Experiments on blocking and morosus Br. (Phasmida,...

/ . Embryol. exp. Morph. Vol. 36, 2, pp. 383-394, 1976 3 8 3

Printed in Great Britain

Experiments on blocking andunblocking of first meiotic metaphase in eggs of

the parthenogenetic stick insect Carausiusmorosus Br. (Phasmida, Insecta)

By L. P. PIJNACKER1 AND M. A. FERWERDAFrom the Institute of Genetics, University of Groningen, The Netherlands

SUMMARYThe 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 activatingagent is oxygen from the air, which enters the egg cell through the micropyle. An exposureshorter than one minute is sufficient to release the blockage.

In non-activated (micropyle-less) eggs the first metaphase chromosomes either degenerateor change into an interphase nucleus. This nucleus polyploidizes by endoreduplication, andthen either degenerates or multiplies by amitosis. Similarly more generations of nuclei mayarise resulting in a chaotic development. These nuclei survive better in the anterior region ofthe egg.

The question of whether the cytoplasmic factors which control nuclear behaviour, alsooperate in eggs of C. morosus is discussed.

INTRODUCTION

During oogenesis meiosis advances to a certain stage and then may remainarrested until the sperm enters the egg. The maturation divisions are completedand embryonic development begins only after sperm penetration (Monroy,1967; Fautrez-Firlefyn & Fautrez, 1967). The sperm triggers a mechanism in thecortex leading to the cortical reaction which activates the egg to proceed withmeiosis. In eggs of organisms which reproduce parthenogenetically by femalesexclusively (obligatory thelytoky) a sperm is not involved in the activationprocess. Since a meiotic block occurs in eggs of many thelytokous species, itmay be questioned how such eggs are activated. To answer this question, ex-periments were carried out on eggs of the thelytokous stick insect Carausiusmorosus Br. Their eggs are blocked in first meiotic metaphase during a period of5-5 days preceding the moment of oviposition and continue meiosis immedi-ately thereafter (Pijnacker, 1966). It will be shown that oxygen from the airtriggers 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 kalein cylindrical cages of perforated zinc (diameter 17 cm, height 33 cm) at 20-22 °Cand a daily light period of 16 h (7 a.m. to 11 p.m.) The females lay the eggssimply by dropping them on to the bottom of the cage (at night). However, anegg 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 browncalcareous exochorion = shell (40-45 (im thick) and resembles a laterallycompressed ellipsoid (Pijnacker, 1971). An operculum (lid) is situated onthe anterior pole and the micropyle apparatus is on the narrow dorsal side ofthe egg. The micropyle lies posteriorly in the micropyle apparatus at a distanceof about 0-8 mm from the posterior pole. It perforates the exochorion and theendochorion. The endochorion is a tough membrane (3 /an thick). One to twoper cent of the eggs lack a micropyle apparatus, the micropyle inclusive (Pijnacker,1971). The experiments were carried out on normal eggs and on eggs missingthe micropyle apparatus.

Oviposition in a physiological solution or in a gas was obtained as follows. Thefemales were attached in vertical position to a piece of perspex with waterprooftape. Then they were allowed to deposit the eggs in a cuvet with a physiologicalsolution by submerging the ovipositor, i.e. the last four abdominal segments, inthe solution. Care was taken that the cavity between the valvulae and the sub-genital plate did not contain air. The physiological solution was composed asfollows: 1000 ml distilled water, 3-660 g K2SO4, 2-162 g KC1, 0-994 g NaCl,2-033 g MgCl2.6H2O, 1-095 g CaCl2.6H2O, 0-534 g Na2HPO4.2H2O and0-817 g KH2PO4 (Koch, 1964). (It may be stated here that the females did notoviposit in tap-water.) For oviposition in oxygen and/or in nitrogen the lastfour 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 approxi-mately 1 drop/sec. (Treatment with nitrogen could not last longer than 2 dayssince nitrogen apparently caused the yolk to swell and consequently broke theshell.)

