diploid parthenogenetic mouse embryos produced by heat ... · diploid parthenogenetic mouse embryos...

/ . Embryo/, exp. Morph. Vol. 35, l,pp. 25-39, 1976 2 5

Printed in Great Britain

Diploid parthenogenetic mouse embryos producedby heat-shock and Cytochalasin B

By HANNA BAtAKIER1 AND ANDRZEJ K. TARKOWSKI1

From the Department of Embryology,Institute of Zoology, University of Warsaw

SUMMARYSwiss albino and C57BL/10 eggs from induced ovulations, and spontaneously ovulated

A eggs, were activated in vitro by a heat shock of 44 °C for 5 or 7-5 min and cultured in thepresence of 10 /tg/ml of Cytochalasin B (CB) for 5-8 h. The activation rate was about 70 %in Swiss albino, 40 % in C57BL and 90 % in A eggs. CB suppressed second polar body(2P.B.) formation in over 90 % of activated eggs, with the majority containing two pronuclei.When eggs were placed in CB-free medium their surface became wrinkled and they formedprotrusions of various sizes, which in some eggs detached to form enucleate or pronucleatecytoplasmic fragments; some eggs broke down completely into fragments. In most eggs,however, the surface smoothed out in a few hours and suppression of 2P.B. appeared to bepermanent. The rate of development of these eggs after transplantation to the oviduct wasdelayed in terms both of cell divisions and of the time of blastocyst formation. Out of 41implants collected on the 8th—10th day of pregnancy only two healthy looking egg-cylinderswere found on the 8th and 9th day; both were retarded, at the stage characteristic for the7th day of normal development. The reasons for delayed preimplantation development andlow implantation rate are discussed. The present experiments corroborate earlier observa-tions that parthenogenetic mouse embryos, even if diploid, rarely survive in the uterusbeyond the egg-cylinder stage.

INTRODUCTION

During the last few years there have been several successful attempts toinduce parthenogenetic development in the mouse (reviewed by Graham, 1974;Tarkowski, 1971, 1975). Activation in situ by electric shock (Tarkowski,Witkowska & Nowicka, 1970; Witkowska, 1973a) or in vitro by heat shock(Komar, 1973) or by hyaluronidase (Graham, 1970, 1971; Kaufman, 1973 c)leads most often to the initiation of haploid parthenogenesis. In order to in-crease the proportion of diploid parthenogenones, Graham (1972) developed atechnique of subjecting cumulus-free eggs to hypotonic shock, which gives ahigh yield of potentially diploid eggs due to suppression of the second polarbody. This technique has been also used with success by other authors(Graham & Deussen, 1974; Kaufman & Gardner, 1974; Kaufman & Surani,1974). Another attempt to produce diploid parthenogenones is presented in

1 Authors' address: Department of Embryology, Institute of Zoology, University ofWarsaw, 00-927/1 Warszawa, Poland.

26 H. BALAKIER AND A. K. TARKOWSKI

this paper. We have taken advantage of the observations that Cytochalasin Binhibits second meiotic division of the mouse oocyte (Niemierko, 1975) andcleavage divisions (Snow, 1973; Tarkowski, Witkowska & Opas, in prepara-tion) and that short treatment of eggs with this drug does not impair furtherdevelopment. Diploid parthenogenetic embryos obtained by suppression ofsecond polar body with Cytochalasin B can develop beyond implantation, butso far we have not observed survival beyond the early egg-cylinder stage.

MATERIAL AND METHODS

Eggs were obtained from Swiss albino females (randomly bred colony) andC57BL/1O and A females. With the exception of A females which ovulatedspontaneously and mated with vasectomized males, all others were induced toovulate (5 i.u. of PMSG and HCG given at an interval of 45-48 h) and werekept apart from males.

Eggs were activated in vitro within the excised oviducts by a heat shock of44-0 ± 0-1 °C (Komar, 1973). Eggs from induced ovulations were shocked12-20 h post-HCG and those from spontaneous ovulations between 8.00 and9.00 a.m. Some eggs of A origin were heated for 7-5 min; all other eggs receiveda shock of 5 min duration.

