cytoplasmic polarity develops at compaction in rat and ... · cytoplasmic polarity develops at...

/. Embryol. exp. Morph. Vol. 62, pp. 351-361, 1981 35]Printed in Great Britain © Company of Biologists Limited 1981

Cytoplasmic polarity develops at compaction inrat and mouse embryos

By W. J. D. REEVE1

From the Department of Anatomy, University of Cambridge

SUMMARY

Cells of both rat and mouse morulae can be stained vitally to reveal an asymmetry in theorganization of their cytoplasm. In each cell of the rat 8-ceil embryo a column of organellesdevelops between the nucleus and the embryo periphery as revealed by toluidine blue,acridine orange and horseradish peroxidase (HRP). Although cells of the mouse morula lackthe blatant asymmetric distribution of organelles observed in rat cells, a long pulse (> 3 h) ofHRP to compact 8-cell mouse embryos revealed a distinct restricted localization of theenzyme not evident at earlier pre-compaction stages. The cytoplasmic polarity generated inthese embryos can be demonstrated in cells of intact embryos, and also in cells disaggregatedfrom embryos before vital staining.

INTRODUCTION

The polarization hypothesis (Johnson, Pratt & Handyside, 1981) proposedrecently to explain the generation and maintenance of spatial differentiation inthe early preimplantation mouse embryo postulates the establishment in the8-cell embryo of a radial asymmetry which is expressed in individual cells in theform of an axial polarity. The generation of surface polarity in cells of the 8-cellmouse embryo has been shown already (Handyside, 1980; Ziomek & Johnson,1980; Reeve & Ziomek, 1981). This study demonstrates that cells of 8-cell ratand mouse embryos also show a cytoplasmic polarity absent at earlier stages.

The existence of localized cytoplasmic material in the mammalian embryohas long been proposed (Dalcq, 1957). Vital staining with toluidine blue(Izquierdo, 1955) and acridine orange (Austin & Bishop, 1959), and studies bytransmission electron microscopy (TEM) (Izquierdo & Vial, 1962; Schlafke &Enders, 1967) have shown a very clear reorganization of cytoplasmic componentsat the 8-cell-stage of the rat embryo. Thus, whereas organelles of earlier stageshave a relatively uniform cytoplasmic distribution (Sotelo & Porter, 1959),those of the 8-cell embryo are restricted largely to a column extending from thenucleus of each cell to the embryo periphery (Izquierdo & Vial, 1962; Schlafke& Enders, 1967; Dvorak, 1978). In contrast, the mouse embryo has been re-ported to display no such overt cytoplasmic polarization (Calarco & Brown,

1 Author's address: Department of Anatomy, Downing Street, Cambridge CB2 3DY, U.K.12-2

352 W. J. D. REEVE

1969), although microtubules may orientate parallel to areas of cell contact, andmitochondria occupy the cortical region (Ducibella, Ukena, Karnovsky &Anderson, 1977). This report confirms that in the mouse 8-cell embryo, unlikethat of the rat, no blatant asymmetry is observed in the pattern of staining bytoluidine blue and acridine orange. In contrast, long pulses of horseradishperoxidase (HRP) did result in a polarized distribution of ingested enzyme in thecytoplasm of cells of compact morulae of both rat and mouse.

MATERIALS AND METHODS

Embryo collection

Female HC-CFLP mice (4-5 weeks; Hacking & Churchill) were super-ovulated with intraperitoneal injections of 5 i.u. of pregnant mare's serum(PMS: Folligon, Intervet), followed after 44-48 h by 5 i.u. of human chorionicgonadotrophin (hCG: Chorulon, Intervet). Females were paired with HC-CFLPmales, and vaginal plugs taken as an indication of mating. Mice were killed bycervical dislocation and embryos were flushed from the oviducts, at times between49 and 68 h post-hCG, with phosphate-buffered medium 1 supplemented with0-4 % (w/v) bovine serum albumin (PB1 +0-4 % BSA) (Whittingham & Wales,1969), and were cultured at 37 °C in medium 16 with 0-4 % (w/v) BSA (Whit-tingham, 1971) in 5 % CO2 in air.