Pricking eggs was carried out with a sharp needle. The exo- and endochorionas well as the vitelline membrane (the surface layer of the egg cell) were punc-tured till the yolk protruded. The hole was kept as small as possible. Normal eggswere pricked in the micropyle, micropyle-less eggs in one of the two narrowsides at about 0-8 mm from the posterior pole (i.e. the micropyle-equivalentregion).

For cytological investigations the operculum was removed and the underlyingendochorion 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 385and endochorion were removed with a needle. At that moment the dorso-ventralaxis of the egg cell was marked with a cut dorsally or ventrally in the yolk. Thenthe egg cell was stained with the Feulgen reaction at pH 1-5 after 12minhydrolysis in 1 N-HCI at 58 °C. Slices of yolk to be studied were cut off, squashedin 45 % acetic acid, and mounted by the freezing method. Marking the dorso-ventral axis made selective slicing possible. However, dorso-ventral markingcould not be carried out on eggs without a micropyle apparatus since theyhave no visible dorso-ventral axis. The eggs were investigated cytologically overthe 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 metaphaseblockage with normal eggs are given in Table 1 (expts. 1-10) and those with eggslacking micropyles in Table 2 (expts. 11-14).

The control eggs showed a release of the first metaphase blockage after ovi-position (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 masculin-ized females (see Bergerard, 1961; Pijnacker, 1964) which showed egg-reten-tion. Such eggs must have remained in the abdomen for a pre-ovipositionperiod longer than normal. None of 81 extirpated eggs showed any sign ofactivation, demonstrating that internal activators are not present. This is alsosubstantiated by the fact that 38 of 43 ripe eggs dissected out of the oviductsand the terminal follicles of ovaries of normal ovipositing females had a normalembryonic development during an incubation period of 14 days. The occurrenceof embryonic development in these eggs also proves that normal activation is notcaused by the oviposition act itself. Thus, development begins as soon as theeggs become exposed to air. This also follows from expts. 2-5. Eggs laiddirectly 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 incubatedin physiological solution the blockage is released (expts. 3-5). Though it wastechnically impossible to collect eggs at an exactly known time within a minuteafter 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 ofair, nitrogen and oxygen (expts. 6-9), it was found that release from the meta-phase arrest occurred only when oxygen was present. It can thus be concludedthat oxygen is the agent which activates the egg to proceed with meiosis immedi-ately after oviposition.

The investigation on eggs without a micropyle (expts. 11-14) supports thepreceding results. Under normal conditions micropyle-less eggs are not activated(expt. 11). However, when a hole is made in the exochorion, endochorion and


Table 1. Release or maintenance of first metaphase blockage in eggs ofCarausius morosus

Experiment

123

4

5

6789

10

Incubation conditions of eggs after oviposition

21 days normal21 days in physiological solution1-15 min normal -> 3 days in physiologicalsolution used in expt. 2

1-15 min normal -> 3 days in physiologicalsolution

0-1 min normal -> 3 days in physiologicalsolution

1 day in 1 vol. O2 + 3 vol. N2 mixture3 days in O22 days in N21-12 h in N2 -> 1 day normal0-12 h in physiological solution -*• pricking eggsin the solution -»• 3 days in physiologicalsolution

No. ofeggs

6052

26

18

6525212220

23

Eggs withrelease ofblockage

(%)

1004

96

100

75100100

595

4

Table 2. Release or maintenance of first metaphase blockage in micropyle-less eggs of Carausius morosus

Experiment Incubation conditions of eggs after oviposition

Eggs withrelease of

No. of blockageeggs (%)

11 47 weeks normal 337* 1.212 0-3 days normal ->• pricking -»• 8 days normal 26 8813 0-3 days normal -> pricking -• 10 days in

physiological solution 14 2114 0-3 days normal -> pricking eggs in physiological

solution ->• 10 days in physiological solution 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 asair can enter the egg cell, the egg becomes activated. It moreover means thatoxygen enters normal eggs through the micropyle. However, it could be askedwhether pricking itself influences activation. This does not occur as shown byexpts. 10, 13-14.