After the shock eggs with the cumulus oophorus intact were placed in tubeswith 0-5-1 ml of Whitten's medium (Whitten, 1971) containing 10//g of Cyto-chalasin B (CB)/ml. This concentration was worked out in our earlier experi-ments on suppression of cleavage divisions (Tarkowski et af., in preparation)and was found effective also by Snow (1973). The tubes were gassed with 5 %CO2 in air and kept at 37 °C. Observations of Niemierko (1975) and otherpilot experiments carried out in this laboratory showed that when newly ferti-lized or activated eggs are kept in CB for less than 4 h many of them subsequentlyundergo fragmentation. Heat-activated eggs were therefore subjected to CBfor 5-8 h. Subsequently cumulus was removed with hyaluronidase and theeggs placed in drops of CB-free Whitten's medium under liquid paraffin for\-A h. During this period the eggs were examined under the inverted micro-scope for signs of activation and type of reaction. Some of these eggs weresubsequently fixed and mounted as permanent preparations (Tarkowski &Wroblewska, 1967).

The activated eggs with second polar body (2P.B.) suppressed were trans-planted to the oviducts of Swiss albino recipients on the first day of pseudo-pregnancy (day of vaginal plug = first day) or to sexually immature females(4-5 weeks old) using the technique of Tarkowski (1959). For transplantationwe used only eggs which were collected 14-16-5 h post-HCG (in the majorityof experiments, 15-16 h).

From a number of recipients the transplanted parthenogenetic eggs wererecovered after 72-96 h (4th and 5th day) and cultured for another day in

Diploidparthenogenetic mouse embryos 27

Whitten's medium under liquid paraffin. Colcemid (1 /^g/ml) was added toculture drops 2-3 h before making air-dried preparations (Tarkowski, 1966).In order to evaluate whether or not the rate of preimplantation developmentof parthenogenones is delayed, the stage of development and the number ofcells were estimated in F1 (Swiss ? x A ( J ) embryos obtained on the 4th day afternight matings and after delayed matings (7.00-9.00 a.m.), i.e. at the time atwhich activation was performed.

Another group of recipients (only mature pseudopregnant females) werekilled on the 8th—10th day and inspected for signs of implantation. Most im-plantation swellings recovered on the 9th day and all from the 10th day weretorn open and inspected under the dissecting microscope. Eight-day and someof the 9-day implantations were examined in routine haematoxylin and eosinhistological preparations.

As control experiments, eggs harvested 12, 16 and 20 h post-HCG weresubjected to heat shock and cultured in CB-free medium, and non-heat-treatedeggs were cultured in medium containing CB. The control eggs were fixed afterthe same period of time as the experimental ones and mounted in permanentpreparations for examination.

RESULTS

Control experiments

Reaction of non-heated eggs to culture in CB

As can be seen in Tables 1 and 2, over 90% of eggs cultured in the intactcumulus oophorus remained in metaphase II. The incidence of activated eggsdid not exceed 3%, but a number of eggs underwent complete fragmentationor were degenerate. Thus culture in CB has no appreciable effect per se onactivation.

Reaction of eggs to heat shock alone {culture in CB-free medium)

In order to evaluate properly the effect of CB on activated eggs, over 800Swiss albino eggs were subjected to heat shock alone and fixed for examinationas permanent preparations 4-9 h later (Table 1). With the exception of eggstreated 12 h post-HCG, the overall activation rate in 15-16 and 20-h-old eggswas about 70%, which corresponded well to the activation rate in the experi-mental series (cf. Tables 1 and 2). Because of the low activation rate in the12-h group, eggs used in experiments with CB were 13-16-5 h old. The presentobservations confirmed the fact, noted already by other authors, that withadvancing age the eggs become more susceptible to activation but at the sametime the incidence of 'fragmentation' increases. As already shown by Komar(1973), eggs subjected to heat shock react most often by extrusion of 2P.B. andformation of a haploid pronucleus and by immediate cleavage. Becauseimmediate cleavage is sometimes irregular (the two cells are of uneven size

to ooT

able

1.