Female Wistar rats (250 g body weight; Olac Limited) were paired withWistar males, and examined the next morning when spermatozoa in the vaginalsmear indicated day 1 of the pregnancy. Rats were killed by an ether overdose,and embryos flushed from the oviducts with PB1+0-4% (w/v) BSA or pre-gassed culture medium, kept below pH 7-4. Four-cell embryos were obtained lateon day 3, and 8-cells during the afternoon of day 4. Embryos were cultured ineither a modification of the standard mouse medium of Biggers, Whitten &Whittingham (1971) with 25 % (v/v) foetal calf serum (Mayer & Fritz, 1974) orin T6' modified Tyrode's medium with 0-1 % (w/v) BSA (Wood & Whittingham,1980).

Zonae pellucidae of both rat and mouse embryos were removed by a 15- to30-sec incubation in prewarmed (37 °C) acid Tyrode's solution (pH 2-5) + 0-4 %(w/v) polyvinylpyrrolidone (Nicolson, Yanagimachi & Yanagimachi, 1975).

Disaggregation

Disaggregation into single cells was accomplished by pipetting embryos witha flame-polished micropipette after incubation in either trypsin/EDTA orcalcium-free medium, both pre-equilibrated for at least 30 min at 37 °C in 5 %CO2 in air. After disaggregation, cells were restored immediately to the culturemedium. The disaggregating media were as follows.

(i) Trypsin/EDTA

Rat embryos were incubated in 0-5% (w/v) trypsin + 0-2% (w/v) EDTA

Cytoplasmic polarity at compaction in rat and mouse embryos 353

(Gibco) in calcium-free medium 16. After 5 min, when decompaction wascomplete, the medium was drawn off and replaced with a large volume ofeither PB1 +0-4 % (w/v) BSA, or pregassed culture medium.

(ii) Calcium-free medium

Mouse embryos were incubated for 10-30 min in calcium-free medium 16 +0-6 % (w/v) BSA.

Toluidine blue

Cells or embryos were placed in a 1/50000 (w/v) solution of toluidine blue inTyrode's medium for 20 min before fixation in a solution formed from water andsaturated aqueous solutions of mercuric chloride, phosphotungstic acid andammonium molybdate in the volumetric ratio of 6:3:1:2 (Izquierdo, 1955).After fixation for at least 1 h, embryos and cells werewashed in water, dehy-drated through a graded alcohol series, cleared in toluene, and mounted inDepex (Gurr Limited).

Acridine orange

Embryos and cells were incubated in a range of concentrations of acridineorange (Gurr Limited) between 1/20000 (w/v) and 1/250 (w/v) in PB1 + 0-4 %(w/v) BSA. The fluorescent staining pattern was observed directly, and 1/1000(w/v) acridine orange gave optimum differential fluorescence. The viability ofcompacted 8-cell embryos (67 h post-hCG) was examined by culture, eithercontinuously or for 2 h followed by restoration to control medium, in 1/1000(w/v) acridine orange in medium 16 + 0-4% (w/v) BSA at 37 °C in 5 % CO2

in air.

Horseradish peroxidase (HRP)

Embryos and cells were incubated in 2 mg/ml HRP (Sigma Type II) inmedium 16 + 0-4% (w/v) BSA (mouse) or T6' modified Tyrode's medium +0-1 % (w/v) BSA (rat) at 37 °C for 3-10 h in 5 % CO2 in air. Cells were rinsedin PB1 +0-4 % (w/v) BSA, and fixed in 4 % (w/v) paraformaldehyde (Anderson& Co. Ltd) in phosphate-buffered saline (PBS) at 4 °C for 1 h, before furtherwashing and storage in PB1 +0-4 % (w/v) BSA at 4 °C. Cells were stained forHRP by the aminoethylcarbazole (AEC; Sigma) method (Pearse, 1968). Twomg AEC were dissolved in 10 ml 50 mM acetate buffer pH 5-0 to which a drop of3 % (v/v) hydrogen peroxide was added immediately before use. After stainingfor 10-60 min, rat embryos were mounted in glycerine jelly (Cavanaugh, 1964),while, for better resolution, mouse cells were examined in wells of a tissue-typing slide (Baird & Tatlock) in drops of PB1 +0-4 % (w/v) BSA under oil.

