/. Embryol. exp. Morph. Vol. 36, 2, pp. 383-394, 1976
Printed in Great Britain
383
Experiments on blocking and
unblocking of first meiotic metaphase in eggs of
the parthenogenetic stick insect Carausius
morosus Br. (Phasmida, Insecta)
By L. P. PIJNACKER1 AND M. A. FERWERDA
From the Institute of Genetics, University of Groningen, The Netherlands
SUMMARY
The eggs of the parthenogenetic stick insect Carausius morosus, which remain arrested in
first meiotic metaphase until oviposition, must be activated in order to develop. The activating
agent is oxygen from the air, which enters the egg cell through the micropyle. An exposure
shorter than one minute is sufficient to release the blockage.
In non-activated (micropyle-less) eggs the first metaphase chromosomes either degenerate
or change into an interphase nucleus. This nucleus polyploidizes by endoreduplication, and
then either degenerates or multiplies by amitosis. Similarly more generations of nuclei may
arise resulting in a chaotic development. These nuclei survive better in the anterior region of
the egg.
The question of whether the cytoplasmic factors which control nuclear behaviour, also
operate in eggs of C. morosus is discussed.
INTRODUCTION
During oogenesis meiosis advances to a certain stage and then may remain
arrested until the sperm enters the egg. The maturation divisions are completed
and embryonic development begins only after sperm penetration (Monroy,
1967; Fautrez-Firlefyn & Fautrez, 1967). The sperm triggers a mechanism in the
cortex leading to the cortical reaction which activates the egg to proceed with
meiosis. In eggs of organisms which reproduce parthenogenetically by females
exclusively (obligatory thelytoky) a sperm is not involved in the activation
process. Since a meiotic block occurs in eggs of many thelytokous species, it
may be questioned how such eggs are activated. To answer this question, experiments were carried out on eggs of the thelytokous stick insect Carausius
morosus Br. Their eggs are blocked in first meiotic metaphase during a period of
5-5 days preceding the moment of oviposition and continue meiosis immediately thereafter (Pijnacker, 1966). It will be shown that oxygen from the air
triggers a reaction in the egg cell which activates the first metaphase to proceed.
1
Author's address: Vakgroep Genetica, R.U. Groningen, Biologisch Centrum, Vleugel A,
Kerklaan 30, Haren (Gn), The Netherlands.
25-2
384
L. P. PIJNACKER AND M. A. FERWERDA
MATERIALS AND METHODS
The thelytokous stick insect Carausius morosus is bred on leaves of curly kale
in cylindrical cages of perforated zinc (diameter 17 cm, height 33 cm) at 20-22 °C
and a daily light period of 16 h (7 a.m. to 11 p.m.) The females lay the eggs
simply by dropping them on to the bottom of the cage (at night). However, an
egg may be carried around in the subgenital plate for several hours. If necessary,
the exact timing of oviposition was assured by removing the subgenital plate.
The egg {ca. 2-7 mm long, ca. 2 mm high, ca. 1-8 mm wide) has a brown
calcareous exochorion = shell (40-45 (im thick) and resembles a laterally
compressed ellipsoid (Pijnacker, 1971). An operculum (lid) is situated on
the anterior pole and the micropyle apparatus is on the narrow dorsal side of
the egg. The micropyle lies posteriorly in the micropyle apparatus at a distance
of about 0-8 mm from the posterior pole. It perforates the exochorion and the
endochorion. The endochorion is a tough membrane (3 /an thick). One to two
per cent of the eggs lack a micropyle apparatus, the micropyle inclusive (Pijnacker,
1971). The experiments were carried out on normal eggs and on eggs missing
the micropyle apparatus.