Eff

ect

of h

eat-

shoc

k w

ith

or w

itho

ut C

B o

n Sw

iss

albi

no e

ggs,

est

imat

ed o

n fi

xed

and

stai

ned

eggs

(%

)

Tre

atm

ent

Hea

t-sh

ock,

4—

9 h

cult

ure

in C

B-f

ree

med

ium

Hea

t-sh

ock,

5-8

h c

ultu

rein

CB

, 0-4

h c

ultu

re i

nC

B-f

ree

med

ium

Age

post

-H

PCr

(h)

12 15-1

6(S

e-ri

es 1

)J16 (S

e-ri

es I

I) X

15-1

6(t

otal

)20 13

-16

Tot

al11

U.

Ul

eggs

114

303

191

494

236

320

Non

-cl

L-l

l

vate

d

64

0

8-6

23

0

14-2 6-4

6-3

Exp

erim

ent

De-

gene

-ra

ted

0 9-6

10

6-3

3-8

15 3

Fra

g-m

pn

f _

JH

Cil

l

ing 3-5

14-2 9-9

12-6

23-3 6-6

Tot

al

32-5

1000

67-7

1000

66

010

006

70

1000

66-5

1000

71-9

1000

Act

ivat

ed

2P.B

.*su

ppre

ssed

2P

Rf

0 13-7 40

10

0

6-4

80

0

1 P

R

2-7

5-9

3-2

4-8

0 8-3

2P.B

.i

1 P

R

54-1

35-6

571

43-8

47-8 0-9

eggs 2P

.B.

pres

ent

orab

sent

,m

any

nucl

ei(s

ub-

nucl

ei)

10-8 7-8

7-9

7-9

7-6

9-6

Imm

e-di

ate

L-l

Cci

v-

age

32-4

36-6

27-8

33-2

29-3 0-9

Enu

c-lo

o „

JCcl

*

tion 0-5

0-3

8-9

0-4

f Tot

al

eggs —

156 —

156 —

163

Con

trol

Art

i-va

ted — 2-6 — 2-6 — 31

K

Non

-

vate

d —

96-8 —

96-8 —

96-3

Frag

-11

Id

1 L

ing — 0-6 — 0-6 — 0-6

w ;> hi

> 0 > • H & ^\

* 2P

.B. =

seco

nd p

olar

bod

y.t

PR

= p

ronu

cleu

s.t

Serie

s I

and

II w

ere

perf

orm

ed i

n id

entic

al m

anne

r, b

ut s

ever

al m

onth

s ap

art.

Str

ain

Tab

le 2

. Eff

ect

of h

eat-

shoc

k an

d C

ytoc

hala

sin

B o

n Sw

iss

albi

no a

nd C

57B

L/1

0 eg

gs, e

stim

ated

on

eggs

in t

he li

ving

sta

te 1

-4 h

aft

er t

he s

hock

(va

lues

in

%)

Exp

erim

ent

Act

ivat

ed e

ggs

Age

post

-H

CG

(h)

2P.B

.* s

uppr

esse

dC

ontr

olIm

med

- r

Tot

al

No

n-

No

. of

2P

.B.

iate

T

otal

N

on-

Fra

gmen

t-no

of

acti

- D

egen

er-

Fra

g-

PR

un-

+

cl

eav-

no

of.

A

cti-

ac

ti-

ing

or d

e-eg

gs

vate

d at

ed

men

ting

T

otal

2

PR

f 1

PR

kn

own

1 P

R

age

eggs

va

ted

vate

d ge

nera

ted

S:

Sw

iss

albi

no

C57

BL

/10

14-1

6-5

15 16-1

6-5

Tot

al

1031

102

106

208

18-2

72-5

29-2

50-5

3-3

3-8

—

10

2-8

1-4

11-3 6-3

74-7

1000

26-5

100-

0

56-6

1000

41-8

1000

89-6

59-3

85

0

77-0

5-2

33-3 8-3

16

1

4-2

3-7

50

4-6

460

2-8

0-3

0-6 3-7

1-7

2-3

34

—

93-7

73-5

3-5

26-5

* 2P

.B. =

se

cond

pol

ar b

ody.

t P

R=

pr

onuc

leus

.


and may be accompanied by enucleate cytoplasmic fragments) classification ofsuch eggs either as undergoing immediate cleavage or fragmentation becomessomewhat arbitrary.

