system of classification was developed for the metaphase nuclei. This classification encompassed
mitotic stages ranging from early through late metaphase. The overall classification described below took into account other stages of mitosis besides metaphase
when this was deemed necessary.
(1) Normal interphase nuclei.
(2) Normal metaphase chromosomal complex (Type I).
(3) Essentially normal metaphase chromosomal complex, but with some
incipient scattering of the individual chromosomes (Type II).
(4) Metaphase chromosomal complex, showing extensive scattering and/or
fragmentation of the individual chromosomes (Type III).
(5) Metaphase chromosomal complex in which the chromosomes appear
clumped in a mass; identification of individual chromosomes is no longer
possible (Type IV).
(6) Cells with deeply stained nuclei, in which individual chromosomes could
no longer be identified.
In most cases, the progression of the nucleus from Type I to Type IV was
consistent enough to regard it as a sequential series of events associated with
'metaphase aging'. Under certain conditions, however, a form of chromosome
degradation occurred which Type I and Type II complexes literally disintegrated. The ultimate result of such a process was a nucleus without recognizable
chromosomes; what remained was interpreted as chromosome fragments. This
condition, which was a prelude to general nuclear dissolution, will be referred
to in subsequent discussion as chromosomal disintegration, a state which
apparently is unrelated to that of 'metaphase aging' mentioned above. Occasionally it was difficult to separate Type I and Type II; in such cases, the two
types were considered together. The same, at times, applied to Type III and
Type IV.
Besides these groupings, all of which involved the post-prophase nucleus, there
was one other, concerned with the interphase nucleus. Here the progressive
condition of increasing morbidity, aside from the ever-greater affinity for
nuclear strains, ultimately led to a change in nuclear shape; to an exaggerated
degree it became spherical or ovoid. Sometimes it was difficult to differentiate
these necrotic nuclei from Type IV nuclei, which, it will be recalled, represented
the last step in the induced 'premature aging' of the metaphase nucleus.
Exceptions to this possible source of classification error were the more lightly
stained necrotic prophase nuclei, in which one could discern the nucleolus as well
as the more deeply stained nucleoplasm and nuclear membrane; such cells
could be easily distinguished from Type IV nuclei.
The frequencies of interphase and of normal and abnormal post-interphase
nuclei were obtained by counting and appropriately classifying these nuclei.
36-2
560
T. K O N I S H I
AND
I. L. K O S I N
Types I I I
B
A
Type III
E
D
Types III IV
G
H
Type IV
K
M
N
Cytology of non-incubated chick blastoderm
561
Counts were based on several sections of each blastoderm examined; the average
number of nuclei counted per blastoderm was 5750, the actual numbers ranging
between 3400 and 9200.
The four chromosomal configurations (I-IV) are illustrated in Fig. 1.
RESULTS
It is clear from the data summarized in Table 1 that the frequency of mitotic
nuclei with chromosomal configurations that showed marked signs of 'metaphase aging' (Types III and IV) rose with increasing duration of pre-incubation
storage. This was true regardless of treatment. Ambient temperature proved to
be the most important single factor determining, on the nuclear level, the morphological status of the blastoderm; neither enrichment of the air in the storage
chamber with CO2 not the use of plastic bags for storing eggs in any way
altered the overriding importance of temperature.
As already indicated, changes observed in the chromosomal configuration of
mitotic nuclei of blastoderms undergoing aging either at 5 °C or at 15-5 °C could
be represented by a reasonably predictable morphological shift from a condition
of 'normality' (Types I and II) to that of 'abnormality' (Types III and IV),
associated with environmentally induced aging (Fig. 1). The aging process in
interphase nuclei, as already mentioned, was visually represented by the condition in which nuclei became progressively more susceptible both to overstaining
and to a distinct change in shape. Examples of such aging metaphase and interphase cells are shown in Figs. 2 and 3. A further examination of Table 1 reveals
the frequency of the different types of chromosomal configuration related to
hatchability, reported in a previous study (Kosin & Konishi, 1973). Eggs stored
in normal air for 4 weeks at 15-5 °C showed a preponderance of Type IV configurations and their hatchability was likely to be markedly lowered (deduced
from the hatchability of eggs treated comparably by Kosin & Konishi, 1973).
This chromosomal pattern was repeated when eggs were kept at 15-5 °C in
CO2-enriched air, with or without the use of plastic bags. At the same time,
keeping eggs up to 4 weeks at 24 °C in normal air (with or without plastic bags)
resulted in relatively few Type IV nuclei; the bulk of the configurations were
Types II and III.