354 W. J. D. REEVE

Indirect immunofluorescence

HRP-treated embryos were incubated for 5 min in 25 fi\ drops of a rabbitantiserum (RAMS) to mouse species antigens (Gardner & Johnson, 1975;Handyside, 1980) diluted 1 in 15 in PB1+O4% (w/v) BSA + 0-02% (w/v)sodium azide, followed by thorough washing in PB1+BSA + azide, and asimilar incubation in fluorescein-conjugated goat anti-rabbit IgG (FITC-GARigG; Miles Labs) diluted 1 in 15 in PB1+BSA + azide. The embryos werewashed again, and disaggregated into single cells which were fixed in 4 % (w/v)paraformaldehyde in PBS at 4 °C for 1 h, before being stained for HRP.

Immunosurgery

Inner cell masses (ICMs) were isolated by immunosurgery (Solter & Knowles,1975). Blastocysts were incubated for 5 min in RAMS (diluted 1:10 with PB1)at 37 °C, washed extensively in PB1 +0-4 % (w/v) BSA, and incubated for 30min in guinea-pig complement (Flow Labs.) (diluted 1:10 with PB1) at 37 °C.After thorough washing, the inner cell masses were separated from the lysedtrophectodermal cells by drawing the embryos through a finely pulled Pasteurpipette.

Cell counting

Embryos and inner cell masses were prepared and fixed according toTarkowski (1966). Nuclei were stained with a millipored 1 % (w/v) suspensionof Giemsa (Raymond A. Lamb). Cell numbers were not recorded for the verysmall minority of spreads which showed either excessive clustering or dispersionof nuclei.

Light microscopy

A Zeiss Universal microscope, which was fitted with incident source HBO50, III RS condenser and Zeiss filter set 487709, was used to examine cells foracridine orange staining and HRP-stained cells for FITC-labelling. KodakTri-X 35 mm film was used for both bright-field and fluorescence photography.Cells mounted in Depex or glycerine jelly were examined with a Zeiss UltraphotII microscope, and photographed on Pan F film.

Transmission electron microscopy

Embryos were fixed for 1 h at room temperature in 2-5 % (v/v) glutaraldehydein 0-1 M sodium cacodylate buffer at pH 7-4. Embryos were washed with thebuffer, and postfixed in 1 % (w/v) osmium tetroxide in 0-1 M sodium cacodylatebuffer. After dehydration through a graded alcohol series, embryos were in-filtrated and embedded in Spurr resin (Spurr, 1969). Sections, 50 nm thick, werecut with a glass knife, and stained with uranyl acetate (Gibbons & Grimstone,1960) followed by lead citrate (Reynolds, 1963). Sections were viewed in aSiemens Elmiskop I microscope.


RESULTS

1. Toluidine blue

Vital staining of rat embryos with toluidine blue revealed uniform cytoplasmicstaining in 4-cell blastomeres (Fig. 1) and a polarized pattern in 8-cell blasto-meres (Fig. 2). The metachromasia with toluidine blue is considered by Izquierdo& Vial (1962) to be associated with vesicles which are rich in mucopolysac-charides and acid hydrolases (Stastna, 1974). At the 8-cell stage there is asegregation of organelles into a column extending from the nucleus of each cellto the embryo periphery (Fig. 3). Five different staining patterns were identifiedin single cells disaggregated from embryos and then stained (Fig. 4), Whenclassified according to staining pattern, cells from 8-cell embryos showed a highincidence of cytoplasmic polarity compared with cells from 4-cell embryoswhich lacked cytoplasmic columns (Table 1). However, when all cells of indi-vidual embryos were examined, non-polarized cells were detected in manyembryos in which the majority of cells were polarized (W. J. D. Reeve, un-published observations). Culture of both intact embryos and single cells invitro before staining resulted in a slight decrease in the incidence of polarity(Table 1). Tn contrast to the rat embryo, cells from 8-cell mouse embryos stainedwith toluidine blue offered only a hint of polarity which was lost after fixation.

2. A cridine orange

Acridine orange stains the DNA of the nucleus green, and lysosomes andribonucleic acids are considered to be associated with the orange cytoplasmicstaining (Austin & Bishop, 1959; Allison & Young, 1969). Staining of intact8-cell rat embryos and their dissociated cells revealed asymmetries of cytoplasmsimilar to those observed with toluidine blue. After being stained with acridineorange, dissociated rat cells were sorted on the basis of cytoplasmic polarization,and then further stained with toluidine blue. The two stains were localized insimilar patterns. Thus, of 16 cells which stained with acridine orange to reveala column stretching from the nucleus to the cell surface, all showed columnsafter staining with toluidine blue; seven cells had nuclear caps or ringswhen stained with both acridine orange and toluidine blue; and of four cellsshown by acridine orange to have a scattered distribution of organelles, stainingwith toluidine blue showed two to have nuclear caps, one to have a uniformstaining pattern and one to be unscorable. In contrast, only a minority ofdissociated cells of both pre-compact and compact mouse embryos revealed anasymmetry of cytoplasmic staining with acridine orange over a range of con-centrations. Just 16 % of 53 cells from pre-compact 8-cell embryos and 9 % of64 cells from compact 8-cell embryos revealed a columnar cytoplasmic stainingpattern. There was a similar low incidence of cytoplasmic stain concentrated innuclear caps, nuclear rings and dispersed cytoplasmic aggregates. In contrast,