In Table 1 we included separately the results of two identical series of experi-ments carried out several months apart, in order to show that even when allthe parameters under control remain constant and the number of eggs in-vestigated is reasonably high (200 and 300) there may be significant differencesin the proportions of eggs entering various routes of development. For instance,the incidence of suppression of 2P.B. varied between 19-6% and 7-2% and ofextrusion of 2P.B. between 35-6% and 57-1 %.

Contrary to the similar experiments by Komar (1973), subnuclei were formedin less than 10 % of eggs. We are unable to explain this difference.

Experiments with CB

Immediate reaction of eggs to heat shock and CB

Eggs subjected to this double treatment were examined in two ways. First,320 Swiss albino eggs after being transferred for 0-4 h to CB-free mediumwere fixed and examined as permanent preparations (Table 1). Second, 1031Swiss and 208 C67BL/10 eggs were transferred for 1-4 h to CB-free mediumand were examined in the living state only (Table 2). After examination someeggs from this group were selected for transplantation (see below). From bothseries of experiments it is evident that CB effectively suppresses formation ofthe 2P.B. (over 90%), the most common reaction being formation of twopronuclei. As mentioned above, suppression of 2P.B. can occur after heatshock alone, but the frequency of this phenomenon never exceeds 20 %.

In C57BL/10 eggs examined in the living state only, the activation rate amongeggs shocked 16-16-5 h post-HCG was twice as high as among eggs treatedat 15 h. The significance of this difference is uncertain. As in Swiss albino eggs,in the majority of C57BL/10 eggs, 2P.B. was suppressed by CB.

Spontaneously ovulated eggs of A origin subjected to 5 min heat shockbetween 8.00 and 9.00 a.m. reacted only in 10% (4 out of 40). However, over90% were activated when the duration of the shock was prolonged to 7-5 min(60 out of 65). Data concerning activation of A eggs are not included in Tables1 and 2.

As long as the activated eggs stay in CB their surface remains completelysmooth. However, when placed in CB-free medium the surface becomeswrinkled and forms protrusions of various size (Figs. 1, 2) which may - thoughnot necessarily - eventually detach to form small cytoplasmic globules lyingfreely in the perivitelline space. Sometimes an indentation is formed in thepresumptive region of the second polar body formation so that the egg acquiresa bean-like shape (Fig. 1). In such a case the pronuclei may be lying on the topof the two protrusions which are formed on both sides of the indentation

Diploid parthenogenetic mouse embryos 31

Fig. 1. Swiss albino eggs kept in CB for 5 h 10 min and subsequently in CB-freemedium for 3 h 20 min. The surface of eggs is still wrinkled. The upper right eggshows an indentation in the presumptive region of the second polar body forma-tion; the two protrusions are occupied by pronuclei. The egg shown in the middlehas extruded several small enucleate fragments and a large one with a pronucleus.x 200.Figs. 2, 3. Swiss albino eggs kept in CB for 6 h .10 min and in CB-free mediumfor 30 min.

Fig. 2 Five activated eggs and one non-activated (top middle). The surface of theactivated eggs is nearly smooth and the eggs are only slightly deformed. In themiddle egg of the lower row the two pronuclei have already moved to the centre.Note that the non-activated egg is round and has a completely smooth surface,x 200.

Fig. 3. Another group of eggs from the same experiment. The surface of eggs issmooth but the two pronuclei still remain close to the egg surface. The egg on theright has retained the first polar body, x 400.


(Fig. 1). If the furrow is not formed the two pronuclei very often lie side byside and stay close to the egg surface (Fig. 3). Similar surface reactions areobserved when cleaving mouse eggs are subjected to CB at the time of cleavageand subsequently placed in CB-free medium (Snow, 1973; Tarkowski et ai,in preparation).

With time the surface of activated eggs steadily smooths out (Fig. 2). How-ever, a number of eggs undergo severe fragmentation or extrude cytoplasmicfragments which may even contain pronuclei (Fig. 1). This explains a smallnumber of eggs with 2P.B. or undergoing immediate cleavage which arerecorded in Tables 1 and 2. These phenomena occur only after the eggs areremoved from CB.