Changes in the nuclei became more dramatic when eggs were stored for a
FIGURE 1
Semi-diagrammatic representation of chromosomal configurations as seen in the
blastoderms of both stored and non-stored chicken eggs. Roman figures in the left
margin identify the four types described in the text. Within each type, going from
left to right, are shown examples of progressive degradation of the pattern. Aside
from configuration J, which was classified as either late metaphase or early anaphase,
all others are metaphase plates. As noted in the text, no spindles were observed in
the mitoses of aged blastoderms.
562
T. KONISHI AND I. L. KOSIN
Table 1. Prevalence of types of chromosomal configuration,
according to storage and treatment duration
Treatment
No storage. Egg still warm
after laying
No storage. Egg already cooled
to room temperature
Storage in air at 15-5 °C
Storage in air at 24 °C
Storage in air at 5 °C
Storage in 1-5% CO2 at
15 5 °C
Storage in air (within plastic
bag) at 15-5 °C
Storage in 1-5% CO2 (within
plastic bag) at 15-5 °C
Storage in air (within plastic
bag) at 24 °C
Storage in air at 15-5 °C
Storage in air at 24 °C
Length of
storage
(weeks)
No. of
blastoderms
Frequency (%) of Types
0/
I&II
III
IV
/o
Fertiles
hatched t
t
0
9
72-4
22-7
4-9
No data
0
67
24-2
63 1
12-7
93
\l
\l
41
42
12
12
2
8
33
31
29
30
15
9
11
11
11
13
2
I\4
I142
(2
u
i2
14
2 days
2 days
20-7
790
80
971
2-9
31
39-2
54-4*
6-4
33
Disintegrating ; nuclei
0
0
940 No data
60
0-6
260
73-4
80
0
95-2
20
4-8
0-2
240
86
75-8
0
95-6
54
4-4
0-2
44-2
55-6
89
0
97-2
39
2-8
36-8
61-2*
52
20
Disintegrating ; nuclei
0
38-4
45 0
16-6
95
79-7
4-2
92
16-1
(I 27-6
11 521)
0-3
0
* Some were intermediate between Type II and Type III.
t Data for eggs comparably treated but subsequently incubated (Kosin & Konishi, 1973).
length of time at 24 °C. First, the level of mitotic activity at that temperature was
greatly increased in all relevant treatment groups, with the concomitant presence
of metaphase, anaphase and telophase cells. Longer storage led to a rapid
breakdown in the mitotic apparatus and, eventually, to chromosomal disintegration ; this is illustrated in Fig. 4.
The quantitative data on mitotic activity are summarized in Table 2. Activity
was low in blastoderms fixed immediately after laying. It is not clear whether or
not a few hours' delay in fixing the specimens (as in the case of those eggs permitted first to cool down to room temperature - 20-21 °C) resulted in the
increased frequency of mitotic nuclei. On the other hand, it is clear that prolonged storage did lead to a sharp rise in the frequency of mitotic nuclei. When
the temperature of the storage chamber was 15-5 °C, close to that which is
regarded as optimal for storing eggs, the accumulation of mitotic nuclei did not
continue beyond the first 2 weeks of storage. This was true both in normal and
in CO2-enriched air, but not for treatments involving the use of plastic bags.
Cytology of non-incubated chick blastoderm
Fig. 2. Typical plates in stored and non-stored eggs, x 1440. A, B, Type II configuration. Non-stored eggs. C, Type III configuration. Egg stored two weeks at 15-5 °C.
D, Type IV configuration. Egg stored four weeks at 15-5 °C. E, Type III (arrow) and
Type IV configurations. Egg stored 4 weeks at 15-5 °C in an atmosphere containing
1-5% COa.
Fig. 3. Necrotic interphase nuclei in stored eggs, x 1440. (A) Egg stored 4 weeks at
15-5 °C. Note three deeply stained nuclei. (B) Egg stored 2 weeks at 5 °C. Note the
exaggerated round shape of several nuclei and the gradations in their strainability.
563
564
T. KONISHI AND I. L. KOSIN
B
Fig. 4. (A, B). Disintegrating metaphases in an egg stored 4 weeks at 24 °C. Fragments of what are interpreted as the remains of cytolyzed nuclei can also be seen
(arrows).
Mitotic activity in the latter was markedly depressed if the air in the storage
chamber was supplemented by CO2.