Oviposition in a physiological solution or in a gas was obtained as follows. The
females were attached in vertical position to a piece of perspex with waterproof
tape. Then they were allowed to deposit the eggs in a cuvet with a physiological
solution by submerging the ovipositor, i.e. the last four abdominal segments, in
the solution. Care was taken that the cavity between the valvulae and the subgenital plate did not contain air. The physiological solution was composed as
follows: 1000 ml distilled water, 3-660 g K2SO4, 2-162 g KC1, 0-994 g NaCl,
2-033 g MgCl 2 .6H 2 O, 1-095 g CaCl 2 .6H 2 O, 0-534 g Na 2 HPO 4 .2H 2 O and
0-817 g KH 2 PO 4 (Koch, 1964). (It may be stated here that the females did not
oviposit in tap-water.) For oviposition in oxygen and/or in nitrogen the last
four abdominal segments pierced a layer of parafilm which closed the cuvet.
Then the gas was introduced by a narrow glass-tube with a speed of approximately 1 drop/sec. (Treatment with nitrogen could not last longer than 2 days
since nitrogen apparently caused the yolk to swell and consequently broke the
shell.)
Pricking eggs was carried out with a sharp needle. The exo- and endochorion
as well as the vitelline membrane (the surface layer of the egg cell) were punctured till the yolk protruded. The hole was kept as small as possible. Normal eggs
were pricked in the micropyle, micropyle-less eggs in one of the two narrow
sides at about 0-8 mm from the posterior pole (i.e. the micropyle-equivalent
region).
For cytological investigations the operculum was removed and the underlying
endochorion and vitelline membrane carefully punctured before fixation,
enabling the fixative to enter the yolk. Fixation occurred in 1:3 acetic acid/
alcohol mixture for at least 2 h. After the fixative had hardened the yolk the exo-
Blocking of first meiotic metaphase
385
and endochorion were removed with a needle. At that moment the dorso-ventral
axis of the egg cell was marked with a cut dorsally or ventrally in the yolk. Then
the egg cell was stained with the Feulgen reaction at pH 1-5 after 12min
hydrolysis in 1 N-HCI at 58 °C. Slices of yolk to be studied were cut off, squashed
in 45 % acetic acid, and mounted by the freezing method. Marking the dorsoventral axis made selective slicing possible. However, dorso-ventral marking
could not be carried out on eggs without a micropyle apparatus since they
have no visible dorso-ventral axis. The eggs were investigated cytologically over
the whole period of the (last) incubation period.
RESULTS
A. Activation experiments on first metaphase eggs
The results of the experiments on release or maintenance of first metaphase
blockage with normal eggs are given in Table 1 (expts. 1-10) and those with eggs
lacking micropyles in Table 2 (expts. 11-14).
The control eggs showed a release of the first metaphase blockage after oviposition (expt. 1). However, it could be questioned whether internal factors,
such as chemicals released by the accessory sex glands, could act as activators.
Therefore ripe eggs were removed, by dissection, from the oviducts of masculinized females (see Bergerard, 1961; Pijnacker, 1964) which showed egg-retention. Such eggs must have remained in the abdomen for a pre-oviposition
period longer than normal. None of 81 extirpated eggs showed any sign of
activation, demonstrating that internal activators are not present. This is also
substantiated by the fact that 38 of 43 ripe eggs dissected out of the oviducts
and the terminal follicles of ovaries of normal ovipositing females had a normal
embryonic development during an incubation period of 14 days. The occurrence
of embryonic development in these eggs also proves that normal activation is not
caused by the oviposition act itself. Thus, development begins as soon as the
eggs become exposed to air. This also follows from expts. 2-5. Eggs laid
directly into the physiological solution (expt. 2), being non-poisonous (expt.
3), are not activated. But, when the eggs are first laid in air and then incubated
in physiological solution the blockage is released (expts. 3-5). Though it was
technically impossible to collect eggs at an exactly known time within a minute
after oviposition, expt. 5 indicates that the air needs to act for a fraction
(25 %) of a minute only. Investigating the influence of the main components of
air, nitrogen and oxygen (expts. 6-9), it was found that release from the metaphase arrest occurred only when oxygen was present. It can thus be concluded
that oxygen is the agent which activates the egg to proceed with meiosis immediately after oviposition.