The behaviour of eggs appeared to depend on the duration of treatment withCB. After 5-h treatment the egg surface was very wrinkled and they requireda few hours to acquire normal appearance; the incidence of fragmentation washighest in this time group. After 8-h treatment the surface was smooth ornearly smooth and the eggs needed a much shorter time to regain their normalappearance. For practical reasons treatment of 6-h duration was usuallyapplied. Whether the longer times of exposure of eggs to CB were more harmfulfor their survival could not be determined in the present study. To make surethat the eggs with 2P.B. suppressed would remain in this state after transplanta-tion, the eggs were kept in CB-free medium for as long as was feasible (the ideabeing to complete the experiment in one day and thus to reduce the length ofculture to a minimum) and only those which looked normal, i.e. which did notextrude cytoplasmic fragments and clearly contained one or two pronuclei,were selected for transfer.

Contrary to the activated eggs, the surface of the non-activated eggs remainedall the time completely smooth. The different behaviour of these two types ofeggs permits them to be easily segregated even if the pronuclei are not yetclearly visible (Fig. 2).

Preimplantation development of transplanted eggs

Healthy-looking Swiss albino eggs with 2P.B. suppressed and with twopronuclei (occasionally with one pronucleus) were transplanted to pseudo-pregnant or sexually immature recipients and recovered on the 4th or 5th day.The embryos were subsequently cultured for 24 h. On the 4th day the mostadvanced embryos were morulae (Fig. 4). Blastocysts were first observed onthe 5th day; some of them transformed from morulae after 24 h in vitro (Fig. 5).All morulae and blastocysts containing analysable metaphase plates (15 out of27 studied) were diploid.

The tempo of development of parthenogenetic embryos was clearly retarded:formation of blastocysts was delayed until the 5th day and the mean cell numberof 4-5- and 5-5-day-old parthenogenones corresponded to that of 3-5-day-oldcontrols from delayed matings (Table 3). On the 4th day the mean cell number


Figs. 4, 5. The same group of seven Swiss albino parthenogenetic embryos afterrecovery from the recipient on the 4th day (Fig. 4) and after 24 h in culture(Fig. 5). During the culture phase one egg remained at the 8-cell stage and twomorulae transformed into blastocysts. x 200.Fig. 6. An egg-cylinder recovered on the 8th day following transfer of activatedeggs of A strain. The section is nearly perpendicular to the long axis of the cylinder.Pro-amniotic cavity is clearly visible, x 200.Fig. 7. A C57BL/10 egg-cylinder recovered on the 9th day. The section is againnearly perpendicular. Although the embryo is much larger than that shown in Fig. 6,it represents a similar stage of development. By the time of examination Reichert'smembrane must have undergone disruption, x 200.

and the proportion of embryos that had reached the blastocyst stage was con-siderably lower in embryos originating from females mated in the morningfollowing ovulation than from females mated at night (Table 3). It follows fromthis that the proper control material for parthenogenetic embryos are theembryos developed from eggs fertilized as a result of delayed matings whichtake place at the time when artificial activation is carried out. However, evenif this correction is taken into account the parthenogenones still fall behindnormal embryos.

3 EMB 35

Tab

le 3

. Pre

impl

anta

tion

de

velo

pmen

t of

Sw

iss

albi

no c

ontr

ol fe

rtil

ized

eg

gs a

nd o

f tr

ansp

lant

ed d

iplo

id p

arth

enog

enic

egg

s

Mat

eria

l

Co

ntr

ol

embr

yos

?$

mat

ed a

tn

igh

t w

ith

Ac?

c?

Co

ntr

ol

embr

yos

$$

mat

ed w

ith

AcJ

c? 7

.00

- 9.

00a.

m.

(del

ayed

mat

ing

s)

Par

then

og

enet

icem

bry

os

Day

of

auto

psy

4th

4th

4th

5th

No.

of

fem

ales

6 7 5 5

No.

of

tran

s-fe

rred

eggs

— — 64 48

i. ^

KJ•

d

ll

mo

rula

e

c To

tal

47 42

18 28-1

%16 33

-3%

(M°)

an

dcy

sts

(B)

A M

14 29-8

%

27 64-3

%

18 12

l/\J

V t

l \~>

\£

blas

to-

B

33 70-2

%

15 35-7

%

0 4

afte

r 24

hcu

ltur

e in

vit

roA

M

B

—

—

—

—

10

8

7 9

Day

of

deve

lop-

men

t

4th

4th

5th

6th

Cel

l nu

mbe

r in

M

< Mea

n±

S.E

.