A curious contradiction appeared in the treatments where the storage temperature was 24 °C: when eggs were kept at that temperature without the use of
plastic bags, the accumulation of mitotic figures continued for 4 weeks. When
bags were used, the relative standings of the two 2-week periods were reversed
Cytology of non-incubated chick blastoderm
565
Table 2. Frequency of mitotic cells according to storage
duration and treatment
No. of
blastoderms
Mitotic cells
(% of total cells
counted ± S.E.)
0
9
0-90 ±015
0
67
1-38 ±0-23
I\42
i2
41
42
12
12
5-20 ±0-63
4-75 ±0-52
500 ±0-57
8-50 ±0-96
3-40 ±0-92
6-35 ±0-75
5-45 ±0-59
4-39 ±0-39
503 ±0-63
2-50 ±0-27
2-80 ±0-74
8-60 ±0-68
5-40 ±0-63
4-70 + 0-89
4-70 ±0-80
Length of
storage
(weeks)
Treatment
No storage. Eggs still
warm after laying
No storage. Eggs already
cooled to room temperature
Storage in air at 15-5 °C
Storage in air at 24 °C
Storage in air at 5 °C
Storage in 1-5% CO2 at
15 5 °C
Storage in air (within
plastic bag) at 15-5 °C
Storage in 1-5% CO2 (within
plastic bag) at 15-5 °C
Storage in air (within
plastic bag) at 24 °C
Storage in air at 15-5 °C
Storage in air at 24 °C
2
8
I2
33
31
29
30
15
9
11
.11
11
13
14
{I2 days
2 days
Table 3. Frequency of mitotic stages according to storage treatment
Frequency distribution (%)
of mitotic stages
Treatment
No storage. Eggs still warm
after laying
No storage. Eggs already
cooled to room temperature
Storage in air at 24 °C
Storage in air: 24 h at 24 °C,
2 4 h a t 15-5 °C
Storage in air at 15-5 °C
Length
of
No. of
storage blasto(days) derms
r
Metaphase
Anaphase
78-4
8-6
0
9
0
13
2
—
13
14
100
2
11
100
100
83-6
Anaphase
Telophase telophase
130
21-6
0
0
0
8-3
0
81
0
0
0
16-4
0
0
with respect to the level of mitotic activity; the proportion of mitotic cells was
greatly increased after the first 2 weeks, although by the end of the second 2-week
period it dropped again.
566
T. K O N I S H I A N D I. L. K O S I N
Optimum storage temperature (15-5 °C)
I->M
Death at interphase
/
Cytolytic
_ y _ _ J*" activity
4 weeks
2 weeks
High storage temperature (24 °C)
Cytolytic activity
Death at
interphase
4 weeks
Low storage temperature (5 °C)
Death at interphase
0
1 2
.
Days
\
Laying
3
2 weeks
4 weeks
*•
Duration of storage
Fig. 5. Diagrammatic representation of the chain of events postulated and observed
to take place in the nuclei of blastodermal cells of eggs subjected to three levels of
storage temperature. I, interphase; M, metaphase; A, anaphase (and possibly telophase). Solid line curves = observed events; broken lines curves = postulated
events. The intensity and the nature of mitotic activity as well as the ultimate fate
of the affected nuclei are shown on the vertical axis. The scale is arbitrary though
comparable among the three treatments.
While mitotic nuclei were present in the blastoderms of all eggs, especially
those subjected to storage after laying, most of these were in metaphase. Mitotic
stages beyond metaphase were encountered only under two conditions: (1) in
blastoderms fixed immediately after laying, i.e. when the egg was still warm to
the touch, and (2) in eggs kept at 24 °C for the first 48 h, even when they were
allowed to cool in the period between laying and actual storage (Table 3).
Cytology of non-incubated chick blastoderm
567
DISCUSSION
Active cell division, indicated by the presence of cells in various stages of
mitosis, characterizes an early chicken blastoderm exposed to temperature
conducive to its further development. However, even a partial suspension of this
activity, as happens, for example, during pre-incubation storage when blastodermal cells undergo 'immature aging' (cf. Gelfant & Smith, 1972), can lead to
abnormal embryogenesis and subsequently to high embryonic mortality. The
longer the suspension, the more pronounced is the effect (Arora & Kosin, 1968).
This has been further corroborated by the present study.