The investigation on eggs without a micropyle (expts. 11-14) supports the
preceding results. Under normal conditions micropyle-less eggs are not activated
(expt. 11). However, when a hole is made in the exochorion, endochorion and
386
L. P. PIJNACKER AND M. A. FERWERDA
Table 1. Release or maintenance of first metaphase blockage in eggs of
Carausius morosus
Experiment
1
2
3
4
5
6
7
8
9
10
Incubation conditions of eggs after oviposition
21 days normal
21 days in physiological solution
1-15 min normal -> 3 days in physiological
solution used in expt. 2
1-15 min normal -> 3 days in physiological
solution
0-1 min normal -> 3 days in physiological
solution
1 day in 1 vol. O2 + 3 vol. N2 mixture
3 days in O2
2 days in N2
1-12 h in N2 -> 1 day normal
0-12 h in physiological solution -*• pricking eggs
in the solution -»• 3 days in physiological
solution
No. of
eggs
Eggs with
release of
blockage
(%)
60
52
100
4
26
96
18
100
65
25
21
22
20
75
100
100
5
95
23
4
Table 2. Release or maintenance of first metaphase blockage in micropyleless eggs of Carausius morosus
Experiment
Incubation conditions of eggs after oviposition
11
12
13
47 weeks normal
0-3 days normal ->• pricking -»• 8 days normal
0-3 days normal -> pricking -• 10 days in
physiological solution
0-3 days normal -> pricking eggs in physiological
solution ->• 10 days in physiological solution
14
No. of
eggs
337*
26
Eggs with
release of
blockage
(%)
1.2
88
14
21
15
7
* Eggs without any sign of chromosomes or nuclei (Table 3) have not been included.
vitelline membrane, development starts (expt. 12). This means that as soon as
air can enter the egg cell, the egg becomes activated. It moreover means that
oxygen enters normal eggs through the micropyle. However, it could be asked
whether pricking itself influences activation. This does not occur as shown by
expts. 10, 13-14.
Although it cannot be excluded that some unknown factor(s) is (are) responsible for the exceptions which occur in the experimental results, it can be
assumed that at least some of these exceptions may be due to technical failures.
Blocking of first meiotic metaphase
387
B. Cytological investigations
The normal eggs of expt. 1 are in first metaphase of meiosis (Fig. 1, approximately 66 autobivalents) immediately after oviposition (see also Pijnacker, 1966).
The spindle is situated against the cortex in the ventral posterior side of the egg
just opposite to the micropyle. First anaphase begins almost immediately after
oviposition (Fig. 2). The two maturation divisions last 14-24 h (Fig. 3, 4). The
polar bodies degenerate and, since fertilization does not take place (parthenogenesis), only the pronucleus develops further. The latter starts migration
along the cortex towards the dorsal side of the egg. During this migration it
divides mitotically (approximately 66 chromosomes) in the ventral egg half within
35 h after oviposition. One of the daughter nuclei remains in situ, begins to
divide and initiates a ventral development. The other nucleus migrates further in
the dorsal direction and arrives in the region beneath the micropyle within 40 h
after oviposition, where it begins with a dorsal development (Figs. 5-7). Cell
membranes appear after 5-7 days of development which marks the onset of
blastoderm formation. The cells of the ventral development and the dorsal
development make contact during the second week of development. About
that time the cells lying near the micropyle define the germ anlage. During the
third week a distinct germ band is formed. (The total embryonic development
lasts 3-4 months.)
This schedule of development was also observed in the developing eggs dissected out of ovaries and in those of expts. 2-10, the non-developing eggs of
these experiments remaining in first metaphase. However, the following remarks
can be made. The first metaphase chromosomes of eggs treated with physiological solution (expts. 2, 10) obtain a swollen and granular appearance within
2-4 days (Fig. 8). The spindle disappears and, consequently, the equatorial
plane. After 3 weeks (expt. 2) the individual chromosomes cannot be distinguished any longer and form a mass of fibrillo-granular structures. A similar
development began in the non-activated eggs of expt. 5. Two eggs of expt. 2
showed a sign of development after one week of incubation, i.e. one egg contained four groups of sticky chromosomes and the other one despiralized chromosomes in 'first anaphase'. The developing egg of expt. 10 was found in first
anaphase at the end of the experiment; the one of expt. 8 was in first telophase
after 3 h in N 2 .