3561

W. J. D. REEVE2

it-

50 50

Fig. 1. The 4-cell rat embryo shows uniform cytoplasmic staining after immersion intoluidine blue.Fig. 2. An 8-cell rat embryo stained with toluidine blue reveals a column extendingfrom the nucleus of each cell to the embryo periphery.Fig. 3. The region of intense staining corresponds to columns of organelles seen inTEM (x 17000), N, nucleus; Z, zona pellucida; C, column of organelles.


4a 4b Ac

f

4c/ 4e

, 30 Mm

Fig. 4. Rat 8-cell embryos were disaggregated to single cells and stained with tolu-idine blue. Five categories of staining pattern were recognized, (a) Tight column.Metachromasia extends in band (width less than nuclear diameter) from the nucleusto the periphery of the cell, (b) Loose column. The column is wider than the nucleus.(c) Nuclear cap. The metachromasia extends less than half-way from the nucleus tothe cell periphery, (d) Nuclear ring. Metachromasia surrounds the nucleus, (e)Uniform stain. Stain occurs throughout the cytoplasm.

53 % and 66 % of the cells from pre-compact and compact 8-cell embryos,respectively, showed a uniform cytoplasmic pattern of staining.

3. Horseradish peroxidase

Incubation with HRP revealed a polarized HRP accumulation in compact ratembryos (Fig. 5) and a similar pattern was also found in cells from compactmouse morulae (Figs 8, 9). All cells in any one embryo showed very similarpatterns of stain distribution. Thus the staining patterns of intact embryos wereclassified as either uniform, localized or aggregated. Embryos showing alocalized pattern of staining appeared to have one restricted mass of stain nearthe nucleus of each cell, while embryos classed as aggregated appeared to have amore diffuse distribution of HRP-containing vesicles. Mouse embryos, whichwere either 4-cells (Fig. 6) or pre-compact 8-cells (Fig. 7) at the termination ofpulses of HRP of up to 10 h duration, did not show a restricted localization ofHRP-containing vesicles. The incidence of localized HRP-containing vesicles

Tab

le 1

. C

ytop

lasm

ic s

tain

ing

patt

erns

w

ith

tolu

idin

e bl

ue o

f ce

lls

diss

ocia

ted

from

ra

t em

bryo

s be

fore

sta

inin

goo

Mea

n n

um

ber

of c

ells

per

embr

yo ±

S.D

.

Cyt

opla

smic

sta

inin

g*

Tre

atm

ent

Nu

mb

er o

f T

ight

L

oose

N

ucle

ar

Nuc

lear

%

Uns

core

d(n

umbe

r of

em

bryo

s)

scor

ed c

ells

co

lum

n co

lum

n ca

p ri

ng

Uni

form

or

los

t ce

lls

% P

olar

ized

f

Imm

edia

te d

isag

greg

atio

n 4.

0 ±

0 37

0

0 0

38

62

8 38

and

stai

ning

(1

0)

Inta

ct e

mbr

yos

cult

ured

4.

0 ±

0 35

0

0 12

14

74

3

26in

vit

ro 1

0 h

befo

re

(9)

disa

ggre

gati

on a

nd

stai

ning

Imm

edia

te d

isag

greg

atio

n 8.

0 ±

1.3

43

1 19

47

12

8

14

21

86an

d st

aini

ng

(81)

Sin

gle

cell

s cu

ltur

ed

8.4

±1

.6

143

11

37

18

15

19

33

81in

vit

ro 7

h b

efor

e st

aini

ng

(33)

Inta

ct e

mbr

yos

cult

ured

9.