It will be recalled (cf. Table 2) that the proportion of mitotic cells in the total cell
population increased markedly with the age of blastoderms in non-incubated
eggs; the bulk of the mitotic cells were in metaphase. Further mitotic activity in
aging (stored) blastoderms evidently was blocked even at near-optimum storage
temperature. The coincidental increase in the proportion of metaphases exhibiting Type IV configuraton was regarded by us as a sequential degradation process
associated with environmentally induced aging. Thus, the fact that the incidence
of Types III and IV increased consistently with the age after laying of stored
hatching eggs is considered to reflect a growing senescence of the blastoderm, and
its impending death. The dramatic rise in the proportion of Type IV nuclei in
4-week-old non-incubated blastoderms or in eggs held for 2 weeks at a low
storage temperature (5 °C) illustrates this point well. Barring sudden disintegration of mitotic nuclei, a process which was only seldom seen in eggs stored at
15-5 °C, the progression from Type I to Type IV was seen to be time-related: the
longer the storage period, the more likely was the process to be encountered.
This was true regardless of other factors, i.e. temperature, presence or absence of
excess CO2, use of plastic bags. This concept of progressive chromosomal
degradation through a series of sequential steps, mainly involving the metaphase
stage, is presented in Fig. 5.
In newly laid eggs, so long as their internal temperature was still high enough
to permit further blastodermal growth, successive waves of mitoses occurred,
each finding a temporary resolution in interphase. Not unexpectedly, such eggs
showed a high incidence of Type I and Type II nuclei, indicating their recent
activation into the post-interphase mitotic process.
Although the total frequency of mitotic figures in non-stored blastoderms was
considerably below the level found in the blastoderms of their stored counterparts, in the former even a casual examination revealed the presence of all of the
major mitotic stages. The presence of active mitosis, and the high frequency of
Type I and Type II nuclei, coupled with hatchability data based on correspondingly treated eggs, are regarded by us as a' normal' pattern. Any deviation from it
can be expected to lead to depression of the affected blastoderm's developmental
potential. It should be recalled that the degree of blastodermal 'normality' was
not at all affected by the CO2 content of the air in the storage chamber.
568
T. KONISHI AND I. L. KOSIN
Cristofalo (1970) suggested that senescent mammalian cells are incapable of
division because of an inability to initiate the synthesis of DNA. Earlier evidence
from this laboratory (Arora & Kosin, 1967) concerning the depressed capacity
for DNA synthesis in blastodermal cells of stored avian eggs is in agreement with
this postulate. The occurrence of post-metaphase nuclei in eggs stored for 48 h at
24 °C (cf. Table 3), some 10 °C above optimal storage temperature, does not contradict this concept. The temperature of 24 °C is close to a level that permits some
growth in the blastoderm, even though the ultimate effect of such suboptimal
growth on the organism is negative (Harrison & Klein, 1954; Kosin & Sato, 1960).
The possibility that chromosomal abberations are involved in cellular and
organ deterioration has been considered by numerous investigators (e.g. Emanuelson, 1961; Allison & Paton, 1965; Curtis, Leith & Tilley, 1966; Bloom, 1969;
Michaels, Albright & Patt, 1971; Hofsaess & Meacham, 1971; Zartman, 1972;
Carrano & Heddle, 1973). Evidence supporting this was found in the present
study, even though use had to be made of a somewhat arbitrary scheme for
classifying visually detectable changes in chromosomal configuration at metaphase. On the other hand, the expected evidence of chromosomal fragmentation
in nuclei which at best could be characterized as static, if not actually moribund,
did not appear. A conclusion seems justified, therefore, that individual chromosomes in the aging nuclei are resistant to the otherwise regressive effects of
extended storage. The same cannot be said for the mitotic spindles; they were
observed only either in the blastoderms of eggs which were subjected to fixation
immediately after laying, or in eggs kept following laying at 24 °C for 48 h. The
absence of spindles in the cells of blastoderms aging under conditions which did
not permit full mitosis (even when the metaphase configuration of chromosomes
led one to expect to see spindles), was regarded as another sign of the growing
morbidity of the affected cells. The spindle apparatus is known to be extremely
susceptible to a wide range of environmental factors (Mazia, 1961).
It will be recalled that many nuclei in senescing blastoderms, particularly
those kept in CO2-enriched air or at 5 °C in normal air, were deeply stained.
These were classified as pycnotic. Because of the absence in these blastoderms of
the step-like retrogressive changes which we came to associate in this study with
nuclei blocked at metaphase, such nuclei were interpreted as having been in
interphase at the time of their death. If this interpretation is correct, then raising
the CO2 level of the air in the storage compartment, or keeping the temperature
at a drastically reduced level, e.g. 5 °C, should interfere with the mitotic process
of blastodermal cells in eggs subjected to such treatments, which, of course, they
did. Only few pycnotic nuclei were seen in the blastoderms of either newly laid
eggs or those kept at 24 °C. In other words, these pycnotic nuclei were almost
completely absent when the mitotic cycle was not impeded. It appears likely,
therefore, that when cells, for whatever reason, are left without the benefit of at
least temporary rejuvenation through DNA synthesis and subsequent mitosis,
they rapidly degrade and become necrotic.