The observations on the micropyle-less eggs of expt. 11 have been summarized in Table 3 and Figs. 9-14. The phases of development could be divided into
seven categories which were found during definite periods after oviposition. (It
may be remembered here that the normal embryonic period lasts 3-4 months.)
Categories 2 and 3 are present from 3 days after oviposition. In category 6 the
chromatin is present as either one single highly polyploid interphase nucleus or
several highly polyploid nuclei. The latter nuclei may be surrounded by smaller
nuclei which may be polyploid to a lesser degree (Fig. 13). The polyploid
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L. P. PIJNACKER AND M. A. FERWERDA
Blocking of first meiotic metaphase
389
Table 3. Course of development in micropyle-less eggs of
Carausius morosus
Category
1
2
3
4
5
6
7
Phase of development of
chromosomes/nuclei
Bivalents in normal
first metaphase
Dispersed sticky bivalents, no spindle
Bivalents as if in first
prometaphase, no
spindle
Sticky, poorly stained,
chromosomes
Swollen granular
chromosomes, sometimes as one mass
(Polyploid) nuclei in a
restricted region of
the yolk surface
(Polyploid) nuclei
throughout the yolk
Eggs without chromosomes or nuclei/10
weeks (%)
0
Eggs with
anterior
No. of weeks after
position of
oviposition
chromosomes/
—, No. of*
10 20 30 40 50
nuclei (%)
eggs
82
24
33
6
110
39
29
16
31
42
30
80
18
20 | 42 1 45 | 60 | 68 |
176
* Four eggs with exceptional development have not been included (see text).
nucleus may acquire irregular shapes through protrusions and constrictions.
Polyploidy is established by endoreduplication, and the increase in nuclei is
caused apparently by amitosis since mitosis was not observed. Pycnosis may
occur in some or all the nuclei. The nuclei observed in the eggs of category 7
develop as in category 6 eggs, but they are found in the surface of the entire yolk
as well as more centrally in the yolk (Fig. 14). This development, which has a
chaotic appearance, is pronounced in the anterior region of the yolk.
Twenty per cent of eggs were found without chromosomes or nuclei during
the first 10 weeks after oviposition (Table 3). A similar percentage was found for
FIGURES 1-6
Fig. 1. Side view of first metaphase. x 1900.
Fig. 2. Side view of first anaphase. x 1900.
Fig. 3. Side view of first telophase. x 1900.
Fig. 4. Side view of second metaphase and degenerating first polar body, x 1900.
Fig. 5. Prophase of superficial cleavage mitosis, x 1900.
Fig. 6. Side view of metaphase of superficial cleavage mitosis, x 1900.
390
L. P. PIJNACKER AND M. A. FERWERDA
10
13
14
Blocking of first meiotic metaphase
391
normal eggs if selective slicing was not carried out (meiotic phases were scored
in 98 % of marked eggs (n = 50) and in 83 % of unmarked eggs (n = 100)).
In micropyle-less eggs older than 10 weeks the percentages are considerably
higher which means that complete resorption of chromatin can have taken place
in these eggs.
The descriptions of the phases of development indicate that micropyle-less
eggs may develop in two ways. One way leads to resorption of the first metaphase chromosomes via sticky structures. It involves the following categories:
cat. 1 -> cat. 2 -»- cat. 4 -» resorption. In the other eggs an interphase nucleus is
formed which becomes polyploid. (In young eggs these processes must take place
rapidly, since cat. 5 was not found in weeks 4-11.) This nucleus either starts to
degenerate or multiplies by amitotic processes. The next generation(s) of nuclei
may also undergo polyploidization, degeneration, or amitosis which may result
in a chaotic development of nuclei. The sequence of categories in this development is:
*i
* i
± c
i c / resorption
cat. 1 -» cat. 3 -> cat. 5 -> cat. 6 < . _
> cat. 7 -» resorption
In normal eggs the first metaphase is situated against the cortex just opposite
to the micropyle in the ventral posterior part of the egg. In the micropyle-less
eggs 24 % of the first metaphases (Table 3) occupies an anterior position against
the cortex up to the region beneath the operculum. The cause of this abnormal
position is unknown. The percentage is lower in eggs of categories 2 and 4,
which show the degeneration of the first metaphase chromosomes, and is higher
in eggs of categories 3, 5 and 6 (and 7), the chromosomes of which undergo
polyploidization first. It thus appears that the nuclei survive better in the anterior region of the egg. Keeping in mind the results of expts. 1-9, the better
survival of nuclei may be due to the presence of air which enters through the
F I G U R E S 7-14
Fig. 7. Side view of telophase of superficial cleavage mitosis, x 1900.