6 ±

3.1

23

1 7

23

22

16

32

27

68in

vit

ro 7

h b

efor

e (3

3)di

sagg

rega

tion

an

d st

aini

ng

Inta

ct e

mbr

yos

reta

ined

14

.1 ±

2.6

80

10

44

18

21

7

19

93in

viv

o fo

r 7

h be

fore

(7

)di

sagg

rega

tion

an

d st

aini

ng

* U

se o

f th

ese

stag

ing

crit

eria

may

cau

se a

n un

dere

stim

ate

of t

he i

ncid

ence

of

pola

riza

tion

sin

ce a

cyt

opla

smic

col

umn

perp

endi

cula

r to

the

foca

l pl

ane

can

no

t al

way

s be

ide

ntif

ied.

t %

Pol

ariz

ed =

%

of

scor

ed c

ells

whi

ch l

acke

d a

unif

orm

pat

tern

of

cyto

plas

mic

sta

inin

g.

w


increased in the more overtly compact embryos (Table 2). Whereas 64 % of pre-compact 8-cell embryos had cells which showed a uniform distribution of HRP-containing vesicles, only 14 % of compact embryos had cells which did so.

Disaggregation of pre-labelled individual compact embryos showed thatrestricted localization can occur in all 8 cells of an embryo, although one or twocells in any one embryo may not appear polarized. Double staining with HRPand fluorescent antibody demonstrated that the localization of HRP lies underthe fluorescent pole described previously (Fig. 9) (Handyside, 1980; Ziomek &Johnson, 1980; Reeve & Ziomek, 1981). The incidences of the different patternsof localization of HRP-containing vesicles (Fig. 10) were similar for cellslabelled in situ before disaggregation, and for cells from embryos completelydisaggregated before incubation with HRP (Table 3).

4. Viability of embryos

Mouse embryos cultured in toluidine blue lysed within 1 h, and acridineorange also proved harmful to development. Compacted 8-cell embryos (67 hpost-hCG), whether exposed to acridine orange for 2 h or continuously, retaineda compact appearance at 100 h post-hCG when control embryos were expandedblastocysts. At 125 h post-hCG, all embryos pulsed earlier for 2 h with acridineorange had formed abnormal blastocysts with extruded cells, while morulaestill in acridine orange had lysed. Prolonged culture in HRP did not affectdevelopment so adversely (Table 4).

DISCUSSION

Cells of rat and mouse 8-cell embryos show evidence of a cytoplasmic polarityabsent at earlier stages of development. The results for the rat embryo confirmand extend previous observations, but hitherto no equivalent polarity in cellsof the mouse embryo has been described.

There is abundant cytological evidence from light and electron microscopicstudies on rat embryos to support the results obtained by staining with toluidineblue and acridine orange. In the rat 4-cell embryo, organelles lose the scattereddistribution of earlier stages (Sotelo & Porter, 1959), and tend to localize at theperiphery and around the nucleus (Mazanec & Dvorak, 1963; Schlafke &Enders, 1967; Stastna, 1974). At the 8-cell stage, each cell shows a definitecytoplasmic segregation involving the organization of almost all organelles intoa column extending from the nucleus to the embryo periphery. The columncontains most of the mitochondria, the small regions of the Golgi apparatus, theendoplasmic reticulum (mostly agranular) and a heterogeneous assortment ofvesicles (Izquierdo & Vial, 1962; Schlafke & Enders, 1967; Stastna, 1974). Thesecytoplasmic columns are revealed by vital staining with both toluidine blue(Fig. 2) (Tzquierdo, 1955) and acridine orange (Austin & Bishop, 1959). Outsidethe column, extensive areas of more dense homogeneous cytoplasm contain a

360 W. J. D. REEVE

\

50 um

'v.

50

50 50

, 30 um

f f0 30 Mm .


Table 2. Incidence of HRP staining patterns in intact %-cell mouse embryos

Stage ofembryos

Pre-compact

Peri-compact

Compact

* All cells in any

Totalnumber ofembryos

91

69

65

one embryo

Uniform(%)

58(64)29

(42)9

(14)

Staining pattern*Cytoplasmicaggregates

(%)

21(23)25

(36)20

(31)

showed very similar patterns of stain

Restrictedlocalization

(%)

12(13)15

(22)36

(55)

distribution.