Although it cannot be excluded that some unknown factor(s) is (are) respon-sible for the exceptions which occur in the experimental results, it can beassumed 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, approxi-mately 66 autobivalents) immediately after oviposition (see also Pijnacker, 1966).The spindle is situated against the cortex in the ventral posterior side of the eggjust opposite to the micropyle. First anaphase begins almost immediately afteroviposition (Fig. 2). The two maturation divisions last 14-24 h (Fig. 3, 4). Thepolar bodies degenerate and, since fertilization does not take place (partheno-genesis), only the pronucleus develops further. The latter starts migrationalong the cortex towards the dorsal side of the egg. During this migration itdivides mitotically (approximately 66 chromosomes) in the ventral egg half within35 h after oviposition. One of the daughter nuclei remains in situ, begins todivide and initiates a ventral development. The other nucleus migrates further inthe dorsal direction and arrives in the region beneath the micropyle within 40 hafter oviposition, where it begins with a dorsal development (Figs. 5-7). Cellmembranes appear after 5-7 days of development which marks the onset ofblastoderm formation. The cells of the ventral development and the dorsaldevelopment make contact during the second week of development. Aboutthat time the cells lying near the micropyle define the germ anlage. During thethird week a distinct germ band is formed. (The total embryonic developmentlasts 3-4 months.)

This schedule of development was also observed in the developing eggs dis-sected out of ovaries and in those of expts. 2-10, the non-developing eggs ofthese experiments remaining in first metaphase. However, the following remarkscan be made. The first metaphase chromosomes of eggs treated with physio-logical solution (expts. 2, 10) obtain a swollen and granular appearance within2-4 days (Fig. 8). The spindle disappears and, consequently, the equatorialplane. After 3 weeks (expt. 2) the individual chromosomes cannot be dis-tinguished any longer and form a mass of fibrillo-granular structures. A similardevelopment began in the non-activated eggs of expt. 5. Two eggs of expt. 2showed a sign of development after one week of incubation, i.e. one egg con-tained four groups of sticky chromosomes and the other one despiralized chromo-somes in 'first anaphase'. The developing egg of expt. 10 was found in firstanaphase at the end of the experiment; the one of expt. 8 was in first telophaseafter 3 h in N2.

The observations on the micropyle-less eggs of expt. 11 have been summar-ized in Table 3 and Figs. 9-14. The phases of development could be divided intoseven categories which were found during definite periods after oviposition. (Itmay 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 thechromatin is present as either one single highly polyploid interphase nucleus orseveral highly polyploid nuclei. The latter nuclei may be surrounded by smallernuclei which may be polyploid to a lesser degree (Fig. 13). The polyploid

Blocking of first meiotic metaphase 389

Table 3. Course of development in micropyle-less eggs ofCarausius morosus

Phase of development ofCategory chromosomes/nuclei

No. of weeks afteroviposition

0 10 20 30 40—, No. of*50 eggs

Eggs withanterior

position ofchromo-

somes/nuclei (%)

1 Bivalents in normalfirst metaphase

2 Dispersed sticky biva-lents, no spindle

3 Bivalents as if in firstprometaphase, nospindle

4 Sticky, poorly stained,chromosomes

5 Swollen granularchromosomes, some-times as one mass

6 (Polyploid) nuclei in arestricted region ofthe yolk surface

7 (Polyploid) nucleithroughout the yolk

Eggs without chromo-somes or nuclei/10weeks (%)

82

33

110

29

31

24

6

39

16

42

30

18

80

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 iscaused apparently by amitosis since mitosis was not observed. Pycnosis mayoccur in some or all the nuclei. The nuclei observed in the eggs of category 7develop as in category 6 eggs, but they are found in the surface of the entire yolkas well as more centrally in the yolk (Fig. 14). This development, which has achaotic appearance, is pronounced in the anterior region of the yolk.

