/. Embryol. exp. Morph. Vol. 50, pp. 131-144, 1979
Printed in Great Britain © Company of Biologists Limited 1979
137
The temporal stability of the Drosophila oocyte
By ROBERT WYMAN 1
From the Department of Biology, Yale University
SUMMARY
Individual female Drosophila melanogaster were maintained without yeast for up to 18 days.
Some of the females did not lay any eggs during this holding period. The females produce
mature oocytes (stage 14) by day 3 and from day 5 onward the females each held about 60
mature oocytes. The nulliparous females were mated and it was shown that the first egg
produced by females held up to 18 days before mating could be viable and develop into a
normal, fertile adult. On day 2 each female contained about 500 oocytes in post-oogonial
stages of oogenesis. The first 500 fertilized eggs were collected from females that had been
held nulliparous for 7-10 days before mating. No period of significantly reduced viability
was found. It is concluded that the developmental program of Drosophila eggs can be
interrupted between oogenesis and fertilization for at least 15 days without loss of the
ability to produce normal, fertile adult progeny.
INTRODUCTION
The Drosophila ovary as reviewed by King (1970) may be likened to an
assembly line for a biochemical product. Each ovary contains about 16 ovarioles
and each ovariole contains about 16 oocytes in different stages of development.
The oocytes are lined up in single file in strict order of age. The newborn
cystoblasts are at the anterior end of the ovariole while the mature oocytes are
at the oviduct end. The total time of egg maturation, from oogonium to oviposition, is about 8 days. In a healthy inseminated female each ovariole completes about two eggs a day; hence with 32 ovarioles the female can lay an egg
every 20 min or so (King, 1970).
Egg laying begins on the 2nd or 3rd day after emergence of the adult. Uninseminated females will normally not hold eggs much beyond this time (Wilson,
King & Lowry, 1955). The unfertilized eggs, when laid, degenerate rapidly.
This might be taken to mean that the production line process cannot be stopped.
The mature egg might have a short time in which it must be fertilized, after
which it is no longer capable of supporting normal development. Also a halt in
the assembly line may damage the viability of oocytes at an earlier stage of
maturation. In both cases it would be advantageous for the fly to lay each egg
shortly after its maturation whether or not it is fertilized.
1
Author's address: Department of Biology, Yale University, New Haven, CT 06520,
U.S.A.
138
R. WYMAN
It is known that the mature oocyte synthesizes protein rapidly and that this
rate does not change when the eggs are fertilized and laid (Zalokar, 1976). In
fact there is no change until the blastoderm stage. This is the approximate
stage when the zygotic genes start making mRNA and protein. Protein synthesis
continues also in unfertilized eggs, laid by virgin females, at about the same rate
as in the normally developing preblastoderm embryo (Zalokar, 1976). The
unfertilized eggs visibly degenerate by about 6 h after oviposition. It is possible
that biochemical processes are irrevocably set in motion during oocyte maturation which lead to normal embryogenesis if the egg is fertilized at the proper
stage of this process, or to egg death if fertilization does not occur at the
proper time. The present investigation tests this possibility.
In the experiments presented here we introduced a delay of up to 15 days
between the completion of oogenesis and fertilization. Even after this delay, the
egg can still develop into a normal adult. We conclude that the developmental
program can be interrupted for at least 15 days and yet the Drosophila oocyte
will remain stable and developmental^ competent.
METHODS
Flies used were Drosophila melanogaster, Oregon-R strain from the stocks of
Dr D. Poulson, Yale University. Since ether interferes with reproductive
activities, no anesthesia was used at any stage in the experiments. To facilitate
handling of unanesthetized flies, all flies used were homozygous for curved, and
therefore flightless.
Culture dishes were Corning plastic Petri dishes either 35 x 10 mm or 60
x l 5 m m . These dishes were half filled with cornmeal, molasses, agar and
killed yeast medium (Table 3 in Doane, 1967).' Yeasted' dishes had, in addition,
a layer of live baker's yeast spread on top of the medium.