few mitochondria and vesicles, and large amounts of proteinaceous lamellaeconsidered to be storage material used in cleavage (Dvorak et al. 1975; Dvorak,Travnik & Stankova, 1977). The columns are stable, and persist even in isolatedcells cultured for several hours (Table 1). There are, however, problems in theuse of the rat embryo for a dynamic study of polarization. First, the supply ofembryos is limited as superovulation in the rat is not an established technique.Second, published data on the culture of preimplantation rat embryos isscarce, and successful culture difficult. Although culture of 8-cell embryos toblastocysts has been reported as 80 % or more successful (Folstad, Bennet &Dorfman, 1969; Mayer & Fritz, 1974), the media used by these authors and alsothe T6' medium of Wood & Whittingham (1980) never gave a success rate above60 % (W. J. D. Reeve, unpublished results). Lastly, development in vitro of therat embryo over the 4-cell stage is particularly difficult (Suzuki & Iizuka, 1969;Mayer & Fritz, 1974), thus preventing culture over the period in which cyto-plasmic polarity is generated.

The compact mouse embryo lacks the blatant cytoplasmic segregationdescribed for the rat 8-cell embryo when examined by TEM (Calarco & Brown,

FIGURES 5-9

Fig. 5. A rat morula stained with HRP to show cytoplasmic polarity. Individual cellswhich show a polarized distribution of HRP are arrowed. (The other cells also showa restricted localization of the enzyme, but are not in focus.)Fig. 6. A mouse 4-cell embryo shows a uniform cytocortical distribution of HRP.Fig. 7. A pre-compact 8-cell mouse embryo shows widespread distribution of HRP.Fig. 8. A compact 8-cell mouse embryo shows the restricted cytoplasmic localizationof HRP peripheral to the nucleus of each cell. Two cells are arrowed.Fig. 9. Cells of an 8-cell mouse embryo stained with (a) HRP and (b) fluorescentligand before embryo disaggregation. Each pole of fluorescent ligand-binding over-lies the restricted cytoplasmic localization.

362 W. J. D. REEVE

Table 3. Incidence of staining patterns in cells of compact S-cell mouse embryosstained with HRP (68-72 h post-hCG) before or after disaggregation

Total Tight Loosecell local- local- Nuclear

Treatment number ization ization ring Aggregates Uniform Unscorable

Disaggregationbefore staining

Disaggregationafter staining

81

80

21

25

26

23

8

7

17

18

6

3

3

4

1969), although showing a surface polarization of microvilli (Ducibella et al.1977; Reeve & Ziomek, 1981), numerous microtubules orientated parallel to theapposed membranes of blastomeres, and mitochondria localized to the cortex(Ducibella et al. 1977). Neither toluidine blue nor acridine orange stainingpatterns provided conclusive evidence of cytoplasmic polarity in blastomeres ofcompact 8-cell mouse embryos.

In contrast, when HRP was used as a vital stain, cells of both rat and mouse8-cell embryos showed a pronounced cytoplasmic polarity. HRP differs fromacridine orange and toluidine blue in its active uptake by cells, and its depend-ence on cellular mechanisms for transport, ultimate localization and meta-bolism. It has proved a useful tracer in studies of endocytosis (reviewed Silver-stein, Steinman & Cohn, 1977) as it is non-toxic and its enzymic activity can bedemonstrated histochemically (Graham & Karnovsky, 1966). HRP uptake is byfluid pinocytosis, and washing before fixation ensures that adsorbed enzyme isremoved from the cell surface (Steinman & Cohn, 1972; Steinman, Silver &Cohn, 1974). In published data on preimplantation rabbit (Hastings & Enders,1974) and rat (Schlafke & Enders, 1972) embryos, HRP pulses never exceeded60 min, and provided mostly information on uptake patterns at the cell surface.Few vesicles were observed before the 8-cell stage, and endocytosis increasedby the blastocyst stage, at which vesicles were restricted mainly to the supra-nuclear region.

Four-cell mouse embryos showed a uniform distribution of HRP reactionproduct in the cytocortex, but poor cytoplasmic staining, after prolonged pulsesof HRP (Fig. 6). However, after pulses as short as 3 h, some 8-cell embryos wereshown to have localization of reaction product between the nucleus of each celland the embryo periphery (Figs. 8, 9). The increase in the incidence of dispersedaggregates and of restricted localization of HRP stain in cells was associated withcompaction (Table 2). The restricted localization of HRP-containing vesiclesshown by a minority of pre-compact 8-cell embryos is consistent with the genera-tion of surface polarity before overt cell flattening (Ziomek & Johnson, 1980;Reeve & Ziomek, 1981). Interestingly, in the rat embryo, compaction as assessed

Tab

le 4

. T

oxic

ity

of H

RP

for

cu

ltur

ed 2

-cel

l m

ouse

em

bryo

s (4

9 h

post

-hC

G)

Cul

ture

con

ditio

ns

Con

trol

+ H

RP

Initi

al n

umbe

r2-

cell

embr

yos

31 29

Num

ber o

fbl

asto

cyst

s

26 17

*IC

M/b

last

o

Bla

stoc

yst

mea

n ce

ll no

. ± S

.D.