Twenty per cent of eggs were found without chromosomes or nuclei duringthe 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.


13

10

14

Blocking of first meiotic metaphase 391normal eggs if selective slicing was not carried out (meiotic phases were scoredin 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 considerablyhigher which means that complete resorption of chromatin can have taken placein these eggs.

The descriptions of the phases of development indicate that micropyle-lesseggs may develop in two ways. One way leads to resorption of the first meta-phase chromosomes via sticky structures. It involves the following categories:cat. 1 -> cat. 2 -»- cat. 4 -» resorption. In the other eggs an interphase nucleus isformed which becomes polyploid. (In young eggs these processes must take placerapidly, since cat. 5 was not found in weeks 4-11.) This nucleus either starts todegenerate or multiplies by amitotic processes. The next generation(s) of nucleimay also undergo polyploidization, degeneration, or amitosis which may resultin a chaotic development of nuclei. The sequence of categories in this develop-ment is:

* i * i ± c i c / resorptioncat. 1 -» cat. 3 -> cat. 5 -> cat. 6 < . _> cat. 7 -» resorption

In normal eggs the first metaphase is situated against the cortex just oppositeto the micropyle in the ventral posterior part of the egg. In the micropyle-lesseggs 24 % of the first metaphases (Table 3) occupies an anterior position againstthe cortex up to the region beneath the operculum. The cause of this abnormalposition is unknown. The percentage is lower in eggs of categories 2 and 4,which show the degeneration of the first metaphase chromosomes, and is higherin eggs of categories 3, 5 and 6 (and 7), the chromosomes of which undergopolyploidization first. It thus appears that the nuclei survive better in the an-terior region of the egg. Keeping in mind the results of expts. 1-9, the bettersurvival of nuclei may be due to the presence of air which enters through the

FIGURES 7-14

Fig. 7. Side view of telophase of superficial cleavage mitosis, x 1900.Fig. 8. First metaphase chromosomes of egg incubated in physiological solutionduring 2 weeks, x 1900.Fig. 9. Sticky first metaphase chromosomes in a micropyle-less egg 11 weeks afteroviposition. x 1900.Fig. 10. First metaphase chromosomes again in prometaphase configuration in amicropyle-less egg 4 weeks after oviposition. x 1900.Fig. 11. Nearly resorbed sticky first metaphase chromosomes in a micropyle-less egg32 weeks after oviposition. x 1900.Fig. 12. Granular mass of first metaphase chromosomes in a micropyle-less egg 17weeks after oviposition. x 1900.Fig. 13. Polyploid nuclei surrounded by (degenerating) smaller nuclei in a micropyle-less egg 21 weeks after oviposition. x 1900.Fig. 14. Chaotic development in micropyle-less egg 31 weeks after oviposition.xl90.


suture between operculum and shell. The possibility of an 'opening' in theshell in the anterior part of the egg is sustained by the fact that most of thedegenerating eggs of categories 6 and 7 exhibited desiccation of yolk in theanterior region after the 14th week.

In expt. 11 four eggs were observed which showed signs of normal develop-ment (not included Table 3). Their meiotic stages were (1) first anaphase inan 1- to 2-day-old egg, (2) first telophase in a 4- to 5-day-old egg, (3) a secondtelophase in a 5- to 6-day-old egg, and (4) chaotic development with mitoses in15-week-old egg.

Activated eggs (expt. 12) develop as the control eggs of expt. 1 during the 8days after pricking. This means that normal developmental information hasbeen stored in the micropyle-less eggs. However, development may be slower.The mitoses show the somatic number of chromosomes and were also observedin the pricking region. Three eggs showed deviating development 8 days afterpricking. One egg was still arrested in first metaphase, the other two containedpolyploid nuclei among normal nuclei. In the eggs of expts. 13 and 14 thechromosomes behaved similarly to those of expts. 2 (and 10). Three eggs ofexpt. 13 and one of expt. 14 were in first telophase after 10 days of incubation inthe physiological solution.