Flies were kept at 20 °C. Eggs laid by the flies were incubated at 25 °C until
emergence of the adult to confirm egg viability. Viability in all experiments was
measured as number of adults eclosing/total eggs oviposited, expressed as a
percentage.
RESULTS
Controls
Fifty-five virgin females were collected within a few hours after emergence;
each was placed in a separate yeasted dish with three males. Of these females
13 % laid eggs on day 2, 80 % on day 3 and 2 % on day 4. The viability of the
first batch of eggs laid by these females was 88 % (321 adults/366 eggs). We
conclude from the above that mature, viable eggs are produced by females by
day 3 after eclosion.
To control for the possibility that the males present in the above experiment
accelerated the maturation of the eggs, the following experiment was per-
Aged Drosophila oocytes
139
formed. Forty virgin females were collected within a few hours after emergence
and placed individually in yeasted dishes. On the third day three males were
introduced into each dish. Three-quarters of the females laid eggs within 8 h; all
laid eggs within 24 h. We conclude that virgin females when isolated also
produce mature eggs by day 3 after emergence.
Retention of eggs by virgin females
When held isolated for long periods in yeasted dishes, virgin females eventually lay unfertilized eggs. Fifty virgin females were collected upon emergence
and placed individually in yeasted dishes. They laid their first eggs (unfertilized) between day 3 and day 11. Day 7 was the median day of the first egg
laying.
Since these experiments required that females be kept for more than 7 days
before laying any eggs, the following procedure was used to prevent egg laying.
Individual virgin females were collected within a few hours after emergence and
placed in yeasted dishes. By day 3 we could be sure that these females carried
mature eggs. On day 4 these females were placed in new, unyeasted dishes.
Thereafter they were transferred every other day to new unyeasted dishes. This
procedure greatly reduced the probability that the unmated females would lay
eggs. The few females that did lay eggs were discarded. In this manner females
were obtained which had not laid eggs for up to 18 days after emergence.
Oocytes are held in mature state
The abdomens of aged, nulliparous females become swollen with eggs. The
abdomens of nulliparous females were dissected; almost the whole volume was
taken up by mature oocytes (stage 14 of King, 1957). There were very few
oocytes in stages 9 through 13 (yolk accumulating stages). Younger stages were
present; their total number was slightly fewer than that found in normally
mated young females. The number of mature (stage 14) oocytes found in the
females averaged 48-7 ±9-8 (N = 17) on day 4 and 58-9 ±15-5 (N = 11) on
day 5. Females held nulliparous until day 12 or 13 had 58-6 ± 13-2 (N = 24)
mature oocytes. The largest number of stage-14 oocytes counted in one female
was 91. No degenerating stage-14 oocytes were seen. Thus the females carry a
maximum of about 60 stage-14 oocytes by the fifth day and then maintain
these oocytes until at least the 12th or 13th day.
Viability of aged oocytes
Aged nulliparous females were transferred to yeasted dishes and males were
introduced. Egg laying began from 3 to 24 h after the transfer. There was no
evidence that the females ever became egg-bound. When the females started
laying eggs, they laid them very rapidly. The first batch of eggs (about nine
eggs/female) was collected. The total viability of the first batch of eggs laid by
140
R. WYMAN
Table 1. Viability offirst batch of eggs laid by previously nulliparous
females, when laid on the nth day
Day
...
No. of females
No. of eggs laid
No. eggs surviving
to adult
%
6
7
8
9
10
11
12
13
14
.15
18
All
10 18
179 348
12
132
13
67
16
98
14 20
87 103
11
74
15
111
4
59
5
17
138
1275
140 310
79 89
106
80
39
58
66
67
61
70
37
50
47
42
30
51
14
82
918
72
67
65
Table 2
Nulliparous females were held unmated until day n. They were then mated and
their first batch of eggs was collected. The viability of this batch of eggs was then
measured as the percentage developing to adulthood. The number of females whose
eggs were at least 75% viable is shown in the top row, while those with poor
viability (<75%) are shown in the middle row. The percentage of mothers with
highly viable eggs is shown in the bottom row.
Day
...