24-8

±2-

1

23-3

±1-

5

16-9

±2-

22

10

±3-

1

M A

Ran

ge

15-4

7

13-4

5

8-29

14-2

9

and

B a

t B A

Mea

n+

S.E

.

47-2

±2-

3

30-2

±0-

4

29-4

±3-

031

-8±

6-2

the

end

Ran

ge

30-7

8

27-3

2

15-4

1

20-5

3

of e

xper

imen

t

Ove

rall

Mea

n+

S.E

.

40-5

±2-

3

25-7

±1

1

22-4

±2-

427

-0±5

-5

A

Ran

ge

15-7

8

13-4

5

8-41

14-5

3

X w n >—1 w d > H t* o

Tab

le 4

. Im

plan

tati

on

of p

arth

enog

enet

ic e

mbr

yos

in S

wis

s al

bino

pse

udop

regn

ant r

ecip

ient

s

No.

and

% o

f im

plan

tatio

ns

No.

of

No

. of

No

. of

horn

sD

onor

str

ain

tran

sfer

red

eggs

tr

ansf

ers*

w

ith i

mpl

ants

Tot

al 16 7-1

°/

2 4-2%

23 32-9

%

In s

ucce

ssfu

ltr

ansf

ers

16-7

%

14-3

%

34-8

%

No.

of

embr

yos f

3 2 3

No.

of

fem

ales

auto

psie

don

a g

iven

day

12x8

2x

95x

102

x8

1x

91x

102

x8

3x

91x

10

Swis

s al

bino

C57

BL

/1O

226 48 70

19

* E

ggs

wer

e tr

ansf

erre

d ei

ther

int

o on

e or

into

bot

h ov

iduc

ts.

t A

ll e

mbr

yos

wer

e re

tard

ed

in d

evel

opm

ent

and

only

tw

o lo

oked

hea

lthy

(one

A e

mbr

yo r

ecov

ered

on

the

8th

day

and

one

C57

BL

/1O

rec

over

ed o

n th

e 9t

h da

y).

3 I 1


Development after implantation

Altogether 41 implantation swellings were available for examination (Table4). The implantation rate was generally very low but clearly depended on thedonor strain. The highest rate was observed after transplantation of C57BL/10eggs (32-9%); Swiss albino eggs and A eggs implanted only in 7-1% and4-2% respectively.

However, although C57BL/10 eggs were superior to Swiss and A eggs as faras the implantation rate was concerned, their subsequent survival was equallypoor (Table 4). Embryos could not be found in any swellings collected on the9th and 10th day and inspected under the dissecting microscope. In histologicallyanalysed implants eight embryos were found - five on the 8th day and three onthe 9th day. Only two embryos - one A and one C57BL/1O from the 8th and9th day respectively - looked normal and healthy. However, even these twoembryos were retarded, as they were in the stage of an egg-cylinder typical forthe 7th day of normal development (Figs. 6, 7). The remaining embryos wereclearly abnormal and checked in development at the stage of an early 6-daycylinder.

DISCUSSION

Heat shock of 44-0 °C followed by short-term culture in the presence of10 ^g/ml of Cytochalasin B appears to be another effective method of producingdiploid parthenogenetic mouse embryos (cf. Graham, 1972; Graham & Deussen,1974; Kaufman & Gardner, 1974; Kaufman & Surani, 1974). CB suppressessecond polar body formation in nearly 100% of activated eggs. Depending onthe strain used (Swiss albino or C57BL/10) and the way of examination (fixedand stained eggs or living eggs) the percentage of eggs with two pronucleivaried between 80 and 90, and of those with one diploid pronucleus between5 and 16. The incidence of eggs containing small subnuclei as well as one ortwo pronuclei was estimated to be about 10%. Since the same frequencies wereobserved in eggs cultured in CB-free medium, formation of subnuclei must bea result of heat shock rather than CB. These eggs are potentially hypodiploid.We have also observed that in many eggs the two pronuclei were of differentsize, which suggested that at the 2nd meiotic division the chromatids wereunequally distributed. This phenomenon was observed with the help of chromo-somal studies of the first cleavage of eggs activated in vitro with hyaluronidaseand hypotonic shock (Graham & Deussen, 1974). However, when 2P.B. issuppressed, as was the case in the present experiments, aneuploidy of pronucleihas probably no further implications, as diploidy will be restored at the firstcleavage. The fact that all morulae and blastocysts containing analysablemetaphase plates were diploid proves that suppression of 2P.B. due to CB waspermanent and confirms earlier observations that both pronuclei contributechromosomes to one metaphase plate of first cleavage (Kaufman, 1973&;