Cytology of non-incubated chick blastoderm
569
The fact that the blastoderms of eggs stored for 4 weeks at 24 °C had many
moribund or dead nuclei probably reflects the effect of enzymically induced cytolysis fostered by high storage temperature. However, such blastoderms showed
no necrotic interphase nuclei of the type found in eggs kept for extended periods
at optimal storage temperature. This was taken to mean that at 24 °C the supply
of G2 interphase cells (cells which have completed DNA synthesis but which
have not yet proceeded with mitosis) initially available to the blastoderm at the
time of laying, gradually became exhausted through a progression of repetitive
incomplete cell cycles. Because this level of storage temperature does not permit
normal embryogenesis, an attractive hypothesis suggests itself: most of the
nuclei in the affected blastoderm proceed only to metaphase, fewer to anaphase
and still fewer to telophase. Thus, through a gradual attrition of cells capable of
normal mitosis, the likelihood of mitosis occurring would diminish with storage.
This would naturally be accompanied by a rise in the frequency of necrotic cells.
Asmundson demonstrated (Asmundson, 1947) that the hatching capacity of
eggs which were allowed to cool before incubation was higher than that for
eggs incubated immediately after laying. With regard to this point, our study
showed that post-metaphase mitotic stages were absent in eggs stored for 48 h
at 15-5 °C (cf. Table 3), which suggests that a period of relative mitotic quiescence is important for the ultimate fate of the blastoderm. It is possible that
during short-term storage at 12-15 °C preceding incubation, the higher proportion of interphase cells usually seen under such conditions permits the
organism to store energy reserves (for example, ATP) and to re-enter DNA
synthesis.
Our previous observations in avian blastoderms subjected to pre-incubation
storage, together with the results of the present study, suggest to us that the
extent and pattern of mitotic activity is a function of temperature. A higher CO2
content of the air in the storage environment obviously has no beneficial effect
on the mitotic status of these blastoderms. We postulate, therefore, that the
blastoderm survives best at 'optimal storage temperature' because those cells
which at the beginning of storage were in metaphase can complete the mitotic
cycle and divide; the resultant daughter cells remain in interphase. By the same
token, the post-interphase nuclei of the original cell population (i.e. those that
have completed the G2 stage) would proceed with mitosis but only to late
metaphase. Until conditions once again become conducive to normal growth, at
about 38-5 °C, the nuclei remain blocked from further morphologically discernible activity. If storage is unduly extended, an increasingly larger proportion
of cells would exhaust their energy supply, become moribund and eventually die.
When enough cells die, the blastoderm also dies. During the first week of
storage at optimal temperature, the cells can be expected to suffer little damage
to their (and the blastoderm's) developmental potential, because by that time
they are likely to have dissipated only a relatively small amount of the energy
passed on to them from previous successfully completed cytokineses. On the
570
T. KONISHI AND I. L. KOSIN
At laying
24-48 h.
storage 15-5 °C
2 weeks
storage
4 weeks
storage
No data
Fig. 6. Diagrammatic representation of progressive degradation of nuclei in blastoderms of eggs subjected to three levels of storage. Roman numerals (as subscripts)
refer to Type I-IV chromosomal configurations. I, interphase; M, metaphase; A,
anaphase (and possibly telophase); DC, death due to cytolysis; Dal, death at
interphase.
other hand, as storage is extended, the ensuing catabolic activity within cells
leads to cytolysis and ultimately to blastodermal death. High storage temperature
is detrimental to the blastoderm because under such conditions cytolytic
activity, a result of catabolism, can be expected to arise early in the storage
period. The process of enzymically induced degradation would then affect cells
in interphase, those which have just managed to complete cytokinesis as well as
those in metaphase and beyond. By contrast, low temperature, while drastically
depressing mitotic activity would bring about the death through early cytolysis
both of the interphase cells (those carried over from a time which was more
favorable for completion of mitosis) and of those blocked at metaphase. In
essence, the cells at low temperature die quickly not because they have used up
their supply of energy, but because they are not able to use the energy available
to them. A schematic presentation of this possible chain of events is shown in
Fig. 6.
This investigation was supported in part by funds from Medical and Biological Research,
State of Washington Initiative no. 171.
Cytology of non-incubated chick blastoderm
571
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ALLISON,
{Received 15 November 1973, revised 5 February 1974)