No. with 2*75% viability
No. with < 7 5 % viability
/o
6
7
8
9
10
11
12
13
14
15
18
8
2
80
14
4
78
9
3
75
5
8
38
8
8
50
9
5
64
11
9
55
6
5
55
7
8
47
2
2
50
4
1
80
females (n = 138) aged between 6 and 18 days (at the time of first egg laying)
was 72% (918 adults from 1275 eggs).
This is significantly below the viability (88 %) of eggs laid by females who
were fertilized and laid eggs on day 2 or 3. Table 1 shows the viability of the
first batch of eggs when laid by a female on the wth day. There seems to be a
tendency for the viability to decrease gradually after day 8. However, the small
number of eggs collected from the few females who were mated on the 18th day
showed high viability (82 %). At all ages the loss of egg viability was due to
some females whose eggs had a low viability, while the eggs of other females
retained a high viability (Table 2). A few of the females first laid a group of
eggs that did not hatch (possibly unfertilized) and then laid eggs that did hatch.
Viability of the oldest oocytes
Because of the single file development of the eggs in the ovarioles, the first
eggs laid should be the oldest. It was therefore important to determine if the
first egg laid by each female (or the first two eggs, one presumably from each
ovary) developed to adulthood. Since dishes, when collected, usually had more
than one egg in them, the viability of the first egg could only be determined
when all eggs in a dish reached adulthood. Of the 85 females which laid their
first eggs between 10 and 18 days after emergence, the entire first batch of eggs
Aged Drosophila oocytes
141
Table 3
Nulliparous virgin females were mated at 7-10 days of age. Eggs were collected
daily thereafter until 500 eggs were collected from each female. Table shows number
of these eggs developing to adulthood. Day of laying refers to days after mating.
Day of laying ...
No. eggs laid
No. adults eclosing
O'i
.0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15
243 432 573 552 592 566 597 556 488 531 375 237 110 52 30
195 346 460 462 478 458 478 398 336 401 280 164 79 36 22
80 80 80 84 81 81 80 72 69 76 75 69 72 69 73
of 32 of them reached adulthood (32/85 = 38 %). An additional 17 (20 %) had
only one death.
Of the five females which did not lay eggs and were not mated until the 18th
day, all of the first batch of eggs of four females reached adulthood.
Viability of oocytes held in immature stages
The foregoing experiments have shown that stage-14 oocytes can be held for
many days. However it is possible that the holding procedure damages eggs of
earlier stages. To check the effect of holding on all these stages we collected and
checked the viability of the first 500 eggs laid by each of 11 aged females. Eggs
were collected daily. Table 3 shows the viability of eggs laid by females who
were 7—10 days old at the time of laying their first egg. No period of greatly
reduced viability was found. The slight decrease in viability for day 8 and
beyond may be related to the age of the mothers who were 15-18 days old on
the eighth day of laying.
Stability of polar plasm
The germ cells of Drosophila arise from the polar plasm and pole cells of the
egg while somatic cells derive from the cortical cytoplasm and the blastoderm
cells. By noting the emergence of adults from aged oocytes, it is possible to
conclude that the requisite substances for somatic cell development are stable
during the period of aging. It was still necessary to prove that the same is true
for the germ cells. Consequently a parental generation was set up and the
females held nulliparous until 6-^-8 days after emergence. They were then, mated
and the first F± offspring were collected. It was the viability of the germ cells of
this Fx generation that was in question. Thus single Fx males and females were
mated (five pairs). All of these crosses were fertile and yielded males and
females of an F2 generation.
DISCUSSION
A simple method for reducing the probability that a virgin female will lay
her unfertilized eggs was used to obtain females who did not lay eggs for many
days after emergence. When held singly in yeasted dishes, virgin females started
IO
EMB 50
142
R. WYMAN
laying eggs at an unpredictable age, with a median of 7 days. However, when
transferred to unyeasted dishes, the virgin females could be held for up to
18 days before they started laying eggs.