Fig. 8. First metaphase chromosomes of egg incubated in physiological solution
during 2 weeks, x 1900.
Fig. 9. Sticky first metaphase chromosomes in a micropyle-less egg 11 weeks after
oviposition. x 1900.
Fig. 10. First metaphase chromosomes again in prometaphase configuration in a
micropyle-less egg 4 weeks after oviposition. x 1900.
Fig. 11. Nearly resorbed sticky first metaphase chromosomes in a micropyle-less egg
32 weeks after oviposition. x 1900.
Fig. 12. Granular mass of first metaphase chromosomes in a micropyle-less egg 17
weeks after oviposition. x 1900.
Fig. 13. Polyploid nuclei surrounded by (degenerating) smaller nuclei in a micropyleless egg 21 weeks after oviposition. x 1900.
Fig. 14. Chaotic development in micropyle-less egg 31 weeks after oviposition.
xl90.
392
L. P. PIJNACKER AND M. A. FERWERDA
suture between operculum and shell. The possibility of an 'opening' in the
shell in the anterior part of the egg is sustained by the fact that most of the
degenerating eggs of categories 6 and 7 exhibited desiccation of yolk in the
anterior region after the 14th week.
In expt. 11 four eggs were observed which showed signs of normal development (not included Table 3). Their meiotic stages were (1) first anaphase in
an 1- to 2-day-old egg, (2) first telophase in a 4- to 5-day-old egg, (3) a second
telophase in a 5- to 6-day-old egg, and (4) chaotic development with mitoses in
15-week-old egg.
Activated eggs (expt. 12) develop as the control eggs of expt. 1 during the 8
days after pricking. This means that normal developmental information has
been stored in the micropyle-less eggs. However, development may be slower.
The mitoses show the somatic number of chromosomes and were also observed
in the pricking region. Three eggs showed deviating development 8 days after
pricking. One egg was still arrested in first metaphase, the other two contained
polyploid nuclei among normal nuclei. In the eggs of expts. 13 and 14 the
chromosomes behaved similarly to those of expts. 2 (and 10). Three eggs of
expt. 13 and one of expt. 14 were in first telophase after 10 days of incubation in
the physiological solution.
DISCUSSION
The foregoing results show that the eggs of the parthenogenetic stick insect
Carausius morosus, which remain arrested in first meiotic metaphase until oviposition, must be activated to develop. The egg contains all the necessary elements for completing the maturation divisions and initiating embryonic development, only some agent to trigger the system is required. The activating agent is
oxygen from the air which enters the egg cell through the micropyle. A short
exposure (< 1 min) is sufficient to release the blockage. Cytologically the first
visible sign is an initiation of the anaphase-movement of the chromosomes
immediately after activation. A signal thus travels from the micropylar region
on the dorsal side of the egg to the first metaphase spindle in the cortical cytoplasm on the ventral side (straight distance 1500 fim). However, it remains to be
resolved how the oxygen exerts its influence on the egg cell, and which kind of
signal reaches the spindle. The mechanism of action may primarily be a removal
or inactivation of a cytostatic factor, if present, as in frog oocytes (Masui &
Markert, 1971). Since in non-activated micropyle-less eggs the first metaphasemay
persist for 18 days, and since meiotic and mitotic divisions do not even occur
until 47 weeks after oviposition, such a cytostatic factor must be very stabile.
It can be stated that the structural organization of the cortex in the region of
oxygen entrance is not of any importance to the response of the egg cell to oxygen, since it can be damaged by pricking without the cell losing its ability to
react. Further, it is interesting to note that the egg reacted with normal matura-
Blocking of first meiotic metaphase
393
tion divisions and normal superficial cleavage divisions to pricking, though
slight cortical damage provokes mitotic abnormalities in amphibian eggs
(Brachet & Hubert, 1972).