Witkowska, 1973# and personal communication; Graham & Deussen, 1974).We believe, therefore, that in experiments aimed at inducing diploid partheno-genesis, aneuploidy - strictly speaking hypodiploidy - is much more likely toarise when subnuclei are formed than when there is uneven distribution ofchromatids between the two pronuclei.

The tempo of preimplantation development of transplanted eggs (number ofcleavages and the time of transformation of morulae into blastocysts) was inthe present study rather low and unsatisfactory. There are four factors whichmight have been responsible for this state of affairs. First, damage produced toeggs by heat shock; second, harmful effect of treating the eggs with CB; third,shock imposed on eggs at the one-cell stage by handling and culturing themfor several hours in vitro; and fourth - probably of minor importance - theeffect of second handling and culturing for the last 24 h of the experiment.Although all these four factors might have been important, we believe that thesecond and the third factors were of major importance. Support for this viewcomes from the observations by Niemierko (1975) that when newly fertilizedmouse eggs are subjected to CB in vitro for 5-6 h before being transplanted tothe oviduct, the survival of embryos is affected and the rate of development isclearly delayed so that blastocysts do not form until the 5th day.

The low rate of implantation of the Swiss albino and A eggs was probablydue to different causes. The A eggs were heated for 7-5 rather than 5 min,which might have had an adverse effect on their survival. As far as Swiss albinoeggs are concerned their preimplantation development is generally slow, andthese embryos were handicapped when transferred to the synchronized recipientsof their own strain. In other words, in the combination of transfer 1 -» 1, theparthenogenetic embryos might not have been able to 'catch up' with theuterus, the more so since their preimplantation development was retarded.

Unlike eggs of many other strains including Swiss albino, fertilized C57BL/10eggs develop fast (cf. table 1 in McLaren & Bowman, 1973 and Table 3 in thepresent paper) and probably for this reason the parthenogenetic ones weremore successful in implantation. Additional evidence for this supposition comesfrom the observation that the decidual swellings induced by C57BL/10 partheno-genetic embryos were regularly larger than those induced by Swiss albinoand A embryos.

The inability of diploid parthenogenones to survive in the uterus beyond theegg-cylinder stage remains a mystery. Our present results are concordant withearlier observations on post-implantation development of experimental par-thenogenones presented by Tarkowski et al. (1970), Graham (1972), Witkowska(19736), Mintz & Gearhart (1973) and Kaufman & Gardner (1974) as well asof spontaneous parthenogenones (Stevens & Varnum, 1974). The most relevantto the problem under consideration are the papers by Graham and by Kaufman& Gardner, because these authors dealt exclusively with diploid eggs ratherthan a mixed population of haploids, diploids and n/2n mosaics, so the mortality


of embryos which they observed could not be due to haploidy. As suggestedalready in 1970 by Tarkowski et ah, diploidy per se is not a sufficient conditionfor the survival of parthenogenones beyond the egg-cylinder stage. At presentthere is no satisfactory explanation for the death of these embryos at thisparticular stage (for a more detailed discussion see Graham, 1974; Tarkowski,1975). The possibility that embryonic death is due to lethal genetic factorswhich at this stage start to operate at the cellular level can be rejected becauseboth experimentally produced and spontaneously arising mouse partheno-genones when transplanted to ectopic sites can grow and give rise to variousdifferentiated tissues (Graham, 1970; Stevens & Varnum, 1974; lies et ah 1975).Also, parthenogenetic origin does not affect trophoblast, in that giant cells arepresent in embryos growing in the uterus as well as in ectopic 'growths'.

We wish to thank Mrs A. Szarska for skilful technical assistance and Mr P. Halkiewiczfor preparing photographic documentation.

This work was partly financed by the Zoological Committee of the Section of BiologicalSciences of the Polish Academy of Sciences.

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{Received 16 April 1975, revised 2 July 1975)

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