In control experiments it was found that females of the strain used, when
held singly in yeasted dishes, with or without males, produce mature eggs by
day 3 after emergence. By day 5 the swollen abdomen of each virgin nulliparous
female is filled with about 60 mature (stage 14) oocytes. Thereafter this number
is maintained; degenerating stage-14 oocytes were not seen. The females also
hold immature oocyte stages. When finally mated, after being held nulliparous
for many days, these females started to lay eggs rapidly. The first two eggs laid,
presumably one from each ovary, were the eggs that had matured first and
therefore were the ones that had been held the longest in the mature state. It
was shown that these first eggs were viable when they came from mothers who
had been aged up to 18 days prior to mating and egg laying. Thus eggs may be
held in a mature state for at least 15 days inside the female without losing their
ability to develop into a normal adult. If those eggs had been laid unfertilized
they would have degenerated rapidly. It is not known how holding in vivo
prevents this degeneration.
Although we call stage-14 oocytes mature because they seem morphologically complete, they may go through some last stage of preparation by the
ovary only after insemination of the female. In that case, the statement that
'fully mature' eggs can be held for many days without loss of viability would be
untrue. Until means of artificial, external fertilization are possible this argument
cannot be completely ruled out. In our experiment, the transfer to a yeasted
dish with males is the first source of a stimulus to the female which could induce
these putative final steps. We have observed that the minimum time between
transfer of an aged female and the laying of viable eggs is 3 h. We can state with
certainty that eggs which are not more than 3 h from 'full maturity' can be
held for many days without losing their developmental capability.
The total viability of the first batch of eggs laid by aged females was somewhat less than that of the control group. This was due to some females whose
eggs had a low viability. The eggs of other females retained a high viability.
Thus the loss of viability was not a necessary concomitant of aging, but was
characteristic of some females. Possibly the loss in viability was due to the
generally poor health of some of the older mothers. It is concluded that aging
of the mature oocytes, per se, does not cause a marked reduction in viability.
Using King's (1970) estimates, a normally mated female has about 500 cysts
and oocytes in post-oogonial stages when opposition starts (day 3). We have
shown that a female prevented from laying eggs for 7-10 days can still lay 500
eggs with normal viability (Table 3). We do not know if all these eggs are the
same ones that were present on day 3. Upon dissection we found that about 60
of the oocytes mature to stage 14, and since they are never seen to degenerate,
must be held in that stage until fertilized and laid.
Aged Drosophila oocytes
143
Younger stages do not accumulate. King (1970) believes 'that some control
mechanism operates in Drosophila which causes a cessation of production of
further egg chambers by the germarium once the ovarioles are packed with
mature eggs'. The alternative is that the immature oocytes degenerate and are
replaced by younger oocytes. Holzworth, Spector & Gottlieb (1974) found only
two necrotic oocytes in 600 ovaries, including virgins aged up to 10 days.
However, King, Rubinson & Smith (1956) state that about 15 % of egg chambers
degenerate at stage 8. This implies the possibility of a birth and death cycle of
oocytes with a decision point at stage 8. If the ovariole is full the oocyte degenerates; otherwise it develops to stage 14. The virtual absence of eggs in stages 9-13
in females that hold their eggs (King et al. 1956; King, 1957; King & Sang,
1959; David & Merle, 1968; results of this paper) supports this hypothesis.
From the results of the present research, it must be concluded that the
Drosophila egg is a stable entity capable of being held for many days without
losing its developmental capability. Drosophila cleavage nuclei are totipotent
(lllmensee, 1972). They migrate into the egg cortical cytoplasm and form cell
membranes at about the 6000 nuclei stage (Turner & Mahowald, 1976; Zalokar
& Erk, 1976). Transplantation experiments show that at this time the developmental fate of these cells is already at least partly determined (Chan & Gehring,
1970). Since the zygotic nuclei do not begin mRNA or protein synthesis until
this stage (Zalokar, 1976), whatever substances are responsible for the determination seen at the blastoderm stage must be produced by maternal genes
and placed in the egg during oogenesis. The biochemical nature of these substances is unknown. From the results of this paper, it may be concluded that the
maternal determinating substances are stable, or can be stably maintained by
the egg for at least 15 days.
It is my pleasure to thank Stanley Poole and Karen Valentino for their assistance in
carrying out the project. Supported by USPHS NS-07314.
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{Received 21 July 1978, revised 6 November 1978)