DISCUSSION

The foregoing results show that the eggs of the parthenogenetic stick insectCarausius morosus, which remain arrested in first meiotic metaphase until ovi-position, must be activated to develop. The egg contains all the necessary ele-ments for completing the maturation divisions and initiating embryonic develop-ment, only some agent to trigger the system is required. The activating agent isoxygen from the air which enters the egg cell through the micropyle. A shortexposure (< 1 min) is sufficient to release the blockage. Cytologically the firstvisible sign is an initiation of the anaphase-movement of the chromosomesimmediately after activation. A signal thus travels from the micropylar regionon the dorsal side of the egg to the first metaphase spindle in the cortical cyto-plasm on the ventral side (straight distance 1500 fim). However, it remains to beresolved how the oxygen exerts its influence on the egg cell, and which kind ofsignal reaches the spindle. The mechanism of action may primarily be a removalor inactivation of a cytostatic factor, if present, as in frog oocytes (Masui &Markert, 1971). Since in non-activated micropyle-less eggs the first metaphasemaypersist for 18 days, and since meiotic and mitotic divisions do not even occuruntil 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 ofoxygen entrance is not of any importance to the response of the egg cell to oxy-gen, since it can be damaged by pricking without the cell losing its ability toreact. Further, it is interesting to note that the egg reacted with normal matura-

Blocking of first meiotic metaphase 393tion divisions and normal superficial cleavage divisions to pricking, thoughslight cortical damage provokes mitotic abnormalities in amphibian eggs(Brachet & Hubert, 1972).

Chromosome condensation in oocytes may be controlled by a chromosomecondensation factor which rapidly disappears at activation (Ziegler & Masui,1973). If in C. morosus such a factor exerts its influence during oogenesis up toactivation in first metaphase, it apparently remains effective in the non-activatedmicropyle-less eggs in which the first metaphase only degenerates from 17 weeksafter oviposition onwards, indicating its stability, but it vanishes in those non-activated eggs in which the first metaphase changes into an interphase nucleus.How the influence of the condensation factor is broken down in the latter casecan only be guessed. The responsible factor may be the air filtering very slowlythrough the shell, particularly in the region of the operculum.

The occurrence of endoreduplication in non-activated micropyle-less eggsmeans that the egg cell contains all the necessary elements for DNA synthesis,even for many cycles. In Xenopus eggs nuclear DNA synthesis is controlled bya cytoplasmic factor which appears during oocyte maturation and initiates DNAsynthesis after activation (Gurdon, 1967). If this cytoplasmic factor is present inC. morosus eggs, it persists in the non-activated micropyle-less eggs until itbecomes 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 onthe control of the complex processes which occur during early embryonic devel-opment. However, the present paper shows that such investigations can becarried out on eggs with hard shells, particularly on those of a parthenogeneticorganism like C. morosus, and that they can give us an answer at least to thequestion as to whether the principles in cytoplasmic control of nuclear behav-iour are of general validity. Whether the C. morosus eggs can also be used forisolating factors which control the early development, will be a matter of futureresearch.

REFERENCES

BERGERARD, 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 earlyamphibian 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 earlycleavage stages of the egg. Int. Rev. Cytol. 22, 171-204.

GURDON, J. B. (1967). On the origin and persistence of a cytoplasmic state inducing nuclearDNA synthesis in frogs' eggs. Proc. natn. Acad. Sci. U.S.A. 58, 545-552.

KOCH, P. (1964). In vitro-Kultur und entwicklungsphysiologische Ergebnisse an Embryonender Stabheuschrecke Carausius morosus Br. Wilhelm Roux Arch. EntwMech. Org. 155,549-593.

MASUI, Y. &MARKERT, C. L. (1971). Cytoplasmic control of nuclear behaviour during meioticmaturation of frog oocytes. / . exp. Zool. Ill, 129-146.


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 Carau-

sius 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 brainnuclei. Devi Biol. 35, 283-292.

{Received 24 May 1976)

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