(no.

bla

stoc

ysts

)

84-5

± 3

2-2

05

)68

-0 ±

18

1(9

) mea

n ce

ll i

Inne

r ce

ll m

ass

(IC

M)

mea

n ce

ll no

. ± S

.D.

(no.

IC

Ms)

19-5

±7-

2(1

1)21

-3 ±

6-8

(8)

num

ber

of I

CM

s~J "*

m

ean

cell

num

ber

of b

last

ocys

ts.

ICM

/bla

stoc

yst*

0-23

0 31

Out

grow

th

V V

•§ r Hi s

364 W. J. D. REEVE

10a 106

• * »

10c 10c/ 10e

30 urn

Fig. 10. Dissociated cells of mouse 8-cell embryos showed several patterns of locali-zation of HRP vesicles, (a) Tight localization, (b) Loose localization, (c) Nuclearring, (d) Aggregates, (e) Uniform.

by cell flattening occurs at the 4-cell stage, although the cytoplasmic organellesbecome organized into columns only at the 8-cell stage. The polarity of HRPlocalization in the intact 8-cell mouse embryo does not depend on differences inthe area of exposed cell surface, since it is also shown by dissociated cells in-cubated in HRP (Table 3).

There is no obvious ultrastructural basis for the restricted localization ofHRP stain. The HRP reaction product is thought to coincide with the Golgiapparatus (Steinman et al. 1974; Piasek & Thyberg, 1979), but information onthe Golgi apparatus of the rodent embryo is confined almost entirely to the rat.The quantity of Golgi changes little during cleavage stages (Dvorak et al. 1977).


Although early cleavage stages were shown to have Golgi zones near both thecell membrane and nucleus, in the 8-cell embryo the membranous componentsare aggregated and small Golgi zones are present throughout the organelle-richregions of cytoplasm (Schlafke & Enders, 1967). However, Stastna (1978)reported no obvious relationship between the Golgi apparatus and the nucleus orblastomere surface in cleavage-stage embryos. The Golgi complex of the pre-implantation mouse embryo is not prominent. The occasional small regions ofstacked cisternae in early cleavage stages develop to larger regions of stackedcisternae in the morula (Calarco & Brown, 1969). The Golgi complex of theblastocyst of both the rat (Schlafke & Enders, 1963, 1967; Stastna, 1972, 1974,1978) and the mouse (Calarco & Brown, 1969; Nadijcka & Hillman, 1974)embryo is located in a juxta-nuclear position.

The demonstration that mouse 8-cell blastomeres are cytoplasmically polarizedon an identical axis to the surface polarity already described (Handyside, 1980;Ziomek & Johnson, 1980; Reeve & Ziomek, 1981) suggests a reorganization ofcell structures and function at this developmental stage. Such a reorganization isconsistent with the first postulate of the polarization hypothesis (Johnson, Pratt& Handyside, 1981) which suggests that the operation of axial polarity in 8-cellblastomeres involves the allocation of ICM- and trophectoderm-like propertiesto basal and apical portions, respectively, of the cell. Distinct cell lineages couldthen be generated by subsequent divisions of the polarized cells. An asymmetriccellular distribution at division has already been demonstrated for features ofsurface polarization (Johnson & Ziomek, 1980), and the stability and lack oftoxicity of HRP may permit a similar analysis of the conservation of cytoplasmicpolarity.

I am grateful to John Bashford and Ian Edgar for technical help, and to Drs M. H. Johnson,C. A. Ziomek and A. H. Handyside for valuable discussion. This work was supported bygrants from the Medical Research Council and the Ford Foundation to Dr Johnson, and byan MRC research studentship.

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{Received 1 August 1980, revised 20 October 1980)

cytoplasmic polarity develops at compaction in rat and ... · cytoplasmic polarity develops at...

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