Chromosome condensation in oocytes may be controlled by a chromosome
condensation factor which rapidly disappears at activation (Ziegler & Masui,
1973). If in C. morosus such a factor exerts its influence during oogenesis up to
activation in first metaphase, it apparently remains effective in the non-activated
micropyle-less eggs in which the first metaphase only degenerates from 17 weeks
after oviposition onwards, indicating its stability, but it vanishes in those nonactivated eggs in which the first metaphase changes into an interphase nucleus.
How the influence of the condensation factor is broken down in the latter case
can only be guessed. The responsible factor may be the air filtering very slowly
through the shell, particularly in the region of the operculum.
The occurrence of endoreduplication in non-activated micropyle-less eggs
means that the egg cell contains all the necessary elements for DNA synthesis,
even for many cycles. In Xenopus eggs nuclear DNA synthesis is controlled by
a cytoplasmic factor which appears during oocyte maturation and initiates DNA
synthesis after activation (Gurdon, 1967). If this cytoplasmic factor is present in
C. morosus eggs, it persists in the non-activated micropyle-less eggs until it
becomes effective after despiralization of the first metaphase chromosomes,
i.e. after the condensation factor has disappeared.
In general, eggs with a calcareous shell are not suitable for investigations on
the control of the complex processes which occur during early embryonic development. However, the present paper shows that such investigations can be
carried out on eggs with hard shells, particularly on those of a parthenogenetic
organism like C. morosus, and that they can give us an answer at least to the
question as to whether the principles in cytoplasmic control of nuclear behaviour are of general validity. Whether the C. morosus eggs can also be used for
isolating factors which control the early development, will be a matter of future
research.
REFERENCES
J. (1961). Intersexualite experimental chez Carausius morosus Br. (Phasmidae).
Bull. biol. Fr. Belg. 95, 273-300.
BRACHET, J. & HUBERT, E. (1972). Studies on nucleocytoplasmic interactions during early
amphibian development. I. Localized destruction of the egg cortex. /. Embryol. exp. Morph.
27, 121-145.
FAUTREZ-FIRLEFYN, N. & FAUTREZ, J. (1967). The dynamism of cell division during early
cleavage stages of the egg. Int. Rev. Cytol. 22, 171-204.
GURDON, J. B. (1967). On the origin and persistence of a cytoplasmic state inducing nuclear
DNA synthesis in frogs' eggs. Proc. natn. Acad. Sci. U.S.A. 58, 545-552.
KOCH, P. (1964). In vitro-Kultur und entwicklungsphysiologische Ergebnisse an Embryonen
der Stabheuschrecke Carausius morosus Br. Wilhelm Roux Arch. EntwMech. Org. 155,
549-593.
MASUI, Y. &MARKERT, C. L. (1971). Cytoplasmic control of nuclear behaviour during meiotic
maturation of frog oocytes. /. exp. Zool. Ill, 129-146.
BERGERARD,
394
L. P. P I J N A C K E R AND M. A. FERWERDA
MONROY, A. (1967). Fertilization. Comprehensive Biochem. 28, 1-21.
PIJNACKER, L. P. (1964). The cytology, sex determination and parthenogenesis of Carausius
morosus Br. Thesis, University of Groningen.
PIJNACKER, L. P. (1966). The maturation divisions of the parthenogenetic stick insect Carausius morosus Br. (Orthoptera, Phasmidae). Chromosoma 19, 99-112.
PIJNACKER, L. P. (1971). The origin of abnormal micropyle apparatus of eggs of Carausius
morosus Br. (Cheleutoptera, Phasmidae). Neth. J. Zool. 21, 366-372.
ZIEGLER, D. & MASUI, Y. (1973). Control of chromosome behavior in amphibian oocytes.
I. Theactivity of maturing oocytes inducing chromosome condensation in transplanted brain
nuclei. Devi Biol. 35, 283-292.
{Received 24 May 1976)